HIV-1 V3-SPECIFIC NEUTRALIZATION: VALENCY, REVERSIBILITY, AND THE STATE OF THE VIRION DETERMINE IN VITRO EFFICACY

Abstract

Bimolecular characteristics such as valency, kinetics, and the reversibility of the reactions between immunoglobulin, the virus, and the target cell are necessary for the neutralization reaction of HIV-1 to be biologically effective, especially in an efficacious vaccine induced humoral immune response. This thesis investigates these parameters by studying the in vitro ability of IgG or its Fab fragments to neutralize HIV-1. Quantitative linear syncytium-forming infectivity assays, SDS PAGE, amino acid analysis, centrifugation equilibrium dissociation studies, and gp120 ELISA were used to collect data on studies of two highly purified and quantified neutralizing murine V3 specific monoclonal antibodies (MoAb) and their Fabs (0.5B, 50.1) on two homologous strains of HIV-1 (HX-10, MN) and one closely related HIV-1 strain (IXB-3). The variety of neutralizing activities observed were dependent on viral strain, the state of the virus (i.e. cell-free or cell-associated), and valency of the antibody. Virus could be neutralized by Fab alone, and in some cases, virus (HIV-603_3/0.5B) was neutralized by Fab or IgG (0.5B) equally well. Bivalency of neutralization by IgG was also dependent on the state of the virion. In one case (HIV-1) an increase in blocking activity (ratio) of 100x was observed when virus was cell-associated rather than cell-free. Conversely, HIV-1110 demonstrated a 43x greater blocking ratio when the cell-free state was compared to the cell-associated state of the virion. Despite these differences, concentrations of IgG, 10-8 M (HIV-1), 10-7 M (HIV- 11ix-10) , and in one case (HIV-110/0.5B Fab) at 10-6 M, were to result in an irreversible blocking of infectivity. This loss of infectivity was not due to IgG or Fab-induced shedding of gp120. When virus (HIV-603_3) was allowed to decay (16 Hr), a transient shift in cell-associated blocking ratios for IgG (0.513) was observed and is in agreement with previous findings for sCD4. However, at high concentrations of soluble gp120 together with low molar concentrations of IgG/Fab in the decayed viral stocks, competition/interference with neutralization was observed. In one case (HX6-3/0.50 IgG) during increasing molar titrations, a 4x decrease in the blocking ratios was observed. This result appears to be a demonstration of IgG-induced stearic inhibition on the viral surface. In general, all these findings point to a structural barrier to V3-specific IgG mediated neutralization of HIV-1. These types of analysis should have an important application in future in vitro studies designed to evaluate and predict in vivo efficaciousness.