Flow cytometric analysis of the interaction of the generated antibodies with diverse pneumococci showed that antibodies to PspA 245/00 and 94/01 were able to increase complement deposition on the widest range of pneumococci tested. The complement deposition on the different pneumococci appeared to be also influenced by the serotype. We observed that some serotypes exhibited an increased complement deposition in the absence of anti-PspA antibodies, as demonstrated previously with serotype 6B strains [31]. We tested the ability of these antisera to induce the complement deposition in pneumococcal

strains bearing family 2 PspAs (data not shown), and no increase in complement deposition was observed. This result is in accordance with our previous

findings Enzalutamide in vivo [21], and suggests that, although some family 1 molecules can broaden cross-reactivity within this family, this effect is not extended to family 2. Our results demonstrated a significant variability in the cross-reactivity DAPT of antisera generated against PspAs of the same clade, which correlates with differences in antibody mediated complement deposition on pneumococci. In order to correlate the results of cross-reactivity with protection, we evaluated the ability of the two most cross-reacting sera to promote the opsonophagocytosis of different pneumococcal strains by peritoneal phagocytes. Since it has been difficult to show killing using the classical OPA by anti-PspA antisera (unpublished data), we have optimized this assay in order to overcome the protective effect of the capsule. Using peritoneal cells too recovered from mice stimulated with a polyclonal T-cell activator, we were able to demonstrate the ability of anti-PspA antibodies to induce complement mediated phagocytosis of pneumococci of different serotypes.

The results demonstrate that both sera were able to induce complement-mediated phagocytosis leading to a minimum reduction of 30% on the number of pneumococci. This effect was observed for pneumococci of diverse capsular types, including serotypes 1, 3 and 6B, demonstrating the viability of this adapted opsonophagocytic assay for measuring the protective role of anti-PspA antibodies, which can overcome the inhibitory effects of different capsule types. Although these two sera were generated against PspAs of different clades, both were equally efficient against all family 1 strains. These results are in accordance with the complement deposition assay, in which both sera were able to increase complement deposition onto pneumococci containing PspA clades 1 and 2. This cross-reactive effect within strains bearing family 1 PspA has been previously reported using anti-PspA1 antibodies [21] and [22]. Moreno et al.