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Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide.Pre-and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre-to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults.These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides.

Streptococcus pneumoniae is a human bacterial pathogen responsible for serious infections including pneumonia. The currently licensed polysaccharide vaccine provides 60 to 80% protection in young adults, but in the elderly the vaccine efficacy is drastically reduced despite normal antibody levels. We hypothesized that the reduced vaccine efficacy in the elderly results from altered variable gene family usage. We have analyzed the light chain gene usage in 20 young (20 to 30 years of age) and 20 elderly (65 to 86 years of age) adults in response to pneumococcal polysaccharide 4 (PPS4) and PPS14. We generated a variable light chain library using B cells specific for PPS4 and PPS14 from each vaccinated individual. We determined complete sequences and somatic mutation frequencies in all isolated variable light chain fragments. Six gene families, 1, 2, 3, 4, 1, and 3, were identified in response to PPS4 and PPS14 in both age groups. Comparison of young and elderly adults demonstrated significant differences in 4, 1, and 3 gene usage in response to PPS4 and PPS14. With aging, there was a significant increase in 4 gene usage and a significant decrease in 1 and 3 gene usage in response to both PPS4 and PPS14. Although both V1 and V3 gene products demonstrated extensive mutations, there was no age-related difference in mutational frequency per gene family. These findings suggest an age-related change in light chain gene usage in response to PPS4 and PPS14.The incidence of pneumococcal pneumonia is significantly increased in individuals Ͼ65 years of age (13). Several studies (8,18) have demonstrated a significant impairment in the immune response to pneumococcal polysaccharides in the elderly, particularly those Ͼ77 years of age, associated with a markedly decreased efficacy of the pneumococcal vaccine (6, 23).The molecular mechanisms responsible for the decreased immune response in the elderly remain poorly understood. Nicoletti et al. (15) studied the immune response to phosphorylcholine (PC) in aged mice vaccinated with Streptococcus pneumoniae. These studies demonstrated a significant functional impairment in the immune response to PC despite normal antibody concentrations. Molecular analysis of the anti-PC-specific antibody repertoire in aged mice revealed a significantly altered V gene usage compared to young immunized mice (14).To date, studies examining the structure-function relationship of antipneumococcal antibodies have been performed using human monoclonal antibodies (1, 22, 26), combinatorial libraries (12,17,27,28), and monoclonal antibodies derived from a transgenic mouse strain with human immunoglobulin loci (2, 19). All studies, with the exception of those using the transgenic mouse, have analyzed the V repertoire using peripheral blood mononuclear cells obtained from young high responders. Although these studies have enriched our knowledge concerning the human immune response to pneumococcal polysaccharide (PPS), they were not designed to represent the in vivo antibody repertoire response to PPS in the...

The primary reservoir for Streptococcus pneumoniae is the human nasopharynx, and colonization is often the initial step in pathogenesis. Recently we have demonstrated that pneumococcal colonization primes the immune response to subsequent vaccination with the pneumococcal conjugate vaccine (CPV). In this study we wished to determine if colonization stimulates the production of B cell memory that is activated following vaccination with CPV. To test this hypothesis, we colonized mice with S. pneumoniae serotype 14, adoptively transferred their B cells and CD4 + T cells into naïve recipients, and vaccinated the recipients with CPV. Our results indicate that pneumococcal colonization stimulates the production of memory B cells which are responsible for enhancing the immune response to CPV vaccination.

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Unlike adults, young children cannot mount an antibody response to T cell-independent (TI) antigens such as Pneumococcal polysaccharides (PPS) and the basis for this impairment remains elusive. The ontogeny of B cells early in life is IL-7-independent, whereas in adults it is IL-7-dependent. Despite having TI B cell subsets, young wildtype or adult IL-7-/- mice are impaired in anti-PPS response and do not survive Pneumococcal challenge. In contrast, enforced expression of IL-7 restores protective anti-PPS responses in young mice, indicating that IL-7-dependent B cells are critical for TI responses. Thymic stromal lymphopoietin (TSLP) is structurally similar to IL-7 and utilizes IL-7Rα for signaling, therefore it may also contribute to the development of TI B cell responses. Indeed, we found that young TSLP transgenic mice generate an anti-PPS response and protective immunity comparable to that of adult mice. TSLP can be induced upon activation of TLR2 and TLR9. Borrelia hermsii, an agent of relapsing fever, activates TLR2 and TLR9 signaling and induces a protective TI antibody response even in young mice, suggesting that TSLP induced during this infection could play a role in anti-B. hermsii responses. Indeed, IL-7Rα-/- mice (deficient in both IL-7 and TSLP signaling) have a more severe impairment in anti-B.hermsii responses and suffer more prolonged bacteremia than IL-7-/- or TSLPR-/- mice. These data indicate that TSLP can restore functional TI responses in the young.