Synthesis of a trimeric gp120 epitope mimic conjugated to a T-helper peptide to improve antigenicity.

A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.

[Experimental study of candidate vaccines against variable or quasi-species pathogenes: multiepitopic synthetic peptide antigenes and new receptor-guiding adjuvants].

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

Chemoenzymatic Synthesis of HIV-1 Glycopeptide Antigens.

Glycosylation is one of the most common posttranslational modifications of proteins and can exert profound effects on the inherent properties and biological functions of a given protein. Structurally well-defined homogeneous glycopeptides are highly demanded for functional studies and biomedical applications. Various chemical and chemoenzymatic methods have been reported so far for synthesizing different N- and O-glycopeptides. Among them, the chemoenzymatic method based on an endoglycosidase-catalyzed ligation of free N-glycans and GlcNAc-tagged peptides is emerging as a highly efficient method for constructing large complex N-glycopeptides. This chemoenzymatic approach consists of two key steps. The first step is to prepare the GlcNAc peptide through automated solid-phase peptide synthesis (SPPS) by incorporating an Asn-linked GlcNAc moiety at a predetermined glycosylation site; and the second step is to transfer an N-glycan from the corresponding N-glycan oxazoline en bloc to the GlcNAc peptide by an endoglycosidase or its efficient glycosynthase mutant. In this chapter, we provide detailed procedures of this chemoenzymatic method by demonstrating the synthesis of two HIV-1 V3 glycopeptide antigens carrying a high-mannose-type and a complex-type N-glycan, respectively. The described procedures should be generally applicable for the synthesis of other biologically important N-glycopeptides.

HIV-1 peptide vaccine candidates: selecting constrained V3 peptides with highest affinity to antibody 447-52D.

The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.

Mother to child transmission of HIV-1 in a Thai population: role of virus characteristics and maternal humoral immune response.

The objective of this study was to investigate factors influencing mother to child transmission of HIV-1 in Thailand, where HIV-1 CRF01_AE, the major subtype in Southeast Asia, predominates. Samples from 84 HIV-1 infected, anti-retroviral treatment-naïve, non-breast feeding mothers, 28 who transmitted HIV-1 to their babies (transmitters) and 56 who did not (non-transmitters), were studied for maternal humoral immune response and virus characteristics. Maternal humoral immune response was measured by lymphocyte phenotyping; neutralizing antibodies to laboratory HIV-1 MN strain and two clinical isolates; peptide binding antibody to gp41 and V3 from strains CRF01_AE, B, and MN; autologous antibodies; and quasispecies diversity. Virus characteristics studied were viral load, co-receptor usage, and viral replication capacity. No significant difference between transmitters and non-transmitters was found for any parameter of maternal humoral immune response. However, viral load and viral replication capacity were significantly higher in transmitters versus non-transmitters and were not correlated with each other. This suggests that viral replication capacity may be a transmission factor independent of viral load, which is already well established as a risk factor for transmission of HIV-1. All except four viral isolates used the CCR5 co-receptor. This is one of few studies of vertical transmission in a population where HIV-1 CRF01_AE predominates. The data suggest that in this population the maternal humoral immune response was not important in preventing transmission at parturition, but that virus characteristics were key factors, and that viral replication capacity may contribute to birth-associated mother to child transmission of HIV-1.

Synthetic multivalent V3 glycopeptides display enhanced recognition by glycan-dependent HIV-1 broadly neutralizing antibodies.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.