DESIGN, SYNTHESIS, AND ANTIBODY BINDING STUDIES OF HIV-ASSOCIATED CARBOHYDRATE ANTIGENS
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The development of an effective prophylactic human immunodeficiency virus (HIV) vaccine is a critical global health priority. However, to-date, all efforts to design an HIV vaccine have been met with a paucity of success. The design of an effective HIV vaccine is challenging, however, the recent isolation of potent broadly neutralizing antibodies (bNAbs) capable of neutralization across multiple HIV strains suggests that a properly designed HIV immunogen could develop into an effective vaccine. The work presented here describes the synthesis of a series of HIV antigens for functional studies as well as potential immunogens. Five projects are described herein, probing both the role of carbohydrates in defining the epitopes of anti-HIV carbohydrate reactive proteins, as well as approaches to reconstitute these carbohydrate-based epitopes in a synthetic format. In Chapter 2, a series of oligomannose-virus-like particle (VLP) conjugates representing the bNAb 2G12 epitope were synthesized and used to probe the specific binding preferences of 2G12. The synthetic glycan-protein conjugates were highly antigenic toward 2G12. Chapter 3 describes a general method developed to chemoenzymatically synthesize differentially glycosylated HIV-related glycopeptides in a site-defined manner. The method was used to synthesize of series of glycopeptide antigens of the bNAb PG9. The method developed was applied in in Chapter 4 toward the synthesis of multivalently displayed glycopeptides on a VLP scaffold as PG9 immunogens. The multivalent glycopeptide display significantly enhanced the antigenicity compared to monomeric glycopeptides. In Chapter 5 the synthesis of an oligomannose library containing all intermediate oligomannose glycans Man1-9GlcNAc2 is described, including the associated glycan-protein conjugates. Preliminary binding studies against a panel of mannose-reactive lectins and anti-HIV bNAbs revealed binding preferences consistent with the given oligomannose display. Similarly, in Chapter 6, a library of highly-branched, bisected Galactose/N-acetylglucosamine terminal glycan-protein conjugates were synthesized. The conjugates were designed to present potential cryptic N-glycan HIV epitopes for use in serum screening studies to identify novel glycan binding proteins. The work presented has yielded important information regarding the reconstitution of HIV glyco-epitopes using synthetic protein conjugates. Together, these insights should facilitate the rational design of immunogens that are better able to mimic the native gp120 epitopes.