Influence of strain viability and antigen dose on the use of attenuated mutants of Salmonella as vaccine carriers

Influence of strain viability and antigen dose on the use of attenuated mutants of Salmonella as vaccine carriers. 34]), strains harboring deletions in the adenylate cyclase (genetic locus (15), a two-component regulatory system (virulence (15b, 23) and survival within macrophages (13, 14). causes a typhoid-like disease in mice and has been used extensively to generate attenuations which can be evaluated for their safety and immunogenicity in the murine model. We have shown that vaccination with attenuated salmonellae can lead to specific antibody Cetirizine responses in the genital tract of mice (19, 31, 32) and at least one human female volunteer (25). This suggests that PhoPc bacteria expressing HPV16 virus-like particles (VLPs) resulted Cetirizine in HPV16-specific conformational and neutralizing immunoglobulin A (IgA) and IgG in genital secretions (26). However the PhoPc strain contains a point mutation in the gene (15a, 16, 23a) and can revert at high frequency (24). Therefore, we decided to evaluate the HPV16-specific antibody responses elicited by strains harboring attenuations (PhoP? and strains (9), new isogenic strains were constructed. Expression of HPV16 L1 and HPV16 VLPs in the four isogenic strains.4989 ((isogenic strains: i.e., the PhoPc (CS022 [24]) and PhoP? (CS015 [22]) strains and 4989 and 4990 (this paper). Western blot analysis of bacterial lysates revealed only minor differences in the levels of HPV16 L1 expression (57-kDa band in Fig. ?Fig.1A)1A) among the different strains examined. Densitometric analysis of the Western blot Cetirizine bands obtained from five different preparations Cetirizine of each recombinant strain are shown in Fig. ?Fig.1B.1B. The maximal difference observed in HPV16 L1 expression was 2.5-fold between 4990 and 4989. We were not able to detect HPV16 L1 in the supernatant of cultures, suggesting that none of these strains secreted the HPV16 VLP antigen. We have previously shown that this anti-HPV16 L1 antibodies measured with our enzyme-linked immunosorbent assay (ELISA) are conformational and only recognize assembled HPV16 VLPs (26). It was therefore important to establish whether, despite similar levels of HPV16 L1 expression, the assembly of VLPs could differ between the recombinant strains. For all those strains, HPV16 VLPs could be extracted by sonication of the bacteria, suggesting that they were not located in inclusion bodies. To determine the amount of VLPs assembled in strains. Salmonellae were grown overnight at 37C and lysed by boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer made up of 5% SDS. Bacterial lysate equivalent to 3 107 CFU was separated by SDS-PAGE, and an immunoblot with an anti-HPV16 L1 monoclonal antibody (MAb), CAMVIR-1, is usually shown (A). The 57-kDa protein band identified as L1 is usually indicated by an arrow. Scanning of the L1 protein bands obtained in five impartial experiments was performed by using NIH Image software. The results are shown as the means of the pixel densities of the L1 protein bands expressed in models per 1011 CFU Cetirizine (B). VLP production was assessed in overnight cultures. After sonication, the bacteria were centrifuged to discard the debris, and the supernatants were analyzed Pecam1 for the presence of HPV16 VLPs. The amounts of VLPs were determined by sandwich ELISA. Microtiter plates were coated with an anti-HPV16 VLP conformational MAb, H16.E70, another biotinylated anti-HPV16 VLP conformational MAb, H16.V5, was used as the secondary antibody, and HPV16 VLPs produced in insect cells (26) were used as a standard. The.