Project 1 Summary & Aims

Project 1 Summary

This Project proposes to establish a pipeline for the preclinical development of highly active and safe microbicides to prevent rectal transmission of HIV-1 infection. For this Project, the term ‘preclinical’ includes drug-development efforts ranging from in vitro testing of pre-selected candidate compounds in cell lines, single cell suspensions of PBMCs or mucosal mononuclear cells (MMCs). Those candidates that demonstrate efficacy without toxicity/reduction in cellular viability will be promoted to evaluation using an ex vivo model of rectal explants for both toxicity and efficacy. Setting a stepwise selection approach, doses of safe and effective candidates will subsequently be considered for in vivo macaques efficacy studies using the MDP’s pre-selected group of microbicidal agents: PMPA, UC-781 and TMC-120. The primary endpoint of this project will be to identify the best rectal microbicide product possible, given the available time and resources, for human trials. Secondary goals will be to characterize differences in viral targets (for different viral strains) in the rectal mucosa and the relative efficacy of the 3 initial microbicidal candidates in inhibiting infection in macaques. Tertiary goals will be to characterize immuno-inflammatory responses to viral infection and describe expected changes following drug exposure with the aim of deriving confidence intervals for data analysis and sensitivity as well as enabling assay correlations and selection of the most predictive assays for future use in pipeline drug assessments.

Specific Aim #1. Definition of potential target cells for HIV-1 infection and/or dissemination within rectal mucosa to guide drug development. Using rectal explant cultures obtained from surgery and biopsy specimens we will define the population of susceptible target cells within rectal mucosa and the phenotype of cells emigrating from rectal tissue with the capacity to disseminate HIV-1 draining lymph nodes. This is important as migration of DC and/or CD4 T cells to draining lymph nodes, rich in susceptible target cells, may circumvent that activity of any topically applied microbicide, impacting drug targets and duration of effect. Furthermore, we will define the influence of viral phenotype and genotype on the dynamics of infection within these model systems in order to determine which HIV-1 populations need to be targeted by candidate microbicide compounds. Finally, we will evaluate the influence of seminal plasma on all of the above parameters. The goal of this component is to define which cellular populations in rectal mucosa need to be targeted by candidate drugs in order to prevent HIV-1 infection and dissemination. Results from these studies will inform in vitro and animal efficacy studies (Aims 2-4).

Specific Aim #2. Evaluate the efficacy of potential rectal microbicide candidates, alone and in combination against HIV-1 infection using cell-based assays and explant cultures. We will evaluate the anti-HIV-1 activity of compounds alone and, later, in combination using our identified RT inhibitor candidate microbicides to prioritize agents and combinations with potent anti-viral activity without toxicity. Initial studies will focus on the three RT compounds: PMPA, UC-781 and TMC-120. Later studies will be broadened to include a wider range of novel microbicide candidates. Compounds will first be screened for synergistic activity using cell lines and primary cells. Only those agents demonstrating significant activity alone and/or in combination will be assessed for efficacy in the human rectal explant model to determine their ability to inhibit localized HIV-1 infection. Additional studies will be carried out to evaluate the efficacy of candidate drugs, combinations and formulations against key HIV-1 phenotypes determined above. The goal of this component is to identify the best candidates and/or combinations to progress to bio-compatibility studies.

To evaluate the tissue bio-compatibility of potential rectal microbicide candidates, alone, and in combination, using cell based assays and rectal explant cultures. The biocompatibility of compounds, combinations and formulations will be assessed by measurement of potential toxicity and production of cytokines and chemokines using primary cells and rectal explants. The goal of this component is to identify candidate compounds, combinations and formulations with detailed, in vitro correlates of safety profiles. Once identified candidates will progress to the next stage of in vitro evaluation.

To evaluate the efficacy of candidate microbicides against rectal challenge with SIV and infectious RT-SHIV of Rhesus macaques. We propose to assess the protective effect conferred by lead candidates (PMPA, UC781 and TMC-120) and combinations against rectal challenge using an infectious RT-SHIV in rhesus macaques. RT-SHIVs are simian/human chimeric viruses in which the reverse transcriptase gene of the pathogenic SIVmac239 strain has been replaced with its corresponding HIV counterpart. These modified viruses will be important as UC-781 and TMC-120 are ineffective against reverse transcriptase of SIV. Furthermore, the choice of an RT-SIV will directly determine the efficacy of candidate compounds against HIV RT in an in vivo model. In proof of concept studies, viral challenge will be performed 15 minutes post drug application, and in a smaller number of animals, viral inoculation will be delayed over a range of time points post drug administration to determine the period of protection provided. In addition, rectal tissue from uninfected animals will be harvested at necropsy and used in comparative explant studies to cross validate human rectal explant studies. The goal of these investigations is to predict potential efficacy of candidate microbicides against rectal HIV transmission in humans, providing valuable information for selecting lead candidates for clinical trials (as in Project 4).

Project 1 Aims: Milestones