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Expression of Meningococcal antigens in Salmonella strains and assessment of their immunogenicity

The antigens pET clones obtained from Siena were introduced into a range of E. coli host strains. Under conditions, which facilitated expression of the cloned antigens, the plasmid vectors segregated heavily from the bacterial hosts unless antibiotic selection was maintained.

To assess expression in Salmonella strains, NMB 0992 was cloned with an intact signal sequence into a medium copy number plasmid, under the control of the dps promoter (induced during the stationary phase in vitro and within macrophages). The new plasmid was then transformed into E. coli and Salmonella hosts. Once again, the plasmid was lost in vitro in the absence of antibiotic. Western blotting confirmed expression of NMB 0992 in a number of host strains. So, expression of meningococcal antigens in Salmonella appears to present a number of technical challenges in terms of maintaining stable expression of antigen in the absence of antibiotic selection. This would clearly compromise in vivo immunization experiments. More stable expression might be achieved by expression of antigens from the Salmonella chromosome. These data led us to concentrate subsequent efforts on immunization studies with purified meningococcal proteins and mucosal adjuvants.

Three immunization experiments were carried out in BALB/c mice, immunized intranasally, using purified meningococcal antigens and a range of mucosal adjuvants (CT, LT, LTR72, LTK63). Qualitative differences were apparent between the immune responses induced following immunization with the three meningococcal antigens selected for study. All induced clear cellular immune responses. However, in contrast with NMB 0992 and NMB 1994, NMB 1985 was a very poor inducer of either local or systemic antibody responses. These results are in close agreement with those obtained by Kingston Mills at the Dublin group. One of the criteria used for selection of the antigens was that they had all been shown to induce bactericidal antibodies. In our studies however, bactericidal antibody production was only observed following immunization with NMB 1994. This may reflect differences between the protocol used for initial screening of proteins carried out in CHIRON (i.p. inoculation in presence of FCA) and the mucosal delivery employed in the experiments described above.

LTR72 produced responses which in general were quantitatively similar to, or greater than, those induced by LT. Even at a ten-fold higher dose than that used for LT or LTR72, LTK63 was a far less potent adjuvant. In conclusion, our studies reveals that, although technical problems linked to instable expression of antigens in Salmonella, the all three meningococcal antigens selected for the study induce clear cellular immune responses after mucosal immunization in mice, strengthen the idea that mucosal immunization can be an effective alternative for vaccine delivery.

More information on the MUCIMM project can be found at:

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