HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice

Abstract

Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.

Keywords: CD4bs; glycan; gp160.

Fig. 1.

Combination immunotherapy with 3BNC117, PG16, and 10-1074. (A) Plasma viral loads for HIV-1_YU2–infected hu-mice treated with combination immunotherapy were monitored at regular intervals by quantitative RT-PCR. Each line represents a single animal. Treatment began at day 0. Antibodies were administered s.c. at 1.0 mg per antibody, twice per week, for a total of 6 wk. Green line, average viral load for untreated mice (Fig. S4). (B) Fold change in viral load (shown in A) from the baseline measurement for each animal. Red line shows the geometric mean viral load change at each time point. Green line, as above. (C) Combination immunotherapy resulted in a significant drop in viral load in all animals (n = 6) after 6 wk of continuous treatment (*P < 0.05, Wilcoxon signed rank test, two-tailed). (D) Cell-associated HIV-1 DNA per 10⁶ human peripheral blood mononuclear cells (PBMC) over time. Each line represents a single mouse from the same experiment as in A–C. (E) Fold change in cell-associated HIV-1 DNA (shown in D) from the baseline measurement for each animal. Red line shows the geometric mean change in cell-associated HIV-1 DNA levels at each time point. (F) Combination immunotherapy resulted in a significant drop in cell-associated HIV-1 DNA in all animals after 6 wk of continuous treatment (*P < 0.05, Wilcoxon signed rank test, two-tailed).

Fig. 2.

Termination of ART in the presence of combined immunotherapy. (A) Plasma viral loads. HIV-1_YU2–infected hu-mice were treated with ART beginning at day 0 for a total of 3 wk. Five days after starting ART, mice were additionally treated with combination immunotherapy using bNAbs 45-46^G54W, 10-1074, and PG16 (0.5 mg each twice weekly s.c.). ART was terminated and combination immunotherapy was continued for an additional 4 wk, then withdrawn. Each line represents a single mouse; viral load measurements were taken at the indicated time points (symbols). Blue shading, ART only; purple shading, overlapping ART and immunotherapy; pink shading, immunotherapy only. Green line, as in Fig. 1A. (B) Individual mice from A after antibody therapy was stopped. Each plot shows a single animal. Blue lines/symbols, plasma viral load; red lines/symbols, gp120-binding human IgG in plasma. (C) Mutations in gp120 cloned from rebound viral RNA after antibody withdrawal. Each line represents a single clone; red and green marks indicate nonsynonymous and synonymous mutations relative to HIV-1_YU2, respectively. (D) TZM-bl neutralization IC₅₀ values for the indicated bNAbs against pseudovirus carrying representative mutations found in gp120 clones from C for each indicated animal.

Fig. 3.

Combination of ART and single antibodies. Viral load measurements (symbols) from mice treated with ART and one of four monoclonal bNAbs are shown. Each line reflects a single mouse. Treatment was initiated at day 0 for all groups. Mice were treated for 5 d with ART alone and then with the combination of ART and the indicated antibody for 3 wk (45-46^G54W, 10-1074, or PG16) or 4 wk (3BNC117). ART was then discontinued and bNAb monotherapy continued for an additional 3 wk (3BNC117) or 4 wk (45-46^G54W, 10-1074, and PG16). Shading as in Fig. 2A; blue symbols/black lines, mice that remain controlled during immunotherapy after ART termination; red symbols/lines, mice that escape bNAb therapy; green line, as in Fig. 1A.

Fig. 4.

Viremia in hu-mice after stopping antibody therapy. Mice controlled with antibody treatment (Fig. 3) were followed for up to 80 d after stopping antibody injection. Each graph represents a single mouse. Blue lines, viral load; maroon lines, plasma antibody concentration; red shading, end of antibody treatment period. Viral rebound occurred in nearly all cases when antibody concentrations were low or undetectable, with one exception (ID number 399, viral load shown in red line). Three of 17 mice failed to rebound (viral load >3 × 10³ copies per milliliter).

Fig. 5.

Viral gp120 sequences during and after immunotherapy. (A) Mutations in plasma virus gp120 sequences cloned from mice that escaped antibody therapy after ART was stopped (Fig. S6). Each line represents a single clone; red and green marks as in Fig. 2C. All escape variants suffered mutations at sites known to confer resistance to the respective antibody (highlighted in blue vertical shading; bold numbers refer to the HXBc2-aligned residue). (B) Mutations in gp120 sequences cloned from mice that rebounded after antibody levels became subtherapeutic did not contain mutations at sites known to confer resistance, with the exception of ID number 399 (Fig. S8).

Fig. 6.

AAV-directed immunotherapy. (A) HIV-1_YU2–infected hu-mice were treated first with ART for 5 d and then with ART plus biotinylated 10-1074 antibody for 16 d (10-1074:bio, 0.5 mg twice per week s.c.). ART was then terminated and 10-1074:bio continued for an additional 12 d. Mice with viral loads below the limit of detection (solid blue or red symbols/lines) 12 d after stopping ART were injected with 2.5 × 10¹¹ genomic copies of a 10-1074–expressing AAV (AAV_10-1074) and passive 10-1074:bio was stopped. Viral loads were monitored for an additional 67 d. Only one of seven mice escaped AAV_10-1074 therapy, ID number 683. Open symbols/dashed lines, mice above the limit of viral load detection 12 d after stopping ART; green line, as in Fig. 1A. (B) Individual mice treated with AAV_10-1074. Each graph represents a single mouse. Blue or red lines/symbols, viral load; yellow lines/symbols, plasma 10-1074 concentration measured by gp120-specific anti-human IgG1 ELISA. Asterisks reflect the time point at which 10-1074:bio was no longer detectable on a gp120-specific anti-biotin ELISA. (C) Mutations in gp120 sequences cloned from the one mouse that escaped AAV_10-1074. Each line represents a single clone; red and green marks as in Fig. 2C. Consensus mutations were found at two residues in the 10-1074 binding site (highlighted in blue vertical shading; bold numbers refer to the HXBc2-aligned residue). (D) Pseudovirus neutralization by 10-1074 of HIV-1_YU2 control and mutant virus from mouse ID number 683 that escaped 10-1074 immunotherapy.