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Vaccine 29 (2011) 2590–2600

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

urther evidence of antigenic drift and protective efficacy afforded by aecombinant HVT-H5 vaccine against challenge with two antigenicallyivergent Egyptian clade 2.2.1 HPAI H5N1 strains

. Rauwa,∗, V. Palyab, S. Van Borma, S. Welbya, T. Tatar-Kisb, Y. Gardinc, K. Moore Dorseyd,.M. Alye, M.K. Hassane, M.A. Solimane, B. Lambrechta, T. van den Berga

Avian Virology and Immunology Unit, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, 1180 Ukkel, Brussels, BelgiumCEVA Phylaxia, Szallas utca 5, H-1107 Budapest, HungaryCEVA Santé Animale, BP 126, 33501 Libourne, FranceCeva Biomune, 8901 Rosehill Rd Lenexa, KS 66215-3513, USAAnimal Health Research Institute, Nadi El-Seid Street, Doki, P.O. Box 264, 12618 Giza, Egypt

r t i c l e i n f o

rticle history:eceived 23 September 2010eceived in revised form 7 December 2010ccepted 18 January 2011

a b s t r a c t

In this study, we have compared the protection afforded by a recombinant turkey herpesvirus vaccineexpressing the H5 gene from a clade 2.2 H5N1 strain (rHVT-H5) and a Mexican-origin H5N2 inactivatedvaccine, alone or in combination, against two antigenically divergent H5N1 Egyptian strains isolatedin 2007 and 2008. Our results confirm the existence of a major antigenic drift among the Egyptian

vailable online 1 February 2011

eywords:nfluenzahicken5N1

H5N1 strains such that, although protection against the “classical” 2007 HPAI H5N1 Egyptian strain couldbe obtained with both types of vaccines, only vaccination with the rHVT-H5 vaccine protected againstchallenge with the “variant” 2008 HPAI H5N1 Egyptian strain.

© 2011 Elsevier Ltd. All rights reserved.

gyptaccination

. Introduction

H5N1 highly pathogenic avian influenza (HPAI) virus emergedn Egypt in early 2006, and then spread very rapidly within the

hole country with a huge number of outbreaks both in rural andntensively reared poultry [1]. The main approaches for control-ing the outbreaks have been the culling of all poultry suspectedf harbouring the virus and the vaccination of birds [2,3]. Around0–35 million birds have been culled so far, which has helpedo control the infection in commercial farms. Initially, emergencyaccination was used to protect grand-parent and parent flocks.owever, stamping out policy and targeted vaccination were insuf-cient to control the situation with the outbreaks spreading rapidlyo the country was declared endemic in July 2008. Consequently,ince the virus had become entrenched in the country, the deci-

ion to use mass vaccination against HPAI in Egypt was made asn emergency measure based on positive impact of such blan-et vaccination in Vietnam and the People’s Republic of China [4].accination of domestic poultry against H5N1 has been used on

∗ Corresponding author. Tel.: +32 0 2 379 13 21; fax: +32 0 2 379 13 37.E-mail addresses: farau@var.fgov.be, fabienne.rauw@var.fgov.be (F. Rauw).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.01.048

a large-scale in Egypt since the veterinary authorities made thedecision to vaccinate all commercial flocks starting in May 2007,followed by vaccination of household poultry. However, accord-ing to Peyre et al. [4], the vaccination coverage was limited withan average of 50–60% in the commercial farms and 25–30% in vil-lage household poultry in the best vaccinated Governorates. Thismight be one of the reasons why the impact of AI vaccination ondisease control remains quite limited in Egypt, i.e., outbreaks arestill reported regularly despite continuous vaccination of poultryand spill-over to humans remains a permanent risk.

But the shortcomings of the vaccination strategy may also beattributed in part to a lack of sufficient support in terms of fundingand communication, the absence of an efficient monitoring system,and inadequate training of field technicians as emphasized recently[5], as well as to limitations in the use of inactivated vaccines,which are the only registered vaccines in Egypt. Indeed, inacti-vated vaccines must be administered individually by the parentalroute which is laborious, time consuming and harbours the risk

of transmission of the field virus by the vaccination crews. Theefficacy of inactivated vaccines depends particularly on the vac-cine characteristics, the presence or absence of passive immunityand the age of the targeted host [6]. Besides poor administrationof the vaccines, genetic and antigenic drift of the circulating viral

ine 29

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trains have also been listed among the reasons for vaccination fail-res in Egypt but, until recently, this phenomenon had not beenlearly demonstrated. Indeed, important sequence diversity in theaemagglutinin gene of H5N1 isolates circulating in Egypt has beenhown [7]. The Egyptian strains isolated between 2006 and 2008elong to the subclade 2.2 of the H5N1 virus of Eurasian origin1,8] but have diverged significantly compared to other viruseselonging to the same subclade from Africa, Europe and Asia, thus

ustifying their further classification in a third-order 2.2.1 clade8–12]. In addition to genetic variation, recent Egyptian strainsemonstrated significant antigenic drift within clade 2.2.1 [9,10].he impact of this antigenic diversity on vaccine efficacy has beenoorly investigated so far.

Turkey herpesvirus (HVT) has been used extensively as a vaccinegainst Marek’s disease [12–14], and is usually administered at day-ld. The HVT vaccine is provided either under the cell-associated orhe cell-free form, which is lyophilized. The relatively fast growthnd easy production of HVT in chicken embryo fibroblast (CEF) cul-ure are two important factors that have contributed to its widese as a vaccine. Among other advantages, the HVT vaccines can bedministered by the in ovo route 3–2 days before hatching or by theubcutaneous (s/c) or intramuscular route to day-old chicks [15,16].lso, HVT vaccine is well-known to be fully safe and poorly sensi-

ive to interference with maternally derived antibody (MDA) whennder the cell-associated form, which is the reason why it has beenroposed as a vector for IBD [17] and other diseases [18–20]. Finally,ontrary to the inactivated vaccines, vector-based vaccines havehe capacity to multiply and induce strong cell-mediated immu-ity, which has been shown to be important for protection and foreduction of excretion of the challenge virus [21]. They are alsoapable to induce immunity in the presence of passive immunityirected either against the HVT vector or the insert. These features,

n addition to long-term immune protection due to its persistentiraemic infection [22], make HVT a promising live viral vector forhe expression of avian influenza antigens.

The present study confirmed the existence of a major drift inhe Egyptian HPAI H5N1 strains and its negative impact on vaccina-ion efficacy, as demonstrated by failure of a Mexican H5N2-basednactivated vaccine to protect against the newly emerging 2008gyptian strain. A new AI vaccination regimen, including a recom-inant HVT-AI vaccine expressing the HA gene from a recent clade.2 HPAI H5N1 combined or not with the inactivated vaccine, wasroposed. The induced HA specific humoral immunity and protec-ion against mortality and clinical signs as well as reduction of viralxcretion were followed. This paper shows that this rHVT-H5 vac-ine has a promising potential to protect against two antigenicallyivergent clade 2.2.1 HPAI H5N1 Egyptian strains.

. Materials and methods

.1. Chickens

SPF chickens were provided by Charles River Laboratory, Biovo-ohacs, Hungary. After hatching, all birds were kept in biosecurity

evel 3 (BSL-3) isolators and animal experiments were conductednder the authorization and supervision of the Biosafety andioethics Committees at the Veterinary and Agrochemical Research

nstitute (Belgium), following National and European regulations.

.2. Vaccines and challenge strains

The New Flu Kem H5N2 vaccine (inact-H5N2) was producedy Ceva-Mexico, Cuarnavaca, and is an oil emulsion inactivatedaccine containing antigens of both AIV (H5N2 Low Pathogenicvian Influenza (LPAI) virus A/Chicken/Mexico/232/1994 strain)

(2011) 2590–2600 2591

and NDV (Newcastle Disease Virus) La Sota strain. It was previouslyshown that the NDV component has no influence on the responseagainst the AI antigens in the vaccine [23]. The cryopreserved cell-associated rHVT-H5 (Vectormune® AI, Acc #359-07) was providedby CEVA-Phylaxia Veterinary Biologicals Co Ltd. (Hungary). It wasconstructed with the FC-126 strain of HVT as the vector and the HAgene from the H5N1 clade 2.2 HPAI A/Swan/Hungary/4999/2006strain. Vaccines were administrated subcutaneously in the neck ata dose of 5000 pfu in 200 �l at day-old for the rHVT-H5 vaccine orat one dose in 500 �l at 1, 10 or 12 days of age for the inactivatedH5N2 vaccine (Table 1).

The two HPAI H5N1 viruses selected for the challenge were iso-lated in poultry farms in Egypt at the end of 2007 and beginningof 2008 and provided by the OIE/FAO reference laboratory for NDand AI of Padova, Italy. The A/Chicken/Egypt/1709-1VIR08/2007and A/Chicken/Egypt/1709-6/2008 strains were considered repre-sentative of the HPAI H5N1 viruses circulating in Egypt in 2007and 2008, respectively. They belong to the genetic clade 2.2.1but cluster separately [24]. A/Chicken/Egypt/1709-1VIR08/2007is a representative of the “Egypt group B” cluster of “classical”Egyptian isolates circulating in the country since 2006 [9], whileA/Chicken/Egypt/1709-6/2008 clusters with the antigenically devi-ating “Egypt group E” isolates that have at least 11 amino acidmutations compared to the rest of the Egyptian H5N1 isolates [9].Chickens were challenged at 3 weeks post-vaccination (pv) by theoculo-nasal route with 100 �l (50 �l in eye, 50 �l in nose) of inocu-lum containing 106 EID50 of HPAI H5N1 virus.

2.3. Viral excretion

The quantification of the AIV challenge strain in the oropha-ryngeal swabs was determined by quantitative real-time reversetranscription-polymerase chain reaction according to current pro-cedures in our laboratory [25–27]. The numbers of viral genomecopies were quantified in a Taqman QRT-PCR targeting theinfluenza A matrix (M) gene [28]. Quantification was done relativeto a standard curve based on tenfold dilutions of an in vitro tran-scribed RNA template. A Ct value of 40 was chosen as the cut-offfor AI detection and samples with a higher Ct value were consid-ered negative for virus, in accordance with the standard curve. Theresults were expressed as the number of M gene copies per ml ofswab samples.

Moreover, the quality of the sample and the RNA extraction pro-cedure were validated using �-actin as previously described [26].

2.4. Serological analysis

The haemagglutination inhibition (HI) tests were conductedaccording to standard procedures, using 4 haemagglutination(HA) units per well, as previously described [27]. Four unitsof HA antigens were tested to check for cross-reactivity ofthe serums toward different recent H5N1 and vaccine strains,i.e., the two antigenically divergent Egyptian HPAI H5N1strains used for challenge (A/Chicken/Egypt/1709-1VIR08/2007and A/Chicken/Egypt/1709-6/2008); the Hungarian HPAI H5N1strain (A/Swan/Hungary/4571/2006), which provided the HA genefor the construction of the rHVT-H5 vaccine; and the Mexican LPAIH5N2 strain (A/Chicken/Mexico/232/94) included in the New FluKem H5N2 vaccine. The HI geometric mean titres were expressedas reciprocal log2 and inhibition of haemagglutination in a dilutionof >23 was taken as specific to AIV.

2.5. Experimental design

Two studies were conducted on SPF chickens and the differ-ent experimental designs are summarized in Table 1. In the first

2592 F. Rauw et al. / Vaccine 29 (2011) 2590–2600

Table 1Vaccination and challenge scheme.

Exp Groups Nb of chick Vaccination Challenge

Vaccine Age day Strain Age day

I IChallenged rHVT-H5 10 rHVT-H5 1 H5N1 Egypt 2007 21Sentinel rHVT-H5 5 rHVT-H5 1 – –

IIChallenged inact-H5N2 10 Inactivated H5N2 10 H5N1 Egypt 2007 31Sentinel inact-H5N2 5 Inactivated H5N2 10 – –

IIIChallenged unvaccinated 9 – – H5N1 Egypt 2007 21

IVChallenged rHVT-H5 10 rHVT-H5 1 H5N1 Egypt 2008 21Sentinel rHVT-H5 5 rHVT-H5 1 – –

VChallenged inact-H5N2 10 Inactivated H5N2 10 H5N1 Egypt 2008 31Sentinel inact-H5N2 5 Inactivated H5N2 10 – –

VIChallenged unvaccinated 9 – – H5N1 Egypt 2008 21

VIIChallenged unvaccinated 9 – – H5N1 Egypt 2007 31

VIIIChallenged unvaccinated 9 – – H5N1 Egypt 2008 31

II IChallenged rHVT-H5/inact-H5N2 10 rHVT-H5

Inactivated H5N2112

H5N1 Egypt 2008 33

Sentinel rHVT-H5/inact-H5N2 5 rHVT-H5Inactivated H5N2

112

– –

IIChallenged inact-H5N2/inact-H5N2 10 Inactivated H5N2

Inactivated H5N2112

H5N1 Egypt 2008 33

Sentinel inact-H5N2/inact-H5N2 5 Inactivated H5N2Inactivated H5N2

112

– –

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Challenged unvaccinated 10Sentinel unvaccinated 5

rial, two groups of 30 birds were vaccinated either at day-oldith the rHVT-H5 vaccine or at 10 days old with the inactivated5N2 vaccine. The third group of 36 birds was left unvaccinated.ach of the two vaccinated groups was further divided into tworoups of 15 birds for the challenge at 3 weeks pv, meaningt 21-day-old for the rHVT-H5 and at 31-day-old for the inacti-ated H5N2 groups. The challenge was performed with the 2007nd 2008 Egyptian H5N1 strains on 10 out of the 15 vaccinatedhickens, the 5 remaining birds being used as sentinels. The unvac-inated group was divided into four groups of 9 chickens, whichere challenged at the age of 21 or 31 days with each challenge

train.In the second trial, SPF chickens were divided into three groups

f 15 birds. The first group was primed at day-old with the rHVT-5 vaccine and boosted at 12 days of age with the inactivated5N2 vaccine, while the second group was vaccinated twice with

he inactivated H5N2 vaccine at 1 and 12 days of age. The lastroup was left unvaccinated and used as control. The chickens werehallenged with the 2008 Egyptian H5N1 strain 3 weeks after theecond vaccination, meaning at 33 day of age. Again, the challengeas performed on 10 out of 15 chickens, the 5 remaining chickens

eing used as sentinels.In both experiments, each chicken was identified individually.

fter challenge, birds were monitored daily for clinical signs (pros-ration, diarrhea, respiratory distress, and for neurological signsike loss of balance, torticollis and incoordination). Birds showing

evere clinical signs were humanely euthanized. To monitor viralxcretion, oropharyngeal swabs were taken on several days post-nfection (pi) as previously described [29]. Swabs were stored at80 ◦C before processing. To assess seroconversion, blood samplesere taken before and after challenge, at 3 weeks pv and 2 weeks

– H5N1 Egypt 2008 33– – –

pi, respectively. Serums were harvested and stored at −20 ◦C untilfurther analysis.

2.6. Statistical analysis

Statistical analyses of data were performed using Minitab 13and STATA 10 software (statistical programmes for Windows 2000)and differences were considered as significant at P < 0.05. The sero-logical results (HI titres), the challenge virus shedding data andthe mean death time were analysed using Minitab 13 software(statistical programmes for Windows 2000) and differences wereconsidered as significant at P < 0.05. After checking the validityhypotheses (homogeneity of the within-groups’ variances by theLevene’s test, and normality of the distribution of the criterion foreach group by the Ryan-Joiner’s test), one-way ANOVA was car-ried out on this criterion in order to compare the different groups.In case of a significant global group effect, pairwise comparisonswere carried out by Tukey’s tests. When normality or homogeneityof variance tests failed, the non-parametric Kruskal–Wallis test wasused. In case of group effect, pairwise comparisons were carriedout by Kruskal–Wallis test, using the Bonferroni’s method to adjustthe risk ˛ (adjusted risk ˛′ = ˛/c where c is the number of pairwisecomparisons). Similar statistical analyses were performed on theHI titres in order to compare the results obtained by the differentHA antigens for the same serum sample.

The qualitative criterion “mortality” was analysed by the

Fisher’s exact test STATA 10 software. Because of the small valueof expected frequencies (np < 5) for cells of contingency table, aFisher’s exact test was performed. In the case of global significantdifference between groups, pair wise comparisons were carried outperforming new contingency tables and using Fisher’s exact test.

F. Rauw et al. / Vaccine 29 (2011) 2590–2600 2593

Table 2AHA coding sequence nucleotide identity scores between the classical and recent Egyptian HP H5N1 strains and the two Mexican LP H5N2 and Hungarian HP H5N1 vaccinestrains.

Strains Vaccine Egyptian field strains

Inact-H5N2 rHVT-H5 Classical H5N1 Variant H5N1

A/Chicken/Mexico/232/1994 (AY497096)a 1 0.799 0.798 0.783A/Swan/Hungary/4999/2006 (FJ445226) 0.799 1 0.985 0.979A/Chicken/Egypt/1709-1VIR08/2007 (EU17849) 0.798 0.985 1 0.985A/Chicken/Egypt/1709-6/2008 (EU17857) 0.783 0.979 0.985 1

a Genbank accession number.

Table 2BHA1 amino acid identity scores between the classical and variant Egyptian HP H5N1 strains and the two Mexican LP H5N2 and Hungarian HP H5N1 vaccine strains.

Strains Vaccine Egyptian field strains

Inact-H5N2 rHVT-H5 Classical H5N1 Variant H5N1

A/Chicken/Mexico/232/1994 (AY497096)a 1 0.826 0.817 0.791A/Swan/Hungary/4999/2006 (FJ445226) 0.826 1 0.982 0.959

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A/Chicken/Egypt/1709-1VIR08/2007 (EU17849) 0.817A/Chicken/Egypt/1709-6/2008 (EU17857) 0.791

a Genbank accession number.

he risk ˛ was adjusted using the Bonferroni method (adjusted risk′ = ˛/c where c is the number of pair wise comparisons).

.7. Quantification of transmission

A stochastic SEIR (susceptible-exposed-infected and infectiousemoved) model formed the basis for the estimation of the epidemi-logical parameters of interest. The gist of the statistical analyses isiven by Van der Goot et al. [30] and Veldhuis et al. [31]. The param-ter log ˇ was estimated by modelling the number of new infectionspon contact, using the offset function ln(I�t/N), and complemen-ary log–log function. A back transformation of log ˇ was requiredo obtain ˇ. This parameter ˇ was then used to estimate R followinghe formulae R = ˇ × 1/˛, with infectious time period 1/˛. The con-dence intervals (CI) and P-values used to determine whether theesult were significant or not were the ones obtained for the param-ter log ˇ. The goodness of fit of the model was assessed with theIC criteria. The CI of the parameter R, was calculated based on the

ollowing property Var(ˇ × 1/˛) = (Eˇ)2Var(1/˛) + (E1/˛)2Var(ˇ).

. Results

.1. Sequence diversity of Egyptian isolates compared withaccine strains

The HA coding nucleotide sequence and HA1 amino acid identitycores are presented in Tables 2A and 2B, respectively, for the vac-ine and challenge strains. The sequence similarity between the HAenes of the 2007 and the 2008 Egyptian H5N1 strains was 98.5%t the nucleotide and 96.5% at the amino acid levels. Eight of the1 observed mutations of the 2008 “variant” virus are located onredicted antigenic peptides (P74S, N120S, A184E) or mapped epi-opes (H110R, R140G, S141P, F144Y, M226V) when compared tohe 2007 “classical” isolate and using the AA numbering describedn the H5N1 epitope mapping study by Duvvuri et al. [32]. An addi-ional mutation (N165H) results in the loss of a glycosylation siteear a mapped epitope, while the two remaining mutations (S123P,162K) are just upstream or downstream of mapped epitopes.

he Mexican H5N2 vaccine strain had 79.8% nucleotide and 81.7%mino acid sequence identity with the 2007 H5N1 Egyptian virus.he phylogenetic distance from the 2008 Egyptian H5N1 strain wasven more important with nucleotide and amino acid sequencedentities of only 78.3% and 79.1%, respectively. Conversely, the HA

0.982 1 0.9650.959 0.965 1

of the Hungarian H5N1 strain included in the rHVT-H5 vaccine had98.5 and 98.2% similarity at nucleotide and amino acid levels withthe 2007 and 97.9% and 95.8% sequence identities with the 2008Egyptian H5N1 virus, respectively.

3.2. Protection against morbidity and mortality

In the first trial, unvaccinated chickens challenged with the2007 Egyptian H5N1 strain, showed typical signs of HPAI includingdepression, ruffled feathers and nervous signs at 2 days post-infection (dpi). Birds started dying on 2 dpi and all animals werefound dead by the next day. Neither clinical signs nor mortalitywere observed among the rHVT-H5 and the inact-H5N2 vaccinatedgroups (data not shown). When the 2008 Egyptian H5N1 strainwas used for challenging, a similar clinical picture was observed inthe unvaccinated group (Fig. 1A), with all birds dying after 3 days.Within the inact-H5N2 group, the challenged and sentinel chickensshowed clinical signs from 3 to 6 dpi, respectively, and all the chal-lenged birds were found dead by 10 dpi while all sentinel chickensdied by 12 dpi. The inact-H5N2 vaccine did not prevent mortal-ity but provided a delay in day of death (Fig. 2C). On the contrary,chickens vaccinated with the rHVT-H5 vaccine and challenged withthe 2008 Egyptian H5N1 strain remained healthy throughout the14 days long post-challenge observation period and no mortalitywas observed.

In the second trial, a similar pattern of mortality was obtainedin the challenged unvaccinated control group and the clinical signswere delayed to 5 dpi in the sentinel birds with all animals dying by6 dpi (Fig. 1B). In the group where the birds were vaccinated twicewith the inact-H5N2 vaccine, 60 and 80% of challenged and sen-tinel vaccinated animals died, respectively, and the survived birdsshowed clinical signs. Among the rHVT-H5/inact-H5N2 vaccinatedgroup, only 2 challenged and one sentinel animals showed clinicalsigns of HPAI from 4 dpi and were found dead by 8 dpi.

3.3. Serology

Before challenge, the serological titres of all unvaccinated chick-ens were below the threshold of positivity (≤23), regardless of theHA antigen used, and were therefore considered as negative in bothtrials (data not shown). The HI titres of the chickens vaccinated intrials I and II are represented in Fig. 2.

2594 F. Rauw et al. / Vaccine 29 (2011) 2590–2600

Fig. 1. Protection against mortality after challenge with 2008 Egyptian HP H5N1 strain on vaccinated SPF chickens. In trial I (A), birds were vaccinated at day-old with rHVT-H5vaccine or at 10-day-old with inactivated H5N2 vaccine. In trial II (B), animals were vaccinated at 1 and 12 day-old with rHVT-H5 and inactivated H5N2 vaccines, respectively,or twice with inactivated H5N2 vaccine. Unvaccinated animals are used as the control group. Ten chickens per group were challenged at 3 weeks pv, the remaining birdsbeing used as contact sentinels. Data represent survival rate at different days pi. The mean death time was given for all mortality data sets (C). Mean ± standard deviationwith no common letters differ significantly (P < 0.05).

F. Rauw et al. / Vaccine 29 (2011) 2590–2600 2595

Fig. 2. HI antibody titres of SPF chickens vaccinated with the rHVT-H5 and/or the inactivated H5N2 vaccine and challenged with the 2007 (A) or 2008 (B) Egyptian H5N1strain. Ten birds per group were challenged at 3 weeks pv with 105 EID50 by oculo-nasal route. Data represent mean ± standard deviation of HI titre (log2) at 3 weeks pv (beforechallenge) and at 2 weeks pi (after challenge), which corresponds to the last dilution showing an inhibition of haemagglutination of 4 haemagglutination units of AIV strains.The HI geometric mean titres were expressed as reciprocal log2 and titres >3 log2 were considered positive (this cut-off is indicated by the dotted line). Mean ± standarddeviation at time points with no common letters or with * superscript differ significantly (P < 0.05) by used vaccine or HA antigen, respectively.

2 ine 29 (2011) 2590–2600

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Table 3AViral shedding by the oropharyngeal route after challenge with 2007 and 2008 Egyp-tian HP H5N1 strains on SPF chickens vaccinated at day-old with rHVT-H5 or at10-day-old with inactivated H5N2 vaccines (trial I).

Groups Days post-infection with 2007 Egyptian H5N1strain

3 7

IChallenged rHVT-H5 3.48 ± 2.29aC8/10b 0.26 ± 0.87A1/10Sentinel rHVT-H5 3.74 ± 1.20C 5/5 0.36 ± 0.49A 2/5

IIIChallenged unvaccinated 9.23 ± 0.92A 9/9 S.M.

IIChallenged inact-H5N2 5.44 ± 1.19B 10/10 0.40 ± 1.25A1/10Sentinel inact-H5N2 4.37 ± 0.41BC 5/5 0.00 ± 0.00A 0/5

VIIChallenged unvaccinated 9.13 ± 0.58A 6/6 S.M.

P-value 0.000 0.906

Groups Days post-infection with 2008 Egyptian H5N1strain

3 7

IVChallenged rHVT-H5 4.23 ± 0.79C10/10 3.04 ± 1.59B3/10Sentinel rHVT-H5 2.63 ± 1.18D 1/5 0.00 ± 0.00B 0/5

VIChallenged unvaccinated 8.83 ± 0.35A 9/9 S.M.

VChallenged inact-H5N2 6.17 ± 1.23B10/10 5.93 ± 1.51A 2/2Sentinel inact-H5N2 4.13 ± 1.08C 5/5 6.51 ± 1.22A 3/3

VIIIChallenged unvaccinated 8.46 ± 0.42A 9/9 S.M.

P-value 0.000 0.000

S.M. = specific mortality.a Data represent mean ± standard deviation of Matrix gene copies in ml of swabs

596 F. Rauw et al. / Vacc

.3.1. HI tests results using the Egyptian challenge strains asntigens

In the first trial, at 3 weeks pv, 73% of the rHVT-H5 vaccinatedirds showed positive HI titres when the 2007 Egyptian H5N1 strainas used as HA antigen, while only 26% showed positive HI titres

n the inact-H5N2 vaccinated group. The rate of seroconversionas reduced to 26% and 13%, respectively, when the 2008 Egyptian5N1 HA antigen was used. The rHVT-H5 vaccinated group showedhigher HI titre using the 2007 Egyptian H5N1 HA antigen, com-ared to the 2008 Egyptian H5N1 HA antigen. In the second trial, therime/boost rHVT-H5/inact-H5N2 vaccination regimen resulted in00% seroconversion of the vaccinated animals, which showed aitre equal to 25 against the 2008 strain. By contrast, the inact-5N2/inact-H5N2 group remained negative when tested against

his HA antigen.

.3.2. HI tests results using the Mexican LPAI H5N2 vaccine strains antigen

As expected, all the chickens from the inact-H5N2, rHVT-5/inact-H5N2 and inact-H5N2/inact-H5N2 groups were positiveith high HI titres when tested with the homologous Mexi-

an H5N2 HA antigen. Three weeks after a single inact-H5N2accination, titres averaged 27, while after the prime/boost rHVT-5/inact-H5N2 and inact-H5N2/inact-H5N2 vaccinations, titres

eached 210. Within these three vaccinate groups, the HI titregainst the H5N2 HA antigen were higher than the ones obtainedith the H5N1 HA antigens. By contrast, the majority of the chick-

ns within the rHVT-H5 vaccinated group were below the thresholdf HI positivity with the H5N2 HA antigen. The humoral immuneesponse of the inact-H5N2/inact-H5N2 group was significantlyigher when the homologous Mexican H5N2 HA antigen was usedhan the one measured in the other vaccinate groups.

.3.3. HI tests using the Hungarian clade 2.2 H5N1 as antigenIn the first trial, 80% of the rHVT-H5 vaccinated chickens were

ositive when tested with the homologous Hungarian H5N1 HAntigen. The HI titres against the homologous Hungarian H5N1A antigen were higher compared to the ones when the otherA antigens were used. In half of the inact-H5N2 vaccinatedirds positive HI titres could be measured when the Hungarian5N1 virus was used as HA antigen, confirming the closer relat-dness between the Hungarian H5N1 and Mexican H5N2 strains.n the second trial, the two prime/boost vaccination regimensesulted in 100% seroconversion, however the humoral immuneesponse of the rHVT-H5/inact-H5N2 group (≈210) was signifi-antly higher than the one measured in the inact-H5N2/inact-H5N2ne (≈27).

After the challenge, a significant increase of the HI titre wasbserved in most of the vaccinated birds which survived the chal-enge regardless to which HA antigen was used for serological tests,ndicating a boosting effect due to the challenge.

.4. Virus excretion after challenge

All unvaccinated chickens shed AI virus as measured by real-ime RT-PCR after challenge (Table 3A). The inact-H5N2 andHVT-H5 vaccinations reduced significantly the 2007 and 2008gyptian virus shedding, as revealed by the statistical comparisonf the average gene copy number between the groups. In the firstrial, among the vaccinated birds challenged with the 2007 Egyp-ian strain, the excretion at 3 dpi was significantly lower within the

HVT-H5 group than in the inact-H5N2 vaccinated group. Four daysater, there was no significant difference between the two groups.n the contrary, 7 days after challenge with the 2008 Egyptian

train, all the birds in the inact-H5N2 group still excreted virushile only 30% in the rHVT-H5 group. At 3 and 7 dpi, the virus

(log10). Mean ± standard deviation at time points with no common superscript differsignificantly (P < 0.05).

b Frequency (number positive/total tested chicken) of virus detection in 1 mlswabs.

shedding by the rHVT-H5 group was significantly lower than in theinact-H5N2 group.

In the second trial, the prime–boost vaccination with rHVT-H5/inact-H5N2 vaccines and the double vaccination with theinact-H5N2 reduced significantly the excretion of virus when com-pared to the unvaccinated group (Table 3B), but there was nosignificant difference between the two vaccinated groups. In bothtrials, shedding was observed in the sentinel vaccinated birds in allgroups also, but was limited if animals were vaccinated accordingto one of the prime–boost vaccination regimen investigated in thepresent study.

The transmission parameters were also analysed and are pre-sented in Table 4. These results indicate a marked reduction in thetransmission ratio of the 2008 Egyptian H5N1 strain provided bythe rHVT-H5 vaccine (R < 1) in comparison with the inact-H5N2vaccine.

4. Discussion

In addition to quarantine measures, culling of infected poul-try flocks and improved biosecurity measures, vaccination is animportant tool to prevent AI and to limit outbreaks [2,3,29].Ideally, vaccination should prevent infection, but more realisticgoals are to prevent the clinical manifestation of the disease andreduce virus shedding. Vaccination has been implemented in Egypt

since 2006 together with culling, quarantine, improved biose-curity and surveillance. During the first large scale vaccinationcampaign, AI-inactivated vaccines based upon LPAI virus strainA/Chicken/Mexico/232/1994 were used. These vaccines were then

F. Rauw et al. / Vaccine 29 (2011) 2590–2600 2597

Table 3BViral shedding by the oropharyngeal route after challenge with the 2008 Egyptian HP H5N1 strain of SPF chickens vaccinated at 1 and 12 day-old with the rHVT-H5 andinactivated H5N2 vaccines, respectively, or twice with the inactivated H5N2 vaccine (trial II).

Groups Days post-infection with H5N1 Egypt 2008

2 4 7

IChallenged rHVT-H5/inact-H5N2 2.73 ± 2.21aB8/10b 3.22 ± 2.50AB7/10 1.14 ± 2.03A3/10Sentinel rHVT-H5/inact-H5N2 0.00 ± 0.00C 0/5 1.87 ± 2.56BC 2/5 1.04 ± 1.27A 2/3

IIChallenged inact-H5N2/inact-H5N2 2.67 ± 2.19B 6/9 5.14 ± 2.09A 8/9 1.34 ± 2.69A 1/4Sentinel inact-H5N2/inact-H5N2 3.04 ± 1.88B 4/5 0.72 ± 1.62C 1/5 0.00 ± 0.00A 1/1

IIIChallenged unvaccinated 7.26 ± 0.60A 10/10 S.M. S.M.Sentinel unvaccinated 4.48 ± 0.31A 5/5 4.56 ± 2.05AB 5/5 S.M.

P-value 0.000 0.010 0.946

S.M. = specific mortality.a s (log

sbs.

fHsantHAvahaio

vpiTlt9bastasp82i

TEg

Data represent mean ± standard deviation of Matrix gene copies in ml of swabignificantly (P < 0.05).

b Frequency (number positive/total tested chicken) of virus detection in 1 ml swa

ollowed by European origin H5N2 or reverse genetics Chinese5N1 inactivated vaccines [32]. Nevertheless, despite these exten-

ive vaccination campaigns, the H5N1 HPAI has continued to spreadmong many Egyptian poultry farms and the virus underwent sig-ificant changes during its circulation. Indeed, from a single origin,he initial clade 2.2 virus has evolved toward a distinct sublineage of5N1 termed 2.2.1 that consists of viruses mostly from Egypt [9,12].mong factors already mentioned in the introduction, the observedaccination failures could be explained by a significant genetic andntigenic drift observed in the field. In order to investigate this, weave compared the protection afforded by an inactivated vaccinend a recombinant vaccine against two selected Egyptian strainssolated in 2007 and 2008, and considered as representative strainsf the “classical” and “variant” lineages, respectively [9].

The goal of the present study was to determine if an innovativeaccination strategy including a rHVT-H5 vaccine could increaserotection and reduce viral shedding, and presumably, the spread-

ng and the transmission of a HPAI Egyptian strain isolated in 2008.he existence of antigenic variants among the H5N1 viruses circu-ating in Egypt was confirmed in this study. At the amino-acid level,he HA1 of the two selected 2007 and 2008 Egyptian strains showed6.5% homology, diverging from each other by 11 amino acids onlyut, interestingly, these mutations appeared to be located mostlyt the level of important epitopes of the HA. This was demon-trated by the lower HI titres and lack of protection afforded byhe phylogenetically more distant H5N2 vaccine against the “vari-nt” 2008 H5N1 Egyptian strain. This indicates most probably that

ome epitopes have been modified or lost by substitutions affectingrimarily the outer surface of the globular head of the HA1. Indeed,of the observed amino acid mutations observed in the “variant”

008 H5N1 Egyptian virus compared to the “classical” 2007 H5N1solate are located on peptides that were predicted as antigenic

able 4stimation of the transmission parameter ˇ (transmission rate) and calculation of R (repiven between brackets.

Exp Groups Transmission rate (ˇ

Infection with H5N1 Egypt 2007I rHVT-H5 vaccinated 1.01 (−0.88–0.91)

Inact-H5N2 vaccinated 0.97 (−1.03–0.97)

Infection with H5N1 Egypt 2008I rHVT-H5 vaccinated 0.11 (−4.19–0.27)

Inact-H5N2 vaccinated 1.04 (−0.85–0.93)II rHVT-H5/inact-H5N2 vaccinated 0.70 (−1.25–0.53)

Inact-H5N2/inact-H5N2 vaccinated 0.78 (−1.14–0.64)Unvaccinated 1.04 (−0.85–0.93)

10). Mean ± standard deviation at time points with no common superscript differ

and mapped as epitopes, while an additional mutation resulted inthe loss of a glycosylation site near a mapped epitope, and the tworemaining mutations are just upstream or downstream of othermapped epitopes ([32],G. Cattoli, personal communication).

Nevertheless, it seems that these antigenic mutations do notplay pivotal role in the pathogenicity of the two HPAI H5N1 Egyp-tian strains as infection of SPF chickens with either of the twostrains was highly lethal and the kinetics of clinical signs and mor-tality were similar. In our study, although a satisfactory clinicalprotection could be obtained by both types of vaccines used individ-ually against the “classical” 2007 HPAI H5N1 Egyptian strain, onlythe single rHVT-H5 vaccination afforded clinical protection againstchallenge with the “variant” 2008 HPAI H5N1 Egyptian strain. A sin-gle vaccination with an inactivated H5N2 Mexican vaccine delayedthe onset of mortality but did not prevent it. Although a recent studyof Kim et al. [33] demonstrated sufficient protection against labora-tory challenge with the 2008 HPAI H5N1 Egyptian virus followingvaccination with a Mexican-origin H5N2 inactivated vaccine, pro-tection in the field was insufficient. But in this mentioned study,an alternative H5N1 challenge virus from 2008 was used, at a sig-nificantly lower challenge dose. In the context of high sequencevariability in the circulating HA genes and insufficient knowledge ofthe antigenic significance of this variation it is difficult to interpretto what degree the observed sequence differences have contributedto the outcome of our experiment. Another recent study using sim-ilarly a Mexican-origin H5N2 inactivated vaccine [24] also showedsufficient protection under controlled experimental conditions. It

should be noted, however, that in the later study, remarkably highhomologous titres were obtained after vaccination (up to 210) anda lower infectious dose of A/Chicken/Egypt/1709-6/2008 was usedfor the challenge. Nevertheless, in our hands, a booster vaccinationwith the same H5N2 inactivated vaccine, although increasing the

roduction ratio), based on the SIR model, with a GLM. 95% confidence intervals are

) Infectious period (day) (1/˛) Reproduction ratio (R)

1.85 (0.52–3.17) 1.86 (0.14–3.59)1.20 (0.73–1.67) 1.16 (0.28–2.05)

1.80 (0.86–2.74) 0.20 (−0.07–0.47)2.60 (1.16–4.04) 2.70 (0.53–4.88)2.15 (0.68–3.62) 1.50 (0.07–2.94)1.85 (0.91–2.79) 1.44 (0.23–2.66)2.00 (2.00–2.00) 2.08 (0.67–3.49)

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598 F. Rauw et al. / Vacc

omologous HI titres up to 210 (similar as those in the referredtudy), still failed to induce satisfactory protection against highhallenge infectious doses.

As a consequence of the considerable antigenic variation amonghe same subtype [34,35], antisera raised against one AI strain canhow varying reactivity with other virus strains from the sameubtype. This was clearly demonstrated in our study by the sero-ogical HI tests using four different HA antigens of the H5 subtypecorresponding to the two vaccines and the two challenge virusesA antigens) but with different amino acid sequences. As coulde expected, our results showed that the rHVT-H5 and the inact-5N2 vaccines induce a higher humoral immune response to their

espective homologous H5 antigens. Nevertheless, the HI titres tohe homologous H5 antigen remained lower following the rHVT-H5accination than those observed after the inact-H5N2 vaccination.his could be explained by the different immune responses inducedy the two different vaccines. Indeed, as the HVT replicates in aighly cell-associated manner in lymphocytes, it has been sug-ested that this delivery system is a strong inducer of cell-mediatedmmune response [36] and slower humoral antibody response, ashown previously with a rHVT-ND vaccine [21]. On the contrary, thenactivated vaccines are known to induce mostly humoral immu-ity [37,38].

In addition, to corroborate these findings, estimation of trans-ission parameters ˇ (transmission rate) and calculation of(reproduction ratio: average number of secondary infections

aused by one typical infectious individual), based on the stan-ard stochastic susceptible-infectious-removed (SIR) model, werebtained from a generalized linear mixed model (GLM) devel-ped in SAS 9.2. software. In comparison to other methods usedor the estimation of transmission parameters, such as the finalize (FS) model or the transitional state (TS) model, this longitu-inal model accounts for the time intervals between observations30,31]. Interestingly, although we confirmed that vaccination withn inactivated heterologous H5N2 vaccine was able to reduceransmission of the 2007 classical Egyptian H5N1 strain, only theHVT-H5 vaccine could efficiently block transmission of both the007 as well as the 2008 variant Egyptian H5N1 strains.

Our study indicates that AI protection is dependent on theystemic humoral immune response and on the genetic related-ess between the HA of the vaccine and the field strain. Indeed,he HI titres measured against the “variant” 2008 H5N1 Egyptiantrain in both the rHVT-H5 and the inact-H5N2 vaccinated chickensere lower than obtained against the “classical” 2007 HPAI H5N1

gyptian strain. These serological results correlated well with thehallenge results of birds vaccinated with the inact-H5N2 vaccine:ower protection against challenge with the “variant” 2008 H5N1gyptian strain than against the “classical” 2007 Egyptian H5N1train. This indicates that the antigenic distance between the H5N2exican inactivated vaccine seed virus and the Egyptian H5N1 is

ritical, as only 3.5% divergence at the amino acid level between thewo Egyptian HAs had very significant consequences. This also indi-ates that the HA1 amino acid homology should be considered mores an indication than a rule for prediction of protection as someubstitutions might play a key role in the antigenic profile. Vaccine-nduced protective immunity against AI is primarily the result of anA specific antibody response that blocks viral attachment. Suchntibody response is specific to each of the 16 different HA subtypes32]. The current results suggest the use of HA antigen homologouso the circulating field virus in the serology as an indicator of protec-ion [39,40]. Although serology can have some predicting value forrotection of inactivated AI vaccines provided that an epidemiolog-

cally relevant antigen is used, additional data are needed to assesshe correlation between protection and HI titres. This study alsoupports the development of antigenic cartography for inactivatedaccine seed strain selection [41]. The heterologous prime/boost

(2011) 2590–2600

rHVT-H5/inact-H5N2 strategy investigated during this study rein-forced the humoral immunity. While both inact-H5N2/inact-H5N2and rHVT-H5/inact-H5N2 vaccination schemes induced high HItitres to the Mexican H5N2 antigen, the second vaccination regi-men was able to induce similarly high HI titres to the HungarianH5N1 antigen, suggesting a broader antigenic specificity of anti-body. Moreover, among the four vaccination schedules investigatedin the present study, the rHVT-H5/inact-H5N2 prime/boost strat-egy induced the highest seroconversion specific to the “variant”2008 Egyptian H5N1 strain.

Nevertheless, our study also indicates that protection could notbe explained only by the humoral immunity specific to the HA anti-gen of the challenge strain. Firstly, high titre to a heterologous HAantigen other than that of the challenge virus also provided someprotection against HPAI infection. This indicates that a lack of anti-body specific to the HA antigen of challenge virus could partially becompensated by a high heterologous antibody titre as suggested byothers [24]. Secondly, other types of immune responses are likelyto be involved, such as cell-mediated immunity [32,42]. Indeed,the rHVT-H5 vaccinated chickens were protected against the HAantigens of the 2008 strain without detectable HI antibody againsteither homologous or heterologous HA antigens, while those vac-cinated with the inactivated vaccine died. This could be explainedby the higher efficiency of the rHVT-H5 vaccine to induce cell-mediated immune response than the inactivated vaccine. In orderto investigate this further, much attention will be given to themeasurement of the AI-specific cell-mediated immunity by thedevelopment of an AI antigen recall system (manuscript in prepa-ration), as previously published for NDV [21,29,43,44].

The efficacy of AI vaccines should not only be considered withregard to clinical protection but also with regard to the reduc-tion of excretion after challenge in order to stop the circulationof the virus. In this sense, the single vaccination with the rHVT-H5vaccine appeared to be more efficient regarding reduction of shed-ding than the inact-H5N2 vaccine. The closer antigenic relationshipbetween the rHVT-H5 vaccine and the challenge virus could affectthe mucosal viral replication and its resulting viral excretion. Asignificant positive correlation between HA sequence similarity ofvaccines and challenge viruses and the ability to reduce the levelof excretion by the oropharyngeal route has been reported previ-ously [45]. Finally, the second study indicated a higher reduction inthe number of excreting chickens after a prime/boost vaccinationstrategy, which was able to prevent the transfer of challenge virusto the sentinel birds.

In conclusion, considering the enzootic situation in Egypt, thereis a clear need for an adequate vaccine that can be used at day-oldfor mass vaccination because there is a continuous race betweenthe induction of immunity by vaccination and the time of challengein the field. The current paper shows that even two vaccinationswith the inactivated vaccine including the LPAI H5N2 seed strainwas not sufficient to afford satisfactory protection (as defined bythe clinical protection and reduction of viral excretion) of chickensagainst the more recent 2008 “variant” Egyptian HPAI H5N1 fieldstrain. Extensive vaccination of chickens with H5N2 based inacti-vated vaccine has been shown to produce the emergence of vaccineescape mutants in Mexico [46] and might be responsible for a simi-lar situation in Egypt with H5N1 [9,33]. Therefore, future successeswill mandate inactivated vaccine seed virus more closely match-ing the field virus, because genetic drift occurs in countries andregions experiencing immune pressure from enzootic infection orextensive vaccine use in birds. In response to antigenic variants,

vectored vaccines might allow an easier and more rapid develop-ment and testing of new vaccine strains that will be more adaptedto the field situation. In this context, the studied HVT vector vac-cine with HA inserts from the recent HPAI H5N1 strain appeared

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ery promising to use as a single clade 2.2 rHVT-H5 vaccinationt day-old against the recent H5N1 strains circulating in Egypt.dditional studies are needed to investigate whether the rHVT-5 vaccine performs better because of its better antigenic matchith current Egyptian isolates and/or because of its immunogenicroperties (like the induction of a stronger cell-mediated immuneesponse). It appears that the rHVT-H5 vaccine is able to preventajor outbreaks caused by the 2008 Egyptian H5N1 in chickens but

dditional transmission studies are necessary in order to confirmhe efficacy of this vaccine in reducing virus excretion and trans-

ission. Further advantage of this vaccine could be the possibilityf administration both as day-old and in ovo vaccination throughutomated methods without the interference of passive immu-ity. As a result of this vaccination strategy, priming an immuneesponse to AI will occur at very early age. It is also well knownhat HVT establishes a persistent infection in chickens, offering thedvantage of delivering foreign antigens to the immune system ofaccinated birds during an extended period of time. However, fur-her studies with commercial chickens, which are immunologicallyifferent from SPF birds and might possess AI maternal derivedntibody, are required to determine the usefulness of the rHVT-H5lone or in a prime/boost scheme in the field.

cknowledgements

We thank G. Cattoli and C. Terregino for providing us the Egyp-ian strains, for making their HA sequences rapidly available and forelpful input and discussions. The authors gratefully acknowledge. Lemaire and N. Dumont for qualified technical work (serologicalnd molecular tests) and C. Delgrange for technical assistance inird experiments (animal handling and sampling assistance).

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