Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals

Abstract

Influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population. A key aspect of antibody-mediated anti-influenza virus immunity is adaptation to antigenically distinct epitopes on emerging strains. We examined factors contributing to reduced influenza vaccine efficacy in the elderly and uncovered a dramatic reduction in the accumulation of de novo immunoglobulin gene somatic mutations upon vaccination. This reduction is associated with a significant decrease in the capacity of antibodies to target the viral glycoprotein, hemagglutinin (HA), and critical protective epitopes surrounding the HA receptor-binding domain. Immune escape by antigenic drift, in which viruses generate mutations in key antigenic epitopes, becomes highly exaggerated. Because of this reduced adaptability, most B cells activated in the elderly cohort target highly conserved but less potent epitopes. Given these findings, vaccines driving immunoglobulin gene somatic hypermutation should be a priority to protect elderly individuals.

Keywords: elderly population; immunoglobulin genes; influenza vaccine; monoclonal antibodies.

Figure 1.. VH mutation analysis of influenza-reactive plasmablast antibody genes.

(A) To analyze intraclonal diversity, de novo mutations of antibody genes from the same clonal pool were compared (i.e., intraclonal mutations in red). Shared mutations (in blue) were not considered for intraclonal diversity but both shared and de novo mutations were considered for total VH number of mutations. (B-C) Antibody VH genes intraclonal mutations represented as variable distribution with quartiles (blue dotted line: median). Statistical significance: Mann–Whitney U test. (B) Amino-acid differences by antibody pair in each group (young adults n = 2465; elderly adults n = 340). (C) Intraclonal mutations by subject in each group (young adults n = 26; elderly adults n = 7). (D-E) Total number of mutations for antibody VH genes represented as variable distribution (blue dotted line: median). Statistical significance: Mann–Whitney U test. (D) Total number of mutations by antibody VH gene in each group (young adults n = 757 influenza VH sequences; elderly n = 112). (E) Total number of mutations by subject in each group (young adults n = 26; elderly adults n = 7). n.s. not significant.

Figure 2.. Newly introduced mutations confer the capacity to adapt to drifted and shifted influenza viruses

(A-B) Plasmablast clonal lineage (12 mAbs) induced after the monovalent 2009 pandemic influenza vaccine. (A) Phylogenetic tree depicting the relationship between the variable gene sequences of the clonal lineage. (B Approximated binding K_D determined by ELISA, HAI titers, and neutralization titers to H1N1 A/Texas/36/1991 and A/California/07/2009 virus strains. (C-D) Plasmablast clonal lineage (4 mAbs) induced after TIV seasonal vaccine in a young adult. (C) Phylogenetic tree depicting the relationship between the variable gene sequences of the clonal lineage (D) Approximated binding K_D to rHA determined by ELISA, HAI titers, and neutralization titers to B/Florida/4/2006 (past strain) and B/Brisbane/60/2008 (vaccine strain) viruses. (E-F) Plasmablast clonal lineage (8 mAbs) induced after TIV seasonal vaccine in an elderly individual. (E) Phylogenetic tree depicting the relationship between the variable gene sequences of the clonal lineage. (F) Approximated binding K_D to rNA determined by ELISA and neutralization titers to B/Florida/4/2006 (past strain) and B/Brisbane/60/2008 (vaccine strain) viruses. The assays were performed in duplicate 2-3 times for each antibody; in B, D and F. shown are mean +/− SD.

Figure 3.. Most influenza virus-reactive antibodies from elderly individuals are low potency and target conserved epitopes on HA and on other viral proteins.

(A-B) HAI and in vitro neutralization by plaque reduction neutralization assay (n = 102 mAbs in each group). Statistical significance: Fisher’s exact test. The assays were performed 2-3 times for each antibody. The number in the middle of the pie charts represents the total number of mAbs tested. (A) Proportion of neutralizing antibodies in each group. (B) Proportion of HAI⁺ neutralizing antibodies in each group. (C) Intraclonal mutations based on HAI status. Amino acid differences by antibody pair (young adults HAI⁺MN⁺ n = 1066, HAI⁻MN⁺ n = 26; elderly adults HAI⁺MN⁺ n = 18, HAI⁻MN⁺ n = 303) represented as variable distribution with quartiles. Statistical significance: Mann–Whitney test. n.s. not significant. (D) Proportion of highly potent neutralizing antibodies. The concentration 3.33 μg/ml was used as a cut-off value based on the median PRNT₅₀ of all neutralizing antibodies. Statistical significance: Fisher’s exact test. The number in the middle of the pie charts represents the total number of mAbs tested. (E) Competition for conserved epitopes on HA. Biotinylated mAbs CR9114 (stalk-reactive, MN⁺) and CR8059 (head HAI⁻MN⁺) were tested for binding to rHA protein by ELISA with or without the presence of a competitor mAb. Only HA-reactive mAbs were used as competitors. The experiment was done in duplicate 2-3 times. The average percentage of competition was calculated. Represented here are different degrees of inhibition (> 90%, 70%-90%, 50%-70%, 30%-50% or < 30%). An inhibition greater than 50% was considered a positive competition. “n.d.”, not determined. (F) Proportion of antibodies targeting conserved epitopes on HA in each group. HA-reactive MN⁺ antibodies were divided into HAI⁺MN⁺, HAI⁻MN⁺ competing and HAI⁻MN⁺ non-competing mAbs. The number in the middle of the pie charts represents the total number of mAbs tested. Statistical significance: chi-square test. (G-H) Proportion of HA-, NA- and NP-reactive antibodies. (G) All mAbs in each group (n = 102 for each group) were compared. (H) Antibodies induced by the Fluzone vaccine (n = 35 for the young cohort and n = 102 for the elderly cohort) were compared. Statistical significance: Fisher’s exact test.

Figure 4.. Elderly individuals rely on cross-reactive memory B cells generated early in life.

(A) Proportion of cross-reactive antibodies capable of binding to the number of rHAs listed in the legend. MAbs reactive to H1N1 vaccine strains were tested against six other H1N1 rHAs and one H5N1 rHA; mAbs reactive to H3N2 vaccine strains were tested against five other H3N2 rHAs. Statistical significance: chi-square test. (B) Proportion of cross-reactive antibodies capable of binding to HA of historic strains (A/Puerto Rico/8/1934 for H1N1 and B/Lee/1940 for B strain). All H1N1 and B strains HA-reactive antibodies were included. We did not include H3N2 strains as the first strain appeared in 1968 when a majority of the young subjects were already born. Statistical significance: Fisher’s exact test. (C) Approximated K_D values for cross-reactive antibodies by rHA ELISA. Vaccine strains: A/Solomon Islands/6/2006 and A/Brisbane/59/2007 (H1N1) and B/Brisbane/60/2008 and B/Malaysia/2506/2004. Historic strains were A/Puerto Rico/8/1934 (H1N1) and B/Lee/1940. The more “contemporary” past strains were A/Texas/36/1991 (H1N1) and B/Yamagata/16/1988. The assays were performed in duplicate three times for each antibody and shown are representative K_D values. Statistical significance: paired Friedman test. (D-E) Cross-reactivity to past influenza H1N1 and H3N2 rHAs tested by ELISA. The assays were performed in duplicate three times for each antibody. ELISA binding affinities represented by K_D (M) were plotted as a heatmap. HAs clustered by amino acid sequence phylogeny, multiple alignments performed using CLUSTALW algorithm. Rooted tree constructed using neighbor-joining method and visualized using FigTree v1.4.0 software. The antibody in blue cross-reacts to influenza A and B strains. (D) H1N1 vaccine strains: A/Brisbane/59/2007 and A/Solomon Islands/6/2006. (E) H3N2 vaccine strains: A/Wisconsin/57/2005 and A/Uruguay/716/2007.

Figure 5.. Mechanistic model showing the first response of young adults to the pandemic 2009 H1N1 strain compared to the general response of elderly individuals to any influenza strain.

In young adults, first exposure to the 2009 H1N1 pandemic strain resulted in a biased response towards highly conserved epitopes on the stalk or head domains of HA (Wrammert et al., 2011; Andrews et al., 2015). Upon subsequent exposure to this strain, the biased response to these conserved epitopes was lost, and the divergent globular head was then predominantly targeted, similar to the past responses to H1N1 strains prior to 2009 and to all H3N2 and B strains. This biased response to conserved epitopes upon first exposure to that novel strain and the mechanism driving this response represents an interesting corollary to that of the aged cohort to any strain. That is, young adults in 2009 had memory B cells that could only target the conserved epitopes, simply based on a lack of immune history. Subsequently they could adapt to the novel epitopes and since have a typical response biased for the globular head of the 2009 H1 strain and all strains. Because the elderly population has lost the capacity to adapt by somatic hypermutation in recent years, their response becomes analogous to first exposure to that highly divergent pandemic strain, but now indefinitely, and to all recent influenza variants arising.