Virological Control by the CD4-Binding Site Antibody N6 in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

Abstract

Passive immunotherapy against HIV-1 will most likely require broadly neutralizing antibodies (bnAbs) with maximum breadth and potency to ensure therapeutic efficacy. Recently, the novel CD4 binding site antibody N6 demonstrated extraordinary neutralization breadth and potency against large panels of cross-clade pseudoviruses. We evaluated the in vivo antiviral activity of N6-LS, alone or in combination with the established V3-glycan antibody PGT121, in chronically simian-human immunodeficiency virus (SHIV)-SF162P3-infected macaques. A single dose of N6-LS suppressed plasma viral loads in 4 out of 5 animals at day 7, while the combination of both antibodies suppressed all animals. The combination of both antibodies had no additive antiviral effect compared to a single dose of PGT121, potentially reflecting the nearly 10-fold-higher potency of PGT121 against this SHIV. Viral rebound occurred in the majority of suppressed animals and was linked to declining plasma bnAb levels over time. In addition to the effect on plasma viremia, bnAb administration resulted in significantly reduced proviral DNA levels in PBMCs after 2 weeks and in lymph nodes after 10 weeks. Autologous neutralizing antibody (nAb) responses and CD8⁺ T-cell responses were not significantly enhanced in the bnAb-treated animals compared to control animals, arguing against their contribution to the viral effects observed. These results confirm the robust antiviral activity of N6-LS in vivo, supporting the further clinical development of this antibody.IMPORTANCE Monocloncal antibodies (MAbs) are being considered for passive immunotherapy of HIV-1 infection. A critical requirement for such strategies is the identification of MAbs that recognize the diversity of variants within circulating but also reservoir viruses, and MAb combinations might be needed to achieve this goal. This study evaluates the novel bnAb N6-LS alone or in combination with the bnAb PGT121, in rhesus macaques that were chronically infected with SHIV. The results demonstrate that N6-LS potently suppressed plasma viral loads in the majority of animals but that the combination with PGT121 was not superior to PGT121 alone in delaying time to viral rebound or reducing peripheral blood mononuclear cell (PBMC) or lymph node proviral DNA levels. The occurrence of viral escape variants in an N6-LS-monotreated animal, however, argues for the need to maximize breadth and antiviral efficacy by combining bnAbs for therapeutic indications.

Keywords: antiviral activity in vivo; autologous immune responses; bnAbs; broadly neutralizing antibodies; cellular reservoir; effect on tissue viral reservoir; immunotherapy.

FIG 1

Serum PGT121, N6-LS, and N6 concentrations in naive, uninfected rhesus macaques. A single intravenous infusion of 10 mg/kg of either MAb was given on day 0. The half-life (t_1/2) for PGT121 was 13.4 days (standard error [SE], 3.8); for NG-LS, 8.2 days (SE, 1.7); and for N6, 2.8 days (SE, 0.4). The dotted line represents the assay limit of detection.

FIG 2

N6-LS and PGT121 therapeutic study schedule. PB, peripheral blood; LN, lymph node; bx, biopsy specimens.

FIG 3

Therapeutic efficacy of N6-LS, PGT121, or the cocktail of both. (A to D) Plasma viral RNA (log₁₀ copies per milliliter) in rhesus monkeys chronically infected with SHIV-SF162P3 after infusion on day 0 (vertical dotted lines) of N6-LS (A), PGT121 (B), N6-LS plus PGT121 (C), or PBS (placebo control) (D). (E) Median viral load declines in all groups were similar, ranging from 0.131 to 0.188 log₁₀ RNA copy/ml/day. The error bars indicate standard error of the mean (SEM). (F) Viral rebound was measurably delayed in the PGT121 and N6-LS/PGT121 groups compared to N6-LS alone. The assay sensitivity limit was 150 RNA copies per ml.

FIG 4

(A to C) Serum N6-LS concentrations in animals that had received N6-LS alone (A), PGT121 levels in animals that had received PGT121 alone (B), and N6-LS and PGT121 levels in animals that had received the cocktail of both (C). The half-life for N6-LS alone was 6 days, for PGT121 alone was 10 days, for N6-LS in the combination was 10 days, and for PGT121 in the combination was 12 days. The dotted lines represent the assay limit of detection. (D) Correlation of the AUC for each MAb in each animal and the time to viral rebound for each animal. (E) Kinetics of viral RNA levels and serum antibody concentrations per animal throughout the study (top row, all animals that received N6-LS alone; middle row, all animals that received PGT121 alone; bottom row, animals that received the combination). The error bars indicate standard deviations.

FIG 5

(A to D) Proviral DNA was significantly reduced after 2 weeks in PBMCs in monkeys that received monoclonal antibodies (N6-LS, PGT121, or both) (A) versus placebo controls (B), and proviral DNA levels were also significantly lower in lymph node mononuclear cells (LNMC) after 10 weeks in MAb-pretreated monkeys (C) versus placebo controls (D). The error bars indicate standard deviations. (E) Proviral DNA levels for each animal in PBMCs and lymph node mononuclear cells.

FIG 6

HIV-1 envelope sequence analysis before and after N6-LS, PGT121, or combination MAb infusion. HIV-1 envelopes were cloned from plasma samples. The logogram shows Env gp120 regions (amino acid positions: V3 region, residues 296 to 334; loop D, residues 276 to 283; CD4 binding site, residues 362 to 374; and V5 region, residues 458 to 469, according to HXBc2 numbering) indicating sequence changes from week −1 (Pre) to the time of viral rebound (Post). The frequency of each amino acid is indicated by its height. Red residues represent mutations that differ from the consensus sequence. Viruses from 2 animals in the PGT121 group did not amplify. A mutation in position 279 (D/E) was observed in the animal MGI, which showed virological failure following N6-LS infusion.

FIG 7

Anti-SHIV-specific adaptive immune responses before and after MAb/placebo administration. (A to C) Autologous serum neutralization titers against tier 1B viruses in all bnAb-treated animals (A), by bnAb or placebo group (B), and against the challenge stock SHIV-SF162P3 (C) as determined by TZM-bl assay. The numbers of antigen-specific T cells were measured in PBMCs by in vitro stimulation with HIV-1 Env, SIV Gag, and Pol peptide pools, followed by fluorescence-activated cell sorter (FACS) detection of intracellular IFN-γ, IL-2, and TNF-α. The numbers of responding cells were summed across the cytokines within CD8⁺ T cell subsets and are reported as percentages. (D and E) Animals that received bnAbs were compared to untreated animals at week −1 and week 4, and significant differences in total (D) and protein-specific (E) responses are indicated. (F) The proportions of cells that expressed Boolean combinations of IFN-γ, IL-2, and TNF-α are depicted as pie charts, averaged across bnAb versus placebo groups at the indicated weeks.