UC Santa Cruz

Over three decades have passed since Human Immunodeficiency Virus (HIV) was identified as the causative agent of AIDS. With committed distribution and use of antiretroviral therapy, the AIDS crisis ebbed to a point where it is no longer a major concern for a majority in first world countries. However, for the select regions in the world where the disease endures as a major public health concern, there remains considerable incentive to develop a safe, effective vaccine against HIV. The RV144 HIV vaccine clinical trial completed in 2009 has been the only study to show protection against HIV infection. However, the modest efficacy (31.2%, P=0.04) indicated that the vaccine design used in the trial would need to be improved for higher efficacy before widespread use. Since the results of the RV144 clinical trial, various lines of research have elucidated methods that could be explored to improve vaccine efficacy. Follow-up studies of the RV144 trial indicated that an antibody response against regions in the HIV envelope protein (gp120) variable domains (V1V2 and V3) inversely correlated to infection. The identification of broadly neutralizing antibodies (bNAbs) against gp120 was a welcome discovery providing evidence that a human antibody response could effectively neutralize a vast majority of HIV strains. Perhaps most surprising was the observation that multiple families of bNAbs targeting regions in the V1V2 and V3 domains bound oligo-mannose N-linked glycans on gp120. This observation was particularly relevant, as the original gp120 immunogens of the RV144 trial displayed predominantly sialic acid-terminal N-linked glycans. The work in this dissertation describes three major lines of research that converge upon improving upon the efficacy of gp120 based HIV vaccines. First, the identification of a monoclonal antibody that binds a glycan-dependent antibody within the V1V2 domain suggests that antibodies to such epitopes can be raised by immunization with gp120 molecules, providing further support for the viability of a gp120 based vaccine design. Secondly, we show that the addition of key, conserved glycosylation sites to the A244 and MN rgp120 vaccine immunogens of the RV144 clinical trial, in conjunction with modification of the incorporated glycoform, results in new binding by multiple families of bNAbs previously thought not to bind monomeric gp120 proteins. We further built upon the development of glycan-optimized gp120 immunogens by showing that a CHO-derived cell line, in which the Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase had been inactivated, could be stably transfected to produce glycan-modified clade C rgp120 immunogen. The resulting immunogen would be more amenable for translation to large-scale production, thereby making relevant gp120 based immunogens more accessible for widespread investigation. Finally, we investigated the potential for glycosylated V1V2 and V3 based gp120 protein fragments to improve the magnitude of the antibody response to regions associated with protection in the RV144 trial. Cumulatively, these studies contribute both potential reagents for, as well as a deeper understanding of, a second-generation of gp120 based vaccines for use in clinical trials.