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

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.

Figure 1. Cross-reactive HA antibody responses in the mouse model measured by ELISA

Serum IgG antibodies against all subtypes of HA were measured by ELISA. Pooled sera of 10 mice per group were measured in technical duplicates and the geometric mean value was used for graphical representation. A heat map overlay on top of a phylogenetic tree was used to illustrate the antibody titers induced by repeated influenza virus infection. The scale bar represents 4% amino acid difference. A) HA titers after a single H1N1 (NC99) infection in mice. B) HA titers after two consecutive H1N1 (NC99+NL09) infections in mice. C) HA titers after a single H3N2 (Phil82) infection in mice. D) HA titers after two consecutive H3N2 (Phil82+Vic11) infections in mice.

Figure 2. Cross-reactive NA antibody responses in the mouse model measured by ELISA

Serum IgG antibodies against NA were measured by ELISA. Pooled sera of 10 mice per group were measured in technical duplicates and the geometric mean value was used for graphical representation. A heat map overlay on top of a phylogenetic tree was used to illustrate the antibody titers induced by repeated influenza virus infection. The scale bar represents 8% amino acid difference. A) NA titers after a single H1N1 (NC99) infection in mice. B) NA titers after two consecutive H1N1 (NC99+NL09) infections in mice. C) NA titers after a single H3N2 (Phil82) infection in mice. D) NA titers after two consecutive H3N2 (Phil82+Vic11) infections in mice.

Figure 3. Cross-reactive antibody titers post infection negatively correlate with phylogenetic distance

To illustrate a correlation of antibody titers with phylogenetic distance, antibody titers measured by ELISA after sequential influenza virus infection were plotted on the y-axis and the percent amino acid difference to the HA of the strain used for the 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. The geometric mean titer against all non-H1 group 1 HAs is plotted as a light blue line. The geometric mean titer against all non-H3 group 2 HAs is plotted as a light red line. A) Mouse group 1 HA titers after two consecutive H1N1 (NC99+NL09) infections. 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 two consecutive H1N1 (NC99+NL09) infections. ELISAs were performed on individual sera of 3 guinea pigs and geometric mean titers are shown. C) Ferret group 1 HA titers after two consecutive H1N1 (NC99+NL09) infections. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown. D) Mouse group 2 HA titers after two consecutive H3N2 (Phil82+Vic11) infections. 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 two consecutive H3N2 (Phil82+Vic11) infections. ELISAs were performed on individual sera of 2 guinea pigs and geometric mean titers are shown. F Ferret group 2 HA titers after two consecutive H3N2 (Phil82+Vic11) infections. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown.

Figure 4. Human antibody responses post pH1N1 infection are broader than post H3N2 infection

A) Geometric mean fold-induction of individual antibody responses (n=9) post pH1N1 infection was plotted as a heat-map on a phylogenetic tree of HA. The scale bar represents 4% amino acid difference. B) The geometric mean fold-induction and 95% confidence intervals post pH1N1 infection of 9 individuals was plotted in a bar graph and sorted from highest to lowest induction. Group 1 HAs are shown as blue bars, group 2 HAs are shown as red bars and influenza B HA is shown in green. C) Three dimensional surfaces of the pre- and post-pH1N1 infection antibody titers were generated by plotting the relative distances of HAs to each other based on amino acid differences. Both x-and y-axes show amino acid differences and geometric mean titers (n=9) are plotted on the z-axis. The post-infection surfaces were plotted in color (blue for group 1 HAs, red for group 2 HAs) and the corresponding pre-infection surfaces are plotted in gray. D) Geometric mean fold-induction of individual antibody responses (n=10) post H3N2 infection was plotted as a heat-map on a phylogenetic tree of HA. The scale bar represents 8% amino acid difference. E) The geometric mean fold-induction and 95% confidence intervals post H3N2 infection of 10 individuals was plotted in a bar graph and sorted from highest to lowest induction. Group 1 HAs are shown as blue bars, group 2 HAs are shown as red bars and influenza B HA is shown in green. F) Three dimensional surfaces of the pre- and post-H3N2 infection antibody titers were generated by plotting the relative distances of HAs to each other based on amino acid differences. Both x-and y-axes show amino acid differences and geometric mean titers (n=10) are plotted on the z-axis. The post-infection surfaces were plotted in color (blue for group 1 HAs, red for group 2 HAs) and the corresponding pre-infection surfaces are plotted in gray.

Figure 5. Titers in the general human population show distinct age related patterns

A) Schematic of selection of age groups that were tested. To investigate the effect of pre-exposure to different viruses, three distinct age groups were selected depending on the influenza viruses that most likely circulated during the childhood of the individuals. The oldest age group (49–64 year olds, ‘experienced’) was born when either a drifted version of the 1918 H1N1 virus or the H2N2 pandemic virus circulated. All individuals in this group should therefore have had exposure to H2N2 viruses. The slightly younger age group (33–44 year olds, ‘middle-aged’) was born after H2N2 went extinct and was replaced by the group 2 H3N2 virus and has therefore not been previously exposed to H2N2. 18–20 year olds (‘young’ cohort) were used as a young control group with only a limited exposure to group 1 and group 2 influenza A virus strains. B–D) To illustrate the differences of antibody profiles in the different age groups, three dimensional antigenic landscapes were plotted. All HAs are shown in their relative difference to each other based on amino acid sequences on the x- and y-axes. The geometric mean titers were plotted on the z-axis. B) Geometric mean titers of 18–20 year old individuals (n=30). C) Geometric mean titers of 33–44 year old individuals (n=30). D) Geometric mean titers of 49–64 year old individuals (n=30).

Figure 6. In vitro and in vivo protective effect of sera from the general human population

To test if antibody titers measured by ELISA also translate to measurable neutralizing responses in vitro and protective responses in vivo, re-assortant viruses that contain the same PR8 backbone and an irrelevant neuraminidase were generated for H1, H3, H4, H4, H7 and H9. A) Individual microneutralization titers against group 1 viruses (H1, H5, H9) are shown for sera from 18–20 year olds (blue dots, n=30), 33–44 year olds (red squares, n=30) and 49–64 year olds (teal triangles, n=30). A bar that represents the geometric mean titer is shown for each group. The gray line indicates the limit of detection for the assay. For each virus, groups were compared with an ordinary one-way ANOVA, followed by a Tukey’s multiple comparisons test. Statistical significance is indicated as follows: ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. B) Proportion of individuals with a neutralization titer of 1:40 or higher against group 1 viruses (H1, H5, H9) for each age group (n=30 per group). C) Individual microneutralization titers against group 2 viruses (H3, H4, H7) are shown for sera from 18–20 year olds (blue dots, n=30), 33–44 year olds (red squares, n=30) and 49–64 year olds (teal triangles, n=30). A bar that represents the geometric mean titer is shown for each group. The gray line indicates the limit of detection for the assay. For each virus, groups were compared with an ordinary one-way ANOVA, followed by a Tukey’s multiple comparisons test. Statistical significance is indicated as follows: ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. D) Proportion of individuals with a neutralization titer of 1:40 or higher against group 2 viruses (H3, H4, H7) for each age group (n=30 per group) E–F) Serum transfer studies in mice (n=5 per group per virus) were performed with pooled serum from each age group (n=30 per group) with subsequent challenge with viruses containing H3, H4 or H7 HAs. Mice that received immunoglobulin depleted serum served as a control. To measure the level of protection, lungs were extracted on day 3 and day 6 post-challenge and virus titers measured by plaque assay. A bar that represents the geometric mean titer is shown for each group. The gray line indicates the limit of detection for the assay. For each virus, groups were compared to the immunoglobulin depleted group with an ordinary one-way ANOVA, followed by a Dunnet’s multiple comparisons test. Statistical significance is indicated as follows: ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.