Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease worldwide. Fragments of the cell wall and the cytolytic toxin pneumolysin have been shown to contribute substantially to inflammatory damage, although the interactions between pneumococcal components and host-cell structures have not been elucidated completely. Results of a previous study indicated that cell-wall components of pneumococci are recognized by Toll-like receptor (TLR)2 but suggested that pneumolysin induces inflammatory events independently of this receptor. In this study we tested the hypothesis that pneumolysin interacts with surface proteins of the TLR family other than TLR2. We found that pneumolysin stimulates tumor necrosis factor-␣ and IL-6 release in wild-type macrophages but not in macrophages from mice with a targeted deletion of the cytoplasmic TLR-adapter molecule myeloid differentiation factor 88, suggesting the involvement of the TLRs in pneumolysin recognition. Purified pneumolysin synergistically activated macrophage responses together with preparations of pneumococcal cell walls or staphylococcal peptidoglycan, which are known to activate TLR2. Furthermore, when compared with wild-type macrophages, macrophages from mice that carry a spontaneous mutation in TLR4 (P712H) were hyporesponsive to both pneumolysin alone and the combination of pneumolysin with pneumococcal cell walls. Finally, these TLR4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with pneumolysin-positive pneumococci than were control mice. We conclude that the interaction of pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus.

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell–dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1−/− mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-γ or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.

Acquired immunity to Streptococcus pneumoniae (pneumococcus) has long been assumed to depend on the presence of anticapsular antibodies. We found, however, that colonization with live pneumococci of serotypes 6B, 7F, or 14 protected mice against recolonization by any of the serotypes and that protection from acquisition of a heterologous or homologous strain did not depend on anticapsular antibody. Further, intranasal immunization by live pneumococcal colonization or by a killed, nonencapsulated wholecell vaccine protected antibody-deficient mice against colonization, suggesting independence of antibodies to any pneumococcal antigens. Protection by intranasal immunization with whole-cell vaccine was completely abrogated in T cell-deficient mice, and in mice that were congenitally deficient in CD4 ؉ T cells or depleted of these cells at the time of challenge. In contrast, mice congenitally deficient in, or depleted of, CD8 ؉ T cells were fully protected. Protection in this model was observed beyond 2 months after immunization, arguing against innate or nonspecific immune mechanisms. Thus, we find that immunity to pneumococcal colonization can be induced in the absence of antibody, independent of the capsular type, and this protection requires the presence of CD4 ؉ T cells at the time of challenge.Streptococcus pneumoniae ͉ cell-mediated immunity ͉ vaccine A lmost 1 million children in the developing world die of infections due to Streptococcus pneumoniae (pneumococcus) each year (1). Pneumococcus is considered an ''extracellular'' bacterial pathogen, i.e., it is killed upon ingestion by phagocytic cells. Ingestion is facilitated by antibody (Ab) to its capsular polysaccharides (PS), of which there are at least 90 different serotypes. The two existing pneumococcal vaccines are based on injected mixtures of PS. Plain (unconjugated) PS vaccine contains 23 serotypes but is not efficacious in children Ͻ2 years old and therefore fails to protect those at highest risk. Protein-conjugated PS contains seven serotypes and protects infants (2), but it is difficult to manufacture (resulting in repeated shortages), is expensive, needs refrigeration, requires multiple injections, and does not include many of the capsular serotypes that cause pneumococcal disease in the developing world. Furthermore, serotype replacement, whereby pneumococcal serotypes not included in the conjugate vaccine become more prevalent causes of colonization and disease, has already been observed in clinical trials (3) and in epidemiologic studies (4) after implementation of conjugate vaccine immunization programs. Therefore, despite the success of the capsule-based vaccines, alternative strategies are urgently needed.The success of serum therapy (passive transfer of anticapsular Ab from hyperimmune animals) and the efficacy of PS and PS-protein conjugate vaccines have clearly demonstrated that anticapsular Ab alone is sufficient to treat or prevent pneumococcal disease. Furthermore, studies in animals (5) and humans (6) clearly demonstrate that antica...

A whole-cell killed unencapsulated pneumococcal vaccine given by the intranasal route with cholera toxin as an adjuvant was tested in two animal models. This vaccination was highly effective in preventing nasopharyngeal colonization with an encapsulated serotype 6B strain in mice and also conferred protection against illness and death in rats inoculated intrathoracically with a highly encapsulated serotype 3 strain. When the serotype 3 challenge strain was incubated in the sera of immunized rats, it was no longer virulent in an infant-rat sepsis model, indicating that the intranasal immunization elicited protective systemic antibodies. These studies suggest that killed whole-cell unencapsulated pneumococci given intranasally with an adjuvant may provide multitypic protection against capsulated pneumococci.Streptococcus pneumoniae (pneumococcus) annually causes 10 million deaths worldwide, including the deaths of 1 million children in low-income countries (26). Type-specific immunity, based on the capsular polysaccharides (PS), is well established (20). The licensed 23-valent PS vaccine, however, is not efficacious in children younger than 2 years. The newly licensed heptavalent PS conjugate vaccine protects against 90% of pneumococcal invasive disease in infancy in the United States (28) but includes fewer serotypes than the PS vaccine and omits several that are prevalent worldwide (10). Other drawbacks of the conjugate vaccine include a limited effect on otitis media (2, 11), high costs, and the potential for serotype replacement, which has already been suggested in recent clinical trials (11, 17; R. Dagan, N. Givon, P. Yagupsky, N. Porat, J. Janco, I. Chang, et al., Program Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. S52, 1998).Several investigators have identified protective antigens common to pneumococci of many or all serotypes. Several such "species" antigens in purified or vectored form have shown protection in animal models (4-6, 8, 18, 19, 23, 25), but it is uncertain whether, when, and at what cost any of these will be developed as an effective vaccine for humans, particularly in low-income countries. As an alternative presentation of species antigens, we have studied unencapsulated whole cells, which should present a number of such antigens in native configuration unoccluded by capsule. In addition, the intranasal route of immunization might elicit mucosal immunity and, with suitable adjuvant, systemic immunity as well. Finally, of importance to low-income countries, a mucosally administered whole-cell preparation has the possible advantage of low cost of production and administration, without the need for sterile injection devices. In the present study we tested killed, unencapsulated cells applied intranasally with cholera toxin (CT) as an adjuvant (R. Malley, S. Pelton, A. Stack, R. Saladino, D. E. Briles, and P. Anderson, 2nd Int. Symp. Pneumococci Pneumococcal Dis., abstr. P25, 2000), using two animal models: nasopharyngeal colonization of mice with type 6B and lethal intrat...

The bactericidal activity of Streptococcus pneumoniae toward Staphylococcus aureus is mediated by hydrogen peroxide. Catalase eliminated this activity. Pneumococci grown anaerobically or genetically lacking pyruvate oxidase (SpxB) were not bactericidal, nor were nonpneumococcal streptococci. These results provide a possible mechanistic explanation for the interspecies interference observed in epidemiologic studies.

Serotype-specific immunity to Streptococcus pneumoniae is conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulated S. pneumoniae of serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4 ؉ T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy against S. pneumoniae.

The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H 2 O 2 ), not LytA, and that the gene responsible for H 2 O 2 production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H 2 O 2 in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing ⌬spxB mutants that do not produce H 2 O 2 . Likewise, addition of H 2 O 2 to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While ⌬lytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H 2 O 2 has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the ⌬spxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.Streptococcus pneumoniae is a gram-positive human pathogen that colonizes the nasopharynx of most children during infancy. Despite the availability of effective antibiotics and vaccines, pneumococci remain a major cause of morbidity and mortality, causing pneumonia, bacteremia, and meningitis. It is estimated that over 850,000 children die annually of pneumococcal infections worldwide (3).S. pneumoniae grown in broth dies spontaneously when reaching stationary phase. How this process occurs and why it would persist in pneumococci are controversial and unresolved issues. Almost a century ago, McLeod and Gordon suggested that this phenomenon is due to accumulation of self-produced H 2 O 2 (18); however, their hypothesis was not widely accepted. In 1970, Tomasz et al. (39) reported that several antibiotic groups induce their bactericidal effect through the induction of lytA and that with loss of LytA function, pneumococci fail to lyse and lose viability at a substantially lower rate. Despite a subsequent report demonstrating that a LytA-independent component was responsible for cell death (19), the phenomenon of spontaneous death, or "suicide," has been conventionally attributed to autolysis by the major autolysin of S. pneumoniae, an N-acetylmuramoyl-L-alanine amidase (LytA) (11,14). LytA appears to be constitutively expressed (9, 11), but its activity is likely under the control of a complex and only p...

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.