Transient CD4+ T cell depletion during suppressive ART reduces the HIV reservoir in humanized mice

Abstract

Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently available approaches. This study evaluated antibody mediated transient CD4+ T cell depletion as a strategy to reduce the latent HIV reservoir. Anti-CD4 antibodies effectively depleted CD4+ T cells in the peripheral blood and tissues of humanized mice. We then demonstrate that antibody-mediated CD4+ T cell depletion of HIV infected ART-suppressed animals results in substantial reductions in cell-associated viral RNA and DNA levels in peripheral blood cells over the course of anti-CD4 antibody treatment. Recovery of CD4+ T cells was observed in all tissues analyzed except for the lung 26 days after cessation of antibody treatment. After CD4+ T cell recovery, significantly lower levels of cell-associated viral RNA and DNA were detected in the tissues of anti-CD4 antibody-treated animals. Further, an 8.5-fold reduction in the levels of intact HIV proviral DNA and a 3.1-fold reduction in the number of latently infected cells were observed in anti-CD4-antibody-treated animals compared with controls. However, there was no delay in viral rebound when ART was discontinued in anti-CD4 antibody-treated animals following CD4+ T cell recovery compared with controls. Our results suggest that transient CD4+ T cell depletion, a long-standing clinical intervention that might have an acceptable safety profile, during suppressive ART can reduce the size of the HIV reservoir in humanized mice.

Fig 1. Human CD4⁺ T cells are robustly depleted in animals treated with anti-CD4 antibody.

(A) Experimental design of anti-CD4 antibody treatment in humanized mice. (B) The frequency of human CD4⁺ T cells in peripheral blood was longitudinally monitored by flow cytometric analysis. The frequency (C) and the absolute number (D) of human CD4⁺ T cells in the tissues of humanized mice was determined by flow cytometric analysis at necropsy. The frequency of naïve (E, CD27⁺CD45RA⁺), central memory (F, CD27⁺CD45RA^-), and effector memory (G, CD27^-CD45RA^-) CD4⁺ T cells from anti-CD4 antibody-treated and control animals was determined by flow cytometry at necropsy. BM, bone marrow; LIV, liver; LNG, lung; LN, lymph nodes; SPL, spleen, ORG, human thymic organoid. Combined: all individual tissues from all animals are graphed together. Blue arrows in A and B show the timing of 4 anti-CD4 antibody administrations (6 mg/kg) to the anti-CD4 antibody treated group. Anti-CD4 antibody-treated animals (n = 6) are shown in red; control animals (n = 3) are shown in black. Data are expressed as mean ± SEM. Statistical analyses were performed using unpaired two-sided Mann–Whitney U-tests. Statistical significance was considered when P < 0.05.

Fig 2. Peripheral blood CD4⁺ T cells are effectively depleted in ART-suppressed, HIV-infected humanized mice receiving anti-CD4 antibodies and rapidly recover following cessation of antibody treatment.

(A) Experimental design of HIV infection and anti-CD4 antibody treatment in humanized mice. The frequency of human CD45⁺ hematopoietic cells (B), CD3⁺ T cells (C), CD19⁺ B cells (D), CD3^-CD19^- myeloid cells (E), CD4⁺ T cells (F) and CD8⁺ T cells (G) in peripheral blood was longitudinally monitored by flow cytometric analysis. Blue arrows show the timing of 4 anti-CD4 antibody administrations (6 mg/kg) to the anti-CD4 antibody treatment group. Anti-CD4 antibody treated animals (n = 6) are shown in red; control animals (n = 7 in A; n = 4 in B-G) are shown in black. Data are expressed as mean ± SEM. Statistical analyses were performed using unpaired two-sided Mann–Whitney U-tests. Statistical significance was considered when P < 0.05.

Fig 3. Anti-CD4 antibody administration reduces cell-associated viral RNA and DNA levels in peripheral blood.

(A) Plasma viral load (HIV-RNA copies/ml) was quantified in longitudinal plasma samples using a qRT-PCR assay following HIV-1 infection. (B) Levels of cell-associated viral RNA in PBMCs were longitudinally quantified by qPCR. (C) Levels of cell-associated viral DNA in PBMCs were longitudinally quantified by qPCR. PBMC: peripheral blood mononuclear cell. The shaded gray area in A represents ongoing ART administration. The dotted line in panel A indicates the limit of detection (693 copies/ml). Samples in B and C with undetectable values are set as 1 copy per 10⁵ hCD45⁺ cells. Blue arrows show the timing of 4 anti-CD4 antibody administrations (6 mg/kg) to the anti-CD4 antibody treatment group. Anti-CD4 antibody treated animals (n = 6) are shown in red; control animals (n = 7 in A; n = 4 in B and C) are shown in black. Data are expressed as mean ± SEM. Statistical analyses were performed using unpaired two-sided Mann–Whitney U-tests. Statistical significance was considered when P < 0.05.

Fig 4. CD4⁺ T cell depletion results in a reduction in the levels of intact HIV proviruses and latently infected cells.

Copies of cell-associated viral RNA (A) and DNA (B) levels in purified CD4⁺ T cells isolated from pooled MNCs were quantified by qPCR. (C) Copies of intact virus per million purified CD4⁺ T cells isolated from pooled MNCs were measured by quantitative viral outgrowth assay (QVOA). (D) The frequency of intact virus, 5’ defective virus and 3’ defective virus in purified CD4⁺ T cells isolated from pooled MNCs was measured by the intact proviral DNA assay (IPDA). MNC: mononuclear cell; Intact, intact virus; 5’ def, 5’ defective virus; 3’ def, 3’ defective virus. Anti-CD4 antibody treated animals (n = 6) are shown in red; control animals (n = 6) are shown in black. T and C in the X axis represent Test and Control, respectively. Data are expressed as mean ± SEM. Statistical analyses were performed using unpaired two-sided Mann–Whitney U-tests. Statistical significance was considered when P < 0.05.

Fig 5. Systemic recovery of CD4⁺ T cell levels after antibody treatment termination in ART-suppressed, HIV-infected mice.

The absolute number of human CD3⁺ T cells (A), CD4⁺ T cells (B) and CD8⁺ T cells (C) in the tissues of anti-CD4 antibody-treated and control animals was determined by flow cytometry at necropsy. BM, bone marrow; LIV, liver; LNG, lung; LN, lymph nodes; SPL, spleen, ORG, human thymic organoid. Combined: all individual tissues from all animals are graphed together. Anti-CD4 antibody treated animals (n = 6) are shown in red; control animals (n = 4) are shown in black. Data are expressed as mean ± SEM. Statistical analyses were performed using unpaired two-sided Mann–Whitney U-tests. Statistical significance was considered when P < 0.05.