Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Preparation of meningococcal antigens

The whole-genome sequence of N. meningitidis serogroup B strain MC58 was used to identify novel vaccine candidates. Putative novel antigens were expressed in E. coli as His-tag or GST fusions, purified, and used to immunize mice. Analysis of sera allowed for the identification of proteins that are surface-exposed and able to induce a bactericidal antibody response.

Among the novel identified antigens considerable as vaccine candidates against MenB, three proteins were selected as model antigens for these studies: Orf1 (NMB1985), Orf40 (NMB0992), 961 (NMB1994). These antigens have been be used to evaluate the efficacy of different live delivery systems and the potentiality of mucosal vaccination against meningococcus.

All the three proteins are:
- Surface-exposed in MenB;
- Well expressed in E. coli as fusion proteins;
- Able to induce a bactericidal antibody response (an important property because it correlates with protection in humans);
- Homologous to known bacterial virulence factors;
- Surface-localized in E. coli using E. coli as a model system for heterologous bacterial surface-expression (native-expression).

Orf1 is a protein of 1457 aa homologous to Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae. In MenB, as well in E. coli expressing the full-length gene, it is exported to the outer membrane, processed and secreted in culture supernatant. Orf40 (591 aa) is homologous to Hsf and Hia adhesins of Haemophilus influenzae. In MenB it is found on the outer membrane as a protein of about 200 kDa (oligomers). In E. coli expressing the full-length gene, Orf40 is surface-localized, expressed as monomer and possibly forms also multimers.

961 (405 aa) is homologous to YadA of enterophatogenic Yersinia, a non-pilus associated adhesin and to UspA2 of Moraxella catarrhalis, involved in serum resistance. The sequence homology is limited to the carboxyl terminal region, while an overall similarity can be observed at the level of the secondary structure. In fact, 961, YadA and UspA2, have a carboxyl terminal membrane anchor formed by four amphipatic beta-strands and internal alpha-helical regions which probably form coiled-coils. In MenB and in E. coli expressing the native form, 961 are localized in the outer membrane and forms very stable oligomers. These genes were expressed in E. coli under the control of T7 promoter.

Orf1 and Orf40 were produced and purified as cytoplasmic–insoluble His-tag fusions, while 961 was produced as different forms (GST/His-fusions, untagged protein, domains, cytoplasmic proteins, secreted forms), most of which are able to elicit a bactericidal activity. The three MenB antigens, as well the novel mucosal adjuvants derivatives of LT, LTK63 and LTR72, have been provided, as genes and as purified recombinant proteins, to different consortium partners (Imperial College of Sciences, Institute Pasteur and University of Siena) for expression in Salmonella, BCG and Streptococcus gordonii.

More information on the MUCIMM project can be found at http://www.altaweb.it/mucimm/.

Kontakt

Pizza MARIA GRAZIA, (Senior Scientist)
Tel.: +39-057-72430878
Faks: +39-057-7243564
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