Use of Biolayer Interferometry to Identify Dominant Binding Epitopes of Influenza Hemagglutinin Protein of A(H1N1)pdm09 in the Antibody Response to 2010-2011 Influenza Seasonal Vaccine

Abstract

The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010-2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines.

Keywords: dominant binding epitope; humoral response; influenza vaccine.

Figure 1

Influenza pH1N1 vaccine-induced Ab response to the HA1 and HA2 domains of HA. (A) The binding of rH1/rHA1/rHA2 to the pre-vaccination (S1) and post-vaccination sera (S2) diluted at 1:10 was determined by the f-AbBA. Each symbol represents one measurement of Ag-Ab binding. Horizontal lines show the medians of binding by the group. The results are representative of two independent experiments. (B) HI titers of the 20 paired sera to pH1N1 virus CA/08 were determined by the HI assay. Each dot represents one measurement of HI titer for one or multiple serum samples. Results are representative of three independent experiments. (C) Correlations of the HI titers to the BLI binding of rH1/rHA1/rHA2 for S1 and S2 were analyzed. Statistical significance was determined using a two-tailed Wilcoxon matched-pairs signed rank test for (A,B) and a two-tailed Spearman correlation coefficient (r) test for (C). Significance levels of **** p < 0.0001 are shown in (A,B), and r plus p values are shown in (C).

Figure 2

Use of rHA1 as Ag increased the assay sensitivity. (A) The binding of rH1/rHA1 of CA/07 WT or K163Q mutant to post-vaccination sera #13 was determined. Each bar represents the median and standard deviation of results from three independent experiments. Statistical significance was determined using a two-tailed t-test with significance levels of **** p < 0.0001. (B) Normalization of binding levels of K163Q mutants to WT of rH1 or rHA1 showed more reduction in the percentage of WT binding using rHA1 K163Q as Ag.

Figure 3

Epitope mapping of post-vaccination sera to pH1N1 vaccine. Dominant binding residues of HA for each indicated serum sample were determined using a panel of 35 (A) or 11 (B) rHA1 mutants. Each bar represents the median and standard deviation of results from three independent experiments. “*” indicates mutation that causes ≥50% reduction or increase in binding compared to the wild type. The dashed vertical lines indicate the 50% cutoff. Statistical significance was determined using one-way ANOVA with Dunnett’s test for multiple comparisons with significance levels of **** p < 0.0001.

Figure 4

The f-AbBA-2-defined dominant binding epitopes of CA/07 HA in Ab response to pH1N1 vaccine. The sequences and locations of residues as part of dominant binding epitopes are shown on the 3D structure of trimeric pH1N1 HA (PDB ID 3M6S) with different colors indicating corresponding antigenic sites: Sa (red), Ca2 (magenta), and RBS (green). Potential epitopes are indicated by ovals with dashed lines.