Research Project
8329194
DESCRIPTION (provided by applicant): Women bear the brunt of the global AIDS epidemic due in part to their inherent social and biological vulnerability. Biologically, the cervical and vaginal mucosae are vulnerable targets for HIV transmission. In healthy women of childbearing age, mucosal antibodies and microflora, including Lactobacillus jensenii, help protect these surfaces and serve to block the colonization and infection by intruding pathogens. Epidemiological studies suggest that loss of vaginal lactobacilli is associated with an increased risk of heterosexual HIV-1 transmission and other sexually transmitted infections. Osel, Inc. and Profectus BioSciences are collaborating to exploit the properties of these natural protective agents by engineering a native human vaginal Lactobacillus, L. jensenii 1153, to express potent and broadly neutralizing single domain antibodies/nanobodies directed against HIV. Osel recently showed proof-of-concept for this recombinant Lactobacillus approach, by expressing an HIV-1 entry inhibitor from L. jensenii that reduced SHIV virus transmission in macaques by 63% (Lagenaur et al., Mucosal Immunol, (Jul 6, 2011)). The initial goal of this approach is to develop a Lactobacillus strain capable of delivering an antibody locally to prevent HIV infection at the vaginal or rectal mucosa. This strain can be formulated into a cost-effective product capable of persistent protection that is coitally-independent and controlled by women. We intend to evaluate this approach in two phases, and propose the following aims in Phase I: PHASE I PROPOSAL 1. Express secreted neutralizing single domain antibodies in the human vaginal Lactobacillus isolate, L. jensenii 1153. The genes encoding single domain antibodies will be subcloned into expression vector pOSEL175 and transformed into L. jensenii 1153. The resulting bacterial strains will be examined for antibody expression. The secreted proteins will be characterized using immunoprecipitation, gel electrophoresis, and gp120 ELISA. Full-length antibodies that bind to gp120 will be selected to for further characterization. 2. Characterize HI neutralizing activity of Lactobacillus-expressed single domain antibodies. The secreted antibodies in culture media will be purified using affinity chromatography and tested for neutralization of primary HIV-1 isolates in cell line-based and PBMC-based assays. The single domain antibodies that exhibit the best combination of HIV neutralization, (based on neutralization of isolates in the Profectus Virus Panel, scored by the number of isolates neutralized at >90% at 10 ¿g/ml concentration) will be selected for optimization of expression in L. jensenii 1153. 3. Optimize of plasmid-based expression of single domain antibodies by L. jensenii 1153. The expression cassettes of the leading single domain antibody candidates will be optimized for high level expression in L. jensenii 1153 and the expression levels compared by Western blot analysis. The candidate with the best combination of HIV neutralization potency and expression level will be selected for further strain development. 4. Construct integration vectors and integrate single domain expression cassettes into the L. jensenii 1153 chromosome. We will construct integration vectors and integrate the optimal antibody expression cassettes into the pox1 gene of L. jensenii 1153 via homologous recombination. The strains generated will be evaluated for antibody expression level, bioactivity against HIV, phenotypic characteristics compared to the parental strain and genetic stability. Technical feasibility in Phase I will be achieved by expressing intact and functionally active single domain antibodies in L. jensenii at concentrations high enough to block HIV-infectivity in vitro. In Phase II, we would complete development of the chromosomally integrated strain and test its ability to vaginally colonize rhesus macaques and produce sufficient levels of single domain antibody in vivo to protect against viral challenge. A proof of concept SHIV challenge study would then be conducted in animals colonized with the antibody- secreting strain to demonstrate protection from virus infection. Studies would also be conducted on formulation and manufacturing process development, and safety testing. If successful, this approach could provide a safe, yet inexpensive means to deliver passive immunity against HIV to the vaginal mucosa and to address the urgent need for female-controlled approaches to prevent sexually transmitted viral infection. PUBLIC HEALTH RELEVANCE: Our goal is to develop a novel female-controlled preventative against HIV by harnessing the vaginal microflora. We have developed technologies to bioengineer human vaginal Lactobacillus to express potent single domain antibodies in situ, thus creating a live microbicide. This project has the potential to reduce HIV acquisition in women of childbearing age in a coital-independent way.
