Doctoral thesis from the Department of Immunology, the Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

Mucosal immunity against mycobacterial infection

Muhammad Jubayer Rahman

Stockholm 2010

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All previously published papers were reproduced with permission from the publishers Printed in Sweden by Universitetsservice AB, Stockholm 2010 Distributor: Stockholm University Library © Muhammad Jubayer Rahman, Stockholm 2010 ISBN 978-91-7447-081-9

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"It is a good morning exercise for a research scientist to discard a pet hypothesis every day before breakfast - it keeps him/her young."

Konrad Lorenz (1903 - 1989) 1973 Nobel Laureate in Medicine

TO MY FAMILY

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Summary

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB.

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LIST OF ARTICLES

This thesis is based on the following original articles (manuscripts), which will be referred to

by their Roman numerals:

I. Arko-Mensah J*, Rahman MJ*, Julián E, Horner G, Singh M, Fernández C. Increased

levels of immunological markers in the respiratory tract but not in serum correlate

with active pulmonary mycobacterial infection in mice. Clin Microbiol Infect.

2009;15(8):777-86.

*Authors contributed equally to this work

II. Rahman MJ and Fernández C. Neonatal vaccination with Mycobacterium bovis

BCG: potential effects as a priming agent shown in a heterologous prime-boost

immunization protocol. Vaccine. 2009;27(30):4038-46.

III. Rahman MJ, Dégano IR, Singh M and Fernández C. Influence of maternal

gestational treatment with mycobacterial antigens on postnatal immunity in an

experimental murine model. PLoS One. 2010;5(3):e9699.

IV. Rahman MJ, Chuquimia OD, Singh M and Fernández C. Immunization with

mycobacterial antigens: a role for innate immunity in antigen presentation.

(Preliminary manuscript)

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LIST OF ARTICLES (not included in this thesis)

The following original articles are relevant but not included in this thesis. The articles will be

cited by their Roman numerals:

V. Arko-Mensah J*, Rahman MJ*, Dégano IR, Chuquimia OD, Fotio AL, Garcia I,

Fernández C. Resistance to mycobacterial infection: a pattern of early immune

responses leads to a better control of pulmonary infection in C57BL/6 compared with

BALB/c mice. Vaccine. 2009;27(52):7418-27.

*Authors contributed equally to this work

VI. Olleros ML, Vesin D, Martinez-Soria E, Allenbach C, Tacchini-Cottier F, Pache JC,

Marchal G, Rahman J, Fernández C, Izui S, Garcia I. Interleukin-12p40

overexpression promotes interleukin-12p70 and interleukin-23 formation but does not

affect bacille Calmette-Guérin and Mycobacterium tuberculosis clearance.

Immunology. 2007;122(3):350-61.

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CONTENTS ................................................................................ Page Introduction-------------------------------------------------------------------------10 Tuberculosis (TB)- a global health problem………………………………………10 Tuberculosis- clinical forms……………………………………………………… 10 Mycobacterium tuberculosis - the etiologic agent of TB………………………… 11 Host-pathogen interactions ------------------------------------------------------------------ 12 Mycobacterial survival strategies………………………………………………… 12 Adaptation of macrophages to infection …………………………………………. 13 Susceptibility to infection………………………………………………………… 14 Immunology of TB------------------------------------------------------------------------------- 15 Innate immunity --------------------------------------------------------------------------- 16 Mycobacterial interactions with innate receptors………………………. 16 Macrophages……………………………………………………………. 19 Dendritic cells…………………………………………………………... 19 Epithelial cells…………………………………………………………...20 Natural killer (NK) cells ……………………………………………….. 21 Adaptive Immunity------------------------------------------------------------------------ 22 Cell-mediated immunity to mycobacteria………………………………. 22 CD4 T cells………………………………………………………………22

CD8 T cells………………………………………………………………23 Th17 cells……………………………………………………………….. 24 Gamma delta (γδ) T cells……………………………………………….. 25 Regulatory T cells (Treg)………………………………………………… 25

Humoral immunity……………………………………………………… 26 Cytokines and Chemokines…………………………………………………….. 28 Mucosal immunity in the respiratory tract……………………………………. 31 Vaccination against TB------------------------------------------------------------ 33 The BCG vaccine-------------------------------------------------------------------------- 33 Neonatal immunity and maternal-fetal interactions--------------------------------35 Failure of BCG and progress in new TB vaccine development-------------------37

Subunit vaccines…………………………………………………………38 DNA vaccines……………………………………………………………39 Auxotrophic vaccines…………………………………………………… 40

Recombinant (r) BCG……………………………………………………40 Combination vaccines (prime-boost)…………………………………… 41 Immune correlates of protection (multifunctional T cells)------------------------ 42 Route of vaccination (mucosal versus systemic)------------------------------------- 43 Diagnosis of TB--------------------------------------------------------------------- 46 Classical methods-------------------------------------------------------------------------- 46 Microscopy and culture…………………………………………………. 46 Tuberculin skin test…………………………………………………….. 47 Radiology or chest X-ray……………………………………………….. 47 New methods-------------------------------------------------------------------------------- 47 BACTEC radiometric system……………………………………………47 Microscopic-observation drug-susceptibility (MODS) assay………….. 48 Nucleic acid amplification assay……………………………………….. 48 Immune-based tests…………………………………………………….. 48

Biomarker (s) of infection………………………………………………. 51 The present study------------------------------------------------------------------- 52 Aims------------------------------------------------------------------------------------------ 52 Methodology-------------------------------------------------------------------------------- 53 Results and discussion-------------------------------------------------------------------- 54 Paper I....................................................................................................... 54 Paper II…………………………………………………………………. 58 Paper III………………………………………………………………… 62 Paper IV………………………………………………………………… 65 Concluding remarks and future perspectives-------------------------------- 68

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Acknowledgements----------------------------------------------------------------- 70 References---------------------------------------------------------------------------- 72

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List of abbreviations AFB Acid fast bacilli Ag Antigen APC Antigen presenting cell BAL Broncho-alveolar lavage BALT Bronchus-associated lymphoid tissue BCG Bacillus Calmette-Guérin BCGBMM BCG infected bone-marrow derived macrophages BCGAg antigen pulsed bone-marrow derived macrophages CFP-10 10-kDa culture filtrate protein CR Complement receptor CT Cholera toxin CTL Cytotoxic T lymphocyte DC Dendritic cell DC-SIGN Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ELISA Enzyme-linked immunosorbent assay ESAT-6 6-kDa early secretory antigenic target HIV Human immunodeficiency virus i.n. Intranasal IP-10 Interferon gamma-induced protein i.v. Intravenous IFN-γ Interferon gamma IL Interleukin iNOS Inducible nitric oxide synthase kDa kiloDalton LALT Larynx-associated lymphoid tissue LAM Lipoarabinomannan MALT Mucosa-associated lymphoid tissue MDR Multidrug-resistant MHC Major histocompatibility complex NALT Nose-associated lymphoid tissue nHBHA Heparin-binding hemagglutinin native NO Nitric oxide NOD Nucleotide oligomerization domain PCR Polymerase chain reaction PI3P phosphatidylinositol 3-phosphate PknG protein kinase G PPD Purified protein derivative PRR Pattern recognition receptor rBCG Recombinant Bacillus Calmette-Guérin RD Region of deletion rHBHA Heparin-binding hemagglutinin recombinant sTNFR Soluble tumor necrosis factor receptor TB Tuberculosis TCR T cell receptor TLRs Toll-like receptors TNF Tumor necrosis factor WT Wild-type XDR Extensively drug resistant

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Introduction

Tuberculosis- a global health problem: Tuberculosis (TB) is one of the leading infectious

diseases in humans, caused by one of the most devious pathogens, Mycobacterium

tuberculosis (M. tuberculosis). It is estimated that one third of the world’s population is

infected with M. tuberculosis. Estimates from the World Health Organization indicate that 8-9

million new cases are reported every year (1). Even only 5-10% of the infected individuals

progress to develop active TB, an annual instance of death due to TB is two million (2).

Despite the fact that TB itself has been a threat for the world population, co-infection with

human immunodeficiency virus (HIV) has further increased the risk of TB disease

progression and deaths. Multidrug-resistant (MDR) TB has already been a problem in TB-

history and recently, extensively drug-resistant (XDR) TB has added more risk in the control

of TB. At least 45 countries around the world have been identified with positive cases of

XDR-TB (3). On the other side of the picture, several effective regimes have been developed

over the past century to combat TB-pathogens including the TB-drugs and Mycobacterium

bovis Calmitte Guerin (BCG) vaccine, which are likely the key components. Unfortunately,

the control of TB is still a challenge, which simply translates into the urgent need of effective

regimes available.

Generally it is considered that vaccines are the most cost-efficient measures for the

control of diseases if they work successfully. The BCG vaccine is the only recommended

vaccine for humans against TB, developed in 1921, and found to be successful against only

miliary TB in neonates and toddlers at a very low cost (4). Unfortunately, the BCG vaccine

has been proven to be very inconsistent in protection against the most common form of adult

pulmonary TB (5, 6).

Tuberculosis- clinical forms: Lungs are the first habitat for the M. tuberculosis after being

inhaled by a person. M. tuberculosis has a particular tropism for the lungs. Several possible

outcomes can be seen when a person first encounters M. tuberculosis. First, the organisms can

be immediately destroyed by the host innate immune barriers and the host remains uninfected.

Second, people may contain TB germs in their body for their lifetime without showing

clinical symptoms, which is called latent infection. Finally, only approximately 10% of the

latently infected individuals may develop active TB with common clinical symptoms, i.e.

chronic cough, fever, night sweats and weight loss.

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There are two different types of TB in humans in general: pulmonary and extra-

pulmonary. Pulmonary TB is the most common form of TB that develops in the lungs.

Manifestations of disease varied significantly from young children to adolescence to adults.

Apparently, young children do not show classical symptoms of TB and that lead to difficulties

in diagnosis and treatment. On the other hand, adult individuals show more clear symptoms in

advanced pulmonary TB, which can primarily be diagnosed by X-ray radiography.

Enlargement of mediastinal lymph nodes, bronchial obstruction may cause air trapping,

hyperinflation, and even emphysema. A complete bronchial obstruction can be visualized by

the typical radiographic shadows.

Extrapulmonary TB develops in organs other than the lungs. Disseminated forms of

TB, a type of extrapulmonary TB, are more common in young children than adults.

Mycobacteria can be released into the blood stream or lymphatics from the primary infected

site (lungs) and may cause disseminated TB in any parts of the body. Children, elderly people

and patients with HIV infection are at greater risk of progressing disease more promptly due

to their suboptimal immune function. The most common forms of disseminated TB are

observed in kidney, bones, cervical lymph nodes, skin, stomach and meninges. Children at 2-

6 months of age contract meningeal TB, which affect brain and central nervous system and

can be fatal if leave untreated.

M. tuberculosis - the etiologic agent of human TB: M. tuberculosis is a bacterial species,

which belongs to the genus of Mycobacterium, family of Mycobacteriaceae and order of

Actinomycetaceae. Robert Koch in 1882 first discovered M. tuberculosis and with further

characterisation of this organism led to the understanding of causation of TB, which was later

named Koch’s postulates in 1890.

Mycobacteria are slow-growing, divide every 15-20 hours, aerobic, non-motile, non-

sporulated rods. Most of the mycobacteria live and propagate in natural habitats such as water

or soil and rarely cause disease. Only a few of them cause disease in mammals and are known

as intracellular pathogens. M. tuberculosis, M. bovis, M. africanum, and M. microti are

collectively called M. tuberculosis complex. All the members of M. tuberculosis complex are

pathogenic. M. tuberculosis and M. africanum are pathogenic for humans and M. bovis is

usually pathogenic for animals but also can be transmitted to humans. M. microti is a

pathogen of voles but is avirulent in humans and mice. The cell wall structure of

mycobacteria is very different from other fast-growing bacteria. The complex mycobacterial

cell wall is structured differently, consisting of unusual amounts of lipid moieties i.e.

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peptidoglycolipids (mycosides), cord factors and sulpholipids. M. tuberculosis complex

organisms can be stained with carbolfuchsin and appeared to be rod-like shape when

visualized under a microscope. Using acid or alcohol for decolourisation, mycobacteria could

retain the colour and hence, they are also called ‘acid-fast bacilli’.

Host-pathogen interactions Airways infection with M. tuberculosis leads to sampling of organisms by the primary host

cells such as alveolar macrophages. As a consequence, bacteria become arrested inside of a

phagosome and later being delivered to the lysosome by phago-lysosome fusion in order to be

killed by the host cell. M. tuberculosis could interfere with the host killing machineries and

establish their fate inside the host either following a state of ‘dormancy’ or causing active

disease (7).

Mycobacterial survival strategies

There are several strategies that have been described in relation with mycobacterial survival

within a host. Intrinsically, mycobacterium could withstand the host killing machineries due

to the unique features of the mycobacterial cell wall. Depletion of the cell wall components

reduces bacterial virulence activity, suggesting the importance of the cell wall integrity

towards survival of mycobacteria inside the host (8).

Formation of phosphatidylinositol 3-phosphate (PI3P) is important in regulating a

normal host cell trafficking event. PI3P functions as a docking site for proteins, which is

required for the maturation of phagosomes into lysosomes. (9, 10). Mycobacteria could inhibit

the process of accumulation of PI3P on phagosomal membrane and thereby block

phagosome-lysosome fusion. It was postulated that M. tuberculosis toxin lipoarabinomannan

(LAM) could block the cytosolic increase of Ca2+ and inhibit a novel Ca2+/Calmodulin-PI3K

hVPS34 cascade, which is essential for the synthesis of PI3P on phagosomes (11). As shown

in Fig. 1, once inside the phagosome, M. tuberculosis secretes virulence factors such as SapM

(an eukaryotic-like acid phosphatase) and serine/threonine kinase PknG to inhibit phago-

lysosome fusion (7). It is thought that SapM may hydrolyse PI3P and PknG may

phosphorylate host molecules, thereby preventing the formation of phago-lysosome formation

(7). Host tryptophan aspartate containing coat protein (TACO), also called P57, recruitment

enhanced when phagosomes harbour live mycobacteria but the TACO protein levels dropped

when phagosomes contain killed bacteria, suggesting that TACO is another component that

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interferes with the lysosomal delivery (12, 13). Active retention of TACO leads to the

activation of calcium-dependent phosphates calcineurin, which is being considered as a

critical factor for blocking lysosomal delivery although the precise mechanism is still

unknown.

Adaptation of macrophages to infection

In most infected individuals, who do not develop active TB, a delicate balance is established

between the host immune response and the M. tuberculosis virulence, which is termed

‘granuloma formation’ (14, 15). The structure of granulomas is a cluster of M. tuberculosis

living inside macrophages surrounded by other cells. Within this granuloma, mycobacteria are

kept in check so that they are not able to cause disease. The exact biology of granuloma

formation is still not completely understood, however, it is believed that mycobacteria in such

a condition can be actively dividing or be silent even within a same individual (16, 17). Under

appropriate activation, macrophages process antigens and present them to T lymphocytes.

Activated T cells produce cytokines and chemokines and that result in further activation of

macrophages or an influx of other immune cells to the site of granuloma. A discontinuation of

Fig. 1: Macrophage antimicrobial activity and escape mechanisms for the survival of M. tuberculosis. Once inside the host cell, M. tuberculosis secretes SamP and PknG to be able to inhibit the phagosome-lysosome fusion. Accumulation of TACO protein around the phagosome interferes with the phagosome-lysosome fusion. LAMP1 and V-ATPase are lysosomal proteins. The Toll-like receptor (TLR) signaling can modulate phagosome-lysomal fusion through p38MAPK pathway and also TLR signaling can increase the production of antimicrobial peptide LL-37 via the upregulation of vitamin D receptor. (Cell Host Microbe. 2008,3:399-407, reprinted with permission from Elsevier Inc.)

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this check-in process due to any suboptimal host response contributes to M. tuberculosis

release and dissemination to other organs, which can be ended up with active TB (18).

Susceptibility to infection

Many people exposed to M. tuberculosis bacilli do not contract infection. Also, the infection

with M. tuberculosis in humans poses only a 10% lifetime risk of developing active TB. Since

the vast majority of the infected people do not develop active TB, this perhaps indicates that

the large interindividual variability in the induction of immune responses and/or the genetic

predisposition are probably associated in part with the variable outcome of infection.

Previous studies have gained insights into this complex phenomenon and unveiled many

environmental factors such as first-contact epidemics that increase the susceptibility (19),

poor economy, malnutrition, stress, overcrowding, which all enhance the susceptibility to TB

in humans (20). Apart from all those factors, there have been many studies performed in

humans (21, 22) or animals (23-26) revealing that host genetic factors play a significant role

in the outcome of the M. tuberculosis infection. Searching for genetic components of

susceptibility to M. tuberculosis infection in humans has been a difficult task. Recently,

forward genetic approaches in mice and humans have been used to explain the molecular

basis for predisposition to mycobacterial diseases (27).

Genome wide screening of both resistant and susceptible mouse strains with M.

tuberculosis infection identified 18 genes in resistant and 120 genes in susceptible strains that

are regulated selectively (28). Further characterization of some of those genes revealed that

macrophages from susceptible strains induced more inflammatory responses and caused tissue

damage (28). A similar study has also found a group of genes responsible for tissue fibrosis

showing very high levels of changes in gene transcripts in the susceptible mouse strains (29).

Structure of the lung pathology has been found to be different in the resistant mouse strains

compared to the susceptible strains, where a large aggregate of lymphocytes was found close

to the granulomas. Only a few lymphocytes with other immune cells were present

surrounding the granulomas in the susceptible strains suggesting that a generalized defect in

the presence of lymphocytes might contribute to the susceptibility of infection (30).

Thus far, a vast majority of the work in humans focused on some of the candidate

genes and their association with TB susceptibility. The natural resistance-associated

macrophage protein gene 1 (NRAMP1), a homologue of a gene (Nramp 1) on mouse

chromosome 1 has been reported to be very critical in controlling TB. Polymorphisms in

NRAMP1 have been demonstrated to be a risk factor for adult (31) and paediatric TB (32). A

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metaanalysis of studies with NRAMP1 polymorphisms revealed that polymorphisms were

associated with pulmonary TB in African and Asian populations but not in populations of

European descent (33). Conflicting results have been obtained from recent studies on SP110

variants (a nuclear body protein) and TB susceptibility in patients from West Africa (27). An

association between MHC genes, for example, alleles encoding an aspartic acid at codon 57

of HLA-DQ β-chain and pulmonary TB was reported. HLA-DQ β57-Asp showed reduced

ability to bind with a peptide from early secreted antigenic target 6 (ESAT-6) protein (34).

Polymorphisms in the gene encoding DC-SIGN (dendritic cell-specific intercellular adhesion

molecule-3 (ICAMP-3)-grabbing non-integrin) have also been reported to be associated with

the adult pulmonary TB in a South African population (35).

The susceptibility to mycobacterial infection can also be assessed by ‘genetic

mutation’- that targets one or more genes of interest and results in malfunction of proteins

after expression. In fact, genetic mutations have been recorded in many infected individuals,

which even can transmit from person to person followed by Mendelian inheritance. Genes

that were identified to be responsible for the susceptibility to mycobacterial infection centre

around interleukin 12 (IL-12) and interferon gamma (IFN-γ) axis include IFN-γR1 and IFN-

γR2, two chains of IFN-γ receptor; IL-12β, encoding the p40 subunit of IL-12; IL-12 βR1, the

β1 subunit of the IL-12 receptor; and signal transducer and activator of transcription-1

(STAT-1) (36). Mutations in IFN-γR2 and IL-12p40 have been found to be associated with

the susceptibility to M. tuberculosis infection but most of the others are demonstrated in

relation with M. bovis or environmental mycobacterial infection (36).

Immunology of TB In normal circumstances, exposure to foreign molecules turns on a series of defence reactions

in the body in order to develop an effective protection. The immune system is classified into

two distinct branches based on the nature of their responses; one called innate immune

system, non-specific and another called adaptive immune system, specific. M. tuberculosis

infection can induce both innate and adaptive immune responses in humans as well as in

experimental animal models. Since the 90% of the exposed individuals do not develop active

TB, this observation suggests that the immune system plays pivotal roles in controlling

disease.

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Innate immunity

The innate immunity is the first-line of defence against pathogens that comes quick in a non-

specific manner and has no recall power. The innate immune system is activated upon

recognition of microbial components by a number of receptors on host cells. For the detection

of M. tuberculosis, macrophages use several receptors, which include mannose receptor

(MR), complement receptor (CR), class A scavenger receptor, dectin 1 (C-type lectin), DC-

SIGN, TLRs and the nucleotide oligomerization domain (NOD)-like receptors (Fig. 2).

Mycobacterial interactions with innate receptors

Mannose receptors interact with the mannose-capped lipoarabinomannan (ManLAM) on M.

tuberculosis that facilitate attachment and internalization of bacilli by macrophages. M.

tuberculosis ManLAM blocks phagosome-lysosome fusion and thereby enhances survival of

M. tuberculosis in human macrophages.

Complement receptors promote phagocytosis of M. tuberculosis by macrophages.

Activation of the alternative complement pathway by M. tuberculosis promotes opsonisation

mediated by the complement components C3b and iC3b (37). This allows the recognition of

bacilli by CR1, CR3 and CR4. In the absence of CR1, patients with TB disease have increased

levels of immune complexes that enhance the severity of the disease (38). However, studies in

murine models of TB showed that CR3 deficiency did not have any significant effect on

phagocytosis or alteration of the course of the disease (39). Alternatively, in the absence of

CR3, M. tuberculosis could gain entry into the host cells by other phagocytic receptors and

establish infection (39).

DC-SIGN is a C-type lectin, initially found on the dendritic cells and later it was also

found on alveolar macrophages (40). DC-SIGN interacts with LAM, lipomannan, and

arabinomannan antigens of M. tuberculosis (41-43). DC-SIGN, one particular pattern

recognition receptor (PRR), can induce immune responses by modulating TLR-induced

activation at the level of the transcription factor NF-kappaB (44). It has been described that

DC-SIGN not only could interact with M. tuberculosis but also with other pathogens e.g. M.

leprae, Candida albicans, measles virus, and HIV-1 and activate the NF-kappaB signalling

pathway. Upon interaction with M. tuberculosis DC-SIGN on DC triggers a cascade of

signalling pathway including activation of serine and threonine kinase Raf-1, which

subsequently promotes acetylation of the NF-kappaB subunit p65 provided that NF-kappaB is

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also activated by TLR-induced signalling (44). Acetylation of NF-kappaB leads to increased

and sustained levels of IL-10 to enhance anti-inflammatory responses.

TLRs are very important in the course of interaction with mycobacterial components.

TLRs are a type of PRRs that interact with microbes/pathogen associated molecular patterns

and that subsequently help in phagocytosis and induction of immune responses. Ten members

of TLR family have been identified in humans (45). Different components of microbes are

recognized by different TLRs, for example, lipopeptides interact with TLR2, which forms

dimer with TLR1 or TLR6; lipopolysaccharide is recognized by TLR4; flagellin by TLR5 and

CpG DNA by TLR9. In TB, a mycobacterial component particularly lipoprotein is recognized

by TLR2 and the role of TLR2 has been described as central in many cases (46, 47). TLR1/6,

9 and also TLR4 have been found to interact with mycobacterial components (48, 49). The

19kDa lipoprotein, a secreted antigen of M. tuberculosis, soluble TB factor, protein-free

Fig. 2: Innate receptors and ligands for recognition of M. tuberculosis (Immunol Rev. 2007 ;219:167-86, reprinted with permission from John Wiley & Sons Inc.).

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short-term culture filtrate of M. tuberculosis signal through TLR2 and similarly,

mycobacterial cell wall components such as LAM, lipomannan, phosphatidyl-myo-inositol

(PIM) interact with TLR2 (49).

CD14, a coreceptor of TLR4, is present on the macrophages, dendritic cells, or

neutrophils. CD14 has no cytoplasmic signalling domain. CD14 has been found to interact

with mycobacterial AraLAM and activated cells in a TLR2-dependent manner (50). Bone-

marrow derived macrophages from TLR2-/- and TLR4-/- infected with live BCG have

confirmed the involvement of TLR2 signalling and to a lesser extent TLR4 signalling for the

production of tumor necrosis factor (TNF) and IL-12 (51).

TLR signalling is also very important in the vitamin D activation pathway. Vitamin D

could induce antimicrobial activity against M. tuberculosis as has been suggested by Rook et

al in 1986 (52). Recently, it has been shown that activation of TLR2/1 augments vitamin D

receptor and 25-hydroxyvitaminD3-1α-hydroxylase, which are important for the conversion

of the pro-form of vitamin D to an active form. The activation of vitamin D pathway in a

TLR-dependent manner leads to the synthesis of antimicrobial peptides for example,

cathelicidin in humans (53). Low levels of vitamin D and the risk of developing TB have been

demonstrated in human studies (54).

The nucleotide oligomerization domain (NOD)-like receptor (NLR) also interacts with

the mycobacterial components. NLRs are cytoplasmic proteins that belong to a TLR-related

protein family, which have a C-terminal leucine-rich domain, central nucleotide-binding

domain and N-terminal protein-binding domain (36). There are two types of NLRs; NOD1

and NOD2 that could interact with peptidoglycans, muramyl dipeptides and diaminopimelate-

containing N-acetylglucoseamine-N-acetyl muramic acid tripeptide. Mycobacterial muramyl

dipeptide interacts with NOD2 and induces cytokine production, this synergizes with the

19kDa-mediated activation of TLR2 and cytokine production (55). Ferwerda et al suggested

that defective expression of NOD2 results in 80% reduction of the cytokine production by

mononuclear cells after stimulation with M. tuberculosis and the lack of function of either of

TLR2 or NOD2 causes the loss of synergism (55). Therefore, it has been proposed that NOD2

pathway is an independent and nonredundant in the recognition of M. tuberculosis (55).

Activation of NLRs causes downstream signalling via two pathways: activation of caspase-1

and the NF-κB pathway, which ends up with the production of α-4 defensins or cryptdins. α-4

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defensins or cryptdins have bactericidal activity against M. tuberculosis (56). Despite the fact

that polymorphism of NOD is linked to several inflammatory diseases, and NOD deficiency

has in vitro effects on M. tuberculosis recognition, animals are not susceptible to M.

tuberculosis infection due to the lack of NOD2 (57).

Innate cells

Macrophages

Macrophages are known as the primary habitat for mycobacteria. Monocytes become

activated and differentiated into macrophages. Macrophages are one of the most important

professional antigen-presenting cells (APCs), which can phagocytose, process and present

antigens to the T lymphocytes in an association with major histocompatibility complex

(MHC) molecules. One hundred years ago Metchnikoff, who received the Nobel Prize in

1908, discovered that macrophages are able to phagocytose and have a potential role in the

host-defense mechanism. It is proposed that alveolar macrophages engulf mycobacteria after

entering the host via the nasal route and subsequent activation of macrophages attracts more

macrophages from the bloodstream. Macrophages harbour many receptors on their cell

surface and inside which are important for the interaction with M. tuberculosis. Depending on

the type of receptor-ligand interaction, macrophages generate different types of immune

responses. In vitro experiments have shown that pretreatment of bacilli with immune sera

enhances attachment and phagocytosis of bacilli and also accelerates phagosome-lysosome

fusion (58). In contrast, CR3-mediated nonopsonic internalization of pathogenic mycobacteria

(M. kansasii) does not trigger the formation of oxygen intermediates (59) and blocks the

maturation of phagosomes, and prevents phagosome-lysosome fusion (60). TLRs mediated

sensitization of macrophages promotes activation of the NFκB signaling pathway and

enhances IL-12, TNF and NO synthesis. These mediators stimulate the microbicidal pathway

to kill the ingested mycobacteria.

Dendritic cells

DCs are considered as the frontline sentinels of the body defence system. Like the

macrophages, DCs are also known as professional APCs as they can engulf, process and

present antigens on the surface of other cells with the help of MHC molecules. Recognition of

mycobacterial components by DCs occurs by interaction with C-type lectin receptors, DC-

SIGN, and TLR receptors. Interestingly, phagocytosis of M. tuberculosis by interacting

ManLAM with DC-SIGN is not associated with the inhibition of phago-lysosome fusion as

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suggested by Kang et al, which is in contrast to the observation when macrophages engulf

mycobacteria by engaging the MR with M. tuberculosis ManLAM (61). TLR9 in DCs could

recognize mycobacterial DNA released from the ingested bacteria and promote IL-12

secretion, which is not the case for macrophages infected with M. tuberculosis (62). M.

tuberculosis infection causes rapid remodelling at the IL-12p40 promoter and thereby

increases IL-12p40 transcription only in the DCs in a TLR9 dependent manner. However, in

the macrophages this occurs in a TLR2-dependent manner (62). The critical role of DCs in M.

tuberculosis infection is probably the initiation of the immune responses (63). DCs sample

mycobacteria/secreted antigens in the lungs and carry them to the regional draining lymph

nodes and that is essential for the initiation of immune responses (64, 65). However, it is not

proven whether the activation of T cells in the draining lymph node occurred by the direct

interaction with the lung-derived bacteria-infected DCs.

Epithelial cells

Epithelial cells form a single layer lining over the alveolar lumen and are thought to be the

first cells that M. tuberculosis adheres to during invasion into the host tissues. There are two

major types of cells that are important for the maintenance of alveolar epithelium; a thin,

squamous, type I cells that cover 95% of the epithelium and cuboidal type II cells. Earlier

studies have shown that mycobacteria could infect and multiply inside the type II alveolar

cells (66). Identification and characterization of heparin-binding hemagglutinin adhesin

(HBHA) from M. tuberculosis and M. bovis have revealed that mycobacteria use HBHA to

adhere on the surface of the epithelial cells by interaction with sulphated glycoconjugates

(67). It remains to be unveiled if epithelial cells have any role in the induction of immune

responses. Saiga et al demonstrated that Lipocalin 2 (Lcn2), also known as neutrophil

gelatinase-associated lipocalin, produced by epithelial cells and macrophages during the early

phase of mycobacterial respiratory infection, is important for the host defence against M.

tuberculosis (68). Lcn2-deficient mice are susceptible to intratracheal route of M. tuberculosis

infection. Lcn2 seize iron and therefore inhibits the mycobacterial growth in the epithelial

cells but not in alveolar macrophages (68). Debbabi et al have demonstrated in murine models

that type II alveolar cells become activated upon infection with M. tuberculosis and express

cell surface class II MHC, CD54, and CD95 molecules (69). Also, type II cells can present

mycobacterial antigens to immune CD4 T cells isolated from mice infected with M.

tuberculosis (69).

21

Neutrophils

Neutrophils are the most abundant white blood cells belong to the polymorphonuclear family.

Neutrophils are circulating in the bloodstream but in response to acute infection migrate to the

site of infection and show microbicidal activity (70). Neutrophils are the reservoirs of

granules with high concentration of antimicrobial activity. Two major types of neutrophil

granules are characterized: primary granules that contain α-defensins, myeloperoxidase, and

serprocidins, and secondary granules that contain lactoferrin, cathelicidin, and neutrophil

gelatinase-associated lipocalin. Upon inflammatory responses, neutrophils undergo apoptosis

and macrophages phagocytose the apoptotic neutrophils in order to clear toxic substances

from the body. Tan et al have shown that M. tuberculosis infected macrophages can readily

phagocytose apoptotic neutophil and acquire antimicobacterial activity (71). Experimental

animal models of TB have shown that neutophils are found in the lungs at early times of

infection as well as some days after initial infection (72, 73). Defective neutrophil functions

or depletion of neutrophil activity exacerbate myobacterial growth in different organs and

reduce IFN-γ levels and nitric oxide synthase activity (72), suggesting the protective role of

neutrophils in the host defence mechanism. Recently, it has been described that interaction

between neutrophils and DCs could occur through Mac-1 expressed on neutrophil and DC-

SIGN expressed on DC, which promotes DC maturation by TNF secretion (74, 75). Apart

from being involved in immune protection, accumulation of neutrophils can be harmful for

the host due to pathology because susceptible animals with M. tuberculosis infection have

larger and longer accumulation of neutrophils in TB lesions (76).

Natural killer (NK) cells

NK cells are cytotoxic lymphocytes, which play an important role in the innate immune

system. NK cells are activated by cytokines and once activated bind to the Fc portion of

antibodies and perform antibody-mediated cellular cytotoxicity in order to lyse infected cells.

Regulation of NK-cell activity is maintained by so called ‘activating and inhibitory receptors’

that help to differentiate between the infected and normal cells. Studies by Junqueira-Kipnis

et al have suggested that NK cells have only minimal role in protection against M.

tuberculosis (77). NK cells are increased in numbers during the early stage of infection,

however, depletion of NK cell activity does not show significant effect on pulmonary

bacterial load (77). However, it has been found that human NK cells could contribute to

immune defences against M. tuberculosis by producing IL-22 cytokine. IL-22 cytokine

producing human NK cells promote phagosome-lysosome fusion and result in the inhibition

22

of mycobcaterial growth (78). Feng et al have shown that NK cell-driven IFN-γ production

functions in a T-cell independent way in T-cell deficient (RAG-/-) mice infected with M.

tuberculosis (79), suggesting a possible significant role of NK-cell mediated immunity in

HIV-infected individuals.

Adaptive immunity

The adaptive immunity is highly specific to a particular antigen previously encountered by the

immune system. Two major components are involved in maintaining this host adaptive

immunity: one is called cell-mediated which includes T cell activation and effector

mechanisms and another is humoral immunity where B cells and antibodies are involved. M.

tuberculosis infection in humans is intracellular and thereby cell-mediated immune responses

are regarded to be very important in host protection. Humoral/antibody-mediated immune

responses may also contribute to the immune resistance to TB (80, 81).

Cell mediated immunity to mycobacteria

CD4 T cells

CD4 T cells, also known as helper T cells, play a significant role in directing/regulating the

adaptive immune system. Initiation of T-cell mediated immune responses begins upon

activation of T cells by the innate immune cells e.g. APCs. Without the cooperation from the

innate cells T cells remain naive. The importance of CD4 T cells in protection against TB is

tremendous because HIV-mediated decrease of CD4 T cell number results in progressive

primary infection, reactivation of latent infection and debilitate the condition of patients with

TB disease (82-85). In mouse models of TB, the requirement of CD4 T-cell mediated immune

responses has also been demonstrated by using CD4 T cell knockout animals or by passive

transfer of CD4 T cells (86, 87). Passive transfer of CD4 T cells is associated with an early

protection against M. tuberculosis (88). A defective CD4 T-cell function or MHC II molecule

increases the susceptibility to M. tuberculosis infection, revealing the central role of CD4 T

cell in protection (89). Cytokines produced by CD4 T cells are of critical importance for the

regulation of Th1 or Th2 type of immune responses. Th1 cells secreting IL-2 and IFN-γ, are

associated with cell-mediated immunity, whereas Th2 cells that produce typically IL-4, IL-5,

IL-10 and IL-13 are responsible for the regulation of humoral immunity.

Upon antigen presentation, CD4 T cells become activated and produce the key

cytokine IFN-γ. Macrophages are activated by IFN-γ and produce antibacterial components

i.e. reactive oxygen and reactive nitrogen intermediates in order to kill the bacteria, which is

23

the major effector mechanism of cell-mediated immunity against TB. CD4 T cells have been

reported to express cytotoxic activity (90). CD4 cytotoxic T lymphocytes (CTL) preferentially

lyse their targets via Fas-Fas ligand interaction, whereas the major cytotoxic effect of CD8

CTL is mediated by perforin and granzymes. Although some CD4 CTL may kill the targets

by perforin and granzymes, this pathway is of limited significance (90).

CD8 T cells

CD8 T cells or cytotoxic T cells recognize peptide antigens in the context of MHC class I

molecules. Although it is believed that CD8 T cells are less important than CD4 T cells in

protection against TB, defective function of CD8 T cells due the loss of function of β2

microglobulin mice succumb to M. tuberculosis infection (91). M. tuberculosis mediated

activation of CD8 T cells enhances IFN-γ, granulysin, Fas-L, and perforin synthesis, which

act through different mechanisms on infected cells and kill the bacteria (92). In human TB,

CD8 CTL have been found to act directly on the infected cells and kill the mycobacterial

pathogen. It has been suggested that granulysin alters the integrity of bacterial cell and in

combination with perforin, reduces the viability of M. tuberculosis (93). Other evidence

showed that a subset of CD8 T cells could express CCL5 chemokine together with perforin

and granulysin and the presence of CCL5 attracts M. tuberculosis infected cells which

enhances the clearance of M. tuberculosis from the host (94).

Although perforin and granzyme have direct mycobactericidal activity, gene knockout

experiments with perforin and granzyme have shown that there is no discernible influence of

the lack of expression of perforin or granzymes on the course of M. tuberculosis infection and

pathology (95) tested in mice. This suggests that other mechanism of protection offered by

CD8 T cell probably exists and that it might be cytokine dependent. A passive transfer of CD8

T cells from the control mice to the infected mice improved the course of M. tuberculosis

infection and it failed if the cells were taken from IFN-γ gene knockout mice, indicating the

importance of IFN-γ cytokine produced by CD8 T cells in protection (96).

M. tuberculosis antigen processing and presentation to CD8 T cells are likely to be

operated by three different mechanisms (called cross-processing) other than the classical

MHC class I antigen presentation (97). First, exogenous antigens can be taken up by the APCs

for MHC class I antigen processing and presentation; second, M. tuberculosis infected cells

may produce exosomes containing mycobacterial antigens, which can be presented by

bystander APCs for MHC class I cross processing; third, apoptosis of M. tuberculosis infected

24

cells releases apoptotic vesicles containing the mycobacterial antigens, which are taken up by

bystander APCs for MHC class I cross processing.

In addition to the classical MHC class I A, B, C mediated recognition, studies in

humans with M. tuberculosis infection explained that recognition of mycobacterial peptide

antigens by CD8 T cells could be possible via a novel nonpolymorphic MHC class Ib antigen-

presenting pathway (98). Lewinsohn et al (99) assessed the frequency of classically and non-

classically restricted CD8 T cell clones among 96 M. tuberculosis positive CD8 T cell clones

and observed that the classically restricted CD8 T cell clones are very few (4%) compared to

the non-classically restricted CD8 T cell clones (96%) suggesting that the classically

restricted CD8 T cells comprise only a small part of the total M. tuberculosis specific CD8 T

cells.

Th17 cells

More than 20 years ago, Mosmann and Coffman (100) described two subsets of Th cells (Th1

and Th2) based on their distinct patterns of cytokine expression as explained above. More

recently, a new subset of Th cells named Th17 has been characterized primarily based on their

ability to produce IL-17 cytokine (101). IL-17 cytokine was described in mice, rats and

humans in the mid-1990s and proposed that IL-17 could play an important role in tissue

inflammation. New experimental findings have further shown that Th17 cells are important

not only for the resistance to fungal infection and for the mucosal immunity but also can be

instrumental for the development of organ-specific autoimmunity (102). IL-23 cytokine has

been proposed to be important for the Th17 responses (101). Activation of human DCs

through the combination of ligands for TLR2 and NOD2 is required for the production of IL-

23 which is in contrast to the IL-12 production where additional activation by IFN-γ-priming

and/or costimulation with TLR7/8 ligand (R848) are necessary (101).

IL-23, which is required for the generation of Th17 responses, may participate in the

vaccine-induced protection against M. tuberculosis infection. Experimental murine models of

TB have shown that IL-23 production accelerates IFN-γ producing CD4 T cell responses and

the establisment of an IL-17-producing CD4 T cell population in the lungs. It has been shown

that vaccination with I-Ab-restricted ESAT-6 [1–20 amino acids] epitope induces IL-17-

producing CD4 T cell population in the lungs and upon challenge with M. tuberculosis, Th17

cells express chemokines CXCL9, CXCL10 and CXCL11, which are required for the

recruitment of IFN-γ producing CD4 T cells in the lungs (103).

25

Gamma delta (γδ) T cells

γδ T cells are a group of cells that contain a distinct T cell receptor (TCR) molecule, called γδ

TCR, which is composed of γ chain and δ chain. Unlike αβ T cells that recognize processed

antigens in an association with the MHC molecules, γδ T cells recognize natural and synthetic

non-peptide antigens (104-106). Little is known about the conditions for γδ T-cell activation

but their role in host immune responses could be involved during the early stage of infection

by collaborating between the innate and adaptive immune system (107). γδ T cells function

independently in the airway, do not need help from αβ T cells (108). In humans with

mycobacterial infection, the frequency of mycobacterial antigen specific γδ T cells was found

to be increased and their role in IFN-γ production and cytotoxic activity has been reported

previously (109, 110). γδ T cells do express Fas-FasL and perforin to a similar extent as CD4

and CD8 T cells express in both patients with M. tuberculosis infection and healthy controls

(111).

Studies in mouse models of TB have suggested that γδ T cells might have anti-

inflammatory activity against M. tuberculosis. Depletion/defective functionality of γδ T cells

accelerates inflammatory damage in the lungs with M. tuberculosis infection (112). γδ T cells

express IL-23 receptor and thus can produce IL-17 in response to IL-1β and IL-23 cytokines

(113), which might promote cell migration into the site of infection (114).

Apart from being cytokine producers, γδ T cells are also able to function as

professional APCs and give sufficient costimulatory signals to αβ T cells for the induction of

proliferation and differentiation (115). Thus, γδ T cells are called as alternative type of

professional APCs (116) and novel initiators for adaptive immunity (117).

Regulatory T cells (Treg)

Treg cells are primarily known as suppressive T cells, function to control immune responses

against self antigens. Treg cells represent 5-10% of CD4 T cells and are characterized by a

specific transcription factor, forkhead box p3 (Foxp3). A large portion of the Treg cells

expresses CD25 marker and thus the phenotype of Treg cells is denominated as CD4+CD25+

cells. It has become clear that the Treg cells not only control self-immune responses but also

respond to foreign antigens (118).

Given that a delicate balance may exist between the virulence of M. tuberculosis and

the host responses following infection, it has become important to assess the functional

capacity of regulatory cells in M. tuberculosis infection. It has been reported by Ribeiro-

26

Rodrigues et al that the frequency of CD4+CD25+ and CD4+CD25high T cells was increased in

patients with active TB and remained elevated at completion of six months of therapy (119).

The cytokines IFN-γ and IL-2 are suppressed in patients with active TB but IL-10 and TGF-

β1 levels increase, therefore overproduction of IL-10 and TGF-β1 has been implicated for the

decreased T-cell function in TB (119). Although following antituberculosis treatment IL-10

and TGF-β1 levels drop, T-cell responses remain suppressed suggesting that additional

mechanisms might control T-cell responses during active TB. TGF-β1 has been shown to act

on the conversion of CD4 T cells into the Treg cells and also TGF-β1 is required for survival,

retention and function of the Treg cells (120). However, there is no clear indication that CD25+

T cells produce IL-10 and TGF-β1. The role of Treg cells in the suppression of IFN-γ

production has been established by cell depletion experiments where the depletion of CD25+

T cells increased IFN-γ production by CD4 T cells compared to the undepleted cells (119).

Moreover, increased levels of Treg cells have been observed in patients with extrapulmonary

TB (121). Extrapulmonary TB manifests due to the failure of the Th1-type of immune

responses and that causes mycobacteria to migrate to another place. A balanced immune

response is needed for the control of infection or extensive pathology. The majority of the

infected individuals who do not develop active TB are believed to be fine-tuned with the

immune responses. It is considered that the Treg cells down-regulate the immune responses

following eradication of pathogens to avoid the development of pathology, however, this may

lead to the establishment of chronic infection with M. tuberculosis.

Humoral immunity

B cells are able to perform the role of APCs and are recognized as the key players for the

induction of humoral or antibody-mediated immune responses against antigens. B-cell

activation can take place in two ways: T-cell dependent or T-cell independent manner. In the

T-dependent activation, antigen-presenting macrophages activate helper T cells with matching

receptors and promote T cell and B cell interaction. This process induces memory B-cell

formation and also triggers proliferation and differentiation into antibody forming plasma

cells. In the T-independent activation, B cells can be activated by thymus independent

antigens. For example, many repeating carbohydrate epitopes that are present on bacterial

surfaces can cause cross-linking of antibodies on the surface of B cells and result in activation

of the B cells. Circulating antibodies or cell bound antibodies function in the complement

27

activation, neutralization of toxins, opsonisation of pathogens and finally enhance the

elimination of pathogens by phagocytosis.

Since M. tuberculosis is an intracellular pathogen, the role of antibodies in protection

against TB is generally regarded as nonprotective. However, antibodies might be very

important in neutralization and prevention of invasion of pathogens especially at the mucosal

surface. TB is primarily a respiratory mucosal disease, therefore, antituberculous antibody

research gained renewed interest and it has already been shown that antibody could provide

protection against TB (122, 123). In the absence of B cells, mice had exacerbated

immunopathology and a large number of neutrophil accumulation which coincides with

increased bacterial burden (124, 125). Adoptive transfer of B cells to the B-cell deficient mice

could reduce the pathology and increase serum antibody levels, suggesting an

immunoglobulin mediated ‘endocrine’ regulation (125). In the BCG-infected experimental

models, mycobacteria-specific antibodies have been shown to increase uptake and killing of

BCG bacteria by neutrophils and macrophages (126). Antimycobacterial antibodies have an

effect on the cell-mediated immunity as demonstrated by de Valliere et al (126). They

observed that in vitro antibody-coated BCG bacteria were taken up by DCs more efficiently

and could stimulate CD4 and CD8 T cells for IFN-γ production. Rodríguez et al have shown

that IgA-deficient mice were more susceptible to M. bovis BCG infection (127). Upon

immunization IgA-deficient mice were unable to produce IgA and also cytokine responses

were reduced compared to the wild-type control mice (127).

Among several mycobacterial antigens, mycobacterial arabinomannan, HBHA and

16kDa α-crystallin have been reported to induce protective antibody responses (122, 123,

128-130). It remains to be unveiled how antibodies could contribute to the host protection.

Maglione and Chan have proposed a possible mode of action of antibodies together with Fcγ

receptors (Fig. 3) (131). Upon interaction with stimulatory Fcγ receptors on macrophages,

Th1 responses can be enhanced due to the increased production of IFN-γ. Because upon

infection with M. tuberculosis mice deficient in inhibitory FcγRIIB receptors have been found

to develop increased Th1 responses evidenced by the increased IFN-γ producing CD4 T cells

(132). On the other hand, B cells have a significant impact on IL-10 cytokine production since

an increased level of IL-10 production has been shown to be associated with B-cell deficiency

(133) and defective function of Fcγ-stimulatory receptor (125, 134). Thus, it is assumed that

the excessive levels of IL-10 might be linked with immunopathology during an infection with

M. tuberculosis (125).

28

B-cell function seems to be varied during the course of M. tuberculosis infection as

suggested by Maglione and Chan (131). B cells contribute to optimal granulomatous response

during the acute condition of infection and help in the containment of bacilli. During the

latent infection, it is thought that B cells promote local host immune responses against M.

tuberculosis and that might restrict reactivation of infection.

Cytokines and chemokines

Cytokines and chemokines are essential components of the immune system. Cytokines and

chemokines contribute to the formation of a cell network by delivering signals to the cells and

helping them to the induction of effector functions.

Cytokines

Cytokines such as IL-12, IFN-γ, IL-2 and TNF play an essential role in the maintenance of

protective immunity against TB. Experiments in animal models with the loss of function of a

cytokine gene or an inhibition of cytokine function by administration of a specific antibody

revealed the need of cytokine function for the host protective immunity (135-138). Similarly,

a defective cytokine receptor function is also associated with the lack of protective host

responses against TB (139, 140). IL-12 is produced by macrophages or DCs upon

phagocytosis of M. tuberculosis. IL-12-regulated IFN-γ production by T cells or NK cells is a

key pathway for the control of M. tuberculosis infection (141, 142). IL-12p40 but not IL-

Fig. 3: Proposed mechanism of antibody-mediated modulation of immune responses in TB (Eur J Immunol. 2009;39:676-86, reprinted with permission from John Wiley & Sons Inc.).

29

12p35 is essential for the regulation of IFN-γ production since gene knockout of the IL-12p40

subunit causes reduced IFN-γ production and mice succumb to infection (142). IL-12p40 is

critically involved in the migration of DCs to the regional lymph nodes to activate T cells

(143). IFN-γ activates macrophages, CD4 and CD8 T cells for the induction of effector

functions in order to kill the pathogen. Activation of macrophages starts the synthesis of nitric

oxide (NO), and related nitrogen intermediates (RNIs) upon the action of inducible nitric

oxide synthase (iNOS), which facilitate the killing process. In murine models, RNIs mediated

antimycobacterial effects are well documented (144, 145), however, it is still uncertain how

much of this is relevant to human TB. Upon antigen presentation by macrophages, activated T

cells produce IL-2, which causes expansion of the antigen specific T cells.

TNF is an important cytokine in the control of TB. TNF regulates the effective

granuloma formation and thereby prevents bacterial dissemination. TNF influences the

expression of adhesion molecules and also chemokines that attract immune cells to the

infected tissues (146). In absence of TNF, effective granuloma formation is impeded and

bacterial growth is rapidly increased, which reduced the survival time of the mice (147, 148).

TNF functions by forming a trimer with its receptor - either with TNF receptor 1 (TNFR1, 55

kDa) or TNFR2 (75 kDa), present on the cell membrane and expressed by almost all

nucleated cells. TNFRs belong to the TNF receptor superfamily. The extracellular domain of

both receptors is cleaved by metalloproteases and then the soluble form (sTNFR) binds to

TNF and neutralizes TNF-mediated activities. It has been described that the complete

neutralization of TNF activity by sTNFR prevents the induction of cell-mediated immunity in

mice and therefore mice succumb to BCG infection (149). Lower sTNFR levels result in

higher bactericidal activity of macrophages by enhancing iNOS activity (149).

IL-4, one of the cytokines that drives the immune system to induce Th2 type response,

is considered as the harmful candidate for the host protective immunity against TB. IL-4 has

been implicated with the poor efficacy of BCG vaccine in the developing world where a pre-

existing enhanced IL-4 immunity in people with helminth infection is characterized (150).

Elevated levels of IL-4 have been observed in patients with pulmonary TB (151, 152). The

increased IL-4 levels seem to be correlated with serum IgE concentration and with the extent

of cavitation (151, 152). In mouse models of TB, IL-4 has been shown to downregulate iNOS

activity (153). IL-4 delta 2 (IL-4δ2), a splice variant and inhibitor of IL-4 has been described

recently and found that both are increased in active TB and only IL-4δ2 is elevated in

individuals with latent infection (154, 155).

30

Chemokines

Chemokines, also known as chemotactic cytokines, are small proteins, 8-10 kDa in size. The

main function of chemokines is to call immune cells to a specific place (infected site) in order

to fight against the pathogens. Changes in the expression pattern of chemokine receptors

(CCR) determine whether the cells will migrate to another place. Chemokine receptors belong

to four different groups; CXC, CC, CX3C and XC that correspond to the 4 distinct

subfamilies of chemokines they bind. Dendritic cells upon antigen stimulation downregulate

CCR5 and CCR1 expression and increase CXCR4 and CCR7 expression which promote

migration of DCs to regional lymph nodes in order to activate T cells (156). M. tuberculosis

infection leads to the synthesis of chemokines by cells as early as 2 h postinfection. Upon M.

tuberculosis infection, human macrophages produce a range of CC chemokines CCL2, CCL3,

CCL4 and CCL5 (alternate names MCP1, MIP1α, MIP1β and RANTES) (157). Higher levels

of MCP1, RANTES, IL-8 but not MIP1α were detected in bronchoalveolar lavage of TB

patients (157). Increased levels of CCL5 correlate with the higher number of CD4 T cell in

bronchoalveolar lavage of TB patients (158). Studies in murine models with CCR2 knockout

gene have revealed that cell migration is substantially delayed as well as IFN-γ and NO

production upon low dose infection with M. tuberculosis (159). However, there was no

difference observed in the bacterial growth between the knockout and wild-type control mice.

Granuloma formation was not hampered in the knockout animals. In contrast, high dose

infection promoted uncontrolled bacterial growth in the lungs (160). Excessive production of

chemokines can also be detrimental for the host as demonstrated by Rutledge et al.

Transgenic mice overexpressing CCL2 are more susceptible to M. tuberculosis infection (156,

161).

A critical component involved in the regulation of CC chemokine function is the D6

chemokine receptor or decoy receptor. This receptor is not specific for only one chemokine. It

can recognize 15 different CC chemokines. Interaction between D6 and CC chemokines

results in the formation of a receptor-ligand complex, which is internalized by the cells and

causes degradation of the CC chemokines, recycling the decoy receptor to the cell membrane

(162). In this way, the decoy receptor controls CC chemokine-driven inflammatory responses.

M. tuberculosis infection in decoy receptor knockout mice causes excessive inflammation and

reduces survival time (162).

31

Mucosal immunity in the respiratory tract

More than 50 years ago Bull and McKee reported that specific immunity and protection

against pneumococcal organisms could be generated in the nasal route after local instillation

of organisms (163). The mucosal immune system has been an area of recent interest assuming

that it is the most potential contributor of the host immunity since many infectious agents

including M. tuberculosis come in contact with the host through the mucosal surfaces. Lungs,

gastrointestinal tract and urogenital tract are covered by mucous membrane. M. tuberculosis

gets access to human hosts via nasal mucosa and comes across the respiratory mucosa in order

to be settled in the lungs. Therefore, lungs appear to be an important place where induction of

immunological responses against TB is required.

The upper respiratory tract comprising mucosal tissues and the lung parenchyma

represents thin-walled alveoli. In the upper respiratory tract, epithelial lining containing

ciliated cells and locally produced IgA antibodies prevent the entry of foreign invaders. IgA is

the predominant immunogloubulin in most external secretions that exceeds that of all other

immunoglobulin classes combined (164, 165). In humans, IgA comprises two subclasses:

IgA1, dominant in serum and IgA2, mainly found in secretions. B cells produce IgA1 in

response to protein antigens and IgA2 in response to polysaccharide antigens. In external

secretions, both IgA1 and IgA2 are found in a different form called secretory IgA (sIgA),

which is a polymeric form and is stable in the external secretions. The second compartment of

the respiratory tract is largely associated with lymphoid tissues, containing both T and B cells.

The lymphoid tissues within the bronchial walls form discrete structures, which resemble the

Peyer's patches in the gut. This site is called bronchial-associated lymphoid tissue (BALT).

The presence of BALT in adult human is controversial (166, 167), however, children do have

BALT structure. Surveillance of the respiratory surfaces for antigens is primarily mediated by

the resident airway mucosal DCs which capture antigens, upregulate CCR7 and migrate to the

draining lymph nodes in order to present antigens to naive T cells. Antigen-specific T cells

proliferate and differentiate into effector T cells that could home to the mucosal effector sites

and contribute to the pathogen clearance.

Antigen-specific CTL responses can be induced at the mucosal surfaces as reported by

Gallichan et al (168). The CTL are long-lived, generated locally after mucosal but not

systemic immunization and could migrate to the systemic compartments. Besides, the

mucosal CTL responses, IFN-γ producing CD4 T cells have been found to be important for

the mucosal protection against a variety of mucosal pathogens (169). Understanding of the

mucosal immunity against M. tuberculosis has become an important event for the next

32

generation of TB vaccines. As a result, many studies have focused on mucosal vaccination

using mycobacterial antigens in order to stimulate predominantly lung immunity, which is

believed to be more effective against infection than that induced after systemic vaccination

(169). Circumstantial evidence indicates that mucosal sensitization improves the level of

protection and this could be due to the heightened cell-mediated immune responses found in

the lungs (170-172). In addition, many studies reported on the passive transfer of IgA via

intranasal route and improvement of protection against TB (122, 123, 173), which imply the

potential role of IgA-mediated immunotherapy against TB.

Fig. 4: Uptake of antigens and presentation to immune cells in the lung compartments. (Nature Rev. 2008;8:142-152, reprinted with permission from NPG ).

33

Vaccination against TB Frequently, vaccinations in humans are performed during the first years of life starting soon

after birth. In fact, vaccines given to the neonates pose a huge challenge against the success

due to the suboptimal maturation of the neonatal immune system. Vaccination against TB was

introduced about a century ago. Unfortunately, failure of the BCG vaccination has been

recorded and attempts are being taken to develop a new successful vaccine in order to ensure

life-long protection against TB.

The BCG vaccine

‘Mycobacterium bovis Bacille Calmette-Guérin’ is the complete name of the BCG vaccine,

which was first introduced in 1921. Edmond Nocard first isolated a virulent strain of M. bovis

called ‘lait Nocard’ which was transferred to the Institut Pasteur at Lille in 1901. Albert

Calmette and Camille Guerin at the Institut Pasteur started culturing ‘lait Nocard’ organism

with potato slices cooked in beef bile supplemented with glycerol for a duration of 3 weeks.

This process was continued to a total of 230 passages by changing the culture medium every 2

weeks until 1921. The organisms lost their virulence over the culture period as tested in

different animal models.

The first BCG vaccination with the attenuated form of bacteria was performed in a

newborn whose grandmother had pulmonary TB in 1921. This vaccinated individual

remained free of TB throughout his life (174). From 1924 to 1926, BCG Pasteur strain was

distributed to 34 countries and in 1927 more than 26 other countries were reported to receive

the Pasteur strain. Since then many substrains of BCG were generated at different laboratories

at various conditions and therefore today it has raised the question on the identification of the

actual BCG strain in use (175). It is clear that BCG substrains are different from each other

and also have different characteristics from the original strain. Comparative genomic analyses

have first identified a gene coding for a protein MPB64, not found in some BCG substrains

(176, 177). Subtractive genomic analyses were performed later to identify more specifically

the deletion regions (RDs) among the substrains delivered before 1925 and after 1926

separating the BCG vaccine into ‘early’ and ‘late’ substrains. Strains that were distributed

before 1925, had a RD1 deletion containing esat6 and cfp10 genes and strains delivered after

1926 had RD2 deletion which codes for mpb64 and cfp21 (Fig. 5).

34

The use of gene probe based experimental techniques helped to discover two copies of

insertion sequence IS6110 in all the early substrains except Gothenburg substrain however,

only one copy of IS6110 was found in the ‘late’ substrains. More recent investigation

discovered that the deletion of insertion sequence is even not within the RD regions (175).

Biochemical studies uncovered the differences among BCG substrains with regard to

their lipid structures. Some of the substrains e.g. Gothenburg, Moreau and Tokyo strains have

complex structure of mycolic acids containing methoxy groups attached to the mycolic acid.

However, BCG Danish, Glaxo and Pasteur strains do not have such structure.

Considering the genetic diversity and biochemical properties that the BCG substrains

acquired during the attenuation process, it is probably not surprising that the protective

efficacy of the BCG substrains declined after many passages. Studies reported that ‘early’

substrains including BCG Moreau and Tokyo are moderately immunogenic in animal studies

than the ‘late’ substrains, which include BCG Danish and Pasteur (175). According to the

genealogical tree of the BCG substrains, the ‘late’ substrains lost more regions than the ‘early’

substrains, however, this does not contribute to the immunogenicity or protective efficacy of

the BCG vaccines (178, 179). As a result, World Health Organization recommended the most

commonly used BCG substrains for future vaccination against TB (180).

Fig. 5: Evolutionary framework of BCG strains. (Vaccine 1999;17:915-922 , reprinted with permission from Elsevier Science Ltd.).

35

Neonatal immunity and maternal-fetal interactions

Immunization during the early life is required to protect infants from infectious diseases.

However, during the early life, neonatal immune system is not sufficiently ready to respond to

vaccine antigens and this leaves the neonates susceptible to infections. Since the neonates are

often biased to develop Th2 type immunity and in some cases show unresponsiveness or

immune tolerance, it may be of importance to consider special delivery protocols of

vaccines/adjuvants to divert the immune responses. Induction of Th1 responses in the

neonates has been made by using CpG-containing oligonucleotides (181), Freund’s complete

adjuvant (182) or Titermax (183). The preferential polarisation of Th2 responses and failure

to induce sufficiently high Th1 responses might be explained by the limited microbial

exposure, lack of costimulatory activity by immune cells and suboptimal APC-T cells

interaction in the neonatal life. In vitro studies have shown that neonatal T cells produce low

levels of IL-2 and proliferate poorly in response to anti-CD3 stimulation, however, in the

presence of anti-CD28 antibody and exogenous IL-6, neonatal T cells produce large amounts

of IL-2 (184), implying a greater requirement of accessory cell signals for neonatal T cells.

The reduced expression of MHC class II molecules in monocytes has been reported in utero

and this could contribute to the impaired APC function (185). Similarly, human neonatal DCs

have impaired synthesis of type I IFNs upon exposure to microbial components (186).

Decreased expression of IL-12 by neonatal APCs and CD40-ligand by neonatal T cells has

also been reported (187). A low level of CD40/CD40-ligand signals and insufficient amounts

of IL-12 could limit the priming of the Th1 cells.

Given the immune interactions between mother and fetus, investigators discovered

several mechanisms, which may explain why the mother does not reject foreign fetus. One of

the critical events is the failure of the trophoblast cells to express HLA class I and II

molecules (188) and that may aid the fetal semiallogeneic graft in evading maternal

immunologic responses. During the early pregnancy, nonclassical HLA molecules such as

HLA-G and HLA-E have been found to be expressed by the cytotrophoblast cells. HLA-G has

been shown to be important for the survival of fetus in the mother. The binding of HLA-G to

the inhibitory receptors of maternal-uterine NK cells prevents NK cell-mediated cytolytic

activity thereby, fetus remains unaffected (188). Moreover, a Th2 but not Th1-cytokine

environment is critical for a successful pregnancy. It is now known that Th2 cytokine

environment contribute to implantation of the embryo, development of the placenta, and

survival of the fetus to term (189).

36

In addition, immunological contribution from the mother to the fetus or to the

neonates could also block immune activation process in the neonates. For example, studies

showed that maternal antibodies could suppress vaccine responses against tetanus, diptheria

toxoids (190, 191), haemophilus influenzae type b (Hib) conjugates (192, 193) and hepatitis A

antigens (194). This could be overcome by repeated vaccination with diptheria-tetanus-

pertusis-polio or Hib vaccines. The transfer of passive immunity from the mother to the baby

via placenta could be present in the neonates until the first couple of months. These antibodies

disappeared by six months of age (195) and therefore have less influence on the repeated

vaccination. Of note, the presence of maternal antibodies has no influence on neonatal T-cell

proliferation and cytokine production as observed in mouse models with tetanus toxoid or

measles vaccines (196, 197).

It is possible that the neonatal immune cells are not ready to respond sufficiently to

vaccine antigens since both qualitative and quantitative differences are noticed among innate

and adaptive immune cells from newborns and adults (186, 198). However, mounting

evidence indicates that the condition of the vaccines including delivery or presence of strong

adjuvant could shape the neonatal immune system (187). BCG vaccination during the

neonatal age is one of the classic examples that could induce sufficiently adult-like immune

responses (199). Moreover, immune activation or priming of the immune system could also

occur as early as during the gestational period when fetal immune cells could be activated by

antigens transferred from the mother to the fetus through placenta (200, Paper III). This

prenatal exposure to antigens could impact on higher immune responses during the postnatal

life, which has been reported (201, Paper III). Prenatal exposure to mycobacterial antigens

enhanced postnatal immune responses, which were similar to that observed in adults (201).

37

Failure of BCG and progress in new TB vaccine development

Unfortunately, the BCG vaccination has been reviewed unsuccessful against adult pulmonary

TB but effective against childhood tuberculous meningitis and miliary disease. Therefore,

BCG vaccination is still recommended in many countries with high prevalence of TB. There

is no single cause that could explain why the BCG vaccination failed to deliver enough

protection. A combination of many factors perhaps contributes to the lower efficacy of the

BCG vaccination. For example, genetic differences within and among host populations,

varying levels of malnutrition among host populations, virulence differences among M.

tuberculosis strains, endogenous reactivation of persistent infection versus exogenous re-

infection, effects of environmental mycobacteria on the host immune response to BCG, and

methodological differences among the clinical trials. In addition, the BCG-derived immunity

wanes with time, which might not be due to the decreased levels of Th1 immunity or to the

factors mentioned above. It has been suggested that other mechanisms like the induction of

inappropriate Th2 immunity by helminth infection (202) or regulatory T-cell activity (203)

are likely to contribute to the poor Th1 immunity.

Since the protection offered by the BCG vaccination against adult pulmonary TB is

variable, many approaches have been pursued to achieve a new successful vaccine, which

includes auxotrophic vaccines, rBCG, DNA vaccines and subunit vaccines. The most

commonly used experimental animals are mouse and guinea pig that have been vaccinated

with the newly developed vaccines in order to obtain some basic information such as the

ability to control bacterial burden, lung pathology and to improve survival period. A summary

of the TB vaccines tested to date is depicted in Table 1.

38

Table 1: List of TB vaccines tested in animal models for evaluation of protective efficacy Vaccine type Name of the vaccine Protection

compared with BCG (Mouse)

Survival compared with BCG (Guinea pig)

References

1. HBHA =BCG 204 2. Ag85B <BCG 205 3. ESAT-6 <BCG 206

Single antigens

4. TB10.4 <BCG 205 5. Ag85B-ESAT-6 ≥BCG* <BCG 207, 208 6. Ag85B-TB10.4 ≥BCG* 209

Fusion antigens

7. Mtb72F (MTB32, 39) <BCG 210 8. STCF =BCG* 211 Su

buni

t vac

cine

s

Antigen complex 9. CFP-10 <BCG 212

10. DNA-85A 12. DNA-85B 13. DNA-ESAT-6

<BCG 213, 214 DNA vaccines 14. rAd vector--Ag85A,

Ag85B, and TB10.4

<BCG 215

Auxotrophic 15. Auxotrophs BCG <BCG 216 Live vaccine

Recombinant BCG (rBCG)

16. Over-expressing Ag85 17. Coexpressing Ag85B-ESAT-6 18. ∆ureCHly+rBCG

>BCG >BCG >BCG

217, 218 219 220

DNA prime-fusion protein boost

19. DNA-Ag85A prime …Ag85B-ESAT-6 boost

=BCG 221

Viral vector prime- fusion protein boost

20. MVA-Ag85A prime…Ag85B-ESAT-6 boost

=BCG 221

BCG prime-protein boost

21. BCG… Ag85A boost 22. BCG… Ag85B-ESAT-6 boost 23. BCG… Ag85B-TB10.4 boost

>BCG >BCG (i.n.)

>BCG

222 170 223

BCG prime-viral vector

24. BCG… MVA85A boost 25. BCG…Ad85A

>BCG (i.n.)

>BCG 221 172

BCG prime-DNA boost

26. BCG…ESAT-6 >BCG 224

Com

bina

tion

vacc

ine

(pri

me-

boos

t)

BCG/protein prime-protein boost

27. BCG/Ag85B-ESAT-6--- Ag85B-ESAT-6

>BCG 225

*Not significantly better; i.n. intranasal, Ag, antigen; STCF, short-term culture filtrate; CFP-10, culture filtrate protein, 10 kDa; rBCGDUre:CHly+, rBCG expressing listeriolysin Subunit vaccines

Besides showing high efficacy, a vaccine has to be safe for human use and it is generally

considered that subunit vaccines are safe than live or attenuated vaccines. Though subunit

vaccines are considered to be safe, one of the shortcomings of subunit vaccination is that the

candidate antigen alone could not stimulate enough the host immune system and therefore

39

induces suboptimal immune responses. Thus, most subunit vaccines require adjuvant.

Unfortunately, progress of subunit vaccine development is impeded due to the lack of safe

and effective adjuvants. However, searching for a safe and effective adjuvant for human use is

continuing and very recently squalene-containing adjuvants have been found to be safe in

human when used in two H1N1 vaccines developed by Novartis and Glaxo-Smith Kline

(226). Also, IC31(R) adjuvant has been found to be safe in humans when administered with

Ag85-ESAT-6 fusion antigen (227). The development of subunit vaccines against TB is being

focused much in the recent times since the genomic sequences of M. tuberculosis are

available now (228) and it is believed that a key antigen of the M. tuberculosis might induce

protective immune responses. Several single mycobacterial protein antigens including Ag85

complex (30-32 kDa), ESAT-6, CFP-10 and TB 10.4 (belong to the esat-6 gene family), 19

and 38 kDa lipoproteins, HBHA have been tested in preclinical studies using mouse models

and have been found to be promising although none of them induced better protection than the

BCG vaccination. Vaccination with HBHA has been shown to be as effective in controlling

bacterial burden as the BCG vaccination (204). Therefore, HBHA has been demonstrated as a

potent vaccine candidate given also the fact that HBHA specific immunity was found in the

healthy infected individuals but not in the individuals with active disease (229).

In order to prioritize the subunit vaccination, instead of adding one single antigen in

the vaccine formulation, polyprotein antigens have been used, which showed great promise

for future vaccination. This has been used in vaccines against other infectious diseases such as

malaria (230-232), leishmaniasis and hepatitis. Fusion or polyprotein antigens in the vaccines

could induce better protection than any of the components of the fusion antigen tested

individually. Mycobacterial Mtb72F, a 72-kDa polyprotein genetically linked in tandem in the

linear order Mtb32(C)-Mtb39-Mtb32(N), Ag85B-TB10.4 and Ag85B-ESAT-6 are most used

fusion antigens that conferred protection comparable to that induced by the BCG vaccination

(208, 209, 233). All three candidates have entered clinical trails.

DNA vaccines

DNA vaccination is one of the new strategies that has drawn focus on new TB vaccine

development. Instead of immunizing with the foreign antigen, a gene of interest is inserted

into a vector and finally the vector is taken up by the host cells. Using host cell machineries

the gene of interest is expressed and the host develops immunity against this specific antigen.

DNA vaccine against mycobacteria was first introduced in 1994. The J774 macrophage cell

line was transfected with the M. leprae Hsp65 gene and administered into syngeneic

40

(BALB/c) mice (234). This way of immunization induced protection against M. tuberculosis

infection (235). In mice, DNA vaccines targeting mycolyl transferase enzyme, Ag85A or 38-

kDa lipoprotein have been shown to induce some level of protection against M. tuberculosis

infection (213, 214, 236). DNA vaccination could generate both humoral and cellular immune

responses as described by Huygen et al (236) and the immune responses were found to be

mixed Th1/Th2 types (237). Although many attempts were taken in the preclinical settings

using mouse models and the outcomes were promising, experiments in the larger animals like

guinea-pigs or non-human primates showed disappointing results with DNA vaccination

(238).

Auxotrophic vaccines

Immunocompromised individuals are at greatest risk of developing disseminated TB after

receiving the conventional live BCG vaccine. Therefore, auxotrophic mutants of BCG or M.

tuberculosis have been introduced in order to reduce the virulence without affecting the

immunogenicity in vaccinated animals. Auxotrophic mutants of BCG, require specific amino

acid to grow, reported to be safe in experimental animals (216). Auxotrophic vaccines from

M. tuberculosis have also been made during the past years, however, there are concerns about

reversion of virulence and thus their use requires more investigation. Although auxotrophic

vaccines are believed to be safe at least in the experimental animal models, a major problem

seems to be the short period of immunological memory (239) due to the limited duration of

replication in the host (216, 240).

rBCG vaccines

Recombinant DNA technology has created an opportunity to reinsert the missing genes in the

BCG vaccine as well as in inducing the expression of one target gene. Several strategies have

been followed to develop vaccines against TB. The rBCG expressing RD1 genes comprising

ESAT-6 and CFP-10 has been shown to be more protective than the wild-type BCG (241).

Bao L et al developed two rBCG vaccines containing ESAT-6 fused with HSP-60 and ESAT-

6 with a secretory sequence. Both of them showed enhanced protection against M.

tuberculosis infection in animal models (242) however, none of them showed significantly

better level of protection compared with the wild-type BCG vaccination.

rBCG designed for overexpression of one single gene has been found to be attractive.

For example, rBCG30 constructed with mycobacterial Ag85B, which is a 30kDa enzyme

involved in outer cell wall synthesis, appears to enhance survival time significantly better than

41

the conventional BCG vaccine upon challenge with a highly virulent strain of M. tuberculosis

(218). Phase I clinical trial with rBCG30 vaccine has been completed in 35 healthy adult

volunteers and found to be safe and immunogenic (243).

In preclinical studies, rBCG expressing listeriolysin (rBCGDUre:CHly+) was reported

to induce better protection than the conventional BCG vaccination (220). Listeriolysin O is

expressed after vaccination with rBCGDUre:CHly+. Listeriolysin induces perforation in the

membrane of early phagosome and thereby promotes leakage of some BCG into the

cytoplasm. The basic principle of this type of vaccine is to enhance both CD4 and CD8 type T

cell responses by augmenting MHC I and MHC II dependent antigen presentation. It has been

postulated that listeriolysis not only promotes pore formation but also apoptosis of the

infected macrophages. Releasing antigens form apoptotic blebs that are taken up by DCs and

increase antigen presentation to T cells followed by a mechanism called ‘cross-priming’.

Listeriolysin functions in an acidic environment. However, BCG secreting urease could block

the acidification process. Therefore, the urease gene was deleted in the rBCGDUre:CHly+

vaccine in order to facilitate the acidification. When tested in immunocompromised SCID

mice rBCGDUre:CHly+ was found to be safe (220).

Combination vaccines (prime-boost)

Prime-boost vaccination strategy is probably the best approach in terms of achieving high

level of protection as observed in many pre-clinical studies. Different forms of prime-boost

vaccination have been tested so far i.e. protein-protein, DNA-protein, BCG-protein antigens.

As many people around the world already received the BCG vaccine, and their immune

system is already primed, the future vaccination strategy should consider boosting of the

BCG-derived immunity. In the combination of BCG priming and boosting with protein

antigens, protein antigens could be delivered together with adjuvants or in the form of naked

DNA or viral vector. Skeiky and Sadoff (244) have explained how the immune responses are

generated following priming with BCG or rBCG and boosting with recombinant protein in

adjuvant or viral vector (Fig. 6). This heterologous prime-boost vaccination could

preferentially induce antigen-specific memory T-cell expansion against some epitopes shared

by both the priming and the boosting antigens.

42

Whichever the combination of prime-boost vaccination was followed, priming with BCG and

boosting with another antigen (single or tandem) displayed significantly higher protection

than the BCG vaccination alone. McShane et al progressed with recombinant modified

vaccinia virus Ankara (MVA) expressing Ag85A (MVA85A) as a BCG-boost vaccine in

clinical trials. This was the first candidate subunit vaccine in clinical trials to boost BCG

immunity and found to be efficient in enhancing and prolonging antimycobacterial immunity

(221, 245-247). A subunit BCG-boost vaccine comprising Ag85B and TB10.4 (HyVac4)

delivered as a fusion molecule and formulated with the adjuvant IC31 has been shown to

provide higher protection than BCG vaccination alone in the more stringent guinea pig model

of pulmonary TB (223). This vaccine was reported to be safe and will be progressed for

clinical studies. Single mycobacterial antigen HBHA has been evaluated in mouse models as

BCG-boost vaccine and found to increase immune responses and protection (Paper II, 248).

Immune correlates of protection (Multifunctional T cells)

There are a number of different types of T cells that has been named as naive T cells, memory

T cells and within the memory-cell population it is further classified as effector memory T-

cells or central memory T cells. All these groups of cells are differentially regulated and

Fig. 6: How does prime-boost vaccination work? Modified from Nat Rev Microbiol. (2006;4:469-476, reprinted with permission from NPG)

43

thereby produce different cytokines, suggesting their heterogeneous effector functions and

also their ability to homing different tissues (249, 250).

Multifunctional T cells are primarily characterized by their ability to produce more

than one cytokine concomitantly. Multifunctional T cells are not only restricted to the CD4 T

cell population, CD8 T cells can also be multifunctional. CD8 T cells with multifunctional

characteristics correlated better with the control of HIV-infection (251) because IL-2, TNF

and IFN-γ triple positive T cells produced more IFN-γ than double or single cytokine

producing cells implying better effector functions of polyfunctional T cells.

Multifunctional T cells have been suggested to be important in protection although very little

is known about their mechanism of action. This has been investigated with much attention

especially in vaccine research. The hypothesis is that detection of a single cytokine producing

cell population, which might have limited functional activity is probably insufficient in the

process of characterizing immune correlates of protection. Several studies have already

addressed the importance of multifunctional T cells in hepatitis B virus vaccine (252) and

HIV vaccine (253) development, vaccinia-induced responses (254) and also in Leishmania

major infection (255). Assessment of the frequency of Th1 cytokine producing cells, mainly

CD4 T cells expressing IFN-γ, has been considered the principle correlate of immune

protection against TB but many studies have recently showed that this was insufficient (4,

256). It has been demonstrated in animal models of TB vaccine studies that the presence of

multifunctional T cells coexpressing multiple cytokines correlated better with the vaccine-

induced protection when animals were challenged with M. tuberculosis (257). Contrasting

results have been obtained by Tchilian et al; systemic multifunctional T cells do not correlate

with the protection in a murine model of prime-boost immunization study (258). Forbes et al

compared mucosal and systemic routes of vaccination in a murine model of TB and found that

multifunctional Th1 cells in the lungs but not in the spleen correlate with the protection (259).

Thus it remains to be determined the exact phenotype of the multifunctional T cells and their

subtypes which could be of importance in the characterization of immune correlates of

protection.

Route of vaccination (mucosal versus systemic)

Mucosal vaccination promotes induction of local and systemic immune responses but

vaccination via systemic routes (subcutaneous or intramuscular) mostly generates systemic

immune responses, which are not sufficiently effective against pathogen invasion at the

mucosal sites. A vast majority of pathogens invade via mucosal surfaces and therefore it is

44

considered that new-generation vaccines should target mucosal route for the induction of local

as well as systemic immunity. Examples include enteric infections caused by Helicobacter

pylori, Vibrio cholerae, enterotoxigenic Escherichia coli (E. coli), Shigella spp., respiratory

infections caused by M. tuberculosis, Mycoplasma pneumoniae, influenza virus and

respiratory syncytial virus; and sexually transmitted genital infections caused by HIV,

Chlamydia trachomatis, Neisseria gonorrhoeae and Herpes simplex virus (169). It is

becoming clear that the development of a broader range of mucosal vaccines requires the

development of effective and safe mucosal adjuvants. The best-studied and potential mucosal

adjuvants in experimental animal models are cholera toxin (CT) and E. coli heat-labile

enterotoxin (260, 261). Non-toxic forms of CT such as cholera toxin B subunit (CTB), active

cholera toxin A1 subunit linked to specific APC-binding protein derived from Staphylococcus

aureus protein A (CTA1-DD) have been developed and proven to be a very efficient and safe

adjuvant (169). Bacterial DNA or synthetic oligodeoxynucleotides containing unmethylated

'CpG motifs' (CpG ODN) has been found to be promising as a mucosal adjuvant when CpG

ODN was linked to the B subunit protein of CT (262).

The mucosal route of vaccination against TB including i.n. or oral delivery of antigens

or attenuated M. tuberculosis has been tested in many preclinical studies. Oral mucosal

vaccination has several disadvantages such as it requires much higher doses of antigens,

increases the exposure to low pH and a broad range of enzymes. In contrast, i.n. mucosal

vaccination could significantly reduce these limitations and sensitize better the immunological

compartments at the nasopharyngeal tissue and other innate organs all the way down to the

respiratory tract. Studies in mouse models have shown that the mucosal (i.n.) route of

vaccination is more effective than the systemic route of vaccination even when BCG is used

(263). Instead of delivering live organisms via nasal route, delivery of purified single or

fusion antigens, and DNA vaccines have received much research focus as a potentially useful

concept. Rodríguez A et al have shown that i.n. immunization induces both mucosal and

systemic immune responses while intraperitoneal immunization induces only systemic

immune responses (264). Santosuosso et al demonstrated that i.n. but not intramuscular

vaccination with adenoviral-based vaccine (AdAg85A) showed protection against pulmonary

M. tuberculosis challenge (265). In a prime-boost immunization protocol, protection by BCG

prime immunization was effectively boosted by AdAg85A administered i.n. and that

correlated with the increased levels of IFN-γ producing CD4 and CD8 T-cell responses in the

airway lumen (172). Subcutaneous or intramuscular boosting with BCG or AdAg85A

respectively failed to impart such protection. Similarly, fusion antigens Ag85B-ESAT-6

45

delivered via i.n. induced strong T cell responses in the spleen, blood, draining lymph nodes

from the nasal cavities whereas subcutaneous immunization with the same antigen failed to

induce similar immune responses in the lymph nodes (170). Likewise, mucosal administration

of Ag85B-ESAT-6 increased protection significantly against M. tuberculosis infection in the

BCG-primed mice compared with the BCG-vaccinated nonboosted mice (170).

46

Diagnosis of TB Yet, there is no reliable technique available for the diagnosis of TB. Thus, a secure and quick

test is of utmost importance in order to interrupt the transmission of TB. Methods that are

being used today in the diagnostic laboratories or under development show limitations in

producing accurate results. Although a number of diagnostic tests are in use none of them are

optimal - either slow to perform, less sensitive or difficult to establish in resource-limited

countries. Diagnosis of TB infection has been truly dependent on microbiological tests since

the M. tuberculosis was identified. Currently, immunological and PCR-based tests are

available which show an improvement in the detection process. Since a successful penetration

of M. tuberculosis in human host could lead to either latent infection or active disease,

development of diagnostic techniques have also focused separately on these two different

outcomes.

Classical methods

Microscopy and culture

Microscopic visualization of M. tuberculosis is extremely fast, inexpensive and results can be

delivered within hours. This method has been extensively used during the past decades to

diagnose mainly active TB disease. A patient with active TB usually suffers from coughing

and produces sputum and it was observed that mycobacteria are present in the sputum. Ziehl

and Neelsen introduced a special bacteriological stain, called acid-fast stain, for microscopic

visualization of the bacilli. Unfortunately, several shortcomings hinder its use for accurate

diagnosis of TB. It requires 5x103 bacilli/ml concentrations in the sputum samples to be able

to detect them (266), which is nearly impossible to get from the children because they do not

produce enough sputum. Acid-fast staining test cannot be used to monitor drug susceptibility

or resistance which therefore delays the treatment procedure. Some modifications prior to

staining including cytocentrifugation or overnight sedimentation have been found to increase

the concentration of bacilli in the smear and results in higher sensitivity (267).

Conventional culture methods to isolate M. tuberculosis from the clinical samples are

most definitive diagnostic procedures for TB. Mycobacteria can grow in egg based solid

medium such as Lowenstein-Jensen medium or agar based, Middlebrook 7H10 or 7H11

medium and also in liquid medium such as Middlebrook 7H9 broth. Various types of samples

such as sputum, bronchial washings or non pulmonary samples can be used to cultivate

mycobacteria in the laboratory. The major difficulty of laboratory cultivation of mycobacteria

47

is slow growth-rate, which essentially takes an average of 4 weeks and examination of drug-

resistance or susceptibility tests requires additional 4 weeks.

Tuberculin skin test

Tuberculin skin test is performed by intradermal injection of purified protein derivative (PPD)

tuberculin. This test is widely used to diagnose latent infection and the response against PPD

is dependent on T-cell function. PPD tuberculin is a glycerol extract obtained from the culture

of tubercle bacillus. In 1890, Robert Koch first described the PPD tuberculin. After injecting

the PPD into the forearm, an induration is measured in millimetres within 48-72h. Although

this test is simple and easy to perform and has been used in many countries around the world

unfortunately the test results can be positive for the people infected with mycobacteria other

than the M. tuberculosis. More importantly, BCG-vaccinated people react against PPD and

become positive in the test. On the other hand, people with anergy (unable to respond to

foreign molecules) most commonly with HIV infection failed to show enough immune

reaction and could be diagnosed negative in the test (268).

Radiology or chest X-ray

Chest X-ray allows visualization of infiltrates or consolidations and/or cavities in the upper

lungs. Abnormalities on the radiograph do not indicate TB disease however; it can be

suggestive of TB if a person is positive for PPD skin test. Chest X-ray can also be useful in

monitoring the treatment progress of the disease. Radiology or chest X-ray is used in

conjunction with PPD skin test and microscopy for acid-fast bacilli.

New methods

BACTEC radiometric system

The BACTEC (Becton-Dickinson) radiometric system introduced relatively faster detection

of mycobacterial growth compared to the conventional culture assays. In this assay system,

radioisotope, 14C labelled palmitic acid containing 7H12 medium is used to culture the

mycobacteria. Palmitic acid substrate is used up by the bacteria and produce 14CO2, which is

detected in an ionic chamber with electronic detector in the BACTEC instrument. Instead of

waiting for bacterial colony formation, released 14CO2 from the culture is used to calculate

growth index. BACTEC radiometric system can be used efficiently to test drug-susceptibility.

Risk of having exposure of radioisotopes and also the equipment costs preclude its

implementation as a routine diagnostic protocol in the developing world.

48

Microscopic-observation drug-susceptibility (MODS) assay

In order to obtain result as early as possible with high sensitivity, a modified method with the

combination of microscopic and culture techniques has been evaluated recently (269). More

importantly, this combined method is very useful to monitor drug-susceptibility when the

culture is positive. MODS assay has been developed based on the bacterial growth in liquid

medium since bacteria in the liquid medium grow faster than the solid agar. Also, it is easy to

visualize microscopically early the characteristic cord formation of M. tuberculosis in the

liquid medium. Moore et al found that the sensitivity of MODS assay is higher (97.8%) than

the automated mycobacterial culture (89%) or Lowenstein-Jensen culture (84%) techniques

(269). Accomplishment of the MODS test including drug-susceptibility test is faster (2 weeks)

than automated mycobacterial culture (6-7 weeks) or Lowenstein-Jensen culture (13-14

weeks) techniques. Although MODS assay is a fairly cheap and simple method for the

diagnosis of TB, it requires a very skilled laboratory technician to identify the organism

microscopically. Since the culture needs to be handled often, it poses biosafety risk for the

laboratory staffs.

Nucleic acid amplification assay

Nucleic acid amplification assay by polymerase chain reaction (PCR) is a modern approach

for rapid diagnosis of TB. This method allows exponential increase of the copy of target DNA

from the specimens through a process of multiple cycles of denaturation, annealing and

polymerization. The PCR assay is simple and rapid; results can be obtained within a day of

DNA isolation from the clinical samples. Among several target genes identified for the

detection of M. tuberculosis, IS6110 is the most common one, which is present up to 20 times

in the genome. Some other targets including 65 kDa heat-shock protein gene, the 126 kDa

fusion protein gene, 16S-23S spacer region and the gene encoding the β-subunit of RNA

polymerase have also been tested for the identification of M. tuberculosis (270). Although the

PCR-based methods are highly sensitive and specific, major challenges are remained in the

implementation of this technique that include skilled workers, PCR machines and biosafety

level-3 facilities, which are not cost effective for many developing countries.

Immune-based tests

Serological: Detection of specific immune complexes, antibodies in serum or circulating TB-

antigens have been a new attempt for the rapid diagnosis of TB. The most common format of

serological tests is enzyme-linked immunosorbent assay (ELISA) or immunochromatographic

49

tests. An accurate serological test would be advantageous over the conventional microscopy

or culture-based assays. Serological tests are quick to perform and especially needed for the

patients who rarely produce sputum (children) and also are smear negative. The most

common format of serological tests relies on the detection of antigen-specific antibody

responses in sera of TB patients. A meta-analysis of 254 studies has recently been performed

to evaluate the performance of single antigens and multiple-antigen combinations for the

serodiagnosis of pulmonary TB (271). A total of 13 distinct antigens (recombinant 38kDa,

native 38kDa, MPT51, malate synthase, CFP-10, TbF6 polyprotein, Ag85B, α-crystallin, 2,3-

Diacyltrehalose, 2,3,6-triacyltrehalose, sulfolipid I, cord factor, TbF6 plus MPT32) and

several multiple-antigen combinations were included. Compared with the single antigen-

based serological tests (median sensitivity 53%) multiple-antigens used for the detection of

antibodies showed higher sensitivities (median sensitivity 76%). Examination of the

immunoglobulin classes revealed that detection of IgG and/or IgA antibodies provided higher

sensitivities than that used for IgM detection (271, 272). Unfortunately, the detection of

antigen-specific antibodies in sera was not sufficiently sensitive/specific when compared with

the gold standard culture-based methods. Furthermore, individuals exposed to environmental

mycobacteria or upon BCG vaccination develop antibodies against common antigenic

epitopes. This generates difficulties in the interpretation of the results. Hence, antigens such

as ESAT-6 and CFP-10, which are present in M. tuberculosis but not in BCG have been used

to detect antibodies. Unfortunately, the use of ESAT-6 and CFP-10 is also questioned due to

the fact that these two proteins are not exclusive for M. tuberculosis (273) since orthologues

of ESAT-6 and CFP-10 are present in M. leprae and M. smegmatis. Recently, Rv3425, a

member of the PPE family of proteins, encoded by RD11 of M. tuberculosis has been

demonstrated as a potential antigen for serological diagnosis because Rv3425 is missing from

BCG and all virulent M. bovis strains tested (274). Rv3425 specific IgG responses were

significantly higher in patients with active disease compared to the BCG-vaccinated healthy

controls (274). The detection of circulating TB antigens using monoclonal antibodies has been

a promising strategy for the diagnosis of active TB. Among many others, El-Masry et al

developed a modified ELISA, which is called Fast-Dot ELISA (FD-ELISA), for the detection

of circulating 20kDa TB antigen (275). FD-ELISA has been reported to be simple, rapid and

highly sensitive (90.8%) for the detection of TB antigen in serum samples of TB patients.

However, further studies are required to improve reproducibility of the FD-ELISA-based

diagnosis in a large population.

50

T cell-based cellular immune response: The oldest and widely used method based on the cell-

mediated immune response is the tuberculin skin test. A major drawback of this test is that it

produces false positive results in people exposed to the environmental mycobacteria or

vaccinated with BCG. Thus, efforts have been made to develop new tests based on the release

of IFN-γ by T cells upon in vitro stimulation with antigens specific to M. tuberculosis.

Antigens, ESAT-6 and CFP-10 have been used for such stimulation and a great success was

observed in the detection of latent infection with more accuracy than the PPD skin test (276).

This diagnosis method is based on the assumption that T cells from sensitized individuals

produce IFN-γ when they re-encounter antigens of M. tuberculosis. The most promising and

readily available test based on the IFN-γ release assay is QuantiFERON-TB Gold, which is

the advanced version of QuantiFERON-TB. QuantiFERON-TB Gold test was certified in

2005 by United States Food and Drug Administration. A positive test indicates that the M.

tuberculosis infection is likely and a negative test indicates that infection is unlikely. Analysis

from several studies have found that QuantiFERON-TB Gold test has 97.7% specificity (277)

in the diagnosis of latent TB infection but it showed variable sensitivity (55-88%) in the

diagnosis of active TB infection (277). As stated previously, antigens ESAT-6 and CFP-10

are not truly specific for M. tuberculosis, therefore it is likely that the test results might be

influenced in some cases if the subjects are exposed to other mycobacteria. On the other hand,

a significant number of expertise, trained technicians and necessary equipments are required

to run the test smoothly within a short window period 16-24h and interpret the results. The

test is still under evaluation in the diagnosis of TB in children and patients with HIV

infection.

Non-invasive methods: Non-invasive, needle-free, immune-based methods for the diagnosis

of TB are of utmost importance in the developing world to limit the spread of the infectious

diseases. In this case, saliva samples from TB patients can be checked for the presence of

specific sIgA. This might be possible since previous studies have shown in mouse models that

antigen-specific IgA antibodies were increased after mycobacterial infection or i.n.

immunization with mycobacterial antigens (264, 278). In a human study, Araujo et al

succeeded in the detection of 38kDa-specific sIgA in saliva samples of TB patients.

Unfortunately, this study reported very low sensitivity (36.1%) (279). Another non-invasive

immune-based method has been described by Hamasur et al for the rapid diagnosis of TB

based on the detection of mycobacterial LAM in urine samples of TB patients (280). A catch-

up ELISA and a dipstick test were used to detect LAM antigen at concentrations of 1 ng/ml

51

and 5 pg/ml respectively. The sensitivity was 81% and the specificity was 87%. Few

laboratories in the developing countries are well equipped with the necessary safety cabinets

to be able to work safely, thus, this diagnostic method based on the detection of antigen in

urine samples would be extremely valuable.

Biomarker (s) of infection

There have been continuous efforts to rectify the available diagnostic methods for an accurate

diagnosis of TB. Biomarkers are biological features or substances that can be used as

indicators of infection. This definition of biomarker has widened the search for specific

parameters. Recently, IFN-γ-inducible protein 10 (IP-10/CXCL10) has been evaluated as a

potential marker for active TB. It has been reported that IP-10 is secreted by lymphocytes and

monocytes after antigenic stimulation of whole blood cells and could be useful as a biomarker

for active TB in children (281). IP-10 is not a specific marker of TB since IP-10 levels are

also increased in patients with autoimmune disease (systemic lupus erythematosus, thyroid

disease), acute coronary syndrome, cerebral malaria and allergy (282-285). Volatile

metabolites from M. tuberculosis organisms can be used as biomarkers of infection.

Compounds such as methyl phenylacetate, methyl p-anisate, methyl nicotinate and o-

phenylanisole have been detected in cultures before colonies were appeared. Phillips M et al

demonstrated the significance of volatile compounds in the diagnosis of TB based on the fact

that detection of volatile compounds could differentiate between ‘sick’ and ‘healthy’ subjects

including infected and non-infected individuals (286). Cytokine levels in broncho-alveolar

lavage (BAL) could be good markers of infection. TNF, IFN-γ and IL-2 levels in BAL of

patients with smear-negative pulmonary TB are increased significantly compared to the other

pulmonary disease (287). Moreover, other candidate biomarkers for TB infection such as

lactoferrin, CD64, and the Rab33A (member of the Ras-associated GTPase) have recently

been suggested (288). Expression of CD64 on monocytes has been found higher in TB

patients than M. tuberculosis-infected healthy subjects. Similarly, a higher gene expression of

lactoferrin and Rab33A was reported in TB patients. However, these molecules whether or

not specific for TB are yet to be defined. Nevertheless, these molecules may play a role as

part of a panel of diagnostic biomarkers.

52

PRESENT STUDY

Aims

TB remains a global problem even though it is a curable disease. The goal of eliminating TB

is largely dependent on the development of simple and rapid diagnostics, drugs and vaccines.

In order to improve the diagnosis and vaccination against TB, the overall aim of this study

was to understand the mucosal immune responses in the respiratory tract upon mycobacterial

infection or vaccination with mycobacterial antigens in experimental murine models.

Our specific objectives were:

To detect immune responses locally (lungs) and systemically (blood) in mice upon

mycobacterial infection and to identify immunological parameters or biomarkers

associated with the infection (Paper I)

To investigate the priming effect of neonatal BCG-vaccination in a heterologous

prime-boost immunization protocol (Paper II).

To examine the effect of maternal gestational treatment with mycobacterial

antigens on postnatal immunity in the offspring (Paper III).

To determine the importance of innate immunity in mycobacterial antigen

recognition, processing and presentation for the induction of immune responses

and protection (Paper IV).

53

Methodology

The methods used for paper I-IV are described in the respective ‘Materials and methods’

section of each paper. The following methods were used:

-ELISA (Enzyme-linked immunosorbent assay)

-Flowcytometry

-Gel-electrophoresis

-Fluorescence microscopy

-Isolation of mononuclear cells from mouse lung and spleen

-In vitro antigen restimulation

-In vitro restimulation with BCG- or antigen-pulsed bone-marrow derived macrophages

-Statistical analyses

All in vivo experiments were conducted in mice. All experiments were done in accordance

with the ethical guidelines available at Stockholm University.

54

Results and Discussion Paper I

Increased levels of immunological markers in the respiratory tract but not in serum correlate

with active pulmonary mycobacterial infection in mice

The mucosal immune system is highly compartmentalized; origin of activation of immune

cells determines the fate of effector and memory cells. Cells activated in the NALT and

Peyer’s patches preferentially home to the nasal mucosa and to the intestinal mucosa

respectively (289). This unique characteristic of mucosal immune responses led us to

hypothesize that detection of immune biomarkers in the respiratory mucosal secretions but not

in serum would correlate better with mycobacterial infection since nasal mucosa is the natural

path for penetration of the host by M. tuberculosis.

The major findings of this study are:

a) Active mycobacterial infection, but not exposure to non-replicating mycobacteria resulted

in elevation of IL-12, IFN-γ and sTNFR in BAL independently of the route of infection.

Serum levels were not equally conclusive.

b) Reactivation of controlled BCG infection resulted in increased bacteria growth in the lungs

and in increased levels of sTNFR in BAL.

c) Active infection, but not exposure to non-replicating mycobacteria resulted in production of

antigen-specific IgG or IgA in BAL.

One of the major problems of the immune-based diagnosis is that it fails to discriminate

between active infection and the exposure to mycobacteria or BCG vaccination in individuals.

In order to improve the situation, in this study we chose some immune markers namely

sTNFRs (sTNFR1 and sTNFR2) and cytokines (TNF, IL-12 and IFN-γ) and assessed whether

the levels of any of these markers correlated with active but not exposure to mycobacterial

infection. To test this, we modelled the natural route of infection by infecting mice i.n. with

live (replicating) or dead (heat-killed, non-replicating) BCG. To mimic the exposure of

mycobacterial antigens, we also treated mice with BCG-lysate (soluble antigens). The

important observation from this study was that i.n. infection with live BCG but not with non-

replicating BCG or BCG antigens induced elevated levels of sTNFRs, IL-12 and IFN-γ in the

BAL (Fig. 1 in Paper I). A strong positive correlation was observed between the bacterial

55

growth in the lungs and the levels of sTNFR1 and sTNFR2 (r 0.7 and r 0.68, respectively)

(Fig. 1d-e, in Paper I). Unlike sTNFRs, levels of IL-12 showed a poor association with

bacterial growth (r 0.48). In contrast, although the levels of IFN-γ increased after infection

and decreased when the bacterial growth was controlled, the peak of IFN-γ levels did not

coincide with the peak of bacterial growth (Fig.1c, in Paper I). To evaluate whether the

responses were restricted to the i.n. route of infection, we measured cytokines and cytokine

receptor after infecting mice intravenously (i.v.). Similar results were obtained even after i.v.

infection. In contrast to the data obtained from BAL, results from the serum analysis showed

that infection with live BCG as well as treatment with non-replicating BCG caused increased

levels of IL-12 and sTNFRs in serum, however, there was no correlation found between the

serum cytokine or sTNFRs and the bacterial growth in the lungs (Fig. 2a-c, in Paper I).

In general, upon exposure to M. tuberculosis, lung macrophages in the host

phagocytose the bacilli and thereafter host immune responses are initiated to eliminate the

bacilli. In response to infection, macrophages produce cytokines such as IL-12 and TNF and

T cells are recruited to the site of infection and produce IFN-γ. A combination of TNF and

IFN-γ mediated activation of macrophages leads to the formation of granuloma (290). The

findings from Paper I strongly emphasise on the fact that the measurement of immune

responses particularly cytokines or sTNFRs in the respiratory mucosal secretions but not in

the blood might be better correlates of infection. The relevant supports came from the

experiments when we tested non-replicating BCG or BCG-lysate i.n. for the induction of

immune responses. Daugelat and colleagues have reported that infection with live- but not

killed-BCG resulted in the activation of a type of T cells, which are able to recognize secreted

antigens (291). The difference between the live- and dead-BCG lies on the fact that live BCG

could replicate and secrete antigens and therefore stimulate a wide range of immune cells

compared to the non-replicating BCG. In addition, the responses in the lungs after live-BCG

infection were even detectable in the blood but did not correlate with the bacterial burden

neither in the spleen nor in the liver.

In recent times, several studies have focused on the detection of immune parameters

locally in the lungs, which might be good correlates of infection. Expression of IL-12 and

IFN-γ mRNA in the BAL (292) and elevated levels of cytokines such as IFN-γ, IL-12, IL-1β,

IL-8 and TNF produced by broncho-alveolar cells were described to be associated with active

pulmonary TB (293). In addition to the cytokine levels, cytokine receptors, such as sTNFR,

have been reported to be a sensitive marker of infection. Increased shedding of sTNFR1 into

56

serum is predominantly associated with TB rather than HIV infection when examined in

subjects categorized into TB only (TB+HIV-), TB and HIV co-infection (TB+HIV+), HIV

infection only (TB-HIV+), or neither infection (294). The levels of sTNFR1 are reduced even

after anti-tuberculosis treatment (294). Guler et al reported that transgenic mice expressing

high serum levels of sTNFR1 exhibited reduced bactericidal activity, undifferentiated

granulomas and succumbed to BCG infection (149). Despite the fact that serum levels of

sTNFR are also found to be possible markers for infection, unfortunately exposure to

mycobacterial antigens alone could also induce sTNFR levels in serum, suggesting that serum

analysis might not provide true results.

Upon infection with M. tuberculosis it is estimated that about one-third of the world’s

population is latently infected and among them the annual risk of reactivation of infection is

approximately 0.1%. However, it has been estimated that only 10% of those with latent

infection will ever develop active disease in their lifetime (295). The nature of the reactivation

of latent infection is explained by the failure of the equilibrium between the host responses

and the bacterial growth. Live bacillus within the granuloma can persist for many years as a

clinically inactive state (296, 297). Even if the latently infected people pose relatively lower

threat compared to the people with clinically active TB disease, however, diagnosis of the

latent infection as well as diagnosis of active disease converted from the latent infection is of

utmost priority. We therefore, modelled the latent infection and the reactivation of infection in

our current study. Results showed that treatment of mice with dexamethasone (DXM), a

corticosteroid that suppresses the effector functions of T cells, reactivated the bacterial growth

in the lungs from a state of infection, which was experimentally considered latent. Analysis of

sTNFRs in BAL and serum indicated that sTNFR levels increased significantly in BAL 2-

weeks after the DXM treatment (Fig. 3b–c, in Paper I), and this correlated strongly with the

bacterial growth in lungs (r 0.9 and r 0.7 for sTNFR1 and sTNFR2, respectively). In contrast,

sTNFR levels in serum were not increased and were comparable to the untreated group. Thus,

our observation from such reactivation model also suggests that sTNFR level in BAL is a

good correlate of active infection. Elevated levels of pro-inflammatory cytokines are a general

characteristic of inflammatory responses upon infection. Therefore, TNF production is a

prerequisite for the granuloma formation, important for restriction of the mycobacterial

growth and dissemination. This supports the fact that neutralization of the TNF activity could

lead to mycobacterial growth and we reasoned that elevated levels of sTNFRs resulted in TNF

neutralization (149). Of note, TNF in BAL was undetectable in this study using BALB/c

mouse strain. Similar observations were made for the BALB/C strain when we compared

57

BALB/c with C57BL/6 mice after i.n. infection with BCG (Paper V, not included in this

thesis). Compared to the BALB/c, C57BL/6 mice produced significantly higher amounts of

TNF.

It is, however, important to acknowledge the fact that the measurement of cytokines or

cytokine receptors for instance sTNFRs either in BAL or in serum is not related with the

disease specificity. As reported earlier, sTNFR levels are not only increased in mycobacterial

infections, but also in many other inflammatory diseases (298-300). Therefore, we reasoned

that detection of specific antibodies and cytokines or soluble cytokine receptors could be used

as biomarkers for distinguishing active infection from latent infection. Detection of

mycobacteria-specific antibodies in serum of TB patients by ELISA is simple, inexpensive

and straightforward. Unfortunately, serodiagnosis is still unreliable due to the fact that

serodiagnosis alone is unable to differentiate between the active and the exposure to the

infection. Antibody responses are mounted during the active infection as well as after being

exposed to mycobacterial antigens since this does not require the presence of live bacilli.

Results from our study also confirmed this fact that the antigen-specific antibody levels in

serum are not true indicators of active infection since the increased antibody levels were

observed after treatment of mice with non-replicating BCG or BCG-antigens. In contrast,

antigen-specific antibody levels in BAL samples were nearly undetectable after treatment

with non-replicating BCG or BCG-antigens. Importantly, we found that antigen specific

antibody levels in BAL were increased after infection with live BCG although there was no

correlation between the bacterial growth and the antibody levels. This may be expected since

the immune system becomes activated by the bacterial presence and starts generating

antibody responses, which probably sustained for a certain time. On the other hand, the

control of bacterial growth does not necessarily depend on the antibody responses. The cell-

mediated immune response is most likely involved in the control of mycobacterial infection.

Unfortunately, the correlation between active infection and cell-mediated immune responses

are also subject to question, because mycobacterial exposure, but not active infection, could

induce cell-mediated immune responses. In conclusion, we suggest that single immune

parameters are not sufficient to be considered as diagnostic biomarkers of mycobacterial

infection even when assessed in the local lung environment, however, this shortcoming could

be substantially eliminated by combining two immune parameters such as measurement of

TNFRs and antigen-specific antibodies.

58

Paper II

Neonatal vaccination with Mycobacterium bovis BCG: potential effects as a priming agent

shown in a heterologous prime-boost immunization protocol

A new vaccine against TB is urgently needed for the control of the disease. In recent times,

there have been significant advances in the development of new TB vaccines particularly in

the improvement of the BCG vaccination. The best model for the improvement of BCG

vaccination is probably the heterologous prime-boost strategy (258, 301-304). In this

protocol, priming with BCG and boosting with either DNA-based vaccine or adjuvanted

protein vaccine have been reported to be very efficient in providing protection in animal

models of TB. In this study, we evaluated a single mycobacterial protein, HBHA as a BCG

boost vaccine. HBHA is one of the most potent mycobacterial antigens tested in murine

models for immunization and found to induce protection against M. tuberculosis challenge

which is similar to the protection provided by the BCG vaccination (204). We addressed how

the neonatal BCG vaccination could contribute as a priming agent to a heterologous prime-

boost immunization protocol for HBHA.

The major findings from this study are

a) Neonatal BCG vaccination primed the immune system for native (n) HBHA and therefore

upon HBHA boosting, immune responses to HBHA were improved.

b) Priming with BCG and boosting with nHBHA (BCG/nHBHA) significantly improved

protection against BCG infection independently of the route of nHBHA immunization and the

coadministration of adjuvant.

c) Immune responses induced after a shorter prime-boost interval decline with time,

suggesting that repeated boosting with an optimal prime-boost interval is required.

d) Boosting with non-protective form of rHBHA induced protection in the BCG-primed mice.

The neonatal immune system is not fully matured and often biased to Th2 type response. The

challenge of neonatal vaccination arises due to the poor response of neonates to most

vaccines. Neonatal BCG vaccination is one of the few examples that can shift the immune

system from a Th2 to Th1 type response (305). Although the BCG-induced immunity wanes

with time, BCG vaccination can induce even longer protective immunity than subunit

vaccines (212). In order to maintain the BCG-derived immunity, revaccination with BCG has

been pursued in different animal models as well as in humans and it revealed that repeated

59

BCG administration did not add benefit rather found to be detrimental (172, 306-309).

Revaccination with BCG induces excessive proinflammatory cytokine production and results

in lesions formation (306).

Prime-boost vaccination strategy has been focused much for the TB vaccine

development although it is already in use for humans. Retrospectively, some of the vaccines

such as Diphtheria, Tetanus and Pertussis (DPT) are given to the paediatric population three

times untill six months of age followed by a final boost between four to six years of age (304).

The basic mechanism of this strategy lies on the fact that the first dose of the vaccine primes

the immune system and the booster dose with the same vaccine further increases the immune

responses and therefore the vaccine-derived immunity lasts long. Recently, it has been

discovered that same vaccines given multiple times (called homologous prime-boost

vaccination) are less effective than same vaccines delivered in different forms (called

heterologous prime-boost vaccination) (304). Heterologous prime-boost technique was first

introduced by Shiu-Lok Hu and co-workers in 1992 in the context of HIV vaccine

development. Since then many studies were conducted by using this approach for the

induction of cellular immunity to a variety of pathogens including M. tuberculosis (170, 310-

313).

In this study, following vaccination with BCG/HBHA combination we observed that

BCG vaccination not only primed the immune system to HBHA but also BCG itself acted as

an immune adjuvant. We found that introduction of CT adjuvant in nHBHA vaccine did not

show a better protection against infection than the nHBHA formulated without CT (Fig. 3 in

Paper II). Interestingly, priming with BCG improved protective immune responses even for

rHBHA, which was previously reported to be non-protective (204). We observed that lung

lymphocytes from the BCG-primed and HBHA-boosted mice produced large amounts of IFN-

γ upon stimulation with BCG-infected bone-marrow derived macrophages (BMMBCG), a

methodological approach suggested to correlate with the level of protection (314, 315). IFN-

γ levels upon restimulation with HBHA were also induced but were unable to correlate with

the level of protection. This perhaps could be explained in two ways: upon BCG infection,

BMM present mostly the protective epitope to the lymphocytes from HBHA-immunized

mice, which is not the case with HBHA stimulation. In another way, the condition with

BMMBCG facilitates antigen presentation in an alternative way, namely cross-presentation.

Investigation made by Pramod K. Giri and Jeffrey S. Schorey demonstrated that BMMBCG

deliver fully-functional antigens-loaded exosomes and that stimulate both CD4 and CD8 T

cells via class I and class II mediated antigen presentation pathways (316). The classical way

60

of assessing the antigen-specific recall immune responses is to restimulate the immune cells

with the same antigens used for immunization. Unfortunately, this method does not provide

correct information regarding the status of the immunized animals whether protective or non-

protective. Of note, it has been demonstrated previously that, cells isolated from rHBHA-

immunized animal were able to produce substantial amount of IFN-γ after in vitro

restimulation with rHBHA however, this did not correlate with the protection (204).

Similarly, another mycobacterial antigen (27-kDa) used for vaccination has been reported to

induce higher levels of IFN-γ but not to offer protection (317). In this regard, many studies

have reported the potential use of multifunctional-T cells as immune correlates of protection

(257).

Even if rHBHA has been found non-protective upon systemic delivery, recent study

showed that mucosal delivery of rHBHA offered limited dissemination of mycobacteria from

the lungs to the spleen (318). In our study, vaccination with BCG followed by rHBHA

delivered either i.n. or s.c. induced a certain level of protection as the bacterial growth was

reduced both in the lungs and in the spleen. Presently, we cannot explain the mechanism of

protection induced after rHBHA boosting in the BCG-primed animals. Most probably rHBHA

acted as a carrier molecule for the protective epitopes generated after BCG vaccination.

Induction of protective immune responses in the respiratory compartment is probably

the most effective way to prevent pulmonary TB since the respiratory tract is the natural route

of M. tuberculosis infection (171, 319, 320). Therefore, our main aim has been centered on

the possibility to induce a strong protection against TB based on the lung immunity. Indeed,

several studies have already described the potential benefits of mucosal delivery of booster

antigens into the BCG-primed animals including the more sensitive guinea-pig models (172,

321). Despite the fact that mucosal delivery of antigens holds the great promise for future

vaccination, an implementation of such strategy is under challenge in the case of adjuvanted-

antigen delivery. Today, there is no secure adjuvant available for human use. Considering

this, we primarily aimed to compare i.n. versus s.c. administration of HBHA booster antigen

in the BCG-primed mice which showed a comparable level of immune responses after

restimulation with BMMBCG and that correlated with the protection (Fig. 2 and 3 in Paper II).

Since the s.c. route of immunization was as effective as the i.n. route which is in contrast to

the other reports, we examined whether HBHA boosting in the absence of adjuvant could

maintain the similar efficacy. Results from our study demonstrated that immune responses

and the ability to control bacterial infection were remained unchanged which were

comparable to that achieved by adjuvanted-vaccine delivery.

61

Does neonatal BCG vaccination contribute by priming the immune system in advance

or offer generalized adjuvant effects? It has been postulated that treatment with the live BCG

possesses a more general adjuvant effect and thus able to influence the response to unrelated

antigens (322). We continued our investigation to delineate the role of BCG vaccination prior

to the boosting with HBHA. Since BCG contains the HBHA antigen it is therefore expected

that the immune system could be primed by BCG vaccination. In this study, we also re-

examined the BCG-derived adjuvanticity by using ESAT-6, not present in BCG, in a

heterologous prime-boost vaccination protocol. We also asked whether the BCG-mediated

adjuvanticity is attributable to the status (live or killed) of the bacteria in the BCG-vaccine.

Results showed that even if ESAT-6 is not present in BCG, priming with live BCG but not

with HK-BCG and boosting with ESAT-6 enhanced ESAT-6-specific recall IFN-γ responses

(Fig. 5 in Paper II) indicating that live bacteria could act as an immune adjuvant when

injected prior to the antigen. In contrast, both live and HK-BCG were able to increase the

recall IFN-γ responses to HBHA (Fig. 5 in Paper II) suggesting that for HBHA, BCG has a

dual mode of activity: specific in the case of killed-BCG, and an additional adjuvant effect

when given as live bacteria. These results are in agreement with the previously published data

where it has been demonstrated that live but not killed BCG is an immunostimulant (323) and

due to this property BCG is used as an immunotherapeutic agent against bladder cancer (324).

Collectively, our study unveiled how neonatal BCG vaccination could be of

importance in the response to a booster antigen whether or not the antigen belongs to the

BCG. Additionally, a combination of BCG and HBHA vaccination in the order of BCG-

priming and HBHA-boosting offers better immune response and protection regardless of the

route of vaccine delivery and adjuvant dependency. Since the immune responses decline with

time even following vaccination with BCG/HBHA, thus, repeated boosting with HBHA is

necessary for the maintenance of immunological memory.

62

Paper III

Influence of maternal gestational treatment with mycobacterial antigens on postnatal

immunity in an experimental murine model

It is thought that the major constraint for a successful vaccination in the neonates is the

immature immune system. In most cases, upon vaccination neonates remain unresponsive to

the vaccine antigens or develop Th2 type response. A tendency of developing limited Th1

immune responses could contribute to the severity of infection with intracellular pathogens in

the early life. Therefore, to prevent the risk of contracting diseases during the early life,

several vaccination strategies are currently in progress. Most recently, early life vaccination,

as early as during the fetal life, has been suggested; provided that the fetal immune system

could be primed in advance and that neonates become ready for postnatal vaccination. In

paper III, we have examined whether the fetal immune system could be primed in advance by

gestational treatment with mycobacterial antigens and thus postnatal vaccination could be

done efficiently.

The major findings from this study are

a) In utero exposure to mycobacterial antigens during the 2nd week of pregnancy led to

antigen transportation from the mother to the fetus through the placenta.

b) Immunization with the same mycobacterial antigen during the postnatal life improved T-

cell responses compared to the offspring born to the mother untreated during the gestational

period

c) Upon challenge with BCG, offspring could control infection better owing to an early

exposure of mycobacterial antigen in utero.

The placenta is a highly specialized organ that forms an interface between the maternal and

the fetal circulation and regulates the transport of materials from the mother to the fetus and

wastes in the other direction. Recently, this transportation route has got attention with the

probability that mother-derived substances delivered to the fetus could influence

immunological activation of the fetal immune system (325-327). However, it is assumed that

the placental barrier only allows certain material exchange, which perhaps reduces the

chances of the fetal immune system to be exposed to a broad range of substances. Therefore,

uncertainty remains in the case of protein antigen transportation through the placenta. On the

other hand, it has been shown that maternal immunological experience (transfer of IgG) could

63

be delivered to the fetus through the placenta and this way fetus as well as neonates could be

protected (328, 329). Increasing number of evidence suggesting that allergens can be

transported through the placenta (330) and the immune system is primed in utero by allergens

(326). Therefore, in this study, we addressed whether the fetal immune system is exposed to

protein antigens (foreign) and hence could be primed earlier. In this study, we showed that

mycobacterial protein antigens were transported through the placenta after administered to the

mother at the 2nd week of pregnancy. Fluorescent quantum dot conjugated with protein

antigens were injected to the mother and several hours postinjection fluorescence signals were

detected in the whole fetus and placenta samples. A time point of gestational period could be

important for the maturation of placenta as well as for the development of fetal cells. In mice,

two weeks of gestational period seem to be enough for the maturation of placenta.

We have investigated the effects of antigen transfer from the mother to the fetus on the

immune responses generated in the offspring. In order to examine this antigen-specific effect

we have immunized offspring mice with the same antigens used for maternal treatment. As

previously stated, in addition to transplacental antigen transportation, antigens could also be

transported via milk. We examined this effect by including a group of newborn mice born to

the treated mother and nursed by the untreated mother. Antigen-specific T cell and B cell

responses in the offspring mice were examined. For T cell activation, lung lymphocytes from

the immunized mice were restimulated in vitro with the antigens and IFN-γ responses were

determined. For B cell responses, antigen specific IgM and IgG antibody levels were

measured in sera of immunized mice. We observed that antigen-specific T-ell responses (IFN-

γ levels) were significantly higher in the mice born to the treated mother than the offspring

born to the untreated control mother (Table 1, in Paper III). Similarly, significantly higher

levels of T-cell responses were observed in the offspring mice born to the treated mother but

nursed by the untreated foster mother. Antigen-specific antibody responses were remained

unchanged in the offspring mice born to the treated mother as compared with their control

littermates. These results indicated that gestational treatment with mycobacterial antigens

resulted in priming of the fetal immune system and that enhanced the T-cell responses

following postnatal immunization. We also examined antibody responses in the pregnant

mother post-treatment with antigen and found that there was not any detectable antibody

response in the serum. Since the gestational treatment was performed in the absence of

adjuvant this perhaps explained why the treated mother had no detectable antibody response.

Thus, the results from this study indicated that there was no influence of maternal antibody

response on the T cell responses in the offspring.

64

Why was the T cell compartment but not the B cell compartment sensitised following

gestational treatment? An analysis of murine T- and B-cell ontogeny revealed that B-cell

development is delayed compared to the T-cell development. Fetal thymocytes are able to

proliferate in response to T-cell mitogens by day 17 of gestation (331) and also a few matured

CD4 and CD8 T cells can be found immediately before birth. In contrast, the B-cell

development starts after birth. This species-specific T- and B-cell ontogeny might explain the

reason why we have seen the activation only in the T-cell compartment after gestational

treatment. Compared to the mouse T- and B-cell ontogeny, in humans functional T and B

cells are found before birth (331). An increasing number of studies in human models proposed

that fetal or neonatal immune responses are increased by the prenatal priming (199, 332) and

this might occur due to the transfer of antigens through the placenta. However, in those

studies, there was no direct evidence showing that antigens were transported through the

placenta. Our current study has provided evidence for the placental-antigen transportation and

its effects on postnatal immune responses.

It is, however, not clear how fetal T cells were primed following gestational treatment

with antigens. Priming of the T cells requires antigen presentation by APCs. Therefore, the

APCs in utero should have attained certain level of maturity to present antigen to the T cells.

Very little is known about the phenotype and functional development of APCs during the fetal

life. In humans, it has been shown that the percentage of MHC Class II-positive fetal

monocytes was increased over the gestational period although it was lower compared to the

adult monocytes (185). Moreover, the percentage of CD40+ or CD86+ fetal/neonatal

monocytes was comparable to the adult (185).

The BCG vaccine against adult pulmonary TB induces partial protection but its

efficacy is more satisfactory against disseminated forms of TB. Thus, the BCG vaccination is

still recommended in many countries around the world where the risk of contracting infection

is high. In general, the BCG vaccine is safe for humans with the exception of a few reports

where disseminated BCG disease was detected post BCG vaccination (333). Disseminated

BCG disease is very uncommon and might be seen in immunocompromised infants or adult

individuals with HIV infection. This poses a risk against the continuation of BCG vaccination

in immunocompromised individuals. Thus, the findings from this current study might be

useful for the development of a future TB vaccine targeting a particular group of people, who

are at greater risk of receiving the BCG vaccine.

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Paper IV

Immunization with mycobacterial antigens: a role for innate immunity in antigen presentation

TLR2 has been identified as an important mediator of macrophage activation in response to

M. tuberculosis (334). Three structurally different components of mycobacterial cell wall

fractions enriched with LAM, mycolylarabionogalactan-peptidoglycan complex or total lipids

induce TNF production upon recognition by macrophages via TLR2 (334). TNF production is

important for granuloma formation and induction of protective immune responses (334). In

the absence of TLR2, macrophages have decreased ability to produce TNF in response to

mycobacterial infection and animals are susceptible to mycobacterial infection (335, 336). In

Paper IV, we aimed at investigating the role of TLR2 for the induction of antigen-specific

immune responses upon immunization. We immunized wild-type (WT) and TLR2-/- mice

with mycobacterial 19kDa (TLR2-agonist) or Ag85A (irrelevant for TLR2). We measured

antigen-specific humoral and cellular immune responses. We also examined whether TLR2 is

necessary to activate APCs and to promote antigen presentation in vitro.

The major findings from this study are

a) Antigen-specific antibody responses were comparable between the WT and TLR2-/- mice.

b) Immunization with 19kDa induced more Th1 type response as reflected by the increased

levels of IgG2a in serum.

c) Recall IFN-γ responses to 19kDa were significantly lower in the TLR2-/- mice compared to

the WT-type mice.

d) Spleen cells from TLR2-/- mice secreted comparable levels of IFN-γ in response to

BMMBCG.

Our data from this study showed that antigen-specific antibody responses in serum were

enhanced both in the WT and TLR2-/- mice (Table 1, Paper IV). 19kDa is a culture filtrate

protein of M. tuberculosis and by virtue of its potent immunostimulatory properties,

immunization with 19kDa induced potent humoral and cellular immune responses, which

have also been described previously (336, 337). Purified 19kDa antigen induces

proinflammatory cytokine IL-12 production in the macrophages and could influence the

development of Th1 type response in the host (338). Consistent with this notion, we found

that mice immunized with 19kDa had more Th1 type response than mice that received

Ag85A. The immune responses induced in the TLR2-/- mice raised the question about how

66

the 19kDa antigen was taken up by the cells and how the immune system was primed in the

absence of TLR2. There has been data indicating that 19kDa lipoprotein of M. tuberculosis

functions as a major adhesin, interacts with MR and promotes phagocytosis of mycobacteria

(339). The 19kDa antigen that we used for immunization was a purified recombinant protein

without posttranslational modifications, hence we speculate that in the absence of TLR2,

19kDa antigen can be internalized by other possible receptor-mediated endocytic

mechanisms, which include membrane immunoglobulin or Fc-receptor-mediated interaction,

micropinocytosis or macropinocytosis (340).

We attempted to determine the cellular immune responses by restimulating spleen

cells from the immunized mice with the antigens. Upon in vitro restimulation primed cells

become activated and secret IFN-γ in the culture supernatants. We observed that cells from

the Ag85A-immunized mice produced comparable levels of IFN-γ independently of the type

of mouse strains. By contrast, a noticeable difference was found in the case of the 19kDa-

immunized mice. In the absence of TLR2, cells produced less IFN-γ, suggesting two

possibilities: either the T cells were not properly primed in vivo or the antigen presentation in

vitro was not sufficient. We addressed these two questions in the subsequent in vitro

experiments by coculturing spleen cells from TLR2-/- mice with BMM pulsed with antigen

(BMMAg) from WT mice and vice versa. Our data from this experiments showed that both

spleen cells and BMM from TLR2-/- mice were functionally active when cocultured with

BMM and spleen cells from WT-type mice respectively. These results indicated that probably

the immunological synapse between the T cells and the BMM was not sufficiently formed in

the absence of TLR2. If this is true, the T-cell receptor (TCR) and MHC-peptide complex was

not formed or the necessary costimulatory signals were not generated. However, the former

might not be possible since the IFN-γ responses were not completely abolished in the absence

of TLR2. Thus, we examined the expression of costimulatory molecules in the BMM upon

activation with 19kDa and the data revealed that CD86 costimulatory molecules were not

upregulated in the TLR2-/- BMM, indicating that TLR2-dependent activation was necessary.

By contrast, TLR2-dependent activation of BMM was not seen when BMM were pulsed with

BCG (BMMBCG). Surface expression of CD86 and CD80 was increased in both WT-type

TLR2-/- BMMBCG. Hence, spleen cells from TLR2-/- mice were activated in the presence of

TLR2-/- BMMBCG. These results suggested that APCs from TLR2-/- mice could be

differentially activated when pulsed with BCG instead of 19kDa antigen.

67

Although the 19kDa antigen is a potent Th1-promoting agent, several in vitro

investigations have shown that prolonged exposure to the 19kDa antigen through TLR2

inhibited antigen processing by reducing MHC II expression (341, 342) and also interfered

with IFN-γ-induced mycobacterial killing (343). Likewise, it has been shown in vivo that

immune responses were modulated by 19kDa after immunization with rBCG overexpressing

the 19kDa antigen (rBCG19N) (338, 344). Upon overexpression of 19kDa, responses to

BCG-antigens were polarised towards Th2 type (338). In contrast, responses to 19kDa antigen

remained Th1 type (338). Consistent with the previous observations, we found Th1-

dominated humoral and cellular immune responses following immunization with the 19kDa

antigen. In contrast to the in vitro data that support the inhibitory role of the 19kDa antigen,

we however, did not observe any inhibitory function of 19kDa in the BMM pulsed with

19kDa. Therefore, cell surface expression of MHC II, CD86 and CD80 was not inhibited.

Since 19kDa is a TLR2 ligand, we rather found TLR2-dependent activation of BMM after

pulsing with 19kDa.

Together, our results from this study suggested that even a lower level, immune

responses could be generated in the absence of TLR2. TLR2-mediated innate responses were

important for the presentation of the 19kDa antigen and activation of T cells. Thus, in the

absence of TLR2, 19kDa-specific responses were substantially compromised.

68

Concluding remarks and future perspectives

The current studies have focused on two issues; improvement of the diagnostic procedures

and the development of better vaccines which are likely the key components in the overall

strategy for the control of TB. We reasoned that understanding of the local (respiratory

mucosal) immune responses would be more rational since the respiratory tract is the natural

route of M. tuberculosis infection. In Paper I, we have shown that the detection of cytokines

or cytokine receptors together with antigen-specific antibodies IgG or IgA in the respiratory

tract but not in the serum distinguishes active infection from latent infection or exposure with

mycobacterial antigens. Cytokines or cytokine receptors correlate better with active infection

and specific antibody responses are needed for the identification of the causative organisms.

Based on the findings from Paper I we envisage that it will be of clinical importance to

examine these immunological parameters in patient with active or latent infections. In

humans, obtaining bronchoalveolar lavage samples represents a challenge to the implication

of this strategy. Therefore, one approach to meeting this challenge and clinical entropy might

be to use human saliva or sputum samples for the detection of immunological biomarkers.

To ameliorate TB vaccination strategies, in Paper II, we attempted to understand how

the BCG vaccination works after neonatal administration and whether the immune responses

could be enhanced by boosting with mycobacterial HBHA. The results indicate that BCG

vaccination has a dual role: BCG-vaccination primes the neonatal immune system for HBHA

and thus boosting with HBHA further improves immune responses and protection in the

respiratory tract; BCG-vaccination has a generalized adjuvant effects to BCG-related or

unrelated antigens. Priming with BCG followed by boosting with HBHA improves the

immune responses and protection independently of the route of vaccination and the use of

adjuvant. This heterologous prime-boost protocol shows that the magnitude of the immune

responses drops with time, suggesting that repeated booster immunizations are needed to

maintain the memory. Since the neonatal BCG-vaccination is still recommended for the place

where the risk of childhood TB is high, it is logical to use the BCG as a priming vaccine. In

the future studies using more sensitive animal models, a comprehensive analysis is required to

further assess the importance of the BCG as a priming vaccine. Essentially, the route of

vaccination and the need of adjuvants are needed to be re-examined in the course of

generating immune responses and protection in the lungs.

In Paper III, we sought to examine whether neonatal vaccination with mycobacterial antigens

could be improved by prenatal priming. Summarizing the results from Paper III, we conclude

69

that maternal gestational treatment with mycobacterial antigens primes the fetal immune

system; therefore, postnatal immunization with the same antigens enhanced immune

responses and protection in the offspring mice. We provide evidence that mycobacterial

antigens can be transported from the mother to the fetus through the placenta. In a future

perspective, it would be of great importance to get an in-depth knowledge on the maternal-

fetal immunological interactions in relation to the antigen presentation to the T cells and

generation of memory T cells during the fetal stage. Species differences are obvious regarding

the ontogeny of immune cells, thus one would have to examine whether placental-antigen

transportation could prime the fetal immune system in humans. It would be of great interest to

examine the influence of maternal-immune mediators on the prenatal priming and thus to

unfold immune-regulatory mechanisms in connection with the neonatal unresponsiveness. It

is likely that the neonatal BCG vaccination in the immunocompromised individuals results in

BCG-itis (regional disease) or BCG-osis (disseminated disease), therefore the strategy with

the prenatal priming with mycobacterial antigens holds a great promise particularly for the

new generation of TB vaccines.

In Paper IV, we addressed the role of innate immunity in the course of generating adaptive

immune responses. In this preliminary manuscript, we conclude that immunization with

mycobacterial antigens enhances antigen-specific humoral and cellular immune responses

even in the absence of TLR2. Memory T cells from TLR2-/- mice are not sufficiently

activated upon in vitro restimulation with 19kDa antigen presumably it requires TLR2-

dependent activation of APCs. Formation of TCR and MHC-peptide complex followed by the

expression of necessary costimulatory molecules might be required for the generation of

immunological synapse. Future studies should investigate how innate immune responses

could contribute to the formation of immunological synapse between the antigen-specific T

cells and the APCs.

70

Acknowledgements

This study was carried out at the Department of Immunology, Stockholm University. I am

grateful to all who were involved in these studies, in particular my supervisor Prof. Carmen

Fernández for her strong support from the first day in Sweden. I thank Carmen very much for

opening a PhD position for me and assisted me in my growth scientifically over the past

years.

I thank Prof. Marita Troye-Blomberg, Prof. Klavs Berzins, Prof. Eva Severinson and Associate Prof. Eva Sverrenmark for all the important suggestions towards improving the quality of the work. Apart from the scientific discussion you all have given a nice friendly environment to the immunology department and I feel proud of that.

My special thanks to a former PhD student John Arko-Mensah, who worked in the TB project with me and we shared first authorship in some articles. I appreciate John very much for his relentless cooperation in the laboratory. I thank Olga Flores who is currently a PhD student for providing me help whenever I asked for. I also thank Irene Roman for her active participation and strong will towards achieving results. I am grateful to Eshter Julián, a former postdoc in our group, for her excellent care at the beginning of my degree project.

I appreciate Eva Nygren and all the staffs at the animal house for their important support in the animal house. Especially, I got remarkable help from Eva Ngren with all the breeding experiments.

I thank Manijeh Avedi for helping me with FACS antibodies.

I thank Magaretha Hagstedt, Ann Sjörlund, Gelana Yadeta, Anna Leena for their

invaluable assistance. I will never forget your contribution.

I would like to extend my warmest thanks to Ebba, Maria, Pablo, charles and Shahid. You

were as nice to me as you are always. I found you extremely cooperative.

Thanks to all past and present colleagues at the department especially Shanie, Yvonne and Salah for sharing the knowledge of Flow cytometry. Thanks also to katharina, Stefania, Anna, Petra, Halima, Manijeh, Lisa, Nora, Ylva, Nancy, Khosro, Jacqueline, Gishanti, Amre, Nnaemeka, Jacob, Gudrun, Sandra, Christian, Ulrika, Magdi, and Camilla for the nice discussion at the department.

I appreciate beyond words the families who over the years supported me for everything. A special thanks to my wife for her patience and constant encouragement. Thank you Ilma, my little daughter, with a smile always, a special gift from the Almighty. You have got an unbelievable understanding which helped your pappa working at home. You never annoyed me while I was writing rather brought my hand to the computer whenever you saw me resting.

71

My special thanks to Qazi Khaleda Rahman who actually paved the way for us in Sweden for higher study. I thank Apu Bhai who is very nice person that I ever found. Thanks to Ariane for your cute and lovely gesture that gave me immense pleasure. I thank Dr. Atikul Islam and Dr. Zarina Kabir for helping my family on several occasions.

I owe a debt of gratitude to my parents and sisters for their continuing support, grounding and love. These studies were financially supported by grants from Hja¨rt-Lungfonden, European Commission, FP6 and FP7, LSHP-CT-2005 018736 and HOMITB200732 respectively.

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