Vaccination of chimpanzees against infection by thehepatitis C virus.

Abstract

A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatoceflular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [El (gp33) (4) and leads to the development of chronic hepatitis and liver cirrhosis in "50%o and 10% of cases, respectively (5). A significant proportion of patients with liver cirrhosis will also develop primary hepatocellular carcinoma (6). The prevalence of HCV infection around the world is generally between 0.4 and 2% (7-10), although a much higher level has been reported in Egypt (14%; see ref. 11). Therefore, HCV constitutes a major cause of chronic liver disease throughout the world. With the recent development of recombinant-based diagnostic assays for the detection of circulating HCV antibodies (2, 3), the risk of being infected with HCV after transfusion of blood or cellular components has been substantially reduced (12,13). However, community-acquired infection is much more common and occurs at various frequencies in high-risk groups such as i.v. drug users, health-care workers, and sexual and household contacts ofhepatitis patients, although -40%o of cases in the United States appear to have no known risk factor for acquisition of infection (14). Thus, the development of an HCV vaccine to prevent transmission within the community is highly desirable.HCV is distantly related genetically to both the pestiviruses and flaviviruses and, like these relatives, appears to process virion structural proteins from the N-terminal region of the polyprotein precursor encoded by the positivestranded RNA genome (15). The host signal peptidase mediates the cleavage of a basic, presumed nucleocapsid protein (C; -20 kDa) from the N terminus of the polyprotein precursor followed by two glycoproteins (El, glycoproteins (gp33 and gp72) under nondenaturing conditions from the endoplasmic reticulum. A fraction of the purified material was shown to exist in the form of a large E1/E2 oligomeric complex (22). We now report on the efficacy of this purified preparation in vaccinating chimpanzees against experimental infection with HCV-1. MATERIALS AND METHODSVaccine Preparation. A Stu I-Bgl II cDNA restriction fragment ofthe HCV-1 genome (nt 63 to +2901; aa 1-967; ref. 15) encoding the complete C (20 kDa), El (gp33 kDa), and E2 (gp72 kDa) proteins along with a C-terminally truncated NS2 product was cloned into the Sma I site of plasmid SC59 downstream of a hybrid early/late vaccinia promoter (S. Chakrabarti and B.M., unpublished work). BSC40 cells preinfected with wild-type WR vaccinia were transfected with the SC59 recombinant and thymidine kinase-negative recombinants, selected, and purified through three rounds ofplaque purification (23). Spinner cultures of HeLa cells (109 c...

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

“…Previously, GNA was used as an enrichment step for the isolation of recombinant E1E2 and E2 proteins (35,37,43). This method was also used successfully to obtain high-purity E1E2 for vaccination of human volunteers in a phase I HCV vaccine trial (38).…”

“…To date, virtually all successful viral vaccines have been based on the induction of nAbs, usually via the targeting of viral coat proteins (58). For HCV, 1a E1E2 was shown previously to reduce the carrier rate in immunized chimpanzees and generate sterilizing immunity in some individuals in response to homologous and heterologous 1a virus challenges (35,36,57). Furthermore, E1E2 is the only candidate vaccine to demonstrate broad nAb responses from vaccinated human volunteers (38,39,41,42).…”

“…However, we anticipate that the purification scheme for Fc-tagged rE1E2 will generate significantly better yields upon scale-up and offer several advantages for manufacturing. WT E1E2 has been isolated previously using the mannose-binding lectin GNA (35,37,43). GNA was originally described to have specificity for Man␣ (1, 3) and Man-containing oligosaccharides (47), and members of this family interact with viral glycoproteins (48).…”

“…The aim of the current study was to investigate a new method to purify E1E2 in an effort to improve upon the existing Galanthis nivalis lectin agarose (GNA)-based purification scheme thus facilitating vaccine production and global delivery (35,37,43). We were able to express and isolate a novel E1E2 antigen from an Fc-tagged precursor protein from Chinese hamster ovary (CHO) cells.…”

“…Sterilizing immunity was observed in some of these vaccinated animals (35)(36)(37). A phase I dose-ranging clinical trial was conducted on healthy human volunteers and demonstrated the induction of anti-E1E2 antibodies (determined by an enzyme immunoassay) and strong T-helper cell responses (38).…”