Background: An HIV vaccine would certainly benefit from a potent neutralizing antibody response. However, no vaccine candidate has demonstrated the ability to induce broad neutralizing responses. Therefore, a deeper knowledge of neutralization is required. The mechanisms of antibody-mediated neutralization of HIV-1 remain unclear. For TCLA isolates, studies suggest that neutralization follows a multiple hit kinetic and occurs by steric hindrance, following Ab-coating of the viral particle. Here we have studied the stoichiometry of neutralization of the primary HIV-1 isolate JR-CSF by the broadly neutralizing anti-CD4 binding site antibody b12.

Methods: Pseudo-typed viruses bearing various proportions of wild type and escape mutant HIV-1 JR-CSF envelope were generated by transfection of 293 T-cells. The escape mutant envelope used contains a mutation in the C2 region that diminishes b12 binding. Viruses bearing such envelope are resistant to neutralization, in contrast to viruses bearing wild-type envelope. In order to study the stoichiometry of b12-mediated neutralization, the pseudo-typed viruses were studied for their sensitivity to neutralization by the antibody. Neutralization curves were generated for each chimeric virus population. Neutralization was assessed by measuring infection of U87-CCR5 target cells using a luciferase reporter gene.

Results: Our results demonstrate that neutralization of the HIV-1 primary isolate JR-CSF by the antibody b12 follow multiple hit kinetics and occurs when the number of gp120 molecules on the virus bound by antibody reaches a certain threshold. This threshold corresponds to about 1–2 antibody molecules bound per envelope trimer on the surface of the virion. The experiments were repeated using the monovalent antibody fragment Fab b12 in order to study the importance of bivalent binding in neutralization; similar results were obtained.

Conclusion: The results suggest that, similar to the mechanism proposed for antibody neutralization of TCLA isolates, neutralization of primary isolates is the result of antibody-coating of virions.