Mammalian cell expression and immunogenicity of gpE1/gpE2. Chiron Corporation, lead by M. Houghton, has extensively studied HCV vaccine candidates over the past decade . These studies have lead them to focus upon a recombinant gpE1/gpE2 heterodimer vaccine produced in CHO cells, which showed promising results in the chimpanzee model, and which is now being tested in a Phase I clinical trial. Challenge of immunized chimpanzees with homologous or heterologous HCV. Chimpanzees (Pan troglodytes) were immunized i.m. with 30-80 ug recombinant HCV-1 gpE1/gpE2 prepared from mammalian cells, along with MF75 adjuvant (an oil-water emulsion containing squalane and pluronic acid) and 100 ug of muramyl tripeptide. After HCV-H challenge, all but one cleared the ensuing acute viremia without progressing to the chronic, carrier state. Of 12 vaccinated chimpanzees challenged with homologous HCV-1, only 2 developed the chronic carrier state versus 7/10 in the control group. These data provide the first indication for the ability of an HCV vaccine to be effective against heterologous strains of the virus and provides encouragement for the development of at least a partially effective vaccine against .this important pathogen. These vaccinees produced antibodies to gpE2 that blocked the binding of recombinant gpE2 to human CD81, a cell surface receptor for HCV, and also effectively blocked the binding of infectious HCV-1 to CD81. These findings suggest that complete gpE1 and gpE2 assembled as heterodimer is required for effective elicitation of antibody that blocks binding to CD81. These studies strongly indicate the importance of anti-gpE2 antisera in the protection from infection or from progression to the chronic carrier state in the chimpanzee model. Although we recognize the importance of CTL responses for clearing HCV infection, it is clear that serum antibody against envelope protein contributes significantly to protection. Therefore, we believe that recombinant gpE1/gpE2 will be an important component of the ultimate vaccine that will be developed for human use. Expression of a viral glycoprotein in plant cells: AIV hemagglutinin. We tested the expression of four different constructs encoding the HA of AIV TK-Wi68 in tobacco suspension cultured cells to examine the use of plant cells for production of an oral vaccine against avian influenza for poultry. Our results showed the plant cell produced HA was correctly processed, assembled trimeric forms in vivo, and elicited virus-neutralizing antibodies in chickens. These data provide compelling evidence that plant cells can faithfully produce an animal virus envelope glycoprotein. Geminivirus-based gene amplification for enhanced expression in plants. We have developed a novel system for enhanced expression of foreign genes in plants using geminivirus elements to mediate controlled gene amplification . Transgenic geminiviral system. We constructed a regulated expression vector for Rep using an alcohol-inducible promoter of the alcA gene, , in order to control expression of the Rep protein in transgenic plants. We achieved significant enhancement of expression for an extended period (7 days). These data show a strong potential for the use of the BeYDV-derived gene amplification system to maximize foreign gene expression in transgenic plants. p19 viral suppressor of gene silencing. PTGS in plants can be inhibited using viral suppressors such as the tomato bushy stunt virus p19 protein . By transient co-expression of p19 with 3 different antigen expression cassettes in tobacco leaves, we have confirmed that RNA-mediated silencing is suppressed for up to 10 days post-transfection, with consequent multi-fold increases in antigen accumulation (data not shown). Since p19 is likely to be toxic if constitutively expressed in transgenic plants, we have now constructed an alcA-p19 cassette for alcohol-inducible expression.
|Diamos, Andrew G; Rosenthal, Sun H; Mason, Hugh S (2016) 5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves. Front Plant Sci 7:200|
|Chen, Qiang; He, Junyun; Phoolcharoen, Waranyoo et al. (2011) Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin 7:331-8|
|Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang et al. (2010) High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng 106:9-17|