Lu: Creating a Better Envelope
HIV-1 envelope (Env) plays a central role in virus entry into permissive cells, which serve as the host for viral propagation and as the prime target for protective antibody responses. The partial success of the RV144 trial has generated considerable excitement around "prime boost" strategies for HIV vaccines, which include a recombinant Env protein immunogen, thereby, inspiring an increased demand for high quality Env proteins as a key component of HIV vaccine development. The production of Env proteins in large amounts in mammalian cells is a very costly process. Furthermore, it is challenging to make Env proteins with correct glycosylation patterns and conformational structure, two features thought to be necessary to elicit high quality antibodies and protective immunity.
There are two major roadblocks for the use of recombinant Env proteins as part of HIV vaccines: 1) it is not clear how different patterns of glycosylation will influence the immunogenicity of Env and 2) the high cost of Env production in traditional mammalian cell lines greatly reduces the opportunity to quickly evaluate and implement any HIV vaccine that contains a recombinant Env protein component. The goal of this proposed project is to produce high quality and low cost recombinant HIV-1 Env proteins with optimized N-glycans.
Seeking to take advantage of technology that enables yeast (Pichia pastoris) to modify proteins such that they possess one unique glycosylation pattern, Dr. Shan Lu (in collaboration with Merck GlycoFi, developers of the technology) proposes to generate three Env proteins that each have a unique and homogenous glycosylation pattern to assess the impact of specific glycosylation patterns on the immunogenicity of HIV-1 Env immunogens.
Although high risk, a proof of concept study showing that a correctly folded Env protein from humanized yeast cells with one of the aforementioned glycosylation patterns induces a humoral immune response comprised of high quality protective antibodies in rabbits will be transformative. The results could potentially be rapidly translated into pre-clinical vaccine trials. Further, "humanized" yeast that can make properly folded and glycosylated Env proteins in a cost effective manner could accelerate the development of more prime-boost HIV vaccines.
1. To understand the structural and biochemical features of Env with modified N-glycans
2. To conduct immunogenicity studies in small animal models
3. To characterize the level and quality of antibodies elicited by Env with modified N-glycans.
Six HIV-1 Env gp120 proteins with distinct N-linked glycan structures have been generated by Merck GlycoFi using four glycoengineered
Pichia pastoris strains. The Env proteins were purified to sufficient homogeneity to support analyses both
in vitro and
in vivo. Extensive biochemical and biophysical analyses of these novel gp120 proteins have been conducted. Glycan analysis confirmed that the respective strains produced HIV-1 Env gp120 proteins with high level homogeneity of predicted glycan profiles. Assays have been established for characterizing the antigenicity of these novel gp120 proteins by well-characterized human mAbs.
Rabbit immunization studies have tested the overall immunogenicity of two high mannose gp120 protein antigens produced from the above system in addition to the complex glycan gp120 protein produced from Chinese Hamster Ovary (CHO) cells as the control. The effect of adjuvant was also examined. The quantity and quality of antibodies elicited by modified Env vaccines were evaluated through a panel of diverse assays. Results show that recombinant gp120 proteins produced from humanized yeast cells are immunogenic and elicit gp120-specific antibody responses. Most significantly, sera elicited by the high mannose gp120 protein immunogens revealed an increased diversity of epitopes than the sera elicited by the traditional gp120 proteins produced in CHO cells.
Data from these studies confirmed the original hypothesis that glycan patterns directly influence the antigenicity and immunogenicity of Env immunogens, and such findings provide useful information in the selection of optimized gp120 immunogens for HIV vaccine development.