Immunogenicity of Avian Influenza H7N9 Virus in Birds
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1 Immunogenicity of Avian Influenza H7N9 Virus in Birds Identification of Viral Epitopes Recognized by the Immune System Following Vaccination and Challenge Darrell R. Kapczynski US DEPARTMENT OF AGRICULTURE, Agricultural Research Service South Atlantic Area, Athens, Georgia
2 Avian Influenza (AI) Orthomyxoviridae family. Enveloped type A, B, C birds affected by A and B designated A/turkey/Virginia/1/2002 Negative-sense RNA 8 segments Small RNA virus Type A influenza virus Surface spikes hemagglutinin & neuraminidase proteins 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes Ex. H5N1, H9N2, H7N7 Vary in disease production (chickens): Low pathogenicity (LP): local -mild respiratory disease and egg drop High pathogenicity (HP): systemic - deadly disease (some H5 & H7 AIV) Paul Digard, Dept Pathology, University of Cambridge.
3 Background for Avian Influenza Avian influenza virus (AIV) is endemic in wild birds Transmission of AIV from wild birds to poultry species (ducks, chickens, turkeys) occurs commonly AIV on rare occasions may become adapted to and become endemic in poultry species (chickens and turkeys) AIV once adapted to chickens and turkeys can be difficult to eradicate Has been demonstrated to jump species barriers
4 H7N9 Avian Influenza Infections 2013: China -First report March 31, cases (115 deaths) as of 2/28/2014 (25 % fatality) Low pathogenic for birds ecdc.europa.eu
5 H7N9 Gene Origins Molecular characteristics HA: PKGR / GLF Cleavage site-> low pathogenic Gln226Leu-enhances binding of HA to mammalian α2-6 sialic acid NA: Deletion of residues in stalk region-mutation associated with adaption to domestic poultry and with replication of the virus in the respiratory tract. PB2: Glu627Lys-associated with adaption to mammals
6 Surveillance Data Source: Zhang Zhongqiu 5/30/2013
7 Location of Positives Samples
8 H7N9 predicted to be poorly immunogenic High Low De Groot, et al. Low immunogenicity predicted for emerging avian-origin H7N9: Implication for influenza vaccine design. Vaccines & Immunotherapeutics 2013; 9: ; PMID:
9 Avian Influenza Vaccines: Poultry Vaccination not routine in most of the world No universal vaccine for avian influenza viruses Anti-hemagglutinin antibodies are protective Types of Vaccines Oil-emulsified Inactivated Whole Avian Influenza Virus (95.5% vaccine used) Recombinant live virus vectors with AI hemagglutinin gene insert (4.5% used): Recombinant Fowlpox virus (rfpv) Recombinant Newcastle Disease virus (rndv) *Recombinant herpesvirus turkey (rhvt) *Recombinant duck virus enteritis (rdve)
10 Epitope Mapping Against H7 HA From A/Anhui/1/2013 H7N9 J Stevens et al. Science 2006;312:
11 Technology Combinatorial Approach laser printing of amino acid toner particles with a 24- color peptide laser printer melting initiates coupling based on Fmoc chemistry peptide microarray resolution of 800 peptides/cm 2 digital printing flexibility, routine double coupling Bayer et al., Science 2007, Stadler et al., Angew Chem Int Ed Engl. 2008
12 PEPperCHIP Epitope Mapping Antigen Sequence Overlapping Peptides Target Antibody Secondary Antibody Peptide Microarray Microarray Scan (Double Spots) Monoclonal Sample Polyclonal Sample Epitope = Consensus Motif Data Analysis
13 Adjuvant Development goal: epitope mapping of B-cell responses upon immunizations with different adjuvants microarray setup: antigen translated into overlapping peptides in duplicate samples: >50 rabbit IgG antibodies outcome: fingerprint analysis of epitopes and immunoreactivity (ELISA: yes/no) Shukla et al., PLoS One 2012; 7(8), e43612
14 Pre-Immune/Immune Analysis goal: in-depth analysis of immune responses upon immunization, vaccination or drug administration microarray setup: given antigens translated into overlapping peptides with maximum peptide overlap samples: various pre-immune/immune sera (mice, chicken, human) with different immune time points outcome: time-resolved fingerprint of B-cell responses (immunization, anti-drug antibodies) Pre-Immune Immune 4 Immune 2
15 Epitope Mapping against H7 HA From A/Anhui/1/2013 H7N9 Microarray Content: The sequence of H7 HA was elongated by neutral GSGSGSG linkers at the C- and N-terminus to avoid truncated peptides and translated into 15 aa peptides with a peptide-peptide overlap of 14 aa. The resulting peptide microarrays contained 560 different peptides printed as duplicates (1,120 peptide spots) and were framed by Flag and HA control peptides (86 spots each). Antigen Sequence: GSGSGSGMNTQILVFALIAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNIPRICSKGKR TVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKEAMGFTYS GIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKSPALIVWGIHHSVSTAEQTKLYG SGNKLVTVGSSNYQQSFVPSPGARPQVNGLSGRIDFHWLMLNPNDTVTFSFNGAFIAPDRASFLRGKS MGIQSGVQVDANCEGDCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEIPKGRGLF GAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFNEV EKQIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMDKLYERVKRQLRENAEEDGTGCFEIFHK CDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVKLSSGYKDVILWFSFGASCFILLAIVMGLVFICVKNGN MRCTICIGSGSGSG Samples: Prechallenge Control Sham-H7N9 Challenged Vaccinated-H7N9 Challenged
16 Controls Peptide Array Scans- Secondary Antibody Only Peptide Array Scans Prechallenge Bird Goat anti-chicken IgG (H+L) DyLight680 antibody, 1:5000 Pre-Challenge, 1:1000
17 Peptide Array Scans Sham-H7N9 infected Bird
18 Intensity Plots Sham-H7N9 infected
19 Peptide Array Scan Low dose vaccine / H7N9 challenge Post-Ch, 1:1000
20 Intensity Plots Low dose vaccine / H7N9 challenge
21 Peptide Array Scans High dose vaccine/ H7N9 challenge
22 Intensity Plots High dose vaccine/ H7N9 challenge
23 FUTURE WORKS Map location on HA protein that the antibodies are recognizing Produced polyclonal antibodies to each individual peptide Examine function of antibodies at each location Tharakaramanetal.,GlycanReceptorBindingoftheInfluenzAVirusH7N9Hemagglutinin,Cell(2013
24 Conclusions In the field, the H7N9 virus does not cause any disease or apparent production loss in the poultry population. H7N9 is low pathogenic for poultry. Vaccination reduced clinical signs of disease and shedding. The high dose H7N9 vaccine provided best protection from high challenge dose used here. Current US H7 master seed isolates fully protective. We observed strong and clear polyclonal responses in sera agaisnt HA epitopes of H7N9. The most complex response was observed for serum from birds vaccinated with a high dose of vaccine and challenged. Dominant epitopes were identified. The most immunogenic site in H7 HA was obviously presented with clear antibody responses in all birds. Vaccine studies under way. Could a vaccination program reduce or prevent transmission to humans?
25 SEPRL David Swayne David Suarez Erica Spackman Mary Pantin- Jackwood Aniko Zsak Diane Smith Scott Lee Cam Greene HaiJun Jiang Roger Brock Ronald Graham Acknowledgements Dr. Darrell Kapczynski USDA/ARS/SEPRL 934 College Station Rd Athens, Georgia, USA Volker Stadler Lisa Steinbrück Carsten Harber Southeast Poultry Research Southeast Laboratory Poultry Research Laboratory
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