Rapid analysis of epitope-paratope interactions between HIV-1 and a 17-amino-acid neutralizing microantibody by electrospray ionization mass spectrometry.
Progress in therapeutic or prophylactic immune intervention in HIV-1 infections may only come about with a detailed understanding at the molecular/atomic level of how antibodies neutralize (inactivate) virus infectivity. Currently information on the molecular aspects of antibody-virus interaction comes predominantly from X-ray crystallography, a process that is dependent on the production of suitable crystals. NMR can also be valuable but is complex and time consuming, while mass spectrometry has been limited to matrix-assisted laser-desorption ionization (MALDI) analysis of peptides eluted from the cognate antibody. Here, we have used electrospray ionization mass spectrometry (ESI-MS) to detect directly the interactions of a novel 17-amino-acid microantibody (MicroAb) that has HIV-1-inhibitory activity, and peptides representing the V3 regions of primary HIV-1 strains isolated from Brazil (clade B) and Africa (clade A). The MicroAb is based on the third complementarity-determining region of the heavy chain (CDR-H3) of a murine monoclonal IGGI (F58) specific for the V3 loop of the gp120 envelope glycoprotein of HIV-1. ESI-MS proved to be rapid (taking < 3 h for the entire analysis), sensitive (analytes at 2 mmol/ml), and accurate (RMM estimation to 0.01-0.1%). With it, we showed that the MicroAb forms complexes with the V3 peptides, implying that its antiviral activity is mediated by binding directly to the virus particle. In addition, through controlled protease digestion of the V3 peptides, we concluded that the CDR-H3 MicroAb bound to RKXXXIGPGR, a region similar to the epitope of the whole IgG as determined by ELISA. We believe that the approach exemplified here will be applicable generally to the identification of groups involved in receptor-ligand interactions.
Millar
A L
AL
Department of Biological Sciences, University of Warwick, Coventry, UK.
Jackson
N A
NA
Dalton
H
H
Jennings
K R
KR
Levi
M
M
Wahren
B
B
Dimmock
N J
NJ
D000595:Amino Acid Sequence; D000939:Epitopes / Q000276:immunology*; D015483:HIV Antibodies / Q000737:chemistry / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology; D015497:HIV-1 / Q000276:immunology* / Q000472:pathogenicity; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology
D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments