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 v irus type 2 (HIV-2) contains 22 cysteine residues. The positions of th e 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 con taining from 13 to 47 amino acids were synthesized by the solid-phase method according to 9-fluorenylmethoxycarbonyl strategy, except for pe ptide 5, which was assembled according to t-butoxycarbonyl (Boc) strat egy. Analysis of all the crude peptides showed that the expected pepti des 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%. Fo r 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 w ere purified by HPLC and characterized by fast atom bombardment mass s pectrometry. This analysis showed that a small quantity (<10%) of dime ric peptides (2 and 8) and cyclic peptides containing oxidized methion ine or tryptophan residues (4, 9 and 10) were formed. To assess the re levance of conformation for the antigenicity of disulfide-bonded loops of HIV-2 gp125, the antigenicity of linear and cyclic peptides was te sted against a set of 76 HIV-2 positive human sera by enzyme-linked im munosorbent assay. Peptides 2, 4 and 9, mimicking the V1, V2 and V3 re gions of the external envelope glycoprotein (gp 125) of HIV-2, were th e most highly reactive with HIV-2 positive human sera tested at the di lution of 1:50. Cyclic peptides generally were recognized more than li near peptides, as shown by their greater inhibition (2 to 10 times mor e) of antigen-antibody complexes. Structure-antigenicity of peptide V3 , the most reactive peptide (75% of the HIV-2 positive sera tested), w as analyzed further. Cyclic peptide 9C had a higher affinity for anti- gp125 antibodies than linear peptide 9L. In addition, circular dichroi sm showed that linear and cyclic peptides 9 had a similar structure, b ut when analyzed in aqueous solution or in trifluoroethanol (TFE), the structural difference shown with antibodies was not confirmed. No sig nificant difference was observed between the antigenicity of linear an d 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 s era. This result agrees with the low immunogenicity of conserved regio ns. (C) Munksgaard 1998.