Impact of alemtuzumab-mediated lymphocyte depletion on SIV reservoir establishment and persistence

Abstract

Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low.

Fig 1. CD52 expression on lymphocytes in blood.

CD52 expression as measured by mean fluorescent intensity (MFI) on CD4⁺ naïve (T_N), CD4⁺ memory (T_M), CD8⁺ T_N, CD8⁺ T_M, CD20⁺ B cells and CD3-NKG2A⁺ NK cells in the blood of SIV-naïve RM (n = 28).

Fig 2. Alemtuzumab depletes circulating CD4⁺ naïve and memory T cells in a pilot study.

(A) Schematic representation of the study protocol showing SIVmac239 infection, ART initiation 12 dpi, alemtuzumab treatment at 294-, 301-, 308- and 322-days post-infection (dpi). (B) Mean (+SEM) plasma viral load (pvl) profiles of alemtuzumab-treated RM (n = 6) versus untreated controls (n = 3) prior to antibody infusion. (C—F) Change in absolute counts and change in the proliferative fraction (Ki67+) of CD4+ memory (T_M) and naïve (T_N) T cell subsets in blood following alemtuzumab (n = 6) or no treatment (n = 3). Results are shown as mean (+SEM) change from baseline of percentages of baseline absolute counts or percentages of Ki-67. (G and H) Quantification of cell-associated SIV DNA levels in PBMCs (copies per 10⁶ cell equivalents) in alemtuzumab-treated RM and untreated controls. RM D6 with no post-ART rebound viremia is shown in green. (I and J) Quantification of cell-associated SIV DNA levels in peripheral lymph nodes (LN) and small intestine (copies per 10⁶ cell equivalents) in alemtuzumab-treated RM. Threshold sensitivity varied as a function of the number of cells available for analysis; values below threshold are indicated by the gray area. (K) Kaplan-Meier analysis of SIV rebound kinetics in RM treated with alemtuzumab (n = 6) or no treatment (n = 3). (L) Individual pvl profiles of RM in each treatment group following ART release at 530 dpi. The dotted line indicates a pvl threshold of 15 RNA copies/ml. RM D6 received the anti-CD8α depleting mAb MT807R1 at 10, 5, 5, and 5mg/kg on days 252, 255, 259 and 262 post-ART release. The WRS test was used to determine the significance of differences in AUC between the two treatment groups (p-values ≤ 0.05 are shown).

Fig 3. Alemtuzumab depletes CD4⁺ an CD8⁺ T cells in blood and lymph nodes.

(A) Schematic representation of the study protocol showing SIVmac239 infection, ART initiation 7 dpi and alemtuzumab (n = 8) or human IgG control mAb (n = 4) treatment at 294, 301, 308, 315 dpi. (B and C) Mean (+SEM) plasma viral load profiles and comparison of SIV DNA (left panel) and SIV RNA (right panel) levels in PBMC and peripheral lymph node (LN) (copies per 10⁶ cell equivalents) 3-4 days after ART initiation of alemtuzumab-treated RM (n = 8) versus human IgG-treated controls (n = 4). Threshold sensitivity varied as a function of the number of cells available for analysis; values below threshold are indicated by the gray area. (D and E) Change in absolute counts (left panel) and change in the proliferative fraction (right panel) of CD4⁺ memory (T_M) and naïve (T_N) T cell subsets in blood following alemtuzumab (n = 8) or human IgG control mAb (n = 4). Results are shown as mean (+SEM) change from baseline of percentages of baseline absolute counts or percentages of Ki-67. (F) Quantification of the number of CD4⁺ cells per 1 × 10⁵ cells in T cell zone, B cell follicle and medullary cords of in LN before and after alemtuzumab treatment. LN biopsies were obtained between 7–13 days after the last dose of alemtuzumab. Each data point represents the average number of CD4⁺ cells derived from quantitative measures from 2–3 LN sections from a single time point from an individual RM. (G) Change in absolute counts (left panel) and change in the proliferative fraction (right panel) of CD8⁺ T_M in blood following alemtuzumab (n = 8) or human IgG control mAb (n = 4). Results are shown as mean (+SEM) change from baseline of percentages of baseline absolute counts or percentages of Ki-67. The two-sample WRS test was used to determine the significance of differences in AUC between treatment groups, and the pairwise WRS test to compare cell counts between timepoints (p-values ≤ 0.05 are shown).

Fig 4. Alemtuzumab treatment during full ART suppression fails to delay post-ART SIV rebound dynamics.

(A) Individual plasma viral load (pvl) profiles monitored by high-sensitivity assay (LOD of 1 RNA copy/ml) following alemtuzumab or (B) human IgG control mAb (n = 4) during ART. Values below assay threshold are indicated by the gray area. (C) Comparison of SIV DNA levels in PBMC, peripheral lymph node (LN), mesenteric LN, spleen and colon (copies per 10⁶ cell equivalents) between alemtuzumab-treated RM (n = 8) and human IgG-treated controls (n = 4) at 36- and 67-weeks pi. Assay threshold sensitivity varied as a function of the number of cells available for analysis; values below threshold are indicated by the gray area. (D) Kaplan-Meier analysis of SIV rebound kinetics in RMs treated with alemtuzumab (n = 8) or human IgG control (n = 4). (E and F) Individual pvl profiles of RM in each treatment group following ART release at 533 dpi. The dotted line indicates a pvl threshold of 15 RNA copies/ml. (G) Comparison of SIVmac239M clonal reactivation rates in plasma by high-throughput sequencing after ART cessation between RM in each treatment group.

Fig 5. Alemtuzumab at time of ART initiation depletes CD4⁺ and CD8⁺ T cells and impedes plasma viral load suppression.

(A) Schematic representation of the study protocol showing SIVmac239M infection, ART initiation 7 dpi and alemtuzumab (n = 8) or human IgG control mAb (n = 6) treatment at 7, 14, 21, 28 dpi. (B and C) Mean (+SEM) plasma viral load profiles and comparison of SIV DNA (left panel) and SIV RNA (right panel) levels in PBMC and peripheral lymph node (LN) (copies per 10⁶ cell equivalents) 3-4 days post ART initiation of alemtuzumab-treated RM (n = 8) versus human IgG-treated controls (n = 6). Assay threshold sensitivity varied as a function of the number of cells available for analysis; values below threshold are indicated by the gray area. (D and E) Change in absolute counts (left panel) and change in the proliferative fraction (right panel) of CD4⁺ memory (T_M) and naïve (T_N) T cell subsets in blood following alemtuzumab (n = 8) or human IgG control mAb (n = 6). Results are shown as mean (+SEM) change from baseline of percentages of baseline absolute counts or percentages of Ki-67. (F) Quantification of the number of CD4⁺ cells per 1 × 10⁵ cells in T cell zone, B cell follicle and medullary cords of in LN before and after alemtuzumab treatment. LN biopsies were obtained between 7–13 days after the last dose of alemtuzumab. Each data point represents the average number of CD4⁺ cells derived from quantitative measures from 2–3 LN sections from a single time point from an individual RM. (G) Change in absolute counts (left panel) and change in the proliferative fraction (right panel) of CD8⁺ T_M in blood following alemtuzumab (n = 8) or human IgG control mAb (n = 6). Results are shown as mean (+SEM) change from baseline of percentages of baseline absolute counts or percentages of Ki-67. The two-sample WRS test was used to determine the significance of differences in AUC between treatment groups, and the pairwise WRS test to compare cell counts between timepoints (p-values ≤ 0.05 are shown).

Fig 6. Alemtuzumab at time of ART initiation had a limited impact on SIV rebound dynamics.

(A) Comparison of SIV DNA levels in PBMC, peripheral lymph node (LN), mesenteric LN, spleen and colon (copies per 10⁶ cell equivalents) between alemtuzumab-treated RM (n = 8) and human IgG-treated controls (n = 6) at 36- and 67-weeks pi. Threshold sensitivity varied as a function of the number of cells available for analysis; values below threshold are indicated by the gray area. (B) Kaplan-Meier analysis of SIV rebound kinetics in RMs treated with alemtuzumab (n = 8) or human IgG control (n = 6). (C and D) Individual plasma viral load (pvl) profiles of RM in each treatment group following ART release at 533 dpi. The dotted line indicates a pvl threshold of 15 RNA copies/ml. The gray bar indicates CD8 depletion with 50mg/kg of mAb MT807R1 on days 200, 214 and 228 post-ART release. RM G1 and G5 with no post-ART rebound viremia are shown in green. (E) Comparison of SIVmac239M viral barcode clonal reactivation rates in plasma by high-throughput sequencing after ART cessation between RM in each treatment group.

Fig 7. Time to SIV rebound correlates with levels of virus production at time of ART initiation.

Scatterplots of time to SIV rebound versus plasma viral loads (pvl) at time of ART on 7 dpi (left panel) and area under curve (AUC) of pvl between 0–84 dpi (right panel) in RM infected with 500IU of SIVmac239M, placed on ART 7 dpi and treated with alemtuzumab at time of ART or during stable ART suppression (n = 16); or a human IgG control antibody at time of ART or during stable ART suppression (n = 10). Spearman rank correlation coefficient r with unadjusted p values testing association between paired samples are shown.

Fig 8. Alemtuzumab-treated RM without post-ART rebound viremia had relatively low virus production at time of ART initiation.

Comparison of peak plasma viral load (pvl) and total viral burden, as measured by AUC of pvl between 0–84 dpi, in alemtuzumab-treated RM and no post-ART rebound viremia (n = 3) relative to alemtuzumab-treated RM with post-ART rebound viremia (n = 19). Note that all control RM (n = 14) manifested SIV rebound following ART cessation. The two-sample WRS test was used to determine the significance of differences between treatment and outcome groups (p-values ≤ 0.05 are shown).