Short Communication: Lack of Effect of Maraviroc Intensification on Blood and Gut Reservoir

Study design and recruitment

A prospective study whereby 10 individuals with chronic HIV infection with sustained virological suppression on ARV for >12 months and a CD4+ T cell count less than 500 cells/mm³ were intensified with a 24-week course of standard dose maraviroc. Blood samples were collected at weeks 0, 4, 12, and 24. At baseline and week 24, individuals underwent flexible sigmoidoscopy, in which 8–10 rectal biopsies were taken and fixed overnight in 10% normal buffered formalin at 4°C.

Laboratory tests

HIV reservoir and residual viraemia

Low copy viral load was measured with an internally controlled ultrasensitive quantitative RT-PCR able to detect 3 copies/ml. Purified peripheral blood CD4 T cells were analyzed by quantitative PCR (qPCR) for HIV-1 DNA (total and integrated) and cell-associated HIV-1 RNA unspliced transcripts as reported elsewhere. ¹²

For preparation of the gut biopsies for HIV reservoir quantitation, a commercial kit was used (Cat. No. 56404; Qiagen, Hilden, Germany), and the manufacturer's protocol modified as follows to ensure maximum yield of DNA. Biopsy sections were removed from paraffin blocks using a scalpel and placed into 1.5 ml micro centrifuge tubes. Samples were washed repeatedly with xylene and ethanol until the precipitate had disappeared, then dried at room temperature. Biopsy sections were incubated at 37°C and DNA was extracted using a commercial kit (QIAAMP DNA FFPE Tissue Extraction Kit, ID 56404; Qiagen). The resulting DNA was eluted into 200 μl and analyzed by qPCR using the same assays for peripheral blood CD4 cells as already detailed.

Immune activation and lymphocyte subsets

Peripheral blood mononuclear cells isolation were stained with the anchor markers (CD3- VioBlue, CD4 (VIT4)-VioGreen, and CD8-APC) and a live/dead marker near IR APC-Cy7 plus either an activation panel [CD25 (3G10)-PE, CD38-PE-Vio770, CD69-FITC, and anti-HLA-DR-PerCP] or an exhaustion panel (TIGIT-PE, TIM-3-FITC, LAG-3-PerCPeF710, and PD1-PE-Cy7). Cells were run on a MACSQuant and analyzed with FlowJo software v10 (Miltenyi Biotec).

Immunohistochemistry

Rectal biopsy sections were stained for CD4 (goat polyclonal; Novus Biologicals) and CD8 antibodies (rabbit polyclonal; Abcam). Images were analyzed in ImageJ and the distribution of cells was determined as described by Milano et al. ¹³

Statistics

For each outcome measure, the results at baseline and week 24 were compared using the paired t-test. Wilcoxon rank test was used to assess the impact of tropism on each parameter.

Patient characteristics

The median age was 46 years. All patients were male with subtype B virus and were receiving two nucleoside reverse transcriptase inhibitors (NRTIs) combined with either a non-NRTI (n = 5) or protease inhibitor (n = 5). The mean CD4 T cell count was 321 cells/μl (SD 112), CD4:CD8 0.51 (SD 0.27), and nadir CD4 117 cells/ml (SD 135). All patients were maintained an HIV viral load <50 copies/ml throughout the study. Individual plots of viral reservoir, microbial translocation, and immune activation are shown in Table 1 and mean changes in parameters between week 0 and 24 are shown in Figure 1.

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FIG. 1.

Individual change in key parameters between week 0 and 24.

Baseline to week 24

Tropism

Six out of 10 individuals had R5 virus. Tropism (R5 vs. X4 or X4/R5) did not correlate with changes in reservoir, immune activation, immune exhaustion, or microbial translocation (all p values > .05).