VARIATIONS IN TRIM5α AND CYCLOPHILIN A GENES AMONG HIV-1 ELITE CONTROLLERS AND NON CONTROLLERS IN UGANDA

Abstract

Background: Human immunodeficiency virus type-1 (HIV-1) elite controllers represent a unique population that controls viral replication in the absence of antiretroviral therapy (ART). Various genetic, immunologic and virologic factors have been implicated as mechanisms involved in their viremic control. Host restriction factors such as TRIM5α and Cyclophillin A have been reported to influence HIV susceptibility and disease progression and that polymorphism in their genes may contribute to the viral suppression phenotype exhibited by HIV-1 elite controllers. However, little is known about the occurrence of these polymorphisms among Ugandan HIV-1 elite controllers and non-controllers. Objective: To characterize the variations in TRIM5α and CypA genes among Ugandan HIV-1 elite controllers and non-controllers. Methods: Upon obtaining ethical clearance from SBS-HDREC, samples were retrieved from the ELITE study sample repository. These were thawed and CD4+ T cell isolated using CD4+ T Cell enrichment magnetic kit. After 2-hour incubation, CD4+ T cells were cultured under 4 conditions; a) unstimulated, b)Anti-CD3 & CD28, Anti-CD3/28/Imiquimod, and c) Imiquimod alone. Cells were stained for surface markers (CD3, CD4, CD38, and PD-1) and analyzed by flow cytometry, while the supernatant was stored for use in measuring cytokines(Th1, Th2, and Th17 cytokines) by Luminex. RNA was extracted using Quick-RNA™ Whole Blood kit, quality assessed using QIAxcel gel analyzer and RT qPCR done using QuantiTect Probe RT-PCR Kit in a Rotor gene Q real-time PCR machine. mRNA was quantified using delta CT relative quantification method. DNA was extracted using Qiagen Blood Genomic DNA Kit, PCR amplified and cleaned with exosap and stored at -200C awaiting DNA sequencing. Results: Upon stimulation, non-controller cells expressed significantly more PD-1 compared to the elite controllers, with the differential expression being significantly more in CD3/CD28 stimulated cells(p=0.0315), unstimulated (p=0.0401) and Anti-CD3/28/imiquimod (p=0.0565). There was no statistical difference in the expression of CD38. The genes of the intrinsic cellular defense including TRIM5α and CypA were more expressed among elite controllers, but this differential expression was not significant both in the Anti-CD3/28 stimulated (TRIM, p=0.6695 & CypA p=0.6340) and Anti-CD3/28/Imiquimod stimulated (TRIM5α, p=0.4879 & CypA p=0.7632). Conclusion and recommendation: This study highlights differential PD-1 expression among elite controllers and non-controllers. This may have implications for potential immune checkpoint therapies as adjuncts in HIV-1 therapy. Additionally, the nondifferential expression of TRIM5α and CypA genes may imply that elite controllers have unique TRIM5α and CypA that have more capacity of innate immune signaling, not necessarily expressing more of the protein. Further investigations need to be done to establish the uniqueness of TRIM5α and CypA proteins expressed by the elite controllers