EFFECT OF HIV-1 PEPTIDE PRESENTATION ON THE AFFINITY CONSTANTS OF 2 MONOCLONAL-ANTIBODIES DETERMINED BY BIACORE(TM) TECHNOLOGY

We studied two monoclonal antibodies (MAbs 9-11 and 41-1) which are sp ecific for dominant and conserved epitopes located on HIV-1 transmembr ane Gp41. These MAbs recognize both Gp41 and a synthetic HIV-1 envelop e peptide (39GC) which is a fragment of Gp41. The interactions between MAbs 9-11 and 41-1 and 39GC either coupled to a sensor chip or to alk aline phosphatase were investigated using BIAcore(TM) technology. The association and dissociation rate constants as well as the affinity co nstants were determined. BIAcore(TM) technology allows real-time deter mination of the interaction between two molecules without the need for any labeling, neither isotopic nor enzymatic. The peptide 39GC was im mobilized by coupling to dextran on the BIAcore(TM) biosensor through a disulfide bond with a cysteine residue added to the N-terminus of th e synthetic peptide. The two native cysteine residues located in the l oop of Gp41 were protected by ethylcarbamoyl residues (CONHC2H,); this chemical modification prevented the formation of the S-S bridge and i n particular the internal loop. We specifically studied the interactio n between the MAbs and either the protected peptide or the peptide who se cysteine residues had been deprotected in situ by alkaline treatmen t. The results showed that MAb 41-1 recognized 39GC either protected ( Ka = 7.6 x 10(6) M(-1))or unprotected (Ka = 1.48 x 10(8) M(-1)), where as MAb 9-1 1 recognized only the unprotected form (Ka = 2.18 x 10(8) M (-1)), Our results suggest that the epitope MAb 9-11 is directed again st a part of the peptide sequence which includes the two native cystei nes. The difference in affinity observed for MAb 41-1 between the prot ected and the unprotected forms of 39GC was found to be due to a Lower rate of dissociation for unprotected 39GC; these results illustrate t he importance of peptide conformation on antibody recognition and migh t be explained by a conformational change due to reconstitution of the internal loop following deprotection of the thiol groups. MAbs 9-11 a nd 41-1 also recognized 39GC conjugated to alkaline phosphatase and de protected. We observed a difference between the rate constants for MAb 41-1 binding to free peptide and its binding to the peptide-enzyme co njugate which might be due to changes in peptide flexibility. In contr ast, the rate constants of MAb 9-11 were the same in both experiments, suggesting that the rigidity of the internal loop prevents changes in 9-11 epitope conformation.