CCR5 promoter variants among Ugandan HIV-1 elite and viremic 1 controllers: a laboratory ‑ based cross ‑ sectional study 2

Mechanisms for HIV control among HIV-1 elite and viremic-controllers are not fully understood. In Uganda, 25 Studies have reported individuals who without Antiretroviral therapy have the inherent ability to control HIV 26 progression to AIDS for a period of greater than 5 years. However, reasons for this phenotype are not 27 understood. The study objective was to determine the distribution of CCR5 co-receptor on CD4+ T-cells and 28 its associated promoter variants among HIV-1 elite and viremic-controllers.

are still unknown in Uganda. Exploring variations in this promoter region is essential to identify 90 protective mutations among Ugandan HIV-1 elite and viremic controllers that can be associated 91 with delayed HIV progression to AIDS. Therefore, this study determined the distribution of CCR5 92 and its associated promoter variants among the elite and viremic controllers in Uganda. Data generated 93 provides insights into mechanisms that could be responsible for the different clinical HIV courses 94 of disease seen among this study population.

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Cryopreserved PBMC samples collected from ART naïve HIV infected individuals followed for a 104 duration greater than 5 years were used in this study. Study participants were enrolled from 105 Makerere University Joint AIDS Program (MJAP), Mulago ISS clinic. 106 The Immunology assays were conducted at Makerere University, College of Health Sciences  in 1 ml of freeze media, and each sample aliquoted and stored in 2 cryo-vials.

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Following thawing, CD4+ T cell were isolated using the EasySep TM Human Isolation Kit (Stem 152 Cell Technologies, Catalogue no. 19052). The stem cell Isolation protocol was followed. But 153 briefly, cells were centrifuged at 1500rpm for 10 minutes, decanted and the pellet re-suspended in 154 1ml of 2% FBS containing 0.5% EDTA. The samples were transferred into FACs tubes from where 155 50μl of the enrichment cocktail were added and then incubated at room temperature for 10 minutes.

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Thereafter, 100 μl of the magnetic beads were added and the sample incubated at room temperature incubated at room temperature for 5 minutes. In one continuous motion, the sample (isolated CD4+ 159 T cells) was poured into a second tube after the 5 minutes' incubation. The isolated CD4+ T cells 160 were washed in 1ml PBS, centrifuged at 1500rpm for 10 minutes. These were re-suspended in 2ml 161 R-10 media, stained for counting with trypan blue and then incubated at 37 0 C on a 24 well plate 162 for 2 hours in a CO2 incubator. The cells were also stained for purity using anti-CD3, and anti-

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CD4 and ran on a BD FACS Canto II (BD Biosciences, Franklin lakes, New Jersey, USA).

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Samples with an average purity of 98% and above determined after staining for flow cytometry 165 were considered for stimulation. Prior to stimulation, the cells were rested in a 24-well-plate at 166 37 0 C in a C02 incubator.      259 Because CCR5 is expressed only on activated CD4+ T cells (23, 24), we activated CD4+ T cells 260 in vitro using Anti-CD3 (eBioscience Clone CD28.2) and anti-CD28 (eBioscience clone OKT3). 261 We then performed flow cytometry to study CCR5 expression. Flow cytometry data was analyzed 262 using Flow Jo and the gating strategy used to determine CCR5 expression ( Fig. 2A).

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The rare occurrence of delta 32 bp deletion within Africa (12), has led to a number of studies to in some regions and detrimental in others, thus this study was set out to explore which CCR5 307 promoter variants are associated with the different phenotypes in this study. We used previously stored PBMCs which were thawed and then DNA extracted using Qiagen Blood Genomic DNA 309 Kit (QIAamp DNA kit; Qiagen, Inc., Valencia, California, USA). The DNA was PCR amplified 310 and then sequenced. 311 We found that rs1799987 single nucleotide polymorphisms (SNPs) were predominant among elite 312 controllers and viremic controllers (71% and 61% respectively) while rs41469351was more among 313 non-controllers (68%). Furthermore, we also identified two Novel mutations; 1070 T>G (14.3%) 314 and 785 A>G (14.3%) among elite controllers (Table 5).

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Of significance was the finding that showed that elite and viremic controllers had statistically 334 significant reduction in CCR5 densities compared to non-controllers. This shows that even though