Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy

Abstract

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.

Keywords: AIDS/HIV; T cells.

Figure 1. pVL suppression and CD4 depletion in blood.

(A) SIV RNA was quantified in longitudinal plasma samples using a qRT-PCR assay with a threshold quantification limit of 15 vRNA copies/mL. Shown are the values for the first 94 weeks of the study, which include pre-cART time points and approximately 93 weeks on cART (gray shaded region). (B and C) Longitudinal cell counts in blood for (B) total CD4⁺ T cells and for (C) naive (CD95^–), central memory (CM, CD95⁺CD28⁺), and effector memory (EM, CD95⁺CD28^–) CD4⁺ T cell subsets are shown. Data shown in C are represented as means ± SEM for all 5 animals within each group. Red arrows show the timing of 6 anti-CD4 Ab administrations to the CD4-depleted group. CD4-depleted experimental group animals are shown with red plots; control animals are shown with blue plots.

Figure 2. CD4 depletion in tissues.

(A and B) The frequency of CD4⁺ T cells as a percentage of total CD3⁺ T cells in rectal (A) and LN (B) tissues was determined by flow cytometry at the indicated time points. Values were normalized to the pre-CD4–depletion time point (30 weeks after cART initiation) for CD4-depleted (red) and control (blue) animals. (C) The frequency of naive (CD95^–), central memory (CD95⁺CD28⁺), and effector memory (CD95⁺CD28^–) CD4⁺ T cells as a percentage of total CD3⁺ T cells in LNs was determined by flow cytometry at the indicated time points for CD4-depleted (red) and control (blue) animals. (D, left) Representative images showing immunohistochemical staining for CD4 (brown) and CD68/CD163 (red) in LN sections from a CD4-depleted animal and a control animal at the indicated time points. B cell follicles are indicated with dashed borders. Scale bars: 200 μm. (D, right) Quantitation of immunohistochemical CD4 staining in T cell zone regions and B cell follicles of LN tissues collected at the indicated time points from CD4-depleted (red) and control (blue) animals. The data shown in A, B, and D were statistically analyzed using a 2-sample 2-sided t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Cell- and tissue-associated vDNA levels.

vDNA quantification in PBMCs (top panel), rectal pinch biopsy samples (center panel), and LN biopsy samples (bottom panel) are shown for CD4-depletion (red symbols) and control (blue symbols) group animals at the indicated time points. Determined vDNA levels were normalized based on input diploid genome cell equivalents (Cell Eq) assayed for each sample determined by the duplex quantification of the copy number of a host cell gene within the same sample extraction. Open symbols represent samples in which no vDNA was detected, with the symbol plotted at the threshold sensitivity limit for that sample based on the number of cell equivalents assayed.

Figure 4. Time to rebound.

Shown are Kaplan-Meier curves depicting comparison of time to first vRNA-positive blood sample (>15 vRNA copies/mL plasma) following cART cessation for CD4-depleted (red) and control (blue) group animals (n = 5 per group). The data were statistically analyzed using a Mantel-Cox test.

Figure 5. Viral rebound after cART cessation.

SIV RNA was quantified in longitudinal plasma samples using a qRT-PCR assay with a threshold quantification limit of 15 vRNA copies/mL. Shown are the values spanning the final 2 weeks of cART (gray shaded region) and the time period following cART discontinuation for each of the 10 study animals. For each animal, deep sequencing was used to determine the number and relative proportions of the viral clonotypes, shown in inset plots, present at the circled time point. Each bar in the inset plots represents a single clonotype, with its log relative proportion shown. The clonotype proportions and viral loads at the circled time points were used to calculate a viral reactivation rate for all animals other than ZK08. For ZK08, because only a single clonotype was detected at the 2 time points indicated by dashed circles, the viral reactivation rate was estimated using a different probabilistic model (see methods). Control animals are shown with blue plots; CD4-depleted experimental group animals are shown with red plots.

Figure 6. Comparison of calculated viral reactivation rates.

Viral reactivation rates (i.e., the number of days between each productive viral genome reactivation) were calculated (27, 29) for each study animal based on the relative frequency of rebounding viral barcode clonotypes and the viral growth rate during the exponential growth phase of viral rebound. Data were statistically analyzed using a 1-tailed Wilcoxon’s rank-sum test.