Application and development of molecular biological meth

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Summary: The goal of this project is to apply and develop molecular biological methods and animal models that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination and to assess the oncogenic and infectivity risks associated with residual cell-substrate DNA. Reverse transcriptase (RT) assays measure the presence of retroviruses. The conventional RT assay was insenstive and not quantitative. This changed with the advent of PCR-based RT PBRT) assays, which are a million-fold more sensitive than conventional RT assays. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro, and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown. We have demonstrated that mixed pseudotypes [i.e., pseudotypes containing envelope glycoproteins (Envs) from two different viruses] can form between measles virus and HIV-1 Envs. This raises the possibility that such virions can form in vivo. This may have implications for HIV pathogenesis, since HIV particles containing an Env of measles virus would be able to infect non-CD4 cells; such infected cells would have unknown consequences on HIV pathogenesis. We have developed Q-PCR assays to detect the genomes of primate polyomaviruses (SV40, JCV, BKV). Assays for these viruses will be a first step in our program to establish quantitative assays for the detection of adventitious agents. With these Q-PCR assays, we are quantifying the levels of the human polyomaviruses BKV and JCV DNA in human blood with the aim of determining whether the primate polyomavirus SV40 is present in human blood. We have shown that antibodies to SV40 can neutralise BKV and antibodies to BKV can neutralise SV40. We are developing a reporter-gene neutralisation assay for SV40, BKV, and JCV in order to test whether exposure to one polyomavirus can affect the probability of being infected with another polyomavirus. We have initiated studies to develop quantitative assays to assess the biological activity of residual cell-substrate DNA. For many years, DNA resulting from cell substrates has been considered to pose a risk to vaccine recipients receiving products manufactured in neoplastic cells. This is one of the main reasons for these cells not being used for vaccine production to date. The risk is perceived to be either from an oncogenic potential and an infectious potential. We are developing quantitaive assays to assess both types of risks. In collaboration with NCI and CDER, we are exploring animal models to asssess DNA oncogenicity. We have developed an in vitro system to quantify the infectivity of retroviral DNA.