Immune history profoundly affects broadly protective B cell responses to influenza

Abstract

Generating a broadly protective influenza vaccine is critical to global health. Understanding how immune memory influences influenza immunity is central to this goal. We undertook an in-depth study of the B cell response to the pandemic 2009 H1N1 vaccine over consecutive years. Analysis of monoclonal antibodies generated from vaccine-induced plasmablasts demonstrated that individuals with low preexisting serological titers to the vaccinating strain generated a broadly reactive, hemagglutinin (HA) stalk-biased response. Higher preexisting serum antibody levels correlated with a strain-specific HA head-dominated response. We demonstrate that this HA head immunodominance encompasses poor accessibility of the HA stalk epitopes. Further, we show polyreactivity of HA stalk-reactive antibodies that could cause counterselection of these cells. Thus, preexisting memory B cells against HA head epitopes predominate, inhibiting a broadly protective response against the HA stalk upon revaccination with similar strains. Consideration of influenza exposure history is critical for new vaccine strategies designed to elicit broadly neutralizing antibodies.

Figure 1. Plasmablasts induced by influenza vaccination are often derived from memory B cells

(A, B) Four subjects were immunized in each of two consecutive years and mAbs made from vaccine-induced plasmablasts were tested for reactivity to influenza strains present in the vaccine. Shown are the numbers of mAbs that were clonally related in both of the seasons by individual (colored black) (A) and in total (B). (C-F) Phylogenetic trees depicting the variable gene sequences rooted in the germline sequence of vaccine-positive clonotypes isolated from 3 subjects over 2 years. mAb from year one (Y1) are in blue and from year two (Y2) in red. In (F), members of the same influenza+ clone were found in memory B cells (Mem) isolated from blood collected 21 days after vaccination in year one and year two and plasmablasts (PB) from year two.

Figure 2. A subset of subjects generated HA stalk-biased plasmablast response

(A, B) Proportion of H1N1 rHA+ mAbs in each subject capable of neutralizing viral replication (A) or inhibiting turkey red blood cell agglutination including all mAbs (B, left panel) or only clonally distinct mAbs (B, right panel). The black line separates the subjects based on level of HAI+ mAbs (C) Neutralizing but HAI negative mAbs were tested for binding to H5N1 rHA and the ability to compete for binding to Cal09 rHA with the HA stalk-binding mAb sc70-1F02. Each bar represents the neutralizing titer (PRNT₅₀), binding affinity to H5N1, or percent inhibition for each HAI negative mAb. HA stalk-binding mAbs are represented by black bars. Only one representative member of clonally related mAbs is shown. (D-E) The subjects were divided into two groups as indicated in (B) and the proportion of neutralizing rHA-binding mAbs that bind the HA stalk in each subject (D) or within each group is shown (E). mAbs that were HAI negative but did not bind H5N1 or compete with sc70-1F02 are labeled as unknown. mAbs capable of inhibiting hemagglutination are labeled as HA head-binding. The number in the middle of the pie charts represents the total number of mAbs tested from that response group. Statistical significance of the difference between the two groups was determined using the Mann Whitney test (D) or Chi-square test (E). All data is representative of 2-3 replicates per mAb.

Figure 3. A subset of subjects generated a broadly neutralizing plasmablast response

(A, B) All rHA-binding and neutralizing clonally distinct mAbs were tested for the ability to bind rHA by ELISA from 8 different H1N1 strains. Shown is the proportion of clonally distinct mAbs in each response group capable of binding the number of rHAs listed in the legend (A) or percentage of mAbs in each group (black: stalk-biased group; gray: head-biased group) that binds each of the H1N1 strains and H5N1 as listed on the X-axis with (B, left panel) or without (B, right panel) H5N1+ mAbs included in the analysis. Statistical significance of the difference between the two groups was determined using a (A) Chi-square test or (B) Fisher's exact test (*p<0.05; **p<0.005). Exact p values can be found in supplementary material (source data). All data is representative of 2-3 replicates per mAb (C) The binding avidity of all clonally distinct H1N1+ mAbs against Cal09 whole virus was determined by ELISA. Each dot represents the median of the –log transformed apparent kD of all clonally distinct mAbs in each subject. (D) The median number of mutations in the VH gene of each clonally distinct H1N1+ mAb for each subject. (E) The percentage of H1N1+ mAbs in each subject that was IgG+. All other H1N1+ mAbs were IgA+. For (C-E) the black line represents the median value of the subjects in each response group. A Mann-Whitney test showed no statistical significance between response groups.

Figure 4. The plasmablast response upon revaccination is HA head-biased

(A, B) Proportion of H1N1 rHA-binding mAbs in each subject able to neutralize viral replication in MDCK cells (A) and inhibit viral-induced hemagglutination (B). (C) Neutralizing, but HAI negative mAbs were tested for their ability to bind to H5N1 rHA and compete with sc70-1F02 for binding to Cal09 rHA is in Figure 2. Each bar represents one neutralizing HAI negative mAb, black bars represent HA stalk-binding mAbs. Only one representative of clonally related mAbs is shown. (D) Proportion of neutralizing mAbs in the revaccinated group able to bind the HA stalk. The number in the center represents the total number of neutralizing mAbs. (E) mAbs from the revaccinated group were tested for their ability to bind rHA from 8 different H1N1 strains. Shown is the proportion of neutralizing and clonally distinct mAbs from the revaccinated group (grey line) and 2009 stalk-biased group (black line) able to bind each H1N1 strain as indicated in the X-axis. The panel on the right represents the proportion of mAbs able to bind each strain after excluding H5N1+ mAbs from the analysis. Statistical significance was determined using Fisher's exact test (*p<0.05, **p<0.005). Exact p values can be found in supplementary material (source data). All data is representative of 2-3 replicates for every mAb.

Figure 5. Pre-vaccine serum Ab levels correlate with the specificity of the plasmablast response

Sera collected on the day of vaccination was tested by ELISA for the level of antibodies able to bind H1N1 strains. Pre-vaccine serum was not collected from three subjects (SFV018, 030-09 and 039-10) so we were unable to include data from these subjects. (A) The percentage of H1N1+ mAbs produced from each subject able to bind Cal09 but not Bris-2007 was plotted against the Cal09 EC50 of pre-vaccine serum collected from that same individual. Each dot represents one subject vaccinated either for the first or second time with Cal09. Degree of correlation was determined using the nonparametric Spearman correlation coefficient and p value. The Cal09 EC50 is the mean of three independent measurements for each sample. (B, C) Direct comparison of Cal09 EC50 in pre-vaccine sera (B) or birth year (C) of subjects in both 2009 groups and the revaccinated group. Statistical significance was determined using the Mann-Whitney test. (D) The pre-vaccine serum antibody EC50 for 9 H1N1 strains was determined in each subject. Shown is the mean EC50 with SEM from 3 independent replicates in each vaccine group. Statistical significance of differences between vaccine groups for each H1N1 strain is detailed in Fig. S4.

Figure 6. HA stalk-specific mAbs bind with lower affinity

(A-B, D) 35 HA head-reactive mAbs, 33 sc70-1F02 competing and 16 noncompeting HA stalk-specific mAbs were tested by ELISA for binding to Cal09 rHA or whole virus. Each line in (A) and (D) represents the paired apparent kD of binding of each mAb to rHA or whole virus. In (B) each dot represents the apparent kD of one mAb with the median of each group shown by a horizontal line. Data representative of 2-3 independent experiments. Statistical significance was determined using the Wilcoxon test (A and D) or Mann-Whitney test (B). (C) Testing of a subset of HA-stalk versus HA-head reactive antibodies by surface plasmon resonance to determine, association (Ka), dissociation (Kd), and affinities (KD). Shown is mean of two independent experiments. Statistical significance was determined using an unpaired two-tailed t-test.

Figure 7. HA stalk-specific are polyreactive

(A-B) Each mAb was tested for binding by ELISA to dsDNA, LPS or Insulin. For (A) Shown is a comparison of the area under the binding curve (AUC) for each mAb tested against each antigen. The data points for the well-characterized stalk-binding mAbs CR9114 and F10 are indicated in purple and green, respectively. For (B) mAbs that had an OD above 0.5 at the highest concentration were consider positive for binding that particular antigen (fig. S8A). A mAb was considered polyreactive if it bound all three antigens. Shown is the percentage of polyreactive mAbs in each group as indicated. Statistical significance was determined using Fisher's exact test. Data representative of 2-3 independent experiments. (C) Binding of the HA stalk-reactive mAbs to HEp-2 cells by immunofluorescence at a concentration of 50ug/ml. The data points for CR9114 (purple) and F10 (green) are marked. HE-p2 binding of high-affinity anti-nuclear antibody 3H9 is shown in red as a comparison. (D) The polyreactivity of HA stalk-reactive antibodies was compared with (+) and without (−) the presence of physiological concentrations (5%) of human serum albumin. Binding of the prototypical lupus-associated autoantibody mAb 3H9 (in red) was also tested as a comparison. Values represent the average of three replicates. Statistical significance was determined using paired t-tests. (E-F) Binding to dsDNA, LPS and insulin was compared between mAbs encoded by VH1-69 that bound influenza, but not the HA stalk, or bound the HA stalk (21 mAbs/group). Shown is a comparison of the AUC (E) as shown in A or percent polyreactive (F), determined as in B. Binding curves are representative of 3 independent experiments. The AUC is the mean of 3 independent experiments.