Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study

Abstract

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.

Keywords: clinical research/practice; immunosuppressant - mechanistic target of rapamycin: everolimus; immunosuppression/immune modulation; infection and infectious agents - viral: human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS); infectious disease; organ transplantation in general; translational research/science.

© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1.. CD4+ T cell counts and everolimus levels in HIV-infected solid-organ transplant recipients on suppressive ART.

Peripheral blood was collected prior to starting everolimus, 2 and 6 months on everolimus, and 6 months following discontinuation of study drug (study month 12). A majority of participant received 6 months of everolimus therapy either substituted for or in addition to calcineurin inhibitor-based immune suppression. Participant 6 discontinued everolimus at study month 2 due to diarrhea. Participant 10 continued on everolimus therapy through the 12 month sample time point. Shaded areas represent periods of everolimus exposure. Dotted lines represent the therapeutic target range for everolimus concentrations.

Figure 2.. Measures of HIV persistence prior to, during and following everolimus therapy.

(A) CD4+ T cell-associated HIV-1 DNA, (B) unspliced CD4+ T cell-associated RNA measures for each time point are shown. Changes between baseline (study month 0) all other time points were observed using Freidman’s test with Dunn correction for multiple comparisons (N=9, as participant 6 did not have sampling on month 6). Cell associated DNA and RNA levels for participants who maintained time-averaged drug trough measures of >5 ng/ml are shown in (C) and (D). Low-level plasma HIV RNA measures by single copy assay for each time point are shown in (E). Correlations between fold changes (FC) in CD4+ T cell-associated HIV-1 RNA and time-averaged everolimus trough levels in ng/mL are shown in (F-H). Significant negative correlations between CD4+ T cell-associated HIV-1 RNA FC at M2 and M12 and time-averaged everolimus trough levels between BL (M0) and M2, and BL and M6 (last date of study drug) were observed.

Figure 3.

Percentages of CD4+ and CD8+ T cell subsets expressing CCR5, PD-1 and CD69 prior to, during and following everolimus therapy. No changes in the distribution of naive, central memory, effector memory, or effector memory CD45RA+ cells were observed (A, B). However, increases in the percentage of total, central memory, and effector memory CD4+ and CD8+ T cells expressing the early activation marker CD69 were observed between study month 0 and month 6, the last time of sirolimus (C, D). Although not significant, a visual change in surface expression of CCR5 on various CD4+ and CD8+ T cell subsets are shown in E and F. A significant decrease in PD-1 expression from month 0 to month 6 was observed in CD4+ TEMRA cells (G) but not CD8 TEMRA cells (H). Differences between study month 0 and month 6 were measured using paired, non-parametric Wilcoxon signed-rank tests (* P

Figure 4.

Associations between CCR5 or PD-1 expression and cell-associated HIV DNA and RNA levels are shown. Modest statistically significant negative correlations were observed between HIV DNA and RNA copies/106 CD4+ T cells and the percentage of CD4+ T cells expressing CCR5 (A, C) and between cell-associated HIV RNA levels and the percentage of CD8+ T cells expressing CCR5 (B, D). Significant positive correlations between cell-associated HIV RNA levels and the percentage of both CD4+ and CD8+ T cells expressing the immune checkpoint marker, PD-1 were observed (E, F). Correlation coefficients and P values were calculated using Spearman rank tests.

Figure 5.

Responses to HIV and CMV peptides are shown. Overall, no significant differences in the percentage of IFNγ or CD107a expressing CD4+ or CD8+ T cells were observed for either HIV-Gag (A, B) or CMV-pp65 peptides (C, D) between baseline and month 6 or in repeat measures analysis.

Figure 6.

Differentially enriched pathways at BL (A) and from BL to M2 (B, C) by change in CD4+ T cell HIV-1 RNA are shown. Response to two months of everolimus therapy in terms of changes in CD4 HIV RNA was significantly associated with both positive and negative changes in gene pathway enrichment (A). A greater decrease in mTORC1 signaling was significantly associated with greater decreases in CD4+ T cell associated HIV RNA from baseline to month 2 and month 12 as shown in (B and C) suggesting sustained impact on mTOR signalling and HIV transcriptional silencing up to 6 months following cessation of everolimus (Pt 10 who remained on therapy through M12).

Figure 7.

Differentially enriched pathways from BL to M2 (A) and BL to M12 (B) by time-averaged everolimus trough level (BL through M2). Increased everolimus trough levels during the first two months of treatment were significantly correlated with decreased mTORC1, TNFα and TGFβ Hallmark pathways among others involved in apoptosis and cell stress response over the first 2 months of mTORC inhibitor therapy. Of note, downregulation of mTORC1 signaling was also seen at M12, 6 months following cessation of everolimus treatment (with the exception of Pt 10 who remained on therapy through M12). Everolimus trough levels from baseline to through M2 and through M6 inversely correlated with CCR5 gene expression at M6 (C).