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Annual and Interim Progress Report Summaries

Principal Investigator: Shan Lu

Project: Creating a Better Envelope

 
Submitted August 31, 2012

In Year 1 of this project, two forms of HIV-1 Env gp120 proteins with distinct N-linked glycosylation profiles (Man5 and Man8) were generated by Merck using glycoengineered Pichia pastoris strains GFI 2.0 and GFI 1.0, respectively. Extensive biochemical and biophysical analyses were conducted. Glycan analysis confirmed that the respective strains produced HIV-1 Env gp120 proteins with the predicted glycan profiles. Analytical size-exclusion chromatography with multi-angle laser light scattering method (SEC-MALLS) indicated that a range of oligomeric species exist for gp120 produced in either strain, but purification processes can fractionate product into monomeric-enriched and higher order oligomers. Also, ELISA and Fortebio assays confirmed that most antigenic epitopes are preserved except N-glycan-dependent ones.

Two pilot rabbit immunization studies (R-MK-01 and R-MK-02) have been performed to assess overall immunogenicity of high mannose gp120 protein antigens. Man5 gp120 protein and complex glycan gp120 protein produced from Chinese Hamster Ovary (CHO) cells were tested for their relative immunogenicity. The effect of adjuvants, Alum and ISCOMATRIX®, was also examined. Man5 gp120 protein was shown to be highly immunogenic, when compared with those derived from the CHO cell expression system. However, the epitope profiles were not identical between rabbit sera elicited by Man5 and CHO gp120 proteins. Neutralizing antibodies were characterized using either 293T-derived pseudoviruses or modified pseudoviruses expressing high mannose Env antigens. Preliminary data indicated that, indeed, glycosylation could impact the levels of neutralizing antibody (NAb) responses. Purified rabbit IgG derived from immunized rabbit sera could recognize specific types of glycan antigens in a glycan array screen.

In collaboration with Loyola University, one rabbit immunized with the Man5 gp120 protein antigen was tested for its ability to produce rabbit monoclonal antibodies that can be used as tools to understand the unique antibody profiles associated with each gp120 with its own glycan pattern.

 
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