Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

Abstract

Background: Current influenza A vaccines primarily induce neutralizing antibodies targeting the variable hemagglutinin (HA) head domain, limiting their effectiveness against diverse or emerging influenza A virus (IAV) subtypes. The conserved HA stem domain, particularly the long α-helix (LAH) epitope, is a focus of universal vaccine research due to its cross-protective potential. Additionally, Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are recognized as important protective immune mechanisms. This study evaluated IgG responses to the HA head, stem, and LAH regions and assessed cross-reactive potential through neutralization, ADCC, and ADCP assays. Methods: IgG responses to the HA head, stem, and LAH regions were measured in vaccinated individuals. Functional assays were conducted for neutralization, ADCC, and ADCP to evaluate the association between antibody levels and immune function. Results: The results showed that HA head-specific IgG increased significantly after vaccination in 50 individuals, whereas stem-specific IgG increased by 72% and LAH-specific IgG by 12-14%. Among the induced antibody subclasses, IgG1 was predominantly increased. Neutralization titers were detected in viruses of the same strain as the vaccine strain, but not in classical or pandemic strains (H5N1, H7N9). HA stem-specific IgG1 antibody titers showed a significant correlation with ADCC/ADCP activity breadth, but no correlation was observed with neutralization breadth. Conclusions: These findings suggest that although current influenza vaccines can induce HA stem-targeted cross-reactive antibodies, their quantity may be insufficient for broad cross-protection, underscoring the need for improved vaccine strategies.

Keywords: antibody-dependent cellular cytotoxicity; antibody-dependent cellular phagocytosis; influenza virus; vaccine.

Figure 1

Characteristics of serum antibodies to each HA region induced by seasonal influenza vaccination. Total IgG, IgG1, and IgG2 antibodies against vaccine strains HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 were quantified by ELISA using pre- and post-vaccination sera from seasonal influenza vaccines. Quantification was performed using ELISA. (A) Total IgG antibody titers against HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 before and after vaccination. (B) IgG1 and IgG2 antibody titers against H1 HA head-specific before and after vaccination. (C) IgG1 and IgG2 antibody titers against H3 HA head-specific antibodies before and after vaccination. Total IgG, IgG1, and IgG2 antibody titers against the H1 stem before and after vaccination. (D) IgG1 and IgG2 antibody titers against H1 stem cells before and after vaccination. Antibody titers were quantified using standard antibodies, and the amounts are shown. Each circle represents the results for an individual participant; lines represent median values. The fold change is shown above the graph as the median antibody titer in the post-vaccination group divided by that in the pre-vaccination group. The data were analyzed using the Wilcoxon matched-pair signed-rank test. Significance is indicated by the following symbols: *** p < 0.001, and **** p < 0.0001.

Figure 2

Cross-reactive neutralizing antibody activity in serum induced by current seasonal influenza vaccination. Post-vaccination sera were subjected to a microneutralization assay using ten different influenza viruses and representative data from two independent experiments. The results obtained were scored as follows: 0 for 20 and below, 1 for 21 to 80, 2 for 81 to 640, 3 for 641 to 1280, and 4 for 1280 and above. Neutralization breath scores were calculated by summing the respective scores for groups 1, 2, and group 1 + 2. Neutralization breath scores were calculated by summing the scores of groups 1, 2, and group 1 + 2. (A) Correlation between neutralization group 1 breath score and total IgG of H1, H3 HA head-specific and H1 stem-specific. (B) Correlation of neutralization group 2 breath score with total IgG for H1, H3 HA head-specific, and H1 stem-specific. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis (n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Figure 3

Cross-reactive ADCC activity induced by current seasonal influenza vaccination in serum. ADCC assays were performed on post-vaccination sera using 10 different influenza viruses, representative data from two independent experiments. The results obtained were scored as follows: 0 for 10 or less, 1 for 11 to 100, 2 for 101 to 500, 3 for 501 to 1000, and 4 for 1000 or more. Breath scores were calculated by summing the scores of group 1, group 2, and group 1 + 2. (A) Correlation between the ADCC group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. (B) Correlation between the ADCC group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. (C) Correlation between ADCC group 1 breath score and H1 stem-specific total IgG and IgG1 levels. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis (n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Figure 4

Cross-reactive ADCP activity induced by current seasonal influenza vaccination in serum. ADCP assays were performed on post-vaccination plasma with 10 different influenza viruses, and representative data were obtained from two independent experiments. The results were scored as follows: 0, 10 and below; 1, 11–100; 2, 101–500; 3, 501–1000; and 4, 1000 and above. Breath scores were calculated by summing the respective scores for groups 1, 2, and 1 + 2. (A) Correlation between the ADCP group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. (B) Correlation between ADCP group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. (C) Correlation between the ADCP group 1 breath score and H1 stem-specific total IgG and IgG1. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis (n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Figure 5

Relationship between cross-reactive neutralization antibody, ADCC and ADCP activity in serum. (A) Neutralization group 1 breath score and ADCC group 1 breath score, neutralization group 2 breath score and ADCC group 2 breath score, neutralization group 1 + 2 breath score and ADCC group 1 + 2 breath score, and the correlation between each breath score. (B) Neutralization group 1 breath score and ADCP group 1 breath score, neutralization group 2 breath score and ADCP group 2 breath score, and neutralization group 1 + 2 breath score and ADCP group 1 + 2 breath score, respectively. (C) ADCC group 1 breath score and ADCP group 1 breath score, ADCC group 2 breath score and ADCP group 2 breath score, and ADCC group 1 + 2 breath score and ADCP group 1 + 2 breath score, correlating with the respective breath scores. (D) Correlation between ADCC group 1 breath score and ADCC group 2 breath score, ADCP group 1 breath score and ADCP group 2 breath score, and their respective breath scores. Data were statistically analyzed using Spearman’s rank correlation coefficient. Lines indicate correlations determined by linear regression analysis (n = 50 for each graph).