Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine

Veterinary Microbiology 189 (2016) 59–67

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Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine Mariette F. Ducateza,* , Jens Beckera,b , Astrid Freudensteinb , Maxence Delverdiera , Mattias Delponta , Gerd Sutterb , Jean-Luc Guérina , Asisa Volzb a b

IIHAP, Université de Toulouse, INRA, ENVT, Toulouse, France Lehrstuhl für Virologie, Institut für Infektionsmedizin und Zoonosen, Ludwig-Maximilians-Universität München Munich, Germany

A R T I C L E I N F O

Article history: Received 15 February 2016 Received in revised form 18 April 2016 Accepted 25 April 2016 Keywords: Influenza virus Vaccine Modified Vaccinia Ankara vector Chicken model

A B S T R A C T

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/ Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction H9N2 influenza viruses are routinely found in wild birds and poultry, mostly in chicken, but also happen to infect mammals and humans. These low pathogenic avian influenza (LPAI) viruses are widespread around the globe since the mid-1990s (Alexander, 2007; Ducatez et al., 2008; Lindh et al., 2014; Monne et al., 2013). In numerous countries where H9N2 infections in poultry have occurred, the disease has become endemic (Lee and Song, 2013; Monne et al., 2013; Negovetich et al., 2011). Examination studies showed that the prevalence of H9N2 in East and South East Asian

* Corresponding author at: Institut National de la Recherche Agronomique (INRA), UMR 1225 Interactions Hôtes-Agents Pathogènes (IHAP), 23 chemin des Capelles, 31076 Toulouse, France. E-mail address: [email protected] (M.F. Ducatez). http://dx.doi.org/10.1016/j.vetmic.2016.04.025 0378-1135/ã 2016 Elsevier B.V. All rights reserved.

countries reaches 7–8% in chickens in live bird markets in South Korea, followed by 16,5% in retail markets in Bangladesh. Almost all samples (94%) that were tested avian influenza virus positive in Bangladesh were of the H9N2 subtype (Negovetich et al., 2011). In addition to the burden the virus causes to poultry industry, repeated zoonotic infections with H9N2 have been reported from 1998 through 2015. Peiris et al. investigated the titers of neutralizing antibodies against H9N2 in blood donor samples in Hong Kong. Two percent of the people tested positive suggesting prior contact with the virus (Peiris et al., 1999). H9N2 viruses classically divide into two main lineages in Eurasia: Y280-like and G1-like viruses (SJCEIRS H9 Working Group, 2013). LPAI H9N2 viruses frequently reassort among their subtype but they have also been shown to “donate” genetic material to other avian influenza virus subtypes. Highly pathogenic avian influenza (HPAI) H5N1 viruses isolated in 1997 harbored for example 6 internal gene segments from H9N2 viruses (Negovetich

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et al., 2011). Similarly the newly emerged LPAI H7N9 viruses in 2013 (responsible for severe zoonotic infections associated with 20% mortality in Human (WHO, 2014)) harbored six internal genes derived from H9N2 strains (Lam et al., 2013). Given that H9N2 viruses infected numerous species around the globe and have caused human infections, they are monitored continuously by the World Health Organization (WHO). A comprehensive study assessed the virulence and transmission of 12 different H9N2 viruses in experimental conditions on normal Human Bronchial Epithelial cells and in ferrets, mice, pigs, and ducks. Viruses from mammalian origin were fitter than their avian counterparts and ranked higher as far as pandemic risk is concerned (SJCEIRS H9 Working Group, 2013). Taken together, H9N2 viruses and their virus progenies threaten avian and human populations thus highlighting the urgent need of a better understanding of the diversity and spread of emerging avian influenza viruses but also of efficient control measures of the virus in poultry. With the increased genetic and antigenic diversity of avian influenza viruses, and especially H9N2 viruses, the challenge is to find cross-reactive vaccines that would protect against several strains at a time of a given subtype. The most potent antibody response targets the most exposed but also the most variable viral protein: the hemagglutinin (HA). At present, the WHO seeks to keep pace with the continuous diversification by producing new vaccine seed strains (WHO, 2015). Economic restrictions, but also the impossibility to stockpile a large number of different vaccination doses fuel the research on new approaches. One of the possible strategies to obtain cross-reactive influenza vaccines is to use “ancestral” antigens. Ancestral sequences are artificial computationally derived amino acid (aa) sequences that would be located at the node of phylogenetic trees. In a previous study we generated ancestral H5N1 vaccines (whole virus inactivated vaccines) that conferred cross-clade protection for morbidity and mortality in the ferret model (Ducatez et al., 2011). The availability of a live candidate vaccine is a promising approach to induce cross-reactive antibodies against several strains of influenza viruses. Modified vaccinia virus Ankara (MVA) is a highly attenuated strain of vaccinia virus that originated from growth selection on chicken embryo fibroblasts (CEF). At present MVA serves as one of the most promising viral vectors for the generation

of recombinant vaccines against infectious diseases and cancer. In the present study, we generated a recombinant MVA candidate vaccine expressing the HA gene of a computationally derived ancestral H9 G1-lineage influenza virus (MVA-H9a). This MVA-H9a vaccine specifically targets the G1 lineage of H9N2 influenza viruses. The immunogenicity and protective capacity of the vaccine was tested in the chicken model. 2. Methods, techniques 2.1. Viruses and vaccine The low pathogenic avian influenza (LPAI) H9N2 isolates A/chicken/Tunisia/12/2010, A/pheasant/UAE/D1521/2011, A/quail/ UAE/D1550/2011, A/environment/Bangladesh/10307/2011, A/Hong Kong/33982/2009, and A/environment/Bangladesh/907/2009 (G1like viruses); A/chicken/Hong Kong/TP38/2003, A/chicken/Hong Kong/G9/97, and A/chicken/Beijing/1/94 (Y280-like viruses), and A/shorebird/DE/249/2006 were used. A vaccine preparation based on a recombinant MVA expressing the HA gene of a computationally derived ancestral H9 G1-lineage influenza virus was used. The details on plasmid construction and generation of recombinant MVA vaccine are presented in the Supplemental material and methods. 2.2. Animal experiment Seventy two SPF white leghorn chickens (PFIE, INRA, Nouzilly, France) were assigned randomly to 8 groups of 8–10 animals. Experimentations were conducted in accordance with European and French legislations on Laboratory Animal Care and Use (French Decree 2001-464 and European Directive CEE86/609) and animal protocols approved by the Ethics Committee “Sciences et santé animale”, committee number 115. The birds were kept in within the animal facilities (biosafety level 2) of the ENVT (Experimental unit agreement number: C3155527) and had access to fed and water ad libitum. MVA-H9 was administrated at a dose of 108 plaque forming units diluted in saline. It was either administered intra-muscularly (IM), by eye drop (ED), or by aerosol (A). For the aerosol vaccination, a compressor nebuliser CompAir Pro NE C29 E (OMRON, Japan) was

Vaccine study: experimental setup

1 day old chick

7-8 weeks old chicken

3 weeks vaccinaon

2-3 weeks (boost)

2 weeks challenge

end

Chicken groups: (i) group IM1: single IM vaccine dose at 3 weeks of age , challenged at 6 weeks of age, (ii) group IM2: 2 IM vaccine doses (at 1 day and 3 weeks of age), challenged at 6 weeks, (iii) group NV1: not vaccinated, challenged at 6 weeks of age, (iv) group NVNC1: not vaccinated, not challenged, (v) group ED: 2 eye drop vaccine doses (at 1 day and 3 weeks), challenged at 5 weeks , (vi) group A: 2 aerosolized vaccine doses (at 1 day and 3 weeks), challenged at 5 weeks, (vii) group NV2: not vaccinated, challenged at 5 weeks, (viii)group NVNC2: not vaccinated, not challenged. Fig. 1. Experimental setup of the vaccine-challenge study.

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used to aerosolize vaccine virus for 20 min. The chicken groups were as follows: (i) group IM1 received a single IM vaccine dose at 3 weeks of age and was challenged at 6 weeks of age, (ii) group IM2 received 2 IM vaccine doses (at 1 day and 3 weeks of age) and was challenged at 6 weeks of age, (iii) group NV1 was not vaccinated and challenged at 6 weeks of age, (iv) group NVNC1 was not vaccinated and not challenged, (v) group ED received 2 eye drop vaccine doses (at 1 day and 3 weeks of age) and was challenged at 5 weeks of age, (vi) group A received 2 aerosolized vaccine doses (at 1 day and 3 weeks of age) and was challenged at 5 weeks of age, (vii) group NV2 was not vaccinated and challenged at 5 weeks of age, (viii) group NVNC2 was not vaccinated and not challenged. The experimental setup is summarized on Fig. 1. Experimentations were conducted in two batches for animal facilities space constraints: groups IM1, IM2, NV1 and NVNC1 birds were housed at the same time period, as were groups ED, A, NV2 and NVNC2 birds. At 5 or 6 weeks of age, chickens were inoculated with 105 tissue culture infectious dose 50 (TCID50) of A/chicken/Tunisia/12/2010 (H9N2) in a 500 ml volume dispensed in the eye, nostrils, and trachea of each bird. After challenge, clinical signs were recorded daily. Oropharyngeal swabs were collected daily from day 1 to day 7 post- inoculation (dpi) for analysis of viral shedding. To determine the presence of H9-specific antibodies, blood samples were collected before vaccine boost, before challenge, and

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at the end of the experiment, 14 days post infection. Tears were also collected pre-boost and pre-challenge for groups ED, A, NV2 and NVNC2 birds sprinkling salt in an eye of each bird (5 birds per group tested at each time point) and collecting the fluid with a micropipette as previously described (Ganapathy et al., 2005). 2.3. Analysis of viral shedding by real-time RT-PCR The swabs were placed in 1 ml PBS containing penicillin and streptomycin and stored at 80  C until analyzed. RNA was extracted using the Qiagen viral RNA minikit following the manufacturer’s instructions. Copy numbers of matrix gene RNAs of A/chicken/Tunisia/12/2010(H9N2) were quantified by real time RT-PCR using the M52C/M253R primers previously described (Fouchier et al., 2000). Real-time RT-PCR was performed using QuantiTect SYBR Green PCR Kit (Qiagen), on a Light Cycler 480 (Roche) with the following cycling conditions: 50  C for 30 min and 95  C for 15 min, followed by 40 cycles of 95  C for 10 s, and 60  C for 30 s. The influenza A M gene PCR fragment (obtained with the M52C/M253R primers) of A/turkey/Italy/977/99(H7N1) was cloned in a pSC-A-amp/kan vector using the Strataclone PCR cloning kit (Agilent). The plasmid was quantified using the Qubit Fluorometric Quantitation device and kit (ThermoFisher Scientific) and diluted to serve as a standard curve.

Fig. 2. Generation and characterizing recombinant MVA. A.) Schematic overview of the MVA genome and its major deletions sites I to VI with the deletion III being the site for insertion of H9a gene sequences. Flank 1 and Flank 2 correspond to MVA DNA sequences adjacent to deletion site III which have been cloned into MVA transfer plasmids targeting gene insertion into the deletion site III by homologous recombination. In the MVA vector plasmid pIIIHredH9a the H9a encoding sequences were placed under the transcriptional control of the synthetic vaccinia virus early/late promoter PmH5. MVA-H9a was isolated in plaque passages by screening for transient co-expression of the fluorescence marker protein mCherry. Repetitive sequences served to remove the mCherry marker gene by intragenomic homologous recombination. B.) Genetic purity and genetic stability of recombinant MVA-H9a. PCR analysis of genomic viral DNA using oligonucleotide primers to confirm the proper insertion of H9a gene sequences and the integrity of the major genomic deletion sites I, II, IV, V and VI. C.) Multiple step growth analysis of MVA-H9a. Recombinant MVA-H9a and wild-type MVA (MVA-WT) can be efficiently amplified in chicken embryo fibroblasts (CEF), multiplicity of infection (MOI) = 0.001 D.) Synthesis of full-length recombinant haemagglutinin upon MVA-H9a infection. Western blot analysis of cell lysates from MA-104 cells infected with recombinant MVA-H9a. Cell lysates were prepared at 4, 24, 48 and 72 h post infection (MOI = 5). Polypeptides were analyzed by SDS-PAGE and immunoblotted using sera from chicken infected with avian influenza (H9N2). Lysates from uninfected cells (MA-104) served as control.

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2.4. Hemagglutinin inhibition and microneutralization assays Sera collected from chickens pre-vaccine boost, pre-challenge and post-infection were treated with receptor-destroying enzyme (RDE), heat-inactivated at 56  C for 30 min, and diluted 1:10 in PBS. RDE treated sera and diluted (1:10 in PBS) tears were tested by hemagglutination inhibition (HI) assay with 0.5% packed chicken red blood cells. HI and Microneutralization (MN) assays were carried out as described previously in the WHO manual on animal influenza diagnosis and surveillance (WHO, 2010). Neutralizing titers were expressed as the reciprocal of the serum dilution that inhibited 50% of viral growth of 100 50% tissue culture infectious dose of virus. Unless indicated, HI and MN assays were carried out using A/chicken/Tunisia/12/2010(H9N2). 2.5. Histological analysis and immunohistochemistry Three H9N2 infected chickens per group were necropsied at day 4 post infection. Tissue samples of trachea, lungs and intestine were taken and stored in 10% neutral formalin. After fixation, tissues were routinely processed in paraffin blocks, sectioned at 4 mm and stained with hemalun and eosin (H&E) for microscopic examination. All elementary lesions were assessed histologically and their intensity was graded as (- to ++ + + + ) for: no lesion, minimal, slight, moderate, marked or severe. Immunostaining was performed on paraffin-embedded sections of trachea with a monoclonal mouse anti-nucleoprotein Influenza A virus antibody (Argene, 11-030, pronase 0,05% retrieval solution, 10 min at 37  C: antibody dilution 1/50, incubation overnight, at 4  C). The immunohistochemical staining was revealed with a biotinylated polyclonal goat anti-mouse Immunoglobulin conjugated with horseradish peroxidase (HRP) (Dako, LSAB2 system-HRP, K0675) and the diaminobenzidine chromagen of the HRP (Thermo Scientific, TA-125-HDX). 3. Results 3.1. Construction and isolation of recombinant MVA-H9a Recombinant MVA-H9a was constructed by using transfer plasmid pIIIH5red to insert the HA gene sequences into the deletion site III of the MVA genome. The strong synthetic early/late VACV specific promoter PmH5 was used to control transcription of the recombinant HA and a marker gene allowed for transient production of the fluorescent protein mCherry. MVA-H9a virus was isolated in plaque passages by screening for transient coexpression of the

fluorescent marker gene mCherry under the transcriptional control of the vaccinia virus late promoter P11. The mCherry marker had been removed by repetitive sequences (del) by intragenomic homologous recombination (marker gene deletion) (Fig. 2A). To confirm genetic integrity and proper insertion of the HA gene within the MVA genome we performed PCR analysis of the viral genomic DNA by using specific oligonucleotide primers targeting sequences adjacent to the MVA deletion site III (Fig. 2B). To further confirm genetic stability, we also performed PCR analysis of the viral genomic DNA by using specific oligonucleotides targeting sequences adjacent to MVAs deletion sites I, II, IV, V, and VI. In vitro growth behavior of the MVA-H9a virus (Fig. 2C) has been analyzed in multiple-step-growth experiments on CEF which are routinely used for the propagation of recombinant MVA viruses. Non-recombinant MVA-WT served as control virus. In CEF the growth of MVA-H9a was comparable to that of MVA-WT as both viruses replicated to similar titers within 72 h, each increasing infectivity by approximately three to four log10 steps. To confirm the synthesis of HA upon infection with the recombinant MVA-H9a virus, total cell lysates from infected CEF were analyzed by Western Blot using H9-specific chicken monoclonal antibodies. We specifically detected a protein with an estimated molecular mass of 65 kDa in lysates from CEF infected with MVA-H9a. Over a time course of 72 h the amounts of HA protein remained stable in cell lysates from infected CEF (Fig. 2D). 3.2. Ancestral MVA-H9 as vaccine antigen The G1-HA ancestral sequence as calculated with ANCESCON is presented as supplementary material. Supplementary Fig. 1 is the phylogenetic tree that enabled calculating the ancestral H9 sequence. Its aa sequence identity with the viruses used in the present study and for which a HA sequence is available (all isolates described in the Material and Methods section except for A/quail/UAE/D1550/2011) ranged between 99.4% (for A/chicken/ Tunisia/12/2010-G1 like) and 87.4% (for A/shorebird/DE/249/2006, a non-G1, non-Y280-like virus). As expected, the H9 ancestral sequence was more similar to G1 than to Y280 HA sequences and it was even further away to A/shorebird/DE/249/2006 (North American lineage) (Table 1). Sera from the IM2 group (from birds that received 2 doses of MVA-H9 vaccine) were tested for their cross-reactivity with our 10 H9N2 viruses by HI. No cross-reactivity (HI titers < 10) could be observed with any of the tested H9N2 viruses except for A/chicken/ Tunisia/12/2010(H9N2) (G1-like virus, closest vaccine counterpart on a genetic level, 80 < HI titers < 1280, Table 1).

Table 1 Hemagglutinin aa sequence identities and cross-reactivity as measured by Hemagglutination inhibition (HI) test between the vaccine (MVA-ancestral H9) and the viruses tested in the present study. Virus

HA aa sequence identity with the H9-ancestral vaccine antigen HI titer with chicken antiserum generated against the (%) MVA-ancestral H9 vaccine (IM2 group sera, post boost)

A/chicken/Tunisia/12/2010 (G1-like)a A/pheasant/UAE/D1521/2011 (G1-like) A/environment/Bangladesh/10307/2011 (G1like) A/Hong Kong/33982/2009 (G1-like) A/environment/Bangladesh/907/2009(G1-like) A/chicken/Hong Kong/TP38/2003 (Y280-like) A/chicken/Hong Kong/G9/97 (Y280-like) A/chicken/Beijing/1/94 (Y280-like) A/shorebird/DE/249/2006 (North American lineage)

99.4 96.5 96.1

80–1280 <10 <10

92.9 97.3 91.4 92.1 92.9 87.4

<10 <10 <10 <10 <10 <10

a

A partial HA sequence only is available for A/chicken/Tunisia/12/2010.

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Table 2 Antibodies titers pre- and post-challenge by vaccine regimen. Bird id (group-bird number)

Pre-boost HI titersa

Pre-challenge HI titersa

Pre-challenge MN titers

Post inoculation HI titer

IM1-1 IM1-2 IM1-3 IM1-4 IM1-5 IM1-6 IM1-7 IM1-8 IM1-9 IM1-10 Mean IM1

– – – – – – – – – – –

80 80 160 80 320 120 160 320 320 80 172

80 80 160 160 80 320 80 40 320 320 164

320 1280 1280 1280 1280 1280 1280 D D D 1142

IM2-1 IM2-2 IM2-3 IM2-4 IM2-5 IM2-6 IM2-7 IM2-8 IM2-9 IM2-10 Mean IM2

60 20 20 15 20 15 160 40 10 15 37.5

 1280 320 160 320 160 320 1280 640 1280 320 608

1280 640 320 320 640 160 1280 640 1280 160 672

640 1280 640 640 1280 640 1280 D D D  914

NV1-1 NV1-2 NV1-3 NV1-4 NV1-5 NV1-6 NV1-7 NV1-8 NV1-9 NV1-10 Mean NV1

– – – – – – – – – – –

<10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

<10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

160 320 640  1280  1280 640 640 D D D  708

A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 Mean A

<10 <10 <10 <10 <10 <10 <10 <10 <10

<10 <10 <10 <10 <10 <10 <10 <10 <10

NT NT NT NT NT NT NT NT NT

160 640 320 320 640 D D D 416

ED-1 ED-2 ED-3 ED-4 ED-5 ED-6 ED-7 ED-8 Mean ED

<10 <10 <10 <10 <10 <10 <10 <10 <10

<10 <10 <10 <10 <10 <10 <10 <10 <10

NT NT NT NT NT NT NT NT NT

320 160 640 160 640 D D D 384

NV2-1 NV2-2 NV2-3 NV2-4 NV2-5 NV2-6 NV2-7 NV2-8 Mean NV2

– – – – – – – – <10

<10 <10 <10 <10 <10 <10 <10 <10 <10

NT NT NT NT NT NT NT NT NT

160 160 160 160 640 D D D 256

NVNC1/2 all birds

–

<10

<10

<10

–: not relevant: birds not vaccinated twice; D: birds autopsied at day 4 post infection so no serum available 2 weeks post-infection; NT: not tested. a For groups A, ED, NV2, and NVNC2 birds, both sera and tears were tested pre-boost and pre-challenge by HI assay and gave the same result: no detectable HI titer.

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Table 3 Kinetics of antibody production after a single IM vaccination with MVA-ancestral H9. Bird id (group-bird number) IM1-1 IM1-2 IM1-3 IM1-4 IM1-5 IM1-6 IM1-7 IM1-8 IM1-9 IM1-10

HI titers 3 dpv

4 dpv

5 dpv

6 dpv

7 dpv

10 <10 <10 <10 <10 10 <10 <10 <10 <10

20 10 10 10 10 10 10 10 10 20

20 20 20 40 20 40 20 20 20 10

40 40 10 40 30 40 40 20 20 20

80 80 20 40 40 80 40 20 30 40

dpv: days post vaccination.

37.5 three weeks post vaccination (pre-boost column, Table 2). An antibody kinetics was carried out for the IM1 vaccinated group in order to assess how early vaccine-induced antibodies were generated. All vaccinated birds had detectable HI titers (10–20) as early as 4 days post-vaccination and the titers increased with time (Table 3). In contrast, no seroconversion was detected in the eye drop and aerosol vaccinated birds even after 2 vaccine doses (pre-challenge time point). No detectable HI titer was observed pre-challenge in tears either, suggesting that the local immunity had not been activated (Table 2). Taken together, while IM vaccinated birds all seroconverted after 1 and 2 vaccinations, eye drop and aerosol vaccinated birds did not generate detectable antibodies nor systemically (in the serum) nor locally (in the tears) (Table 2).

3.3. Post-vaccination antibodies

3.4. Clinical signs post infection and macroscopic lesions at necropsy

HI and MN tests were carried out on sera after prime and boost vaccinations for the 3 vaccine regimens tested in the present study. Tears were also tested by HI for 5 birds per group for the A, ED, NV2, and NVNC2 groups pre-boost and pre-challenge to assess the presence of mucosal IgY. Antibodies were detected pre-challenge in both IM vaccinated groups by HI and MN assays: IM vaccination lead to the production of neutralizing antibodies (Table 2). Titers were significantly higher after a single vaccine dose when birds were older: group IM1 birds vaccinated at 3 weeks of age had a mean HI titer of 172 three weeks post vaccination (pre-challenge column, Table 2) whereas IM2 birds vaccinated at 1 day of age had a mean HI titer of

An absence of clinical signs was recorded irrespective of the vaccine regimen (and in the non-vaccinated groups). At 4 dpi, three birds per group were necropsied in order to compare macroscopic lesions. During the necropsy of the IM vaccinated and their NVC1 control birds, none of the chicken showed macroscopic lesions. Moderate aerosaculitis and laryngotracheitis lesions were observed in the NVC2 group. The A and ED vaccinated birds also showed larynx congestion with very small hemorrhagic foci but these lesions were significantly less severe in intensity and in extension than for the NVC2 group birds. Two weeks post infection, none of the animals showed any lesion at necropsy except for the NVC2 birds that had slight laryngitis.

Fig. 3. Chickens’ virus shedding in the upper respiratory tract after infection with A/chicken/Tunisia/12/2010(H9N2). Unvaccinated, IM vaccinated and locally vaccinated birds are represented with black lines and square shaped symbols, blue lines and triangle shaped symbols, and red lines and circle shaped symbols, respectively. IM1 birds are represented with dark blue line and symbols; IM2 with light blue line and symbols; A with dark red line and symbols; ED with light blue line and symbols. Birds challenged at 6 weeks of age (groups IM1, IM2, and NV1) are represented with solid lines; birds challenged at 5 weeks of age (groups A, ED, and NV2) are represented with dotted lines. *: group ED birds shed statistically significantly less virus than all other birds 4 dpi, ANOVA, p < 0.05.

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3.5. Virus shedding post-infection All birds shed virus post infection with A/chicken/Tunisia/12/ 2010(H9N2) for 4–7 days irrespective of their vaccine regimen (Fig. 3). No difference in virus titers could be observed between IM vaccinated and non-vaccinated birds despite the high titers of neutralizing antibodies detected. The locally immunized birds shed slightly lower virus loads than their counterparts (with group ED birds shedding statistically significantly less virus than all other birds 4 dpi, ANOVA, p < 0.05) and cleared the infection earlier: virus clearance at day 5 and 6 for eye drop, and aerosol vaccinated birds, respectively (only at day 7 for all the other birds, Fig. 3). 3.6. Histological analysis and immunohistochemistry The main histopathological lesions were observed in the trachea and the bronchi. These lesions consisted of diffuse to multifocal subacute tracheitis and multifocal to focal subacute bronchitis (all lesions are summarized in Table 4 and a picture panel is presented in Fig. 4). The main elementary lesions of subacute tracheitis were: loss of ciliature, focal necrosis and exfoliation of the superficial mucosal epithelium, regenerative epithelial hyperplasia, squamous epithelial metaplasia, inflammatory cellular infiltrates in the lamina propria with mononuclear cells (lymphocytes, plasmocytes and macrophages) and a few heterophils. In the lesions of subacute bronchitis, regenerative epithelial hyperplasia and inflammatory cellular infiltrates in the lamina propria with mononuclear cells (lymphocytes,

65

plasmocytes and macrophages) and a few heterophils were also seen. Pathological differences were observed for the different groups. There was no lesion for the two NVNC groups. The most important lesions were seen for the two NVC groups. The trachea of these birds showed a marked loss of ciliature and a moderate to marked inflammatory cellular infiltration in the lamina propria, mainly with mononuclear cells (lymphocytes, plasmocytes and macrophages). A marked subacute bronchitis was also identified (except for one bird). Main elementary lesions were a moderate epithelial hyperplasia and a moderate to marked inflammatory infiltration in the lamina propria with mononuclear cells. For the vaccinated groups, IM groups must be distinguished from A and ED groups. For IM groups, the intensity of tracheal elementary lesions was only minimal to slight and 3/6 birds had no lesion. In the same way, only 2/6 birds showed bronchial lesions (one with a slight subacute bronchitis and the other, a moderate subacute bronchitis). For A and ED groups, the results were very variable. For the trachea as for the lungs, some birds showed a slight lesional intensity and others were like non vaccinated birds. The immunohistochemical staining showed an intense and homogeneous labelling of the nucleus of some epithelial cells, mainly in the trachea and sometimes in the bronchi, in the lesional areas (Table 4,Fig. 4). Taken together, our results show a difference between routes of MVA-ancestral H9 vaccine inoculation in the chicken model. IM vaccination is the only route tested able of producing detectable antibodies. It does however not protect birds against virus replication post challenge. ED vaccination is the only route tested

Table 4 Histopathological lesions and immunohistochemical staining. Chicken id

Tracheaa

Lungs

Caecum

Immunoreactivity

NVNC1-6 to 9 NVNC2-6 to 9 NVC1-6

–

–

–

neg

PT: ++++ DT: +++ PT: +++ DT: +++ PT: +++ DT: +++ PT: ++++ DT: ++++ PT: ++++ DT: +++ PT: ++++ DT: PT: + DT: + PT: + DT: + – – PT: ++ DT: ++ – PT: ++++ DT: +++ PT: ++ DT: PT: +++ DT: ++ PT: ++++ DT: ++++ PT: +++ DT: ++ PT: ++++ DT: ++++

++++

–

pos: epithelial cells (trachea, bronchi, caecum)

++++

–

pos: epithelial cells (trachea, bronchi, caecum)

+++

–

pos: epithelial cells (trachea, bronchi, caecum)

++++

–

pos: epithelial cells (trachea, bronchi, caecum)

+++++

–

pos: epithelial cells (trachea, bronchi)

–

–

pos: epithelial cells (trachea)

–

–

neg

–

++

neg

+++ – +++

+++ – –

pos: epithelial cells (caecum) neg pos: epithelial cells (trachea, bronchi, caecum)

++ +

– –

neg pos: epithelial cells (trachea)

+++

–

pos: epithelial cells (trachea)

+++

–

pos: epithelial cells (trachea)

+++

–

pos: epithelial cells (trachea)

–

–

pos: epithelial cells (trachea)

+++

–

pos: epithelial cells (trachea)

NVC1-7 NVC1-8 NVC2-6 NVC2-7 NVC2-8 IM1-8 IM1-9 IM1-10 IM2-8 IM2-9 IM2-10 A6 A7 A8 ED6 ED7 ED8

a PT: proximal trachea; DT: distal trachea; intensity of the lesions graded as (– to +++++) for: no lesion, minimal, slight, moderate, marked or severe; pos: immunoreactivity, neg: absence of immunoreactivity.

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Fig. 4. Microscopic lesions and evidence of replication of A/chicken/Tunisia/12/2010(H9N2) in trachea epithelium cells of experimentally infected birds as evidenced by hemalun and eosin staining (HE) and immunohistochemical testing (IPx). For each picture, the staining, zoom, organ, and bird number is indicated in parentheses. (A) (HE, x500, trachea, NVNC2-6); (B) (HE, x500, trachea, NVC2-6): diffuse marked subacute tracheitis with total loss of ciliature, regenerative epithelial hyperplasia and inflammatory cellular infiltrate in the lamina propria with mononuclear cells (lymphocytes, plasmocytes and macrophages); (C) (HE, x500, trachea, IM2-9): focal slight subacute tracheitis with partial loss of ciliature and less important inflammatory cellular infiltration; (D) (IPx, x500, trachea, NVNC2-6); (E) (IPx, x500, trachea, NVC2-6): immunohistochemical labelling of the nucleus of epithelial cells; (F) (IPx, x500, trachea, IM2-9): immunohistochemical labelling of the nucleus of epithelial cells.

able to significantly reduce virus replication and to accelerate virus clearance despite absence of detectable pre-challenge antibodies. Histological and immunohistochemistry analyses highlighted a gradation of lesions and positive influenza cells from absent for unchallenged birds to mild for all vaccinated birds and to moderate for unvaccinated and challenged birds. 4. Discussion No cross-reaction could be observed between antibodies generated against the MVA-ancestral H9 antigen and non-A/chicken/Tunisia/12/2010 G1 viruses (Table 1). This could be explained by the low sensitivity of the HI assay (a low level of antibodies may have been missed). The very close genetic proximity between the ancestral H9 sequence and the A/chicken/Tunisia/12/2010(H9N2) HA sequence: 99.4% identity (3 aa substitutions, Table 1) may explain why cross-reaction was observed. None of these 3 mutations located in known antigenic sites as recently identified by Peacock et al. (Peacock et al., 2016). In comparison, HA ancestral antigens generated for highly pathogenic avian influenza H5N1 viruses were broadly cross-reactive (Ducatez et al., 2011). In our H5N1 study, the ancestral HA antigen “D” was also very close genetically to a virus isolate, A/duck/Hunan/795/02(H5N1), with 3 aa difference only. Antibodies generated against A/duck/Hunan/795/02(H5N1) were however cross-reactive with several HPAI H5N1 viruses tested, which is not the case for serum generated against A/chicken/ Tunisia/12/2010(H9N2) (data not shown). In addition, the ancestral H5N1 vaccines generated were inactivated whole viruses: antibodies generated against these latter vaccines did not only target the HA but also the neuraminidase and internal proteins while the MVA-ancestral H9 vaccine may only induce anti-H9 and anti-MVA antibodies. The present ancestral H9 antigen is therefore difficult to compare with previously generated H5N1 ancestral antigens and more studies are warranted to better assess the gain of using

an ancestral antigen strategy as a vaccine for H9N2 influenza viruses. While Veits et al. showed the benefit of using the MVA vector to protect birds against HPAI H5N1 infection with a reduction in virus titer, we only saw a significant virus shedding reduction from 4 dpi with eye drop vaccinated birds (Veits et al., 2008). The main difficulty of our present model is the quasi absence of clinical signs induced by A/chicken/Tunisia/12/2010(H9N2) even in our nonvaccinated and challenged groups. To come closer to field conditions, further studies may consider using a non-SPF chicken model: commercial broilers are for example much heavier than the SPF birds and by nature their respiratory metabolism shows much higher intensity. In addition, adding a co-infecting pathogen to the model may trigger clinical signs and would better mimic the field situation. Other H9N2 vaccine studies in the chicken model highlighted different levels of clinical signs and virus load reduction. A killed vaccine based on A/chicken/Korea/ADL0401(H9N2) reduced egg drop and re-established feed consumption (Shin et al., 2015). The r4M2e/H70c vaccine was protective in chickens but low H9N2 virus titers were detected for a week in vaccinated birds (Dabaghian et al., 2014). Tavakkoli et al. reported a vaccine study based on two H9N2 viruses that generally (but not systematically) lowered the virus load in the respiratory tract of inoculated birds but did not stop virus replication (Tavakkoli et al., 2011). Finally, the Intervet Nobilis influenza H9N2 vaccine (strain A/CK/UAE/415/99) was shown to reduce mortality in chickens (in a lethal challenge experiment) and to reduce macroscopic lesions of the pancreas in turkeys where no clinical signs were induced (DEFRA, 2010). It is reported that 2.5% of the birds immunized with the Intervet Nobilis influenza H9N2 vaccine did not seroconvert pre-challenge but were still protected from the disease (DEFRA, 2010). This finding is also reported in the ferret model with HPAI H5N1 infections (Ducatez et al., 2013). Here we observed this phenomenon with the ED group: virus shedding was reduced by eye drop

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vaccination despite absence of detectable antibodies. However we also observed the opposite phenomenon: IM vaccinated birds harbored neutralizing antibodies, which were unable to reduce the virus replication after challenge. Further studies should focus on antibodies and correlates of protection in the chicken model to understand the precise role of antibodies in preventing disease but also virus replication. Surprisingly, the only vaccine route able to induce antibodies was the IM route. However, IM vaccination is not the preferred administration route in the field when dealing with thousands of birds. We expected aerosol and eye drop vaccine administrations to better induce local immunity and to induce detectable antibody titers in the serum and tears. Further studies are warranted to attempt explaining our challenge findings: (i) screen for IgA specifically and better assess the mucosal immune response, (ii) assess the cellular response, (iii) a challenge study with a homologous ancestral H9 virus would be a good control, (iv) a comparative vaccine/challenge experiment with a commercial H9N2 vaccine, with a recombinant ancestral H9, and with an empty MVA vector would enable a proper estimation of the benefit of using the MVA-H9 vaccine strategy. This latter experiment should indeed give us insights on the launch of anti-MVA versus anti-H9 immune responses and may explain our vaccine study findings. It is unfortunately unknown whether the MVA-ancestral H9 is able to replicate or not in the chicken. The MVA vector was shown not to replicate in mammalian models (Sutter and Moss, 1992) but does efficiently grow in CEF. Actually, MVA as a vaccine does not need to replicate to induce an efficient T cell response. The T cell response to the vaccine may thus explain in part its observed benefit with local administrations provided the vaccine is replicative. Conflict of interest None. Authors contribution MFD, JLG, GS and AV designed the study; MFD, JB, AF, MDelv, MDelp, and AV performed the experiments; MFD, JB, MDelv, GS, JLG and AV analyzed the data; MFD, JB, GS, JLG and AV wrote the manuscript. Acknowledgments The authors want to thank Ambre Grand-Moursel and Céline Bleuart for technical assistance. We thank A. Grahm (Pasteur Institute, Tunis, Tunisia) for providing A/chicken/Tunisia/12/2010 (H9N2), R. Webby and J. Debeauchamp (Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA), M. Peiris and Y. Guan (Hong Kong University, Hong Kong SAR, China) for providing the remaining H9N2 isolates. The project was supported by the EU project FLUNIVAC (grant number 602604) and by the French ministries of Agriculture, Research and Education. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2016.04.025.

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