Figure 6. Protection induced by immunization with recombi- nant fragments of the Spr1875 protein. The diagram illustrates the position of the various fragments relative to the whole protein. (A) Protection induced by R5-GST in comparison with R4-GST. Groups of CD1 mice were immunized with R4-GST, R5-GST or with GST, used as a negative control, and challenged with the D39 strain (16105 CFU). Results represent cumulative data from 3 experiments. A total number of 28 animals were used for each group. (B) Groups of CD1 mice were immunized with R1-GST, R2-GST, R3-GST or GST used as a negative control, and challenged with the D39 strain (16105 CFU). Results represent data from one experiment involving 14 animals per group. *, statistically different (p,0.05) from D39 strain, as assessed by Kaplan- Meier estimator of survival. doi:10.1371/journal.pone.0036588.g006 Sip of Streptococcus agalactiae, which is considered a promising candidate for an S. agalactiae vaccine [12,13], contains one N- terminal LysM domain. In the first part of this study, we focused on the expression of the spr1875 gene and on the impact of Spr1875 upon pneumococcal virulence. Flow cytometry assays demonstrated that Spr1875 is displayed on the surface of pneumococcal strains belonging to different serotypes, although the presence of the polysaccharide capsule largely masked the antigen. This is consistent with other studies reporting that the pneumococcal capsule may mask to varying degrees several surface proteins and adhesins [14,15]. Moreover, we found that the spr1875 gene was remarkably conserved amongst serotypes and showed a low degree of polymorphism. Virulence experiments demonstrated that Spr1875 is required for in vivo pneumococcal growth, as evidenced by the decreased ability of a Dspr1875 deletion mutant to produce lethal infection in mice. The effects of such deletion were marked and equalled or exceeded those observed with a DpspA deletion mutant. Therefore our data indicate that Spr1875 may behave as Spr1875, a Pneumococcal Antigen an important virulence factor, although further studies are clearly needed to elucidate the function of this protein. In the second part of the study, we focused on the immunoprotective activity of Spr1875 and on its potential use as a component of a protein-based vaccine. After immunization with the recombinant R4 fragment, we observed significant protection against lethal pneumococcal infection as 50–70% of R4-GST- immunized mice were protected, while only 10–20% of GST- immunized animals survived. In contrast, the whole Spr1875 protein was devoid of immunoprotective activity. This prompted us to analyze in greater detail the nature of the antibody response induced by whole Spr1875. It was found that 80–90% of the reactivity of serum antibodies in Spr1875-immunized animals was directed against the C-terminal portion of the molecule located outside of the R4 fragment. Therefore, only low levels of anti-R4 antibodies were generated by Spr1875 immunization. Moreover, we did not observe protective effects after immunization with a recombinant fragment (R5-GST) encompassing the whole length of the C terminal fragment external to R4. Thus, lack of immunoprotection by the whole Spr1875 protein was linked to the production of antibodies predominantly directed against non- protective, immunodominant epitopes located outside of the R4 fragment. On the contrary, immunoprotection was associated with the induction of anti-R4 antibodies. Little is known on the molecular factors that determine the immunodominance of some epitopes over others, although this phenomenon is known to occur after immunization with virtually any protein antigen [16]. It is generally thought that antigens bearing immunodominant epitopes are attractive vaccine candi- dates. However the opposite may be true. As recently noted [16], from the pathogen standpoint it would be useful if the host antibody response was directed against the non-protective portion of a virulence factor containing potentially protective epitopes. In other words, immunodominance of some protein regions may reflect an immune evasion mechanism. In this case, it would be appropriate, in terms of vaccine development, to redirect the response against the non-immunodominant, protective portion of an antigen, by synthesizing protein fragments that don’t incorporate the immunodominant epitopes. This seemed to be the case using Rib and a proteins of S. agalactiae and the M antigens of S. pyogens, in which non immunodominant regions proved to be of particular interest as vaccine components [16–18]. Interestingly, in the present study, another Spr1875 fragment (designated as R2) showed, in addition to R4, protective activity against pneumococcal infection after immunization. Since the R2 and R4 fragments overlap in a relatively short (54 aa long) portion of the Spr1875 protein, our data raises the possibility that this overlap region contains the protective epitope(s). Further studies are clearly needed to test this hypothesis. Our data showing that transfer of anti-R4 antibodies protected mice from lethal challenge against the encapsulated D39 strain could, at first glance, appear in contradiction with the inability of such antibodies to bind to the surface of in vitro grown D39, due to the masking effect of the capsule. However, it is likely that in vivo, at least temporarily or in specific host microenvironments, capsular material is shed from the bacterial surface and/or capsule expression is down-regulated, resulting in exposure of underlying antigens. In support of this notion, several surface pneumococcal antigens whose ability to interact with host components is well documented, are inaccessible to specific antibodies when grown in vitro [15]. In conclusion, we have identified a novel immunogenic surface protein with an essential role in virulence. Although complete protection was not observed, these data indicate that selected fragments of the 1875 protein may help, in conjunction with other PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36588