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Defining the antibody cross-reactome directed against the influenza virus surface glycoproteins

Nature Immunology volume 18, pages 464–473 (2017)Cite this article

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Abstract

Infection with influenza virus induces antibodies to the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To assess the breadth and magnitude of antibody responses, we sequentially infected mice, guinea pigs and ferrets with divergent H1N1 or H3N2 subtypes of influenza virus. We measured antibody responses by ELISA of an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after infection of humans with influenza virus H1N1 or H3N2 and found markedly broad responses and cogent evidence for 'original antigenic sin'. This work will inform the design of universal vaccines against influenza virus and can guide pandemic-preparedness efforts directed against emerging influenza viruses.

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Figure 1: Cross-reactive antibodies to HA in the mouse model.
Figure 2: Cross-reactive antibodies to NA in the mouse model.
Figure 3: Correlation of cross-reactive antibody titers after infection and phylogenetic distance.
Figure 4: Human antibody responses after infection with pH1N1 or H3N2.
Figure 5: Antibody titers in the general human population versus age.
Figure 6: In vitro and in vivo protective effect of serum from the general human population.

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Acknowledgements

We thank L. Aguado for cloning of several of the HA expression vectors; J. Runstadler (Massachusetts Institute of Technology) for making several avian influenza viruses available to us; F. Amanat for IgG purification; P. E. Leon (Icahn School of Medicine at Mount Sinai) for the H3N8 and H9N4 viruses; C. Marizzi for reviewing the manuscript; BEI Resources for sequences of influenza virus HA and NA; and R. Fouchier (Erasmus MC) for depositing plasmids encoding HA and NA at BEI Resources. Supported by the US National Institutes of Health (U19 AI109946-01 to P.P. and F.K.; and R01 AI117287-01A1 to F.K. and N.M.B.), Centers of Excellence in Influenza Virus Research and Surveillance of the US National Institutes of Health (HHSN272201400008C to A.G.S., P.P., F.K., R.A.M. and N.M.B.), Comisión Nacional de Investigación Científica y Tecnológica (FONDECYT 1121172 and 1161791 to R.A.M.; and PIA ACT 1408 to R.A.M.), and the Chilean Ministry of Economy, Development and Tourism (P09/016-F to R.A.M.).

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Authors and Affiliations

  1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Raffael Nachbagauer, Angela Choi, Ariana Hirsh, Irina Margine, Randy A Albrecht, Adolfo García-Sastre, Nicole M Bouvier, Rafael A Medina, Peter Palese & Florian Krammer

  2. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Angela Choi

  3. Division of Bioinformatics, Hokkaido University Research Center for Zoonosis Control, Kitaku, Japan

    Sayaka Iida & Kimihito Ito

  4. Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile

    Aldo Barrera, Marcela Ferres & Rafael A Medina

  5. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Randy A Albrecht & Adolfo García-Sastre

  6. Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Adolfo García-Sastre, Nicole M Bouvier & Peter Palese

  7. Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile

    Rafael A Medina

Authors
  1. Raffael Nachbagauer
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Contributions

R.N., A.C., A.H., I.M. and F.K. performed serological experiments. R.N., A.C., I.M., N.M.B., R.A.A. and F.K. performed animal experiments. A.B., M.F. and R.A.M. acquired clinical samples and performed all necessary experiments to characterize the clinical samples. R.N., S.I., A.G.S., K.I., R.A.M. and F.K. performed data analysis. R.N., R.A.A., P.P., A.G.S. and F.K. contributed to the overall concept, experimental design and hypothesis and wrote the manuscript.

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Correspondence to Florian Krammer.

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Integrated supplementary information

Supplementary Figure 1 Infection strategy.

To test the antibody responses against influenza viruses, animals were sequentially infected with two divergent strains of the same subtype. For H1N1 infections, animals were primed with a human seasonal strain from 1999 (NC99, A/New Caledonia/20/1999), followed by the 2009 human pandemic H1N1 strain (NL09, A/Netherlands/602/2009) six weeks later. These viruses express divergent HA head domains, but similar HA stalk domains. For H3N2 infection animals were primed with a human seasonal strain from 1982 (Phil82, A/Philippines/2/1982), followed by a 2011 human seasonal strain (Vic11, A/Victoria/361/2011). These viruses are separated by almost 30 years of antigenic drift.

Supplementary Figure 2 Viral titers on day 2 after infection in animal models.

Mice (A, n=5/virus), Guinea pigs (B, n=2/virus) and ferrets (C, n=2/virus) were infected with NC99, NL09, Phil82 and Cal09 and viral titers were measured on day 2 post infection from nasal wash (guinea pigs and ferrets) or nasal turbinates (mice).

Supplementary Figure 3 Heat-map presentation of guinea pig antibody titers after infection.

A) Anti-HA titers of guinea pigs (n=3) after one H1N1 (NC99) infection. B) Anti-HA titers of guinea pigs (n=3) after sequential infection with two divergent H1N1 (NC99, NL09) strains. C) Anti-HA titers of guinea pigs (n=3) after one H3N2 (Phil82) infection. D) Anti-HA titers of guinea pigs (n=2) after sequential infection with two divergent H3N2 (Phil82, Vic11) strains. E-H) Anti-NA titers of the corresponding sera to panels A-D.

Supplementary Figure 4 Heat-map presentation of ferret antibody titers after infection.

A) Anti-HA titers of ferrets (n=2) after one H1N1 (NC99) infection. B) Anti-HA titers of ferrets (n=2) after sequential infection with two divergent H1N1 (NC99, NL09) strains. C) Anti-HA titers of ferrets (n=2) after one H3N2 (Phil82) infection. D) Anti-HA titers of ferrets (n=2) after sequential infection with two divergent H3N2 (Phil82, Vic11) strains. E-H) Anti-NA titers of the corresponding sera to panels A-D.

Supplementary Figure 5 Profiles of the titers of cross-reactive antibodies after a single infection in animal models.

Antibody titers measured by ELISA after single influenza virus infection were plotted on the y-axis and the percent amino acid difference to the HA of the strain used for the (later) second infection was plotted on the x-axis. Each point represents the geometric mean titer measured against a single HA (dark blue dot for H1 HAs, light blue dot for other group 1 HAs, dark red triangle for H3 HAs, light red triangle for other group 2 HAs). A non-linear fit (plateau followed by one phase decay) was performed to illustrate the differences in the breadth of the antibody response in all animals. Points for HAs with titers lower than 103 are plotted at 103. A) Mouse group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on pooled sera of 10 mice and geometric mean titers of technical duplicates are shown. B) Guinea pig group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on individual sera of 3 guinea pigs and geometric mean titers are shown. C) Ferret group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown. D) Mouse group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on pooled sera of 10 mice and geometric mean titers of technical duplicates are shown. E) Guinea pig group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on individual sera of 3 guinea pigs and geometric mean titers are shown. F Ferret group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown.

Supplementary Figure 6 Glycosylation versus antibody titers after infection.

A-C) Antibody titers post 2x infection with either H1N1 (circles) or H3N2 (triangles) against H1 (blue) or H3 (red) HAs were plotted against the number of corresponding glycosylation sites of each HA for all animal models. Mouse samples show the geometric mean of technical duplicates of pooled sera from 10 mice per virus. Guinea pigs show the geometric mean titers of individual animals (H1N1: n=3, H3N2: n=2). For ferrets, the geometric mean titers of individual animals (n=2/virus) is shown. D) Human antibody titers (geometric mean) post pH1N1 (Cal09, n=9) or H3N2 (Vic11, n=10) infection were plotted against the number of glycosylation sites of the corresponding HAs. Circles indicate titers for individuals infected with pH1N1 and triangles indicate titers for individuals infected with H3N2. Symbols for titers measured against H1 HAs are colored red and symbols for titers measured against H3 HAs are colored blue.

Supplementary Figure 7 Profiles of the titers of cross-reactive antibodies after infection of humans with influenza virus.

A) Geometric mean antibody titers of human individuals post pH1N1 infection (n=9) were plotted on the y-axis and the percent amino acid difference to the HA of the infection strain was plotted on the x-axis. Each point represents the geometric mean titer of all individuals measured against a single HA. Dots illustrate group 1 HAs, triangles group 2 HAs and squares influenza B HA. The post-infection titers are shown in color (blue for group 1, red for group 2 and green for influenza B) and the corresponding pre-infection titers are plotted in gray. A non-linear fit (plateau followed by one phase decay) was performed to illustrate the breadth of the antibody response. B) Geometric mean antibody titers of human individuals post H3N2 infection (n=10) were plotted in the same manner as panel A.

Supplementary Figure 8 ELISA of the titers of antibody to HA in humans before and after infection.

A) Heat map representation of pre-pH1N1 infection ELISA antibody titers against HA (geometric mean of 9 individuals). B) Heat map representation of post-pH1N1 infection ELISA antibody titers against HA (geometric mean of 9 individuals). C) Heat map representation of pre-H3N2 infection ELISA antibody titers against HA (geometric mean of 10 individuals). B) Heat map representation of post-H3N2 infection ELISA antibody titers against HA (geometric mean of 10 individuals).

Supplementary Figure 9 Human reactivity to NA before and after infection.

Heat map representations of human geometric mean ELISA antibody titers against NA pre- and post-pH1N1 (A, B, n=9) or H3N2 (C, D, n=10) infections. E-F) Heat map representations of geometric mean fold-induction of antibody titers against NA post H1N1 (E, n=9) or H3N2 (F, n=10) infection in humans. G-H) Geometric mean fold-induction of antibody titers against NA post-pH1N1 (n=9) or post-H3N2 (n=10) infection in humans were sorted by highest induction in a bar graph. Blue bars represent group 1 NAs, red bars group 2 NAs, green influenza B NA and grey the N10 bat isolate. Error bars indicate the 95% confidence intervals.

Supplementary Figure 10 Heat-map presentation of ELISA of titers of human antibodies to HA and NA (geometric mean values), separated by age group.

A) Geometric mean anti-HA titers of 18-20 year olds (n=30). B) Geometric mean anti-NA titers of 18-20 year olds (n=30). C) Geometric mean anti-HA titers of 33-44 year olds (n=30). D) Geometric mean anti-NA titers of 33-44 year olds (n=30). E) Geometric mean anti-HA titers of 49-64 year olds (n=30). F) Geometric mean anti-NA titers of 49-64 year olds (n=30).

Supplementary Figure 11 Serum-transfer experiment.

For each virus challenge, serum samples of 30 individuals per age group were pooled, sterile filtrated and 250 μl of serum intraperitoneally injected into 10 mice for each group. An equal number of mice received immunoglobulin depleted serum as a negative control group. Two hours after the serum transfer, mice were anesthetized and infected with 1 × 105 PFU of virus diluted in PBS (50 μl total volume) intranasally. Five mice each were euthanized on days 3 and 6, their lungs extracted and viral titers measured by plaque assay.

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Nachbagauer, R., Choi, A., Hirsh, A. et al. Defining the antibody cross-reactome directed against the influenza virus surface glycoproteins. Nat Immunol 18, 464–473 (2017). https://doi.org/10.1038/ni.3684

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