Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies

Abstract

During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.

Figure 1. Antibody responses to 2009 seasonal TIV in young and elderly vaccinees.

(A) Fold increase of serum HAI titer of individual vaccinees against the 3 2009 vaccine component strains (H1N1, A/South Dakota/06/2007, an A/Brisbane/59/2007–like strain; H3N2, A/Uruguay/716/2007, an A/Brisbane/10/2007–like strain; B, B/Brisbane/60/2008) approximately 28 days after vaccination. Asterisks denote significant differences. (B) IgA and IgG ELISA binding titer of PPAbs from individual vaccinees collected 1 week after vaccination. The ELISA plates were coated with 2009 TIV. P values were determined by unpaired t test for young vs. elderly or paired t test for IgA vs. IgG within each group. Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons; asterisks denote differences that remained statistically significant after the adjustment. Geometric means of fold increase (A) or GMT (B) are shown as bars and numerical values below.

Figure 2. ASC responses to TIV immunization in the young and elderly.

(A) Frequency of TIV-specific IgA and IgG ASCs. (B) Frequency of total IgA and IgG ASCs. (C) Percent TIV-specific IgA and IgG ASCs, relative to total ASCs. Geometric means of ASC counts per 0.1 million (M) B cells (A and B) or average percentage (C) are shown as bars and numerical values below. P values were determined by unpaired t test for young vs. elderly or paired t test for IgA vs. IgG within each group. Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons between age groups and isotypes; asterisks denote differences that remained statistically significant after the adjustment.

Figure 3. Concentration of total and TIV-specific IgA and IgG in PPAbs generated on day 7 or 8 after TIV immunization.

(A) Concentration of total IgA or IgG in PPAb from each donor. (B) Concentration of TIV-specific IgA or IgG in each individual, determined by multiplying the concentration of total IgA or IgG by the percentage of vaccine-specific IgA ASCs or IgG ASCs (Figure 2C), respectively. Geometric mean concentrations are shown as bars and numerical values below. P values were determined by unpaired t test for young vs. elderly or paired t test for IgA vs. IgG within each age group. Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons of Ig concentration-related parameters between age groups and isotypes (Figures 3 and 5); asterisks denote differences that remained statistically significant after the adjustment.

Figure 4. Yield of IgG and IgA per ASC in young and aged vaccinees.

(A) Yield of secreted Ig was estimated via RMA regression, using the concentration of total IgA or IgG in PPAb (ng/ml) and the total IgA or IgG ASCs in the B cell culture (cell count/ml) for each donor. Fitted RMA regression line is shown for each Ig isotype and age group, along with estimated mean yield per cell and 95% CI. (B) 95% CI of the difference between the estimated mean yield per cell in the 2 age groups. Because the 95% CI for IgG clearly includes 0, no difference in mean yield per cell between groups is indicated. For IgA, since the 95% CI does not include 0, the results suggest that mean yield per cell was significantly greater (P < 0.05) in the elderly. However, given that the lower confidence bound fell just above 0, this finding should be interpreted with some caution.

Figure 5. Avidity of vaccine-specific PPAbs from young and elderly TIV recipients.

The avidity of vaccine-specific PPAb is defined as the concentration of TIV-specific PPAb (Figure 3B) divided by the TIV-specific ELISA titer of PPAb (Figure 1B) for each donor. Geometric mean avidities are shown as bars and numerical values below. P values were determined by unpaired t test for young vs. elderly or paired t test for IgA vs. IgG within each age group. Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons of Ig concentration-related parameters between age groups and isotypes (Figures 3 and 5); asterisks denote differences that remained statistically significant after the adjustment.

Figure 6. Affinity of influenza vaccine virus-specific re-mAbs derived from young and elderly recipients of seasonal TIV.

Binding affinity was measured by ELISA with microtiter plates coated with individual vaccine component viruses. The binding affinity was defined as the minimum concentration of each re-mAb that resulted in an OD_405nm greater than 0.607 in the assay. All re-mAbs (32 from the young group; 43 from the elderly group) with a minimum binding concentration up to 10 μg/ml for 1 of the vaccine component viruses were considered vaccine specific and included for this analysis. The OD_405nm threshold of 0.607 was set at a level that would exclude 95% of random control re-mAb as vaccine-specific, based on ELISA results of 48 such re-mAbs derived from individual naive B cells. Geometric means of minimum binding concentrations are shown as bars and numerical values below.

Figure 7. Heterovariant versus homotypic reactivity of vaccine-induced PPAbs in young and elderly recipients.

(A) Binding titer of 2009 seasonal TIV-induced PPAbs (IgG) against sH1N1 and pH1N1 vaccine antigens. Binding was measured with ELISA plates coated with monovalent sH1N1 or pH1N1 vaccines. GMTs are shown as horizontal bars and numerical values below. Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons; asterisks denote differences that remained statistically significant after the adjustment. (B) Effects of age on the relative avidity of vaccine-induced PPAbs (IgG) against the pH1N1 antigen. Relative avidity was defined as the ratio of titer against sH1N1 to titer against pH1N1 (see Methods). The dashed line demarcates relative avidity of 1. The P value is from a test of the null hypothesis that slope is 0 for weighted linear regression fit on the logarithmic scale. The shaded area defines the 95% Working-Hotelling confidence band of geometric mean relative avidity as a function of age. Data for 1 donor aged >89 years (relative avidity, 0.124) was not plotted because HIPAA rules prohibit publication of exact age information in this age group; if included, the value remained P < 0.0001.

Figure 8. Heterovariant versus homotypic affinity of re-mAbs derived from young and elderly recipients of seasonal TIV.

Re-mAbs with minimum binding concentration up to 10 μg/ml for sH1N1 (17 from the young group; 15 from the elderly group) were included for this analysis. Affinity of re-mAb for binding sH1N1 or pH1N1 viruses was defined as the minimum binding concentration for the corresponding influenza strains, determined by ELISA (see Figure 6 legend). The minimum binding concentrations greater than 10 μg/ml were right-censored. Data were analyzed using a parametric model for interval-censored data with terms for young vs. elderly, sH1N1 vs. pH1N1, and their interaction plus a γ frailty term. The numeral beside each symbol indicates the number of mAbs with the same minimum binding concentration. Estimated geometric means of minimum binding concentration based on the parametric model are shown as horizontal bars and numerical values below. (This parameter could not be determined for pH1N1 in the elderly group because of the distribution of right-censored observations and the other observations.) Criteria for statistical significance were adjusted to control type I error rate at 5% across the multiple comparisons; asterisks denote differences that remained statistically significant after the adjustment.