Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans

Abstract

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galβ1-4GalNAcβ), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

Keywords: Adaptive immunity; Infectious disease; Influenza; Vaccines.

Figure 1. Age-dependent features of serological repertoire to the influenza A vaccine components.

(A) Experimental design. Ten donors were vaccinated in the 2013–2014 and/or 2014–2015 seasons with Fluzone trivalent inactivated influenza vaccines (IIV3). Peripheral blood samples were collected on D7 and D21 after vaccination. Serum IgG was split and affinity purified against vaccine-H1 and the vaccine-H3 components independently, processed into peptides, and then run using LC-MS/MS. (B) Post-vaccination (D21) serum binding titer (EC₅₀) against vaccine-H1 and vaccine-H3 by age group. The mean ± SD is shown for each group (n = 10, n = 6, n = 12), with each data point representing the mean EC₅₀. *P ≤ 0.05, by Kruskal-Wallis test followed by Dunn’s post-hoc test. (C) Vshm rates of all identified vaccine-reactive clonotypes. Dotted lines represent the 25th and 75th percentiles, and dashed lines indicate the median. The mean ± SD for each group is shown (n = 416, n = 242, n = 411). **P ≤ 0.01, by Kruskal-Wallis test followed by Dunn’s post-hoc test. (D) Estimated selection pressure imposed on complementary determining regions (CDRs) and framework regions (FRs) of the identified vaccine-reactive antibody sequences in different age groups using BASELINe. P < 0.004 by binomial test.

Figure 2. Age-dependent increase in the vaccine-H1+H3 CR serological repertoire.

(A) Clonal composition of the anti–vaccine-H1 serum antibody repertoire (rep.) in a representative young donor and a representative elderly donor. Vaccine-H1+H3 CR: antibody clonotypes were detected in both the anti–vaccine-H1 and anti–vaccine-H3 repertoires. Vaccine-H1 specific: antibody clonotypes were detected only in the anti–vaccine-H1 repertoire. (B) Vaccine-H1+H3 CR and monovalent vaccine-specific serum response in all donors. Inner wheel: percentage of the serum repertoire of vaccine-H1+H3 CR (green) and vaccine-H1 specific-antibody clonotypes (blue) or vaccine-H3 specific-antibody clonotypes (orange). Outer wheel: relative abundance of each clonotype estimated from the LC-MS/MS peak area. (C) Scatter plot showing Spearman’s correlation coefficient (and 95% CI of the regression line) between the fraction of the vaccine-H1+H3 CR serum repertoire and age.

Figure 3. Characterization of rHA-binding s-mAbs identified from different donors.

(A) Pie chart summarizing the binding specificity of recombinant s-mAbs. (B–E) HAI titer (B and D) and binding specificity (C and E) of anti-HA s-mAbs against rHAs from contemporary H1 strains or H3 strains are shown. (F) Comparison of the binding breadth of s-mAbs from the young and elderly cohorts. *P ≤ 0.05, by 2-tailed Mann-Whitney U test. The breadth is defined as the number of strains with detectable binding (EC₅₀ ≤10 μg/mL) divided by the total number of tested strains. The mean ± SD is shown for each group (n = 10, n = 7).

Figure 4. Characterization of s-mAbs that bind to sulfated glycans from egg components.

(A) Analysis showed that 17 of 19 vaccine-H1+H3 CR s-mAbs bound to components other than HA. (B) Binding profile of putative avian glycan–specific antibodies. ELISA assays were performed using 100 nM recombinant s-mAbs with their respective antigens. Denaturation was performed by boiling with SDS, and N-deglycosylation was from treatment with PNGase F after denaturation. Trypsinization was performed after reduction and alkylation. The EC₅₀ was determined with denatured vaccine-H3 as the antigen. A₄₅₀, absorbance at 450 nm. (C) Glycan array binding specificity of 2 s-mAbs with the glycan-targeting IGHV3–7 signature. RFU values for the top 10 glycans (red) and unsulfated versions (black) are shown with the mean ± SD for 4 replicates. (D) Sequence alignments of IGHV3–7 putative avian glycan–specific antibodies from different donors.

Figure 5. Estimated contribution of non-HA response in the vaccine-induced serological repertoire.

(A) Representative examples of antigen specificities for expressed serum antibody clonotypes in the repertoire of a young donor (donor 1074), a late middle-aged donor (donor 236), and an elderly donor (donor 29). (B) Overall fraction of the estimated non-rHA repertoire within the vaccine-H1+H3 CR repertoire in 2 elderly donors (donors 1131 and 29). v-H1, anti–vaccine-H1 repertoire; v-H3, anti–vaccine-H3 repertoire. (C) Endpoint binding titer in 8 donors before and after vaccination against the SPF, chemically inactivated chicken embryo allantoic fluid (n = 8). *P ≤ 0.05, by 2-tailed Wilcoxon signed-rank test. (D) Pre- and post-vaccination serum binding titer (EC₅₀) against NP (n = 8). The endpoint titer was determined as the maximum serum dilution that showed a higher signal than the average + (3 × SD) of 4 blank wells by ELISA. **P ≤ 0.01, by 2-tailed Wilcoxon signed-rank test.