For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.
We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte͞macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vectorimmunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.We have developed Moloney murine leukemia virus-based retroviral vectors encoding therapeutic genes and have been studying the induction of immune responses mediated by these retroviral vectors. We have demonstrated that successful induction of immune responses could be achieved in mice (1), nonhuman primates (2), and humans (3), by injection of ex vivo vector-transduced autologous fibroblasts. These studies led us to investigate whether immune responses could be elicited by direct administration of retroviral vector by intramuscular injection. We have recently demonstrated induction of immune responses after direct administration of retroviral vectors intramuscularly in mice, rhesus monkeys, and baboons (4).Direct injection of retroviral vector offers several advantages over the ex vivo approach, primarily the elimination of the extensive efforts involved in generating autologous ex vivo fibroblast cell lines for each patient. With the ex vivo approach, the target antigen is exclusively expressed by transduced fibroblasts; however, direct administration of retroviral vectors can lead to the transduction of cells near the injection site as well as those cells residing in tissues to which vector can be transported. Consequently, it was not clear which cells were transduced by the retroviral vector, and which cells were responsible for presentation of retroviral vector-encoded antigens. In this paper, we have attempted to identify the vector-transduced cells and cells capable of presenting antigen to delineate mechanisms for induction of immune responses after direct injection of retroviral vectors.We have used retroviral vectors encoding HIV env͞rev, E. coli -galactosidase (-gal), chicken ovalbumin, and firefly luciferase to identify the subsets of cells involved in antigen presentation and induction of immune responses. These four different antigen systems were used to verify that the mechanisms of in vivo transduction and induction of immune responses are comparable among retroviral vectors derived from the same packaging cell line and backbone construct regardless of the specific antigen they encode. Furthermore, each of these re...
The immune response against human immunodeficiency virus type-1 (HIV-1) is believed to play a role in controlling the early stages of disease progression. The cellular immune response, in particular cytotoxic T lymphocyte (CTL) activity, may be important for eliminating virally infected cells in HIV-1-infected individuals. Genetic immunization using retroviral vectors provides an effective means of introducing antigens into the antigen presentation pathways for T cell stimulation. A nonreplicating, amphotropic murine retroviral vector containing the HIV-1 IIIB env gene has been used to transduce primary rhesus monkey fibroblasts for the expression of HIV-1 antigenic determinants. Rhesus monkeys were immunized with four doses of either vector-transduced autologous fibroblasts (VTAF) expressing the HIV-1 IIIB ENV/REV proteins or nontransduced autologous fibroblasts (NTAF) administered at 2-week intervals. The animals were evaluated for both the induction of HIV-1-specific immune responses and potential toxicity associated with this ex vivo treatment. The VTAF-immunized monkeys generated CTL responses specific for HIV-1 ENV/REV expressing autologous target cells, whereas, NTAF-immunized monkeys showed negligible CTL activity. The cytotoxic activity was mediated by CD8+, major histocompatibility complex (MHC)-restricted CTL. In addition, antibody responses directed against the HIV-1 gp120 protein were also detected in the sera of VTAF-immunized monkeys. Clinical and histopathological evaluation of immunized monkeys showed no evidence of significant adverse events. Several animals that received either VTAF or NTAF had detectable anti-cytoplasmic antibodies, but were not positive for anti-nuclear antibodies or rheumatoid factor. Subsequent evaluation of renal, synovial, and hepatic tissue samples from these monkeys revealed no autoimmune disease-associated lesions. This study demonstrates the safety and ability of autologous retroviral vector-transduced cells expressing HIV-1 IIIB ENV/REV proteins to stimulate immune responses in a non-human primate model, and provides a basis for this form of genetic immunization in HIV-infected humans.
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