Enhancement of α-Helicity in the HIV-1 Inhibitory Peptide DP178 Leads to an Increased Affinity for Human Monoclonal Antibody 2F5 but Does Not Elicit Neutralizing Responses in Vitro

Abstract

The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for ␣-helical conformation. We have examined DP178 in the context of a model for optimized ␣-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of ␣-helix present was localized to residues Glu 662 through Asn 671 , a region encompassing the 2F5 epitope. Using NH 2 -terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response.The HIV-1 1 GP160 envelope glycoprotein is synthesized as a single precursor that is cleaved by a cellular endoprotease to generate two noncovalently associated subunits, GP120 and GP41 (1). GP120 is the receptor-interacting constituent, which mediates sequential binding to the cellular CD4 receptor and CXCR4 or CCR5 chemokine co-receptors. The GP41 transmembrane subunit mediates fusion between viral and cellular membranes (reviewed in Refs. 2 and 3). Both envelope segments are highly immunogenic but antibodies raised by vaccination with full-length or subunit versions of these proteins, while capable of neutralizing homologous virus, are generally not protective against heterologous challenge (4 -6). In contrast, several broadly neutralizing human monoclonal antibodies (mAb) to diverse envelope epitopes have been identified in recent years. Among the most well characterized of these are 1b12 and 2G12, which bind to GP120 (7-9) and 2F5, which interacts with the COOH-terminal region of GP41 (10). MAb 2F5 has generated much interest because its epitope is well conserved across HIV clades and because of it...

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

“…2 A) but not by the fluorescein-specific scFv COLIN (data not shown), thus confirming the specificity. The D5 scFv also blocked HIV infection in a single-cycle infectivity assay (20) (Fig. 2B), indicating that D5 can inhibit HIV entry in multiple assay formats.…”

“…Measurement of HIV infection of p4-2͞R5 cells by using a chemiluminescent ␤-galactosidase substrate was done as described (20). BaL and HXB2 were purchased from Advanced Biotechnologies (Columbia, MD); 89.6 was grown in peripheral blood mononuclear cells, and vesicular stomatitis virus-G-pseudotyped HIV was made by transfection as described (21).…”

Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state

“…In addition to simple linear or structurally constrained peptide immunogens, recombinant protein immunogens including displays of constrained 2F5 epitope in the contexts of various protein scaffolds have been also pursued [28][29][30][31][32][33][34][35][36][37]. While inducing high antibody titers against the primary amino acid sequence of the epitope, these immunogens have failed to elicit NAbs against primary HIV-1.…”

Quaternary protein mimetics of gp41 elicit neutralizing antibodies against HIV fusion‐active intermediate state

“…10,20,[23][24][25][26] First, synthetic peptide antigens representing monomeric gp41 peptides are conformationally unstable. Since stability and bioactive conformation are intimately associated with trimeric quaternary structure, they are difficult to duplicate using monomeric peptides.…”

“…Consequently, attempts to exploit these peptides as immunogens, alone or with additional variable regions, have been unsuccessful. [23][24][25][26] Golding et al 10 and Louis et al 20 showed that antibodies gp41 Protein Mimetics Elicit Neutralizing Antibodies 321 generated against complexed peptides or engineered trimeric peptides based on the N-HR or C-HR domains possess viral neutralizing activity. These results suggest that targeting the bioactive conformations of the N-HR or C-HR domains is a viable vaccine design strategy.…”