Background:  The mechanisms of antibody-mediated neutralization of HIV-1 remain unclear. Studies suggest that neutralization of T cell line adapted isolates follows a multiple hit kinetic and occurs by steric hindrance, following antibody-coating of the viral particle. We  studied the neutralization by anti-gp120 monoclonal antibodies of the R5 primary HIV-1 isolates JR-CSF and ADA.

Methods:  Using the ability of HIV-1 to generate mixed envelope oligomers when different envelope genes are expressed within the same cell, we generated pseudo-typed viruses bearing various proportions of wild type and monoclonal antibody-escape mutant HIV-1 envelope. The escape mutant envelopes contained mutations either in the V2 and C3, V3 or V4 regions, conferring resistance to neutralization by the anti-CD4 binding site monoclonal antidoby b12, the anti-V3 loop monoclonal antibody 447-52D and the anti-mannose residues  monoclonal antibody 2G12, respectively. To study the stoichiometry of neutralization, the chimeric pseudo-typed viruses were analyzed for their sensitivity to neutralization by the  monoclonal antibodies. Viral inhibition was assessed by measuring infection of U87-CCR5 target cells using a luciferase reporter gene. The amount of replication at the highest  monoclonal antibody concentration across different proportions of wild type and mutant gp120 was analyzed using a simple power-law model fitted by nonlinear least squares.

Results:   Our results show that the binding of one antibody molecule onto one gp120 within a trimer is sufficient to achieve neutralization of the envelope spike, as suggested by the absence of a lag phase in the neutralization curve. However, at the level of the viral particle, our results demonstrate that stoichiometry of neutralization of the HIV-1 primary isolates JR-CSF and ADA varies among antibodies. In order of incremental to few hit stoichiometry, the ranking is 2G12 < 447-52D < Fab b12, b12. Furthermore, the model suggests that variations among  monoclonal antibodies may arise because of differences in the rate at which unbound trimers encounter CD4/co-receptor complexes on the cell surface.

Conclusions:  Results suggest that the stoichiometry of neutralization of HIV-1 primary isolates follows a multiple hit kinetic, is incremental, and may result not only directly from  monoclonal antibody-mediated blocking of bound envelope trimers, but also indirectly, from decreasing the rate of productive interaction with the target cell. Different stoichiometries may arise from different modifications by the  monoclonal antibody to the rate of productive virus-cell interaction.

Keywords: Stoichiometry; Antibodies; HIV-1 primary isolates