We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.
Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.Hepatitis C virus (HCV), a hepatotropic, positive-stranded RNA virus of the Flaviviridae family, is estimated to infect at least 400 million people worldwide and is a major etiologic agent of liver failure and hepatocellular carcinoma (11,66,70). HCV comprises a 9.6-kb genome with a conserved 5Ј untranslated region that functions as an internal ribosome entry site (33, 69). The untranslated region precedes a long open reading frame that encodes a 3,010-amino-acid (aa) polypeptide that is subsequently cleaved into 10 protein products (33). The amino-terminal region of the viral polypeptide is posttranslationally processed by host cell proteases to generate three structural protein products, Core (nucleocapsid) and envelope glycoproteins E1 and E2 (31, 43). Nonstructural proteins that facilitate virus replication (NS2, -3, -4a, -4b, -5a, and -5b) reside in the carboxy region of the polypeptide and are cleaved by virus-encoded proteases comprising 68).Standard therapeutic intervention for HCV infection consists of the administration of interferon (IFN) in combination with ribavirin. However, less than 50% of infected patients respond to this regimen and few alternative therapies exist (13, 14, 26, 52). There is presently no tissue culture system to efficiently cultivate HCV, which not only hampers research efforts aimed at elucidating the molecular mechanisms of virus replication but also impedes attempts at producing candidate vaccines and immunotherapies that target HCV-related disease. Consequently, a number of recombinant subunit-based HCV vaccine strategies, involving genetic immunization and purified proteins, have been attempted, as well as virus vectors expressing the HCV envelope glycoproteins (3,8,25,30,56,63,65). Determining an effective vaccination strategy has proven di...
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