Effect of HIV-1 peptide presentation on the affinity constants of two monoclonal antibodies determined by BIAcore technology.
We studied two monoclonal antibodies (MAbs 9-11 and 41-1) which are specific for dominant and conserved epitopes located on HIV-1 transmembrane Gp41. These MAbs recognize both Gp41 and a synthetic HIV-1 envelope 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 alkaline phosphatase were investigated using BIAcore technology. The association and dissociation rate constants as well as the affinity constants were determined. BIAcore technology allows real-time determination of the interaction between two molecules without the need for any labeling, neither isotopic nor enzymatic. The peptide 39GC was immobilized by coupling to dextran on the BIAcore biosensor through a disulfide bond with a cysteine residue added to the N-terminus of the synthetic peptide. The two native cysteine residues located in the loop of Gp41 were protected by ethylcarbamoyl residues (CONHC2H5); this chemical modification prevented the formation of the S-S bridge and in particular the internal loop. We specifically studied the interaction between the MAbs and either the protected peptide or the peptide whose cysteine residues had been deprotected in situ by alkaline treatment. 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), whereas MAb 9-11 recognized only the unprotected form (Ka = 2.18 x 10(8) M-1). Our results suggest that the epitope MAb 9-11 is directed against a part of the peptide sequence which includes the two native cysteines. The difference in affinity observed for MAb 41-1 between the protected and the unprotected forms of 39GC was found to be due to a lower rate of dissociation for unprotected 39GC; these results illustrate the importance of peptide conformation on antibody recognition and might be explained by a conformational change due to reconstitution of the internal loop following deprotection of the thiol groups. MAbs 9-11 and 41-1 also recognized 39GC conjugated to alkaline phosphatase and deprotected. We observed a difference between the rate constants for MAb 41-1 binding to free peptide and its binding to the peptide-enzyme conjugate which might be due to changes in peptide flexibility. In contrast, 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.
Mani
J C
JC
CNRS UMR 9921, Faculté de Pharmacie, Montpellier, France.
Marchi
V
V
Cucurou
C
C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015374:Biosensing Techniques; D000939:Epitopes; D015483:HIV Antibodies / Q000032:analysis*; D015700:HIV Envelope Protein gp41 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D007118:Immunoassay / Q000379:methods*; D016056:Immunodominant Epitopes / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; C087253:HIV envelope protein gp41 (584-609); D016056:Immunodominant Epitopes; D010446:Peptide Fragments