Stabilization of Gp120 Outer Domain for Hiv-1-1 Vaccine Immunogen Design

The sole envelope glycoprotein spike on the surface of Human Immunodeficiency Virus (HIV-1) is composed of two subunits: gp120 and gp41. The gp120 consists of three domains: the inner domain, outer domain and bridging sheet, to which the viral primary receptor CD4 binds and induces a conformational change in gp120 to expose the co-receptor binding site. Previous studies have found that the outer domain of gp120 is relatively more stable than the inner domain and bridging sheet. When superimposed, the co-crystal structures of CD4-gp120 complex and VRC01-gp120 complex revealed that the CD4-binding site (CD4-BS) antibody VRC01 actually mainly bound to the outer domain of the gp120. The outer domain (designed OD1) of gp120 has been considered as an immunogen candidate but to be ineffective, suggesting that structural-based modifications to the outer domain are required for designing an effective vaccine immunogen. Previous studies in our laboratory have introduced two disulfide bonds to stabilize the outer domain structure and named it OD2, which significantly improved the immunogenicity when compared to the wild-type outer domain (OD1). In this project, based on the OD2 set of experiment, an additional important mutation from serine to tryptophan at position 375 (S375W) in the CD4-binding cavity was introduced to further stabilize the outer domain, which was named OD3. The OD3 immunogen was expressed and purified in E. coli. Subsequently, the purified OD3 immunogen was characterized in vitro and its immunogenicity was tested in guinea pigs. In comparison to OD1 and OD2, the results demonstrate the antibody titers and neutralizing activity were clearly higher in OD3. It is suggested that the S/375W mutation stabilized the outer domain and further improved the outer domain (OD) immunogenicity. In conclusion, this research has used a structure-based approach to design HIV-1-1 vaccine immunogens based on the gp120 outer domain (OD). ii ACKNOWLEDGEMENT I give my sincere thanks to my advisor, Dr. Shi-hua Xiang for the academic training under his guidance, and I also thank him for giving me the opportunity to be involved in projects of different specializations. I thank my four Committee members during my three academic years. I would like to thank Dr. Mark Wilson for the suggestions he made during protein purification and crystallization. I would like to thank Dr. Peter C. Angeletti for his viral evolution course, and for his kindness allowing me to have access to his lab at any time during my work with protein purification. …