A major goal of this study was to evaluate antibody responses to seasonal influenza vaccines in young and elderly individuals by isolating and characterizing plasmablast-derived polyclonal and monoclonal antibodies 7–8 days after vaccination. Of particular interest were the quantity and quality of influenza-specific antibodies. Antibody titer reveals the overall reactivity of an antibody sample and is a function of both quantity and quality of an antibody. Distinguishing the role of these parameters in antibody activity is important, as each one is modulated by distinct biological pathways. The quantity of antibody is determined by the number of ASCs and the yield of antibody from each ASC, whereas the quality, or avidity, depends on the affinity of individual Ig components. This characteristic is a function of the structure of individual antibody molecules and is ultimately determined by the Ig gene sequences.
We demonstrated that the quantity of vaccine-specific IgG was significantly greater in young than in elderly individuals at day 7–8 after vaccination. Mechanistically, this was associated with higher numbers of IgG ASCs in younger subjects. The average yield of secreted IgG per ASC was comparable between the 2 age groups. For IgA, the yield per ASC was higher in the elderly; however, this finding must be verified, given its marginal statistical significance (Figure B). Since the IgG response is the predominant component of the plasmablast and PPAb responses after TIV immunization in both age groups, our results clearly indicate a quantitative difference in the vaccine-specific antibody response between young and elderly subjects. At least for IgG responses, the reduced antibody quantity in elderly individuals was caused by fewer ASCs, and not by a lower yield of antibody per ASC. Therefore, when evaluating new strategies to enhance influenza vaccine efficacy in the elderly population, such as use of vaccine adjuvants and increased vaccine doses, the quantity of ASCs induced should be considered.
We also compared vaccine-specific antibody avidity between the 2 age groups, taking advantage of the PPAb assay combined with ELISPOT to assess concentrations of vaccine-specific antibodies. Furthermore, we generated and analyzed re-mAbs from individual plasmablasts to assess antibody affinity at the monoclonal level. A statistically significant difference was not detected between the 2 age groups in avidity at the polyclonal level (Figure ), or in overall affinity at the monoclonal level for the vaccinating antigens (Figure ). Taken together, our findings suggest that the inferior antibody responses raised after TIV immunization in the elderly are primarily caused by reduced quantity rather than by reduced avidity of vaccine-specific antibodies.
We next examined the heterovariant reactivity of seasonal influenza vaccine–induced antibodies to the pH1N1 strain, an antigen not included in the seasonal TIV. Although the heterovariant titers of vaccine-induced PPAb were significantly lower than the homotypic vaccine-specific titer in young vaccinees, there was no significant difference in the titers of PPAbs from elderly individuals for sH1N1 and pH1N1 (Figure A). Furthermore, we showed that the relative heterovariant avidity of PPAbs for pH1N1 increased significantly with age (Figure B), which indicates that when adjusted to the same concentration, the polyclonal antibodies induced by the sH1N1 vaccine antigen in elderly vaccinees had greater heterovariant reactivity against pH1N1 than did those in young adults. We confirmed these findings at the monoclonal level by demonstrating that the sH1N1-specific re-mAbs had similar affinity for pH1N1 in the elderly group, but significantly lower affinity for pH1N1 in the younger group, compared with their homotypic affinity for the vaccinating sH1N1 antigens (Figure ).
Age-related changes in the humoral response to antigenic exposure have been studied in mouse models and, to a lesser extent, in humans, focusing on the T cell–dependent B cell response that is critical for effective long-term humoral immunity. Age-related decline of functional antibody responses has been attributed to intrinsic defects in B cells as well as their accessory cells (reviewed in refs. 16
). B cells from older mice showed decreased in vitro proliferation in the absence of exogenous IL-4 compared with their counterparts from younger animals (19
), whereas in a study with human samples, difference in in vitro proliferation was not detected between young and elderly subjects in purified B cells cultured with various stimuli (20
). It has been well established that GC formation and kinetics are impaired in aged mice during both primary and secondary responses (21
). Of special interest is the marked reduction in the expression level of CD154 (the CD40 ligand) in aged, activated T helper cells (22
). Because interaction between CD40 and CD154 on antigen-specific T and B cells is required for GC formation and antibody class switching, reduced CD154 level on aged CD4+
helper T cells could result in poor antibody responses in the elderly (23
). In the current study, we demonstrated a significantly reduced quantity of vaccine-specific ASCs in elderly individuals after TIV immunization compared with young vaccinees. Although other possibilities can be envisioned, 2 potential mechanisms could directly contribute to the difference in ASC quantity: (a) reduced CD154 expression on CD4+
helper T cells, resulting in reduced formation of GCs and reduced proliferation of vaccine-activated B cells; and (b) defects in the intrinsic proliferation capability of aged B cells. Future studies should be directed at assessing the extent to which these 2 mechanisms affect the number of influenza-specific ASCs in humans after influenza vaccination.
A functional B cell response to antigenic stimulus relies on the GC-dependent processes of affinity maturation, during which somatic hypermutation (SHM) diversifies the Ig variable regions of proliferating B cells, and class switch recombination (CSR). These processes are mediated by activation-induced cytidine deaminase (AID) (25
). The activity of AID declines with aging in both mice and humans (26
) and is associated with the reduced serum antibody response to influenza vaccine in the elderly (28
). This is in agreement with studies showing that, compared with young counterparts, elderly individuals undergo less de novo SHM of their Ig heavy chain genes, possess reduced functional antibody activity, and have reduced protective efficacy in response to pneumococcal polysaccharide vaccine (29
). In the case of the polysaccharide vaccine, the reduced antibody response in the elderly is related to low antibody avidity rather than low antibody concentration (30
). In this study of the response to influenza vaccine, we did not observe statistically significant differences in overall polyclonal or monoclonal antibody affinity between young and elderly recipients of influenza vaccine. This discrepancy might be due to the facts that the antibody response to the bacterial polysaccharide antigens does not involve immunological memory, but memory is key in influenza vaccination.
Most influenza vaccine recipients, except very young children, have been previously exposed to influenza antigens by natural infection, prior vaccination, or both. Hence, each adult vaccinee will have certain levels of preexisting immunity, including memory B cells that cross-react with the new vaccine antigen to various degrees. Influenza virus–specific memory B cells have been shown to survive 90 years in human beings (34
). Because of a lower activation threshold of memory B cells, they are able to enter division more easily and produce a greater antibody response than naive B cells (35
). By analyzing the Ig gene sequences of activated B cells, we showed that the B cell response in adult recipients of influenza vaccine was predominantly recall of memory B cells rather than activation of naive cells (10
). We propose that the existence of closely related cross-reactive memory B cell clones — resulting from prior exposure in the elderly vaccinees — compensates, at least in part, for the defects in AID reactivity and antibody affinity maturation found in the elderly vaccinees and results in production of vaccine-specific antibodies with similar avidity, although in quantities smaller than those in younger vaccinees. This hypothesis might be addressed in future studies by comparing the Ig gene repertoire and sequences in prevaccination memory B cells and vaccine-induced plasmablasts in the young and elderly recipients of influenza vaccines. Based on the current findings, we propose that the inferior protection efficacy of influenza vaccines in the elderly population is primarily caused by lower quantity, rather than quality, of influenza-specific antibodies induced by vaccination.
The potential effects of age-related changes in the influenza memory B cell repertoire on antibody responses to a new seasonal vaccination were further examined by analysis of the heterovariant reactivity of antibodies induced by the seasonal influenza vaccines. We found that after seasonal influenza vaccination, heterovariant reactivity for the pH1N1 antigen increased with age: in young vaccinees, heterovariant avidity and affinity were significantly lower than homotypic avidity and affinity for the seasonal vaccine antigens, whereas in the elderly, statistically significant differences were not detected (Figures and ). In fact, in all 5 elderly vaccinees older than 78 years, the titer of PPAbs against the pH1N1 antigen was higher than that against the sH1N1 antigen (Figure B). In a previous 2009 influenza vaccine study, prevaccination serum antibody titers against pH1N1 were detected in 34% of individuals born before 1950, but only in 4% of those born after 1980, which suggests that a substantial fraction of the population 60 years or older had been exposed to influenza strains that were circulating before 1950 and were antigenically related to pH1N1 (37
). A few such influenza A/H1N1 strains were recently identified in a study using a mouse model (38
). These findings are supported by the crystal structure of the HA protein from the pH1N1 virus, which shows an antigenic structure extremely similar to those of human H1N1 viruses circulating in the early 1900s (39
). Conceivably, elderly individuals carry a larger population of memory B cells primed by pH1N1-related viruses. We propose that these memory B cells can be activated by seasonal vaccine antigens, proliferate, and develop into plasmablasts with limited SHM, resulting in production of antibodies that recognize the new vaccinating antigen while retaining high affinity for the pH1N1-related antigen. Interestingly, some PPAbs and re-mAbs from the elderly individuals in our study even had higher reactivity against pH1N1 than sH1N1 (Figures and ). However, it remains to be determined whether the greater cross-reactivity of vaccine-induced antibodies in the elderly has any beneficial effect with respect to vaccine-induced cross-protection.
Taken together, our findings suggest that under certain circumstances, immunization with a current influenza vaccine induces an antibody response to previously circulated influenza strains. Of note, although heterovariant binding activity to pH1N1 antigen was clearly detected in vaccine-induced PPAbs and re-mAbs generated during the current work, Hancock et al. did not observe an increase in serum neutralization antibody titers against the pH1N1 virus in the vast majority of adult recipients aged 60 years or older (37
). To address this discrepancy, it will be critical to carry out properly powered analyses of vaccine-specific and heterovariant functional reactivity of PPAbs and re-mAbs. Such studies will help determine whether this discrepancy reflects the difference between total binding activity (measured by ELISA) and functional activity (measured by HAI and virus neutralization assays) or, more intriguingly, intrinsic differences between the antibodies derived from peripheral plasmablasts and those from bone marrow resident plasma cells (15
). If the latter is the case, analysis of the peripheral plasmablast responses could result in more accurate predictors of vaccine efficacy.