Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K D ؍ 30 nM), close to its IC 50 concentration (ϳ3 nM) but much weaker than the affinity of a related inhibitory C-peptide C37 (K D ؍ 0.0007 nM). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF 3 ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the 0.75 nM). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.
The immunogenicity of adenovirus (Ad) vectors is enhanced by virus-specific memory immune responses present in most individuals as a result of past exposure to these ubiquitous pathogens. We previously identified the first human T-cell epitope from the major capsid protein hexon, H910-924, and found that it is highly conserved among different Ad serotypes. Memory/effector T-cell responses to H910-924 were detected in 14 of 18 (78%) healthy adults by an interferon-g ELISPOT assay. Hexon peptide-specific CD4 Tcell lines were generated from three HLA-typed donors and analyzed using a panel of HLA homozygous B-cell lines and monoclonal antibodies to HLA class II loci. These studies reveal that the hexon epitope is restricted by HLA DP4, a class II allele present in 75% of the population. Analysis of overlapping peptides and peptides with single residue mutations identified a HLA DP4-binding motif. Additionally, antibodies to the hexon peptide were detected in all donor sera by dot blot assay and ELISA. Therefore, most individuals exhibit both memory B-and T-cell responses to this highly conserved epitope on hexon, an obligate component of all Ad vectors, including 'gutted' vectors. These data suggest that current strategies for the use of Ad gene therapy vectors will not evade memory immune responses to Ad.
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