Differential antigenic imprinting effects between influenza H1N1 hemagglutinin and neuraminidase in a mouse model

Abstract

Understanding how immune history influences influenza immunity is essential for developing effective vaccines and therapeutic strategies. This study examines the antigenic imprinting of influenza hemagglutinin (HA) and neuraminidase (NA) using a mouse model with sequential infections by H1N1 virus strains exhibiting substantial antigenic differences in HA. In our pre-2009 influenza infection model, we observed that mice with more extensive infection histories produced higher levels of functional NA-inhibiting antibodies (NAI). However, following further infection with the 2009 pandemic H1N1 strain, these mice demonstrated a reduced NAI to the challenged virus. Interestingly, prior exposure to older strains resulted in a lower HA antibody response (neutralization and HAI) to the challenged virus in both pre- and post-2009 scenarios, potentially due to faster viral clearance facilitated by immune memory recall. Overall, our findings reveal distinct trajectories in HA and NA immune responses, suggesting that immune imprinting can differentially impact these proteins based on the extent of antigenic variation in influenza viruses.

Importance: Influenza viruses continue to pose a significant threat to human health, with vaccine effectiveness remaining a persistent challenge. Individual immune history is a crucial factor that can influence antibody responses to subsequent influenza exposures. While many studies have explored how pre-existing antibodies shape the induction of anti-HA antibodies following influenza virus infections or vaccinations, the impact on anti-NA antibodies has been less extensively studied. Using a mouse model, our study demonstrates that within pre-2009 H1N1 strains, an extensive immune history negatively impacted anti-HA antibody responses but enhanced anti-NA antibody responses. However, in response to the 2009 pandemic H1N1 strain, which experienced an antigenic shift, both anti-HA and anti-NA antibody responses were hindered by antibodies from prior pre-2009 H1N1 virus infections. These findings provide important insights into how antigenic imprinting affects both anti-HA and anti-NA antibody responses and underscore the need to consider immune history in developing more effective influenza vaccination strategies.

Keywords: antigenic imprinting; hemagglutinin (HA); immune history; neuraminidase (NA).

Fig 1

Binding, neutralizing, and NAI antibodies induced by sequential homologous viral infection. (A) Experimental design and sample collection. Six mice in each group were inoculated intranasally with a sequential homologous H1N1 virus infection strategy (1  ×  10⁵ PFU). (B–E) Binding antibodies against (B) USSR/77 HA, (C) Chile/83 HA, (D) Beijing/95 HA, and (E) Bris/07 HA were tested by ELISA. (F–I) Neutralizing antibodies against (F) USSR/77 virus, (G) Chile/83 virus, (H) Beijing/95 virus, and (I) Bris/07 virus were assessed by virus neutralization assay. (J–M) NAI antibody against (J) USSR/77 virus, (K) Chile/83 virus, (L) Beijing/95 virus, and (M) Bris/07 virus were measured by ELLA. Data are representative of two independent experiments performed in technical duplicate. FI6v3 is an influenza hemagglutinin stem-specific antibody, and PBS was used as a negative control. Error bars represent standard deviation. P values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 2

Binding and neutralizing antibodies after sequential viral infection. (A) Experimental design and sample collection. Six mice in each group were inoculated intranasally with a sequential H1N1 virus infection strategy (1  ×  10⁵ PFU). (B–E) Binding antibodies against (B) USSR/77 HA, (C) Chile/83 HA, (D) Beijing/95 HA, and (E) Bris/07 HA were tested by ELISA. (F–I) Neutralizing antibodies against (F) USSR/77 virus, (G) Chile/83 virus, (H) Beijing/95 virus, and (I) Bris/07 virus were assessed by virus neutralization assay. Data are representative of two independent experiments performed in technical duplicate. FI6v3 is an influenza hemagglutinin stem-specific antibody, and PBS was used as a negative control. Error bars represent standard deviation. P values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 3

Lung viral titer after single Bris/07 infection and Beijing/95-Bris/07 sequential infection. Lung viral titers were measured on days 1, 2, 3, 6, and 9 after Bris/07 viral infection (A) and sequential Beijing/95-Bris/07 infection (B) (n = 3).

Fig 4

Cross-binding antibodies after sequential viral infection. (A) Experimental design and sample collection. Six mice in each group were inoculated intranasally with a sequential H1N1 virus infection strategy (1  ×  10⁵ PFU). (B–J) Binding antibodies against (B) A/Puerto Rico/8/34 (H1N1) HA, (C) A/California/04/2009 (H1N1) HA, (D) H1N1 mini-HA, (E) A/Japan/305/1957 (H2N2) HA, (F) A/duck/Laos/2006 (H5N1) HA, (G) A/chicken/NL/2014 (H5N8) HA, (H) A/Uruguay/716/2007 (H3N2) HA, (I) A/Anhui/1/2013 (H7N9) HA, and (J) A/Jiangxi/346/2013 (H10N8) HA were tested by ELISA. Data are representative of two independent experiments performed in technical duplicate. FI6v3 is an influenza hemagglutinin stem-specific antibody, and PBS was used as a negative control. Error bars represent standard deviation. P values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 5

HAI and NAI antibodies after sequential viral infection. (A) Hemagglutination inhibiting antibody against Bris/07 H1N1 virus. (B) Neuraminidase inhibiting antibody against Bris/07 H1N1 virus. Data are representative of two independent experiments performed in technical duplicate. PBS was used as a negative control. Error bars represent standard deviation. P values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 6

Anti-NA inhibiting antibodies after sequential viral infection against Bris/07 H1N1 virus. Anti-NA neutralizing antibodies against Bris/07 virus were assessed by virus inhibition assay. Data are representative of two independent experiments performed in technical duplicate. Error bars represent standard deviation. P values were calculated using a two-tailed t-test (^*P < 0.05, ^****P < 0.0001; ns, not significant).

Fig 7

Neutralizing, HAI, and NAI antibodies with sequential infection history after Cal/09 H1N1 challenge. (A) Experimental design and sample collection. Six mice in each group were first inoculated intranasally with sequential H1N1 virus infection strategy (1  ×  10⁵ PFU) and were challenged with Cal/09 H1N1 virus (4  ×  10⁵ PFU). (B) Neutralizing antibodies against Cal/09 H1N1 virus were assessed by virus neutralization assay. (C) Hemagglutination inhibiting antibody against Cal/09 H1N1 virus. (D) Neuraminidase inhibiting antibody against Cal/09 H1N1 virus. Data are representative of two independent experiments performed in technical duplicate. PBS was used as a negative control. Error bars represent standard deviation. P-values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 8

In vivo protection against Cal/09 H1N1 virus after sequential infection. (A) The mean percentage of body weight change post-infection is shown (n = 6). The humane endpoint, which was defined as a weight loss of 25% from initial weight on day 0, is shown as a dotted line. (B) Kaplan–Meier survival curves are shown (n = 6). (C) Lung viral titers on day 3 after infection are shown (n = 3). Solid black lines indicate means ± SD. P values were calculated using a two-tailed t-test (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns, not significant).

Fig 9

Surface residue difference among pre-2009 H1N1 NA and Cal/09 H1N1 NA. (A) Mutations are highlighted in blue on in a sequence alignment among four pre-2009 N1 protein and Cal/09 NA. (B) Surface residues on Cal/09 NA, which differs from four pre-2009 NAs, are highlighted on the Cal/09 NA protein.

Fig 10

Surface residue difference among pre-2009 H1N1 HA and Cal/09 H1N1 HA. (A) Mutations are highlighted in blue in a sequence alignment among four pre-2009 HA protein and Cal/09 HA. (B) Surface residues on Cal/09 HA head domain, which differs from four pre-2009 HAs, are highlighted on the Cal/09 HA protein.