High preexisting serological antibody levels correlate with diversification of the influenza vaccine response

Abstract

Reactivation of memory B cells allows for a rapid and robust immune response upon challenge with the same antigen. Variant influenza virus strains generated through antigenic shift or drift are encountered multiple times over the lifetime of an individual. One might predict, then, that upon vaccination with the trivalent influenza vaccine across multiple years, the antibody response would become more and more dominant toward strains consistently present in the vaccine at the expense of more divergent strains. However, when we analyzed the vaccine-induced plasmablast, memory, and serological responses to the trivalent influenza vaccine between 2006 and 2013, we found that the B cell response was most robust against more divergent strains. Overall, the antibody response was highest when one or more strains contained in the vaccine varied from year to year. This suggests that in the broader immunological context of viral antigen exposure, the B cell response to variant influenza virus strains is not dictated by the composition of the memory B cell precursor pool. The outcome is instead a diversified B cell response.

Importance: Vaccine strategies are being designed to boost broadly reactive B cells present in the memory repertoire to provide universal protection to the influenza virus. It is important to understand how past exposure to influenza virus strains affects the response to subsequent immunizations. The viral epitopes targeted by B cells responding to the vaccine may be a direct reflection of the B cell memory specificities abundant in the preexisting immune repertoire, or other factors may influence the vaccine response. Here, we demonstrate that high preexisting serological antibody levels to a given influenza virus strain correlate with low production of antibody-secreting cells and memory B cells recognizing that strain upon revaccination. In contrast, introduction of antigenically novel strains generates a robust B cell response. Thus, both the preexisting memory B cell repertoire and serological antibody levels must be taken into consideration in predicting the quality of the B cell response to new prime-boost vaccine strategies.

FIG 1

Influenza vaccine-induced serological Ab response varies with vaccine history. (A) Percentage of peripheral blood CD19⁺ B cells at day 7 after vaccination that were CD27^hi CD38^hi plasmablasts as determined by flow cytometry. The left panel compares the percentage of plasmablasts of all donors vaccinated between the 2010-2011 and 2013-2014 influenza vaccine seasons who had or had not been vaccinated the year prior. The right panel compares the plasmablast response to the 2010-2011 vaccine only. (B) The serum antibody EC₅₀ to the three virus strains present in the 2010-2011 vaccine was determined on the day of vaccination (day 0) with the 2010-2011 TIV and at day 14/21 by ELISA. Each dot represents the fold change increase in the binding EC₅₀ to each strain between day 0 and day 14/21 in an individual donor. Donors are divided according which vaccine(s) they received the year prior (2009-2010 season). The line represents the median fold change within each vaccine group. (C) Fold change in serum Ab EC₅₀ between day 0 and day 14/21 in five individuals to the vaccinating H1N1, H3N2, or influenza B virus strain in the indicated year. Each line represents the yearly serological response to the vaccinating influenza strain of a given donor. None of the donors received the 2009-2010 TIV, while four of them (except 007) received the A/Cal monovalent vaccine. (D) Fold change in serum Ab EC₅₀ as described for panel C to the vaccinating H1N1, H3N2, or influenza B virus strain. As in the data shown in panel C, all 2010-2011 donors had received the A/Cal monovalent vaccine but not the 2009-2010 TIV. After the 2010-2011 season all donors had been vaccinated the year prior as well. Each dot represents the fold increase in serum Abs in each donor with the median indicated by the line. All serum Ab EC₅₀ data are the average from three independent experiments. Statistical analysis was determined using a Mann-Whitney test. Vacc, vaccine; Yam, Yamagata lineage; Vic, Victoria lineage; n.s., not significant.

FIG 2

Alternating the influenza B virus lineage in the vaccine magnifies the plasmablast response. (A) An ELISPOT assay was performed on PBMCS at 7 days after influenza vaccination with the 2012-2013 TIV to determine the proportion of IgG⁺/IgA⁺ plasmablasts (PB) capable of binding each influenza virus strain. ELISPOT assay plates were coated with the given year's vaccine or influenza virus rHA and incubated with fresh PBMCs overnight, and spots were counted under each condition. Shown is the proportion of total vaccine-positive plasmablasts capable of binding rHA from the vaccinating 2012-2013 influenza B virus lineage (Yamagata) and H3N2 strain or the influenza B virus lineage (Victoria) and H3N2 strain present in the previous year's vaccine. Each line links the proportion of vaccine-positive plasmablasts that can bind rHA of each of the two strains in an individual. Statistical significance was determined using a paired Wilcoxon test. (B) Plasmablasts present in peripheral blood at day 7 after vaccination with the 2012-2013 or 2013-2014 TIV were tested for specificity to the vaccinating H1N1, H3N2, or influenza B virus rHA or whole virus by ELISPOT assay as described for panel A. Shown is the percentage of vaccine-positive antibody-secreting cells that were specific to the 2012-2013 vaccinating influenza B virus strain in each donor who had or had not been vaccinated in the 2011-2012 season or to the 2013-2014 vaccinating B strain after vaccination with the 2013-2014 TIV in donors who had also received the 2012-2013 TIV. (C) Proportion of vaccine-positive plasmablasts generated in response to the 2013-2014 TIV specific to the H1N1 (H1), H3N2 (H3), and influenza B virus strain in the vaccine as measured by ELISPOT assay. Each bar represents the response in one donor. (D) Percentage of CD19⁺ B cells that were CD27^hi CD38^hi plasmablasts as detected by flow cytometry in the peripheral blood 7 days after either the 2012-2013 or 2013-2014 TIV. Shown is the percent plasmablasts in each donor, with the median indicated by the horizontal line. Statistical significance was determined with a Mann-Whitney test. (E) The proportion of total antibody-secreting cells specific to the H1N1, H3N2, or B strain was determined by ELISPOT assay and multiplied by the percentage of total plasmablasts detected by flow cytometry to calculate the percentage of total B cells that were influenza virus strain-specific plasmablasts at day 7 after vaccination with the 2012-2013 or 2013-2014 TIV. Shown is the percentage of B cells in each donor specific for each strain, as indicated, with the median represented by the horizontal line. Statistical significance was determined with a Mann-Whitney test. (F and G) Immunoglobulin genes from single-cell sorted plasmablasts were cloned and expressed as MAbs and tested for binding to the vaccinating influenza virus strains by ELISA. Shown is the proportion of total influenza virus-specific MAbs able to bind the vaccinating influenza B virus strain as detected by ELISA in the years indicated. Each dot represents the proportion from a given donor. All donors in the 2007-2008 and 2008-2009 seasons were vaccinated the year prior as well. Shown is the percentage of influenza B virus-specific MAbs (Flu MAbs) comparing all donors as a group between years (F) or a paired comparison of the same individual across years in the subset of donors for whom we tracked the immune response over multiple years (G). Statistical significance was determined using a Mann-Whitney test (F) or paired Wilcoxon test (G).

FIG 3

High prevaccine serological Ab titers dampen the B cell response. (A and B) Correlation between the proportion of influenza virus-specific plasmablasts generated 7 days after vaccination with the 2012-2013 or 2013-2014 TIV and memory B cells specific for the indicated strain detected 12 to 14 days after vaccination (A) or the fold increase in serum Abs levels (upper panels) or fold change (FC) in serum HAI titer (lower panels) between day 0 and day 14/21 after vaccination (B). Plasmablasts specific to the indicated viral strain rHA or whole virus was determined by ELISPOT assay 7 days after vaccination by placing freshly isolated PBMCs on an antigen-coated ELISPOT assay plate overnight. To detect influenza virus specificity of memory B cells, PBMCs were incubated for 5 days with B cell mitogens to induce differentiation of memory B cells into antibody-secreting cells. Activated cells were then placed on an antigen-coated ELISPOT assay plate overnight. Shown is the percentage of total vaccine-positive IgG⁺ IgA⁺ antibody-secreting cells specific for the indicated influenza virus strain (A) or the total number of strain-specific antibody-secreting B cells per 1 × 10⁶ PBMCs (B). (C) Correlation between percentage of influenza virus strain-specific plasmablasts induced by vaccination and the percentage of memory cells (Mem) specific to that strain present just before vaccination determined as described for panel A. (D) Correlation between the total number of strain-specific antibody-secreting B cells per 1 × 10⁶ PBMCs and serum Ab EC₅₀ present on the day of vaccination (day 0). The Spearman r (rs) value for non-Gaussian distributions and corresponding P value were used to determine the degree of correlation for all analyses. d, day; PB, plasmablasts.