Implication For Mechanism Of BIV-mediated Cell Fusion & HCV Vaccine Strategy Research Based On The Attenuated Vaccinia Virus

BIV/rabbit system is a promising small-animal model for HIV infection and persistence. However, despite its importance in basic and clinical research, thus far little is known about BIV and its infection. In the present study, using biochemical and biophysical technologies, we have determined and characterized two heptad repeat (HR) regions in BIV Env (only one heptad repeat region was predicted by program because of the low likelihood of BIV Env with those of other lentiviruses, including HIV, SIV and FIV etc) and have proved that the predicted HRs of BIV Env can form a stable trimer of hairpins or six-helix bundle just like those formed by other lentiviruses in the viral membrane fusion process. We have also demonstrated the inhibitory activity of BIV HR2 peptide in the virus-mediated cell fusion; this very activity has been proved to be therapeutically significant and has already led to the discovery of an antiviral drug for AIDS. This study is the first step of our effort to reveal the largely unknown mechanisms of BIV pathology and to bridge researches on BIV and HIV. As we are convinced, it will shed light on study of the fatal disease of AIDS.HCV is a major etiologic agent of hepatocellular carcinoma and there is an urgent need to develop an effective prophylactic and therapeutic vaccines against HCV infection. My research has been focused on the construction and characterization of HCV vaccines based on the modified vaccinia viral vectors including replication-competent modified vaccinia TianTan (MVTT) strain and replication-incompetent modified vaccine Ankara (MVA). The former has been used in millions of Chinese during the small pox eradication. Since MVTT and MVA have distinct replication capacity, we have been studying whether such difference will translate into different levels of immunogenicity in the immunized hosts.Since HCV genotype 1b is dominant worldwide, we have generated a consensus and codon-optimized HCV-1b full-length envelope sequence (COP-HCV-1b-E1E2) based on the published sequences in Genbank, using computer modeling and overlapping PCR techniques. We have found that COP-HCV-1b-E1E2 glycoprotein was expressed in transfected 293 cells at a higher level compared to unoptimized version. The pseudo typed virus bearing COP-HCV-1b-E1E2 glycoprotein in the HIV-1 backbone was able to infect the hepatic cell line Huh7.5 and sensitive to neutralization by HCV-1b E2-specific monoclonal antibody 3/11. To study the immunogenicity of COP-HCV-1b-E1E2, we constructed two recombinant vaccinia viruses, one based on the MVTT and the other on the MVA. The two recombinant vaccinia viruses MVTT-COP-E1E2 and MVA-COP-E1E2 were used to immunize Blab/c mice and rabbits via the intramuscular route. Both recombination vaccinia viruses were able to induce high levels of binding and neutralizing antibodies against the pseudo typed virus bearing autologous or heterologous natural-occurring HCV-1b glycoprotein. It is hoped that the optimized and functional consensus COP-HCV-1b-E1E2 gene delivered by MVTT or MVA will become promising therapeutic as well as prophylactic vaccine candidates against a broad range of HCV infection.