Browsing by Author "Toonstra, Christian"
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Item Chemoenzymatic Synthesis and Antibody Binding of HIV-1 V1/V2 Glycopeptide-Bacteriophage Qβ Conjugates as a Vaccine Candidate(MDPI, 2021-11-21) Zong, Guanghui; Toonstra, Christian; Yang, Qiang; Zhang, Roushu; Wang, Lai-XiThe broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qβ conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qβ), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qβ-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qβ conjugates synthesized represent a promising candidate of HIV-1 vaccine.Item DESIGN, SYNTHESIS, AND ANTIBODY BINDING STUDIES OF HIV-ASSOCIATED CARBOHYDRATE ANTIGENS(2016) Toonstra, Christian; Wang, Lai-Xi; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)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.