Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers

Abstract

Developing an immunogen that elicits broadly neutralizing antibodies (bNAbs) is an elusive but important goal of HIV vaccine research, especially after the recent failure of the leading T cell based HIV vaccine in human efficacy trials. Even if such an immunogen can be developed, most animal model studies indicate that high serum neutralizing concentrations of bNAbs are required to provide significant benefit in typical protection experiments. One possible exception is provided by the anti-glycan bNAb 2G12, which has been reported to protect macaques against CXCR4-using SHIV challenge at relatively low serum neutralizing titers. Here, we investigated the ability of 2G12 administered intravenously (i.v.) to protect against vaginal challenge of rhesus macaques with the CCR5-using SHIV(SF162P3). The results show that, at 2G12 serum neutralizing titers of the order of 1:1 (IC(90)), 3/5 antibody-treated animals were protected with sterilizing immunity, i.e. no detectable virus replication following challenge; one animal showed a delayed and lowered primary viremia and the other animal showed a course of infection similar to 4 control animals. This result contrasts strongly with the typically high titers observed for protection by other neutralizing antibodies, including the bNAb b12. We compared b12 and 2G12 for characteristics that might explain the differences in protective ability relative to neutralizing activity. We found no evidence to suggest that 2G12 transudation to the vaginal surface was significantly superior to b12. We also observed that the ability of 2G12 to inhibit virus replication in target cells through antibody-mediated effector cell activity in vitro was equivalent or inferior to b12. The results raise the possibility that some epitopes on HIV may be better vaccine targets than others and support targeting the glycan shield of the envelope.

Figure 1. Plasma viral loads following SHIV_SF162P3 vaginal challenge of 2G12-treated and control macaques.

A total of nine female Indian rhesus macaques were divided into treatment groups of five animals for i.v. administration of 2G12, two animals to receive the isotype control (Dengue anti-NS1, DEN3), and two additional controls were challenged prior to the beginning of the protection study to confirm viral fitness, but were not treated with antibody. In (A) two 2G12-treated (40 mg/kg) animals became infected: 90154 reached peak viremia of 2×10⁷ on day 21 similar to controls; 95113 showed a one-week delay of infection onset and peak viremia was lower at 5×10⁶. The remaining three 2G12-treated animals were protected against infection and showed no measurable viremia. In (B) all 4 control animals experienced peak viremia between 1×10⁷ and 4×10⁷ on day 21. The quantity of SIV viral RNA genomic copy equivalents (vRNA copy Eq/ml) in EDTA-anticoagulated plasma was determined using quantitative RT PCR . The assay minimum detection is 150 copies of vRNA Eq/ml (2.1 log) with a 99% confidence level.

Figure 2. Comparison of b12 and 2G12 transudated to the vagina following intravenous administration.

Each antibody treatment group consisted of three female Indian Rhesus macaques which were i.v.-administered 5 mg/kg of either b12 or 2G12 following Depo-provera treatment. Vaginal secretions from each animal were absorbed to cellulose wicks. A set of 3 samples per animal was taken at 6 hours, 12 hours, 24 hours, 4 days, and 7 days post i.v. antibody administration. The concentration of antibody in mucosal secretions was determined by ELISA from the clarified supernatant extracted from the wicks. Resulting data were compared to the corresponding antibody standard curve using nonlinear regression. Arithmetic means and standard deviations were calculated for each set of triplicate samples per animal. Data points were calculated from all animals at each timepoint and error bars represent the standard error of means. The typical time for viral challenge in protection experiments is indicated. The differences in the mean concentrations of b12 and 2G12 at each timepoint were evaluated in a student's t test and determined to be non-significant. Analyses performed in GraphPad Prism Software for Mac, Version 5.0a.

Figure 3. Comparison of antibody-dependent cell-mediated viral inhibition (ADCVI) by 2G12 and b12.

Target cells (CEM.NKR-CCR5) were infected with SHIV_SF162P3 and incubated for 48 hours, washed to remove cell-free virus and combined with Rhesus PBMC effector cells and serially diluted antibody. Viral inhibition was measured after incubation for 7 days. 2G12 is somewhat less effective than b12 in mediating ADCVI for a strict concentration comparison. An unpaired Two-tailed t test (P = 0.3285) of b12 and 2G12 ADCVI with an F test comparison of variance reveals no significant difference (P = 0.4154). Analysis performed in GraphPad Prism Software for Mac, Version 5.0a.