It has been demonstrated that the level of cross-reactivity and cross-protection among PspAs correlates learn more with sequence similarity, being low between PspAs of different families and higher within each family. Furthermore, it has been suggested that the level of cross-reactivity and cross-protection varies depending on the PspA clade

[21]. In that study, a PspA from clade 3 elicited antibodies with the lowest cross-reaction, while PspAs 4 and 5 (belonging to family 2) were highly cross-reactive. For family 1 molecules, neither PspA clade 1 nor clade 2 were able to induce antibodies cross-reactive to all family 1 strains tested. Therefore, further research was needed to better understand cross-reactivity within family 1. In the present study, the N-terminal regions of five clade 1 and five clade 2 PspAs were produced, antibodies generated and screened for their cross-reactivity against a panel of Brazilian strains containing clade 1 and 2 PspAs. The immunoblot analysis revealed a high heterogeneity in the level of cross-reactivity of the different antisera; while most cross-reacted mainly within the homologous clade, four PspAs – 245/00, M12, 94/01 and P339 – generated antibodies able to recognize most of the isolates tested. There was FK228 cost no predominance between the PspA clade and the level of cross-reaction;

clades 1 and 2 were equally cross-reactive. The hybridization of the reverse primers in distinct regions within the proline-rich moiety generated fragments with different sizes; all fragments included the entire alfa-helical domain plus the beginning of the proline-rich region, and some were longer, containing most of the proline block, several including the nonproline block. Although there was no clear correlation between the size of the fragment and the level of cross-reactivity by immunoblot – the most cross-reactive fragments included

both long and short proteins – in the more stringent assays – complement deposition and OPA – the two best candidates included the proline-rich region with the nonproline block. This result suggests a possible role for the proline-rich region with the nonproline block in the induction of functional antibodies. This data is in agreement with a recent study demonstrating that immunization of mice with the proline-rich TCL region including the nonproline block was able to protect mice against fatal challenge [28]. Complement mediated antibody-dependent phagocytosis is considered to be an important mechanism of pneumococcal clearance [29]. The ability of anti-PspA antibodies to promote complement deposition on the bacterial surface greatly contributes to their protective effect [11]. It has been demonstrated, however, that the level of complement deposited depends on the similarity between the PspA used to induce the antibodies and that expressed by the pneumococcus [21] and [30].