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Influenza vaccination in primary and secondary immunodeficienciesvan Assen, Sander

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https://research.rug.nl/en/publications/0ca1a54a-cc06-4ad0-8c17-42343c564e0d

Influenza vaccinationin primary and secondary

immunodeficiencies

Layout: Luite van AssenPrint: Ipskamp Drukkers, Enschede, The Netherlands

ISBN (printed): 978-90-367-5035-6ISBN (digital): 978-90-367-5034-9©2011 Sander van Assen

Financial support for studies in this thesis (in alphabetical order):Abbott B.V., EULAR, Jan Kornelis de Cock Stichting, Roche Nederland B.V.

Financial support for publication of this thesis (in alphabetical order):Abbott B.V., Baxter, BD Biosciences, Boehringer-Ingelheim B.V., Gilead Sciences Netherlands B.V., GlaxoSmithKline, GUIDE, MSD, Roche Nederland B.V., Sanofi-Pasteur MSD N.V., Sanquin, ViiV Healthcare

RIJKSUNIVERSITEIT GRONINGEN

Influenza vaccination in primary and

secondary immunodeficiencies

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. E. Sterken,

in het openbaar te verdedigen op

woensdag 21 september 2011

om 16:15 uur

door

Sander van Assen

geboren op 24 september 1970

te Leeuwarden

Promotor: Prof. dr. C.G.M. Kallenberg

Copromotores: Dr. M. Bijl Dr. A. de Haan

Beoordelingscommissie: Prof. dr. J.W.J. Bijlsma Prof. dr. J.T. van Dissel Prof. dr. H.G.M. Niesters

Paranimfen: Sander van der Beek Arto Boeken Kruger

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Contents

Chapter 1 Introduction

Part 1 Influenza vaccination in patients with humoral primary immunodeficiency

Chapter 2 Patients with humoral primary immunodeficiency do not develop protective anti-influenza antibody titers after vaccination with trivalent subunit influenza vaccine Clinical Immunology 2010 Aug;136(2):228-235

Chapter 3 Cell-mediated immune responses to inactivated trivalent influenza-vaccination are decreased in patients with common variable immunodeficiency Clinical Immunology. In press

Part 2 Influenza vaccination in patients with auto-immune inflammatory rheumatic diseases

Chapter 4 Studies of cell-mediated immune responses to influenza vaccination in systemic lupus erythematosus Arthritis & Rheumatism 2009 Aug;60(8):2438-2447

Chapter 5 Effect of a second, booster, influenza vaccination on antibody responses in quiescent systemic lupus erythematosus: an open, prospective, controlled study Rheumatology (Oxford) 2009 Oct;48(10):1294-1299

Chapter 6 Humoral responses after influenza vaccination are severely reduced in patients with rheumatoid arthritis treated with rituximab Arthritis & Rheumatism 2010 Jan;62(1):75-81

Chapter 7 Polyclonal and influenza-specific cell-mediated immune responses are hampered in rheumatoid arthritis patients treated with rituximab Submitted

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Part 3 eULAR recommendations for vaccination in patients with auto-immune inflammatory rheumatic diseases

Chapter 8 Vaccination in adult patients with auto-immune inflammatory rheumatic diseases: a systematic literature review for the European League Against Rheumatism evidence-based recommendations for vaccination in adult patients with auto-immune inflammatory rheumatic diseases Autoimmunity Reviews 2011 Apr;10(6):341-352

Chapter 9 European League Against Rheumatism recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases Annals of Rheumatic Diseases 2011 Mar;70(3):414-422

Chapter 10 Summary, general discussion and future perspectives

Chapter 11 Nederlandse samenvatting Dankwoord List of selected publications

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CHAPT

ER 1INTRODUCTION

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INTRODUCTIONVaccination has been one of the most effective measures to reduce mortality [1, 2]. It has led to the eradication of naturally occurring smallpox and the control of diphtheria, tetanus, pertussis, yellow fever, Haemophilus influenzae b, poliomye-litis, measles, mumps, rubella, typhoid and rabies [2]. The aim of vaccination is to prevent or mitigate diseases by artificially inducing immunity. Active vaccination triggers the immune system to provide (protective) humoral and cell-mediated immune responses against the vaccine-preventable disease, while passive vac-cination refers to the administration of exogenously produced antibodies for pro-tection.

History of vaccination

In 430 BC the Greek already knew that survivors of the plague were resistant to the plague when a new epidemic occurred [3, 4]. In the 10th century the Chinese transferred dried material from smallpox pustules to susceptible individuals in order to prevent them from contracting smallpox. This was done by putting a cotton with the dried material from a pustule in the nostrils. In India non-immune children were wrapped in pox-laden blankets from children suffering from small-pox during a mild smallpox epidemic, in order to transmit a less virulent smallpox virus to the non-immune children. Also subcutaneous instillation of fresh matter from a ripe smallpox pustule in an incision made in a non-immune person’s fo-rearm, referred to as inoculation or variolation, was performed. The latter term was derived from the Latin word varius (meaning “stained”) or varus (meaning “mark on the skin”) [2, 4, 5].

After being practiced in China, India and Africa, in the 17th century variolation was introduced in the Turkish Ottoman Empire, possibly by Circassian traders or Circassian women, who were in great demand in the Sultan’s harem for their extreme beauty [5].

In the beginning of the 18th century variolation was introduced in Europe. Lady Mary Worthley Montagu, the wife of the British ambassador of “The Sublime Por-te” (the Ottoman government in Constantinople) Lord Worthley Montagu, noti-ced during her stay in Turkey in 1717 that the smallpox was much less of a threat than in the UK and attributed this to variolation, or “engrafting”. She had great respect for the disease as her brother had died of it at the age of 20, while she herself survived the illness, but at the cost of permanent scars on her face. To her friend she wrote: ‘the smallpox, so fatal, and so general amongst us, is here enti-rely harmless, by the intervention of engrafting, which is the term they give it…’. Although several years earlier two Ottoman inoculators of Greek origin, Emma-

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nouel Timonius and Jacobus Pylorinos, send papers on variolation to the Royal Society in 1713 and 1716, respectively, it was Lady Montagu who popularized vari-olation in Europe. After variolation of her daughter by Charles Maitland, who also variolated her sons some years earlier in Turkey, and of several others, many members of the upper class had themselves variolated. Although skepticism remained among British doctors, variolation received official approval from the Royal College of Physicians. In 1757, at the age of 8 years, a boy called Edward Jen-ner was inoculated in Gloucester [5].

The same Edward Jenner, a country doctor, was elected a Fellow of the Royal Society in 1789 for his work on cuckoos: he demonstrated that the newly-hatched cuckoo ejected the eggs and nestlings of its foster-parents. In 1796 Jenner started his experiments on inoculating persons with cowpox in order to prevent them from smallpox. Cowpox was removed from the hand of the dairymaid Sarah Nelmes and Jenner inserted this in two superficial incisions in the arm of a boy named James Phipps. Six weeks later the boy was variolated with freshly remo-ved smallpox: no disease ensued, even when the challenge was repeated after several months [3]. Jenner published his findings at his own costs, since the Presi-dent of the Royal Society rejected his manuscript with the addition that he should be concerned for his reputation and his colleague’s esteem [6]. Hereafter his me-thods of vaccination were adopted in most European countries and subsequently in the USA. Although Jenner deserves credits for carrying out the experiments to test the hypothesis that vaccination with cowpox protects for smallpox, he was neither the first who noticed the protective effect of cowpox infection, nor the first to vaccinate with cowpox. The protective effect of cowpox was already known to farmers from North Dorset, England, who observed that dairymaids did not contract smallpox after contracting cowpox from handling cows’ udders. Knowing this, the cattle breeder Benjamin Jesty deliberately infected his wife and two children with cowpox using his wife’s knitting needle in order to protect them for smallpox, and he proved his right when even after several exposures to smallpox his boys did not acquire the disease. This was 22 years before Jenner performed his experiments. These were the first recorded vaccinations. In 1829 John Fosbroke, surgeon, wrote that if Jenner did not have had fortune, fame and high alliance, his merit would have been crushed or faintly supported [7, 8]. Jen-ner was awarded grants by the House of Commons in 1802 of 10.000 pounds and of 20.000 pounds in 1807 for his discovery of vaccination for smallpox. However, during the committee’s deliberations, it was pointed out that inoculation had been practiced previous to Edward Jenner’s experiments. Therefore, the Original Vac-cine Pock Institution invited Benjamin Jesty in 1805. He was cross-examined and presented with a long testimonial and pair of gold mounted lancets. The verbal

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evidence of their examination was published in the Edinburgh Medical and Surgi-cal Journal [2, 4, 7].

After vaccination for smallpox, several other vaccines were developed. It took until the forties of the 20th century until an influenza vaccine was available. An influenza vaccine was most welcome, since the influenza pandemic occurring in 1918, the so-called “Spanish Flu”, led to an estimated 40 million deaths worldwide. Also, many soldiers fighting in World War I became victim of the influenza pande-mic and therefore the US army supported the development of a vaccine.

However, to be able to develop an influenza vaccine, researchers had to deter-mine the causative micro-organism of influenza, which was initially thought to be bacterial. In 1892 Pfeiffer discovered a new organism from the secretions of flu patients, named by Pfeiffer Bacillus influenzae (later Haemophilus influenzae), by others Pfeiffer’s bacillus. Since this organism could only be isolated from a mi-nority of flu patients during the 1918 pandemic, the causal relation with influenza was rejected. The same held true for many other bacteria, among which Dialister pneumosintes that was isolated in 1918 from nasal secretions of influenza patients, but which turned out to be related with periodontal disease, and played no role in influenza [9].

It was Richard Shope who in 1931 was the first to isolate influenza virus from swine during epizootic periods of hog flu [10]. Shortly thereafter, Thomas Francis, an American virologist, isolated influenza type A in 1934 from humans for the first time, and he was also the first to grow influenza type B, in 1940 [11, 12]. Thomas Francis and Jonas Salk, a protégé of Francis and best remembered for his con-tributions to the development of polio vaccine, worked on an influenza vaccine in Ann Arbor for the US army. They developed a vaccine from an influenza type A strain that had led to an outbreak of atypical pneumonia in 1943 and tested the vaccine in the winter of 1943-1944 on male residents of a mental institution. The vaccine turned out to induce resistance to challenge with active virus. Later on, a large randomized, double-blind, placebo-controlled trial, including 6.236 vaccina-ted men and 6.211 controls who received placebo, was performed in the fall of 1943 and demonstrated an attack rate of influenza of 2.22% in the vaccinated group, while this was 7.11% in the unvaccinated group [13]. Since then, nothing much chan-ged with regard to influenza vaccines currently used, except for purification of the vaccine, which resulted in split and subunit influenza vaccines. These are as immunogenic as the whole inactivated virus (WIV) vaccines that Salk and Francis developed, but are less reactogenic and therefore cause less adverse events. Un-til very recently these vaccines were also still grown on the chorio-allantoic mem-branes of developing chick-embryos [14]. The in 1941 developed hemagglutination

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inhibition assay, which uses the capacity of influenza-specific antibodies in the human serum to inhibit agglutination of chicken, turkey and guinea pig erythrocy-tes by hemagglutinin, is still the gold standard for determination of the humoral response following influenza vaccination, in particular to determine seroprotec-tion [15].

Vaccination immunology with a focus on influenza vaccination

Virus-neutralizing immune effector responses are antibody or immune cell me-diated. Most vaccines confer protection by eliciting B-cell responses leading to the production of antibodies directed against infectious or toxic agents. This also holds true for influenza vaccines, which induce the production of influenza neu-tralizing antibodies in order to prevent binding of the virus to epithelium of the respiratory tract. These are mainly IgG antibodies directed against influenza surf-ace antigens. Mucosal influenza-specific IgA antibodies play an important role in the protection against infection with influenza. However, subunit influenza vac-cine induces only production of IgG and/or IgM antibodies. Only the mucosal ap-plied live-attenuated influenza vaccine leads to the synthesis of anti-influenza IgA antibodies.

Besides neutralizing antibodies, cytotoxic CD8+ T-lymphocytes (CTL) can serve as immune effectors following vaccination by recognizing and killing infected cells or by secreting antiviral cytokines. Influenza-specific CTL responses have been shown to mitigate the course of influenza infection [16] and influenza-spe-cific CTL responses following influenza vaccination (as measured by granzyme B production, an effector mechanism of CTL) correlate with protection for labora-tory confirmed influenza in elderly [17]. CTL responses are not induced by surface antigens, and therefore also not by subunit vaccines. In contrast, WIV influenza vaccine and live-attenuated influenza vaccine do elicit CTL responses, since they contain many other influenza antigens besides surface antigens.

To support the generation and maintenance of these B-cell and CTL responses CD4+ T-lymphocytes (T-helper cells) secrete growth factors and give signals. Moreover, T-cell help results in the induction of long term memory leading to a faster, larger and more specific immune response on a second encounter of the antigen [2].

The influenza vaccines used in the studies presented are trivalent subunit influ-enza vaccines, containing mainly hemagglutinin, one of the two surface protein-antigens of influenza, of the two influenza A-strains and the one B-strain for the particular season. To a smaller extent the vaccines contain neuraminidase (the other surface protein-antigen), and only very little internal antigens such as ma-

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trixprotein and nucleoprotein. Influenza is capable of changing composition and therefore also epitopes, as a form of immune evasion, referred to as “antigenic drift”. Therefore, every year the composition of influenza vaccine is evaluated and adjusted to the changes in hemagglutinin and neuraminidase, based on the most recent circulating influenza strains. Since the mutations that lead to the an-tigenic drift can not be predicted, the efficacy of the vaccine may differ from year to year based on the similarity of the vaccine strains and the natural oc-curring influenza strains. Nonetheless, antibody production following influenza vaccination generally correlates well with protection for influenza infection. An anti-influenza antibody titer of ≥40 as determined by hemagglutination inhibition assay is considered protective [15], although it should be noted that this has only been demonstrated in young healthy volunteers, and that for elderly and immuno-compromised persons, for example, it remains unclear whether the same titer can be presumed to be protective.

Following intramuscular administration of influenza subunit vaccine the vaccine antigens will reach the draining lymph node as free proteins or associated with dendritic cells. The vaccine antigens can be recognized by naïve B-cells and bound to their IgM-receptor, leading to activation of the B-cells and migration of the B-cells to the outer T-cell zone of the lymph node. Here the B-cell is exposed to and T-helper cells by activated dendritic cells , which provide help to differentiate into immunoglobulin secreting plasma cells. T-helper cells also drive immunoglobulin class switch from IgM towards IgG, but there is no hypermutation, so affinity of the immunoglobulins is low [2].

To produce more antibodies and antibodies with a better fit (higher affinity), and, thus, a higher capacity to neutralize the influenza virus, a so-called germinal cen-ter reaction needs to be elicited. Antigen-specific B-cells and follicular T-cells are attracted by follicular denditric cells towards B-cell follicles in the germinal cen-ter. Here follicular T-helper cells provide help to B-cells through costimulatory molecules. An optimal interplay between these cells results in clonal expansion of specific B-cells and antibody production, class switch from IgM to IgG and IgA, and affinity maturation of the antibodies by somatic hypermutation. Also diffe-rentiation of germinal center B-cells towards specific antibody secreting plasma cells or memory B-cells takes place. It takes 10-14 days before the highly specific IgG antibodies enter the circulation and these will peak 4 to 6 weeks after vacci-nation. However, after a re-exposure to the vaccine antigen, in case of infection with natural influenza for example, a faster, larger and more specific immune response will result (also referred to as a memory response) [2].

In contrast to B-cell and CD4+ T-cell responses, no or only limited CTL responses

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are induced by influenza subunit vaccine, since the influenza surface protein from the vaccine is presented by antigen presenting cells in the context of a MHC class II molecule. This is recognized by CD4+ T-cells only, not by CD8+ T-cells. Some pre-sentation of antigen in the context of MHC class I might occur through cross-pre-sentation, leading to CTL responses, but this occurs only to a limited extent [18].

Influenza vaccination in immunocompromised persons

As can be expected based on the former section, an adequately functioning immu-ne system is required to respond with protective immunity following vaccination. Paradoxically, this also means that patients with a compromised immune system, who theoretically profit most from vaccination because they are at increased risk of contracting vaccine-preventable infections and to have a complicated course of these infections, may not be able to elicit protective immune responses.

An immunocompromised state can result from a primary (or hereditary) immu-nodeficiency or from a secondary (or acquired) immunodeficiency. With a preva-lence of up till almost 1 in 200 in Caucasians selective IgA-deficiency is the most common primary immunodeficiency [19]. Most individuals with IgA-deficiency are asymptomatic. The primary immunodeficiency most frequently encountered in the clinic is common variable immunodeficiency (CVID). It is defined by reduced levels of total IgG, in combination with reduced levels of IgM and/or IgA and a poor or absent response on vaccination, despite the presence of normal levels of B-cells in the peripheral blood [20]. The onset of disease is usually in the second or third decade, and it is characterized by recurrent infections of the respiratory and gastrointestinal tracts, auto-immune manifestations, sarcoid-like granuloma for-mation and an increased risk of developing malignancy [21, 22]. The pathogenesis is poorly understood, although many defects in humoral and cell-mediated immu-nity have been described. Ten to 15% of the patients with CVID have a known gene defect causing the disease [23]. Other humoral primary immunodeficiencies are X-linked agammaglobulinemia, autosomal recessive agammaglobulinemia, IgG subclass deficiency and specific antibody response deficiency. In order to prevent infections in patients with humoral primary immunodeficiencies intravenous im-munoglobulins (IVIg) every 2 to 4 weeks or subcutaneous immunoglobulins every 1 to 2 weeks, and prophylactic antibiotics can be administered. Influenza vaccina-tion is recommended for patients with CVID, however, efficacy is unknown. Only a limited proportion of patients with CVID developed an adequate immune res-ponse after vaccination with anti-peptide and anti-polysaccharide vaccines [24].

Secondary immunodeficiencies are acquired during life. Most often the cause is iatrogenic, i.e. treatment with immunosuppressive agents, e.g. corticosteroids,

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methotrexate or biologics that target specific parts of the immune system, usu-ally in order to prevent the immune system from attacking self antigens or trans-planted organs. In this thesis (combinations of) immunosuppressive agents are addressed that are used in the treatment of auto-immune inflammatory rheuma-tic diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

SLE is a multisystem, auto-immune, connective-tissue disorder with a broad range of clinical presentations, predominantly occurring in young women, usually in their third or fourth decade. It mainly affects the skin, joints, kidneys, lungs, nervous system, serous membranes and hematopoietic system (but every organ can be affected), and is characterized by a relapsing and remitting disease course [25]. The prevalence in Europe ranges from 25 to 91 per 100.000 persons, but is higher in specific ethnic groups [26]. Treatment depends strongly on the speci-fic organ involvement and might include non-steroidal anti-inflammatory drugs (NSAIDs), and immunosuppressive medication such as systemic corticosteroids, antimalarials, methotrexate, azathioprine, mycophenolate mophetil, cyclophosphamide, and rituximab.

RA is a chronic inflammatory arthropathy associated with articular damage, at-tendant comorbidities, particularly in the cardiovascular system, and with incre-asing disability and socioeconomic decline [27]. It is an auto-immune disease of unknown cause, and has a prevalence of 1 per 100 persons in Caucasians. The disease usually occurs between 30 and 55 years of age and women are affected 2 to 3 times more often than men. The treatment aims to reduce disease activity to a minimum level as determined by the disease activity score, and start of therapy early in the disease course can prevent irreversible joint damage. Disease modi-fying anti-rheumatic drugs (DMARDs) such as methotrexate, leflunomide, sulp-hasalazine and hydroxychloroquine are the first line treatment, followed by anti-TNFα agents. When anti-TNFα agents fail to induce remission, newer biologics are available: anakinra (IL1 receptor antagonist), abatacept (CTLA4-Ig construct), rituximab (anti-CD20 antibodies) and tocilizumab (anti-IL6 receptor antibodies).

Rituximab is different from other biologics in that it does not block cytokines or costimulatory molecules, but leads to a depletion of CD20+ B-cells in the peripheral blood that persists for 6 to 9 months [28]. It is highly effective in sup-pressing the activity of RA, however, it does not seem to result in a large increase in infection risk, although theoretically one expects humoral immune responses to neo-antigens to be impaired during B-cell depletion [29, 30].

Apart from the immunosuppressive treatments, patients with auto-immune in-flammatory rheumatic diseases might be immunocompromised by the disease

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itself. SLE often is accompanied by lymphocytopenia. In RA patients, even if not treated with immunosuppressive medication, morbidity and mortality due to in-fections are increased [31].

Immunocompromised patients are at increased risk of contracting potentially life-threatening vaccine-preventable infections, and therefore vaccination seems indicated as an elegant and effective measure to reduce this infection risk. Un-fortunately, many controversies and uncertainties regarding vaccination in this population remain. First, the incidence of vaccine-preventable infections, among which influenza, in immunocompromised patients is largely unknown, since good studies addressing this are lacking. Second, investigations aiming to determine the efficacy of vaccination are based on surrogate parameters, mainly antibody responses, and not on clinical outcome. Also, for only a minority of the availa-ble vaccines correlates of protection have been determined, and if so, in healthy volunteers, but not for immunocompromised populations. Finally, harms of vac-cination are of utmost importance. Concerns regarding new onset or flares of auto-immune diseases triggered by vaccination have arisen after the publication of many case reports. In this context, the studies in this thesis have been designed and performed.

Aims and outline of the thesis

In the studies presented in this thesis we aimed to address the following aspects of influenza vaccination in patients with primary or secondary immunodeficien-cies:

• Efficacy, assessed by the investigation of humoral and cell-mediated immune responses following influenza vaccination

• Impact of immunosuppressive agents on efficacy• Impact of the timing of influenza vaccination following the treatment with

immunosuppressive agents on efficacy• Impact of booster influenza vaccination on efficacy• Safety In part 1 of the thesis the investigations regarding humoral (chapter 2) and cell-mediated (chapter 3) immune responses following influenza vaccination in pa-tients with humoral primary immunodeficiencies, in particular CVID, are analyzed.

In part 2 we focus on secondary immunodeficiencies. We performed studies on efficacy and safety of influenza vaccination in patients with auto-immune inflam-matory rheumatic diseases, especially SLE and RA. In chapter 4 cell-mediated influenza-specific immune responses in SLE patients were investigated, while in

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chapter 5 the value of a second, booster, influenza vaccination in patients with SLE was evaluated.

Next, studies addressing the influence of rituximab therapy on the efficacy of influenza vaccination in RA patients are described in chapter 6 and chapter 7: in the former with a focus on humoral responses and in the latter on cell-mediated immune responses.

European League Against Rheumatic Diseases (EULAR) recommendations on vaccination in patients with auto-immune inflammatory rheumatic diseases com-plete this thesis (part 3; chapter 9), preceded by the systematic literature review (chapter 8) that summarizes the current evidence on vaccination in patients with auto-immune inflammatory rheumatic diseases, among which articles presented in this thesis, and that served as the basis for the EULAR recommendations.

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PART 1I n f l u e n z a vaccination in patients w i t h h u m o r a l p r i m a r y i m m u n o -deficiency

CHAPT

ER2Patients with h u m o r a lp r i m a r yi m m u n o -d e f i c i e n c yd o n o t d e v e l o pp r o t e c t i v eanti-influenzaa n t i b o d ytiters after vaccinat ionwith trivalents u b u n i ti n f l u e n z av a c c i n e

S. van Assen

A. Holvast

D.S.C. Telgt

C.A. Benne

A. de Haan

J. Westra

C.G.M. Kallenberg

M. Bijl

Clinical Immunology 2010 Aug; 136 (2): 228-235

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AbsTrAcTIntroduction Yearly influenza vaccination is recommended for patients with humoral primary immunodeficiency (hPID). However, humoral respon-ses following vaccination can be expected to be reduced in these patients.

Methods The efficacy of influenza vaccination in patients with hPID was as-sessed in 26 patients with hPID and 26 matched healthy controls (HC) using hemagglutination inhibition assay.

results Following vaccination, geometric mean titers (GMTs) significantly increased for all influenza strains in the HC group, but only for A/H1N1 in the patient group. Fold-increase in anti-influenza titer and seroprotection rates were lower for patients than for HC for A/H3N2 and A/H1N1, leading to postvaccination titer ≥40 in only 29% and 83% vs. 77% and 100%, respectively. Previous vaccination in patients and treatment with IVIg did not result in a higher rate of postvaccination titer ≥40.

conclusion Patients with hPID show hardly any humoral response following influenza vaccination.

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InTroducTIonInfluenza occurs in seasonal outbreaks of varying extent nearly every winter, and is associated with increased morbidity and mortality. Particularly young children, elderly, patients with chronic illnesses (e.g. cardiac, pulmonary or renal disease, diabetes mellitus) and immunodeficient patients are at increased risk for deve-loping complicated influenza [1]. Influenza-related death is mainly due to cardiac events or influenza pneumonia, with or without secondary bacterial infection [2]. Patients with humoral primary immunodeficiencies (hPID; i.e. IgA deficiency (IgAD), common variable immunodeficiency (CVID), X-linked agammaglobuline-mia (XLA), autosomal recessive agammaglobulinemia, IgG-subclass deficiency (IgGSD), and specific antibody response deficiency), frequently experience recur-rent bacterial infections of the upper and lower respiratory tract. Although these patients seem to be at risk for complicated influenza, the prevalence, morbidity and mortality of influenza in patients with hPID are unknown.

Because of the immunocompromised state and the risk of potentially life threa-tening secondary bacterial respiratory tract infections, influenza vaccination is recommended to patients with hPID [3]. On the other hand, humoral responses to vaccination can be expected to be reduced in these patients; absence of humoral responses to vaccination is even part of the diagnostic criteria for several hPID (http://www.esid.org). Nevertheless, peripheral blood mononuclear cells from a subset of CVID patients have been shown to be capable of producing antibodies in response to influenza antigen in vitro, this in contrast to mononuclear cells from X-linked agammaglobulinemia patients [4]. In vivo, antibody responses to polysaccharide and polypeptide vaccines have been demonstrated in respectively 18% and 23% of CVID patients [5]. Data on in vivo responses following influenza vac-cination in patients with hPID are lacking.

To determine if influenza vaccination results in protective antibody titers against influenza in patients with hPID, we investigated the humoral immune response following vaccination with trivalent subunit influenza vaccine.

MeThods

Patients and healthy controls

All patients had to fulfil the European Society for Immunodeficiencies (ESID) clas-sification criteria for an hPID (http://www.esid.org). Patients and healthy controls (HC) were excluded in case of: (i) no informed consent, (ii) age under 18, (iii) cur-rent infection that needed to be treated with antibiotics, (iv) malignancy, (v) preg-nancy, (vi) known allergy to or former severe reaction following vaccination with trivalent influenza subunit vaccine.

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Vaccine

Trivalent influenza subunit vaccine for the season 2006-2007 (Influvac®; Solvay Pharmaceuticals, Weesp, The Netherlands) was used, containing isolated hemag-glutinin and neuramidase proteins derived from egg-cultured virus. The following strains were included in the vaccine: A/Wisconsin/67/2005 (H3N2)-like strain (A/Hiroshima/52/2005 IVR-142 reass.); A/New Caledonia/20/99 (H1N1)-like strain (A/New Caledonia/20/99 IVR-116 reass.); and B/Malaysia/2506/2004-like strain (B/Malaysia/2506/2004).

Procedures

Patients and HC received the influenza vaccine intramuscularly between Octo-ber 2006 and January 2007, which was before the onset of the seasonal influ-enza outbreak in The Netherlands [6]. Immediately before and 21 or 28 days ± 3 days after vaccination blood samples were taken. The latter variation depended on the interval between the intravenous immunoglobulins (IVIg) administration that patients received; 21 days was chosen when the interval between consecu-tive IVIg (Nanogam®, Sanquin, Amsterdam, The Netherlands or Kiovig®, Baxter B.V., Utrecht, The Netherlands) administration was 2 weeks, and 28 days when the interval between consecutive IVIg administrations was 3 or 4 weeks or when patients did not receive IVIg, and in HC. Patients treated with IVIg were vaccina-ted 7 days before the administration of IVIg to distinguish possible side effects of vaccination from those of IVIg administration, and to diminish the influence of immunoglobulin substitution during the distribution phase on the outcome of the anti-influenza antibody titers. Measurements of complete blood count, lymp-hocyte subsets (CD19+ B-cells, CD4+ T-cells, CD8+ T-cells, and CD16/56+ NK-cells) and immunoglobulins (IgG, IgA, IgM) were performed according to standard pro-cedures. Information on previous influenza vaccination was obtained from all par-ticipants, and adverse effects occurring in the first 7 days postvaccination were recorded. Adverse effects were classified into local (itching, pain, erythema, and induration at the site of vaccination), systemic (fever, tiredness, sweating, myal-gia, chills, headache, arthralgia, diarrhoea, flu-like symptoms), and the need to use antipyretic drugs.

The study was approved by the ethics committees of both participating centers.

hemagglutination Inhibition Assay and eLIsA

For the detection of influenza antibodies the hemagglutination inhibition assay (HIA) was used. HIAs were performed with guinea pig erythrocytes and using cell grown influenza viruses as antigens, following standard procedures [7] with

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slight modifications as described elsewhere [8]. Sera were tested for antibodies against all three vaccine strains. The following parameters for efficacy of vac-cination were evaluated: geometric mean titer (GMT); seroconversion, defined as a ≥4-fold titer rise to a postvaccination titer of ≥40; seroprotection, defined as a rise in titer to ≥40 [9]. Hemagglutination inhibition (HI)-titers ≥40 are generally considered to be protective, while a median titer of 28 is estimated to protect 50% of healthy adult vaccinees [10].

To estimate the contribution of anti-influenza antibodies in the IVIg preparations to the levels of anti-influenza antibodies in the patient treated with IVIg, in sam-ples of three batches of IVIg (Nanogam®, Sanquin, Amsterdam, The Netherlands) the levels of anti-influenza antibodies to A/H3N2, A/H1N1 and the B-strain were determined using HIA (procedure described above) and in-house ELISA. For the latter, in short, microtiter plates were coated with 1 μg/ml subunit of A/H3N2 or A/H1N1 and subsequently incubated with IVIg samples. Detection of influenza-specific antibodies was done with mouse-anti-human IgG-HRP and mouse-anti-human IgM-HRP (Southern Biotech, Birmingham, USA) respectively, followed by color reaction with 3’3’5’5’tetramythylbenzidin (TMB) and H2 O2. Absorbance was read at 450-575 nm in an Emax microplate reader and concentration of antibodies was calculated by SOFTmax PRO software (Molecular Devices, Sunnyvale, USA) according to IgG- or IgM-standard curves included on each ELISA plate. The HIA and ELISA were performed on a 1:10 dilution of the IVIg preparations (in PBS) re-sulting in a concentration of 10 g/l, which was comparable with the mean of the total IgG-concentration in patients at the moment of vaccination (11 g/l).

statistical analysis

Data were analysed using SPSS 16.0 for Windows (SPSS Inc.). Wilcoxon signed rank test, Mann-Whitney U test, Chi square test, Fisher’s exact test, and Spear-man’s rank correlation coefficient were used where appropriate. To obtain a po-wer of 90% with an significance level α of 0.05, and considering a drop-out rate of 10%, 26 patients and HC needed to be included, when the expected proportion reaching a postvaccination HI-titer ≥40 was 30% for hPID patients, and 75% for HC.

resuLTs

Patient characteristics

Twenty-six patients from the university medical centers in Groningen (UMCG, n=21) and Nijmegen, The Netherlands (UMC St.Radboud, n=5), and 42 HC, recrui-ted from UMCG-health care workers and their family members, gave informed consent. Twenty-six HC were successfully matched on group level to the partici-

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pating patients, based on sex, age and history for influenza vaccination. Baseline characteristics are described in table 1. Sixty-two percent of patients and HC re-ceived influenza vaccination the year before. CVID was the most frequent cause of the hPID (69%). Compared to HC, patients had lower levels of IgA (p <0.001) and IgM (p <0.001), but not of total IgG (p =0.216), most likely since the vast majority of patients (81%) received IVIg. Baseline counts for CD19+ B-cells, CD8+ T-cells and CD16/56+ NK-cells were comparable for both groups. However, CD4+ T-cell num-bers were lower in the patient group compared to CD4+ T-cell numbers in the HC. For two patients no postvaccination blood samples were available, one diagnosed with XLA and the other with CVID, and both treated with IVIg.

Table 1. Baseline charactaristics of patients and healthy controls

Patients (n=26)

Healthy controls (n=26)

p-value

Sex (male), no. (%) 17 (65) 13 (50) NS

Age (years), mean (SD) 50 (13) 50 (11) NS

Diagnosis no. (%) / IVIg-use no. (%)

CVID 18 (69) / 16 (89)* N/A N/A

IgAD 3 (12) / 0 (0)* N/A N/A

IgGSD 2 (8) / 2 (100)* N/A N/A

SABRD 1 (4) / 1 (100)* N/A N/A

XLA 2 (8) / 2 (100)* N/A N/A

IVIg, no. (%) 21 (81) N/A N/A

Vaccination 2005/2006, no. (%) 16 (62) 16 (62) NS

IgG (g/l), median (range) 10.1 (4.1-17.4) 11.0 (6.8-16.7) NS

IgA (g/l), median (range) 0.19 (0.09-1.60) 1.90 (0-4.20) <0.001

IgM (g/l), median (range) 0.35 (0-1.60) 0.90 (0.40-2.80) <0.001

CD19+ B-cells (x 10^9/l), median (range) 0.18 (0.02-1.51) 0.21 (0.02-0.43) NS

CD4+ T-cells (x 10^9/l), median (range) 0.63 (0.16-3.81) 0.85 (0.33-1.63) 0.042

CD8+ T-cells (x 10^9/l), median (range) 0.48 (0.14-2.57) 0.43 (0.15-0.96) NS

CD16/56+ NK-cells (x 10^9/l), median (range) 0.19 (0.03-0.37) 0.20 (0.02-0.38) NS

CVID, common variable immunodeficiency; IgAD, IgA-deficiency; IgGSD, IgG-subclass deficiency; SABRD, specific antibody response deficiency; XLA, X-linked agammaglobulinemia; IVIg, intra-venous immunoglobulins; NS, not significant; N/A, not applicable. * Percentage of patients with this specific diagnosis treated with IVIg.

efficacy of influenza vaccination

Geometric mean titers

Prior to vaccination no differences in GMT between patients and HC were found for A/H3N2 (p =0.268), A/H1N1 (p =0.072) and B (p =0.848) (table 2). Following vac-cination, GMT in the patient group only increased for A/H1N1 (p =0.011), while in

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the HC group GMT increased for all three influenza strains (A/H3N2, p <0.001; A/H1N1, p <0.001; B; p =0.002) (table 2). The fold-increase in GMT was lower in the patient group compared to the HC group for A/H3N2 (p <0.001) and A/H1N1 (p <0.001) (figure 1), as was the postvaccination GMT in the patient group in com-parison with the HC group for A/H3N2 (p <0.001) and A/H1N1 (p <0.001) (table 2).

In the subgroup of patients with CVID (n=18), the same pattern was found: prevac-cination GMT were comparable with the HC group, but for none of the three in-fluenza-strains GMT rose significantly after vaccination (table 2). In patients with CVID, compared to HC, the fold-increase for A/H3N2 and A/H1N1 (both p <0.001) (figure 1) and postvaccination GMT for A/H3N2 and A/H1N1 (both p <0.001) were lower (table 2).

Table 2. Geometric mean titer (GMT) for all patients, the subgroup of common variable immu-nodeficiency (CVID) patients and patients treated with intravenous immunoglobulins (IVIg), and healthy controls.

Patients Healthy controls

prevac-cination

postvac-cination

p-value prevac-cination

postvac-cination

p-value

All patients n=26 n=24 n=26 n=26

A/H3N2, mean (SD) 18 (1.8) 22 (2.1) * 0.349 22 (2.1) 50 (1.9) <0.001

A/H1N1, mean (SD) 35 (1.7) 50 (1.8) * 0.011 73 (3.2) 236 (2.6) <0.001

B, mean (SD) 16 (1.8) 21 (1.9) 0.152 18 (1.4) 24 (1.8) 0.002

CVID patients n=18 n=17

A/H3N2, mean (SD) 17 (1.8) 17 (1.7) * 0.201

A/H1N1, mean (SD) 36 (1.5) 41 (1.6) * 0.066

B, mean (SD) 16 (1.7) 17 (1.8) 0.785

Patients on IVIg n=21 n=19

A/H3N2, mean (SD) 18 (1.8) 21 (2.1) * 0.812

A/H1N1, mean (SD) 36 (1.7) 44 (1.8) * 0.041

B, mean (SD) 15 (1.8) 19 (1.9) 0.404

* p <0.001 when compared to healthy controls

Seroconversion

The number of patients and HC developing seroconversion was low for all strains. This is most likely due to the high number of patients and HC who re-ceived an influenza vaccination before the previous season (table 1). No dif-ferences were found between seroconversion rates in patients and HC for A/H3N2 and B (data not shown). However, for A/H1N1 seroconversion occurred less often in the patient group than in the HC group (8% vs. 39%; p =0.013).

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Similarly, compared to the HC group, the subgroup with CVID developed seroconversion less often for A/H1N1 (39% vs. 0%; p =0.004) and for A/H3N2 (27% vs. 0%; p =0.031).

Seroprotection

A HI-titer rise from <40 to ≥40, the latter being considered to be protective for in-fluenza [10], occurred less frequently in the patient group in comparison with the HC group for both A/H3N2 (13% vs. 42%; p =0.019) and A/H1N1 8% vs. 31%; p =0.048) (figure 2). The percentages of patients with an HI-titer ≥40 after vaccination, ir-respective of their prevaccination titer, were 29%, 83% and 21% for the A/H3N2-, A/H1N1- and B-strain, respectively, whereas for HC these were 77%, 100% and 27%. These differences between patients and HC were statistically significant for A/H3N2 (p =0.001) and A/H1N1 (p =0.046) (figure 2).

Compared to seroprotection rates in HC, seroprotection rates in the subgroup of patients with CVID were also lower for A/H3N2 (0% vs. 42%; p =0.002) and A/H1N1 (0% vs. 31%; p =0.014), and the percentages of CVID patients with postvaccination anti-influenza titer ≥40 were significantly lower compared to those in HC for both A/H3N2 (12% vs. 77%; p <0.001) and A/H1N1 strains (77% vs. 100%; p =0.019) (figure 2).

Impact of previous vaccination

In order to investigate the impact of previous influenza vaccination on the humo-ral response, antibody responses in subgroups that received influenza vaccina-tion before the previous influenza season were compared with subgroups that did not receive influenza vaccination one year earlier. In accordance with previous findings of us and others, HC not previously vaccinated had at baseline a lower GMT for A/H1N1 (mean (SD) 36 (2.6) vs. 113 (3.0); p =0.010), and a trend to a larger fold-increase in GMT following vaccination (median (range) 6.75 (0-64) vs. 2 (0-16); p =0.053). In patients, however, previous vaccination did not influence baseline GMT or fold-increase in GMT for A/H1N1 following influenza vaccination. No dif-ferences in humoral response against the A/H3N2- and B-strain were found when comparing previously vaccinated to not previously vaccinated persons within both patient group and HC group (data not shown).

Impact of treatment with IVIg

Next, we assessed the influence of IVIg-treatment on the levels of anti-influenza specific antibodies. Prevaccination titers for all influenza-strains were compa-rable between patients receiving IVIg and patients not receiving IVIg (data not shown). In patients receiving IVIg-therapy (n=21) only the HI-titer for A/H1N1 in-creased significantly following vaccination: from 36 (1.7) to 44 (1.8) (mean (SD);

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Figure 1. Fold-increase in anti-influenza hemagglutination inhibition titer following influenza vaccina-tion for A/H3N2 (A), A/H1N1 (B) and B (C) in all hPID patients (All, n=24), common variable immunode-ficiency patients (CVID, n=17) and patients treated with intravenous immunoglobulins (IVIg, n=19), and healthy controls (HC, n=26). * p ≤0,001 when compared to HC

p =0.041). In patients not receiving IVIg, no significant increase was found follo-wing vaccination: 35 (1.9) to 80 (1.6) (mean (SD); p =0.102). It should be noted, howe-ver, that the number of patients without IVIg-therapy was low (n=5). In contrast,

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patients receiving IVIg-therapy performed worse compared to those who did not receive IVIg-therapy for the following parameters: postvaccination HI-titer for A/H1N1 was lower 44 (1.8) vs. 80 (1.6) (mean (SD); p =0.039); the fold-increase in titer was lower, although not statistically significant (1x (1-2x) vs. 4x (1-5.7x) (me-dian (range); p =0.056), the seroconversion rate was lower (0% vs. 40%; p =0.036); and the seroprotection rate was lower (0% vs. 40%; p =0.036). The percentage of patients with and without IVIg suppletion treatment having a postvaccination HI-titer ≥40 for all three strains did not differ. No other differences were found when comparing patients treated with and without IVIg (data not shown).

Also responses between the subgroup of patients on IVIg and HC were compared. No differences in GMT at the time of vaccination between patients on IVIg and HC were found for the three influenza-strains (table 2). Fold-increase in titer and postvaccination titer for A/H3N2 (both p <0.001) and A/H1N1 (both p <0.001) were lower in patients on IVIg compared to HC, as was seroconversion rate for A/H1N1 (0% vs. 39%; p =0.002). Most importantly, a postvaccination titer of ≥40 was less frequently found for both A-strains in patients on IVIg than in HC (A/H3N2: 21% vs. 77%, p <0.001; A/H1N1: 79% vs. 100%; p =0.014) (figure 2).

correlations between b-cells and vaccination responses

By determining correlations, we tried to find a direct relation between prevac-cination B-cell count in the peripheral blood and the various outcome parame-ters for vaccination response. No correlations could be demonstrated (data not shown).

Anti-influenza antibody levels in IVIg

In the samples of all three batches of IVIg, representing the IgG of several thousands of healthy blooddonors, anti-influenza titers were 1:5 for A/H3N2, 1:10 for A/H1N1 and 1:5 for the B-strain as determined by HIA. Moreover, when using ELISA, a median concentration of IgG anti-influenza antibodies for A/H3N2 was 85.1 mg/l (range 81.6-94.4 mg/l) and for A/H1N1 101.5 mg/l (range 87.5-101.8 mg/l). As expected, no IgM anti-influenza antibodies were found in IVIg using ELISA.

safety of vaccination: side effects

Occurrence of side effects did not differ between patients and HC. In both the patient and HC group, pain at the site of injection was the most frequently noticed side effect (36% and 38%) followed by induration at the site of injection (20% and 15%), muscles aches (20% and 8%) and headache (20% and 8%).

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Figure 2. Percentage of patients and healthy controls with a titer ≥40 before vaccination (black bars) and postvaccination (white bars) for A/H3N2 (A), A/H1N1 (B) and B (C) in all hPID patients (All, n=24), common variable immunodeficiency patients (CVID, n=17) and patients treated with intravenous im-munoglobulins (IVIg, n=19), and healthy controls (HC, n=26). * p <0,05 for seroprotection (HI-titer rise from <40 before vaccination to ≥40 after vaccination), compared to HC

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dIscussIonThe current study is, to our knowledge, the first study evaluating humoral re-sponses following influenza vaccination in patients with hPID. We show that in these patients the humoral response to all three influenza-strains in the vaccine is severely hampered for almost all of the outcome measures. This holds true in particular for influenza A/H3N2 and A/H1N1, the widest circulating and most vi-rulent of the influenza-strains. Most alarming is that patients often did not reach protective anti-influenza HI-titers; 71% and 17% of the studied patients did not have a titer ≥40 of for influenza A/H3N2 and A/H1N1 after vaccination, respectively. For the subgroup of patients with CVID (n=18) the numbers are even worse: 88% and 23%. In comparison, HC responded adequately with 23% and 0% not reaching pro-tective postvaccination titers for A/H3N2 and A/H1N1, respectively.

Previous vaccination in healthy individuals generally results in higher prevacci-nation titers. Following subsequent influenza vaccination postvaccination titers might be higher, similar or even somewhat lower, compared to postvaccination titers in previously unvaccinated HC [11, 12]. Our results show that in previously vaccinated patients with hPID neither a significant higher prevaccination titer, nor higher postvaccination titer, compared to not previously vaccinated patients, are present. Therefore, most likely yearly influenza vaccination in patients with hPID does not result in higher levels of protective anti-influenza titers. This might be expected, since in patients with hPID the decreased number and/or function of memory B-cells is caused by an intrinsic defect instead of a reversible cause underlying the hampered response. While patients with X-linked agammaglobu-linemia have absent or severely reduced numbers of circulating B-cells, a consi-derable number of CVID patients have normal numbers of B-cells , but reduced counts of class-switched memory B-cells [13].

Many patients with hPID (81% in the current study) receive treatment with IVIg to reduce frequency and severity of respiratory infections [14]. IVIg is reserved for those patients with recurrent infections, for whom protection against influenza might be considered to be of great importance. When comparing patients treated with IVIg to patients without IVIg-therapy, patients with IVIg-therapy showed a reduced response to influenza vaccination. This probably reflects the more se-vere immunodeficiency in these patients. Moreover, pre- and postvaccination ti-ters were not protective in a large part of the patients treated with IVIg, despite treatment with IVIg every 2 to 4 weeks. Although anti-influenza antibodies have been detected in IVIg preparations [15, 16], as would be expected since 10-20% of the general population contracts influenza every year, our finding demonstrates that passive immunization with IVIg is not protective for influenza.

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We found large differences between HI-titers for the different influenza-strains. The relatively high titers for A/H1N1 might be caused by the fact that the A/H1N1-component in the vaccine did not change since 2001, while the A/H3N2 and B-strains were new in the influenza vaccine for the season 2006-2007 [17]. There-fore, more anti-influenza A/H1N1 antibodies might be present in the general population, as is reflected by the high level of anti-influenza A/H1N1 antibodies in the prevaccination samples of the HC, and therefore probably also in IVIg. Testing of the levels of anti-influenza antibodies in the IVIg preparations using HIA did in-deed show higher titers of anti-A/H1N1 antibodies compared to anti-A/H3N2 anti-bodies. When using ELISA, the higher concentrations of anti-A/H1N1 compared to anti-A/H3N2 antibodies could be confirmed. However, it should be noted that the HI assay has not been validated for testing influenza-specific antibodies in IVIg preparations. Responses to the influenza B-strain were low in both patients and HC, but this is a common phenomenon when HIA is used to measure anti-influenza antibodies directed against influenza B-strains [18].

As active immunization with influenza subunit vaccine and “passive immuniza-tion” with IVIg does not result in protective levels of anti-influenza antibodies in patients with hPID, other strategies against influenza are warranted. Reducing exposure to influenza by vaccination for influenza of household contacts and of health care workers of patients at high risk for severe complications from in-fluenza is recommended by ACIP [19], as there is evidence that this strategy can reduce transmission of influenza [20, 21]. Therefore household contacts of pa-tients with hPID should be vaccinated for influenza if no contra-indications exist. Passive immunization with serum from donors with high levels of anti-influenza antibodies shortly before the start of and during the influenza season might be another option to protect these patients, but this needs to be further explored. Finally, prophylactic antiviral treatment with adamantanes and neuraminidase inhibitors reduces the chances of contracting influenza [22], but should be used cautiously, since virtually all A/H3N2 are resistant to adamantanes, while resis-tance to the neuraminidase inhibitor oseltamivir is highly prevalent in A/H1N1. To prevent further resistance, the use of these medications should preferentially be reserved for treatment or post-exposure prophylaxis only and not for prop-hylaxis in patients with hPID [23]. Finally, the cellular immune responses following influenza vaccination should be evaluated in patients with a hPID, since cellular immunity has been shown to play a role in the prevention of clinically manifest influenza in elderly [24].

In conclusion, patients with hPID show severely impaired humoral response fol-lowing vaccination with inactivated trivalent subunit influenza vaccine. Moreover, passive immunization with IVIg is insufficient for the prevention of influenza.

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(22) Jefferson T, Demicheli V, Rivetti D, Jones M, Di Pietrantonj C, Rivetti A. Antivirals for influenza

in healthy adults: systematic review. Lancet 2006 Jan 28; 367(9507):303-13.

(23) Poland GA, Jacobson RM, Ovsyannikova IG. Influenza Virus Resistance to Antiviral Agents: A

Plea for Rational Use. Clin Infect Dis 2009 Mar 26.



CHAPT

ER3Cell-mediatedi m m u n eresponses to inact ivatedt r i v a l e n ti n f l u e n z avaccinat ionare decreasedin patients w i t hc o m m o n v a r i a b l ei m m u n o -d e f i c i e n c y

S. van Assen

A. de Haan

A. Holvast

G. Horst

L. Gorter

J. Westra

C.G.M. Kallenberg

D.S.C. Telgt

A.M. Palache

K.M. Giezeman

M. Bijl

Clinical Immunology In press

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AbsTrAcTIntroduction Influenza-specific cell-mediated immune (CMI) responses can protect for influenza, but may be decreased in common variable immuno-deficiency (CVID) patients since defects in CMI responses have been de-monstrated in CVID patients.

Methods CMI responses were evaluated in 15 CVID patients and 15 mat-ched healthy controls (HC) by determining frequencies of interferon (IFN)γ-producing PBMC, and frequencies of IFNγ-, interleukin (IL)-2- and tumor necrosis factor (TNF)α-producing CD4+ and CD8+ T-cells before and after influenza vaccination using IFNγ enzyme-linked immunospot (IFNγ-ELISpot) and flow cytometry. Humoral responses were determined using hemagglu-tination inhibition assay.

results In CVID patients the number of spotforming PBMC in the IFNγ-ELISpot did not increase following influenza vaccination, in contrast to HC. In flow cytometry, the frequencies of IFNγ-producing T-cells decreased in CVID patients after influenza vaccination, while in HC the frequencies of IFNγ-production increased.

conclusion CMI responses following influenza vaccination are hampered in CVID patients compared to HC. Additional protective strategies against influenza other than vaccination are warranted.

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InTroducTIonCommon variable immunodeficiency (CVID) is the primary immunodeficiency most frequently encountered in clinical practice. CVID is primarily characterised by a defective humoral immune response. Recurrent sinopulmonary, and less fre-quently, gastrointestinal infections are the most common clinical manifestations, but CVID patients are also at increased risk for the development of auto-immune diseases, granulomatous disease and hematological and solid malignancies [1].

Because of the immunocompromised state of CVID patients, influenza vaccina-tion is recommended [2]. However, compared to healthy individuals vaccination with polysaccharide and protein vaccines is less efficacious [3, 4]. We demonstra-ted hampered humoral responses following influenza vaccination in patients with CVID, resulting in lower seroprotection rates, defined as an anti-influenza titer as determined by haemagglutinin inhibition assay (HIA) ≥40) in these patients com-pared to healthy controls (HC) [5]

Not only humoral [6], but also cell-mediated immune (CMI) responses following influenza vaccination have been shown to correlate with protection for clinical in-fluenza infection [7]. Although CVID is classified as a humoral immunodeficiency, decreased cellular immune function has been demonstrated in a considerable proportion of CVID patients, consisting of numerical and/or functional defects involving T-cells, natural killer (NK)-cells, dendritic cells (DC), macrophages and monocytes [8-10]. Data regarding the CMI response following influenza vaccinati-on in patients with CVID are lacking. However, in view of their hampered humoral responses, cellular responses should be considered as well when discussing the usefulness of influenza vaccination in these patients.

In this study we investigated the influenza-specific CMI response in patients with CVID before, and 7 and 21-28 days after vaccination with trivalent subunit influen-za vaccine in comparison with that in healthy controls. CMI response on vaccinati-on was determined using interferon (IFN)γ enzyme-linked immunospot (ELISpot) and flow cytometry with intracellular cytokine staining (ICS) for IFNγ, interleukin (IL)-2 and tumor necrosis factor (TNF)α. Finally, a relationship between CMI re-sponses and humoral immune responses was studied.

MATerIAls And MeThods


Patients were included at the University Medical Center Groningen (UMCG) and the University Medical Center St. Radboud Nijmegen, both in The Netherlands, and had to fulfil the European Society for Immunodeficiencies classification crite-ria for CVID (http://www.esid.org). HC were recruited from the UMCG personnel.

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Patients and HC were excluded in case of: (i) no informed consent, (ii) age under 18, (iii) current infection that needed treatment with antibiotics, (iv) malignancy, (v) pregnancy, (vi) known allergy to or former severe reaction following vaccina-tion with trivalent influenza subunit vaccine.

Vaccine

Trivalent influenza subunit vaccine for the season 2006-2007 (Influvac®; Solvay Pharmaceuticals, Weesp, The Netherlands) was used, containing isolated hemag-glutinin and neuramidase proteins derived from egg-cultured virus. The vac-cine contained the following strains: A/Wisconsin/67/2005 (A/H3N2)-like strain (A/Hiroshima/52/2005 IVR-142 reass.); A/New Caledonia/20/99 (A/H1N1)-like strain (A/New Caledonia/20/99 IVR-116 reass.); and B/Malaysia/2506/2004-like strain (B/Malaysia/2506/2004).

Procedures

Patients and HC received the influenza vaccine intramuscularly between October 2006 and January 2007, which was before the onset of the seasonal influenza outbreak in The Netherlands. Immediately before, and 7 and 21 or 28 days after vaccination blood samples were taken. The latter variation depended on the inter-val between the intravenous immunoglobulins (IVIg) administration that patients received, to prevent interference of the administration of IVIg with the results of the humoral and cellular immune responses. Twenty-one days was chosen when the interval between consecutive IVIg administrations was 2 weeks, and 28 days when the interval between consecutive IVIg administrations was 3 or 4 weeks or when patients did not receive IVIg, and in HC. Moreover, patients treated with IVIg were vaccinated 7 days before the administration of IVIg to distinguish possible side effects of vaccination from those of IVIg administration, and also to dimi-nish the influence of IVIg on the outcome of the cellular immune responses on day 7. Measurements of complete blood count, lymphocyte subsets (CD4+ T-cells, CD8+ T-cells, CD16/56+ NK-cells and CD19+ B-cells) were performed according to standard procedures. Information on previous influenza vaccination was obtained from all participants.

The study was approved by the ethics committees of both participating centers.

cell-mediated immunity assays

Processing of PBMC and the procedures for the IFNγ-ELISpot assay and flow cy-tometry were as described earlier [11]. Therefore, these procedures will only be described in short.

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Isolation, storage and thawing of PBMC

PBMC were isolated from BD Vacutainer® CPT™ Cell Preparation Tubes containing 0.1 M sodium citrate anticoagulant and blood separation media composed of a thixotropic polyester gel and a FICOLL™ HYPAQUE™ solution (BD, Franklin Lakes, NJ, USA), according to the instructions of the producer. PBMC were stored in li-quid nitrogen until use in RPMI 1640 (Cambrex BioScience, Verviers, Belgium) sup-plemented with 10% human pool serum, 50 µg/ml of gentamicin (Gibco, Paisley, UK) and 10% dimethylsulfoxide. Prevaccination and postvaccination samples from a patient with CVID and a matched control subject were simultaneously thawed and batch-processed.

Influenza antigens used in assays of cell-mediated responses

PBMC were stimulated with β-propiolactone inactivated whole virus (WIV) of A/New Caledonia/20/99 (IVR-116 reass.; A/H1N1) and A/Hiroshima/52/2005 (IVR-142 reass.; A/H3N2).

IFNγ-ELISpot assay

Immobilon-P membrane plates (Millipore,Billerica,MA,USA) were coated with an-ti-human IFNγ (Mabtech, Nacka Strand, Sweden). 2 x 105 PBMC were added per well and incubated for 8 hours in culture medium (CM), RPMI 1640 (Cambrex Bi-oScience, Verviers, Belgium) supplemented with 10% fetal calf serum, 50 µg/ml of gentamicin (Gibco, Paisley, UK) with WIV A/H1N1 (1 µg/ml) and WIV A/H3N2 (1 µg/ml). Concanavalin A stimulation served as a positive control, a negative con-trol consisted of PBMC in CM alone. Stimulation tests were performed in dupli-cate. Biotinylated anti-human IFNγ, streptavidin-alkaline phosphatase and BCIP/NBT-plus substrate (all Mabtech) were used in order to visualise IFNγ-producing PBMC. Spots were counted using an automated reader (automated ELISpot video-analysis system, Sanquin, Amsterdam, The Netherlands).

Flow cytometry

For stimulations, 1.0–1.5 x 106 PBMC were cultured in CM for 18 hours. Staphylo-coccal enterotoxin B (SEB; Sigma-Aldrich, St. Louis, MO) at 5 µg/ml was used as a positive control. WIV A/H1N1 and WIV A/H3N2 were used at final concentrations of 1 µg of total viral protein/ml. Negative controls were not stimulated (CM only). Pa-cific Blue and Pacific Orange dyes (Invitrogen, Carlsbad, CA), in a different com-bination for each stimulus, were used to enable fluorescent T-cell bar coding. The following monoclonal antibodies were used: allophycocyanin (APC)-Cy7-conjuga-ted anti-CD3, peridinin chlorophyll A protein–conjugated anti-CD8 (Becton Dickin-son Pharmingen, San Diego, CA, USA), phycoerythrin–Cy7 (PE-Cy7)–conjugated anti-CD69 (Biolegend, San Diego,CA, USA), PE–conjugated anti-IFNγ, APC-conju-

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gated anti-TNFα, and Fluorescein isothiocyanate-conjugated anti–IL-2 (eBiosci-ence, Hatfield, UK) Analysis was performed on a LSR II flow cytometer (Becton Dickinson).

The WinList software package (Verity Software House, Topsham, ME) was used. Percentages of antigen-specific cells were expressed as the percentage of CD69+ cytokine-producing CD4+ or CD8+ T-cells within the total CD4+ or CD8+ T-cell po-pulation. Samples were excluded if the proportion of viable cells after thawing was <85%, and if the proportion of IFNγ- or TNFα-producing CD4+ or CD8+ T-cells was <2.00% upon stimulation with SEB (positive control). For IL-2 no cut-off could be determined since the proportion of cytokine-producing CD4+ and CD8+ T-cells upon stimulation with SEB was generally low.

Antibody response to influenza

For the detection of influenza antibodies the HIA was used. HIA were perfor-med with guinea pig erythrocytes and using cell grown influenza viruses as an-tigens, following standard procedures [12] with slight modifications as described elsewhere [13]. Sera were tested for antibodies against A/New Caledonia/20/99 (A/H1N1) and A/Wisconsin/67/2005 (A/H3N2).


Since none of the variables were normally distributed, data are presented as me-dian (range). Data were analysed using PASW Statistics 18 (SPSS Inc., Chicago, IL, USA). To determine differences in baseline characteristics between the matched CVID patients and HC, the levels of spotforming cells with IFNγ-ELISpot and the frequencies of cytokine-producing cells with flow cytometry, the Mann-Whitney U test was used. Friedman’s two-way analysis of variance by ranks was used to analyse the presence of differences in CMI responses at the three time points. All cytokine frequencies reported are after background subtraction of the frequen-cy of the identically gated population of cells from the same sample stimulated without antigen. For correlations, Spearman’s rank correlation coefficients were used. A two sided p-value <0.05 was considered statistically significant. A power calculation could not be performed since no data were available with regard to what difference in CMI responses could be expected and would be clinically rele-vant.

resulTs


Fifteen eligible CVID patients were included and successfully matched for sex, age

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and history for influenza vaccination to 15 HC. Baseline characteristics are descri-bed in table 1. Thirteen out of 15 (87%) patients and 10 out of 15 (67%) HC received influenza vaccination the year before. Baseline counts for CD3+ T-cells, CD8+ T-cells, CD16/56+ NK-cells and CD19+ B-cells were comparable for both groups. CD4+ T-cell numbers were lower in patients compared to CD4+ T-cell numbers in the HC, although not statistically significant (p =0.072). Seven and 21-28 days after vaccination this difference persisted (p =0.007 for both time points). Also, CD4+/CD8+ ratio was lower in patients compared with HC at baseline (p =0.014), after 7 days (p =0.049) and after 21-28 days (p =0.015) following influenza vaccination.

Table 1. Baseline characteristics of patients with common variable immunodeficiency and healthy controls

Patients Healthy controls p-value

(n=15) (n=15)

Age, (years), median (range) 55 (30-74) 52 (36-65) NS

Sex, (male), no. (%) 8 (53) 9 (60) NS

IVIg, no. (%) 12 (80) N/A N/A

Vaccination ‘05/’06, no. (%) 13 (87) 10 (67) NS

Lymphocytes, (x 10^9/l), median (range) 1.69 (0.81-8.31) 1.80 (0.80-2.90) NS

CD3+-cells, (x 10^9/l), median (range) 1.23 (0.58-6.51) 1.39 (0.55-2.53) NS



CD16/56+-cells, (x 10^9/l), median (range) 0.14 (0.03-0.29) 0.20 (0.05-0.38) NS


CD4+/CD8+ ratio, median (range) 1.48, (0.33-6.00) 2.00 (1.29-4.95) 0.014

NS, not significant; N/A, not applicable

higher frequencies of IFnγ-producing cd4+ and cd8+ T-cells in patients with cVId

In the IFNγ-ELISpot assay no differences in the proportion of spotforming cells before vaccination were found between patients and HC for both influenza A strains tested (figure 1).

Flow cytometry revealed higher frequencies of IFNγ-producing CD4+ T-cells upon stimulation with SEB in patients compared to HC on all three time points tested, being significant before vaccination (p =0.014, data not shown). Further-more, before vaccination proportions of IFNγ-producing CD8+ T-cells upon sti-mulation with SEB were higher in patients compared to HC (p =0.005, data not shown). Also, when stimulated with A/H1N1 and A/H3N2, the proportions of IFNγ-producing CD4+ T-cells were higher in patients than in HC (for both strains p =0.019) (figure 2, A). Prevaccination frequencies of TNFα- and IL-2- producing

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CD4+ and CD8+ T-cells did not differ except for a lower frequency of TNFα-producing CD8+ T-cells upon stimulation with SEB (p =0.022, data not shown) and a higher frequency of IL-2-producing CD8+ T-cells upon stimulation with A/H1N1 (p =0.015, data not shown) in patients compared to HC.

lower cell-mediated responses to A/h1n1 and A/h3n2 following influenza vaccination in patients with cVId

In patients with CVID the IFNγ-ELISpot assay revealed no differences in the pro-portion of spotforming cells between time points 0, 7 and 21-28 days upon stimu-lation with A/H1N1 (p =0.202) or A/H3N2 (p =0.905), whereas for HC significant changes in spotforming cells between the three time points were present upon stimulation with A/H1N1 (p =0.020) and A/H3N2 (p <0.001), representing a CMI res-ponse in HC following influenza vaccination (figure 1).

When using flow cytometry, in CVID patients a decrease was found in the fre-quencies of IFNγ-producing CD4+ T-cells upon stimulation with SEB when com-paring time points 0, 7 and 21-28 days (p =0.035, data not shown). Upon stimu-lation with A/H1N1 and A/H3N2 this was also shown, although the decrease was not statistically significant for the latter (p =0.026 and p =0.060, respectively) (fi-gure 2, A). On the contrary and as expected, in HC the frequencies of A/H1N1- and A/H3N2-specific IFNγ+CD4+ T-cells increased after influenza vaccination (p =0.028 and p =0.016, respectively) (figure 2, A).

Regarding CD8+ T-cells, in CVID patients the frequencies of A/H1N1- and A/H3N2- specific IFNγ+CD8+ T-cells did not differ between the three time points (p =0.909

Figure 1. Influenza-specific IFNγ-production by PBMC before, and 7 and 21-28 days following influ-enza vaccination in healthy controls and patients with CVID, as determined by IFNγ-ELISpot assay. Bars represent medians, whiskers represent interquartile range. P-value as calculated using Friedman’s Two-Way analysis of variance by ranks.

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and p =0.846, respectively). In HC, for the frequency of IFNγ-producing CD8+ T-cells a rise following vaccination was found upon stimulation with A/H1N1 (p =0.044), but not upon stimulation with A/H3N2 (p =0.264) (figure 2, B).

The frequencies of TNFα-producing CD4+ and CD8+ T-cells were low and did not differ between time points 0, 7 and 21-28 days both in CVID and HC (figure 2, C and D).

With regard to the different assays for the detection of CMI responses, a cor-relation was demonstrated between the fold-increase in spotforming cells in the IFNγ-ELISpot assay and the fold-increase in the frequencies of IFNγ-producing CD4+ T-cells upon stimulation with A/H1N1 at 21-28 days after influenza vaccina-

Figure 2. Frequencies of IFNγ-producing CD4+ and CD8+ T-cells (A and B) and TNFα-producing CD4+ and CD8+ T-cells (C and D) before, and 7 and 21-28 days following influenza vaccination in healthy con-trols and patients with CVID, as determined by flow cytometry. Bars represent medians, whiskers represent interquartile range. P-values as calculated using Friedman’s two-way analysis of variance by ranks. Differences between HC and P were calculated using Mann-Whitney U test. * p =0.019 when compared to healthy controls at baseline, # p =0.050 when compared to healthy controls at time point 21-28, § p =0.018 when compared to healthy controls at time point 21-28.

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tion for HC (r =0.829, p =0.042) and for patients at 7 days (r =0.750, p =0.052) and at 21-28 days (r =0.667, p =0.071). For A/H3N2 only in patients a correlation was found between the fold-increase in spotforming cells in the IFNγ-ELISpot assay and the fold-increase in the frequencies of IFNγ-producing CD4+ T-cells at 7 days following influenza vaccination (r =0.714; p =0.071).

lower anti-influenza antibody titers as determined by hIA in patients with cVId

The humoral response following influenza vaccination in 18 patients with CVID from the original cohort of 26 patients with a humoral primary immunodeficiency has been described elsewhere [5]. Summarizing, prevaccination geometric mean titers (GMTs) in patients with CVID and HC were comparable. However, for none of the influenza strains GMTs rose significantly after vaccination in patients with CVID. Patients with CVID had, for both A/H1N1 and A/H3N2, lower fold-increase in GMT (both p <0.001), postvaccination GMT (both p <0.001), seroconversion rates (p =0.004 and p =0.031), seroprotection rates (p =0.014 and p =0.002) and propor-tion of patients with protective postvaccination anti-influenza hemagglutination inhibition-titer of ≥40 (p =0.019 and p <0.001), compared to HC.

Inverse correlation between baseline cMI responses to A/h1n1 and A/h3n2 and fold-increase in influenza-specific cMI responses following influenza vaccination

As earlier described by others [14], we found an inverse correlation between influenza-specific CMI responses at baseline and the fold-increase in influenza-specific CMI responses following influenza vaccination. For patients as well as for HC the fold-increase in spotforming cells in the IFNγ-ELISpot assay was in-versely correlated with the prevaccination proportion of spotforming cells upon stimulation with A/H1N1 at 7 days (for CVID r =-0.5152; p =0.130: for HC r =-0.4615; p =0.110) and 21-28 days (for CVID r =-0.8364; p =0.022: for HC r =-0.7636; p =0.009), and upon stimulation with A/H3N2 at 7 (for CVID r =-0.4667; p =0.180: for HC r =-0.8077; p <0.001) and 21-28 days (for CVID r =-0.8364 (p =0.022); for HC r =-0.6545 (p =0.034) following influenza vaccination. Although these correlations were not always statistically significant, these results all point in the same direction.

no correlation between influenza-specific cell-mediated and humoral im-mune response

Next, in order to assess a possible relationship between the influenza-specific CMI responses and influenza-specific antibody responses following influenza vac-cination, we computed correlations between the fold-increase in anti-influenza

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GMT and the fold-increase in spotforming cells in the IFNγ-ELISpot assay as well as the fold-increase in IFNγ-producing CD4+ and CD8+ T-cells by flow cytometry upon stimulation with A/H1N1 and A/H3N2 at 7 and 21-28 days following influenza vaccination. No correlations were found (data not shown).

dIscussIonTo our knowledge, this study is the first to investigate the CMI response follo-wing influenza vaccination in patients with CVID using two different assays: IFNγ-ELISpot assay and flow cytometry with ICS. Earlier we showed that humoral re-sponses following influenza vaccination are severely hampered in patients with CVID [5].

Following influenza vaccination the patterns of the influenza-specific CMI respon-ses were different in patients with CVID and HC. In HC the proportion of IFNγ-producing cells increased, as determined by ELISpot assay as well as by flow cy-tometry analysis. This held in particular true for CD4+ T-cells, as expected, since subunit material is presented by antigen presenting cells to CD4+ T-cells in the context of MHC-class II molecules. Only a small part is presented to CD8+ T-cells, in the context of MHC-class I molecules by cross-presentation. This might explain the increase in the frequency of IFNγ-producing CD8+ T-cells in HC following in-fluenza vaccination. In contrast, in CVID patients IFNγ-producing cells did not in-crease in frequency following influenza vaccination in the ELISpot assay and even a decrease in the proportion of A/H1N1 and A/H3N2-specific IFNγ-producing CD4+ T-cells could be demonstrated using flow cytometry. These findings suggest that influenza-specific CMI responses are impaired in patients with CVID and fits with studies demonstrating a decreased CMI response after vaccination with recall and neo-antigens in these patients [15, 16].

Several causes may underlie the difference in influenza-specific CMI response in CVID and HC. First, defects in antigen-presenting cells have been described in CVID: DC are decreased in number [17-19], have reduced expression of surface molecules associated with maturity [20], express markedly reduced levels of the costimulatory molecules that are critical for T-cell stimulation [21], have defective TLR9 activation [22] and are impaired in differentiation (in part due to the defici-ency of circulating natural IgG antibodies) [23]. Monocytes of patients with CVID produce less IL-6 and TNFα in response to vaccination with 23-valent polysaccha-ride pneumococcal vaccine [24].

Second, T-cell abnormalities may explain the absence of an increase in CMI res-ponse following influenza vaccination in CVID: reduced numbers of (naïve) CD4+ T-cells and CD8+ T-cells, decrease in regulatory T-cells, reduced thymic output,

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increased T-cell activation, altered cytokine production (among which elevated levels of IFNγ-producing CD4+ and CD8+ T-cells, as we also found at baseline using FASC/ICS), disruption of CD4+ and CD8+ T-cell receptor repertoires, increased CD4+ and CD8+ T-cell turnover and increased levels of spontaneous apoptosis have all been found [8, 25-29]. Finally, 80% of the included CVID patients were treated with IVIg, which can inhibit T-cell proliferation and cytokine-production, although the exact mechanism remains to be elucidated [30]. IVIg-treatment in patients with CVID does not seem to play a role in their restriction of T-cell re-pertoires [8].

Our findings of a decrease in CD4+ T-cell count following vaccination and a reduc-tion in the proportion of IFNγ-producing CD4+ and CD8+ T-cells upon stimulation with SEB, A/H1N1 and A/H3N2 might be caused by activation-induced apoptosis of CD4+ and CD8+ T-cells, triggered by influenza vaccination or upon their reactiva-tion ex vivo by influenza virus. Influenza virus, but also its major surface protein hemagglutinin, the major constituent of the trivalent subunit influenza vaccine used in this study, can induce activation-induced apoptosis [31, 32]. Also increased pre-activation of T-cells in vivo , as has been demonstrated in CVID patients, make these cells more prone to apoptosis upon their ex vivo culture, thus reducing fre-quencies of influenza-specific T-cells. Furthermore, brefeldin A, used in the flow cytometry assay to inhibit secretion of cytokines and allowing their intracellular detection by monoclonal antibodies, has been shown to provoke apoptosis in hu-man tumor cells. A similar effect may have been induced in the (apoptosis-prone) T-cells, possibly leading to their accelerated apoptosis. This could also explain decreases in IFNγ-production in the flow cytometry assay, but not in the ELISpot assay. Moreover, this may not only be due to the use of brefeldin A but also due to the more extensive handling of the PBMC samples in the flow cytometry assay, as compared to the ELISpot assay.

We could demonstrate correlations between the fold-increase in spotforming PBMC by ELISpot and the fold-increase in the frequencies of INFγ-producing CD4+ T-cells by flow cytometry following influenza vaccination, but not for the individu-al parameters of CMI responses at the same time points. This latter finding most likely depends on the fact that both tests address different aspects of cell-me-diated immunity and therefore also differ in sensitivity [33]. Also an inverse cor-relation between CMI response at baseline and the fold-increase as determined by IFNγ-ELISpot was found. This has previously been demonstrated by others and is suggested to result from the interactions of memory CD4+ T-cells, CD56dim NK-cells and DC, where low memory CD4+ T-cells lead to better DC-function [14].

Although CD4+ T-cell help to B-cells is necessary for adequate antibody responses,

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B-cells of CVID patients have been shown to have low B-cell activation in res-ponse to stimuli from T-cells [34]. This might be an explanation for the absence of a relationship between humoral immune responses and CMI responses in the CVID patients in the current study. However, this can not explain the absence of a correlation in HC.

A limitation of this study is the low number of patients and HC included. This pre-vented us from analysing the influence of the clinical classification of the CVID patients, of IVIG-treatment and of previous vaccination on the CMI response.

Although influenza vaccination does not lead to the development of protective levels of anti-influenza antibodies [5] and no increase in CMI response using IFNγ- ELISpot and flow cytometry in CVID patients in this study, other parameters of cell-mediated immunity that we did not address (e.g. granzyme B-production by CD8+ T-cells) might lead to protection from influenza in these patients. Of note, correlates of protection from influenza with regard to CMI responses are lac-king. Therefore, influenza vaccination should be offered to patients with CVID, but additional measures for CVID patients to prevent influenza infection are also warranted, such as vaccination for influenza of household contacts and of health care workers of CVID patients [2, 35, 36]. Although prophylactic antiviral treatment with adamantanes and neuraminidase inhibitors reduces the chances of contrac-ting influenza [37], these should preferentially be reserved for treatment or post-exposure prophylaxis only since virtually all A/H3N2 are resistant to adamantanes, while resistance to the neuraminidase inhibitor oseltamivir is highly prevalent in A/H1N1. Passive immunization of CVID patients with serum from donors with high levels of anti-influenza antibodies shortly before the start of and during the influ-enza season might be another option to protect these patients, but this needs to be further explored.

conclusIonIn patients with CVID humoral immune responses as well as CMI responses fol-lowing influenza vaccination are hampered. Additional measures to influenza vac-cination are warranted to prevent patients with CVID from contracting (compli-cated) influenza.

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PART 2I n f l u e n z a vaccination in patients w i t h a u t o - i m m u n e i n f l a m -m a t o r y rheumatic d i s e a s e s

CHAPT

ER4S t u d i e s o f c e l l -m e d i a t e di m m u n eresponses to i n f l u e n z a vaccinat ion in systemic l u p u se r y t h e -m a t o s u s

A. Holvast

S. van Assen

A. de Haan

A.L.W. Huckriede

C.A. Benne

J. Westra

A.M. Palache

J.C. Wilschut

C.G.M. Kallenberg

M. Bijl

Arthritis & Rheumatism 2009 Aug;60(8):2438-2447

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ABSTRACTIntroduction Both antibody and cell-mediated responses are involved in the defense against influenza. In systemic lupus erythematosus (SLE) pa-tients a decreased antibody response to subunit influenza vaccine has been demonstrated. However, cell-mediated responses have not yet been as-sessed.

Methods Fifty-four SLE patients and healthy HC (HC) received subunit influenza vaccine. Peripheral blood mononuclear cells and sera were ob-tained before and one month after vaccination. Cell-mediated responses to A/H1N1 and A/H3N2 were evaluated using interferon (IFN)γ ELISpot and flow cytometry. Antibody responses were measured using the hemagglu-tination inhibition test.

Results Prior to vaccination, SLE patients had fewer IFNγ spotforming cells against A/H1N1 compared to HC and a lower frequency of IFNγ+CD8+ T-cells. After vaccination, the numbers of IFNγ spotforming cells increased in both patients and HC, though it remained lower in patients. Also frequen-cies of CD4+ T-cells producing tumor necrosis factor α and interleukin-2 were lower in patients after vaccination, compared to HC. As expected for a subunit vaccine, vaccination did not induce a CD8+ T-cell response. For A/H3N2-specific responses, results were comparable. Diminished cell-mediated responses to influenza vaccination were associated with the use of prednisone and/or azathioprine. Patients showed a lower increase in A/H1N1-specific and A/H3N2-specific antibody titers after vaccination, as compared to HC.

Conclusion In addition to a decreased antibody response, cell-mediated re-sponses to influenza vaccination are diminished in SLE patients, which may reflect effects of the concomitant use of immunosuppressive drugs. This may render these patients more susceptible for (complicated) influenza infections.

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InTRoduCTIonSystemic lupus erythematosus (SLE) is a systemic autoimmune disease charac-terized by a remitting and relapsing course. SLE patients have an increased risk of infection, due to both intrinsic disturbances of immune responses and use of immunosuppressive drugs which are often needed to control disease activity. Indeed, infection-related morbidity and mortality occur more frequently in SLE patients [1].

For influenza, infection-related morbidity and mortality are increased in immuno-compromised patients [2]. As influenza infection has a high incidence, with an estimated 5-20% of the general population infected annually [3], influenza vac-cination is a clinically relevant issue in SLE patients. Influenza vaccination of SLE patients is safe, as it has been shown that influenza vaccination does not induce disease activity [4]. Annual vaccination in SLE is therefore recommended [5].

In the immune response to influenza, both antibody and cell-mediated respon-ses, comprised of CD4+ and CD8+ T-cells, are involved. In SLE, antibody responses to influenza vaccination are diminished [6], but T-cell-mediated responses have not been assessed. The latter are relevant, as it has been shown that in certain groups, such as the elderly, cell-mediated responses to influenza vaccination can be a marker of clinical protection, independent from antibody responses [7]. The most frequently used vaccine formulations are split virus or subunit vaccines. With these vaccines, antigens are primarily presented via MHC II, which induces CD4+ T-cell stimulation [8]. However, they are incapable of inducing MHC class I restricted CD8+ T-cell responses [9]. In addition, subunit vaccines, in contrast to split virus and whole virus vaccines, do not contain any of the internal proteins that may more readily (re)activate influenza-specific CD8+ T-cells.

In SLE, decreased T-helper (Th) recall responses to influenza A and tetanus toxoid antigens have been reported in a subset of patients, as measured by interleukin (IL)-2 production upon stimulation. This decreased function could not be accoun-ted for by the use of immunosuppressives alone, and was shown to be associated with disease activity [10]. In addition, lower levels of cell-mediated cytotoxicity against target T-cells infected with influenza A and B have been found in SLE patients [11].

Based on these data, we hypothesized that SLE patients have lower CD4+ T-cell responses to subunit influenza vaccine and lower CD8+ T-cell recall responses to influenza antigens than HC. Cell-mediated responses against influenza in SLE, prior to and following vaccination, were evaluated. In addition, antibody respon-ses were evaluated, and vaccine safety was recorded.

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MeThodS

Study population

Patients were eligible for the study when they fulfilled at least four of the Ame-rican College of Rheumatology criteria for SLE [12]. Exclusion criteria were preg-nancy and the presence of an indication for yearly influenza vaccination based on concomitant disease according to international guidelines [13]. A control group of healthy individuals was included that was age and sex matched to the vaccinated SLE patients. Pregnancy was an exclusion criterion for participation as HC.

Study design

SLE patients and healthy controls (HC) were included from October to December 2005. Before entry, patients were randomized in a ratio of 2:1 to receive an influ-enza vaccination or to serve as non-vaccinated patient control. At entry (visit 1), patients randomized for vaccination and all HC were vaccinated. Patients and HC were followed up at 28 days (visit 2) and three to four months after inclusion (visit 3). PBMC were isolated from vaccinated participants at visits 1 and 2 (see below). At each visit blood was drawn, and serum was stored at –20° C until use. Also, SLE disease activity index (SLEDAI) [14] was recorded and patients were asked to mark a visual analogue score (VAS) for disease activity on a scale of 0-10, 0 indicating no activity and 10 indicating the highest activity. Information on influenza vac-cination in the previous year was obtained. Adverse effects to vaccination were recorded using a standardized questionnaire which included: itching, pain, ery-thema, induration at the site of vaccination, shivers, myalgia, fever, headache, nausea, arthralgia, diarrhea, use of an analgesic/ antiphlogistic drug. The study was approved by the institutional medical ethics committee, and informed con-sent was obtained from all participants.

Influenza vaccine

A single dose of a trivalent subunit influenza vaccine (Influvac®, 2005-2006, Sol-vay Pharmaceuticals, Weesp, the Netherlands), containing A/New Caledonia/ 20/99 [H1N1], A/NewYork/55/2004 [H3N2] and B/Hong Kong/330/2001, was admi-nistered intramuscularly.


PBMC were isolated from heparinized venous blood by density-gradient centrifu-gation on Lymphoprep (Axis-Shield, Oslo, Norway) immediately after blood was drawn. PBMC were frozen in RPMI 1640 (Cambrex BioScience, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS), 50 µg/ml of gentamicin (Gibco,

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Paisley, UK) and 10% dimethylsulfoxide. PBMC were stored in liquid nitrogen until use. Pre- and postvaccination samples, from a SLE patient and a matched control, were simultaneously thawed and batch-processed. A minimum cell viability of 90%, evaluated by trypan blue staining, was required. Preceding ELISpot assays, PBMC were rested, by overnight incubation at 37° C. Cells were counted before plating, using an automated cell counter (Beckman Coulter, Fullerton, CA, USA).


β-propiolactone whole inactivated virus (WIV) of A/New Caledonia/20/99 (H1N1) and A/Hiroshima/52/2005 (H3N2) were used to stimulate PBMC. A/Hiroshi-ma/52/2005 is a very closely related antigenic variant of the vaccine strain A/NewYork/55/2004.

Interferon (IFn)γ eLISpot assay

Nitrocellulose plates (Nunc, Rochester, NY, USA) were coated overnight at 4° C with 50 µl anti-human IFNγ, 15 µg/ml per well (Mabtech, Nacka Strand, Sweden). Plates were washed and blocked with culture medium (CM; RPMI supplemented with 50 µg/ml gentamicin and 10% FCS) for one hour at room temperature (RT). Subsequently, 2 x 105 PBMC were added per well, in 200 µl, and incubated in CM at 37° C with WIV of A/H1N1 and A/H3N2, at a final concentration of 5 µg total viral protein/ml. Concanavalin A stimulation, 5 µg/ml, was used as a positive control and a negative control consisted of PBMC in CM alone. Stimulation tests were performed in duplo. After 48 hours plates were washed with phosphate buffered saline (PBS), and 50 µl of 1 µg/ml biotinylated anti-human IFNγ (Mabtech) was ad-ded per well for 3 hours at RT. Next, plates were washed again, and 50 µl 1:1000 streptavidin-alkaline phosphatase (Mabtech) per well was added for 1.5 hours at RT. Plates were washed and 100 µl BCIP/NBT plus substrate (Mabtech) was added per well for 10 minutes. Finally, plates were washed with tap water. After drying, spots were counted using an automated reader (automated ELISpot video-ana-lysis system, Sanquin, Amsterdam, The Netherlands). Results are referred to as IFNγ spotforming cells, as IFNγ-producing CD4+ and CD8+ T-cells as well as natu-ral killer (NK) cells, following WIV stimulation, have been described [15].

Flow cytometry

For stimulations, 1.0-1.5 x 106 PBMC were cultured in 200 µl CM, in 5 ml polypropy-lene round-bottom FalconTM tubes (Becton Dickinson and Company (BD), Franklin Lakes, NJ, USA). Staphylococcal enterotoxin B, at 5 µg/ml, (SEB, Sigma-Aldrich, Saint Louis, MO, USA) was used as a positive control. WIV A/New Caledonia (H1N1) and WIV A/Hiroshima (H3N2) were used at final concentrations of 1 µg of total

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viral protein/ml. WIV and negative control (CM only) cultures were incubated in the presence of 10 µg/ml anti CD28/CD49 (BD). Cells were incubated for 18h at 37° C, the final 16 hours in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich). Following incubation, 10 µl 40mM EDTA in PBS was added, tubes were vortexed and incubated for 10 minutes, to facilitate resuspending. Next, 2 ml FACS lysing solution (BD) was added for 10 minutes. Cells were spun down and washed in PBS-1% bovine serum albumin. Subsequently cells were permeabilized in 500 µl PERM-2 (BD) for 10 minutes in the dark in the presence of pacific blue and orange (Invitrogen, Carlsbad, CA, USA), in a different combination for each stimulus, to enable fluorescent T-cell barcoding [16]. PBS-20% FCS was added for 5 minutes. Cells were washed and pooled per PBMC sample. Next, anti-CD3-FITC, anti-CD4-PE-Cy7, anti-CD8-PercP, anti-CD69-APC-Cy7, anti-IFNγ-Alexa 700, anti-tumor ne-crosis factor (TNF)α-APC and anti-interleukin (IL)-2-PE (all from BD) were added, following the manufacturer’s instruction. After incubation for 30 minutes at RT, cells were washed and immediately analyzed on a LSR II flow cytometer (BD). Data for at least 1 x 106 CD3+ cells were collected. Using the Win-List software package (Verity Software House, Topsham ME, USA), positively and negatively stained populations were gated and Boolean gating was applied. First, lymphocy-tes were gated by CD3 expression and sideward scatter patterns. Next, CD4+ and CD8+ T-cell populations were gated as CD4+CD8- or CD4-CD8+, respectively. Then, cells from different stimulation tubes were separated in a pacific blue/orange plot. Finally CD69+/- cytokine+/- quadrants were set for the different stimuli si-multaneously, according to the negative and positive controls. Percentages of antigen-specific cells were expressed as the percentage of CD69+ cytokine+ CD4+ or CD8+ T-cells within the total CD4+ or CD8+ T-cell population.


For quantitative detection of anti-influenza antibodies the hemagglutination in-hibition assay (HIA) was employed, following standard procedures [17]. Influenza A/New Caledonia/20/99 [H1N1] and A/NewYork/55/2004 [H3N2] were provided by Solvay Pharmaceuticals (Weesp, the Netherlands). Seroconversions were defined as a fourfold rise in titer one month after vaccination, and seroprotection was defined as a titer ≥40. Titers <10 (below detection level) were assigned a value of 5 for calculation purposes [18].

Statistical analysis

Data were analyzed using SPSS 14 (SPSS Inc., Chicago, IL, USA). Titers were log transformed prior to testing of geometric mean titers. For comparisons of T-cell cytokine responses, Mann-Whitney U test and Wilcoxon signed rank test were

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used. All T-cell frequencies reported are after background subtraction of the fre-quency of the identically gated population of cells from the same sample stimu-lated without antigen. For correlations, Spearman’s rho was used. Age was nor-mally distributed and tested with Student’s t-test. For all other variables Fisher’s exact test and Mann-Whitney U test were used where appropriate. A two sided P value <0.05 was considered statistically significant. No adjustments for multiple testing were made given the explorative design of the study.

ReSuLTS


Eighty SLE patients gave informed consent to participate and were randomized: 54 for the vaccination group and 26 for the non-vaccination group. Two patients initially randomized for the non-vaccination group were excluded (due to preg-nancy and withdrawal, respectively). Patient groups did not differ in sex, age, and medication use. More patients in the vaccination group had received an influenza vaccination the year before as compared to patients not receiving vaccination and HC (table 1).

Cell-mediated responses against A/H1N1 and A/H3N2 were measured in a subset

Table 1. Baseline characteristics and disease parameters

SLE patients HC

Non-vaccinated Vaccinated Vaccinated

(n=24) (n=54) (n=54)

Sex, males 2 (8.3%) 10 (18.5%) 11 (20.4%)

Age, mean (SD) 45.5 (11.5) 44.8 (13.6) 43.1 (10.9)

Influenza vaccination in previous year 9 (37.5%) 34 (63.0%) * 3 (5.6%) **

Without immunosuppressives 5 (20.8%) 5 (9.3%) N/A

Prednisone 10 (41.7%) 28 (51.9%) N/A

median (range), in users (mg/day) 6.25 (2.5-15) 5 (1.25-15) N/A

Hydroxychloroquine 10 (41.7%) 30 (55.6%) N/A

median (range), in users (mg/day) 400 (200-800) 400 (200–1000) N/A

Azathioprine 6 (25%) 17 (31.5%)

median (range), in users (mg/day) 87.8 (50-125) 125 (75–200) N/A

Other immunosuppressive drugs 0 (0%) 6 (11.1%) # N/A

SLEDAI, median (range) t=0 2 (0-8) 2 (0-12) N/A

VAS, median (range) t=0 2.2 (0-5.6) 1.6 (0-6.6) N/A

SLE, systemic lupus erythematosus; HC, healthy controls; SLEDAI, systemic lupus erythematosus disease activity index; VAS, visual analogue score; N/A, not applicable # Methotrexate, 5 patients used 15 mg per week, 1 used 25 mg per week; * p <0.05 (vaccinated SLE patients versus non-vaccinated SLE patients); ** p <0.001 (HC versus vaccinated SLE patients)

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of vaccinated SLE patients (n=38) and HC (n=38), matched for age and sex. This subset was based on availability of a matched control and proper acquisition of PBMC prior to and one month following vaccination. Mean age (SD) in this sub-group was 43.4 years (10.2); 24% were male.

Lower prevaccination cell-mediated responses to A/h1n1 and A/h3n2 in SLe patients

In ELISpot, prior to vaccination, SLE patients had fewer IFNγ spotforming cells against A/H1N1 and A/H3N2 as compared to HC (figure 1). In flow cytometry, the fre-quency of CD4+TNFα+ T-cells upon A/H1N1 stimulation was lower in SLE patients than in HC (figure 2, A). SLE patients also had a lower frequency of IFNγ+CD8+ T-cells upon A/H1N1 stimulation as well as lower frequencies of IFNγ- and TNFα-producing CD8+ T-cells upon A/H3N2 stimulation (figure 3, A and B).

Lower cell-mediated responses to A/h1n1 and A/h3n2 in SLe patients fol-lowing influenza vaccination

Following vaccination, 68.4% of SLE patients and 71.1% of HC showed a rise in IFNγ spotforming cells against A/H1N1; for A/H3N2 60.5% of patients and 73.7% of HC showed a rise. Rises were similar in SLE patients and HC. After vaccination, the number of IFNγ spotforming cells remained lower in SLE patients, compared to HC (figure 1).

Figure 1. Enzyme-linked immunospot assay of interferon (IFN)γ spot-forming cells per 2 x 10^5 peripheral blood mononuclear cells in patients with systemic lupus erythematosus (SLE) and healthy controls (HC) in response to A/H1N1 and A/H3N2 stimulation before vaccination (t=0 days) and 4 weeks after vaccination (t=28 days). Results are corrected for responses in unstimulated cultures from the same sample. Bars show the median and interquartile range.

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Following vaccination, A/H1N1-specific IFNγ-producing CD4+ T-cells increased in 66.7% of SLE patients and 65.7% of HC. Similarly, A/H1N1-specific TNFα-producing CD4+ T-cells increased in 61.1% of patients and 71.4% of HC. In 71.4% of HC, also IL-2-producing CD4+ T-cells increased (figure 2, B). For A/H3N2, 60% of SLE pa-tients and 61.8% of HC showed an increase in IL-2+CD4+ T-cells following vaccina-tion; 73.5% of HC showed an increase in TNFα+CD4+ T-cells as well (figure 2, B). So, in SLE patients the response to vaccination was restricted to a more limited cyto-kine profile. Moreover, SLE patients reached lower frequencies of TNFα- and IL-2- producing CD4+ T-cells against A/H1N1 compared to HC (p =0.014 and p =0.034, respectively).

As was expected, neither SLE patients nor HC showed changes in percentages of A/H1N1- and A/H3N2-specific CD8+ T-cells upon vaccination. Accordingly, post-vaccination, similar differences in influenza-specific CD8+ T-cells were observed as prevaccination (data not shown).

Adequate responses of Cd4+ and Cd8+ T-cells following SeB stimulation in SLe patients

Upon SEB stimulation, SLE patients and HC showed similar frequencies of IFNγ-, TNFα- and IL-2-producing CD4+ T-cells (figure 4, A) and CD8+ T-cells (figure 4, B). This indicated that T-cells from SLE patients were generally capable of adequate cytokine responses.

Lower antibody response to influenza vaccination in SLe patients

Prior to vaccination, SLE patients had a higher GMT against A/H1N1 as compared

Figure 2. CD4+ T-cell responses against A/H1N1 and A/H3N2. Frequencies of cytokineproducing CD4+ T-cells upon stimulation with A/H1N1 (A) and A/H3N2 (B) in patients with SLE and healthy control subjects, before vaccination and 4 weeks after vaccination. Results are corrected for responses in unstimulated (Unstim.) cultures from the same sample. Bars show the median and interquartile range. SEB =staphylococcal enterotoxin B; IL-2 =interleukin-2 (see figure 1 for other definitions).

A B

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with HC. One month postvaccination, SLE patients and HC reached comparable GMTs to each vaccine strain. However, the fold-increases following vaccination were lower in SLE patients for the A/H1N1 and A/H3N2 strains. Three to four months after vaccination, titers had decreased in both SLE patients and HC; GMTs remained comparable. SLE patients had a lower seroconversion rate for A/H1N1 than HC (p =0.001), but for A/H3N2 seroconversion rates in SLE and HC were si-milar. Prior to vaccination, seroprotection rates were comparable in SLE patients and HC. One month after vaccination SLE patients had a lower seroprotection rate against the A strains compared with HC, which was significant for A/H3N2 (p =0.032). Three to four months after vaccination seroprotection levels had drop-ped in SLE patients as well as HC to comparable levels (table 2).

Taken together, the antibody response in SLE patients was, moderately, decre-ased. This was further substantiated by results in serologically naïve SLE patients and HC (prevaccination titer <10). For A/H1N1,5 of 11 (46%) SLE patients showed such a seroconversion, versus 20 of 25 (80%) HC (p =0.056); for A/H3N2 this oc-curred in 1 of 7 (14%) SLE patients versus 18 of 22 (82%) HC (p =0.003). Finally, we analyzed whether immunosuppressive medication influenced antibody respon-ses. No such influence was found (data not shown).

Correlations between changes in IFnγ spotforming cells following vaccina-tion and seroconversions in both patients with SLe and control subjects

Changes in IFNγ spotforming cells following vaccination correlated with serocon-versions in both SLE patients and HC. The change in IFNγ spotforming cells against

Figure 3. CD8+ T cell responses against A/H1N1 and A/H3N2. Frequencies of cytokine-producing CD8+ T-cells prior to vaccination upon stimulation with A/H1N1 (A) and A/H3N2 (B) in patients with SLE and healthy control subjects. Results are corrected for responses in unstimulated cultures from the same sample. Bars show the median and interquartile range. For interleukin-2 (IL-2) production following stimulation with A/H3N2 in patients with SLE, both the median and the interquartile range were 0. See Figure 1 for other definitions.

A B

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A/H1N1, measured by ELISpot, correlated positively with seroconversion against A/H1N1 (r =0.311, p =0.058 for HC; r =0.348; p =0.032 for SLE patients; r =0.339, p =0.003 for all vaccinees). For A/H3N2 such a correlation was observed in HC (r =0.318, p =0.052), but not in SLE patients. No correlations were observed between CD4+ T-cell cytokine responses and antibody responses in HC or SLE patients.

Prior vaccination did not influence cell-mediated responses, but did lower antibody responses

In a subanalysis, SLE patients (n=13) and HC (n=35) who were not vaccinated in the previous year, were evaluated. Groups did not differ in age; mean age (SD) was 40.2 (8.9) years in SLE patients and 44.5 (9.6) in HC (p =0.164). In the IFNγ-ELISpot assay, SLE patients had fewer spotforming cells prior to vaccination against A/H1N1 (p =0.023) and A/H3N2 (p =0.034) than HC. After vaccination similar dif-ferences were found, though these did not reach significance (p =0.125 for A/H1N1 and p =0.051 for A/H3N2). Also flow cytometry results showed a tendency towards a restricted CD4+ T-cell response in SLE (data not shown).

In this subanalysis no differences in antibody responses (GMTs, fold increases of GMTs, seroconversion and seroprotection rates) were found between SLE pa-tients and HC (data not shown). In addition, a comparison was made between SLE patients who were vaccinated the previous year (n=20) versus those who were not (n=34). Vaccination in 2004 led to a higher prevaccination GMT against A/H1N1 (26.6 versus 10.5; p =0.001) and, subsequently, lowered seroconversion rate (27% versus 75%; p =0.001).

Figure 4. CD4+ and CD8+ T-cell responses to staphylococcal enterotoxin B (SEB). Frequencies of cytokine-producing CD4+ T-cell (A) and CD8+ T-cells (B) in patients with systemic lupus erythema-tosus (SLE) and healthy controls (HCs) upon stimulation with SEB in prevaccination samples. Results are corrected for responses in unstimulated cultures from the same sample. Bars show the median and interquartile range. IFNγ =interferon-γ ; TNF=tumor necrosis factor; IL-2=interleukin-2.

B A

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The use of prednisone and/or azathioprine was associated with lower cell-mediated responses to influenza vaccination

Patients using prednisone and/or azathioprine (PRED/AZA; n=22) were compa-red to patients who did not use these drugs (n=16). In this subanalysis, no diffe-rences were noted prior to vaccination. Following vaccination, patients on PRED/AZA had fewer IFNγ spotforming cells against A/H1N1 and A/H3N2 (p =0.004 and p =0.007, respectively) and lower frequencies of A/H1N1-specific IFNγ-, TNFα- and IL-2-producing CD4+ T-cells (p =0.004, p =0.033 and p =0.036, respectively) as well as A/H3N2-specific IFNγ-producing CD4+ T-cells (p =0.023). No differences in CD8+ T-cell responses to A/H1N1 and A/H3N2 were observed (data not shown). In patients not using prednisone and/or azathioprine, cell mediated responses to influenza vaccination were not significantly lower than in HC (data not shown).

no increase in disease activity following influenza vaccination, but more adverse effects in SLe than in hC

Prior to inclusion (Table 1), and during follow-up, vaccinated and non-vaccinated patient groups did not differ in SLEDAI and VAS scores. At visit 2, median (range) SLEDAI scores were 2 (0–13) in vaccinated SLE patients versus 2 (0–8) in non-vaccinated patients and at visit 3 these were 2 (0–10) versus 2 (0–4), respectively. For VAS scores, median (range) scores at visit 2 were 1.4 (0–8.1) in vaccinated SLE patients versus 2.1 (0–7.4) in non-vaccinated patients, at visit 3 these were 1.8 (0–9.4) versus 2.2 (0–8.9) respectively. Following vaccination, SLE patients more often reported itching (18% vs. 2% in HC; p =0.006), erythema (24% vs. 4%; p =0.003) and induration (30% vs. 11% p =0.026) at the site of vaccination, and arthralgia (16% vs. 4%; p =0.046). All adverse effects were mild and short-lasting.

dISCuSSIonTo our knowledge, this is the first study to evaluate cell-mediated immune re-sponses to subunit influenza vaccine in patients with a systemic autoimmune disease. To do so, we used ELISpot and flow cytometry. ELISpot is the more sensi-tive assay, whereas flow cytometry allows phenotyping and detection of multiple cytokines, which offers additional information on the gamma of the response [19].

Cell-mediated recall responses to influenza were lower in SLE patients. Prior to vaccination, SLE patients had considerably fewer IFNγ spotforming cells than HC against both A/H1N1 and A/H3N2. CD4+ T-cell responses to A/H1N1 were lower in SLE patients, which reached significance for TNFα-producing CD4+ T-cells. Also CD8+ T-cell responses were lower in SLE patients than in HC, for both A/H1N1 (IFNγ-production) and A/H3N2 (IFNγ and TNFα-production).

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Following influenza vaccination, cell-mediated responses to influenza remained lower in SLE patients. Although both SLE patients and HC showed an increase in IFNγ spotforming cells upon vaccination, for A/H1N1 as well as A/H3N2, numbers remained lower in SLE patients. SLE patients showed an increase in cytokine-pro-ducing A/H1N1-specific and A/H3N2-specific CD4+ T-cells following vaccination, however, this increase was restricted with respect to cytokine profile compa-red to HC. Moreover, SLE patients reached lower frequencies of A/H1N1-specific TNFα-producing and IL-2-producing CD4+ T-cells after vaccination. As expected, we did not observe a change in cytokine-producing CD8+ T-cells following vac-cination in either SLE patients or HC.

As CD4+ and CD8+ T-cell responses to SEB were normal in SLE patients, the de-creased cell-mediated response to influenza vaccination could not be attributed to a decreased responsiveness of T-lymphocytes in general. Furthermore, the observed differences in cell-mediated responses were, at least largely, indepen-dent of previous influenza vaccination status as well. The degree of influenza vac-cination in the previous year was higher in SLE patients, but in a subanalysis com-paring previously unvaccinated SLE patients with HC, SLE patients still showed considerably lower responses. Importantly, the use of medications played a major role, as the use of prednisone and/or azathioprine was associated with lower cell-mediated responses against both A/H1N1 and A/H3N2 following vaccination.

A diminished T-helper cell response in SLE patients to influenza has been repor-ted previously [20], as measured by IL-2 secretion in the supernatant of influen-za-stimulated PBMC of unvaccinated patients. We found a decreased CD8+ T-cell recall response in SLE patients to influenza antigens, which is in accordance with a previous study [11]. WIV, as used in this study, is able to induce CD8+ T-cell re-sponses in vivo and to reactivate memory CD8+ T-cells in vitro [21]. However, WIV might be a weaker stimulus of CD8+ T-cells as compared to live virus, due to lower antigen presentation on MHC I.

Importantly, fewer influenza-specific PBMC in SLE may be of clinical relevance. Recently, it was shown that numbers of spotforming cells correlate with clini-cal protection from, culture-confirmed, influenza in young children [22]. These numbers may vary depending on antigen type and influenza strain, as median numbers in our assays were higher than in assays in which hemagglutinin (HA) or vaccine components were used [23-26], and as in the present study A/H3N2-specific cell-mediated responses were lower than A/H1N1-specific responses. WIV contains core antigens in addition to surface antigens. Also, the uptake and presentation of WIV is more efficient [27]. Both factors might contribute to higher responses to WIV compared to HA or vaccine components.

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SLE patients showed normal T-cell cytokine responses to SEB. Previous reports reported a normal capacity of PBMC from SLE patients to respond to different stimuli, though diminished cell-mediated responses may be present during ac-tive disease [28-31]. As our cohort of SLE patients predominantly had quiescent disease, this may explain their normal responses to SEB. In addition, previous studies reported decreased proliferation of PBMC [32-34], whereas others found normal proliferative capacity [35], or heterogeneous results [36].

Diminished cell-mediated responses to influenza vaccination in SLE patients ap-pear to reflect, in particular, effects of immunosuppressive drugs. Effects of previous influenza vaccinations, or natural infections, could not be completely excluded. Whether intrinsic defects are involved, such as a defective antigen-presenting cell function [37, 38], is uncertain.

In SLE, antibody production upon influenza vaccination is lower than in the general population [39]. In the present study, we too found lower antibody responses in SLE patients, as reflected by lower fold-increases in titers, a trend towards lo-wer postvaccination GMTs and fewer seroconversions in serologically naïve SLE patients. Notably, antibody titers are the gold standard for protection and with regard to seroprotection rates, little differences were observed between SLE pa-tients and HC. Influenza vaccination in the previous year was associated with a lower seroconversion rate to A/H1N1; both vaccines contained the same A/H1N1 strain. Effects of previous influenza vaccination on antibody responses remain subject to discussion, as some studies reported decreased antibody responses [40-42], whereas others found similar [43-45] or improved responses [46].

We evaluated relationships between antibody and cell-mediated responses, as CD4+ T-cell help is necessary for antibody responses [47]. However, we did not find a correlation between CD4+ T-cell responses and antibody responses using flow cytometry. We did observe a, modest, correlation in SLE patients between changes in IFNγ spotforming cells against A/H1N1, measured by ELISpot, upon vaccination and seroconversion to A/H1N1. This suggests that in a subset of poor-er responding patients both cell-mediated and antibody responses are affected. Possibly, no correlation between CD4+ T-cell responses and antibody responses was observed due to the lower sensitivity of flow cytometry as compared to ELISpot [48].

Finally, we showed that influenza vaccination did not induce disease activity over a period of three to four months. This confirms previous studies, reviewed in [49].

Our study has some limitations. First, the sample size was relatively small and multiple comparisons were made. However, a proper power analysis was not

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possible as this is the first study to explore cell-mediated responses to influenza vaccination in SLE patients. Second, medication use in vaccinated SLE patients was heterogeneous. Third, more vaccinated SLE patients than HC had received an influenza vaccination in the previous year, which was of influence upon antibody responses. Fourth, there are no well-defined correlates between cell-mediated responses to influenza and the risk of influenza infection, which limits transla-tion of our results to clinical implications. Fifth, phenotypes of cells responding in ELISpot assays are unknown. It can be speculated that NK cells are among the cells which have responded in our ELISpot assay [50].

Despite these limitations, we conclude that the combined data point towards di-minished cell-mediated immune responses to influenza vaccination in a cohort of SLE patients representative for daily practice. Diminished cell-mediated respon-ses may reflect effects of the concomitant use of immunosuppressive drugs. The antibody response to influenza vaccination is also reduced in SLE patients. Clinici-ans should be aware that this combined defect might increase the morbidity and mortality due to influenza virus infection, in particular in patients on prednisone and/or azathioprine. Therefore evaluation of clinical protection from influenza in SLE patients, following influenza vaccination, seems indicated in order to assess if more effective influenza vaccines, or vaccination strategies, are warranted.

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CHAPT

ER5A s e c o n d , b o o s t e r , i n f l u e n z a vaccinat ion has l imiteda d d i t i o n a le f f e c t o na n t i b o d y r e s p o n s e s in quiescents y s t e m i c l u p u se r y t h e -m a t o s u s

A. Holvast

S. van Assen

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Rheumatology (Oxford) 2009 Oct;48(10):1294-1299

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AbsTrAcTIntroduction In systemic lupus erythematosus (SLE), a decreased antibody response upon influenza vaccination has been reported. In this study, we assessed whether a booster vaccination could improve antibody respon-ses, as determined by seroprotection rates, in SLE patients.

Methods SLE patients (n=52) with quiescent disease (SLE disease activi-ty index; SLEDAI ≤4) and healthy controls (HC; n=28) received subunit in-fluenza vaccine in October - December 2007. After four weeks, only SLE patients received a second vaccination. Sera were obtained prior to both vaccinations, and four weeks after the second vaccination. At each visit, SLE disease activity was recorded. The hemagglutination inhibition as-say was used to measure antibody titers. Seroprotection was defined as a titer ≥40.

results Following the first vaccination seroprotection rates and geometric mean titers (GMTs) to each vaccine strain increased in both SLE patients and HC, to comparable levels. Seroprotection rates in SLE patients after the first vaccination were 86.5% to A/H1N1, 80.8% to A/H3N2 and 61.5% to the B strain; GMTs were 92.6, 56.2 and 39.2, respectively. Overall, the booster vaccination did not lead to a further rise of seroprotection rates and GMTs in SLE patients. However, in patients not vaccinated in the previous year, GMTs and seroconversion rate to A/H1N1 did rise following the booster vac-cination. Both influenza vaccinations did not increase SLEDAI scores.

conclusion Additional value of a booster influenza vaccination in SLE is li-mited to patients who were not vaccinated in the previous year.

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InTroducTIonInfections are a frequent cause of death in systemic lupus erythematosus (SLE) patients, accounting for up to 20–55% of all deaths [1]. An increased risk of infec-tion in SLE is related to both intrinsic disturbances of immune responses and use of immunosuppressive drugs which are often needed to control disease activity.

One of the most frequent infections is influenza, with an estimated 5% of the adult population infected annually [2]. In immunocompromised patients, influenza has a higher morbidity and mortality [3]. Vaccination is considered the cornerstone for prevention of influenza related morbidity and mortality, and is recommen-ded in immunocompromised patients [4]. As influenza vaccination does not induce disease activity in SLE, there is an increasing support for annual influenza vacci-nation of SLE patients [5, 6].

However, several studies have reported a decreased antibody response in SLE patients. Seroprotection (titer ≥40) rates were lower in SLE patients than in heal-thy adults, which may limit clinical protection from influenza in (part of) vaccina-ted SLE patients [7]. Several strategies have been developed to increase antibody responses to influenza vaccination, the most important being addition of an ad-juvans, administration of booster vaccinations, increase of antigen dosage in the vaccine and intradermal instead of intramuscular vaccine administration. All have been reported to have additional value in certain patient groups, as compared to conventional vaccination [8-11]. We chose to evaluate a booster vaccination in our SLE cohort, as this strategy has two advantages over the others. First, in contrast to other strategies, the safety profile of conventional subunit vaccine in SLE has been established. This is important as triggering of autoimmunity is a concern in systemic autoimmune disease. Second, this strategy would be easiest to imple-ment within current vaccination practice.

In liver transplantation patients, an increase of the antibody response following trivalent booster vaccination has been shown [11]. Moreover, in SLE, a booster of A/H1N1 solely, one month after a first vaccination, increased geometric mean titer (GMT) [12]. However, there are also patient groups in which a booster vaccination had no additional value, such as dialysis patients, bone marrow transplant reci-pients and severely immunocompromised HIV patients [10, 13-17].

Based on previous data from our group, we hypothesized that influenza vacci-nation would result in a lower seroprotection rate in SLE patients [18], and that administration of a booster vaccination would increase seroprotection rate up to the level of seroprotection reached in healthy adults after a single vaccination. To test this hypothesis, we administered a booster dose of influenza subunit vac-cine to SLE patients with quiescent disease, four weeks after a first vaccination.

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Antibody responses were determined prior to the first and second vaccination, and four weeks after the booster vaccination.

MeThods

Patients and controls

SLE patients were eligible for the study when they fulfilled at least four of the American College of Rheumatology criteria for SLE [19] and had quiescent disease, defined as a SLE disease activity index (SLEDAI 20) ≤4. Exclusion criteria were pregnancy, malignancy and the use of prednisone >30 mg/day. A control group of healthy individuals was included that was age and sex matched to the patients on group level. Pregnancy was an exclusion criterion for participation as healthy control (HC).

study design

We conducted an open, prospective, controlled study. SLE patients and HC were included from October - December 2007. At entry (t=0), patients and HC re-ceived intramuscularly a single dose of trivalent subunit influenza vaccine (In-fluvac® 2007-2008, Solvay Pharmaceuticals, Weesp, the Netherlands), containing A/Solomon Islands/3/2006 [H1N1], A/Wisconsin/67/2005 [H3N2] and B/Malay-sia/2506/2004. After four weeks (t=1), patients received a second, booster, vac-cination. HC were not given a booster vaccination, because this does not increase antibody responses [11, 15, 17, 21]. Serum was obtained at t=0 and t=1 in patients and HC, and four weeks thereafter (t=2) in patients alone. At each visit, the SLEDAI was recorded. Routine measures were used to determine anti-double-stranded DNA (by Farr assay) and complement C3 and C4. From all participants, informa-tion on influenza vaccination in the previous year was obtained. Adverse effects to vaccination were recorded using a standardized questionnaire which included: itching, pain, erythema, induration at the site of vaccination, shivers, myalgia, fever, headache, nausea, arthralgia, diarrhea, use of an analgesic/ antiphlogistic drug. The study was approved by the institutional medical ethics committee, and informed consent was obtained from all participants.


For quantitative detection of influenza antibodies the hemagglutination inhibition assay (HIA) was used. HIA were performed in duplo with guinea pig erythrocytes following standard procedures [22] with slight modifications as described else-where [23]. Seroprotection was defined as a titer ≥40, seroconversion was de-fined as ≥ fourfold rise in titer; titers <10 (below detection level) were assigned a

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value of 5 for calculation purposes [24].

Power and statistical analysis

Data were analyzed using SPSS 14 (SPSS Inc, Chicago, IL, USA). Titers were log transformed prior to testing of GMTs. For testing differences in age between groups Student’s t test was used. Changes of GMT, anti-double-stranded DNA antibodies, complement C3 and C4 were tested using Wilcoxon signed rank test; McNemar tests were used to test changes in seroprotection rates and se-roconversion rates. Between groups, differences in GMT were tested using the Mann-Whitney U test. For all other comparisons, the Chi-square test or Fisher’s exact test were used, depending on the size of the expected counts. A p-value <0.05 was considered statistically significant.

Based on previous results, it was hypothesized that a single vaccination would re-sult in a 60% seroprotection rate against all three vaccine strains together [18], and that this would increase to 78% following a booster vaccination [11]. Seroprotec-tion against all three vaccine strains together was defined as a titer ≥40 against each of the vaccine strains in the same serum sample. For a power of 80% at an alpha of 5% to demonstrate such a difference, 47 SLE patients had to be included. Accounting for a 10% drop-out, this number was raised to 52.

Table 1. Baseline characteristics

SLE patients Healthy controls

(n=52) (n=28)

Sex, males (%) 9 (17.3%) 6 (21.4%)

Age, mean (SD) 45.2 (10.0) 45.2 (11.3)

Influenza vaccination in previous year (2006) 37 (71.2%) 20 (71.4%)

No immunosuppressives (%) 5 (9.6%) N/A

Prednisone 31 (59.6%) N/A

median (range), in users (mg/day) 5 (1.25-25)

Hydroxychloroquine 25 (48.1%) N/A

median (range), in users (mg/day) 400 (200-400) N/A

Azathioprine 15 (28.8%) 15 (28.8%)

median (range), in users (mg/day) 125 (50-200) N/A

Other immunosuppressive drugs 7 (13.5%) # N/A

SLEDAI, median (range) 2 (0-4) N/A

# four patients used methotrexate (one pt 10 mg/week, three pts 15 mg/wk), three patients used mycophenolate mofetil (one pt 1000 mg/day, two pts 2000 mg/day) and one patient used ciclos-porin 200 mg/day (same pt also used methotrexate). N/A: not applicable

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resulTs


Fifty-four SLE patients gave informed consent to participate, of whom one pa-tient withdrew prior to entry and one patient was excluded due to active disease. Fifty two patients completed the study, and their characteristics are summarized in table 1. Their mean age (SD) was 45.2 (10.0) years and 17.3% were males. Seven-ty-one percent of patients had been vaccinated against influenza in the previous year (2006). Median SLEDAI score at entry was 2, and most patients used immu-nosuppressives, especially prednisone, hydroxychloroquine and azathioprine. In the group of HC, age and sex were comparable to those in SLE patients. Also vac-cination history was similar, as most HC had participated in the hospital’s annual influenza vaccination campaign before.

The first influenza vaccination induced comparable seroprotection rates and geometric mean titers in sle patients and hc

Prior to vaccination, seroprotection rate against all three vaccine strains together did not differ between SLE patients (19.2%) and HC (7.1%; p =0.199). Following the first vaccination, this rate tended to be higher in patients than in HC, surprisingly (51.9% versus 28.6%, respectively, p =0.060). For patients, this rate was close to what was expected, but for HC it was much lower than anticipated, largely due to a low postvaccination seroprotection rate against the B strain.

Prior to vaccination, seroprotection rates and GMTs, for each strain, were compa-rable in SLE patients and HC. Following the first vaccination, seroprotection rates and GMTs increased in both patients and HC. Responses to the B strain were lo-wer as compared to those to the A strains. SLE patients reached a seroprotection

Figure 1. Antibody responses. Seroprotection rates (A) and geometric mean titers (GMTs) (B) in systemic lupus erythematosus (SLE) patients and healthy controls (HC). Seroprotection was defined as a titer ≥40. * p <0.05, ** p <0.01, *** p <0.001: after first vaccination vs. prevaccination, # p <0.05: SLE vs. HC, after first vaccination.

A B

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rate of 86.5% for A/H1N1, 80.8% for A/H3N2 and 61.5% for the B strain. Their post-vaccination GMT was 92.6 for A/H1N1, 56.2 for A/H3N2 and 39.2 for the B strain. HC reached comparable seroprotection rates and GMTs (figure 1). Also fold increases in GMT were comparable in SLE patients and HC. SLE patients showed fold incre-ases of 2.7, 2.1 and 1.9 for strains A/H1N1, A/H3N2 and B, respectively. HC showed fold increases in GMT of 2.7, 1.7 and 1.8 for strains A/H1N1, A/H3N2 and B, respec-tively.

A booster influenza vaccination did not increase seroprotection rates and GMTs in sle patients

Primary focus was the effect of booster vaccination upon seroprotection rates in SLE patients. The second vaccination did not further increase these seroprotec-tion rates (figure 1, A). Accordingly, at t=2, the proportion of patients with serop-rotection to all vaccine strains was 55.8%, demonstrating that there was no signi-ficant increase following the second vaccination. Similarly, the second vaccination did not induce a significant rise in GMTs (figure 1, B).

low seroconversion rates in both patients and hc

Seroconversion rates were low in both SLE patients and HC. After the first vac-cination, seroconversion rates to A/H1N1 were 34.6% in SLE patients and 28.6% in HC, for A/H3N2 rates were 25.0% and 10.7% and for B rates were 19.2% and 10.7%, respectively. Following the booster vaccination (t=2 vs. t=1), five (9.6%) SLE pa-tients showed a seroconversion to A/H1N1 whereas none of the patients showed a seroconversion to either A/H3N2 or the B strain. In SLE patients, when using baseline titers (t=0) as reference, for A/H1N1, the seroconversion rate tended to be higher after the second vaccination (t=2 vs. t=0) than after the first vaccination (46.2% versus 34.6%; p =0.070). However, the booster vaccination did not lead to a further increase of seroconversion rates for A/H3N2 and the B strain.

Influence of previous influenza vaccinations

A large part of SLE patients (71.2%) and HC (71.4%) had received an influenza vaccination in the previous year. Vaccination in the previous year led to higher prevaccination seroprotection rates, which reached significance for strains A/H3N2 (p =0.016) and B (p =0.027) in patients, and for A/H1N1 in HC (p =0.038) (figure 2, A). Accordingly, prevaccination GMTs were higher in previously vac-cinated participants; in patients this difference was significant again for strains A/H3N2 (p =0.001) and B (p =0.026), and in HC for A/H1N1 (p =0.004) (figure 2, B). Influenza vaccination in the previous year did not influence titers and se-roprotection rates after the first vaccination, except for the B strain in HC. The

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postvaccination seroprotection rate to the B strain was higher in HC not vacci-nated in the previous year (75%) than in previously vaccinated HC (30%, p =0.044) (figure 2, A and B).

Higher prevaccination titers in patients and HC vaccinated in the previous year lo-wered seroconversion rates after the first vaccination. In patients, this was most pronounced for the A/H3N2 strain. Patients not vaccinated in the previous year showed a 60.0% seroconversion rate to A/H3N2, versus 10.8% in previously vac-cinated patients (p =0.001). In HC, similar differences were observed, reaching significance for the B strain. HC not vaccinated in the previous year showed a

Figure 2. Effects of previous influenza vaccinations upon antibody titers. Seroprotection rates (A) and geometric mean titers (GMT) (B) in systemic lupus erythematosus (SLE) patients and healthy controls (HC), according to vaccination status in the previous year. Seroprotection was defined as a titer ≥40. For A/H1N1 in patients not vaccinated in the previous year, there was a trend towards an increase in GMT following the second vaccination (p =0.055). * p <0.05, ** p <0.01: prior to vaccination, vaccinated in the previous year versus not vaccinated in the previous year. # p <0.05: After first vaccination, vaccinated in the previous year versus not vaccina-ted in the previous year

A

B

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37.5% seroconversion rate to the B strain, versus 0% of the previously vaccinated HC (p =0.017).

Notably, for A/H1N1, vaccinations in the previous year influenced the response to the booster vaccination. In SLE patients not vaccinated in the previous year, the booster tended to increase the GMT to A/H1N1, but not to A/H3N2 and the B strain. Following the booster vaccination, the GMT to A/H1N1 increased from 89.8 to 139.3 (p =0.055). In previously vaccinated patients, the GMT was not influenced (figure 2, B). For seroconversion rate, a similar effect was found; in SLE patients not vaccinated in previous year, the seroconversion rate increased from 46.7% to 80% (p =0.062) but in previously vaccinated patients the seroconversion rate did not change (29.7% vs. 32.4%).

The use of prednisone and/or azathioprine was associated with lower anti-body responses to influenza vaccination

The use of immunosuppressives was heterogeneous, but stable during the du-ration of the study. Previous studies have reported lower antibody responses to influenza vaccination in SLE patients treated with steroids and azathioprine, but not in patients treated with hydroxychloroquine [18, 25-27]. We performed a suba-nalysis in which patients using prednisone and/or azathioprine (PRED/AZA; n=28) were compared with patients using no immunosuppressives or hydroxychloro-quine only (NO-imm/HCQ; n=17); patients using other immunosuppressive drugs then prednisone, azathioprine and hydroxychloroquine were excluded because of low numbers (n=7). PRED/AZA patients were somewhat younger than NO-imm/HCQ patients, but the groups did not differ with regard to influenza vaccination in the preceding year. PRED/AZA patients had a lower antibody response to influ-enza vaccination as compared to NO imm/HCQ patients, reflected by a lower GMT against A/H1N1 and A/H3N2 following the first vaccination, and a lower serocon-version rate for A/H1N1. The second vaccination had a slight additional effect for A/H1N1 within PRED/AZA patients (table 2).

Disease parameters did not increase following the influenza vaccinations and ad-verse effects of both vaccinations were mild in SLE patients

SLEDAI scores and levels of anti-double stranded DNA antibodies did not increase following the vaccinations. Levels of C3 and C4 remained almost stable during this period; slight increases of C3 and C4 levels were observed (Table 3). More SLE patients (19.6%) experienced erythema after both the first and second vaccination, compared to HC (0%; p =0.013). In SLE patients, adverse effects to the first and second vaccination were comparable (data not shown).

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Tab

le 2

. Eff

ects

of

imm

unos

uppr

essi

ve d

rugs

on

anti

body

res

pons

es

SLE

pati

ents

Pre

dnis

one/

AZ

AP

atie

nts

not

usin

g im

mun

osup

pres

sive

s/H

CQ

(n=2

8)(n

=17)

t=0

t=1

t=2

t=0

t=1

t=2

Age

, mea

n ±

SD, y

ears

42.1

± 9.

248

.4 ±

10*

Infl

uenz

a va

ccin

atio

n in

pre

viou

s ye

ar (2

00

6), n

(%)

21 (7

5)12

(70

.6)

SP r

ate,

n (%

)

H1N

114

(50

)23

(82.

1)25

(89.

3)8

(47.

1)16

(94.

1)16

(94.

1)

H3N

210

(35.

7)19

(67.

9)19

(67.

9)13

(76.

5)*

16 (9

4.1)

***

16 (9

4.1)

***

B11

(39.

3)17

(60

.7)

17 (6

0.7

)7

(41.2

)11

(64.

7)10

(58.

8)

GM

T

H1N

139

.572

.592

.8#

3213

0.5

*13

0.5

H3N

220

3941

39.2

*78

.4*

83.3

*

B22

.936

.740

18.4

40.8

43.4

SC r

ate,

n (%

)

H1N

1N

/A4

(14.

3)3

(10

.7)

N/A

11 (6

4.7)

**1 (

5.9)

H3N

2N

/A5

(17.

9)0

N/A

5 (2

9.4)

0

BN

/A3

(10

.7)

0N

/A5

(29.

4)0

t=0

: pri

or t

o va

ccin

atio

n; t

=1: 4

wee

ks a

fter

the

fir

st v

acci

nati

on; t

=2: 8

wee

ks a

fter

the

fir

st v

acci

nati

on, 4

wee

ks a

fter

the

sec

ond

vacc

inat

ion.

A

ZA

, aza

thio

prin

e; H

CQ

, hyd

roxy

chlo

roqu

ine;

SP

rat

e, s

erop

rote

ctio

n ra

te; S

C r

ate,

ser

ocon

vers

ion

rate

; N/A

, not

app

licab

le*

p <0

.05;

**p

<0

.01;

***

p =0

.064

(SLE

pat

ient

s us

ing

pred

niso

ne a

nd/o

r A

ZA

vs

pati

ents

usi

ng n

o im

mun

osup

pres

sive

dru

gs o

r H

CQ

onl

y); #

p <

0.0

5 (t

=2 v

s t=

1).

91

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5

dIscussIonIn SLE, a hampered antibody response to influenza vaccination has been reported in several studies [7]. As seroprotection rates are related to clinical protection from influenza, strategies to improve antibody responses are relevant in SLE. In the present study, we evaluated whether a second, booster, influenza vaccination could increase antibody titers. We did not find such an enhancing effect. Follo-wing the first vaccination, seroprotection rates and GMTs rose for each strain, but these did not rise further following the second vaccination. As an exception, there was a clear trend in the response to A/H1N1 in SLE patients who were not vaccinated in the previous year. This response did increase following the booster vaccination, in terms of GMT and seroconversion rate. The booster vaccination had mild adverse effects and did not increase SLEDAI scores.

Our findings regarding A/H1N1 in patients not vaccinated in the previous year ap-pear to be in accordance with a previous study in SLE patients, in which boosting was performed for A/H1N1 solely and was found to increase GMTs [12]. In this stu-dy, no information is presented regarding previous influenza vaccinations but it is likely that most patients had not received an influenza vaccination before, since there was much uncertainty regarding safety of vaccination in SLE [5].

In liver transplantation patients, a trivalent booster vaccination (28 days after the first vaccination) led to higher GMTs to all vaccine strains. Furthermore, the seroprotection rate against all three strains increased from >68% after the first vaccination to >80% after the booster vaccination [11]. Also in frail elderly, a boos-ter vaccination after 84 days increased GMTs, as detected by ELISA assay [28]. In healthy elderly, increases in seroconversion rate and GMT following a booster vaccination have been reported [21], however, the majority of studies did not find an additional effect [29, 30]. Similarly, in other patient groups such as bone mar-row transplant recipients [16], severely immunocompromised HIV patients [17] and dialysis patients [10, 13-15], booster vaccination did not have additional value. Also in healthy adults, booster vaccination did not increase antibody responses [11, 15,

Table 3. Disease parameters

t=0 t=1 t=2

SLEDAI, median (range) 2 (0-4) 2.5 (0-8) 2 (0-8)

Anti-dsDNA, median (range) (units/ml) 16 (<3-397) 18.5 (<3-275) 18.5 (<3-261)

C3, median (range) (g/l) 0.91 (0.42-1.42) 0.91 (0.35-1.45) 0.93 (0.31-1.45)*

C4, median (range) (g/l) 0.14 (<0.02-0.52) 0.15 (<0.02-0.52) 0.16 (0.02-0.50)**

t=0: prior to vaccination; t=1: four weeks after the first vaccination; t=2: eight weeks after the first vaccination, four weeks after the second vaccination; Anti-dsDNA: anti-double-stranded DNA; C3, C4: complement C3 and C4. * p < 0.05, ** p<0.01: at t=8 weeks vs. t=0 (prior to vaccination)

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17, 21].

Why booster vaccination did not improve the antibody response to influenza in SLE patients remains speculative. First, previous vaccinations appear to limit the effect of a booster vaccination, as reported in this study. Second, it may be ar-gued that booster vaccination can only have effect in patients with a low titer (<40) after a first vaccination. In this study, over 80% of patients had achieved protective titers to the A strains after the first vaccination and for these strains this may have hindered a further increase. This does not apply for the B strain as the seroprotection rate was 61.5% after the first vaccination. Nevertheless, the seroprotection rate to the B strain did not increase either following the booster vaccination.

Responses and titers to the B strain were low in both patients and HC. Generally, antibody titers to B strains are lower than titers to A strains [16, 31, 32]. This may be due to lower immunogenicity of the B strain as compared to the A strains, or a lower sensitivity of the HIA. The HIA for influenza B with whole virus particles, which is standard and was applied here, was previously found to be less sensitive than testing with influenza B virus disrupted with ether [33].

In accordance with previous reports, the use of prednisone and/or azathioprine was associated with lower antibody responses to influenza vaccination in SLE pa-tients [18, 25-27].

As a secondary study question, we evaluated whether a booster vaccination, sup-posed it were effective, would increase antibody responses in SLE patients up to levels reached in HC after a single vaccination. However, we did not observe differences in antibody responses between SLE patients and HC. Patients showed similar responses as in a previous study, but the responses of HC were lower than expected [18]. Although also some previous studies did not find differences between patients and HC [26, 34-36], most have shown antibody responses in SLE patients to be lower than in HC [12, 18, 25, 27, 37]. Furthermore, we found that cell-mediated responses to influenza vaccination, which correlate to clinical protec-tion from influenza infection, are hampered in SLE as well [38].

It is not clear why in the present study, SLE patients and HC did not differ in anti-body responses, but several factors could be involved. First, lack of power, as the study was not powered to study this question. Second, lower immunogenicity of current vaccine strains could have restrained differences between patients and HC. Third, HC had a higher degree of previous influenza vaccinations as compared to a previous study, while they did not differ with regard to age and sex [18]. Pos-sibly, influenza vaccinations in the preceding year hindered antibody responses

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[39-41], in which case previous findings of impaired responses were (partly) due to differences in vaccination history between SLE patients and HC. This implicates that actual differences between SLE patients and HC may be less than expected. In an extensive overview, Beyer et al. reported that especially for the B strain there is a general tendency to a lower postvaccination GMT and seroprotection rate in previously vaccinated groups [31], as we observed in our HC. It has been suggested that vaccines which are antigenically close to a prior vaccine may be partially eliminated by pre-existent cross-reactive antibodies, thus reducing the immune response [42].

Finally, the influenza vaccinations did not affect disease activity, which is in ac-cordance with previous studies [5]. However, it has been reported previously that although SLEDAI scores remain stable after influenza vaccination, levels of auto-antibodies may transiently increase [43].

In this study, a control group of SLE patients vaccinated once was not included, which might be a limitation. Here, SLE patients functioned as their own controls with regard to the effects of the first and the booster vaccination. This increased the statistical power to detect the expected additional effect of a booster vacci-nation, as it enabled a matched samples analysis. This has been done previously [11, 12, 14-17, 21, 32], though indeed some studies included a patient group vaccina-ted once and a patient group vaccinated twice [10, 13, 28-30].

In summary, booster vaccination with subunit influenza vaccine had no additional value in annually vaccinated SLE patients. In this study we did not find differences between SLE patients and HC in the antibody response to subunit influenza vacci-ne. However, the study was not designed and powered to detect such a difference. Therefore, we do not challenge previous studies showing decreased responses in SLE patients after influenza vaccination. As such, other strategies to improve antibody responses, mentioned earlier, should be considered. For example, the use of an MF59-adjuvanted influenza vaccine, which has a higher immunogenicity than conventional trivalent inactivated vaccine in adults with chronic diseases [8]. Another option would be to use an increased vaccine dose. In hemodialysis pa-tients, not using immunosuppressive drugs, a booster influenza vaccination did not have an additional effect upon titers, but a single double-dose vaccine did have additional value [10]. Finally, intradermal application of conventional influ-enza vaccine was reported to have higher immunogenicity in elderly as compared to intramuscular vaccination [9]. Whether these strategies enhance the immune response to influenza vaccination in SLE should be studied in controlled studies.

We conclude that the positive effect of a booster influenza vaccination on antibo-dy responses was limited to SLE patients who were not vaccinated in the previous

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year. These findings are restricted to patients with quiescent disease. Our results implicate that there is no additional value in offering a booster to annually vacci-nated SLE patients. This is of clinical importance, as annual influenza vaccination is recommended in SLE patients. Finally, it should be noted that in SLE patients who were not vaccinated in the previous year, administration of a booster vac-cination may be considered.

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CHAPT

ER6H u m o r a l r e s p o n s e s a f t e r i n f l u e n z a vaccinat ion are severely reduced in p a t i e n t s w i t h rheumatoid a r t h r i t i s t r e a t e d w i t h r i t u x i m a b

S. van Assen

A. Holvast

C.A. Benne

M.D. Posthumus

M.A. van Leeuwen

A.E. Voskuyl

M. Blom

A.P. Risselada

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Arthritis & Rheumatism 2010 Jan;62(1):75-81

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AbsTrAcTIntroduction For patients with rheumatoid arthritis (RA), yearly influenza vaccination is recommended. However, its efficacy in patients treated with rituximab is unknown. The objectives of this study were to investigate the efficacy of influenza vaccination in RA patients treated with rituximab and to investigate the duration of the possible suppression of the humoral im-mune response following rituximab treatment. We also undertook to as-sess the safety of influenza vaccination and the effects of previous influ-enza vaccination.

Methods Trivalent influenza subunit vaccine was administered to 23 RA patients who had received rituximab (4–8 weeks after rituximab for 11 pa-tients [the early rituximab subgroup] and 6–10 months after rituximab for 12 patients [the late rituximab subgroup]), 20 RA patients receiving metho-trexate (MTX), and 29 healthy controls (HC). Levels of antibodies against the three vaccine strains were measured before and 28 days after vaccination using hemagglutination inhibition assay. The Disease Activity Score in 28 joints (DAS28) was used to assess RA activity.

results Following vaccination, geometric mean titers (GMTs) of anti-in-fluenza antibodies significantly increased for all influenza strains in the MTX-treated group and in HC, but for no strains in the rituximab-treated group. However, in the late rituximab subgroup, a rise in GMT for the A/H3N2 and A/H1N1 strains was demonstrated, in the absence of a repopu-lation of CD19+ cells at the time of vaccination. Seroconversion and serop-rotection occurred less often in the rituximab-treated group than in the MTX-treated group for the A/H3N2 and A/H1N1 strains, while seroprotecti-on occurred less often in the rituximab-treated group than in the HC for the A/H1N1 strain. Compared with unvaccinated patients in the rituximab-tre-ated group, previously vaccinated patients in the rituximab-treated group had a higher pre- and postvaccination GMT for the A/H1N1 strain. The DAS28 did not change after vaccination.

conclusion Rituximab reduces humoral responses following influenza vaccination in RA patients, with a modestly restored response 6–10 months after rituximab administration. Previous influenza vaccination in rituximab- treated patients increases pre- and postvaccination titers. RA activity was not influenced.

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InTroducTIonPatients with rheumatoid arthritis (RA) are considered immunocompromised and at increased risk of infection [1]. Therefore, although the exact prevalence, morbi-dity, and mortality of influenza in patients with RA are unknown, yearly influenza vaccination is recommended [2]. Influenza vaccination is safe and results in pro-tective levels of anti-influenza antibodies in most RA patients, even when they are treated with prednisone, disease-modifying antirheumatic drugs (DMARDs), or tumor necrosis factor α–blocking agents [3, 4]. A growing number of RA patients are being treated with rituximab, depleting B-cells for 6–9 months. Theoretically, humoral responses to neoantigens cannot be elicited during B-cell depletion. Anti- influenza antibody response after influenza vaccination has been shown to be blunted in RA patients treated with rituximab [5, 6]. However, the exact level and duration of suppression of the humoral immune response and the influence of previous influenza vaccination on antibody response after treatment with rituxi-mab remain unclear.

In order to make recommendations for the usefulness and timing of influenza vaccination in RA patients treated with rituximab, we investigated humoral re-sponses in RA patients following vaccination with trivalent subunit influenza vac-cine 4–8 weeks or 6–10 months after treatment with rituximab. The responses were compared with responses in RA patients treated with methotrexate (MTX) and with responses in healthy controls (HC). In addition, the influence of previous influenza vaccination on antibody response and the safety of influenza vaccina-tion were assessed.

PATIenTs And MeThods


Patients had to fulfill the American College of Rheumatology (formerly, the Ame-rican Rheumatism Association) 1987 revised criteria for the classification of RA. Two groups of RA patients were defined. The first group of RA patients (the ri-tuximab group) received influenza vaccination either 4–8 weeks after treatment with rituximab (the early rituximab subgroup) or 6–10 months after treatment with rituximab (the late rituximab subgroup). Rituximab was administered intra-venously (IV) in 2 cycles of 1,000 mg with 100 mg IV methylprednisolone, except for one patient who instead received 4 cycles of 375 mg/m2 based on a proto-col for concomitant mixed cryoglobulinemia. The second group (the MTX group) consisted of RA patients who were treated with MTX at a minimum dosage of 10 mg/week, eventually with additional DMARDs. Health care workers served as HC. Patients in the rituximab group were recruited in all 4 participating Dutch univer-

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sity medical centers. RA patients receiving MTX and HC were recruited from the University Medical Center Groningen.

Exclusion criteria were no informed consent, age <18 years, malignancy, preg-nancy, or known allergy to or former severe reaction following vaccination with trivalent influenza subunit vaccine.

Vaccine

We used trivalent influenza subunit vaccine (Influvac® 2007–2008; Solvay Phar-maceuticals, Weesp, The Netherlands) containing purified hemagglutinin and neuramidase of the following strains: A/Wisconsin/67/2005 (H3N2)–like strain (A/H3N2 strain), A/Solomon Islands/3/2006 (H1N1)–like strain (A/H1N1 strain), and B/Malaysia/2506/2004-like strain (B strain).

Procedures

Patients and HC received the influenza vaccine intramuscularly from October 2007 until January 2008. Immediately before and 28 ± 3 days (mean ± SD) after vaccination, blood was drawn for measurement of CD19+ cell count, C-reactive protein level, erythrocyte sedimentation rate, and anti-influenza antibodies. The Disease Activity Score in 28 joints (DAS28) was recorded before and 7 and 28 days after vaccination. Information on previous influenza vaccination was obtained from all participants, and adverse effects occurring in the first 7 days postvac-cination were recorded. The study was approved by the ethics committees of all participating centers.

hemagglutination inhibition assay

The hemagglutination inhibition assay (HIA) was used for the detection of anti-influenza antibodies. HIAs were performed with guinea pig erythrocytes in ac-cordance with standard procedures [7]. The following parameters for efficacy of vaccination based on anti-influenza antibody response were evaluated: geometric mean titer (GMT), fold increase in titer, ≥4-fold titer rise resulting in a postvac-cination level of ≥40 (seroconversion), and titer rise to ≥40 (seroprotection). HIA titers ≥40 are generally considered to be protective in healthy adults [8].


All other data are presented as the median and range, except for GMTs, which are shown as the mean ± SD. Data were analyzed using SPSS 16.0 for Windows (SPSS, Chicago, IL). Analysis of variance, Student’s t-test with Bonferroni correction, the Kruskal-Wallis test, Friedman’s test, Wilcoxon’s signed rank test, the Mann-

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Whitney U test, the chi-square test, Fisher’s exact test, and Spearman’s rank cor-relation were used where appropriate. P-values less than 0.05 were considered significant.

resulTs

Patient and control characteristics

As shown in table 1, there were 23 RA patients in the rituximab group (11 in the early rituximab subgroup and 12 in the late rituximab subgroup), 20 RA patients in

Table 1. Characteristics at baseline of rheumatoid arthritis (RA) patients treated with rituximab (RTX), RA patients treated with methotrexate (MTX), and healthy controls (HC)

RTX MTX HC p-value

(n=23) (n=20) (n=29)

Age (years), mean (SD) 55.5 (7.6) 57.1 (6.7) 46.5 (12.5) 0.004 (RTX vs. HC)

0.477 (RTX vs. MTX)

Sex (F/M), no. (%) 16/7 (70/30) 11/9 (55/45) 23/6 (79/21) 0.192

Influenza vaccination 2006/2007, no. (%)

12 (52) 10 (50) 21 (72) 0.195

Duration RA (years), median (range)

13.8 (1.1-40) 8.7 (0.3-21) N/A 0.098

MTX (mg/week), median (range)

17.5* (10-25) 16.3 (10-25) N/A 0.873

Prednisone (mg/day), median (range)

8.75** (3.8-40) 0 (0-0) N/A <0.001

DMARDs, no (%)

azathioprine 1 (4) N/A

sulphasalazine 1 (5) N/A

leflunomide 1 (5) N/A

Interval after RTX (4-8 wk/6-10 mo), no. (%)

11/12 (48/52) N/A N/A

Previous RTX-cycles, no. (%)

0 11 (48) N/A N/A

1 5 (22) N/A N/A

2 6 (26) N/A N/A

3 0 (0) N/A N/A

4 1 (4) N/A N/A

CD19+-cells (x 10^9/l), median (range)

0 (0-0.09) 0.16 (0-0.24) 0.25 (0.09-0.44) <0.001 (RTX vs. HC)

<0.001 (RTX vs. MTX)

* n=10; ** n=15

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the MTX group, and 29 HC. The mean age of patients in the rituximab group did not differ significantly from that in the MTX group (p =0.477) but was higher than that in the healthy control group (p =0.004). Patients in the rituximab group had higher baseline DAS28 scores than patients in the MTX group (p =0.001) and lower B-cell counts than patients in the MTX group and HC (both p <0.001).


Geometric mean titers (table 2)

As expected, the GMTs of antibodies against the A/H3N2 and B strains prior to vaccination were higher in HC (p =0.002 and p =0.008, respectively, versus the rituximab and MTX groups combined), since more HC had received an influenza vaccination in the 2006–2007 season. Compared with GMTs before vaccination, GMTs following vaccination increased for all 3 influenza strains both in the HC (p =0.001 for the A/H3N2 strain, p <0.001 for the A/H1N1 strain, p <0.001 for the B strain) and in the MTX group (p <0.001 for the A/H3N2 strain, p <0.001 for the A/H1N1 strain, p =0.022 for the B strain). In contrast, no significant increase in GMT after vaccination was found in the rituximab group as a whole. Postvaccina-tion titers were higher for all 3 strains in the HC and for the A/H3N2 and A/H1N1 strains in the MTX group than in the rituximab group. Compared with the rituxi-mab group, the fold increase in titer was larger in the HC for the A/H1N1 strain (p =0.001) and the B strain (p =0.030) and larger in the MTX group for the A/H3N2 and A/H1N1 strains (both p <0.001).

GMT rose after vaccination in the late rituximab subgroup for the A/H3N2 strain (p =0.040) and the A/H1N1 strain (p =0.042), but not in the early rituximab sub-group, resulting in higher postvaccination GMT (p =0.040 for the A/H3N2 strain, p =0.003 for the A/H1N1 strain, p =0.007 for the B strain) and larger fold increase (p =0.041 for the A/H3N2 strain, p =0.043 for the A/H1N1 strain) in the late rituximab subgroup, thereby indicating some recovery of the humoral immune response 6–10 months after treatment with rituximab. At baseline, the peripheral blood CD19+ cell count was comparable for the early and late rituximab subgroups (me-dian [range] 0 x 109/liter [0 to 0.01] versus 0 x 109/liter [0 to 0.08], respectively; p =0.072). However, 28 days after vaccination, significantly more B-cells were present in the late rituximab subgroup than in the early rituximab subgroup (me-dian [range] 0.01 x 109/liter [0 to 0.10] versus 0 x 109/liter [0 to 0]; p =0.004).

Seroconversion

Seroconversion occurred more often in the MTX group than in the rituximab group for the A/H3N2 strain (p =0.011) and the A/H1N1 strain (p =0.020). Seroconversion for any of the 3 influenza strains occurred in only 3 patients in the rituximab group

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(all for the A/H1N1 strain), and all were in the late rituximab subgroup.

Seroprotection

Compared with the rituximab group, seroprotection was achieved more of-ten for the A/H1N1 strain (p =0.025) in the HC and for the A/H3N2 strain (p =0.020) and the A/H1N1 strain (p =0.020) in the MTX group (figure 1). The per-centage of persons with a postvaccination titer ≥40 irrespective of the prevac-cination titer was higher in the HC than in the rituximab group for the A/H3N2 strain (p <0.001), the A/H1N1 strain (p <0.001), and the B strain (p =0.020), and higher in the MTX group than in the rituximab group for the A/H3N2 strain (p =0.025) and the A/H1N1 strain (p =0.010). Seroprotection in the rituximab group occurred in only 6 patients (5 in the late rituximab subgroup versus 1 in the early rituximab subgroup; p =0.108).

Impact of previous vaccination

Compared with previously unvaccinated HC, HC vaccinated the year before sho-wed higher baseline GMT for the A/H3N2 strain (mean ± SD 41.8 ± 1.8 versus 13.5 ± 2.9; p =0.018). Conversely, the fold increase in titer following vaccination in the previously vaccinated HC was lower than that in the unvaccinated HC for the A/H3N2 strain (median 1 [range -1.4 to 8] versus 2.8 [range 1 to 16]; p =0.003) and the

Table 2. Geometric mean titers (GMTs) and fold increase in GMT for influenza A/H3N2, A/H1N1 and B, before (pre) and after (post) vaccination with trivalent influenza subunit vaccine, in healthy controls (HC), rheumatoid arthritis (RA) patients treated with methotrexate (MTX), and RA patients treated with rituximab (RTX), which were further split up in the subgroup early RTX (4-8 weeks after RTX) and late RTX (6-10 months after RTX)

HC MTX All RTX Early RTX Late RTX

(n=29) (n=20) (n=23) (n=11) (n=12)

GMTs, mean (SD)

A/H3N2 pre 27.6 (2.9) * 13.9 (2.8) 13.1 (2.3) 10.0 (1.7) 16.8 (2.7)

post 44.5 (2.2) † § 34.2 (1.9) † § 14.4 (2.5) 9.4 (2.1) 21.2 (2.6) † ¥

A/H1N1 pre 27.0 (3.0) 14.6 (2.5) 15.0 (2.0) 11.3 (1.8) 19.4 (2.0) ¥

post 73.6 (2.2) † § 47.6 (2.8) † § 18.5 (2.7) 10.0 (1.6) 32.7 (2.8) † ¥

B pre 15.7 (2.6) * 7.7 (1,9) 8.9 (2.1) 6.0 (1.6) 12.6 (2.3) ¥

post 29.7 (2.5) † § 13.4 (2.5) † 10.9 (2.4) 6.6 (1.6) 17.3 (2.5) ¥

Fold increase, median (range)

A/H3N2 1.4 (-1.4 - 16) 2 (1 - 11.3) † 1 (-2 - 2) 1 (-2 - 2) 1 (-1.4 - 2) ¥

A/H1N1 2 (-1.4 - 128) † 4 (1 - 16) † 1 (-2 - 8) 1 (-2 - 1.4) 1.2 (-1,3 - 8) ¥

B 1.4 (-1.4 - 32) 1 (-1.4 - 16) 1 (-2 - 5.7) 1 (-1.4 - 2) 1 (-2 - 5.7)

* p<0.05 compared to MTX- and RTX-group; † p<0.05 compared to and prevaccination titer; § p<0.05 compared to RTX-group; ¥ p<0.05 compared to early RTX-group

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Figure 1. Number of anti-influenza titers ≥40 as determined by hemagglutination inhi-bition assay for influenza A/H3N2 (A), A/H1N1 (B) and B (C) after vaccination with triva-lent influenza subunit vaccine, in healthy controls (HC, n=29), rheumatoid arthritis (RA) patients treated with methotrexate (MTX, n=20), and RA patients treated with rituximab (RTX, n=23). Black bars represent prevaccination titer ≥40, white bars represent postvac-cination titer ≥40 in patients with a prevaccination titer <40 (seroprotection)

0

20

40

60

80

100

HC MTX RTX

p=0.020

p=0.076

Num

ber w

ith ti

tre³ 4

0 (%

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0

20

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60

80

100p=0.020

RTX

p=0.025

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ith ti

tre³ 4

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0

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60

80

100p=0.286

p=0.086

RTXMTXHC

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ith ti

tre³ 4

0 (%

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Figure 1A

B

C

A/H3N2

A/H1N1

B

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(%)

Num

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wit

h ti

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B strain (median 1.4 [range -1.4 to 8] versus 2.8 [range 1 to 32]; p =0.023). In the MTX group, higher baseline GMT in vaccinated patients than in unvaccinated patients were shown for the A/H1N1 strain (mean ± SD 31.8 ± 2.1 versus 9.7 ± 2.7; p =0.019) and the B strain (mean ± SD 10.4 ± 2.0 versus 5.7 ± 1.6; p =0.015). In the MTX group, there was a lower fold increase in the previously vaccinated patients than in the unvac-cinated patients for the A/H3N2 strain (median 1.4 [range 1 to 4] versus 4 [range 2 to 11.3]; p =0.003), the A/H1N1 strain (median 2 [range 1 to 5.7] versus 6.7 [range 1 to 16]; p =0.018), and the B strain (median 1 [range -1.4 to 1] versus 3.4 [range 1 to 16]; p =0.001). In the rituximab group, compared with patients who were not pre-viously vaccinated, patients who were previously vaccinated had higher baseline antibody titers against the A/H1N1 strain (mean ± SD 19.4 ± 1.8 versus 11.3 ± 2.0; p =0.036) as well as higher postvaccination antibody titers against the A/H1N1 strain (mean ± SD 30.8 ± 2.6 versus 10.7 ± 2.0; p =0.007).

Seroconversion occurred more often for the A/H3N2 strain in the unvaccinated MTX group than in the vaccinated MTX group (50% versus 0%; p =0.016), but not for the HC or the rituximab group for any of the influenza strains (data not shown).

Previously unvaccinated HC more often developed seroprotection for the influ-enza B strain than did previously vaccinated HC (75% versus 9.5%; p =0.001). Previ-ously unvaccinated patients in the MTX group developed seroprotection for strain A/H3N2 (70% versus 20%; p =0.035) and strain B (40% versus 0%; p =0.043) more frequently than patients vaccinated the year before. However, the number of pa-tients with a postvaccination titer ≥40, irrespective of prevaccination titer, did not differ between previously vaccinated and unvaccinated patients in the rituximab group (data not shown).

Correlations between B-cell numbers and vaccination responses

In the rituximab group, CD19+ B-cells tended to increase 4 weeks after vaccina-tion (from median 0 x 109/liter [range 0 to 0.08] to 0 x 109/liter [range 0 to 0.10]; p =0.058) due to regeneration of B-cells in the late rituximab subgroup; B-cells in the late rituximab subgroup increased following vaccination (from median 0 x 109/liter [range 0 to 0.08] to 0.01 x 109/liter [range 0 to 0.10]; p =0.031) in contrast to B-cells in the early rituximab subgroup (from median 0 x 109/liter [range 0 to 0.01] to 0 x 109/liter [range 0 to 0]; p =0.317). However, in both the early and late rituximab subgroups, there were no correlations between B-cell count and pre-vaccination GMT, postvaccination GMT, fold increase in GMT, rates of serocon-version, and rates of seroprotection (data not shown).

Safety of vaccination - side effects and RA activity

There were no differences between the 3 groups in the occurrence of side ef-

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fects. RA activity, assessed with the DAS28 prior to and 7 and 28 days after vac-cination, was not influenced by influenza vaccination in either the MTX group (me-dian 3.04 [range 0.77 to 5.17] versus 2.93 [range 0.49 to 3.71] versus 2.59 [range 1.00 to 4.22], respectively; p =0.287) or the rituximab group (median 3.95 [range 2.15 to 5.71] versus 3.97 [range 2.15 to 6.26] versus 4.02 [range 2.04 to 6.77], respectively; p =0.834).

dIscussIonThe present study clearly shows that humoral responses to influenza subunit vac-cine in RA patients receiving rituximab are severely hampered compared with those in RA patients receiving MTX and compared with those in HC. This holds true for almost all outcomes. Our results are in line with those from a study in 4 RA patients, which evaluated humoral responses following influenza vaccination 84 days after treatment with rituximab [6]. A larger study by Oren et al that included 14 RA patients receiving rituximab showed only a lower GMT for influenza B strain and reduced rates of achieving a combined end point of seroconversion and se-roprotection for influenza A/H3N2 strain in patients receiving rituximab, compa-red with 29 RA patients receiving various DMARDs and 21 HC [5]. The discrepancy between our results and those of Oren et al might be explained by the larger time span between treatment with rituximab and influenza vaccination in the study by Oren et al (18 months, versus 10 months in our late rituximab subgroup); further, only 7 patients received influenza vaccination in the first 6 months after rituximab in the study by Oren et al.

The hampered response seems temporary since a significant rise in GMT after influenza vaccination in the late rituximab subgroup was found, while there was no increase in GMT in the early rituximab subgroup. Moreover, the only 3 cases of seroconversion in the rituximab group occurred in the late rituximab subgroup, and of the 6 cases of seroprotection in the rituximab group, 5 occurred in the late rituximab subgroup.

Although B-cells are required for the development of humoral immune responses to neoantigens, and depletion of B-cells following rituximab would be expected to reduce humoral immune responses to neoantigens, no correlation could be de-monstrated between B-cell count and the humoral responses following influenza vaccination in the 3 groups studied. This might be attributed to insufficient sensi-tivity of the standard quantitative assessment of B-cells (the lowest measurable B-cell count being 0.01 x 109/liter) [9]. Responders to influenza vaccination in the late rituximab subgroup probably already achieved some level of B-cell repopula-tion that was undetectable using standard methods. Another explanation could be that the number of B-cells in lymphoid tissues (i.e., sites where vaccine-mediated

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immune responses are initiated) is not correctly reflected by the peripheral blood B-cell numbers; CD19+CD20- B-cells have been shown to remain in the bone mar-row after 2 cycles of rituximab in RA patients [10].

Yearly repeated influenza vaccination leads to higher prevaccination anti- influenza antibody titers during the following year [11] and to a reduction in mortality [12]. In the current study we indeed found higher prevaccination GMT and lower fold increase in titer in previously vaccinated HC and patients in the MTX group compared with previously unvaccinated HC and patients in the MTX group. However, in addition to a higher prevaccination titer for the A/H1N1 strain, previously vaccinated patients in the rituximab group had a higher postvaccination titer. Notably, peripheral blood B-cells after recovery from rituxi-mab-induced B-cell depletion mainly consist of immature and naive B-cells, and low numbers of B-cells remain for up to 2 years [13, 14]. Our findings may there-fore point to the persistence of memory B-cells in compartments other than the peripheral blood that are capable of responding to the vaccine, and indicate that repeated yearly vaccination could be of additional value in achieving adequate levels of anti-influenza antibodies following influenza vaccination of RA patients treated with rituximab. Influenza vaccination was safe. Side effects in the study groups were comparable, and influenza vaccination did not increase RA activity. Finally, one should keep in mind that the correlates of protection for influenza following influenza vaccination in immunocompromised patients are not well de-fined. Anti-influenza titers ≥40 determined by HIA are considered protective, and 50% of persons with a titer of 28 are estimated to be protected; however, this has only been validated in young healthy adults [8]. Moreover, cellular immune responses have been shown to be of major importance in vaccination-mediated protection against influenza [15], and these are affected by rituximab as well. Since even titers <28 might provide some level of protection, even small increases in anti-influenza titer can be of clinical relevance. Therefore, the modest rise in titer in the late rituximab subgroup might be valuable.

Our study has some limitations. First, although this is the largest study to evalu-ate the response to influenza vaccination in RA patients treated with rituximab, the number of patients is still relatively small. However, the results were uni-form, and statistical significance was reached for many parameters. Second, the HC were younger than the RA patients, and age is an important factor in influenza vaccination response [2]. Since the age of patients in the MTX and ri-tuximab groups was comparable, and HIA titers were significantly higher in the MTX group than in the rituximab group, the difference in HIA titers between HC and patients in the rituximab group was unlikely to be caused by differen-ces in age. Third, although the use of additional DMARDs was not standardized,

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most of the patients in the rituximab group who had been taking DMARDs were receiving MTX, and only 1 patient was receiving high-dose corticosteroids. In the MTX group, only 2 patients had taken DMARDs other than MTX. Therefore, we do not believe that the unrestricted use of DMARDs influenced the study out-come. Moreover, the allowance of additional DMARDs offers the possibility to extrapolate our data to daily practice, where use of additional DMARDs is com-mon. The difference in corticosteroid use between the MTX and rituximab groups probably did not change the outcome, since even prednisone at a dosage of >7.5 mg/day has been shown not to affect the humoral response following influenza vaccination in RA patients [3;4].

In conclusion, this study shows a severely hampered humoral immune response to trivalent subunit influenza vaccine in RA patients treated with rituximab com-pared with RA patients receiving MTX and compared with HC. This response was slightly restored but still reduced 6–10 months after rituximab treatment. Previ-ously vaccinated patients in the rituximab group achieved higher anti-influenza titers following influenza vaccination for the A/H1N1 strain than did patients in the rituximab group who were not previously vaccinated. We recommend yearly influenza vaccination for RA patients. For those patients who start rituximab tre-atment, preemptive influenza vaccination should be considered.

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(3) Fomin I, Caspi D, Levy V, et al. Vaccination against influenza in rheumatoid arthritis: the effect

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(5) Oren S, Mandelboim M, Braun-Moscovici Y, et al. Vaccination against influenza in patients with

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rituximab. Ann Rheum Dis 2007 Oct; 66(10):1402-3.





(9) Dass S, Rawstron AC, Vital EM, Henshaw K, McGonagle D, Emery P. Highly sensitive B cell ana-

lysis predicts response to rituximab therapy in rheumatoid arthritis. Arthritis Rheum 2008 Oct;

58(10):2993-9.

(10) Teng YK, Levarht EW, Hashemi M, et al. Immunohistochemical analysis as a means to predict

responsiveness to rituximab treatment. Arthritis Rheum 2007 Dec; 56(12):3909-18.

(11) Beyer WE, Palache AM, Sprenger MJ, et al. Effects of repeated annual influenza vaccination on

vaccine sero-response in young and elderly adults. Vaccine 1996 Oct; 14(14):1331-9.

(12) Voordouw AC, Sturkenboom MC, Dieleman JP, et al. Annual revaccination against influenza and

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54(8):2377-86.



CHAPT

ER7Polyclonal andi n f l u e n z as p e c i f i cc e l l - m e d i a t e di m m u n eresponses are hampered in rheumatoida r t h r i t i sp a t i e n t st r e a t e d w i t h r i t u x i m a b

S. van Assen

A. de Haan

A.E. Voskuyl

A. Holvast

G. Horst

J. Westra

M.D. Posthumus

M.A. van Leeuwen

M. Blom

A.P. Risselada

C.G.M. Kallenberg

M. Bijl

Submitted

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AbsTrAcTIntroduction Yearly influenza vaccination is recommended for rheumatoid arthritis (RA) patients. However, humoral immune responses following influenza vaccination are severely hampered in RA patients treated with rituximab. Influenza-specific cell-mediated immune (CMI) responses follo-wing vaccination also contribute to protection against influenza. Therefore, we investigated CMI responses before and after influenza vaccination in RA patients treated with rituximab.

Methods 40 RA patients and 22 matched healthy controls (HC) were vacci-nated with trivalent influenza subunit vaccine. 21 RA patients had received rituximab and 19 RA patients were on methotrexate (MTX). Polyclonal and influenza-specific CMI responses were measured before, 7, and 28 days af-ter vaccination using flow cytometry, CFSE dye dilution proliferation assay and IFNγ-ELISpot.

results Before influenza vaccination, lower frequencies of polyclonally activated IL-2-producing CD4+ and CD8+ T-cells were observed in the rituximab-group compared to the MTX-group. Also, proliferation of polyclonally activated T-cells was lower in the rituximab-group compared with HC. Numbers of Influenza A/H1N1- specific IL-2+CD8+ and TNFα+CD8+ T-cells were lower in the rituximab-group compared to the MTX-group and HC. No differences were seen for A/H3N2-specific cells. After influenza vaccination, increased numbers of influenza A/H1N1-specific IL-2+CD8+ T-cells and an increased proliferative capacity of A/H1N1-specific T-cells were observed in HC. In contrast, in the MTX- and rituximab-group such incre-ased influenza-specific CMI responses were not seen, although responses tended to be higher in MTX-treated patients than in rituximab-treated RA patients.

conclusion Rituximab therapy reduces polyclonal T-cell responses in RA patients. Also influenza-specific CMI responses before and following influ-enza vaccination are hampered.

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InTroducTIonPatients with rheumatoid arthritis (RA) are at increased risk of infection, due to immune dysregulation inherent to the underlying autoimmune disease and the immunosuppressive treatment that these patients generally need to reduce RA disease activity. In a large prospective cohort study with a long follow-up on 3.501 RA patients, pulmonary infections were observed as the cause of death 5.3 times more often than expected [1]. Although the causative microorganisms of pulmo-nary infections in RA patients have not been well documented, mainly common bacteria and viruses causing respiratory infections, including influenza, are con-sidered to be involved.

Influenza is associated with increased morbidity and mortality, in particular in certain risk groups, such as young children, elderly and patients with chronic ill-nesses such as cardiac, pulmonary or renal disease, diabetes mellitus, and pa-tients with an immunocompromised state, among which RA patients treated with immunomodulating agents [2]. Although the exact prevalence, morbidity and mortality of influenza in RA patients are unknown, yearly influenza vaccination is recommended for RA patients [3].

Influenza vaccination is efficacious in most RA patients, even when treated with prednisone, disease modifying antirheumatic drugs (DMARDs) or TNF-α blocking agents [4]. However, as we and others demonstrated, following treatment with rituximab, RA patients are unable to mount adequate anti-influenza antibody re-sponses after seasonal influenza vaccination [5-9].

Besides humoral immune responses, cell-mediated immune (CMI) responses have been shown to be important for the prevention of influenza [10]. However, little is known about CMI responses in patients treated with rituximab. In order to evaluate CMI responses in RA patients treated with rituximab we analysed the polyclonal and influenza-specific CMI responses in these patients before and fol-lowing influenza vaccination.

PATIenTs And MeThods


Patients had to fulfil the American College of Rheumatology (ACR) clinical clas-sification criteria for RA. Two groups of RA patients were defined. One group consisted of RA patients who were treated with rituximab (rituximab-group), either at 4-8 weeks (early rituximab-subgroup) or 6-10 months (late rituximab-subgroup) after treatment. Rituximab was administered in 2 cycles of 1000 mg IV with 100 mg methylprednisolone IV, except for one patient who instead received 4 cycles of 375 mg/m2 based on a protocol for concomitant mixed cryoglobuline-

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mia. The other group consisted of RA patients treated with MTX, at a minimum dose of 10 mg/week orally or subcutaneously, with or without additional DMARDs. This group served as a disease control group (MTX-group). Health care workers and their family members served as healthy controls (HC-group). Patients in the rituximab-group were recruited from the four participating Dutch university me-dical centers in Groningen, Amsterdam, Nijmegen and Utrecht. MTX patients and HC were recruited from the University Medical Center Groningen. Exclusion crite-ria were: (i) no informed consent, (ii) age under 18, (iii) malignancy, (iv) pregnancy, (v) known allergy to or former severe reaction following vaccination with trivalent influenza subunit vaccine.

Vaccine

Trivalent influenza subunit vaccine (season 2007-2008; Influvac®; Abbott BV, Weesp, The Netherlands) was used. The vaccine contained purified hemaggluti-nin and neuramidase proteins from the following strains: A/Wisconsin/67/2005 (H3N2)-like strain (reassortant virus IVR-142, derived from A/Hiroshima/52/2005), A/Solomon Islands/3/2006 (H1N1)-like strain (reassortant virus IVR-145, derived from A/Solomon Islands/3/2006), and B/Malaysia/2506/2004-like strain (derived from B/Malaysia/2506/2004).

Procedures

Before the onset of influenza activity in The Netherlands, patients and HC received the influenza vaccine intramuscularly from October 2007 up till January 2008. Immediately before, and 7 and 28 ± 3 days after vaccination blood was drawn for measurement of lymphocyte subsets (CD4+ T-cells, CD8+ T-cells, CD16/56+ NK-cells and CD19+ B-cells), C-reactive protein and erythrocyte sedimentation rate, and for isolation of PBMC in order to determine CMI responses using interferon (IFN)-γ ELISpot, intracellular cytokine staining (ICS) using flow cytometry, and CFSE dye dilution proliferation assay. DAS28 was recorded before vaccination, and after 7 and 28 days as a validated measure for RA activity. From all partici-pants information on previous influenza vaccination was obtained. The study was approved by the ethics committees of all four participating centers.

cell-mediated immunity assays

Processing of PBMC and the procedures for the IFNγ ELISpot assay and flow cy-tometry were as described earlier [11]. Therefore, these procedures will only be described briefly.

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PBMC were isolated from BD Vacutainer® CPT™ Cell Preparation Tubes containing 0.1 M sodium citrate anticoagulant and blood separation media composed of a thixotropic polyester gel and a FICOLL™ HYPAQUE™ solution (BD, Franklin Lakes, NJ, USA), according to the instructions of the producer. PBMC were stored in li-quid nitrogen until use in RPMI 1640 (Cambrex BioScience, Verviers, Belgium) sup-plemented with 10% human pool serum, 50 µg/ml of gentamicin (Gibco, Paisley, UK) and 10% dimethylsulfoxide. Prevaccination and postvaccination samples from a RA patient treated with rituximab, a RA patient treated with MTX, and a matched HC were simultaneously thawed and batch-processed.


PBMC were stimulated with whole β-propiolactone-inactivated virus (WIV) or subunit of A/Solomon Islands/3/2006 (IVR-145 reass.; A/H1N1) and A/Hiroshi-ma/52/2005 (IVR-142 reass.; A/H3N2).

IFNγ-ELISpot assay

Before testing thawed PBMC were rested for 24 hours in culture medium (CM; RPMI 1640 [Cambrex BioScience, Verviers, Belgium] supplemented with 10% fetal calf serum, 50µg/ml of gentamicin [Gibco,Paisley, UK]), overnight at 37°C. Immobi-lon-P membrane plates (Millipore,Billerica,MA,USA) were coated with anti-human IFNγ (Mabtech, Nacka Strand, Sweden). 2 x 105 PBMC were added per well and incubated for 48 hours with A/H1N1 subunit (1 µg/ml) and A/H3N2 subunit (1 µg/ml) in CM. Concanavalin A stimulation served as a positive control, a negative control consisted of PBMC in CM alone. Stimulation tests were performed in duplicate. After incubation, biotinylated anti-human IFNγ was added, streptavidin-alkaline phosphatase and BCIP/NBT-plus substrate (all Mabtech) were used in order to visualise IFNγ-producing PBMC. Spots were counted using an automated reader (automated Aelvis ELISpot scanning system, Sanquin, Amsterdam, The Nether-lands).

Flow cytometry

For stimulations, 1.0–1.5 × 106 PBMC were cultured in CM. Staphylococcal ente-rotoxin B (SEB; Sigma-Aldrich, St. Louis, MO) was used as a polyclonal T-cell sti-mulus. WIV A/H1N1 and WIV A/H3N2 were used at final concentrations of 1 µg of total viral protein/ml. Negative controls were incubated in CM only. Pacific Blue and Pacific Orange dyes (Invitrogen, Carlsbad, CA), in a different combination for each stimulus, were used to enable fluorescent T-cell bar coding [12]. The fol-lowing monoclonal antibodies were used: allophycocyanin (APC)-Cy7-conjugated anti-CD3, peridinin chlorophyll A protein–conjugated anti-CD8 (Becton Dickinson

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Pharmingen, San Diego, CA, USA), phycoerythrin–Cy7 (PE-Cy7)–conjugated anti-CD69 (Biolegend, San Diego,CA, USA), PE–conjugated anti-IFNγ, APC-conjugated anti-TNFα, and Fluorescein isothiocyanate-conjugated anti–IL-2 (eBioscience, Hatfield, UK). Analysis was performed on a LSR II flow cytometer (Becton Dickin-son). At least 300.000 events were recorded.

The WinList software package (Verity Software House, Topsham, ME, USA) was used. Percentages of antigen-specific cells were expressed as the percentage of CD69+ cytokine-producing CD4+ or CD8+ T-cells within the total CD4+ or CD8+

T-cell population. Samples were excluded if the percentage of viable cells after thawing was <80%, and if the frequencies of IFNγ- or TNFα-producing CD4+ or CD8+ T-cells were <2.0% upon stimulation with SEB.

CFSE dye dilution proliferation assay

PBMC were washed with and resuspended in PBS at a concentration of 1x107/100 µl. Carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA) was added to the PBMC at a final concentration of 5 µg/ml. After PBMC were incubated for 10 minutes at 37˚C while protected for light, 10 ml RPMI + 10% FCS was added followed by two washing steps with RPMI + 10% FCS. The cell pellet was resuspended in CM (RPMI + 10% HPS), making a dilution containing 1x106 PBMC/ml.

Subsequently, 1x105 labeled cells per well were stimulated for 7 days in a 96-wells plate (Greiner bio-one Cellstar 650 180) with CM alone or CM with anti-CD3/CD28 as a polyclonal activation (anti-CD3 hybridoma supernatant clone WT32; anti-CD28 hybridoma supernatant clone 20-4996), A/H1N1 (5 µg/ml) subunit, or A/H3N2 sub-unit (1 µg/ml).

At day 7 cell pellets were washed with PBS and treated with FACS lysing solution (1:10; Becton Dickinson Pharmingen, San Diego, CA, USA). After washing with PBS supplemented with 0.3% heparin, cells were stained with anti-CD3-APC and anti-CD8-PercP, and subsequently analysed on a BD FACSCalibur. The degree of T-cell proliferation was analysed with Modfit LT 3.2 (Verity House Software) software and expressed as proliferation-index, defined as the average number of divisions of proliferating cells.


Data were analysed using SPSS 16 (SPSS Inc., Chicago, IL, USA) and are presented as median (range), except for age, which is presented as mean (SD). Analysis of variance, Kruskal-Wallis test, Friedman’s two-way analysis of variance by ranks, Mann-Whitney U test, and Pearson’s chi square test were used where appropri-ate. Differences between two of the three groups were only addressed with the

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Mann-Whitney U test if the Kruskal-Wallis test revealed a statistically significant difference between the three groups. A p-value <0.05 was considered statisti-cally significant.

resulTs


Twenty-one RA patients were included in the rituximab-group; 10 received influ-enza vaccination 4-8 weeks after their last rituximab infusion and 11 received influ-enza vaccination 6-10 months after rituximab therapy (table 1). Nine patients (43%) received their first cycle of rituximab, 5 (24%) their second, 6 (28%) their third and 1 (5%) her fifth. As controls, 19 RA patients on MTX and 22 HC were included. Medication use is depicted in table 1. Before vaccination the rituximab-group had higher baseline DAS28 scores than the MTX-group (p =0.003). Lower B-cells were found in the rituximab-group than in the MTX- and HC-group (p <0.001) and CD3+ and CD4+ T-cells were also lower in the rituximab-group (p =0.001 and p =0.002). No significant differences between groups were present for age, sex, influenza vaccination in the previous season (2006/2007), CD8+ T-cells, CD16/56+ cells, and the duration of RA.

Prevaccination polyclonal cMI responses

PBMC taken before vaccination were stimulated with Staphylococcal Enterotoxin B (SEB) to induce a strong polyclonal activation of T-cells that express SEB-bin-ding TCR Vβ families. A statistically significant difference between the HC, the MTX-group and the rituximab-group was found regarding the frequencies of IL-2-producing CD4+ and CD8+ T-cells upon SEB stimulation (p =0.009 and p =0.041, respectively, as determined by Kruskal-Wallis test). The differences in the fre-quencies of IL-2+CD4+ and CD8+ T-cells could be attributed to lower frequencies of the RTX-group in comparison to the MTX-group (p =0.020 and p =0.016, respec-tively) (figure 1, A). No differences between groups were found for frequencies of IFNγ- and TNFα-producing CD4+ or CD8+ T-cells upon SEB stimulation.

In the proliferation assay, polyclonal stimulation of T-cells was done using anti-CD3/28 antibodies. The proliferation-index of CD4+ T-cells in response to CD3/28 stimulation was different between the three groups (p =0.050 as determined by Krusal-Wallis test) (figure 1, B), which was due to a decreased response in the rituximab-group: the proliferation-index in HC was not statistically significantly different from the MTX-group, but was lower in the rituximab-group compared to HC (p =0.033) (figure 1, B). The proliferation-index for CD8+ T-cells was compa-rable between the three groups (figure 1, B).

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These results indicate that following treatment with rituximab, CMI responses in RA patients on rituximab are lower than in HC and RA patients treated with MTX, as indicated by the lower frequencies of IL-2 producing T-cells and the lower pro-liferation-index of CD4+ T-cells upon polyclonal activation.

Influenza-specific cMI responses

Baseline influenza-specific CMI responses

Before influenza vaccination no differences could be demonstrated between the three groups for the frequencies of cytokine-producing CD4+ T-cells upon stimulation with WIV A/H1N1 and A/H3N2. However, the proportions of CD8+

Table 1. Baseline characteristics

Healthy controls MTX-group RTX-group p-value

(n=22) (n=19) (n=21)

Age, mean ± SD years 50.6 ± 10.8 57.4 ± 6.6 56.1 ± 7.7 0.095

No. (%) women/no. (%) men 16 (73)/6 (27) 10 (53)/9 (47) 14 (67)/7 (33) 0.394

Received influenza vaccination 2006–2007, no. (%)

15 (68) 10 (53) 10 (48) 0.366

Duration of RA, median (range) years

N/A 7.3 (0.3-21.4) 13.8 (1.1-39.0) 0.085

DAS-28, median (range) N/A 3.05 (0.77-5.17) 3.94 (2.15-5.71) 0.003

MTX dosage, median (range) mg/week

N/A 15 (10-25) 17.5 (10-25) † 0.757

Prednisone dosage, median (range) mg/day

N/A N/A 8.6 (3.75-40) ‡ N/A

Taking DMARDs, no. (%)

Azathioprine dosage, mg/day N/A 0 150 * N/A

Sulfasalazine dosage, mg/day N/A 1000 * N/A N/A

Leflunomide dosage, mg/day N/A 20 * N/A N/A

Interval after rituximab before vaccination, no. (%) 4–8 weeks/no. (%) 6–10 months

N/A N/A 10 (48)/11 (52) N/A

CD19+ cells x 10^9/liter, median (range)

0.25 (0.14-0.44) 0.19 (0.07-0.25) 0 (0-0.08) <0.001


1.29 (0.84-2.71) 1.05 (0.53-1.95) 1.02 (0.38-2.37) 0.001


0.95 (0.62-1.88) 0.76 (0.26-1.44) 0.68 (0.14-1.74) 0.002


0.37 (0.10-0.75) 0.30 (0.08-0.59) 0.28 (0.08-0.64) 0.096

CD16/56+ cells x 10^9/liter, median (range)

0.24 (0.08-0.49) 0.16 (0.06-0.41) 0.18 (0.04-0.49) 0.326

† n=10; ‡ n=13; * n=1; MTX, methotrexate; RTX, rituximab; N/A, not applicable

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T-cells producing IL-2 and TNFα upon stimulation with WIV A/H1N1 did dif-fer between the groups (p =0.010 and p =0.039, respectively, as determined by Kruskal-Wallis test) (figure 2, A). These differences could be attributed to diffe-rences between the frequencies of IL-2 and TNFα-producing CD8+ T-cells in the MTX-group compared to rituximab-group (p =0.018 and p =0.026, respectively, as determined by Mann-Whitney U-test) (figure 2, A) and in HC compared to ri-tuximab (p =0.005 and p =0.027, respectively, as determined by Mann-Whitney U-test). The three groups did not differ significantly in frequencies of cytokine-producing CD8+ T-cells upon stimulation with A/H3N2 (figure 2, B). When using the proliferation assay, neither for CD4+ T-cells nor for CD8+ T-cells differen-ces between the three groups could be demonstrated at baseline upon stimu-lation with A/H1N1 (figure 2, C) or A/H3N2 (figure 2, D). Also the proportion of influenza-specific spotforming cells in the IFNγ-ELISpot did not differ at baseline between HC, RA patients treated with MTX, or RA patients treated with rituxi-mab for both strains (figure 2, E). It should be noted that the numbers of partici-pants for whom the proliferation and ELISpot assays were performed were small.

Influenza-specific cell-mediated immune responses following influenza vaccination

For HC, upon stimulation with A/H1N1 the frequencies of Il-2-producing CD8+ T-cells increased 7 and 28 days following influenza vaccination (p =0.047, as deter-mined by Friedman’s Two-Way ANOVA; table 2). In the MTX-group and the ri-tuximab-group, frequencies of A/H1N1- and A/H3N2-specific cytokine-producing CD4+ and CD8+ T-cells did not change (table 2).

Figure 1. Prevaccination polyclonal cell-mediated immune responses as determined by flow cytome-try (A) and proliferation assay (B). P-values as calculated with Mann-Whitney U-test. Mann-Whitney U-test was only used when differences between healthy controls (HC), methotrexate (MTX)-group and rituximab (RTX)-group were significant when tested with Kruskal-Wallis-test.

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Figure 2. Prevaccination influenza A/H1N1- and A/H3N2-specific CMI responses as determined by flow cytometry (A and B), proliferation assay (C and D) and IFNγ-ELISpot (E). P-values as calculated with Mann-Whitney U-test. Mann-Whitney U-test was only used when differences between healthy controls (HC), methotrexate (MTX)-group and rituximab (RTX)-group were significant when tested with Kruskal-Wallis-test.

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Tab

le 2

. CM

I res

pons

es b

efor

e, 7

and

28

follo

win

g in

flue

nza

vacc

inat

ion

as d

eter

min

ed b

y fl

ow c

ytom

etry

, in

heal

thy

cont

rols

, RA

pat

ient

s tr

eate

d w

ith

met

ho-

trex

ate

(MT

X) a

nd R

A p

atie

nts

trea

ted

wit

h ri

tuxi

mab

(RT

X).

Num

bers

rep

rese

nt m

edia

n (r

ange

) fre

quen

cies

of

cyto

kine

+ CD

4+ or

CD

8+ T-c

ells

..

Hea

lthy

con

trol

s (n

=17)

MT

X (n

=14)

RT

X (

n=13

)

Bef

ore

728

P-v

alue

Bef

ore

728

P-v

alue

Bef

ore

728

P-v

alue

CD

4+ T-c

ells

, A/H

1N1

IFNγ

0.3

9 (0

.04-

1.31)

0.2

5 (0

.05-

1.97)

0.2

7 (0

.04-

1.86)

0.8

980

.42

(0.0

4-1.8

4)0

.26

(0-2

.28)

0.3

5

0.0

9-2.

35)

0.13

30

.33

(0.0

3-1.8

4)0

.18

(0.0

7-2.

46)

0.2

6 (0

.02-

3.37

)0

.794

IL-2

0.0

4 (0

-0.3

1)0

.05

(0-0

.20

)0

.04

(0-0

.73)

0.0

710

.04

(0-1

.01)

0.0

3 (0

-0.13

)0

.04

(0-0

.20

)0

.694

0.0

1 (0

-0.0

9)0

.02

(0-0

.82)

0.0

3 (0

-0.3

8)0

.90

9

TN

Fα0

.05

(0-0

.67)

0.10

(0

-0.3

2)0

.10

(0-0

.43)

0.0

690

.13

(0.0

1-1.1

3)0

.11

(0-0

.32)

0.14

(0

.02-

0.4

1)0

.256

0.0

5 (0

-0.16

)0

.10

(0-1

.25)

0.0

5 (0

-0.2

8)0

.923

CD

4+ T-c

ells

, A/H

3N2

IFNγ

0.5

0

(0.10

-1.7

8)0

.50

(0

.05-

1.78)

0.5

9 (0

.07-

1.92)

0.5

540

.38

(0.0

6-1.5

1)0

.25

(0.0

7-2.

21)

0.3

8 (0

.09-

2.36

)0

.880

0.3

3 (0

.06-

2.60

)0

.39

(0.14

-3.9

3)0

.79

(0.0

8-4.

88)

0.5

84

IL-2

0.0

4 (0

.01-

0.14

)0

.0

(0-0

.09)

0.0

3 (0

-0.3

1)0

.884

0.0

5 (0

-0.13

)0

.02

(0-0

.12)

0.0

4 (0

-0.10

)0

.40

40

.05

(0-0

.13)

0.0

2 (0

.0.12

)0

.04

(0-0

.10)

0.7

26

TN

Fα0

.12

(0.0

3-0

.50

)0

.10

(0.0

1-0

.92)

0.15

(0

-1.5

4)0

.580

0.12

(0

-0.5

5)0

.14

(0-0

.61)

0.13

(0

04-

0.8

1)0

.797

0.0

6 (0

.02-

0.4

2)0

.08

(0.0

2-1.6

9)0

.14

(0-0

.58)

0.9

23

CD

8+ T-c

ells

, A/H

1N1

IFNγ

0.4

0

(0.0

2-3.

15)

0.2

5 (0

.07-

4.0

0)

0.2

4 (0

-2.3

9)0

.627

0.4

1 (0

.07-

3.22

)0

.27

(0.0

1-2.

16)

0.2

7 (0

.08-

3.29

)0

.171

0.12

(0

.04-

3.61

)0

.23

(0.0

3-4.

79)

0.2

0

(0-6

.87)

0.5

00

IL-2

0.0

5 (0

-0.6

5)0

.04

(0-0

.15)

0.0

7 (0

-0.7

3)0

.047

0.0

4 (0

-0.2

2)0

.04

(0-0

.30

)0

.07

(0-0

.69)

0.2

450

.01

(0-0

.07)

0.0

3 (0

.0.4

7)0

.0

(0-0

.23)

0.2

25

TN

Fα0

.06

(0-1

.58)

0.0

7 (0

-0.5

2)0

.08

(0-0

.62)

0.8

190

.09

(0-0

.33)

0.10

(0

-0.2

8)0

.10

(0.0

2-0

.61)

0.3

05

0.0

2 (0

-0.18

)0

.03

(0-0

.51)

0.0

8 (0

-1.3

50

.922

CD

8+ T-c

ells

, A/H

3N2

IFNγ

0.5

6 (0

-1.7

0)

0.5

5 (0

.06-

3.0

1)0

.52

(0-2

.56)

0.2

360

.63

(0.0

9-3.

01)

0.5

8 (0

.03-

3.27

)0

.60

(0

.09-

2.73

)0

.880

0.5

6 (0

.04-

4.28

)0

.39

(0.12

-2.4

6)0

.48

(0.0

6-2.

61)

0.6

62

IL-2

0.0

3 (0

-0.2

4)0

.03

(0-0

.12)

0.0

5 (0

-0.2

9)0

.650

0.0

4 (0

-0.3

7)0

.02

(0-0

.25)

0.0

4 (0

-0.3

7)0

.119

0.0

1 (0

-0.15

)0

.02

(0-0

.44)

0.2

0

(0-0

.38)

0.2

97

TN

Fα0

.06

(0-0

.60

)0

.07

(0-0

.49)

0.0

8 (0

-0.4

7)0

.938

0.0

7 (0

-1.2

0)

0.0

8 (0

-0.2

7)0

.06

(0.0

1-0

.43)

0.3

900

.06

(0-0

.18)

0.0

7 (0

.01-

0.9

6)0

.11

(0.0

1-1.3

0)

0.6

62

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Also, the proliferation-index for CD4+ T-cells rose in HC following influenza vac-cination upon stimulation with A/H1N1 (p =0.042, as determined by Mann-Whitney U-test), but not upon stimulation with A/H3N2 and not for CD8+ T-cells. Further-more, no change in proliferation-index for CD4+ and CD8+ T-cells could be demon-strated 7 and 28 days following influenza vaccination upon stimulation with A/H1N1 or A/H3N2 in the MTX- and rituximab-group (data not shown).

Since it is unknown what increase in influenza-specific CMI response is of clinical relevance, we categorised the proliferation assay results qualitatively as “rise” or “no rise” in proliferation-index. Five out of 5 (100%) HC, 3 out of 6 MTX patients (50%) and 1 out of 4 rituximab patients (25%) demonstrated a rise following vac-cination for CD4+ T-cells for A/H1N1 (figure 3, A), and 4 out of 5 (80%) HC, 4 out of 6 MTX patients (67%) and 3 out of 4 rituximab patients (75%) for A/H3N2 (figure 3, A). For CD8+ T-cells 3 out of 3 (100%) HC, 4 out of 5 MTX patients (80%) and 2 out of 4 rituximab patients (50%) demonstrated a rise following vaccination for A/H1N1 (figure 3, A), and 3 out of 4 (75%) HC, 3 out of 5 MTX patients (60%) and 3 out of 4 rituximab patients (75%) for A/H3N2 (figure 3, A).

In the subgroup of study participants for whom an IFNγ-ELISpot assay could be performed, comparisons of the proportions of spotforming cells did not reveal any significant changes following influenza vaccination in all three groups upon stimulation with WIV A/H1N1 (table 2). From the 7 HC, 6 (86%) had a rise in spotfor-ming cells upon stimulation with A/H1N1 at day 7 following influenza vaccination, compared to 4 out of 6 (67%) MTX patients and 1 out of 5 (20%) rituximab patients (figure 3, B). No difference was found upon stimulation with A/H3N2 (figure 3, B).

Figure 3. Percentage of healthy controls (HC), methotrexate (MTX) and rituximab (RTX) patients with a rise in proliferation index as determined by proliferation assay (A) and in spotforming cells as determined by IFNγ-ELISpot (B) 7 days after influenza vaccination.

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dIscussIonInfluenza-specific CMI responses following influenza vaccination are important for the protection from clinical influenza [10]. This is of particular importance in patients who are unable to mount sufficient influenza-specific humoral immune responses. RA patients treated with rituximab have been shown to have severely hampered humoral responses following influenza vaccination and seroprotection rates for influenza hardly increase in response to influenza vaccination [5-8]. Therefore, we investigated influenza-specific CMI responses before and follo-wing influenza vaccination, using three different techniques. Flow cytometry was used to determine frequencies of IFNγ-, IL-2- and TNFα-producing CD4+ T-cells and CD8+ T-cells upon stimulation with WIV of the two influenza A-strains as pre-sent in the used vaccine. Moreover, IFNγ-ELISpot was performed since this test has been shown to be more sensitive than flow cytometry with intracellular cyto-kine staining [11, 13]. Finally, proliferation of CD4+ and CD8+ T-cells upon stimulation with influenza-subunit was investigated. This is the first study to address pre- and postvaccination polyclonal and influenza-specific CMI responses in rituximab-treated RA patients this extensively.

Before vaccination we demonstrated lower frequencies of IL-2-producing CD4+

and CD8+ T-cells upon stimulation with SEB in the rituximab-group than in the MTX-group, while the MTX-group and HC performed similarly. Proliferation upon stimulation with anti-CD3/28 was also reduced in the rituximab-group compared to HC. Furthermore, rituximab patients had lower influenza-specific CMI respon-ses at baseline. Following influenza vaccination, flow cytometry showed an incre-ase in frequencies of IL-2- and TNFα-producing CD4+ T-cells and IL-2-producing CD8+ T-cells in HC only, although not all increases were statistically significant. IFNγ-ELISpot and proliferation assay further supported the reduced vaccination responses in rituximab-treated RA patients, taking into account that numbers of patients included in these assays were low.

The lower CMI responses that we found at baseline in the rituximab-group upon stimulation with SEB and anti-CD3/28 indicate that RA patients treated with ri-tuximab have a lower ability to mount CMI responses. This likely explains the de-creased influenza-specific CMI responses in these patients. Little is known on CMI responses in RA patients following treatment with rituximab. Delayed type hy-persensitivity T-cell responses to Candida as measured by skin testing 24 weeks after treatment with rituximab have been shown to be comparable between RA patients treated with rituximab and RA patients treated with MTX [14]. Arad et al. investigated influenza-specific CMI responses following influenza vaccination in RA patients treated with rituximab. Only the frequency of IFNγ-producing CD4+ T-cells was addressed using flow cytometry with intracellular cytokine staining [8].

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At baseline, a lower frequency of IFNγ-producing CD4+ T-cells upon stimulation with a mixture containing WIV of the A/H1N1, A/H3N2- and B-strain was found in ri-tuximab patients than in HC. The current study, however, did not find differences in IFNγ-producing CD4+ T-cells at baseline, but demonstrated lower frequencies of IL-2- and TNFα-producing CD4+ and CD8+ T-cells in rituximab patients, as mea-sured by flow cytometry. This discrepancy might have been caused by the fact that methods differed: we measured responses to A/H1N1 and A/H3N2 separately.

Our findings are in line with experimental animal studies, which revealed that in mice B-cell depletion leads to reduced CD4+ T-cell recall responses to keyhole limpet hemocyanin, and CD4+ T-cell expansion was significantly reduced upon im-munization with ovalbumin in these mice. In response to infection with Listeria monocytogenes B-cell depleted mice had a reduced frequency of dividing CD4+ T-cells and CD8+ T-cell proliferation was reduced. Furthermore, in B-cell depleted mice IL-2- and IFNγ-production by CD4+ T-cells was limited. Finally, it was shown that B-cells were necessary in addition to dendritic cells for optimally inducing CD4+ T-cell responses, pointing at the important role of B-cells with regard to antigen presentation. CD8+ T-cells demonstrated the same responses in B-cell depleted mice as in their littermates that were not B-cell depleted [15]. The re-duced responses in CD8+ T-cells that were found in this study, may therefore be caused by other factors. CD8+ T-cells, as well as CD4+ T-cells, can be driven towards apoptosis by glucocorticoids in vitro [16], and 13 of 21 (62%) of the included RA patients that received rituximab used prednisone. Moreover, five of 21 (24%) rituximab patients used a combination of low dose prednisone and MTX. Schuer-wegh et al. investigated the influence of the use of the combination of low dose MTX (7.5-10 mg/day) and low dose prednisone (5-10 mg/day), comparable to the doses used by our patients, on the production of IFNγ, IL-2 and IL-4 by CD4+ and CD8+ T-cells of 20 RA patients. After 12 months of treatment a significant reduc-tion of the absolute numbers of IL-2 and IFNγ-producing CD4+ and CD8+ T-cells compared to pre-treatment values was found [17]. Therefore, the use of the com-bination of MTX and prednisone might have contributed to the reduction in the frequencies of IL-2 producing CD8+ T-cells in our rituximab-treated RA patients.

Only limited influenza-specific CMI responses following influenza vaccination as determined by flow cytometry were found in all participants, including HC. This might have had several causes. Flow cytometry has been shown to be less sen-sitive than IFNγ-ELISpot [11, 13], and, therefore, a rise in IFNγ-producing CD4+ or CD8+ T-cells might have been missed. However, using the more sensitive method of IFNγ-ELISpot, we found a trend towards a rise in spotforming cells in HC, des-pite the small number of included HC (n=6). Second, a large proportion of the stu-dy participants were vaccinated during the previous year. This was in particular

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true for HC, of whom 68% received influenza vaccine during the previous year. It has been shown that persons who received influenza vaccination the year before can have blunted influenza-specific CMI responses [18]. This may have contributed to the limited CMI responses in both patient groups and HC, following influenza vaccination. In contrast to our results, Arad et al. found a rise in the frequency of influenza-specific IFNγ-producing CD4+ T-cells after influenza vaccination in pa-tients treated with rituximab. However, it was not stated whether or not the rise reached statistical significance [8]. Surprisingly, they demonstrated a drop in the frequency of influenza-specific IFNγ-producing CD4+ T-cells after influenza vac-cination in HC, which could not be confirmed by our results.

It has been assumed that blunted CMI responses in patients treated with rituximab account for the occurrence of opportunistic infections that have been documented in these patients. Opportunistic infections are typically found in patients with cel-lular immunodeficiencies. In particular progressive multifocal leucencephalopathy due to JC-polyoma virus has been described, however, the majority of cases oc-curred in patients with lymphoproliferative disorders treated with rituximab [19]. A meta-analysis performed on the overall incidence of severe infections in lymphoma patients treated with CHOP with or without rituximab did not find an increase in risk in the group treated with rituximab [20]. Another meta-analysis including three randomized controlled trials on treatment of RA patients with rituximab also did not reveal a statistically significant increased risk of serious infection due to the use of rituximab. In the included studies most patients treated with rituxi-mab were also treated with MTX 10-25 mg/week [21]. Therefore, despite the loss of humoral responses following rituximab and the blunted CMI responses that we demonstrated, the immune system seems to be sufficiently redundant to com-pensate for these defects. It is well known that many immune defects by itself do not lead to clinical consequences. For example, a large drop in CD4+ T-cells is mandatory in HIV patients before opportunistic infections occur [22], and hypo-gammaglobulinemia not necessarily is accompanied by infections, which depends largely on the level and the class of immunoglobulins that are decreased [23].

We recognize that our study has some limitations. The number of participating patients and HC was relatively small, although no larger studies have been per-formed so far. At the start of the study no data were available regarding CMI responses following influenza vaccination in RA patients treated with rituximab and RA patients treated with MTX. Therefore, a power calculation could not be performed. Also, treatment in patients with rituximab was not standardized with regard to immunosuppressive co-medication. Therefore, the differences found between the rituximab-group and the two control groups might have been influ-enced by this co-medication. Furthermore, a correlate of protection regarding

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influenza-specific CMI responses has not been determined. Therefore, it remains unknown if the differences found in our study have any clinical relevance with regard to the protection for influenza. Finally, more HC received influenza vac-cination the year before, which could have influenced the CMI response following influenza vaccination as well.

In conclusion, we demonstrated that prior to influenza vaccination RA patients treated with rituximab, compared to RA patients treated with MTX and HC, have reduced influenza-specific CMI responses, but also lower CMI responses upon strong polyclonal stimuli. Moreover, in response to influenza vaccination, no in-crease in CMI responses was found in rituximab-treated patients. Although a cor-relate of protection for influenza with regard to influenza-specific CMI responses has to be determined yet, our findings support the recommendation to offer in-fluenza vaccination to RA patients before treatment with rituximab [3].

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PART 3E U L A R recomen- d a t i o n s for vacci- n a t i o n in patients w i t h a u t o - i m m u n e i n f l a m -m a t o r y rheumatic d i s e a s e s

CHAPT

ER8V a c c i n a t i o n i n a d u l t patients with auto-immune inflammatory r h e u m a t i c d i s e a s e s : a systematic l i t e r a t u r e review for the E U L A R e v i d e n c e -b a s e d r e c o m m e n -d a t i o n s for vaccination i n a d u l t patients with auto-immune inflammatory r h e u m a t i c d i s e a s e s

S. van Assen

O. Elkayam

N. Agmon-Levin

R. Cervera

M.F. Doran

M. Dougados

P. Emery

P. Geborek

J.P.A. Ioannidis

D.R.W. Jayne

C.G.M. Kallenberg

U. Müller-Ladner

Y. Shoenfeld

L. Stojanovich

G. Valesini

N.M. Wulffraat

M. Bijl

Autoimmunity Reviews 2011 Apr;10(6):341-352

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ABSTRACTObjectives To present the systematic literature review (SLR), which for-med the basis for the European League Against Rheumatism (EULAR) evi-dence-based recommendations for vaccination in adult patients with auto-immune inflammatory rheumatic diseases (AIIRD).

Methods AIIRD, vaccines and immunomodulating drugs, as well as eight key questions were defined by the multidisciplinary expert committee commissioned by EULAR for developing the recommendations. A SLR was performed using MedLine through to October 2009 and including data from meta-analyses, systematic reviews, randomized trials, and observa-tional studies, excluding case series with ≤5 participants. Articles in English and regarding patients ≥16 years of age, were eligible.

Results Several vaccine-preventable infections (VPI) occur more often in AIIRD patients and most vaccines are efficacious in AIIRD patients, even when treated with immunomodulating agents, except rituximab. There does not appear to be an increase in vaccination-related harms in vacci-nated patients with AIIRD in comparison with unvaccinated patients with AIIRD. However, these studies are underpowered and therefore not conclu-sive.

Conclusion Based on the current evidence from the literature, recom-mendations for vaccination in patients with AIIRD were made. However, more research is needed in particular regarding incidence of VPI, harms of vaccination and the influence of (new and established) immunomodulating agents on vaccinationefficacy.

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InTROduCTIOnPatients with auto-immune inflammatory rheumatic diseases (AIIRD) are at incre-ased risk of contracting infections, associated with considerable morbidity and mortality [1-11]. Certain infections might be prevented by vaccination. The effi-cacy of vaccinations may, however, be reduced in patients with AIIRD. Moreover, safety of vaccination in patients with AIIRD is an important issue, because of the potential risk of AIIRD-flares following vaccination.

In order to prepare recommendations for vaccination in patients with AIIRD the European League Against Rheumatism (EULAR) commissioned a task force [12]. The exercise performed by the task force involved both evidence-based and ex-pert opinion-based approaches. The current review reports the results of the systematic literature review (SLR) that formed the basis for these recommen-dations.

METHOdSThe expert committee formulated eight key questions to be addressed for the SLR, performed by SvA, MB, NAL and OE. Twenty-seven predefined AIIRD con-ditions, 29 vaccines and 17 immunosuppressive medications (table 1) were used as search terms, using Medline (via PubMed; from 1966 to October 2009) and the abstracts from the meetings of EULAR 2008 and 2009 and the American College of Rheumatology (ACR) 2007 and 2008. Only articles in English and concerning patients older than 16 years of age were included. Additional papers considered relevant in the opinion of the experts could be added (figure 1). Meta-analyses, systematic reviews, randomized trials, and observational studies (both analytic and descriptive) were eligible and scored according to table 2. Case reports and case series (≤5 patients) were not included.

RESuLTS

Question 1. Is the risk of infections for which vaccines are available incre-ased in patients with AIIRd in general, and specifically in those with active disease, and in those using immunomodulating agents?

Influenza

Two retrospective studies showed an increased risk of influenza in elderly pa-tients (≥65 years) with rheumatic diseases, vasculitis, chronic renal failure, de-mentia or stroke (all considered at intermediate risk of complicated influenza). Compared to low-risk elderly, the odds ratio was 1.56 (95% confidence interval [CI] 1.23-2.02) for hospital admission for either pneumonia or influenza and 2.67 (CI 2.26-3.16) for death, measured over 6 seasons [13]. In another study 4.5-7% of

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Table 1. AIIRD, immunomodulating agents and vaccines considered in the literature search and recommendations.

AIIRD Immunomodulating agents Vaccines

RA Corticosteroids BCG*

SLE Methotrexate Cholera

Antiphospholipid syndrome Sulfasalazine Diphtheria

Adult Still disease Leflunomide Hepatitis A

SSc Hydroxychloroquine Hepatitis B

SjS Azathioprine Haemophilus influenzae b

MCTD Mycophenolic acid drugs Human papillomavirus

Relapsing polychondritis Cyclosporine Influenza

GCA Tacrolimus Japanese encephalitis

Polymyalgia rheumatica Cyclophosphamide Measles*

Takayasu arteritis Biologicals: Mumps*

Polyarteritis nodosa TNFα blocking agents: Neisseria meningitidis

AAV: Infliximab (A/C/Y/W135, C conjugated)

Microscopic polyangiitis Etanercept Pertussis

Wegener granulomatosis Adalimumab Poliomyelitis

Churg-Strauss syndrome Rituximab (parenteral and oral*)

Behçet disease Tocilizumab Rabies

Goodpasture disease Abatacept Rubella*

Cryoglobulinemic syndrome Anakinra Streptocccus pneumoniae

PM (polysaccharide and conjugate)

DM Tetanus toxoid

Clinically amyopathic DM Tick-borne encephalitis

Sporadic inclusion body Typhoid fever

myositis (parenteral and oral*)

Anti-synthetase syndrome Varicella zoster*

Eosinophilic myositis Yellow fever*

Eosinophilic fasciitis

Spondylathropathies

Periodic fever syndromes

* Live attenuated vaccine. AIIRD, auto-immune inflammatory rheumatic disease; BCG, Bacillus Calmette-Guérin; RA, rheuma-toid arthritis; SLE, systemic lupus erythematosus; Scl, sceroderma; SjS, Sjogren syndrome; MCTD, mixed connective tissue disease; GCA, giant cell arterits; AAV, ANCA-associated vasculitis; PM, polymyositis; DM, dermatomyositis

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Initial search:

Medline *

ACR abstracts 2007 and 2008

EULAR abstracts 2008 and 2009

Articles for detailed review

Potentially appropriate articles

Included articles

Articles from bibliographies

Exclusion of duplicates and by title

Articles suggested by experts

Exclusion by abstract

Exclusion by text

Figure 1. Search strategy for systematic literature review. * Search terms: Auto-immune inflammatory rheumatic diseases: “Lupus Erythematosus, Systemic” [Mesh]; “Antiphosp-holipid Syndrome”[Mesh]; “Arthritis, Rheumatoid”[Mesh]; “Still’s Disease, Adult-Onset”[Mesh]; “Scle-roderma, Systemic”[Mesh]; “Sjogren’s Syndrome”[Mesh]; “Mixed Connective Tissue Disease”[Mesh]; “Polychondritis, Relapsing”[Mesh]; “Giant Cell Arteritis”[Mesh]; “Polymyalgia Rheumatica”[Mesh]; “Takayasu Arteritis”[Mesh]; “Polyarteritis Nodosa”[Mesh]; “microscopic polyangiitis”; “Wegener Granulomatosis”[Mesh]; “Churg-Strauss Syndrome”[Mesh]; “Behcet Syndrome”[Mesh]; “Anti-Glo-merular Basement Membrane Disease”[Mesh]; “Cryoglobulinemia”[Mesh]; “Polymyositis”[Mesh]; “Dermatomyositis”[Mesh]; “Myositis, Inclusion Body”[Mesh]; “anti-synthetase syndrome”; “eosi-nophilic myositis”; “Fasciitis”[Mesh]; “Spondylarthropathies”[Mesh]; “periodic fever syndrome”. Vaccines: “BCG Vaccine”[Mesh]; “ Cholera Vaccines”[Mesh]; “Diphtheria-Tetanus-Pertussis Vaccine” [Mesh]; “Diphtheria-Tetanus-acellular Pertussis Vaccines”[Mesh]; “Diphtheria Toxoid”[Mesh]; “Dipht-heria-Tetanus Vaccine”[Mesh]; “Hepatitis A Vaccines”[Mesh]; “Hepatitis B Vaccines”[Mesh]; “Haemophi-lus influenzae type b polysaccharide vaccine “[Substance Name]; “Haemophilus influenzae type b-polys-accharide vaccine-diphtheria toxoid conjugate “[Substance Name]; “DTPa-HBV-IPV combined vaccine “[Substance Name]; “Hib-MenCY-TT vaccine “[Substance Name]; “Haemophilus influenzae-type b po-lysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine “[Substance Name]; “Haemophilus influenzae type-b polysaccharide-pertussis vaccine “[Substance Name]; “Papillomavi-rus Vaccines”[Mesh]; “Influenza Vaccines”[Mesh]; “Japanese Encephalitis Vaccines”[Mesh]; “Measles Vaccine”[Mesh]; “Mumps Vaccine”[Mesh]; “Meningococcal Vaccines”[Mesh]; “Pertussis Vaccine”[Mesh]; “Pneumococcal Vaccines”[Mesh]; “Rubella Vaccine”[Mesh]; “Tetanus Toxoid”[Mesh]; “Diphtheria-Teta-nus Vaccine”[Mesh]; “Diphtheria-Tetanus-Pertussis Vaccine”[Mesh]; “Tick-Borne Encephalitis vaccine”; “Typhoid-Paratyphoid Vaccines”[Mesh]; “Herpes Zoster Vaccine”[Mesh]; “Yellow Fever Vaccine”[Mesh]. Vaccine-preventable infections: “Mycobacterium tuberculosis”[Mesh]; “Tuberculosis”[Mesh]; “Vibrio cholerae”[Mesh]; “Cholera”[Mesh]; “Corynebacterium diphtheriae”[Mesh]; “Diphtheria”[Mesh] OR “He-patitis A”[Mesh]; “Hepatitis A virus”[Mesh]; “Hepatitis B”[Mesh]; “Hepatitis B virus”[Mesh]; “Haemop-

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hilus influenzae type b”[Mesh]; “Human papillomavirus 6”[Mesh]’; “Human papillomavirus 18”[Mesh]; “Human papillomavirus 11”[Mesh]; “Human papillomavirus 16”[Mesh]; “Influenza, Human”[Mesh]; “Influenza A Virus, H1N1 Subtype”[Mesh]; “Influenza B virus”[Mesh]; “Influenza A virus”[Mesh]; “In-fluenza A Virus, H3N2 Subtype”[Mesh]; “Encephalitis, Japanese”[Mesh]; “Encephalitis Virus, Japanese”[Mesh]; “Measles”[Mesh]; “Measles virus”[Mesh]; “Mumps”[Mesh]; “Mumps virus”[Mesh]; “Neisseria meningitidis”[Mesh]; “Whooping Cough”[Mesh]; “Bordetella pertussis”[Mesh]; “Poliomyelitis”[Mesh]; “Poliovirus”[Mesh]; “Rabies”[Mesh]; “Rabies virus”[Mesh]; “Rubella”[Mesh]; “Rubella virus”[Mesh]; “Streptococcus pneumoniae”[Mesh]; “Pneumococcal Infections”[Mesh]; “Tetanus”[Mesh]; “Clostridium tetani”[Mesh]; “Encephalitis Viruses, Tick-Borne”[Mesh]; “Encep-halitis, Tick-Borne”[Mesh]; “Typhoid Fever”[Mesh]; “Salmonella typhi”[Mesh]; “Encephalitis, Va-ricella Zoster”[Mesh]; “Herpes Zoster”[Mesh]; “Yellow Fever”[Mesh]; “Yellow fever virus”[Mesh] Limits: Humans, English, Adolescent: 13-18 years, Adult: 19-44 years, Middle Aged: 45-64 years, Middle Aged + Aged: 45+ years, Aged: 65+ years, 80 and over: 80+ years

patients with rheumatic diseases, vasculitis, dementia or stroke who were unvac-cinated for influenza were admitted for pneumonia/influenza or death, compared to 0.8% in unvaccinated healthy controls (HC) [14].

Streptococcus pneumoniae

In a prospective cohort of 45 systemic lupus erythematosus (SLE) patients tre-ated with rituximab, cyclophosphamide and methylprednisolone, one patient developed pneumococcal pneumonia and septicaemia [15]. Retrospective cohort studies in SLE showed invasive pneumococcal infection in 1.34% of patients over an unknown period of time, in 1.9% over 25 years and in 2.40% over 9 years, res-pectively [16-18]. However, comparisons with the general population are lacking. No data are available regarding the incidence of pneumococcal infection for AIIRD other than SLE.

Herpes zoster (HZ)

In rheumatoid arthritis (RA), prospective cohort studies found an incidence of HZ ranging from 0.55 up to 11.1/1000 patient-years (PY) [19-22] and in retrospective cohort studies from 5.6 up to 14.5/1000 PY [23-28]. RA per se is a risk factor for HZ (adjusted hazard ratio 1.65 and 1.91 compared to HC respectively in two large databases [25]). Additional risk factors are the use of steroids (relative risk [RR] point estimates 1.41-2.52) [20, 24, 25, 27], use of anti-TNFα (in particular infliximab/adalimumab [RR point estimates 1.38-2.44]) [20, 24, 25, 28] and the combination of TNFα-blocking agents and steroids (RR estimate 2.44) [25]. Non-biologic DMARDs also increase the risk [24, 25], especially cyclophosphamide (4.2-fold), azathioprine (2.1-fold) and leflunomide (1.4-fold) [27]. Furthermore, combination therapy of non-biologic DMARDs and steroids increases the risk 2.39-fold [25]. Etanercept and methotrexate (MTX) do not seem to increase the risk for HZ [21, 29].

In SLE, one prospective cohort study shows an incidence of HZ of 18.3/1000 PY [30]. In retrospective studies the incidence ranges between 16-91.5/1.000 PY [31-

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36]. The percentage of SLE patients developing HZ is 3.2-46.6%, unfortunately the duration of follow-up in most of these studies has not been well documented [31, 33-43]. Compared to the general population the risk is increased by a factor ran-ging from 5 to 16-fold [31-33], and 3-fold compared to patients with musculo-ske-letal diseases [44]. HZ correlates with the use of cyclophosphamide, azathioprine and steroids in combination with other immunosuppressive drugs [30, 31, 33, 42]. The risk may be lower for mycophenolic acid compared with cyclophosphamide (RR 0.36) [45]. Fifty-two-week follow-up safety data from a randomized controlled trial (RCT) showed that 15.4% of SLE patients treated with rituximab, along with background immunosuppressives including steroids, developed HZ, compared with 8% in the placebo-groups [46]. One study showed that there was no increase in incidence of HZ during flares of SLE [30].

In a RCT in which Wegener granulomatosis (WG)-patients were randomized to receive etanercept or placebo along with standard immunosuppressives, the inci-dence of HZ (both groups together) was 45/1000 PY, compared with 1.2-4.8/1000 PY in the general population. A serum creatinine ≥1.5 mg/dl increased the risk of developing HZ by 6.3 [47].

In patients with polymyositis/dermatomyositis (PM/DM) the incidence of HZ was 27.2/1.000 PY in a retrospective cohort study [34]. There was no comparator-group in this study.

Tuberculosis (TB)

The incidence of TB differs strongly according to geographic area, ranging in RA patients from 0 (England, when using DMARDs) to 36.3/1000 PY (Morocco, when using DMARDs) [19, 48-74]. The highest incidence in an European country was 18.93/1000 PY in Spain among RA patients treated with infliximab, before screening for latent TB infection (LTBI) was recommended [51]. Compared to the general population, RA patients have an increased risk for contracting TB (RR point estimates ranging from 2.0 to 10.9 when treated with non-biologic DMARDs [48, 50-52, 55, 58, 62] and from 4 to 90.1 when treated with TNFα blocking agents

Table 2. Evidence-categories

Category Evidence

1A Meta-analysis of randomized controlled trials

1B Randomized controlled trial

2 Prospective controlled intervention study without randomization

3 Descriptive/analytic study (including case-control, cross-sectional, case series)

4 Expert committee reports or opinion or clinical experience of respected authori-ties or both

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[51, 52, 58, 62]. Within the group of RA patients non-biologic DMARDs increase the risk of TB by 1.2 and 3.0-fold [49, 50], in particular MTX (1.7 and 3.4-fold) and leflu-nomide (10.2 and 11.7-fold) [50, 75]. Steroids are also a risk factor (HR estimates 1.4-2.4) [49, 50, 75]. TNFα-blocking agents increase the risk for TB 1.5-8.7-fold, es-pecially infliximab (1.6-19.9-fold) [48, 49, 51, 58, 62-64, 68, 70]. As a result of these findings, in subsequent studies on biologics, all patients were screened for LTBI before inclusion. For RA patients treated with etanercept TB-risk was compara-ble to placebo-treated patients or the general population [21, 72]. Rituximab [60, 71, 76], anakinra [76] and the recently registered anti-IL6 agent tocilizumab [61, 66] do not seem to increase the risk for TB. For tozilizumab this was confirmed in a meta-analysis, where none of 1870 patients treated with tocilizumab (with or wit-hout additional non-biologic DMARD) developed TB [69]. Of 378 patients treated with abatacept three developed TB in a time span of five years, but there was no comparator group [77].

Only retrospective studies are performed regarding the incidence of TB in SLE patients showing incidences ranging from 2.61/1000 PY in Spain to 20/1000 PY in Korea [56, 68, 78-80]. The percentage of patients in the cohorts developing TB ranges from 0.66%/10 years up to 11.6%/5 years [39, 81-88]. Most studies come from South-East Asian countries, where TB is more prevalent than in most European countries. TB occurs more often in SLE patients than in the general population: 7.9 vs. 2.3/1000 PY [56]; 17 vs. 1.1/1000 PY [80]; 20 vs. 3.93/1000 PY [78] and 11.6%/5 years vs. 1.4-2.6%/5 years [81]. Patients who developed TB were on higher daily prednisone doses [78, 86] steroid pulse therapy [78], had a higher cumulative dose of steroids [80, 86], or lupus-nephritis [78]. Two cohort-studies addressing TB-incidence in ankylosing spondylitis (AS) found an incidence of 0 and 0.34/1000 PY respectively [68, 73]. Percentages of AS patients developing TB vary from 0% up to 12%/4 years [51, 53, 68, 72, 89, 90]. One RCT studying the treatment of AS with infliximab found one TB-case in 20 AS patients [91]. Prospective and retrospective cohorts of AS patients treated with infliximab, etanercept and adalimumab, with follow-up up to 5, 7 and 3 years respectively, did not show any cases of active TB [72, 92-98].

In one prospective cohort-study evaluating the treatment of psoriatic arthritis (PsA) with adalimumab the incidence of TB was 3/1000 PY [73].

TB occurred in 1.5%/13 years of patients with giant cell arteritis (GCA) in a retro-spective cohort study [99]. Three prospective cohort studies found that TB was the cause of death in 0.9-1.13% of patients with GCA/polymyalgia rheumatica [100-102].

Among patients with Sjögren’s syndrome (SjS) TB was not found in a retrospec-

143

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8

tive cohort of Spanish AIIRD patients, duration of follow-up was not stated [68].

Human Papillomavirus (HPV)

HPV-infection occurs more often in SLE patients compared with HC (24.6 vs. 10.4%) [103, 104], also with the high-risk subtype HPV-16 (4.7-53% vs. 1.2-6.7%) [103, 105] and the cumulative prevalence rises from 13.1% at baseline (11.7% high-risk sub-type) to 25.5% (21.4% high-risk subtype) after 3 years. Incidence for HPV infection was 17/1000 patient-months. The most common newly acquired HPV-subtype was HPV-16 (1.7/1000 patient-months), followed by HPV-18, -56 and -58 (1.2/1000 patient-months). Only 31.8% of all incident infections was cleared [106]. Risk fac-tors for HPV-infection in SLE patients are the same as those for the general po-pulation, but also ANA>320 [103, 106, 107].

Question 2. do vaccines decrease the risk of infections in patients with AIIRd in general, and specifically in those with unstable disease, and in those using immunomodulating agents?

Influenza vaccine (table 3)

Just one study addressed a clinical endpoint after vaccination as the primary end point, and found a reduction in pneumonitis, acute bronchitis or viral infection in RA patients and SLE patients who received influenza vaccine compared to those who did not [108]. Other studies evaluated the development of a protective level of antibodies (≥40, as measured by the hemagglutination inhibition assay). The majority of these studies have shown similar efficacy of influenza vaccination in RA patients, compared to HC. Neither DMARDs, nor TNFα-blocking treatment diminished the humoral response [109-120]. Only two studies reported a modestly impaired response in anti-TNFα users, not resulting in a lower percentage of se-roprotection [114, 121]. In contrast, rituximab severely hampers the immune res-ponse [120, 122, 123]: influenza vaccination in the first eight weeks after treatment with rituximab did not induce an antibody response. The antibody response was partially restored six to ten months after rituximab [120].

In one RCT [124] and three controlled studies [125-127] a modestly reduced res-ponse to influenza vaccination in SLE patients was found. In two other studies SLE patients showed a similar [110] or a slightly reduced in vitro response to immuni-sation [119], compared to disease controls (RA patients). In SLE several controlled studies on the efficacy of subunit influenza vaccine have shown a similar humoral response in SLE patients compared to HC [115, 128-131]. In most studies the use of immunosuppressive drugs did not affect the vaccination response [124, 126, 128, 131], however, a lower response to vaccination in SLE patients on azathioprine [125, 132], steroids [115], and hydroxychloroquine [127] has also been observed.

144

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8

Patients with WG develop similar humoral responses following influenza vacci-nation compared to HC. Use of immunosuppressive drugs did not to affect the humoral response [133, 134].

Also in 46 patients with systemic sclerosis (SSc) (virosomal) influenza vaccination was effective in terms of humoral and cellular immune response [135].

Pneumococcal vaccine (table 4)

The efficacy of pneumococcal vaccination is difficult to determine as no generally accepted response criteria are available. Moreover, different vaccines (polysac-charide and conjugate vaccine) are available containing antigens with different numbers of pneumococcal serotypes.

In RA, both similar [116, 136-138] and lower [138-141] responses to pneumococcal vac-cination have been reported. TNFα-blocking agents were not demonstrated to reduce efficacy of pneumococcal vaccination [116, 136-138], except for two studies, where a reduced response was demonstrated [137, 141]. Two other papers repor-ted an impaired response to pneumococcal vaccination in RA patients on the com-bination of MTX and anti-TNFα, [136, 138]. These papers also reported an impaired response in those treated with MTX alone [136, 138]. Finally, rituximab reduced the response to pneumococcal polysaccharide vaccine in a study of 69 RA patients vaccinated 28 weeks after rituximab-administration [139].

Similar [17, 142, 143] as well as a reduced/low responses [140, 144-147] were ob-served in several controlled and uncontrolled studies in SLE patients, without in-fluence of the combined use of steroids and azathioprine or cyclophosphamide [140, 143, 144, 146].

In patients with PsA (n=184) or AS (n=5) responses after pneumococcal vaccination were equal in patients with or without use of TNFα-blocking agents [148, 149].

Pneumococcal vaccination resulted in a protective antibody level to at least three out of four tested serotypes in 83% of 18 SSc-patients in an uncontrolled study [150].

Hepatitis B vaccine (table 5)

The efficacy of hepatitis B (HBV) vaccination has been addressed in patients with RA [151], SLE [152], AS [148] and Behçet disease (BD) [153]. In the majority of patients, irrespective of the underlying AIIRD and the use of steroids or DMARDs, a vacci-nation response could be demonstrated. Only use of TNFα-blocking agents seve-rely hampered the response to HBV vaccine in AS patients [148], However, a clear conclusion can not be drawn due to low numbers (n=20 for RA; n=28 for SLE; n=30 for AS; n=13 for BD) and the lack of appropriately controlled studies.

145

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8

Tetanus toxoid vaccine (table 6)

Tetanus toxoid vaccination seems to be efficacious in RA patients in two pros-pective controlled studies [111, 154]. The use of steroids or DMARDs did not reduce efficacy. Also treatment with rituximab did not diminish response to tetanus vac-cine in 69 RA patients when administered 24 weeks after rituximab [139].

In SLE, controlled studies revealed similar responses to tetanus toxoid vaccina-tion compared to HC [154-156]. A larger, uncontrolled study confirmed these fin-dings: >90% of the 73 SLE patients achieved an adequate response [147]. Only one small study showed a diminished response to tetanus vaccination in SLE patients. The use of steroids or DMARDs did not reduce response rates [157].

Haemophilus influenza B (HIB) vaccine (table 7)

In an uncontrolled study HIB vaccination resulted in protection in 88% of the 73 in-cluded SLE patients. A trend towards a lower response was observed in patients on immunosuppressive drugs [147].

Question 3. do vaccines cause any significant harm in patients with AIIRd in general, and specifically in those with unstable disease, and in those using immunomodulating agents?

Influenza vaccine (table 3)

In one RCT including 126 RA patients and one controlled study including 20 RA patients, RA patients who received influenza vaccine did not have an increased disease activity compared to those who were not vaccinated [109, 110]. Also in other pre-post studies RA patients did not experience increased disease activity following influenza vaccination [112-114, 116, 120, 123].

SLE patients who are vaccinated for influenza do not develop more disease fla-res than unvaccinated SLE patients, as demonstrated in one RCT [124] and three non-randomized comparative studies [108, 110, 132]. Other studies in SLE patients found either no flares [125, 126], mild flares (up to 35%) [115, 128] or renal flares with glomerulonephritis (one of 29 SLE patients) [129, 131]. Since these studies did not include disease-controls, the interpretation is difficult: the flares may have represented the natural course of SLE. In a single study systemic side effects of influenza vaccination occurred more often in SLE patients (in 19 of 56 patients) than in HC [125].

No difference in the occurrence of disease flares was found in WG patients recei-ving influenza vaccination compared to WG patients that did not receive influenza vaccination in two prospective studies [133, 134] and one large retrospective study [158].

146

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8

In 46 SSc patients who received (virosomal) influenza vaccine no flares of SSc were registered [135].

Of note, in all aforementioned studies inactivated influenza vaccine was used.

Pneumococcal vaccine (table 4)

No studies have been performed that compared disease activity following pneu-mococcal vaccination in RA patients with those who did not receive pneumococ-cal vaccine. Uncontrolled pre-post studies showed no increase in disease activity following vaccination [116, 136, 139-141].

In all three studies comparing SLE patients who did (n=20, 18 and 38) and did not (n=20, 8 and 23) receive pneumococcal vaccination, disease activity after vacci-nation did not differ significantly [17, 142, 144]. Other studies, comparing SLE pa-tients receiving pneumococcal vaccine with RA patients and/or HC [140, 143-146], or case series of SLE patients receiving pneumococcal vaccine [143, 147, 159] could not demonstrate an increase in SLE activity following pneumococcal vaccination. Case series in patients with PsA [149] and SjS [160] did not show increased disease activity following pneumococcal vaccination.

Hepatitis B vaccine (table 5)

HBV vaccination did not lead to increased disease activity in 22 vaccinated RA patients compared to 22 unvaccinated RA patients [151].

One uncontrolled prospective study regarding HBV vaccination in 28 SLE patients revealed no significant change in SLEDAI-score after each vaccine dose. The number of flares following vaccination was similar to that observed in the year before vaccination (21%) [152].

In a study of 13 Behçet patients three developed oral aphtae following HBV vac-cination but otherwise no increase in disease activity was observed [153].

Question 4. does the timing of vaccination in relation to disease activity and in relation to the receipt of immunomodulating agents affect the effective-ness of vaccination in patients with AIIRd?

There are no studies comparing the efficacy of vaccination in AIIRD patients with stable and unstable disease: generally, studies performed in patients with AIIRD included typically patients with stable, quiescent disease.

Influenza vaccine

Three studies addressing the influence of disease activity on the humoral response in RA patients found that the degree of disease activity did not affect

147

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8

Tab

le 3

. Eff

icac

y an

d sa

fety

of

infl

uenz

a va

ccin

atio

n in

AIIR

D p

atie

nts

Aut

hor/

Year

Des

ign

No.

cas

esEf

fica

cyIn

flue

nce

of IS

on

effi

cacy

Safe

tyLo

E

Cha

lmer

s [10

9]R

CT

64 R

A-P

VN

o di

ffer

ence

No

No

diff

eren

ce1B

1994

22 R

A n

ot-P

V

40

RA

-IS

64

HC

Kai

ne [1

16]

RC

T99

RA

-AD

AN

o di

ffer

ence

No

No

flar

es1B

20

07

109

RA

Den

man

[111

]C

ontr

olle

d20

RA

No

diff

eren

ceN

oN

/A2

1970

39 R

A-D

C

20

HC

Kap

etan

ovic

[121

]C

ontr

olle

d50

RA

-ant

i-T

NF+

MT

XR

educ

ed in

RA

-ant

i-R

educ

ed o

n T

NF+

MT

XN

/A2

200

762

RA

-ant

i-T

NF+

MT

Xan

d R

A-a

nti-

TN

F co

mpa

red

TN

F+D

MA

RD

to R

A-M

TX

37 R

A-M

TX

18 H

C

Kub

ota

[117]

Con

trol

led

27 R

A-a

nti-

TN

FN

o di

ffer

ence

Incr

ease

d on

ant

i-T

NF

N/A

2

20

07

36 R

A-D

C

52

HC

Ore

n [12

3]C

ontr

olle

d29

RA

Red

uced

in R

A-R

TX

Red

uced

on

RT

XN

o fl

ares

2

20

08

14 R

A-R

TX

21

HC

148

Cha

pter

8

Tab

le 3

. (co

ntin

ued)

Nii

[118]

Con

trol

led

27 R

A-a

nti-

TN

FN

o di

ffer

ence

No

N/A

2

20

09

36 R

A-D

C

52

HC

van

Ass

en [1

20]

Con

trol

led

23 R

A-R

TX

Red

uced

in R

A-R

TX

Red

uced

on

RT

XN

o fl

ares

2

20

1020

RA

-DC

29

HC

Fom

in [1

13]

Con

trol

led

82 R

AN

o di

ffer

ence

No

No

flar

es2

20

06

30 H

C

Gel

inck

[114

]C

ontr

olle

d64

ant

i-T

NF*

No

diff

eren

ceR

educ

ed (m

odes

tly)

on

N/A

2

20

08

48 n

on-a

nti-

TN

F*an

ti-T

NF

Her

ron

[115]

Con

trol

led

20 S

LEN

o di

ffer

ence

Red

uced

on

ster

oids

N/A

2

1979

17 R

A

17

oth

er A

IIRD

32

HC

Tur

ner-

Stok

esC

ontr

olle

d28

SLE

Red

uced

in S

LE, M

CT

DN

oN

/A2

[119]

1988

10 R

Aan

d R

A/S

LE

4

MC

TD

2

RA

/SLE

Stoj

anov

ich[

108]

Con

trol

led

23 S

LE-v

acc

Red

ucti

on in

pne

umon

ia,

N/A

No

flar

es2

200

646

SLE

-DC

acut

e br

onch

itis

or

vira

l

149

Cha

pter

8

Tab

le 3

. (co

ntin

ued)

23 R

A-v

acc

infe

ctio

ns in

SLE

-vac

c

31 R

Aan

d R

A-v

acc

Del

Por

to [1

10]

Con

trol

led

14 S

LEN

o di

ffer

ence

N/A

No

diff

eren

ce2

20

06

10 S

LE-D

C

10

RA

10

RA

-DC

Elka

yam

[112

]C

ontr

olle

d20

RA

-ant

i-T

NF

No

diff

eren

ceN

oN

o fl

ares

2

20

09

23 R

A-D

C

18

SpA

-ant

i-T

NF

17

HC

Will

iam

s [12

4]R

CT

19 S

LER

educ

ed in

SLE

Not

on

ster

oids

No

diff

eren

ce1B

1978

21 S

LE-D

C

36

HC

Ris

tow

[131

]C

ontr

olle

d29

SLE

No

diff

eren

ceN

ot o

n st

eroi

ds/I

S1 g

lom

eron

ephr

itis

2

1978

29 H

C

Bro

dman

[128

]C

ontr

olle

d46

SLE

No

diff

eren

ceN

ot o

n H

CQ

, AZ

A, P

RED

No

diff

eren

ce2

1978

58 H

C

Loui

e [ 1

29]

Con

trol

led

11 S

LEN

o di

ffer

ence

N/A

1 glo

mer

onep

hrit

is2

1978

8 H

C

150

Cha

pter

8

Tab

le 3

. (co

ntin

ued)

Pon

s [13

0]

Con

trol

led

11 S

LEN

o di

ffer

ence

N/A

N/A

2

1979

12 H

C

Abu

-Sha

kra

[132]

Con

trol

led

24 S

LER

educ

ed in

SLE

Red

uced

on

AZ

AN

o fl

ares

2

20

02

24 S

LE-D

C

Mer

cado

[126

]C

ontr

olle

d18

SLE

Red

uced

in S

LEN

oN

/A2

20

04

18 H

C

Hol

vast

[ 125

]C

ontr

olle

d56

SLE

Red

uced

in S

LER

educ

ed o

n A

ZA

No

flar

es2

20

06

18 H

CSL

E m

ore

syst

emic

AE

Wie

sik-

Szew

c-C

ontr

olle

d67

SLE

Red

uced

in S

LER

educ

ed o

n H

CQ

N/A

2

zyk

[127]

20

09

47 H

C

Hol

vast

[161

]R

CT

49 W

GN

o di

ffer

ence

No

No

diff

eren

ce1B

20

09

23 W

G-D

C

49

HC

Zyc

insk

a [13

4]C

ontr

olle

d35

WG

No

diff

eren

ceN

oN

o Fl

ares

2

20

07

28 W

G-D

C

35

HC

Sett

i [13

5]C

ontr

olle

d46

SSc

No

diff

eren

ceN

/AN

o fl

ares

2

20

09

20 H

C

IS, i

mm

unos

uppr

essi

ve d

rugs

; LoE

, lev

el o

f ev

iden

ce; P

V, p

revi

ousl

y va

ccin

ated

; HC

, hea

lthy

con

trol

s; A

DA

, ada

limum

ab; D

C, d

isea

se-c

ontr

ol; A

E, a

dver

se

even

ts; N

/A, n

ot a

ddre

ssed

; vac

c, v

acci

nate

d; M

TX

, met

hotr

exat

e; A

ZA

, aza

thio

prin

e; H

CQ

, hyd

roxy

chlo

roqu

ine;

PR

ED, p

redn

iso(

lo)n

e; C

sA, c

yclo

spor

ine;

ET

A, e

tane

rcep

t

151

Cha

pter

8

Tab

le 4

. Eff

icac

y an

d sa

fety

of

pneu

moc

occa

l vac

cina

tion

in A

IIRD

pat

ient

s

Aut

hor/

Year

Des

ign

No.

cas

esEf

fica

cyIn

flue

nce

of IS

on

effi

cacy

Safe

tyLo

E

Kai

ne [1

16]

RC

T99

RA

-A

DA

No

diff

eren

ceN

oN

/A1B

20

07

109

RA

Elka

yam

[141

]C

ontr

olle

d11

RA

-ant

i-T

NF

No

diff

eren

ceR

educ

ed in

ant

i-T

NF

N/A

2

20

04

5 A

S-an

ti-T

NF

17

RA

-DC

Kap

etan

ovic

[138

]C

ontr

olle

d50

RA

-MT

X/a

nti-

TN

FN

o di

ffer

ence

in R

A-a

nti-

TN

FR

educ

ed o

n M

TX

and

N/A

2

20

06

62 R

A-a

nti-

TN

FR

educ

ed in

RA

-MT

XM

TX

/ant

i-T

NF

37

RA

-MT

X

47

HC

Vis

vana

than

[137

]C

ontr

olle

d20

RA

-ant

i-T

NF

Red

uced

in R

A-a

nti-

TN

FN

oN

/A2

200

736

RA

-MT

X/a

nti-

TN

F

14

RA

-MT

X

Gel

inck

[136

]C

ontr

olle

d52

RA

/Cro

hn-a

ntiT

NF

No

diff

eren

ceR

educ

ed o

n M

TX

and

N

/A2

20

08

41 R

A/C

rohn

-DC

MT

X/a

nti-

TN

F

Bin

gham

[139

]C

ontr

olle

d69

RA

-RT

XR

educ

ed in

RA

-RT

XR

educ

ed o

n R

TX

N/A

2

20

1034

RA

-DC

Elka

yam

[140

]C

ontr

olle

d24

SLE

Red

uced

in S

LE a

nd R

AN

oN

o di

ffer

ence

2

20

02

42 R

A

29

HC

152

Cha

pter

8

Tab

le 4

. (co

ntin

ued)

Klip

pel [

17]

RC

T20

SLE

-vac

cN

o di

ffer

ence

N/A

No

diff

eren

ce1B

1979

20 S

LE-p

lace

bo

Lipn

ick

[143]

RC

T60

SLE

w/o

ISN

o di

ffer

ence

No

No

diff

eren

ce1B

1985

17 S

LE w

ith

IS

Jarr

ett

[144]

Con

trol

led

38 S

LER

educ

ed in

SLE

No

No

diff

eren

ce2

1980

23 S

LE-D

C

17

HC

Cro

ft [1

42]

Con

trol

led

18 S

LEN

o di

ffer

ence

N/A

No

diff

eren

ce2

1984

8 SL

E-D

C

18

HC

McD

onal

d [14

5]C

ontr

olle

d19

SLE

Red

uced

in S

LE a

fter

1 ye

arN

/AN

/A2

1984

5 H

C

Tar

jan[

146]

Con

trol

led

18 S

LER

educ

ed in

SLE

No

No

flar

es2

20

02

9 H

C

Bat

tafa

rano

[147

]U

ncon

trol

led

73 S

LEN

/AT

rend

tow

ards

low

er

3

1998

on P

RED

, AZ

A a

nd C

sA

Fran

co S

alin

asC

ontr

olle

d20

SpA

-ant

i-T

NF

Red

uced

in S

pA-a

nti-

TN

FR

educ

ed o

n an

ti-T

NF

N/A

2

[148

] 20

09

10 S

pA-D

C

153

Cha

pter

8

Tab

le 4

. (co

ntin

ued)

Mea

se [1

49]

RC

T94

PsA

-ET

AN

o di

ffer

ence

Red

uced

on

MT

XN

/A1B

200

490

PsA

-pla

cebo

Mer

cado

[150

]U

ncon

trol

led

18 S

ScG

ood

resp

onse

in 8

3%N

oN

/A3

20

09

IS, i

mm

unos

uppr

essi

ve d

rugs

; LoE

, lev

el o

f ev

iden

ce; H

C, h

ealt

hy c

ontr

ols;

AD

A, a

dalim

umab

; DC

, dis

ease

-con

trol

; N/A

, not

add

ress

ed; M

TX

, met

hotr

exa-

te; A

ZA

, aza

thio

prin

e; P

RED

, pre

dnis

o(lo

)ne;

CsA

, cyc

losp

orin

e; E

TA

, eta

nerc

ept

Tab

le 5

. Eff

icac

y an

d sa

fety

of

hepa

titi

s B

vac

cina

tion

in A

IIRD

pat

ient

s

Aut

hor/

Year

Des

ign

No.

cas

esEf

fica

cyIn

flue

nce

of I

S on

eff

icac

ySa

fety

LoE

Elka

yam

[151

]C

ontr

olle

d22

RA

68%

pro

tect

ion

No

No

flar

es2

20

02

22 R

A-D

C

Kur

uma

[152]

Unc

ontr

olle

d28

SLE

93%

pro

tect

ion

N/A

11%

fla

res

3

20

07

Fran

co S

alin

as

[148]

20

09

Con

trol

led

20 S

pA-a

nti-

TN

F 10

Sp

A-D

CR

educ

ed in

SpA

-ant

i-T

NF

Red

uced

on

anti

-TN

FN

/A2

Erke

k [15

3]C

ontr

olle

d13

Beh

çet

No

diff

eren

ceN

/AN

o2

200

515

HC

IS, i

mm

unos

uppr

essi

ve d

rugs

; LoE

, lev

el o

f ev

iden

ce; H

C, h

ealt

hy c

ontr

ols;

DC

, dis

ease

-con

trol

; N/A

, not

add

ress

ed

154

Cha

pter

8

Tab

le 6

. Eff

icac

y an

d sa

fety

of

teta

nus

vacc

inat

ion

in A

IIRD

pat

ient

s

Aut

hor/

Yea

rD

esig

nN

o. c

ases

Effi

cacy

Infl

uenc

e of

IS o

n ef

fica

cySa

fety

LoE

Den

man

[111

]C

ontr

olle

d20

RA

No

diff

eren

ceN

oN

/A2

1970

39 R

A-D

C

Bin

gham

[139

]C

ontr

olle

d69

RA

-RT

XN

o di

ffer

ence

No

N/A

2

20

1034

RA

-DC

Dev

ey [1

54]

Con

trol

led

24 S

LEN

o di

ffer

ence

N/A

N/A

2

1987

29 R

A

33

HC

Abe

[155

]C

ontr

olle

d20

SLE

No

diff

eren

ceN

oN

/A2

1971

20 H

C

Nie

s [15

7]C

ontr

olle

d9

SLE

Red

uced

in S

LEN

/AN

/A2

1980

9 H

C

Kas

hef

[156]

Con

trol

led

40 S

LEN

o di

ffer

ence

No

N/A

2

200

8 60

HC

Bat

tafa

rano

[147

]U

ncon

trol

led

73 S

LE90

% p

rote

ctio

nT

rend

low

er r

espo

nse

N/A

3

1998

on P

RED

and

AZ

A

IS, i

mm

unos

uppr

essi

ve d

rugs

; LoE

, lev

el o

f ev

iden

ce; H

C, h

ealt

hy c

ontr

ols;

DC

, dis

ease

-con

trol

; N/A

, not

add

ress

ed; A

ZA

, aza

thio

prin

e; P

RED

, pr

edni

so(lo

)ne

155

Cha

pter

8

Tab

le 7

. Eff

icac

y an

d sa

fety

of

H. i

nflu

enza

e b

vacc

inat

ion

in A

IIRD

pat

ient

s

Aut

hor/

Year

Des

ign

No.

cas

esEf

fica

cyIn

flue

nce

of IS

on

effi

cacy

Safe

tyLo

E

Bat

tafe

rano

[147

]U

ncon

trol

led

73 S

LE88

% p

rote

ctio

nT

rend

low

er o

n P

RED

,N

/A3

1998

AZ

A a

nd C

sA

IS, i

mm

unos

uppr

essi

ve d

rugs

; LoE

, lev

el o

f ev

iden

ce; N

/A, n

ot a

ddre

ssed

; AZ

A, a

zath

iopr

ine;

PR

ED, p

redn

iso(

lo)n

e; C

sA, c

yclo

spor

ine

156

Cha

pter

8

this response [112, 113, 123]. One study investigated the influence of timing of the in-fluenza vaccination in relation to the receipt of infliximab in RA and AS patients. A trend towards a lower immune response was observed in RA patients vaccinated three weeks after the administration of infliximab as compared with those vacci-nated on the day of infliximab administration [112]. In another study in RA patients treated with rituximab the humoral response after influenza vaccination was se-verely hampered in the group that received their influenza vaccination four to eight weeks after rituximab, while six to ten months after rituximab a slight reco-very of the humoral response was observed [120].

In SLE, most studies assessing the effect of disease activity on the humoral res-ponse following influenza vaccination did not show a reduced response in patients with active disease [119, 126, 128, 132, 133], although some reported a diminished response [127, 131]. With regard to the influence of immunomodulating agents on the timing of vaccination, no data are available in SLE patients.

Pneumococcal vaccine

In four studies no effect of disease activity on the efficacy of pneumococcal vac-cination in RA patients was shown [137, 138, 140, 141].

Also in SLE, disease activity did not influence the efficacy of vaccination [17, 140, 144], although one open trial demonstrated a trend towards a reduced response in patients with active lupus [147].

Hepatitis vaccine

A single study in RA patients did not show an effect of disease activity on anti-body response to HBV vaccination [151].

Tetanus toxoid vaccine

The efficacy of tetanus booster in RA patients has not been shown to be affec-ted by the degree of disease activity [154]. Also rituximab, when administered 24 weeks before tetanus vaccination, did not reduce the humoral response to teta-nus vaccination [139].

In SLE, the efficacy of tetanus vaccination may be reduced in SLE patients with active disease [147, 155], although data are conflicting [154, 156].

Question 5. does the timing of vaccination in relation to disease activity and in relation to receipt of immunomodulating agents affect important harms of vaccination in patients with AIIRd?

Only two studies addressed the timing of vaccination in patients with AIIRD with regard to harms. In patients with RA or AS, influenza vaccination on the day of

157

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8

infliximab infusion or three weeks afterwards did not result in a significant dif-ference in adverse events and no increase in disease activity was observed [112]. Disease activity did not change following influenza vaccination regardless of whether administered 4-8 weeks or 6-10 months after treatment with rituximab [120].

Question 6. does revaccination with any vaccines increase the effectiveness in patients with AIIRd?

The usefulness of a second vaccination in AIIRD patients was addressed by one study. In SLE patients a booster influenza vaccination four weeks after the first vaccination did not lead to a further rise of seroprotection rates and geometric mean titres (GMTs). However, in the subgroup of SLE patients who have not been vaccinated in the previous year, GMT and seroconversion rate to A/H1N1 did rise following the booster vaccination [161].

Question 7. does revaccination with any vaccine increase significant harms in patients with AIIRd?

There are no data available on the harms of revaccination in AIIRD patients.

Question 8. Is vaccination in patients with AIIRd cost-effective?

There are no data available on the cost-effectiveness of vaccination specifically in AIIRD patients.

dISCuSSIOnThis SLR summarizes the available evidence regarding vaccination is patients with AIIRD. By defining eight key questions we were able to address the most im-portant issues, mainly the indication, the efficacy and the possible harms of vacci-

Table 8. Research agenda

1. Set-up of a vaccination registry for patients with AIIRD with a focus on safety and efficacy

2. Prospective studies evaluating the prevalence and identification of causative micro-organisms of infection in patients with AIIRD, and the outcome of vaccine preventable infections in these patients

3. Evaluation of the impact of therapies on safety and efficacy of vaccination

4. Evaluation of the impact of new therapies on prevalence of (vaccine preventable) infections

5. Research for efficacy and safety of new vaccines (such as herpes zoster, pandemic H1N1 Influ-enza A, HPV)

6. Evaluation of the implementation of vaccination recommendations

7. Research for the safety and efficacy regarding adjuvants and specific risk groups (e.g. preg-nancy)

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8

nation in these patients. Unfortunately, studies investigating the incidence of VPI in AIIRD patients are scarce and no RCT with clinical primary endpoints regarding efficacy were available. Moreover, although vaccination did not lead to signifi-cant harms in the available comparative studies, these studies were too small and had too short follow-up to draw firm conclusions. Safety of vaccination is considered to be of major importance in particular in patients with auto-immune diseases, because of the potential risk for flares of their underlying disease. Although many case series and case reports regarding side effects of vaccination in patients with AIIRD have been published, these were not included since these can not distinguish natural course of the underlying AIIRD from possible adverse effects caused by vaccination. On the other hand, use of live attenuated vaccines in immunocompromised patients is generally considered unsafe, but only case reports are available to support this concern. Finally, cost-effectiveness was con-sidered, but no studies were found to address this issue specifically in AIIRD pa-tients, and results from cost-effectiveness analyses in the general population for these same vaccines may not be applicable for AIIRD.

In conclusion, the currently available evidence was systematically reviewed to make evidence-based recommendations for vaccination in patients with AIIRD [12]. However, because of the lack of adequately large, well-designed studies, there is a need for further research (table 8) regarding the incidence of VPI, the efficacy of vaccination in patients with AIIRD (in particular in relation to treat-ment with traditional and/or new immunosuppressive agents and the timing of vaccination with regard to these immunosuppressive medications), and potential harms of vaccination in patients with AIIRD.

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CHAPT

ER9E U L A R recommen-d a t i o n s f o r v a c c i - n a t i o n i n a d u l t p a t i e n t s w i t ha u t o - i m m u n e inflammatory r h e u m a t i c d i s e a s e s

S. van Assen

N. Agmon-Levin

O. Elkayam

R. Cervera

M.F. Doran

M. Dougados

P. Emery

P. Geborek

J.P.A. Ioannidis

D.R.W. Jayne

C.G.M. Kallenberg

U. Müller-Ladner

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L. Stojanovich

G. Valesini

N.M. Wulffraat

M. Bijl

Annals of Rheumatic Diseases 2011 Mar;70(3):414-422

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AbsTrAcTObjectives To develop evidence-based European League Against Rheu-matism (EULAR) recommendations for vaccination in patients with auto-immune inflammatory rheumatic diseases (AIIRD).

Methods A EULAR task force was composed of experts representing 11 European countries, consisting of eight rheumatologists, four clinical im-munologists, one rheumatologist/clinical immunologist, one infectious disease physician, one nephrologist, one paediatrician/rheumatologist and one clinical epidemiologist. Key questions were formulated and the eligible spectrum of AIIRD, immunosuppressive drugs and vaccines were defined, in order to perform a systematic literature review. We searched Medline from 1966 to October 2009, as well as abstracts from the EULAR meetings of 2008 and 2009 and the American College of Rheumatology meetings of 2007 and 2008. Evidence was graded in categories 1-4, the strength of recommendations was graded in categories A-D, and Delphi voting was ap-plied to determine the level of agreement between the experts of the task force.

results Eight key questions and 13 recommendations addressing vacci-nation in patients with AIIRD were formulated. The strength of each re-commendation was determined. Delphi voting revealed a very high level of agreement with the recommendations among the experts of the task force. Finally, a research agenda was proposed.

conclusion Recommendations for vaccination in patients with AIIRD based on the currently available evidence and expert opinion were formulated. More research is needed in particular regarding incidence of vaccine-preventable infectious diseases and the safety of vaccination in patients with AIIRD.

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InTrOducTIOnPatients with auto-immune inflammatory rheumatic diseases (AIIRD) are at incre-ased risk of contracting infections [1-10]. The increased susceptibility to infection can be attributed to by any of the following: the immunosuppressive effect of the underlying AIIRD, the occurrence of a “locus minoris resistentiae” as a sequel of the AIIRD and the use of immunomodulatory medication to treat the AIIRD. The possible contribution of biologic agents to infection risk is of particular interest, especially since increasingly more indications are being recognized for their use, they are increasingly being used earlier in the course of AIIRD, and newer agents are becoming available [11-23].

Vaccination is an attractive method to prevent certain infections. The efficacy of vaccinations in patients with AIIRD, however, may be reduced, and there is a po-tential risk of flares of the underlying AIIRD following vaccination.

Our aim was to develop recommendations for vaccination in Patients with AIIRD, in line with the European League Against Rheumatism (EULAR)’s standard ope-rating procedures, combining evidence from clinical studies with expert opinion when sufficient evidence was lacking. Our recommendations target all physicians and nurses who are involved in the care for patients with AIIRD.

MeThOds

The expert committee

The committee consisted of eight rheumatologists (OE, MFD, MD, PE, PG, UML, LS, GV), four clinical immunologists (NAL, RC, CGMK, YS), one rheumatologist/cli-nical immunologist (MB), one infectious disease physician (SvA), one nephrologist (DRWJ), one paediatrician/rheumatologist (NW) and one clinical epidemiologist (JPAI), representing 11 European countries.

definitions

In these recommendations the term efficacy represents the capability of a vac-cine to mount a protective immune response, because vaccination studies in pa-tients with AIIRD addressing clinical endpoints are scarce. Moreover, it should be acknowledged that in vitro immune responses may not always correlate well with clinical effectiveness. This should be taken into account when interpreting the available evidence for these recommendations.

development of recommendations

The experts were invited to define the AIIRD, the vaccines and the immunosup-pressive medications, which were to be used as search terms for the systematic

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literature review (SLR). Furthermore, key questions regarding vaccination of pa-tients with AIIRD were formulated.

Medline (via PubMed) was searched from 1966 to October 2009 as well as the ab-stracts from the meetings of EULAR 2008 and 2009, and of American College of Rheumatology 2007 and 2008. As search terms the MESH-terms for the defined AIIRD, immunosuppressive medications and vaccines were combined. Only arti-cles in English and concerning patients older than 16 years of age were included. Other papers that were considered relevant in the opinion of the experts, could be added. The results of the SLR (performed by SvA, MB, NAL, OE) were sent to the committee before the second meeting together with proposals for recom-mendations.

Thirteen recommendations were formulated. For each recommendation, we used a widely-accepted hierarchy for categorizing the available evidence and the strength of the recommendations (table 1). A Delphi exercise with closed voting followed. During this exercise the 13 recommendations were separately voted on and given a score from 0 (absolutely no agreement with the proposed recom-mendation) to 10 (maximal possible support for the recommendation). The means and standard deviations of the scores of the whole group were calculated to de-termine the level of agreement among the experts for each recommendation. Finally, a research agenda was created.

Table 1. Evidence categories and strength of recommendations

Category Evidence

1a Meta-analysis of randomized controlled trials

1b Randomized controlled trial

2 Prospective controlled intervention study without randomization

3 Decsriptive/analytic study (including case-control, cross-sectional, case series)

4 Expert committee reports or opinion or clinical experience of respected authori-ties or both

Strength Based on

A Category 1 evidence

B Category 2 evidence or extrapolated recommendations from category 1 evidence

C Category 3 evidence or extrapolated recommendations from category 1 or 2 evi-dence

D Category 4 evidence or extrapolated recommendations from category 2 or 3 evidence

resulTsTwenty-seven eligible AIIRD, 17 immunosuppressive medications and 29 vaccines (table 2) were identified and eight key questions (table 3) were composed for the SLR. The task force members agreed on 13 recommendations, reaching a high

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level of agreement according to the Delphi scores (table 4).

recommendations

Each recommendation is followed in parenthesis with the grade of the evidence, the strength of the recommendation and the Delphi voting score.

1. The vaccination status should be assessed in the initial work-up of patients with AIIRD. (no grade of evidence possible; strength of recommendation D; Delphi vote 9,50)

In order to make recommendations for the individual patient with AIIRD, it is ne-cessary to know which vaccines the patient received in the past according to ta-ble 5. Catch-up vaccination might be considered for missed vaccinations that are recommended for the general population. Also adverse events and flares of the underlying AIIRD following former vaccinations should be queried, since these might be (relative) contraindications for certain future vaccinations.

2. Vaccination in patients with AIIRD should ideally be administered during sta-ble disease. (no grade of evidence possible; strength of recommendation D; Delphi vote 8,88)

No studies have been performed comparing efficacy and harms between patients with AIIRD with stable and unstable disease. Moreover, almost all vaccination stu-dies in patients with AIIRD addressed patients with quiescent disease. Studies that also included patients with moderate or severe disease activity did not show more frequent side effects or disease flares, or decreased efficacy in patients with AIIRD, compared with healthy controls [24-26]. However, the numbers of pa-tients in these studies were too small to conclude that vaccination during active disease is safe and efficacious. Therefore, based on theoretical risks of disease flare following vaccination in unstable patients with AIIRD, vaccination is prefe-rentially administered during stable disease, according to expert opinion.

3. Live attenuated vaccines should be avoided whenever possible in immunosup-pressed patients with AIIRD. (grade of evidence 4; strength of recommendation D; Delphi vote 9,25)

Live attenuated vaccines (table 2) might lead to (severe) infections in immuno-suppressed patients. It is unknown what level of immunosuppression renders pa-tients to be at risk for infections caused by these vaccines and this risk should be balanced to the risk of (severe) infection the vaccine aims to prevent. Measles-, mumps- and rubella (MMR) vaccine has been administered without subsequent infection to paediatric patients two years after bone marrow transplantation [27] and varicella vaccine has been administered without subsequent infection in HIV-

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Table 2. AIIRD, immunomodulating agents and vaccines considered in the literature search and recommendations.

AIIRD Immunomodulating agents Vaccines

RA Corticosteroids BCG*

SLE Methotrexate Cholera

Antiphospholipid syndrome Sulfasalazine Diphtheria

Adult Still disease Leflunomide Hepatitis A

SSc Hydroxychloroquine Hepatitis B

SjS Azathioprine Haemophilus influenzae b

MCTD Mycophenolic acid drugs Human papillomavirus

Relapsing polychondritis Cyclosporine Influenza

GCA Tacrolimus Japanese encephalitis

Polymyalgia rheumatica Cyclophosphamide Measles*

Takayasu arteritis Biologicals: Mumps*

Polyarteritis nodosa TNFα blocking agents: Neisseria meningitidis

AAV: Infliximab (A/C/Y/W135, C conjugated)

Microscopic polyangiitis Etanercept Pertussis

Wegener granulomatosis Adalimumab Poliomyelitis

Churg-Strauss syndrome Rituximab (parenteral and oral*)

Behçet disease Tocilizumab Rabies

Goodpasture disease Abatacept Rubella*

Cryoglobulinemic syndrome Anakinra Streptocccus pneumoniae

PM (polysaccharide and conjugate)

DM Tetanus toxoid

Clinically amyopathic DM Tick-borne encephalitis

Sporadic inclusion body Typhoid fever

myositis (parenteral and oral*)

Anti-synthetase syndrome Varicella zoster*

Eosinophilic myositis Yellow fever*

Eosinophilic fasciitis

Spondylathropathies

Periodic fever syndromes

* Live attenuated vaccine. AIIRD, auto-immune inflammatory rheumatic disease; BCG, Bacillus Calmette-Guérin; RA, rheuma-toid arthritis; SLE, systemic lupus erythematosus; Scl, sceroderma: SjS, Sjogren syndrome; MCTD, mixed connective tissue disease; GCA, giant cell arterits; AAV, ANCA-associated vasculitis; PM, polymyositis; DM, dermatomyositis

infected children with a CD4-percentage of ≥15% or a CD4-count ≥200/mm3 [28]. Studies are ongoing for herpes zoster vaccine in adult HIV patients with a CD4-count ≥200/mm3 (http://www.clinicaltrials.gov/ct2/show/NCT00851786?term= zostavax+hiv&rank=1) and in elderly patients on treatment with prednisone 5-20

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mg/day (http://www.clinicaltrials.gov/ct2/show/NCT00546819?term=zostavax+corticosteroid&rank=1). The Advisory Committee on Immunization Practices (ACIP) stated, that herpes zoster vaccine may be administered to patients when treated with short-term corticosteroid therapy (<14 days); low-to-moderate dose corti-costeroids (<20 mg/day of prednisone or equivalent); intra-articular, bursal, or tendon corticosteroids injections; long-term alternate-day treatment with low to moderate doses of short-acting systemic corticosteroids; therapy with metho-trexate (MTX; <0.4 mg/kg/week), azathioprine (<3.0 mg/kg/day), or 6-mercapto-purine (<1.5 mg/kg/day) [29]. It must be emphasized that these recommendations are based on expert opinion only and require further investigation.

The EULAR task force on vaccination recommends avoiding the use of live at-tenuated vaccines in immunosuppressed patients with AIIRD whenever possible. MMR, varicella and herpes zoster vaccine might be exceptions to this rule, and may be considered in mildly immunosuppressed patients with AIIRD on a case-by-case basis. Temporary discontinuation of immunosuppressive medication be-fore vaccination with live attenuated vaccines might also be considered, but there are no studies to support this strategy.

4. Vaccination in patients with AIIRD can be administered during the use of disease modifying anti-rheumatic drugs (DMARDs) and tumor necrosis factor (TNF) α-blocking agents, but should ideally be administered before starting B-cell depleting biological therapy. (grade of evidence 2a; strength of recommendation B; Delphi vote 9,13)

The efficacy of vaccination during the use of DMARDs, glucocorticoids and/or TNFα-blocking agents has been studied in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), ANCA-associated vasculitis (AAV) and sys-

Table 3. Key questions

1. Is the risk of infections for which vaccines are available increased in patients with AIIRD in general, and specifically in those with active disease, and in those using immunomodulating agents?

2. Do vaccines decrease the risk of infections in patients with AIIRD in general, and specifically in those with unstable disease, and in those using immunomodulating agents?

3. Do vaccines cause any significant harm in patients with AIIRD in general, and specifically in those with unstable disease, and in those using immunomodulating agents?

4. Does the timing of vaccination in relation to disease activity and receipt of immunomodulating agents affect the effectiveness of vaccination in patients with AIIRD

5. Does the timing of vaccination in relation to disease activity and receipt of immunomodulating agents affect important harms of vaccination in patients with AIIRD?

6. Does revaccination with any vaccines increase the effectiveness in patients with AIRD?

7. Does revaccination with any vaccine increase significant harms in patients with AIRD?

8. Is vaccination in patients with AIIRD cost-effective?

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temic sclerosis (SSc). Influenza, pneumococcal, hepatitis B, tetanus toxoid and Haemophilus influenzae b vaccination were addressed. Most controlled studies showed responses in patients with AIIRD following vaccination comparable to tho-se in healthy controls [24, 25, 30-36, 36-62], while some showed slightly reduced efficacy [38, 43, 45, 47, 49, 61, 63-67]. Of note, azathioprine hampered the response following influenza vaccination in SLE patients, but the majority of patients still develop protective levels of antibodies [63, 68]. The combination of TNFα- bloc-king agents and MTX reduced the response to pneumococcal vaccination in RA patients [45, 67, 69]. Finally, humoral responses following influenza vaccination 2-3 months after treatment with rituximab [38, 41, 69, 70] as well as humoral respon-ses following pneumococcal vaccination 28 weeks after treatment with rituximab [71] are severely hampered. Tetanus toxoid vaccination led to adequate immune responses 24 weeks following rituximab administration [71]. Vaccines should ide-ally be administered before B-cell depleting biological therapy is started, or, when patients are on such a treatment already, at least six months after start, but four weeks before the next course.

5. Inactivated influenza vaccination should be strongly considered for patients with AIIRD. (grade of evidence 1b-3; strength of recommendation B-C; Delphi vote 9,00)

Although the exact incidence of influenza is unknown in patients with AIIRD, their risk of dying from pulmonary infections is increased [4, 5, 72]. Influenza vacci-nation has been shown to reduce admissions for and mortality from influenza/pneumonia in elderly RA patients [73, 74] and is efficacious in RA, SLE, AAV and SSc patients, even when treated with DMARDs, infliximab, etanercept or adali-mumab [24-26, 30-40, 47-50, 63, 67, 68, 75-77], but with rituximab as an exception [41, 70, 78]. Adverse events of influenza vaccination in patients with AIIRD seem comparable with those in healthy controls, although there are no studies that are sufficiently powered with regard to safety. This recommendation regards seaso-nal influenza vaccination as well as pandemic swine flu vaccination, although no studies have been performed on efficacy and safety of swine flu vaccination in patients with AIIRD. Because some of the swine flu vaccines contain the adjuvant MF-59, an oil-in-water emulsion that potentiates the humoral response, it is reas-suring that a large meta-analysis showed no difference in the occurrence of ad-verse events that were of auto-immune origin between persons vaccinated with influenza vaccine with and without MF-59 [79].

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6. 23-valent polysaccharide pneumococcal vaccination (23-PPV) should be strongly considered for patients with AIIRD. (grade of evidence 1b-3; strength of recommendation B-C; Delphi vote 8,19)

As stated above, patients with AIIRD are at increased risk of dying from pulmo-nary infections compared to the general population [2, 4, 5, 72], with pneumococci being considered to be one of the main causative pathogens. Pneumococcal vac-cination induces an adequate to slightly reduced humoral response in patients with RA, SLE, psoriatic arthritis, ankylosing spondylitis and SSc, even when tre-ated with immunosuppressive drugs [36, 42-45, 47, 51-54, 62, 70, 80]. MTX, with or without TNFα-blocking agents [45, 69], and in particular rituximab [71] reduce the humoral response following pneumococcal vaccination. It is unknown if and when revaccination should take place and if the new conjugated pneumococcal vaccines, whether or not in combination with 23-PPV (so-called prime-and-boost strategy), induce more and/or more durable immunity to pneumococci in patients with AIIRD. Pneumococcal vaccination seems safe in patients with AIIRD, but again the available studies were not adequately powered for analyzing safety.

7. Patients with AIIRD should receive tetanus toxoid vaccination in accordance to recommendations for the general population. In case of major and/or con-taminated wounds in patients who received rituximab within the last 24 weeks, passive immunization with tetanus immunoglobulins should be administered. (grade of evidence 2a; strength of recommendation B-D; Delphi vote 9,19)

In RA and SLE patients efficacy for tetanus toxoid vaccination has been demon-strated to be comparable with healthy controls [30, 46, 58, 59]. This also holds true for RA patients on immunosuppressive drugs, including those who have been treated with rituximab 24 weeks earlier [71]. However, since no data are available regarding the efficacy of tetanus toxoid vaccine within 24 weeks after treatment with rituximab [71], we recommend to immunize patients with AIIRD who are tre-ated with rituximab less then 24 weeks earlier passively with tetanus immuno-globulins in case of a serious risk to contract tetanus i.e. in case of major and/or contaminated wounds.

8. Herpes zoster vaccination may be considered in patients with AIIRD. (grade of evidence 3-4; strength of recommendation C-D; Delphi vote 8,00)

Compared to the general population patients with RA, SLE, AAV and polymyositis/dermatomyositis have an increased risk of developing herpes zoster [81-99]. RA is in itself a risk factor and the risk of developing herpes zoster is further incre-ased in patients with AIIRD treated with corticosteroids, TNFα-blocking agents and non-biologic DMARDs, in particular cyclophosphamide , azathioprine and

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Tab

le 4

. Rec

omm

enda

tion

s fo

r va

ccin

atio

n in

adu

lt p

atie

nts

wit

h au

to-i

mm

une

infl

amm

ator

y rh

eum

atic

dis

ease

s w

ith

leve

l of

evid

ence

, str

engt

h of

re

com

men

dati

ons

and

resu

lts

of D

ephi

vot

ing

per

reco

mm

enda

tion

Rec

omm

enda

tion

Cat

egor

y of

Evi

denc

eSt

reng

th o

f re

com

men

dati

onLe

vel o

f ag

ree-

men

t by

Del

phi

voti

ng [V

AS;

m

ean

(SD

)]

Incr

ease

d in

ci-

denc

e of

VP

IEf

fica

cy o

f va

ccin

atio

nH

arm

s of

va

ccin

atio

n

1. T

he v

acci

nati

on s

tatu

s sh

ould

be

asse

ssed

in t

he

in

itia

l wor

k-up

of

pati

ents

wit

h A

IIRD

---

---

---

D9,

50 (0

,97)

2. V

acci

nati

on in

pat

ient

s w

ith

AIIR

D s

houl

d id

eally

be

adm

inis

tere

d du

ring

sta

ble

dise

ase

---

---

---

D8,

88 (1

,26)

3. L

ive

atte

nuat

ed v

acci

nes

shou

ld b

e av

oide

d w

hen-

eve

r po

ssib

le in

imm

unos

uppr

esse

d pa

tien

ts w

ith

A

IIRD

---

---

4D

9,25

(1,13

)

4. V

acci

nati

on in

pat

ient

s w

ith

AIIR

D c

an b

e ad

min

iste

-

red

dur

ing

the

use

of D

MA

RD

s an

d T

NF-

alph

a bl

oc-

k

ing

agen

ts, b

ut s

houl

d id

eally

be

adm

inis

tere

d be

-

for

e st

arti

ng B

-cel

l dep

leti

ng b

iolo

gica

l the

rapy

---

2--

-B

9,13

(1,0

2)

5. In

flue

nza

vacc

inat

ion

shou

ld b

e st

rong

ly c

onsi

dere

d

for

pat

ient

s w

ith

AIIR

D3

1b1b

B-C

9,0

0 (1

,10)

6. 2

3-va

lent

pol

ysac

char

ide

pneu

moc

occa

l vac

cina

tion

sho

uld

be s

tron

gly

cons

ider

ed f

or p

atie

nts

wit

h A

IIRD

31b

1bB

-C8,

19 (1

,38)

7. P

atie

nts

wit

h A

IIRD

sho

uld

rece

ive

teta

nus

toxo

id

v

acci

nati

on in

acc

orda

nce

to r

ecom

men

dati

ons

for

the

gen

eral

pop

ulat

ion.

In c

ase

of m

ajor

and

/or

con-

tam

inat

ed w

ound

s in

pat

ient

s w

ho r

ecei

ved

RT

X

w

ithi

n th

e la

st 2

4 w

eeks

, pas

sive

imm

uniz

atio

n w

ith

t

etan

us Ig

sho

uld

be a

dmin

iste

red

---

22

B-D

9,19

(1,11

)

8. H

erpe

s zo

ster

vac

cina

tion

may

be

cons

ider

ed in

pat

ient

s w

ith

AIIR

D3

---

4C

-D8,

00

(1,5

9)

9. H

PV

vac

cina

tion

sho

uld

be c

onsi

dere

d in

sel

ecte

d

p

atie

nts

wit

h A

IIRD

3--

---

-C

-D8,

44 (1

,41)

181

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9

Tab

le 4

. (co

ntin

ued)

10. I

n hy

posp

leni

c/as

plen

ic p

atie

nts

wit

h A

IIRD

infl

uenz

a

pn

eum

ococ

cal,

Hae

mop

hilu

s in

flue

nzae

b a

nd m

e-

ning

ococ

cal C

vac

cina

tion

s ar

e re

com

men

ded

4--

---

-D

9,50

(0,8

2)

11.

Hep

atit

is A

and

/or

B v

acci

nati

on is

onl

y re

com

men

-

ded

in p

atie

nts

wit

h A

IIRD

at

risk

---

2*3*

D9,

13 (0

,89)

12. A

IIRD

pat

ient

s w

ho p

lan

to t

rave

l are

rec

omm

ende

d

t

o re

ceiv

e th

eir

vacc

inat

ions

acc

ordi

ng t

o ge

nera

l

rul

es, e

xcep

t fo

r liv

e-at

tenu

ated

vac

cine

s, w

hich

sho

uld

be a

void

ed w

hene

ver

poss

ible

in im

mun

osup

-

pre

ssed

pat

ient

s w

ith

AIIR

D

---

---

---

D9,

25 (1

,24)

13. B

CG

vac

cina

tion

is n

ot r

ecom

men

ded

in p

atie

nts

wit

h A

IIRD

3--

---

-D

9,38

(1,0

9)

* fo

r he

pati

tis

B o

nly

VP

, vac

cine

-pre

vent

able

; AIIR

D, a

uto-

imm

une

infl

amm

ator

y di

seas

e; Ig

, im

mun

oglo

bulin

s; H

PV

, hum

an p

apill

omav

irus

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leflunomide [82-85, 90, 91, 98] but not MTX [100]. One study found an increased risk for herpes zoster in SLE patients when treated with rituximab [101]. Lupus disease activity is not a risk factor for herpes zoster [88]. Herpes zoster vaccine has been shown to reduce herpes zoster and post-herpetic neuralgia in patients over 60 years [102], but no studies have been performed in patients with AIIRD. Because of the high burden of herpes zoster in patients with AIIRD, herpes zoster vacci-nation may be considered in these patients, but only when less severely immu-nosuppressed. The ACIP suggested criteria for immunosuppressed patients who can receive herpes zoster vaccine, however, it must be emphasized that these recommendations are not validated but are based on expert opinion, and require further investigation [29]. It seems prudent to administer herpes zoster vaccine only to patients with AIIRD who are seropositive for varicella zoster antibodies, in order to prevent primary varicella infection with the vaccine-strain.

9. Human Papillomavirus (HPV) vaccination should be considered in selected patients with AIIRD. (grade of evidence 3; strength of recommendation C-D; Delphi vote 8,44)

It has been shown that HPV infection occurs more often in SLE patients, also with the high-risk (oncogenic) subtypes of the virus [103-105]. A lower percentage of these infections (31,8%) is spontaneously cleared by SLE patients [106], leading to an increased risk of developing cervical cancer. The risk factors for contracting HPV infection are the same in SLE patients as in the general population [103;105]. The efficacy of HPV vaccination has not been investigated in patients with AIIRD. HPV vaccination is recommended for young females in many countries, and HPV vaccination should be considered for women with SLE until the age of 25 years. The quadrivalent (q) HPV vaccine has been associated with venous thromboem-bolic events (VTE). However, of the 31 cases (0,2/100.000 doses of qHPV vaccine) that had objectified VTE, 90% had a known risk factor for VTE, being antiphospho-lipid syndrome in two cases [107].

10. In hyposplenic/asplenic patients with AIIRD influenza, pneumococcal, Hae-mophilus influenzae b and meningococcal C vaccinations are recommended. (grade of evidence 4; strength of recommendation D; Delphi vote 9,50)

Hyposplenic/asplenic patients are at risk of contracting a so-called “overwhel-ming post-splenectomy infection (OPSI)”. OPSI is caused by encapsulated bac-teria (e.g. S. pneumoniae, H. influenzae b, N. meningitidis) and the mortality of OPSI is up to 70% [108-112]. OPSI can occur as a secondary infection after infection with influenza. No studies have addressed the efficacy of vaccination to prevent OPSI in patients who are hyposplenic/asplenic, but the general consensus is to vaccinate these patients against influenza, S. pneumoniae, H. influenzae b and N.

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meningitides C [113]. When hyposplenic/asplenic patients with AIIRD plan to travel to or live in areas where other meningococcal strains are endemic (A, Y, W135), vaccination for these meningococcal subtypes is also indicated [114]. Prophylactic or on-demand antibiotics and preventive measures for malaria and babesiosis are beyond the scope of these recommendations.

11. Hepatitis A and/or B vaccination is only recommended in patients with AIIRD at risk. (grade of evidence 2a-3; strength of recommendation B-D; Delphi vote 9,13)

Data on incidence of hepatitis A and B infection in patients with AIIRD are lacking. Reactivation of hepatitis B infection in patients with AIIRD has been described following treatment with immunosuppressive medication or immediately after discontinuing immunosuppressive medication (among which anti-TNFα agents). However, no comparative studies have been published. Therefore it is impossible to distinguish whether the immunosuppressive treatment, the disease activity of the AIIRD or the natural course of chronic hepatitis B infection was the cause of the hepatitis flare. Hepatitis B vaccination is efficacious in most patients with AIIRD [55-57]. Vaccination for hepatitis A and/or B is only recommended when the risk of contracting these infections is increased (travel to or residence in endemic countries for hepatitis A and/or B; increased risk of exposure or proven exposure to hepatitis A and/or B (e.g. because of medical profession, infected family mem-ber or contacts) and when protective antibodies against hepatitis A and/or B are absent.

12. AIIRD patients who plan to travel are recommended to receive their vaccinati-ons according to general rules, except for live-attenuated vaccines, which should be avoided whenever possible in immunosuppressed patients with AIIRD. (no grade of evidence; strength of recommendation D; Delphi vote 9,25)

It is unknown whether patients with AIIRD have an increased risk of contracting travel-related vaccine-preventable infections (VPI). In RA patients and SLE pa-

Table 5. Vaccinations to be checked during initial workup (by history taking)

Haemophilus influenzae b

Hepatitis A

Hepatitis B

Human papillomavirus

Influenza

Neisseria meningitides

Rubella (for women of child-baring age)

Streptococcus pneumoniae

Tetanus toxoid

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tients the risk of tuberculosis (TB) is increased (also see recommendation 13) [115-129]. However, the majority of these TB cases represent reactivations from earlier contracted latent TB infection, and BCG vaccination has not been clearly demon-strated to prevent tuberculosis in adults. Influenza is endemic in subtropical and tropical climates during the entire year and is the most frequent VPI among tra-vellers to subtropical and tropical countries [130, 131]. The incidence of influenza in patients with AIIRD is not known. Also the incidence of cholera, diphtheria, hepati-tis A, meningococcal infection, poliomyelitis, rabies, tetanus, tick-borne encepha-litis, typhoid fever and yellow fever is unknown. Studies addressing the efficacy of influenza vaccination (in RA, SLE, SSc and AAV patients) [24-26, 30-41, 47-50, 63, 68, 75, 76] and tetanus toxoid vaccination (in RA and SLE patients) [30, 46, 58, 59] generally showed responses comparable to those in healthy controls .

To protect patients with AIIRD from contracting travel-related VPI, they should receive the vaccinations that are recommended to the general population. Excep-tions are vaccinations with BCG vaccine, oral poliomyelitis vaccine, oral typhoid fever vaccine and yellow fever vaccine, which contain live attenuated micro-or-ganisms and therefore might lead to life threatening infection in immunosup-pressed patients with AIIRD.

13. BCG vaccination is not recommended in patients with AIIRD. (grade of evidence 3; strength of recommendation C-D; Delphi vote 9,38)

The incidence of tuberculosis is increased in patients with AIIRD, in particular when treated with immunosuppressive drugs (DMARDs, corticosteroids [115-119, 121-124, 127-129], and especially TNFα-blocking agents [116-119, 122, 127-129]. The large majority of these cases of active tuberculosis are reactivations of earlier con-tracted latent tuberculosis infections, which cannot be prevented by vaccination. Moreover, BCG vaccination has not been clearly demonstrated to be efficacious

Table 6. Research agenda

1. Set-up of a vaccination registry for patients with AIIRD with a focus on safety and efficacy

2. Prospective studies evaluating the prevalence and identification of causative micro-organisms of infection in patients with AIIRD, and the outcome of vaccine preventable infections in these patients

3. Evaluation of the impact of therapies on safety and efficacy of vaccination

4. Evaluation of the impact of new therapies on prevalence of (vaccine preventable) infections

5. Research for efficacy and safety of new vaccines (such as herpes zoster, pandemic H1N1 Influ-enza A, HPV)

6. Evaluation of the implementation of vaccination recommendations

7. Research for the safety and efficacy regarding adjuvants and specific risk groups (e.g. preg-nancy)

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in preventing tuberculosis in adults. Finally, BCG vaccine contains attenuated my-cobacteria, and vaccination with BCG vaccine has been shown to induce BCG-itis in immunosuppressed patients [132-134].

research agenda

The EULAR Task Force for vaccination in patients with AIIRD agreed on the re-search agenda as represented in table 6.

dIscussIOnThe recommendations for vaccination in patients with AIIRD, as presented abo-ve, are based on the current evidence resulting from the SLR and the opinion of selected experts in the fields of rheumatology, clinical immunology, nephrology, paediatric rheumatology/immunology and infectious diseases from 11 European countries. Unfortunately no randomized controlled studies were available that addressed efficacy of vaccination in patients with AIIRD on clinical endpoints. Therefore the highest strength of these recommendations can be strength B (ta-ble 1). We did not systematically review the literature on vaccines in the general population without AIIRD, but the experts did take into account their knowledge of this wider literature in formulating the recommendations. In general, it should be noted that even for the general population, conceptions about the efficiency and efficacy of vaccination have varied over time. Especially for influenza and pneumococcal vaccines there is strong evidence that adequate immune respon-ses are achieved with vaccines, but this may not always translate to equally high efficiency at the clinical protection level [135-137].

Other infection-preventive measures than vaccination are not addressed in these recommendations, and we suggest a new EULAR task force should be set up to recommend on important issues like general hygienic measures and antibiotic prophylaxis for patients with AIIRD to further reduce infection-related morbidity and mortality in patients with AIIRD.

Our literature search focused essentially on three important aspects of vaccina-tion in patients with AIIRD: the incidence of infectious diseases for which vaccines are available; the efficacy of vaccinations that are indicated; and the harms of vac-cination. We should acknowledge that the grading of the available evidence can differ between the three aforementioned aspects: often little evidence is availa-ble for the incidence of VPI, and most studies are underpowered with regard to adverse events, while efficacy is best studied for most vaccines. The results of the Delphi voting to the traditional level of evidence are therefore of particular importance, since these represent the overall interpretation of the evidence on all aforementioned aspects of vaccination in patients with AIIRD by the panel of

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experts.

The morbidity and mortality for most VPI increase for patients who are more se-verely immunosuppressed, e.g. when treated with a more intensive immunosup-pressive regimen. Therefore we like to stress that the recommendations regar-ding vaccination in patients with AIIRD should be followed more stringently for more immunosuppressed patients. However, because efficacy can be reduced by immunosuppressive treatment, offering vaccination prior to (intensive) immuno-suppressive treatment, in particular rituximab, is started is advisable.

A note of caution is warranted with regard to the safety of vaccination in pa-tients with AIIRD. Although many case reports have been published demonstra-ting flares of AIIRD or new onset auto-immune diseases following vaccination, these adverse events remain rare and a causal relationship has not been proved. Moreover, several controlled studies show no difference in the occurrence of fla-res of AIIRD after vaccination, although these studies have not been powered to address specific adverse events, but efficacy. Because of the lack of sufficiently powered studies focusing on harms, these issues remain an important item on the research agenda.

Finally, there are different vaccination schemes that have been developed and proposed in different European countries. The exact implementation of the cur-rent recommendations may need to take into account local differences in specific countries and settings. Moreover, the recommendations need to be updated on a regular basis (every 3 years, since new evidence will become available with re-gard to current and new vaccines, and current and new immunomodulating tre-atments.

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CHAPT

ER10Summary,g e n e r a l discussion and future perspectives

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SummAryPatients with primary and secondary immunodeficiencies are at increased risk of contracting common and/or opportunistic infections [1, 2]. Moreover, these in-fections more often follow a complicated course, with increased morbidity and mortality [1, 2]. One of the interventions to prevent or mitigate the course of in-fections in these patients is vaccination. However, paradoxically, for an optimal response following vaccination an adequately functioning immune systems is re-quired (also see introduction to this thesis).

In order to make recommendations on the usefulness of influenza vaccination in patients with primary and secondary immunodeficiencies, we investigated the humoral and cell-mediated immune (CMI) responses following influenza vaccina-tion in these patients. Moreover, the timing of vaccination in relation to immuno-suppressive treatment was studied as well as a strategy to optimize the immune response to influenza vaccination: administration of a second, booster, influenza vaccination.

Following chapter 1, where we summarized the history of the discovery of vac-cination, the immune response to influenza vaccination, and the primary and se-condary immunodeficiencies that are addressed in the thesis, part 1 describes the vaccination studies that we performed in patients with humoral primary immuno-deficiencies (hPID). Chapter 2 addresses the humoral immune response following inactivated trivalent subunit influenza vaccination in patients with hPID. Following vaccination, as expected, hPID-patients developed humoral responses that were clearly inferior to those found in healthy controls (HC), as measured by geometric mean titers (GMTs), fold-increase in GMT, and seroprotection rates. In contrast to HC, who responded to all three influenza strains, patients were able to respond with a significant rise in GMT only for the A/H1N1-strain. Moreover, previous vac-cination and treatment with intravenous immunoglobulin (IVIg) did not result in higher postvaccination GMTs or higher rate of seroprotection (defined as titer ≥40).

Since CMI responses are also of importance for the clearance of influenza from infected individuals, in the study presented in chapter 3 we investigated the cell-mediated recall responses following influenza vaccination in patients with com-mon variable immunodeficiency (CVID), a subgroup of the hPID-patients that were included in the study described in chapter 2. Although cellular immune responses are affected in patients with CVID at many different levels, the cellular response following influenza vaccination had never been addressed before by others. We found impaired recall responses following influenza vaccination in the CVID-pa-tients when using interferon (IFN)γ-ELISpot. Flow cytometry with intracellular cy-

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tokine staining revealed a decrease in influenza-specific IFNγ and tumor necrosis factor (TNF)α producing CD4+ and CD8+ T-cells in patients following vaccination compared to baseline, while HC demonstrated an increase in influenza-specific cytokine producing T-cells. In vitro activation-induced cell death of CD4+ and CD8+ T-cells might be the explanation for this fall in T-cell numbers after influenza vac-cination as increased pre-activation of T-cells in vivo has been demonstrated in CVID patients.

Part 2, addressing immune responses to influenza vaccination in patients with se-condary immunodeficiencies, in particular those suffering from auto-inflamma-tory rheumatic diseases (AIIRD), starts with two studies on influenza vaccination in patients with systemic lupus erythematosus (SLE). The first focused on the humoral and CMI responses before and after influenza vaccination (chapter 4). SLE patients showed a decreased antibody response to A/H1N1 and A/H3N2, com-pared to HC. Cell-mediated influenza-specific responses were also found to be lower in SLE-patients in comparison with HC. Using FACS analysis, SLE-patients showed increases in frequencies of influenza-specific CD4+ T-cells for fewer cy-tokines following influenza vaccination as compared to HC. Impaired cell-medi-ated influenza-specific responses were associated with the use of prednisone and/or azathioprine. The second study (chapter 5) investigated a strategy to im-prove the outcome of vaccination in SLE-patients, since an earlier study (chap-ter 4) demonstrated sub-optimal humoral responses in these patients. In this re-vaccination study, the efficacy of administration of a second, booster, influenza vaccination four weeks after the first one was studied. In contrast to previous data, with in part other influenza strains, this study did not find differences in the influenza-specific antibody responses between SLE patients and HC after the first vaccination. The booster vaccination did not result in a further rise in GMT, seroconversion rates or seroprotection rates, except for SLE patients who were not vaccinated in the previous year, who tended to have an additional increase in GMT and seroconversion rate for the A/H1N1 influenza-strain.

Chapter 6 describes a study that was initiated to investigate the influence of tre-atment with rituximab, a B-cell depleting anti-CD20 monoclonal antibody, on the antibody response following influenza vaccination in patients with rheumatoid arthritis (RA). In B-cell depleted individuals no influenza-specific antibodies were produced following influenza vaccination. This was expected, as influenza vac-cine antigens are considered (in part) to be neo-antigens. The antigens are yearly adjusted to the changes in the influenza virus caused by antigenic drift. Vacci-nation within 4 to 8 weeks after administration of rituximab showed no humoral response at all. However, 6 to 10 months following rituximab therapy, when recur-rence of B-cells in the peripheral blood occurred, a significant, but still hampered,

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antibody response to both A-strains in the vaccine could be demonstrated.

Within the same study population that was studied in chapter 6, the CMI respon-ses before and after influenza vaccination (using IFNγ-ELISpot, proliferation as-says and flowcytometry with intracellular cytokine staining) were determined, as described in chapter 7. Before vaccination, flow cytometry demonstrated that polyclonal and influenza-specific CMI responses were reduced in RA-patients tre-ated with rituximab. Moreover, influenza-specific CMI responses following influ-enza vaccination were hampered.

Finally, part 3 presents the systematic literature review (SLR) (chapter 8) that was performed to form the basis of evidence-based recommendations on vaccination in patients with auto-immune inflammatory rheumatic diseases (AIIRD).

After the multidisciplinary expert committee commissioned by EULAR defined the AIIRD, vaccines and immunomodulating drugs to be included in the search, eight key questions were answered. The EULAR recommendations for vaccina-tion in adult patients with AIIRD based on this SLR and expert opinion, using Delphi voting, can be found in chapter 9. Although more research is needed, in particu-lar regarding incidence of vaccine preventable infections (VPI), harms of vacci-nation and the influence of (new and established) immunomodulating agents on vaccination efficacy, 13 recommendations were formulated. Moreover, a research agenda was proposed.

GenerAl diScuSSionIn the studies described in this thesis, we evaluated humoral and CMI responses following influenza vaccination in patients with primary and secondary immuno-deficiencies in order to determine the usefulness of influenza vaccination in these categories of patients. Moreover, (influenza) vaccination can be used as a tool to measure the residual immune response as the resultant of the immunosuppres-sion in immunocompromised patients. When administrating identical antigenic stimuli such as influenza vaccine, the immune response is mainly dependent on host factors.

occurrence of influenza in primary and secondary immunodeficiencies

Influenza infection is clinically asymptomatic in 30-50% of infected cases. It gene-rally presents with an acute illness characterized by fever, chills, sore throat, my-algias, headache and fatigue. Symptoms last an average of 7 days [1, 2]. However, gastro-intestinal symptoms may be most prominent in children, but during the novel influenza A/H1N1 pandemic in 2009 with the influenza virus strain A/H1N1/California/2009 also in adults abdominal complaints frequently occurred as one

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of the presenting symptoms (3).

Influenza is usually a self-limiting disease, but complications may occur in particu-lar in young children, elderly and immunocompromised hosts. Common complica-tions are exacerbations of underlying respiratory or heart disease. Viral pneumo-nia may occur as well as secondary bacterial pneumonia [1-3]. The last pandemic confirmed the vulnerability of young children, patients with underlying cardiopul-monary diseases and immunocompromised patients [3, 4].

For the specific patient groups addressed in this thesis, however, little is known on the occurrence, morbidity and mortalitiy of influenza infection. Patients with hypogammaglobulinemia (chapter 2 and 3) frequently experience bacterial upper and lower respiratory tract infections [5-7], and it seems logical to assume that the risk of contracting influenza and its morbidity and mortality are increased in patients with hypogammaglobulinemia, because humoral immunity following influenza vaccination correlates with protection from or mitigation of influen-za infection [8]. Moreover, an increasing amount of data supports the role CMI responses in the clearance of influenza [9, 10]. CMI responses are hampered in patients with CVID [5, 11-20]. However, no data are available regarding viral pa-thogens causing respiratory infection is these patients. Therefore, not only the mortality and morbidity resulting from influenza infection remains unknown, it is also impossible to estimate the efficacy of influenza vaccination on clinical end-points in these patients.

In patients with secondary immunodeficiencies (SLE and RA, treated with immu-nosuppressive medication) that we studied in chapter 4, 5, 6, and 7 it has been shown that morbidity and mortality from pulmonary infections is increased, but no studies assessed the causative micro-organisms that led to these pulmonary infections. Two studies that included elderly patients with an increased risk of contracting influenza, among whom patients with rheumatic diseases and vas-culitis, found an increased risk for hospital admission for either pneumonia or influenza and for death, compared to low-risk elderly [21, 22]. A subgroup analysis of patients with rheumatic diseases or vasculitis, however, was not performed. Still, patients with rheumatic diseases should be considered at increased risk of influenza and a complicated course of influenza.


The aim of vaccination is to reduce the morbidity and mortality, directly or in-directly provoked by the vaccine-preventable disease. Therefore, studies eva-luating the efficacy of vaccination should ideally use clinical endpoints. In case of influenza vaccination, the reduction of microbiologically confirmed influenza

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infection, the occurrence of influenza-like illness (ILI), influenza-related hospital admission, pneumonia and death have been used as clinical endpoints of studies evaluating the efficacy of influenza vaccination.

On the other hand, ILI can also be caused by many infections other than influenza and for a long time it has been hard to confirm influenza microbiologically, becau-se no sensitive tests for influenza were available (only viral culture and serology). Moreover, vaccine efficacy also depends on the homology between the influenza-strains in the vaccine and the actually circulating influenza-strains. Since influ-enza strains yearly undergo an antigenic change, efficacy of influenza vaccination varies per year and per strain [1, 2]. Therefore, studies investigating efficacy of influenza vaccination on clinical endpoints should investigate their study partici-pants for influenza on a regular basis with a sensitive microbiological test, include large numbers of participants and cover multiple influenza seasons. Such a study is difficult to accomplish, and therefore many studies assessed the efficacy of influenza vaccination by determining surrogate outcome measures.

The most important surrogate parameter is the humoral immune response as determined by hemagglutination inhibition assay (HIA), because this parameter has been shown to correlate with protection from influenza. Hemagglutination inhibition (HI)-titer ≥40 is protective in young healthy adults, and in 90% of healthy young adults HI-titers rise to ≥40 after vaccination [8]. In elderly and most im-munocompromised persons the humoral response following influenza vaccinati-on is suboptimal [23-26]. Moreover, it has never been demonstrated which HI-titer should be strived for in elderly or immunocompromised patients. For example, elderly with very high HI-titers might not be protected from influenza [27-29].

The fact that despite so-called protective antibody levels individuals might not be protected from influenza might be due to an impaired cellular response. The influenza-specific cell mediated immune (CMI) responses following influenza vac-cination are important for the clearance of influenza after infection [10, 27, 29] and can be induced by influenza vaccination, in particular when using whole in-activated virus (WIV)-vaccine or live attenuated influenza-vaccine. In children the production of IFNγ-production has been shown to correlate with protection from influenza [30]. In elderly granzyme-B (as a measure of the effector mechanism of cytotoxic T-lymphocytes), and the ratio between IFNγ and interleukin(IL)-4 (as a measure of the balance between the Th1- and Th2-response) was demonstrated to correlate better with protection from clinical influenza than the HI-titer [27, 29].

In the studies reported in this thesis we evaluated both humoral and CMI respon-ses to determine the efficacy of influenza vaccination in our patient groups with hypogammaglobulinemia, SLE and RA. The humoral immune response was deter-

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mined with the generally accepted HIA, the CMI response by using IFNγ-ELISpot, flow cytometry with intracellular cytokine staining for IFNγ, IL-2 and tumor ne-crosis factor (TNF)α and CFSE-dye dilution proliferation assay. IFNγ-ELISpot has been demonstrated to be sensitive, but only determines the production of one cytokine by PBMC, a mixture of different white blood cells. The advantage of flow cytometry with intracellular cytokine staining is its capability to determine the production of several cytokines in well defined subsets of PBMC, but it is a la-bour-intensive technique and less sensitive than ELISpot [31]. Finally, the CFSE-dye dilution proliferation assay is the most functional assay, assessing an important aspect of the CMI response: the capability of predefined cell types to proliferate in a clonal fashion in response to a specific antigen. However, none of these para-meters of cellular immunity have been shown to correlate with protection from influenza in the investigated study populations.

In patients with hypogammaglobulinemia, especially in patients with CVID, ham-pered humoral and CMI responses were recorded (chapter 2 and 3). Influenza vaccination did not lead to an increase in seroprotection rates. Pre-existent anti-influenza HI-titers might have resulted from earlier infections or treatment with IVIg. SLE patients seem to have humoral responses following influenza vaccina-tion comparable to HC, but might benefit from a second, booster vaccination if not previously vaccinated. CMI responses following influenza vaccination in SLE-patients did increase, but were lower compared to HC, before as well as following vaccination (chapter 4 and 5). Finally, RA patients treated with rituximab, will not respond to influenza vaccination with a rise in HI-titer or increase in seropro-tection rates, unless the interval after the last administration of rituximab is at least 6-10 months. Also these RA patients demonstrated reduced CMI responses at baseline and following influenza vaccination, indicating an important role for B-cells also in CMI responses.

clinical implications of the findings in this thesis

It is hard to determine the clinical implications of influenza vaccination in the pa-tients categories assessed in this thesis realising that the burden of influenza in these patients is unknown. However, as it is generally accepted that immunocom-promised patients “not-otherwise-defined” are at increased risk of contracting influenza and of a complicated course of influenza, our studies do have implica-tions.

The reduced humoral as well as to CMI responses to influenza vaccination in hy-pogammaglobulinemia patients, especially CVID patients, requests for additional measures to prevent influenza in these patients. Influenza vaccination should be

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offered to persons caring for or residing with patients with hypogammaglobuli-nemia and post-exposure prophylactic antiviral treatment should be considered in these patients. When influenza is suspected, these patients should be treated empirically.

Recommendations for vaccination in patients with AIIRD (chapter 9), based on a systematic literature research (chapter 8), have recently been developed for the European League against Rheumatism (EULAR). The articles regarding influ-enza vaccination in patients with SLE, and RA patients treated with rituximab, performed by our research group (chapter 5 and 7), contributed to these recom-mendations. Because of the increased risk of pulmonary infections in AIIRD pa-tients, they should be offered influenza vaccination. A booster can be considered in previously unvaccinated patients, as also supported by the reduced response in AIIRD patients vaccinated with the novel non-adjuvanted A/H1N1-vaccine [32]. Furthermore, vaccination of patients treated with rituximab should be administe-red before the start of rituximab, since rituximab severely hampers the immune response to influenza vaccine.

Besides recommendations on influenza vaccination in patients with AIIRD, also all other in Europe available vaccines were addressed in the EULAR recommenda-tions on vaccination in patients with AIIRD (chapter 9). The next, last, step in the assessment of the clinical implications of vaccination is of course the implemen-tation of these recommendations. Several studies show that, despite recommen-dations and guidelines supporting influenza vaccination in patients with AIIRD, only a small proportion of these patients do receive influenza vaccination [33-36]. Therefore, care givers should be informed about the recent EULAR recommen-dations for vaccination in patients with AIIRD during symposia and conferences. For practical implementation general practitioners, and doctors and nurses spe-cialised in the care for patients with AIIRD, need to offer and actually administer the vaccines. One Dutch study revealed that, despite an indication for influenza vaccination according to the guidelines, 262 of 595 patients were not vaccinated. Half of them was not advised about the vaccination, or was even advised to res-train from vaccination. In contrast, 86% of patients with an indication for influenza vaccination according to the guidelines was vaccinated when advised to [37].

FuTure PerSPecTiveSAlthough some questions have been answered in the studies in this thesis, and practical recommendations based in the currently available evidence have been formulated regarding vaccination in patients with AIIRD, still many questions remain unanswered and some additional questions came up. Therefore, there are many opportunities for further research.

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epidemiology of influenza and its complications in patients with primary im-munodeficiencies and Aiird

As discussed in the general discussion, it is unknown what the extent is of influen-za-related morbidity and mortality in patients with primary immunodeficiencies and AIIRD. Without these data, the efficacy of and also the indication for influenza vaccination remain largely unknown in these patient groups. Also cost-efficacy can not be addressed without this knowledge. Studies investigating the frequen-cy of influenza infection and the morbidity and mortality associated herewith should be performed. Serological studies before and following the influenza sea-son 2009-2010, when the novel influenza A/H1N1 occurred, might provide insight in the epidemiology, although the occurrence of a pandemic is exceptional and because of the large proportion of patients vulnerable for a new influenza virus strain reduces the possibility to extrapolate these data to seasonal flu. Another option would be to determine reference centres treating patients with primary immunodeficiencies and AIIRD that investigate the incidence of particular vacci-ne-preventable infections, among which influenza, during a predefined period. Those reference centers can also evaluate the safety of vaccinations in patients with AIIRD, as limited (however reassuring) data are available, in particular about safety of vaccination in patients treated with newer biologic therapies and/or ac-tive disease.

correlates of protection for influenza

Since large studies assessing clinical endpoint will remain hard to perform in sub-groups of immunocompromised patients, there is a need for good correlates of protection from influenza. Not only should those parameters be determined in healthy adults, also correlates of protection should be defined for immunocom-promised patients, since currently it is uncertain if a HI-titer ≥40 also correlates with protection in these patients. It should also be investigated which parameters for CMI responses following influenza are protective. A good candidate could be granzyme-B [27, 29, 38]. Standardised assays for granzyme-B have recently be-come available. Also the IFNγ/IL-4 ratio should be further explored [27, 29, 38].

more effective immunization of patients with primary immunodeficiency or Aiird

Since influenza-specific immune responses are hampered in the patient groups we investigated in this thesis, more effective immunizations are needed to pro-vide protection from influenza. For example, adjuvants can be added. Alum is well known for its role as an adjuvant. Since it shifts the immune response towards a Th2-response, it leads to better antibody production [39]. Also oil-in-water emul-

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sion adjuvants, i.e. MF59 and AS03, which enhance the recruitment and activation of antigen presenting cells and therefore induces stronger immune responses following influenza vaccination [40], might be used. Moreover, addition of MF59 leads to cross-protection against influenza-strains that were not included in the vaccine [41]. MF59-adjuvanted influenza A/H1N1v vaccination did not result in an increase in disease activity scores in patients with RA, SLE, or ankylosing spondy-litis [42]. A stronger Th1 response can be induced by using whole virus vaccines, unfortunately these are also more reactogenic [38, 43]. Furthermore, antigens can be offered within a constructed virus envelope, so called virosomes or virus-like particles, leading to both humoral and CTL-responses [43-48]. Besides hemagglu-tinin (HA) and neuraminidase (NA), internal proteins of influenza virus, that are more conserved than HA and NA, can the offered in the virosomes for a better CMI response [48]. Stimulation of Toll-like receptors along with the administration of influenza antigens might lead to better Th1 responses and potentially better protection from influenza [49;50]. Finally, intradermal administration of influenza vaccine could be an option to ameliorate the immune response following influen-za vaccination, since resident dendritic cells in the dermis are able to stimulate in-nate immunity thereby increasing the adaptive immune response to vaccination. This has been shown to be safe and feasible in immunocompromised patients in one study, but CMI responses were not assessed [51].

For all these strategies it remains important to realise that the immune system is stimulated more profoundly and therefore the chance of side effects is incre-ased. In particular for patients with (an increased risk of) auto-immune diseases, the benefit of better influenza-specific immune responses may not outweigh the risks of developing auto-immune phenomena in patients with primary immuno-deficiencies or of disease flares in patients with pre-existent AIIRD.

implementation of the recently developed recommendation for vaccination of patients with Aiird

Finally, the evidence-based recommendations that we developed need to be implemented, so that all patients with AIIRD can take advantage of the available vaccines. Little has been published on implementation of influenza vaccination in these specific patient groups. The available evidence shows increasing coverage when a positive vaccination advise is given by the treating physicians and nurses [37].

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NederlANdse sAmeNvATTiNg

introductie

Eind 18de eeuw verscheen de eerste wetenschappelijke publicatie over vaccina-tie, toen nog “variolatie” genoemd. Al ruim voordien, ca. 400 jaar voor Christus, was het de Grieken opgevallen dat iemand die eenmaal de pokken had gehad, dit vervolgens de rest van zijn leven niet meer zou krijgen. Deze waarneming is in feite de basis voor vaccinatie, waarbij van dit principe gebruik wordt gemaakt. Rond het jaar 1000 na Christus probeerde men in China en India door het toedie-nen van een gedroogde vorm van pus van personen met pokken een besmetting op te wekken die niet fataal was, maar wel bescherming voor pokken bood na-dien. Via de Kaukasus bereikte deze strategie Turkije, waar het gunstige effect van variolatie in het begin van de 18de eeuw werd opgemerkt door de echtge-note van de Britse ambassadeur in Constantinopel, Lady Mary Worthley Montagu. Zij liet haar kinderen varioleren en gaf bekendheid aan variolatie onder de Britse upperclass. Edward Jenner, Brits plattelandsdokter en zelf gevarioleerd in 1750 als 8-jarige jongen, was in 1798 de eerste die publiceerde over zijn experimenten waarin hij koepokken i.p.v. de echte pokken toediende aan o.a. een 8-jarige jongen genaamd James Phipps. Dit bleek effectief om pokken te voorkomen, en tevens veel veiliger dan toediening van echte pokken. Jenner was echter niet de eerste die koepokken gebruikte om pokken te voorkomen. Benjamin Jesty, een veehou-der in North Dorset, had al eerder opgemerkt dat melkmeisjes na het oplopen van koepokken geen pokken meer kregen, ondanks blootstelling aan pokken. Jesty bracht, 22 jaar vóór de experimenten van Jenner, met een breinaald koepokken in in de huid van zijn vrouw en zoons en zij bleken daarna beschermd voor pokken tijdens vele daaropvolgende blootstellingen. Nadat Jenner al uitgebreid was ge-lauwerd als ontdekker van vaccinatie, werd in 1805 Benjamin Jesty alsnog geëerd voor zijn bijdrage aan de ontdekking van het beschermende effect van koepokken tegen pokken. De echtgenote van Jesty liet op zijn grafsteen ingraveren dat hij de eerste was die koepokken toediende ter preventie van pokken. Inmiddels is door vaccinatie pokken uitgeroeid en heeft vaccinatie geleid tot een enorme daling in sterfte aan en ziekte door diverse infectieziekten. Hiertoe behoort ook influenza, waartegen in de jaren ’40 van de vorige eeuw een vaccin werd uitgevonden dat nog steeds de basis vormt voor de huidige influenzavaccins.

de werking van vaccinatie

Om te voorkomen dat een micro-organisme het lichaam binnendringt en ziek maakt, beschikt het lichaam over een afweersysteem. Dit afweersysteem is op-gebouwd uit verschillende componenten, die kunnen worden onderscheiden in

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niet-specifieke en specifieke componenten van de afweer. De niet-specifieke af-weer geeft een algemene bescherming tegen een micro-organismen. Voorbeel-den hiervan zijn de huid die geen bacteriën naar binnen laat of de urinestroom naar buiten toe waardoor bacteriën moeilijk via de plasbuis de blaas kunnen be-reiken. Als deze barrières zijn doorbroken zijn er niet-specifieke afweereiwitten (zoals complement) en niet-specifieke afweercellen (zoals macrofagen en neu-trofiele granulocyten) die bescherming bieden, ongeacht de precieze structuur van het micro-organisme dat probeert binnen te dringen. Hiertoe behoren ook zgn. antigeenpresenterende cellen (APCs), die als stofzuigers de indringende micro-organismen opnemen en vervolgens deze in onderdelen aanbieden aan afweercellen van het specifieke afweersysteem. Het specifieke afweersysteem bestaat uit cellen die antistoffen maken specifiek gericht tegen een bepaald mi-cro-organisme (B-lymfocyten, plasmacellen), afweercellen die geïnfecteerde li-chaamscellen aanvallen (cytotoxische T-cellen, ofwel CD8+ T-cellen) en zgn. CD4+ T-cellen, die hulp bieden aan de B-lymfocyten en cytotoxische T-cellen om hun functie optimaal uit te voeren.

Als het specifieke afweersysteem tegen een bepaald micro-organisme al eens eerder een afweerreactie heeft gemaakt, ontstaan er tevens geheugencellen. Hierdoor kan bij een nieuwe blootstelling aan dit micro-organisme het afweer-systeem sneller optreden door binnen een korte termijn de geheugencellen van het specifieke afweersysteem in actie te laten komen. Verder zullen bij een her-haalde blootstelling kwalitatief betere afweercellen en antistoffen geproduceerd worden dan bij een eerste blootstelling. Op dit geheugen is het principe van vac-cinatie gebaseerd. Door het afweersysteem reeds voordat een bepaald micro-organisme is binnengedrongen kennis te laten maken met (onderdelen van) dit micro-organisme door ermee te vaccineren, kan men het afweersysteem alvast laten oefenen om een specifieke reactie hiertegen te maken. Bij werkelijke bloot-stelling aan het micro-organisme heeft het afweersysteem vervolgens sneller en effectiever de verdediging paraat.

Om infectie met influenzavirus te voorkómen zijn vooral de vorming van antistof-fen door het specifieke afweersysteem tegen eiwitten op het oppervlak van het influenzavirus (hemagglutinine en in mindere mate neuraminidase) van belang. Omdat de oppervlakte-eiwitten van influenza (vrijwel) jaarlijks enigszins veran-deren en met grotere tussenpozen geheel veranderen, dient het influenzavaccin elk jaar aangepast te worden aan deze veranderingen. Naast antistoffen blijken ook cytotoxische T-cellen en CD4+ T-cellen een rol te spelen bij het voorkómen van influenza. De cytotoxische T-cellen en CD4+ T-cellen zijn niet alleen tegen oppervlakte eiwitten van influenza gericht, maar ook tegen interne eiwitten en structuren van influenza. Een vaccin dat goed deze cellen stimuleert, maakt dat

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jaarlijkse aanpassing van het vaccin misschien niet meer nodig is, omdat deze interne eiwitten niet een jaarlijkse verandering ondergaan. Bovendien zou een dergelijk vaccin bescherming kunnen bieden aan personen die niet in staat zijn om antistoffen te produceren, bijvoorbeeld omdat de antistofproducerende cel-len onvoldoende aanwezig zijn door een aangeboren afweerstoornis of door me-dicatie zijn verdwenen.

Uit bovenstaande blijkt dat voor het verkrijgen van het beoogde effect van vac-cinatie, nl. een gerichte afweerreactie oproepen, er een adequaat functionerend afweersysteem moet zijn. Dit brengt met zich mee dat bij personen met een min-der goed functionerend afweersysteem vaccinatie ter preventie van een infectie in veel gevallen minder effectief zal zijn dan bij gezonde personen. Anderzijds zijn het juist personen met een verminderd functionerend afweersysteem die veel baat zouden kunnen hebben bij vaccinatie, omdat zij een verhoogd risico lopen infecties te krijgen en deze infecties vaak ernstiger verlopen dan bij mensen met een normale afweer.

doelstellingen van het onderzoek beschreven in dit proefschrift

De onderzoeken beschreven in dit proefschrift zijn uitgevoerd bij personen met een verminderde afweer, waarbij wij verschillende aspecten van de afweerre-actie voor en na toediening van influenzavaccinatie hebben onderzocht. Het be-treft hierbij verschillende categorieën van afweerstoornis. Ten eerste hebben we onderzoek gedaan naar influenzavaccinatie bij patiënten met een aangebo-ren (primaire) afweerstoornis. In dit proefschrift zijn dat patiënten bij wie het afweersysteem onvoldoende antistoffen produceert, een zgn. hypogammaglo-bulinemie. Door dit tekort aan antistoffen is bij deze patiënten de kans verhoogd op het oplopen van bijholteontstekingen, longontstekingen, maag/darm infecties, maar ook op auto-immuunziekten en sommige vormen van kanker. Bovendien blijkt bij een groot deel van de patiënten met een hypogammaglobulinemie ver-oorzaakt door de aandoening “common variable immunodeficiency” er niet alleen een tekort te zijn aan antistoffen, maar ook van niet goed functionerende afweer-cellen. Deze patiënten wordt geadviseerd zich jaarlijks te laten vaccineren tegen influenza, echter de vraag is hoe effectief dat is bij hun ondermaats functione-rende afweersysteem. Daarom werd dit door ons onderzocht. Ten tweede onder-zochten we de effectiviteit en veiligheid van influenzavaccinatie bij patiënten met een verworven (niet-aangeboren, secundaire) afweerstoornis. In dit proefschrift zijn twee patiëntengroepen bestudeerd met verschillende auto-immuunziekten: systemische lupus erythematodes en reumatoïde artritis. Beide ziektes worden veroorzaakt door een ontregeling van het afweersysteem, waardoor de afweer lichaamseigen cellen aanvalt. De ziekten zelf kunnen leiden tot verminderd func-

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tioneren van de afweer, bijvoorbeeld door een tekort aan (bepaalde soorten van) witte bloedcellen of van complementeiwitten. Ook wordt de afweer verminderd bij deze ziekten door het gebruik van afweeronderdrukkende medicijnen, met als doel om de afweer die zich tegen de eigen organen heeft gekeerd tot rust te brengen. Voorbeelden van deze medicijnen zijn prednison, methotrexaat, TNFα-blokkers en rituximab. Rituximab is een medicament dat leidt tot totale verwij-dering uit het bloed van B-lymfocyten, die essentieel zijn voor de productie van antistoffen. Door het gebruik van afweeronderdrukkende medicatie worden deze patiënten echter ook vatbaarder voor infecties. Daarom wordt ook aan patiënten met een secundaire afweerstoornis geadviseerd om zich jaarlijks te laten vac-cineren tegen influenza, maar ook voor hen geldt dat onzeker is hoe effectief dit is. Bovendien is de veiligheid van vaccinatie van speciaal belang bij patiënten met een auto-immuunziekte, omdat verondersteld wordt dat door stimulatie van het afweersysteem door vaccinaties ook de aanwezige auto-immuunziekte kan verergeren.

De doelstelling van het onderzoek beschreven in dit proefschrift was het ver-krijgen van meer inzicht in de effectiviteit en veiligheid van influenzavaccinatie bij patiënten met een primaire (aangeboren) en secundaire (verworven) afweer-stoornis, en tot het komen van goed onderbouwde aanbevelingen voor vaccinatie van deze patiëntengroepen.

Uitkomsten van de in dit proefschrift beschreven onderzoeken

Deel 1 van dit proefschrift beschrijft het onderzoek dat we uitvoerden bij patiën-ten met een primaire afweerstoornis waarbij er sprake is van een aangeboren tekort aan antistoffen, een zgn. hypogammaglobulinemie. Patiënten met een hy-pogammaglobulinemie blijken onvoldoende in staat te zijn om na een influenza-vaccinatie beschermende antistoffen tegen influenza te vormen (hoofdstuk 2). Ook toonden we aan dat de patiënten die lijden aan een “common variable im-munodefeciency”, een vorm van hypogammaglobulinemie, ook in mindere mate in staat zijn een tegen influenza gerichte afweerreactie door afweercellen als cy-totoxische T-cellen en CD4+ T-cellen op gang te brengen na influenzavaccinatie (hoofdstuk 3). Daarom lijkt het verstandig bij deze patiënten niet te vertrouwen op alleen een influenzavaccinatie ter bescherming van influenza, maar aanvullende maatregelen te nemen: vaccineren van de huisgenoten en verzorgenden van deze patiënten en vroegtijdig behandelen met medicijnen tegen influenza bij een ver-denking op een infectie met influenza.

In deel 2 wordt het onderzoek beschreven naar influenzavaccinatie bij patiënten met een secundaire afweerstoornis t.g.v. auto-immuunziekten en de behande-

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ling met afweeronderdrukkende medicatie. Hoofdstukken 4 en 5 betreffen beide studies naar effect van influenzavaccinatie bij patiënten met systemische lupus erythematodes, waarbij in hoofdstuk 4 zowel antistofproductie tegen influenza en afweer door afweercellen werd onderzocht. De antistofproductie na influen-zavaccinatie bleek lager te zijn in vergelijking met gezonde controlepersonen. Ook de afweerreactie tegen influenza door specifieke afweercellen van het im-muunsysteem bleek zich na vaccinatie minder goed te hebben ontwikkeld, in het bijzonder als de patiënten prednison of azathioprine gebruikten, twee afweeron-derdrukkende medicamenten. Met als doel de antistofproductie tegen influenza na vaccinatie te verbeteren, werd in hoofdstuk 5 in plaats van één maal nu twee maal tegen influenza gevaccineerd. De tweede, zgn. boostervaccinatie, vond vier weken na de eerste vaccinatie plaats. Helaas had dit geen toegevoegde waarde bij de onderzochte patiënten, met uitzondering van de patiënten die het jaar ervoor niet voor influenza waren gevaccineerd. Zij hadden wel extra baat bij een tweede influenzavaccinatie.

Een ander groep patiënten met een secundaire afweerstoornis die we onderzoch-ten waren reumapatiënten die waren behandeld met rituximab (hoofdstukken 6 en 7). Rituximab leidt tot het verdwijnen uit het bloed van B-cellen, die een sleutel-rol spelen bij antistofproductie, maar ook een rol vervullen bij het aanbieden van (onderdelen van) micro-organismen en andere lichaamsvreemde substanties aan CD4+ T-cellen. Daarom werden zowel de antistofproductie als de afweer door af-weercellen bestudeerd bij met rituximab behandelde reumapatiënten (hoofdstuk 6). Er werden twee groepen gevormd: een groep van patiënten die 4-8 weken te-voren met rituximab waren behandeld en geen aantoonbare hoeveelheden B-cel-len meer in hun bloed hadden, en een groep patiënten die 6-10 maanden tevoren met rituximab waren behandeld, het tijdstip waarop doorgaans de B-cellen weer in het bloed terugkeren. Patiënten die met rituximab waren behandeld waren niet in staat een beschermende hoeveelheid antistoffen tegen influenza te maken. Bij het deel van de patiënten die 6-10 maanden tevoren rituximab hadden ontvangen was wel enige stijging van de hoeveelheid antistoffen tegen influenza door de vaccinatie, maar nog altijd onvoldoende om bescherming te bieden tegen influ-enza. Ook de afweercellen bleken bij patiënten met rituximab-behandeling niet zo goed te functioneren tegen influenza als bij gezonde personen (hoofdstuk 7). Bo-vendien bleek dat bij de patiënten die rituximab gebruikt hadden de stimulatie van de afweercellen met een algemene stimulus, waarop de afweercellen doorgaans signaalstoffen gaan produceren, te leiden tot een verminderde afweerreactie ten opzichte van gezonde personen. Hieruit kan afgeleid worden dat het verstandig is reumapatiënten te vaccineren met influenzavaccin een aantal weken voordat de behandeling met rituximab wordt gestart.

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Een vertaling van de onderzoeksresultaten van de bovengenoemde studies (en vele andere) naar de praktijk werd gemaakt in deel 3 van dit proefschrift. Namens de European League Against Rheumatism (EULAR) werden door een team van internationale experts op het gebied van reumatologie, klinische immunologie, nierziekten, infectieziekten, kinder-reumatologie/immunologie en klinische epi-demiologie aanbevelingen opgesteld, gebaseerd op de beschikbare wetenschap-pelijke medische literatuur (hoofdstuk 9). Gezocht werd naar literatuur over alle auto-immuun reumatsche aandoeningen in combinatie met alle in Europa beschik-bare vaccins. Bovendien werd de invloed van afweeronderdrukkende medicatie op de effectiviteit en veiligheid van deze vaccins bij deze patiënten nagegaan. Om te komen tot de 13 aanbevelingen werden acht belangrijke vragen opgesteld t.a.v. vaccinatie bij patiënten met een auto-immuun reumatische aandoening. De antwoorden op deze vragen, voor zover mogelijk gebaseerd op de wetenschap-pelijke bewijzen in de medische literatuur, zijn terug te vinden in hoofdstuk 8.

Conclusies en een blik op de toekomst

De respons op influenzavaccinatie is gestoord bij patiënten met een humorale primaire afweerstoornis, patiënten met systemische lupus erythematodes en pa-tiënten met reumatoïde artritis. Dit geldt zowel voor de productie van antistoffen als voor de afweer door specifieke afweercellen. Daarom zouden bij patiënten met een primaire humorale afweerstoornis huisgenoten en verzorgenden ge-vaccineerd moet worden, zodat de kans verkleind wordt dat influenza door deze personen overgebracht wordt op de patiënt. Ook dienen deze patiënten bij een verdenking op infectie met influenza hiervoor behandeld te worden.

Patiënten met systemische lupus erythematodes dienen influenzavaccinatie te ontvangen, waarbij overwogen kan worden om een boostervaccinatie toe te die-nen bij patiënten die in het jaar ervoor geen influenzavaccinatie hebben ontvan-gen.

Vaccinatie voor influenza lijkt de eerste vier tot acht weken na behandeling met rituximab niet zinvol. Daarom zou een influenzavaccinatie toegediend moeten worden vóór het starten van de behandeling met rituximab. Aangezien voor alle patiënten met reumatoïde artritis jaarlijkse influenzavaccinatie wordt aanbevo-len, en rituximab pas wordt toegediend in de fase dat andere reumabehandelin-gen onvoldoende effect hebben gehad, zou dit gemeengoed moeten zijn.

Onderzoek is nooit klaar, over het algemeen werpt het meer vragen op. In de toekomst zou onderzoek zich moeten richten op het verkrijgen van een beter inzicht in de werkelijke hoeveelheid influenza infecties bij patiënten met een afweerstoornis en ook hoe ernstig die gevallen verlopen. Aangezien influenza-

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vaccinatie minder effectief blijkt te zijn bij de patiëntengroepen beschreven in dit proefschrift, zijn nieuwe vaccinatie strategieën nodig om de effectiviteit van influenzavaccinatie bij deze patiënten te vergroten. De ontwikkeling van nieuwe influenzavaccins die op andere wijze het afweersysteem stimuleren of van an-dere toedieningswijzen, om zo een krachtigere respons op te wekken, is in volle gang. Zeker bij patiënten met auto-immuunziekten is nog meer inzicht nodig in de veiligheid van vaccinatie. De huidige beschikbare onderzoeken betreffen te kleine aantallen patiënten om met voldoende zekerheid te kunnen stellen dat vaccinatie bij patiënten met een auto-immuunziekte veilig is, alhoewel deze onderzoeken wel alle in dezelfde richting wijzen, nl. dat de bijwerkingen niet toegenomen zijn. Tot slot verschijnen er met grote regelmaat nieuwe afweerdrukkende medica-menten op de markt, die gebaseerd zijn op nieuwe werkingsmechanismen. Ken-nis van de effectiviteit en veiligheid van vaccinatie tijdens gebruik van deze nieu-were middelen is uiteraard ook noodzakelijk.

Kortom, wordt vervolgd…

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dANkwoord U hebt het dankwoord gevonden, maar nu is de vraag: staat u erin? De kans is klein, omdat ik vind dat hier alleen diegenen bedankt moeten worden die wer-kelijk een substantiële bijdrage hebben geleverd aan het tot stand komen van dit proefschrift. Hiermee volg ik voorschriften van de Leidse faculteit Sociale Wetenschappen, die stellen dat het dankwoord niet bedoeld is om je dank uit te spreken naar “God, hond en jaarclub”. Overigens hebben deze alle drie geen plaats in mijn leven.Om te beginnen wil ik alle patiënten en gezonde vrijwilligers die mee hebben ge-daan aan de in dit proefschrift beschreven studies ontzettend bedanken voor de tijd en moeite die ze hiervoor hebben genomen. Zeker voor een aantal patiënten was dit geen sinecure, maar hun motivatie was bewonderenswaardig.

Dan de personen die mij begeleid hebben bij het onderzoek: van hen wil ik als eer-ste mijn promotor Cees Kallenberg bedanken. Je goede naam en grote kwaliteiten waren bij mijn vertrek uit Nijmegen al door Jos van der Meer genoemd en er was geen woord onwaar. En naast je enorme deskundigheid als onderzoeker (zie de vele andere dankwoorden in door Cees begeleide proefschriften), waarvan ik heb mogen profiteren, wil ik graag benadrukken dat je ook nog eens (ik zou bijna zeg-gen desondanks) een bijzonder aimabele en bindende persoon bent. We zullen je missen na jouw emeritaat, al hoop ik ook daarna nog eens een djogo met jou en Dieneke te mogen delen.

Marc Bijl, jou ben ik zeker de meeste dank verschuldigd. Een betere copromotor had ik mij niet kunnen wensen. Ondanks goede redenen om eens een keer niet thuis te geven, stond je altijd klaar met altijd weer even terechte en consistente commentaren. Ooit hoop ik net zo goed in onderzoek te worden als jij, een toga zou je niet misstaan! Het Martini ziekenhuis zij gefeliciteerd met jouw keuze voor hen.

Aalzen de Haan, mijn tweede copromotor, hopelijk blijf jij voorlopig wel in het UMCG. Veel dank ben ik je verschuldigd voor je optreden als “routeplanner” bij mijn tocht door de voor mij ondoorgrondelijke jungle genaamd immunologie. “Quick and dirty” kregen door jouw invulling een nieuwe, zelfs positieve betekenis.

Verder wil ik Bert Holvast bedanken. Zeker in mijn positie, als parttime promo-vendus, heb ik enorm kunnen en mogen profiteren van al jouw verkennende werk en je hulp bij mijn studies. Hopelijk kruisen onze wegen elkaar weer in onder-zoeksland na het afronden van jouw opleiding tot kinderarts.

Zonder input en output vanuit het immunologie laboratorium was dit onderzoek niet mogelijk geweest. Hannie Westra en Gerda Horst, jullie hebben zowel een be-

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langrijke inhoudelijke bijdrage geleverd als een enorme hoop werk uit mijn klun-zige handen genomen (het meten van de cel-gemedieerde immuunresponsen). Heel erg bedankt! Van het Laboratorium voor Infectieziekten wil ik René Benne, Gioia Smid en Eline Bloemink bedanken voor hun denk- en handwerk bij de he-magglutinatie remmingstest. Ook alle andere analisten die mij hebben geholpen bedank ik: Johan Bijzet, Berber Doornbos-v/d Meer, Minke Huitema, Marcel v/d Leij en Bessel Schaap, en daarnaast studenten Linda Gorter en Kim van Lieshout.

Veel hulp heb ik gehad bij ditjes en DAS’jes van de stafleden, nurse practitioners, AIOS en administratieve medewerkers van de afdeling reumatologie/klinische immunologie, in het bijzonder van Miek van Leeuwen, Marcel Posthumus, Judith Vierdag, Ina Holwerda, Eefke Eppinga en Diana Nijborg, waarvoor mijn dank.

Mijn mede-auteurs dank ik voor hun inbreng in de studies en manuscripten.

Promotie onderzoek doen tijdens een fulltime baan viel niet (altijd) mee. Gelukkig hebben mijn collega’s van de werkgroep infectieziekten mij regelmatig kunnen ontzien waardoor ik net even een paar extra uurtjes had om tot een afronding te komen. Herman, Piet, Tjip, Jan, Rita, Wouter, Cari, Dorien, Ymkje en Kasper (in volgorde van anciënniteit), bedankt! Ik zal jullie steun ook nog hard nodig hebben voor het binnenhalen van de begeerde opleiding in het aandachtsgebied infectie-ziekten.

Rijk Gans, als afdelingshoofd van de interne geneeskunde wil ik je bedanken voor je vertrouwen dat ik de eindstreep zou halen.

Zelf vind ik dit proefschrift esthetisch ook zeer verantwoord. Alle lof daarvoor komt mijn broer Luite toe. Luite, je bent niet alleen altijd heel creatief en perfec-tionistisch, maar ook uitermate gezellig om langdurig mee achter de MacBook te zitten. We moeten op zoek naar een andere reden om elkaar zo regelmatig te blijven zien, spreken en SMS-en.

Sander en Arto, jullie bereidheid om je op te laten tuigen tot mijn paranimfen leidt tot een heugelijk feit: de hereniging van “Medrieco”, de drie aan de Rusthoflaan in het Rotterdamse Crooswijk wonende medico’s. We zien elkaar te weinig, maar wat telt is de kwaliteit!

Ik sluit mijn dankwoord af met Hilma, Sam en Sophie. Ik zal niet beweren dat het proefschrift zonder jullie er nooit was gekomen, maar dan wel zonder jullie liefde, warmte, steun en kritiek. Ik ga hier ook niet zeggen hoe veel ik van jullie houd. Er zijn vele andere manieren om dat te laten merken en daar ga ik nu maar eens werk van maken!

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Submitted

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nies of gag, pol and env sequence data reveal the direction and time interval of HIV-1 transmis-

sion. AIDS 2011 May;25(8):1035-9

(5) van Assen S, Elkayam O, Agmon-Levin N, Cervera R, Doran MF, Dougados M, Emery P, Geborek

P, Ioannidis JP, Jayne DR, Kallenberg CG, Müller-Ladner U, Shoenfeld Y, Stojanovich L, Valesini

G, Wulffraat NM, Bijl M. Vaccination in adult patients with auto-immune inflammatory rheu-

matic diseases: a systematic literature review for the European League Against Rheumatism

evidence-based recommendations for vaccination in adult patients with auto-immune inflam-

matory rheumatic diseases. Autoimmun Rev 2011 Apr;10(6):341-52

(6) van Assen S, Agmon-Levin N, Elkayam O, Cervera R, Doran MF, Dougados M, Emery P, Geborek

P, Ioannidis JP, Jayne DR, Kallenberg CG, Müller-Ladner U, Shoenfeld Y, Stojanovich L, Valesini

G, Wulffraat NM, Bijl M. EULAR recommendations for vaccination in adult patients with autoim-

mune inflammatory rheumatic diseases. Ann Rheum Dis. 2011 Mar;70(3):414-22

(7) Stek CJ, van Eijk JJ, Jacobs BC, Enting RH, Sprenger HG, van Alfen N, van Assen S. Neuralgic

amyotrophy associated with Bartonella henselae infection. J Neurol Neurosurg Psychiatry 2011

Jun;82(6):707-8

(8) van Assen S, Holvast A, Telgt DS, Benne CA, de Haan A, Westra J, Kallenberg CG, Bijl M. Patients

with humoral primary immunodeficiency do not develop protective anti-influenza antibody ti-

ters after vaccination with trivalent subunit influenza vaccine. Clin Immunol 2010 Aug;136(2):228-

35

(9) Alffenaar JW, van Assen S, de Monchy JG, Uges DR, Kosterink JG, van der Werf TS. Intravenous

voriconazole after toxic oral administration. Antimicrob Agents Chemother 2010 Jun;54(6):2741-

2

(10) van Assen S, Holvast A, Benne CA, Posthumus MD, van Leeuwen MA, Voskuyl AE, Blom M, Risse-

lada AP, de Haan A, Westra J, Kallenberg CG, Bijl M. Humoral responses after influenza vaccina-

tion are severely reduced in patients with rheumatoid arthritis treated with rituximab. Arthritis

Rheum 2010 Jan;62(1):75-81

(11) Hagen F, van Assen S, Luijckx GJ, Boekhout T, Kampinga GA. Activated dormant Cryptococcus

gattii infection in a Dutch tourist who visited Vancouver Island (Canada): a molecular epidemio-

logical approach. Med Mycol 2010 May;48(3):528-31

228

Cha

pter

11

(12) Holvast A, van Assen S, de Haan A, Huckriede A, Benne CA, Westra J, Palache A, Wilschut J, Kal-

lenberg CG, Bijl M. Effect of a second, booster, influenza vaccination on antibody responses in

quiescent systemic lupus erythematosus: an open, prospective, controlled study. Rheumatology

(Oxford) 2009 Oct;48(10):1294-9

(13) Holvast A, van Assen S, de Haan A, Huckriede A, Benne CA, Westra J, Palache A, Wilschut J,

Kallenberg CG, Bijl M. Studies of cell-mediated immune responses to influenza vaccination in

systemic lupus erythematosus. Arthritis Rheum 2009 Aug;60(8):2438-47

(14) Holvast A, de Haan A, van Assen S, Stegeman CA, Huitema MG, Huckriede A, Benne CA, Westra

J, Palache A, Wilschut J, Kallenberg CG, Bijl M. Cell-mediated immune responses to influenza

vaccination in Wegener’s granulomatosis. Ann Rheum Dis 2010 May;69(5):924-7

(15) Alffenaar JW, van Assen S, van der Werf TS, Kosterink JG, Uges DR. Omeprazole significantly

reduces posaconazole serum trough level. Clin Infect Dis 2009 Mar 15;48(6):839

(16) Rachinger A, Navis M, van Assen S, Groeneveld PH, Schuitemaker H. Recovery of viremic con-

trol after superinfection with pathogenic HIV type 1 in a long-term elite controller of HIV type 1

infection. Clin Infect Dis 2008 Dec 1;47(11):e86-9

(17) Huits RM, Bremmer R, Enting RH, Sprenger HG, van Assen S. Return of meningeal symptoms in

a patient treated for cryptococcal meningitis. J Neurol 2007 Oct;254(10):1443-4

(18) Meeuse JJ, Sprenger HG, van Assen S, Leduc D, Daenen SM, Arends JP, van der Werf TS. Rho-

dococcus equi infection after alemtuzumab therapy for T-cell prolymphocytic leukemia. Emerg

Infect Dis 2007 Dec;13(12):1942-3

(19) van Assen S, Houwerzijl EJ, van den Dungen JJ, Koopmans KP. Vascular graft infection due to

chronic Q fever diagnosed with fusion positron emission tomography/computed tomography.

J Vasc Surg 2007 Aug;46(2):372

(20) de Vries JJ, van Assen S, Mulder AB, Kampinga GA. Positive blood culture with Plasmodium

falciparum: case report. Am J Trop Med Hyg 2007 Jun;76(6):1098-9

(21) Huits RM, van Assen S, Wildeboer-Veloo AC, Verschuuren EA, Koeter GH. Prevotella bivia necro-

bacillosis following infectious mononucleosis. J Infect 2006 Aug;53(2):e59-63

(22) van Assen S, Bakker SJ. Did syphilis truly strike the kidneys this time? Nephrol Dial Transplant

2005 Jun;20(6):1029-31

(23) van Assen S, Bosma F, Staals LM, Kullberg BJ, Melchers WJ, Lammens M, Kornips FH, Vos PE,

Fikkers BG. Acute disseminated encephalomyelitis associated with Borrelia burgdorferi. J Neu-

rol 2004 May;251(5):626-9

(24) Van Damme PA, Keuter M, Van Assen S, DeWilde PC, Beckers PJ. A rare case of oral leishmani-

asis. Lancet Infect Dis 2004 Jan;4(1):53

(25) van Assen S, Bootsma GP, Verweij PE, Donnelly JP, Raemakers JM. Aspergillus tracheobronchi-

tis after allogeneic bone marrow transplantation. Bone Marrow Transplant 2000 Nov;26(10):1131-

2

(26) van Roijen JH, van Assen S, van der Kwast TH, de Rooij DG, Boersma WJ, Vreeburg JT, Weber

RF. Androgen receptor immunoexpression in the testes of subfertile men. J Androl 1995 Nov-

Dec;16(6):510-6

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