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. 2016 Sep 29;90(20):9383-93.
doi: 10.1128/JVI.01408-16. Print 2016 Oct 15.

Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies

Surender Khurana  1 , Ka Yan Chung  2 , Elizabeth M Coyle  2 , Adam Meijer  3 , Hana Golding  2
Affiliations

Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies

Surender Khurana et al. J Virol. .

Abstract

Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of low-pathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus.

Importance: An outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.

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Figures

FIG 1
FIG 1
Elucidation of the epitope profile of the H7 HA protein recognized by antibodies in individuals following HPAI H7N7 virus exposure in The Netherlands. (A) Distribution of serum HI titers against H7N7 (A/Netherlands/33/03) in 15 individuals following exposure to HPAI H7N7 virus. (B) Alignment of peptides recognized by pooled sera from H7N7-exposed individuals identified by using an H7-HA GFPDL to the H7 HA translated sequence. The HA1 RBD is depicted in yellow. The amino acid designation is based on the HA protein sequence. Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, is shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone with its homologous sequence in the influenza virus HA protein upon alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment.
FIG 2
FIG 2
Total antibody binding, antibody isotype, and affinity of polyclonal plasma for H7-HA1 and HA2 proteins in HPAI H7N7 virus-exposed subjects. (A and B) Steady-state equilibrium analysis of the total binding antibodies to properly folded functional H7-HA1 (A) or H7 HA2 (B) in polyclonal human sera was measured by SPR. HI titers are shown in parentheses for each subject. Dots represent data for serum samples obtained following H7N7 exposure that were diluted 10-fold or 100-fold and injected simultaneously onto H7-HA1 or H7-HA2 immobilized on a sensor chip through the free amine group and onto a blank flow cell free of peptide. Maximum resonance unit (RU) values for antibody binding are shown for individual H7N7-exposed subjects. (C) Isotype of serum antibodies bound to H7-HA1 for sera obtained following H7N7 exposure. Data shown are the mean percent contributions of each isotype to total HA1 binding in individual samples from two independent experiments. (D and E) Total binding antibodies (maximum RU) against H7-HA1 (D) or H7-HA2 (E) in human sera obtained following H7N7 exposure were correlated with the homologous H7N7 virus HI titers. Spearman correlations are reported for the calculation of correlations between total anti-HA1 antibody binding to HA1 (D) or HA2 (E) and HI titers combined across all individuals. The color scheme in panels D and E is the same as that in panels A and B. (F and G) Antibody avidity measurements in polyclonal sera by off-rate constants determined by using SPR. Antibody off-rate constants, which describe the stability of the antigen-antibody complexes, were determined directly from polyclonal serum sample interactions with H7-HA1 (F) or H7-HA2 (G) proteins by using SPR in the dissociation phase and provide a measure of net binding affinity. For accurate measurements, parallel lines in the dissociation phase for the 10-fold and 100-fold dilutions for each human serum sample were required. The off-rate constants were determined from two independent SPR runs. (H and I) Serum antibody off-rate constants for infected individuals (each symbol indicates results for one individual) were plotted. Correlation statistics of affinity measurements and off-rate constants of antibody binding to H7-HA1 (H) or H7-HA2 (I) with H7N7 HI titers did not reach statistical significance.
FIG 3
FIG 3
Epitope repertoire of N7-NA-specific antibodies generated following H7N7 exposure. (A) Schematic alignment of the phage-displayed epitopes in N7-NA recognized by antibodies in pooled plasma obtained following H7N7 exposure. Peptides were identified by panning with an H7N7-NA GFPDL and were aligned to the N7-NA translated sequence. The amino acid designation is based on the NA protein sequence. Bars indicate identified inserts, and the 3 different antigenic domains selected from epitope mapping are depicted as green, orange, and blue bars. The thickness of each bar represents the frequency of repetitively isolated phage inserts (only clones with a frequency of 2 or more are shown), and the scale is shown below the alignment. CTD, C-terminal domain. (B) The conformational epitope in NA (NA residues 105 to 193) (green) and the immunodominant epitope (NA residues 194 to 270) (orange) are shown on the monomeric N7-NA structure (PDB accession number 4QN7), with bound oseltamivir shown in red. (C) Steady-state equilibrium of total binding of antibodies in polyclonal human sera to properly folded functional N7-NA was measured by SPR (HI titers for each subject are shown in brackets). Dots represent data for individual serum samples obtained following H7N7 exposure that were diluted 10-fold and injected simultaneously onto N7-NA immobilized on a sensor chip through the free amine group and onto a blank flow cell free of peptide. Maximum RU values for antibody binding are shown for individual H7N7-infected subjects. Data shown are the means from two independent experiments.
FIG 4
FIG 4
Antibody epitopes in H7N7 internal proteins (H7-GFPDL-6) recognized by pooled sera from H7N7-exposed individuals and immune response to PA-X. (A) Schematic alignment of the identified epitopes recognized by antibodies in pooled polyclonal sera from H7N7-exposed individuals by using GFPDLs (H7-GFPDL-6) expressing all internal proteins of influenza H7N7 A/Netherlands/33/03 virus. Graphical distributions of representative clones with a frequency of ≥2 obtained after affinity selection are shown. The horizontal position and the length of the bars indicate the alignment of the peptide sequence displayed on the selected phage clone to its homologous sequence in the corresponding influenza virus protein. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. The amino acid designation is based on PA, and the PA-X protein sequence shows the common N-terminal sequence between PA and PA-X and the unique 61-amino-acid sequence at the C termini of PA-X due to ribosomal frameshifting. (B, D, and E) Reactivity of serum antibodies from 15 individual H7N7-exposed subjects against a synthetic PA-X peptide (aa 191 to 252) derived from the unique PA-X (aa 192 to 252) protein sequence from H7N7 (B), seasonal H1N1 (D), or seasonal H3N2 (E) determined by an SPR assay. (C) Reactivity of serum antibodies from 20 U.S. adults (exposed to H1N1pdm09) against the same synthetic H7N7 PA-X peptide.
FIG 5
FIG 5
Alignment of the PA-X protein sequence of the H7N7 A/Netherlands/33/03 strain with multiple seasonal and avian influenza virus strains.
FIG 6
FIG 6
Reactivity of sera from ferrets infected with seasonal or avian influenza viruses against PA-X peptides determined by using SPR. Preinfection ferret serum and sera from ferrets infected with wild-type influenza virus strains (H1N1 A/Solomon Islands/3/06, H3N2 A/Uruguay/716/2007, H5N1 A/Vietnam/1203/2004, and H7N7 A/Netherlands/219/03) were analyzed with PA-X peptides (A) derived from H7N7 (B), H1N1 (C), and H3N2 (D) by SPR. wt, wild type.
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References

    1. Morens DM, Taubenberger JK. 2015. How low is the risk of influenza A(H5N1) infection? J Infect Dis 211:1364–1366. doi:10.1093/infdis/jiu530. - DOI - PMC - PubMed
    1. Lee DH, Torchetti MK, Winker K, Ip HS, Song CS, Swayne DE. 2015. Intercontinental spread of Asian-origin H5N8 to North America through Beringia by migratory birds. J Virol 89:6521–6524. doi:10.1128/JVI.00728-15. - DOI - PMC - PubMed
    1. Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y. 2008. Human infection with highly pathogenic H5N1 influenza virus. Lancet 371:1464–1475. doi:10.1016/S0140-6736(08)60627-3. - DOI - PubMed
    1. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, Meijer A, van Steenbergen J, Fouchier R, Osterhaus A, Bosman A. 2004. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in The Netherlands. Lancet 363:587–593. doi:10.1016/S0140-6736(04)15589-X. - DOI - PubMed
    1. Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA, Munster V, Kuiken T, Rimmelzwaan GF, Schutten M, Van Doornum GJ, Koch G, Bosman A, Koopmans M, Osterhaus AD. 2004. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci U S A 101:1356–1361. doi:10.1073/pnas.0308352100. - DOI - PMC - PubMed

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