Robert O. Ralston

Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells

Truncated and full-length versions of the hepatitis C virus protein domain encoding a presumptive envelope glycoprotein designated E2/NS1 were stably expressed in CHO cell lines. Characterization of the processing events involved in the maturation of E2/NS1 revealed that a high-mannose form resident in the endoplasmic reticulum was the most abundant form detected intracellularly. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that the E2/NS1 glycoprotein resided in the endoplasmic reticulum. The full-length form of E2/NS1 appeared to be cell-associated and could not be detected as a secreted product. C-terminal truncated molecules could be detected in the extracellular media as fully processed glycoproteins containing terminal sialic acid additions. These truncated glycoproteins are predicted to be biologically relevant targets of the host immune response and are therefore potential subunit vaccine candidates.

Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors.

A new method of producing vesicular stomatitis virus (VSV) G protein pseudotyped retroviral vectors is described. In this method, stocks of VSV-G pseudotyped vector were reproducibly obtained by infecting an env-, human, retroviral vector producer cell line with a recombinant murine cytomegalovirus (CMV) which expresses VSV-G protein. The recombinant murine CMV, RMCMVG, expressed VSV-G protein under transcriptional control of the human CMV immediate-early promoter. RMCMVG, like murine CMV, can infect human cells, but the infection is limited to the expression of the viral immediate-early genes; no productive replication of murine CMV occurs. Recombinant murine CMV vector infection of non-permissive cells may be useful in situations where high levels of gene expression are desired without concomitant viral vector replication.

The Hepatitis C Virus: Genetic Organization, Persistence, and Vaccine Strategies

The proteins encoded by the hepatitis C virus (HCV) positive-stranded RNA genome have been identified in mammalian cells transfected with cloned HCV cDNA. The putative nucleocapsid protein C, (20kDa) and envelope glycoproteins (El, 33kDa and E2, 70kDa) have been identified along with putative nonstructural proteins 2 (23kDa), 3 (70kDa), 4a (10kDa), 4b (27kDa), 5a (56kDa), and 5b (70kDa). These proteins are processed from a poly protein precursor through the combined action of host and viral encoded proteases. The immune response to HCV has been investigated in patients with chronic hepatitis. Essentially, all patients have circulating antibodies to the envelope glycoproteins, and HCV-specific cytotoxic lymphocytes have been isolated from many individuals. These preliminary data indicate that persistence of HCV is not simply due to the absence of an immune response. Purified recombinant E1 and E2 glycoproteins have been used to immunize seven chimpanzees. Following experimental challenge with homologous HCV-1, five animals were protected from both HCV infection and disease. Although infected, the course of disease may have been inhibited in the remaining two vaccinees. These data encourage the use of an HCV vaccine to prevent infection and the carrier state.