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Expression of Meningococcal antigens in recombinant BCG strains and assessment of their immunogenicity

Recombinant BCG strains expressing the model antigen chicken ova and the N. meningitidis ORF961 (nadA or NMB1994) encoding gene have been successfully constructed. These strains express the gene from an episomal vector and both Moreau and Pasteur rBCG strains have been obtained and grown as vaccine preparations. Expression cassettes have also been integrated into the BCG chromosome by means of a vector derived from mycobacteriophage Ms6.

The “integrative” rBCG-nadA strain is available in the BCG Pasteur background. The integrative rBCG-nadA strain in the BCG Moreau background is ongoing. The two “replicative” rBCG-nadA strains (Moreau and Pasteur) and the integrative rBCG-nadA strain (Pasteur) have been sent to Dr David Lewis to perform immunization studies in mice by the oral and intraperitoneal routes.

In a first experiment, BALB/c mice were immunized i.p. with 2x109 CFUs of rBCG-Pasteur-(pGIP3) and rBCG-Moreau (pGIP3). Animals were bled on day 63 and day 85 following immunization. Sera were sent to Chiron laboratories to evaluate anti-NadA antibodies produced and to test their bactericidal activity. Data indicate that rBCG Pasteur-nadA is more immunogenic than the BCG-Moreau-nadA strain. Anti-NadA titers were quite low but this could be due to the fact that the mice received only one dose of rBCG. Since bactericidal activity seems to correlate with high anti-NadA titers it is then not surprising that none of the sera tested were bactericidal. Another experiment where mice will receive a second dose of rBCG-nadA is needed.

In order to dissect the immune response induced by rBCG, the model antigen ova has been expressed in BCG. Several rBCG-ova strains have been constructed in order to target the heterologous antigen to the cytosol, the cell wall or to secrete the antigen. The constructs (both in replicative and integrative vectors) have been transformed into BCG Pasteur and Moreau background. Work is in progress to localize OVA produced in all strains.

Antigen presentation experiments have been performed using CD4 T-cell hybridomas specific for the OVA I-Ad restricted epitope as well as OT2 OVA-TCR transgenic T cells. Bone-marrow derived dendritic cells (BMDC) infected by all rBCG-ova strains do present the epitope to CD4 T-cells in the two systems. However, presentation using rBCG-ova is less efficient than presentation using Salmonella typhimurium ova strains despite comparable in vitro levels of expression.

More information on the MUCIMM project can be found at:

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Institut Pasteur
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