In the present report, we demonstrated for the first time that BVH-3 confers protection against lethal infection with S. pneumoniae
. As reported by others (1
), BVH-3 shares common sequences with other pneumococcal proteins and could thus be considered a member of a protein family. The previous report, as well as those published by Zhang et al. (31
) and others (Hamel et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol.; Hamel et al., Abstr. 3rd Int. Symp. Pneumo. Dis.), also demonstrated that other members of the family possess protective epitopes. However, no publication provided information on the exact locations of the protective epitopes or that S. pneumoniae
actually simultaneously expresses BVH-3 and BVH-3-related proteins containing distinct protective epitopes. Our results revealed that the homologous amino region of BVH-3 elicits cross-reactive Abs that do not have protective activity despite relatively high Ab concentrations in serum samples from animals. We demonstrated that BVH-3 and BVH-11 (a BVH-3-related protein) protective epitopes are distinct and that the proteins constitute two distinct immune targets for protective Abs.
Ideally, protective-vaccine candidates should show no or minimal molecular and antigenic variation. Altogether, our sequence analyses revealed that the bvh-3
genes encode proteins that are highly conserved among S. pneumoniae
strains. Moreover, Western blot analysis revealed the presence of the proteins in all tested strains (Fig. ). This contrasts with results obtained with PhtA, another BVH-3-related protein, which was not detected in some strains (1
). The high degree of identity of protein-specific sequences containing the surface-exposed protective epitopes throughout representative strains of the species suggests that these molecules could elicit group-protective coverage. Indeed, immunization of mice with BVH-3 or BVH-11 conferred protection against experimental sepsis and pneumonia resulting from challenges with heterologous pneumococcal strains. Data from passive-immunization studies were consistent with those obtained from active-immunization experiments. Moreover, it was observed that protection conferred by rabbit anti-BVH-3 Abs was completely abolished by the addition of soluble BVH-3B (the carboxyl-terminal end) but not BVH-3A (the amino-terminal end), thus establishing the specificity of the protective Abs for epitopes located in the carboxyl half of BVH-3.
Since Ab-mediated opsonophagocytosis may be the major mechanism of protection against pneumococcal infection, the Abs detected by flow cytometry studies of intact cells would likely be the most biologically relevant. Indeed, our data demonstrated that there is good correlation between protection and the accessibility of epitopes. We observed that the C-terminal two-thirds of BVH-3 and half of BVH-11 were protective and surface exposed, while the first 200 amino acids residues were internal and nonprotective. Moreover, passive-protection assays by transfer of immune Abs clearly established that surface-labeling Abs are biologically linked to survival. Our results indicated that transfer of Abs alone could prevent experimental disease. This observation confirmed the role of anti-BVH-3 and anti-BVH-11 immunoglobulins as the major mechanism of protection. In addition, we demonstrated that a systemic immune response to these proteins could induce protection at a mucosal site, such as the lungs.
Since immune responses to BVH-3 and BVH-11 were both protective, it was reasoned that immunization with mixtures of these antigens would provide better protection against S. pneumoniae than the individual counterparts. It was demonstrated that, even though nonprotective, the common, inaccessible region is highly immunogenic. By combining in a vaccine molecule only the surface-accessible regions, the immune response is directed mainly to epitopes necessary for protection. Indeed, vaccination of animals with a chimeric gene product, corresponding to the surface-exposed protective regions of BVH-3 (i.e., BVH-3C) and BVH-11 (i.e., BVH-11B) fused in frame, conferred protection against lethal experimental infection and generated better immune responses. The superiority of the chimeric protein molecule over its BVH-3C and BVH-11B counterparts comes from the capacity of the chimera to induce Abs that recognize surface protective epitopes on two targets on pneumococci. This was demonstrated by several immunoassays including ELISA, Western blotting, and increased surface binding measured by flow cytometry. In addition, the use of competitor antigens to block the paratopes of the Abs clearly demonstrated that both the BVH-3C and BVH-11B vaccine regions are the targets of protective Abs and are thus the effectors of active protection. The chimeric approach has the advantage of focusing the immune response toward functional epitopes and limiting the total amount of protein contained in the vaccine. This could also facilitate the development of a vaccine, since only one recombinant protein needs to be produced and characterized.
The identification of BVH-3 and BVH-11 using human Abs, associated with the observation that human sera were reactive with both molecules, indicates their innate immunogenicity in the natural host. In agreement with these results, Adamou et al. (1
) reported the development of Abs to PhtA and PhtD, two proteins distinct from but related to BVH-3 and BVH-11, during pneumococcal infection in humans. However, the fact that Abs to pneumococcal BVH-3 and BVH-11 recombinant proteins could recognize common epitopes, and that these epitopes are also detected in GBS (data not shown) and possibly in GAS, raises questions about antigenic stimulus associated with Ab production in humans. Interestingly, the Ab reactivity to full-length BVH-3 and BVH-11 is stronger than that observed to BVH-3C and BVH-11B truncates. Thus, it is possible that the common sequence shared by a variety of molecules produced by pneumococci, and possibly by other streptococci, is more abundantly and/or more frequently expressed and stimulates strong Ab responses. Our studies with truncates corresponding to BVH-3- or BVH-11-specific regions provide evidence for in vivo expression of both molecules in S. pneumoniae
. Rapola et al. (26
) reported that contact with pneumococci induces Abs to several pneumococcal proteins, including PsaA, pneumolysin, and PspA. Our results show that humans also naturally develop Abs to BVH-3 and BVH-11 protein epitopes. Further studies are needed to investigate the biological significance of these Abs.
Homology searches with protein fragment sequences corresponding to the carboxyl termini of the proteins (BVH-3C and BVH-11B) established the uniqueness of these sequences. Our flow cytometry data revealed that these regions comprise surface-exposed epitopes, while the common amino termini of BVH-3 and BVH-11 are not accessible to Abs. Genomic analyses indicating hydrophobic leader sequences predicted that these proteins reside on the bacterial-cell surface (1
). We hypothesize that the proteins bind to the cytoplasmic membrane via the hydrophobic portion of the leader peptide sequence and not by a lipidation process of the amino end and that the homologous first 200 amino acid residues cross the peptidoglycan layer. This hypothesis was suggested by (i) the absence of [3
H]palmitic acid labeling of BVH proteins and (ii) results obtained with the BVH-3 proteins of some serotype 9V strains. Although their BVH-3 protein is missing a stretch of 177 amino acids (residues 244 to 420), Abs raised to this truncated version were reactive with surface-accessible epitopes on strains of serotypes different from 9V. These results indicate that the deleted protein section is not involved in cell surface anchoring. In addition, accessibility results similar to those obtained with Abs raised to full-length BVH-3 show that the missing region does not contain accessible epitopes. In agreement with our findings, Adamou et al. reported that BVH-3 and BVH-3-related proteins were not detected in the Triton X-114 fraction phase from which lipoproteins are typically recovered (1
The biological function of the BVH-3 protein is not known. The sequence homology among BVH-3 and BVH-3-related proteins suggests that the proteins might have similar functions through their homologous region and, at the same time, play distinct roles. A similar situation occurs with several pneumococcal proteins sharing a common repeated sequence, the choline-binding domain, which allows the proteins to bind to the phosphorylcholine moiety of the cell wall (10
). Although these proteins are members of the choline-binding protein family, each molecule has a distinct role in the bacterial cell, such as autolysin (LytA), involved in cell division, or PspA, implicated in complement inactivation (10
). It was speculated that the histidine triad motif, HXXHXH, of the BVH proteins may be involved in metal or nucleoside binding and that the binding of zinc ions by the histidine triad motifs would confer the functional conformation on the proteins (1
). It is noteworthy that protein functional analysis suggested that in situations where the bacteria face a zinc-restricted environment, the expression of the BVH protein would be induced and thus result in Streptococcus
adhesion and invasion (23
). Our studies highlighted the fact that histidine triad motifs present in BVH-3 and BVH-11 are not related to protection. Indeed, the truncated proteins BVH-3A and BVH-11A, possessing five and three of these triads, respectively, do not elicit surface-reactive Abs and are nonprotective antigens.
While protection is relative to a pathogen, it is very likely that a combination of preexisting protective Abs and a rapid secondary immune response will be sufficient to protect against S. pneumoniae. Surface proteins, like capsular antigens, could play an important role in protection through the attachment of Abs to the epitopes exposed on the pneumococcal cell surface and subsequent activation of the complement cascade. The protective immunity conferred by conserved protein antigens will have the advantages of being independent of capsular type, being T cell dependent, and eliciting an immunological memory. Continued studies of these proteins will provide new insights into the pathogenesis of these infections and protective immunity. Phase I clinical trials are being conducted to evaluate the safety and immunogenicity of BVH-3 and chimeric recombinant antigen vaccines in humans.