Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop.
BACKGROUND
Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain.


OBJECTIVE
To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro.


METHODS
A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed.


RESULTS
We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro.


CONCLUSIONS
This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.
Duarte
C A
CA
Vaccine Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba. inmdiag@ingen.cigb.edu.cu
Pérez
L
L
Vázquez
J
J
Dueñas
M
M
Vilarrubia
O L
OL
Navea
L
L
Valdés
R
R
Reyes
O
O
Montero
M
M
Ayala
M
M
Gavilondo
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D018604:Epitope Mapping; D015488:HIV Antigens / Q000235:genetics / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004279:DNA, Viral; D015488:HIV Antigens; D015699:HIV Envelope Protein gp120; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D010455:Peptides