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. 2022 Nov 3:13:1026951.
doi: 10.3389/fimmu.2022.1026951. eCollection 2022.

Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies

Aafke Aartse  1   2 , Daniella Mortier  1 , Petra Mooij  1 , Sam Hofman  3 , Marlies M van Haaren  2   4 , Martin Corcoran  5 , Gunilla B Karlsson Hedestam  5 , Dirk Eggink  2   4 , Mathieu Claireaux  2   4 , Willy M J M Bogers  1 , Marit J van Gils  2   4 , Gerrit Koopman  1
Affiliations

Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies

Aafke Aartse et al. Front Immunol. .

Abstract

Several studies have shown that the first encounter with influenza virus shapes the immune response to future infections or vaccinations. However, a detailed analysis of the primary antibody response is lacking as this is difficult to study in humans. It is therefore not known what the frequency and dynamics of the strain-specific hemagglutinin (HA) head- and stem-directed antibody responses are directly after primary influenza virus infection. Here, sera of twelve H1N1pdm2009 influenza virus-infected cynomolgus macaques were evaluated for HA-head and HA-stem domain antibody responses. We observed an early induction of HA-stem antibody responses, which was already decreased by day 56. In contrast, responses against the HA-head domain were low early after infection and increased at later timepoint. The HA-specific B cell repertoires in each animal showed diverse VH-gene usage with preferred VH-gene and JH-gene family usage for HA-head or HA-stem B cells but a highly diverse allelic variation within the VH-usage. HA-head B cells had shorter CDRH3s and higher VH-gene somatic hyper mutation levels relative to HA-stem B cells. In conclusion, our data suggest that HA-stem antibodies are the first to react to the infection while HA-head antibodies show a delayed response, but a greater propensity to enter the germinal center and undergo affinity maturation.

Keywords: HA-head; HA-stem; antibody response; hemagglutinin; influenza A virus; primary response.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
HA-antibody responses to H1N1pdm2009 at day 0, 14, and 56 post-infection (A) Antibody response measured in serum in time against the HA-trimer (blue), HA-head (orange), and HA-stem (purple) domains, calculated as area under curve (AUC), median and 95% CI are depicted (B) Correlation between cumulative viral load in tracheal swabs (AUC) and antibody responses against HA-trimer (blue), HA-head (orange), and HA-stem (purple) domain measured at day 56. (C) Correlation between HA-head and HA-stem antibody responses at day 14 (left) and day 56 (right). Statistical differences between two groups were determined using the Wilcoxon test (p values: *p < 0.05; **p < 0.01; ***p < 0.001) and correlations were determined using a Spearman correlation test (ns, not significant).
Figure 2
Figure 2
Evaluation of HA-specific B cell responses at day 56 post-infection (A) Percentage of the HA-specific B cells of pre- (green) and post-infection (blue) samples. (B) Comparison of the percentage of IgM+, IgG+/IgM+, IgG+, and IgG-/IgM- B cells detected in the total B cell pool (green) and HA-specific B cells (blue). (C) Frequency of HA-head+stem (grey), HA-head (orange), HA-stem (purple), and HA-trimer-only (yellow) binding B cells within the total HA-specific binding B cells. (D) Percentage of IgM+, IgG+/IgM+, IgG+, and IgG-/IgM- B cells observed within the HA-head+stem (grey), HA-head (orange), HA-stem (purple), and HA-trimer-only (yellow) populations of HA-specific cells. (E) Correlation between HA-specific antibody response (AUC) and frequency of HA-specific B cells measured at day 56. (F) Correlation between cumulative viral load in tracheal swabs (AUC) and frequency of HA-specific B cells at day 56. Statistical differences between two groups were determined using the Wilcoxon test (p values: **p < 0.01; ***p < 0.001), bars represent medians with 95% CI, and correlations were determined using a Spearman correlation test (ns, not significant).
Figure 3
Figure 3
Characteristics of HA-specific BCR repertoire and monoclonal antibodies (A) Overlay flowcytometry plot of index-sorted HA-specific B cells of six animals. Original B cells of isolated monoclonal antibodies are highlighted according to ELISA binding pattern, in orange HA-head, in yellow HA-trimer-only, in purple HA-stem, and in grey HA-head+stem. (B) Table presenting the binding domain of HA-specific mAb in ELISA and flow cytometry and the binding domain HA-specific BCRs in flow cytometry. (C) BCR repertoire of IgM germline and HA-specific VH-gene usage. Size of the dots represent relative gene expression per animal. (D) Bar plots representing VH-gene (left) or JH-gene (right) family usage of HA-specific total BCRs, HA-head+stem, HA-head, HA-stem, and HA-trimer-only specific BCRs (upper panel) or isolated monoclonal antibodies (lower panel). (E) Violin plot showing VH-gene SHM-level of pre- (green) and post- infection HA-specific BCRs. Post-infection BCRs are shown in IgM+, IgM+/IgG+, IgG+, and IgM-/IgG- isotypes. (F) Violin plot showing VH-gene SHM-level of HA-head+stem (grey), HA-head (orange), HA-stem (purple), and HA-trimer-only (blue) specific BCRs. (G) Density histogram showing CDRH3-length in amino acids (aa) of total HA-specific BCRs. Dotted line represents the median CDRH3-length. (H) Density histogram showing CDRH3-length distribution of HA-head (orange) and HA-stem (purple) BCRs. Dotted lines represent corresponding medians. Statistical differences between two groups were determined using the Mann-Whitney test (p values: *p < 0.05; ****p < 0.0001).
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