Clinical trials employing rectal microbicides containing antiretroviral drugs have the goal of reducing risk of contracting HIV during sexual activity. The Combination HIV Antiretroviral Rectal Microbicide (CHARM)-01 study is a recent Phase 1, double-blinded, randomized, safety & acceptability, and pharmacokinetic study of rectally-applied tenofovir-based microbicides in healthy adults (aged 37.7 years ± 14.3) completed by the Microbicide Trials Network (MTN). With the hypothesis that gene expression changes in the local immune environment may hallmark the action of tenofovir and potentially alter risk of HIV infection, we used RNA-Seq to assess changes in the rectal mucosa caused by gel usage.
Three tenofovir 1% gel formulations were administered rectally: a vaginal formulation (VF, 3111 mOsmol/kg), a reduced-glycerin vaginal formulation (RGVF, 846 mOsmol/kg), and a rectal-specific formulation (RF, 479 mOsmol/kg). Participants received 4 mL of the gels: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. We isolated total RNA from rectal biopsies from participants (n=14/group) and performed low input Illumina Truseq RNA-Seq on a HiSeq 2500 instrument. Top ranking differentially expressed genes by P value (P<0.05 in T or F tests) were forwarded to Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis.
Multi Dimensional Scaling analysis of the top 500 genes by F-Test P value showed that RGVF had the most significantly different (P=0.001, Kruskal Wallis rank sum test) expression profile from baseline. RGVF increased T cell related proinflammatory responses (IFNG, IL12, CXCL9/10), complement (C2, CASPs), antiviral interferons (OAS1, IFIs/IFITs, MX1), and IL10. This signature was unique to RGVF, however a derivative signature of up- and down-regulated IFNG-response genes, SERPINs, and IL10 was also present in VF and RF. Bioinformatic deconvolution analysis showed that these signatures stemmed from general alterations in B cell and Monocyte/MDC gene activity.
Tenofovir may impact the inflammasome in the rectal mucosa and set a balance between antiviral IFNs and proinflammatory cytokine signaling. This balance may be delicate in determining HIV infection risk outcomes. The above biomarkers may help monitor and identify mechanisms and targets of protection or infection risk in future microbiocide trials. Funding: MTN & NIH/NIAID/DAIDS IPCP Program (5U19AI082637).