Antigenicity of linear and cyclic peptides mimicking the disulfide loops in HIV-2 envelope glycoprotein: synthesis, reoxidation and purification.
The external envelope glycoprotein (gp125) of human immunodeficiency virus type 2 (HIV-2) contains 22 cysteine residues. The positions of the 11 disulfide bridges in HIV-2 gp125 were determined by analogy with the experimental position of the disulfide bonds found in the gp120 of HIV-1. Peptides expected to mimic all 11 disulfide-bonded domains containing from 13 to 47 amino acids were synthesized by the solid-phase method according to 9-fluorenylmethoxycarbonyl strategy, except for peptide 5, which was assembled according to t-butoxycarbonyl (Boc) strategy. Analysis of all the crude peptides showed that the expected peptides were obtained with good yields, between 75% and 85%. Peptides were purified further by high-performance liquid chromatography (HPLC) on an Aquapore RPC30 C8 column. Peptide homogeneity was more than 90%. For each peptide, linear peptides (L) were SH-iodoacetamidated, whereas cyclization of peptides (C) was performed by air oxidation. Oxidation kinetics was followed with the Ellman test and HPLC. Cyclic peptides were purified by HPLC and characterized by fast atom bombardment mass spectrometry. This analysis showed that a small quantity (<10%) of dimeric peptides (2 and 8) and cyclic peptides containing oxidized methionine or tryptophan residues (4, 9 and 10) were formed. To assess the relevance of conformation for the antigenicity of disulfide-bonded loops of HIV-2 gp125, the antigenicity of linear and cyclic peptides was tested against a set of 76 HIV-2 positive human sera by enzyme-linked immunosorbent assay. Peptides 2, 4 and 9, mimicking the V1, V2 and V3 regions of the external envelope glycoprotein (gp 125) of HIV-2, were the most highly reactive with HIV-2 positive human sera tested at the dilution of 1:50. Cyclic peptides generally were recognized more than linear peptides, as shown by their greater inhibition (2 to 10 times more) of antigen-antibody complexes. Structure-antigenicity of peptide V3, the most reactive peptide (75% of the HIV-2 positive sera tested), was analyzed further. Cyclic peptide 9C had a higher affinity for anti-gp125 antibodies than linear peptide 9L. In addition, circular dichroism showed that linear and cyclic peptides 9 had a similar structure, but when analyzed in aqueous solution or in trifluoroethanol (TFE), the structural difference shown with antibodies was not confirmed. No significant difference was observed between the antigenicity of linear and cyclic peptides 1, 8 and 11, mimicking the C1, C2 and C4 regions of HIV-1 gp125. These peptides were weakly reactive with HIV-2 positive sera. This result agrees with the low immunogenicity of conserved regions.
Belhadj Jrad
B
B
Laboratoire d'Immuno-Virologie, Université Paul Sabatier, Toulouse, France.
Bahraoui
E
E
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D015686:Gene Products, env / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000276:immunology*; D054299:env Gene Products, Human Immunodeficiency Virus
D000956:Antigens, Viral; D015686:Gene Products, env; D010455:Peptides; D010456:Peptides, Cyclic; D011498:Protein Precursors; D054299:env Gene Products, Human Immunodeficiency Virus; C066316:gp125 envelope protein, Human immunodeficiency virus 2