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02562 MUCOSAL DELIVERY OF MICROBICIDES BY COMMENSAL BACTERIA: EXPRESSION OF CYANOVIRIN-N IN LACTOBACILLUS Giomarelli, Barbara* Vaginal delivery of microbicides can be achieved using recombinant commensal bacteria. Human strains of Lactobacillus engineered to express HIV-inactivating polipeptides such as cyanovirin-N (CV-N) can colonize the vaginal mucosa, releasing locally the recombinant microbicide. We developed a new genetic system for expression of heterologous proteins in Lactobacillus. It is based on construction of transcriptional and translational fusions with fusion partners of lactobacillary origin. The gene fusions are integrated in the bacterial chromosome within a conjugative transposon, and fusion proteins are either secreted or expressed on the bacterial cell surface. Conjugative transposons carrying the recombinant gene can be transferred to the chromosome of different Lactobacillus species. Previously we obtained a recombinant CV-N expressed in Gram-positive bacteria, which was able to bind gp120 of HIV (Giomarelli et al., AIDS 2002, 16(10):1351-6). In the present work, we also improved the biological activity of the recombinant CV-N, by (i) including a different portion of the fusion partner, and (ii) a linker peptide at the N-terminal end. The new recombinant CV-N showed a gp120-binding activity equivalent to that of native CV-N. Further developments include testing the in vitro HIV-inactivating capability of CV-N expressed by recombinant Lactobacillus, and microbicidal evaluations of CV-N-producing commensals in animal models. Ph.D., Barbara Giomarelli |
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