Dr. Khan's laboratory is involved in understanding the mechanisms of retrovirus latency and activation with focus towards development of strategies for early detection of endogenous retroviruses in cell substrates used in biologics. Additionally, relevant animal models are being developed for pre-clinical product evaluation and to perform prospective risk assessment of retroviruses that are of potential concern in biological products. Current projects include: 1)evaluation of various chemical and biological agents for retrovirus induction from primate vaccine cell substrates and development of sensitive assays for retrovirus detection; 2)studies of latency, replication and transmission of naturally-occurring simian foamy retrovirus isolates; and 3) development of a pre-clinical animal model for AIDS vaccine safety and efficacy. Development of a monkey model for testing antiviral agents against HIV-1. A good animal model for HIV-1 is essential for AIDS vaccine studies and development of efficacious anti-viral agents. Additionally, such a model will provide insight into the mechanism of correlates of virus infection and protection. We have obtained reproducible, long-term HIV-1 infection in pig-tailed macaques and have evaluated virus infection in animals that received high and low doses of HIV-1/LAI. Although, each animal responded uniquely to the infection, in all cases HIV-1 antibodies developed and viral sequences were detected in the PBMCs. No significant clinical symptoms were seen. The results of our study indicate that pig-tailed macaques may be a useful animal model for evaluating the efficacy of novel vaccine strategies. To investigate whether pig-tailed macaques are a good animal model for vaccine studies, we tested the efficacy of the DNA prime with protein boost strategy using our HIV-1 DNA vaccines with subsequent Env protein boost against challenge with homologous virus (HIV-1/LAI). Virus could not be detected in the plasma of animals immunized with a vaccine DNA containing the CMV promoter. PBMC DNA was analyzed by PCR using pol and gag primers to further evaluate virus infection. The results indicate that pig-tailed macaques can be used to demonstrate the efficacy of immunization strategies using HIV/LAI based vaccines against homologous challenge virus. Development and standardization of retrovirus detection assays and safety studies related to cell substrates and biological products. Vaccines and therapeutics can potentially be contaminated with infectious retrovirus due to induction of an endogenous, latent virus in the cell substrate or by recombination between endogenous retroviral sequences to produce a novel virus. Additionally, blood and blood products may contain viruses due to their presence in the host donor. Thus, to assure public health safety in biologics, it is critical to develop sensitive retrovirus detection assays and to evaluate potential risk of retrovirus infection in humans using animal models. 1) Simian foamy retrovirus (SFV) is a safety concern in biological products using simian cells and potentially in blood donors that have been exposed to non-human primates. To evaluate potential risk of SFV infection in humans, naturally-occurring SFVs were isolated from rhesus and pig-tailed macaques. A. In vitro infectivity studies indicated that the naturally-occurring macaque isolates are diverse in replication efficiency and have slower replication kinetics than laboratory-adapted prototype viruses. Current investigations are ongoing to evaluate promoter strength and transactivating factor (tas) gene function. B. Studies are underway to evaluate SFV infection in naive macaques: a)by direct injection, and b) by blood transfer. TaqMan PCR assays were developed to quantify SFV load. The animals are currently being monitored for virus infection and clinical changes. These results will provide information regarding human risk of SFV infection from macaques and address potential safety concerns regarding SFV transmission by blood donors. 2) Endogenous retroviruses can potentially contaminate biological products by activation of viral sequences in the cell susbtrate. To develop a strategy for early cell susbtrate screening for endogenous viruses, we have investigated retrovirus induction by chemicals in a well-characterized mouse cell line using a reverse transcriptase assays for detection of induced retrovirus. The results indicated inducer-dependent endogenous retrovirus activation with increased cellular DNA polymerase production. Modification of the RT assay enhanced retrovirus-specific RT detection. Vaccine cell susbtrates will be evaluated for endogenous retrovirus induction using a highly sensitive PCR based assay (PERT). This project incorporates FY2002 projects 1Z01BK003008-10, 1Z01BK003009-10, 1Z01BK003013-07, and 1Z01BK003014-07.
