Genetic variations within the CCR5 promoter region among elite and viremic controllers in Uganda


 Background: The importance of the C-C chemokine receptor type 5 (CCR5) in HIV infection and disease progression was recognized with the discovery of the ∆32 allele. Individuals homozygous for this mutation lack functional CCR5, and are almost completely resistant to HIV infection while heterozygous individuals display decreased cell surface CCR5, which slows disease progression. However, this mutation is rare in Africa. Genetic variations within the CCR5 promoter region have been associated with regulation of CCR5 gene expression and could attempt to explain the different disease progression statues seen within Uganda. However, the distribution and impact of these variations are not known within this setting where this population exists. A study done in Rakai, reported a prevalence of LTNPs as 9.1% and another study done by Alex Kayondo in 2018, reported a prevalence of 0.26% of HIV controllers in an Urban HIV ambulatory center in Kampala, Uganda. Reasons for their intrinsic resistance to HIV are not fully understood. We hypothesized that variations in the CCR5 promoter gene could affect CCR5 expression thus impacting on the clinical course of HIV. In this study, we determined the median CCR5 expression by CD4 + T cells and the CCR5 promoter genetic variations that could be associated with delayed progression to HIV/AIDS seen among this study group.Results: There was significant reduction in CCR5 densities on CD4 + T cells among elite and viremic controllers compared to non-controllers. CCR5 Promoter polymorphism − 2459 G/G was highly prevalent among Elite and Viremic Controllers and it is associated with delayed progression to AIDS while − 2459 A/A and − 2132C/T were high prevalent among NCs and they are associated with rapid progression to AIDS.Conclusion: The reduction in CCR5 densities and percentage CCR5 + CD4 + T cells could explain the delayed progression to AIDS among these individuals. The reduction could be accounted for by the mutation 2459 G/G reported in the CCR5 promoter region.


Background
HIV has claimed lives of more than 35 million people globally since its discovery, particularly in the WHO African region, home to 70% of the 36.7 million people currently living with HIV (1). Though no documented cure yet exists for HIV, those infected with the virus can enroll in Antiretroviral Treatment (ART), which enables them to live long healthy lives.
One subset of individuals living with HIV, referred to long term non progressors (LTNPs), are able to maintain their CD4 + T cell count above 500 cells/µl for a period greater than five years prior to ART initiation (2). Among these, elite controllers (ECs) have an undetectable viral load (< 50 copies of HIV RNA/ml) while others, viremic controllers (VCs), maintain their viral load between 50 and 2000 copies/ml (3). The existence of this population in Uganda has been reported in two previous prevalence studies. Laeyendecker reported a prevalence of 9.1% of LTNPs in Rakai district (2) while Kayongo, reported a prevalence of 0.26% of ECs in an Urban HIV ambulatory center in Kampala (2,4). Reasons for intrinsic resistance to HIV in these controllers are not fully understood, especially in Uganda and other African populations with high genetic diversity (4).
Studies have identified mutations in CCR5, a major co-receptor for HIV infection that has been linked to delayed disease progression and resistance to HIV (5)(6)(7)(8). The ∆32 mutation with a 32-base pair (bp) deletion in the open reading frame (ORF) of the CCR5 gene confers resistance to HIV in homozygous individuals and retards disease progression in heterozygotes (9). While the CCR5∆32 allele occurs at a variable frequency of 4-15% in Caucasian populations, with an average of 10% in Europe ( (10,11), it is rare to find this mutation among Asian or African populations (12,13).
However, previous investigations have reported single nucleotide polymorphisms (SNPs) within the CCR5 promoter region associated with altered CCR5 expression (14) thereby positively or negatively influencing an individual's susceptibility and rate of disease progression to AIDS (15). Yet, despite the overwhelming evidence of CCR5 promoter polymorphisms' influence on HIV susceptibility and disease progression, data on variations in the CCR5 promoter region among elite and viremic controllers in Uganda is not known. Among different populations where it is found, the distribution of these CCR5 promoter polymorphisms varies greatly (16)(17)(18)). An example being HHC promoter haplotype that has been associated with faster disease progression among African Americans (16), although in the Thai population, this haplotype is reported with slower disease progression (19). The impact of these promoter variants on disease progression are still unknown for Uganda. Exploring variations in this promoter region is essential to identify protective mutations among Ugandan elite and viremic controllers that can be associated with delayed HIV progression to AIDS. Therefore, our study explored the variations in the CCR5 promoter region among elite and viremic controllers in Uganda. Data generated provide insights into mechanisms that could be responsible for the different clinical HIV courses of disease seen among this study population.

Patient Demographic characteristics
In this study, we included 31 participants who were ART naïve chronically infected individuals. These included 14 ECs with HIV viral load <50 viral RNA (viral RNA/ml), 10 VCs with HIV viral load between 50 and 2000 viral RNA (viral RNA/ml) and 7 non-controllers (ART controlled) whose demographic characteristics including years in care, CD4 counts, are summarized below (table 1). Other patient demographics including Body Mass Index (BMI) are summarized in table 6 in the Appendix.

Gating strategy
We developed a gating strategy and used it to analyze the CCR5 expression on CD4 T cells. Fig 1 shows representative flow cytometry dot plots to identify the specific CD4 + and CD8 + T cell subsets from an HIV-1infected participant. (4) Conventional T cells were selected by gating on CD3 + cells from the total lymphocyte population, from which (5) CD4 + T cells were selected. (6) From CD4+ T cells, CCR5+ T cells were selected.

No statistical difference in percentage CCR5+CD4+ T cells among NCs, ECS and VCs.
Considerable evidence suggests that CCR5+CD4 T cells are needed during early stages of HIV infection (20,21). To elucidate whether EC and VC have lower CCR5+CD4+ T cells compared to Non Controllers (NCs), we stimulated CD4 T cells from participants in the different groups and carried out flow cytometry to ascertain the percentage of CCR5+CD4 T cells. Results indicated that ECs and VCs had lower percentage of CCR5+CD4+ T cells compared to NCs, although the difference wasn't significant (Fig 2). These results suggest that the percentage of CCR5+CD4 T cells have no statistical contribution to progression to AIDS among ECs and VCs. The Kruskal-Wallis test was used to compare differences in frequencies of CD4 + among elite, viremic and noncontrollers. p values < 0.05 were considered significant.

CCR5 densities on CD4 T cells are significantly higher in NCs than ECS or VCs.
Because CCR5 densities are independent of the percentage of CCR5+CD4 T cells and they have been associated with high viral loads (22)(23)(24), we carried out experiments to ascertain whether EC, VC and NC have differences in CCR5 densities. Cells were stimulated and flow cytometry was carried out. Results showed significant variation in the Medium Fluorescent Intensity (MFI) of CCR5-expressing CD4+T cells between EC/VC and NC (between EC and NC; P=0.0210, VC and NC; P=0.0312) (Fig 3). However, there was no statistical difference in the MFI of CCR5-expressing CD4 + T-lymphocytes between VC and EC (P=0.3048) (Fig 3). These results suggest that delayed progression to AIDS among EC/VC compared to NC maybe governed by CCR5 densities. The Mann Whitney test was used to compare differences in frequencies of CD4 + among elite, viremic and non-controllers. p values < 0.05 were considered significant.

CCR5 promoter mutations in this cohort
The rare occurrence of delta 32 bp deletion within Africa, has led to a number of studies to explorer additional CCR5 regions for possible causes of the phenotypes seen among African ART naïve individuals who have the capacity to control HIV. Studies have reported several CCR5 promoter polymorphisms associated with either reduced or increased CCR5 expression among different cohorts in Africa. Controversies have arisen where some mutations are protective in some regions and detrimental in others, thus this study was set out to explore which CCR5 promoter variants are associated with the different phenotypes in this study.
Frequency calculated from (number of individuals expressing a given SNP/Total number of individuals in that group, n), i.e. for the elite-controllers, n = 14. For viremic controllers n=9 and for the non-controllers n= 7

Distribution of -2459 G/G, -2459 A/A and -2132 C/T identified in this cohort and have been reported with clinical Significance
-2459 G/G, -2459 A/A and -2132 C/T (25,26) identified in this study have been previously reported in the ClinVar database to either be associated with rapid or delayed progression to AIDS. Their distribution varies across the three groups (Fig 4). -2459 G/G is highly prevalent among the ECs (71%) and VCs (67%), while -2459 A/A (57%) and -2132 C/T (57%) are highly expressed among the NCs. (Fig 4).

Fig 4. Frequencies of CCR5 promoter mutations -2459 G/G, -2459 A/A, -2132 C/T, and -2733 C/T. (A) Frequency of variant -2459 G/G and 2459 A/A, (B) Frequencies of variant -2132 C/T and (C)
Frequencies of variant -2733 C/T. b Frequency were calculated from (number of individuals expressing a given SNP/Total number of individuals in that group, n), i.e. for the elite-controllers, n = 14. For viremic controllers n=9 and for the non-controllers n= 7

Discussion
Our findings indicate that delayed progression to AIDS among Ugandan ECs and VCs could be explained by the reduced number of CCR5+CD4 T cells and CCR5 densities on the surface of CD4 T cells compared to NCs. These differences in CCR5 densities may be due to CCR5 promoter variants that have been reported in this study showing differential distribution among ECs/VCs and NCs.
Although there was no statistical difference in the reported percentage of CD4+CCR5+ T cells between ECs/VCs and NCs, ECs/VCs had lower percentage of CD4+CCR5+ T cells compared to NCs. The reduction in CD4+CCR5+ T cells is in agreement with the findings by Potter et al who showed lower expression of CCR5 CD4 + T lymphocytes in HIV controllers (23,27). This reduction may contribute to the low levels of infection in elite and viremic controllers since CCR5 is required as a co-receptor during initial stages of HIV infection (28).
Of significance were the finding that showed that ECs/VCs had statistically significant reduction in CCR5 densities compared to NCs. This shows that even though there was no statistical significance in the percentage of the CCR5+CD T cells, there were differences in the number of CCR5 expressed on the CD4+ T cell surfaces. These results agree with finding by Reynes et al. who reported data supporting the hypothesis that the rate of evolution of HIV-1 disease in an individual is influenced by the median number of CCR5 co-receptors at the surface of the CD4 T cells of the individual. They demonstrated that CCR5 expression affects virus production and viral load, and individuals with a low viral load have CCR5 densities below the threshold value (22). The low expression of CCR5 on CD4+ T cells could explain why the elite and viremic controllers have the capacity to control the virus since they have reduced CCR5 expression on the surface of their CD4+ T cells.
We identified eleven mutations which could be associated with the clinical outcome seen among the ECs, VCs and NCs. Out of the eleven mutations identified, only -2459 G/G, -2459 A/A and 2132 C/T mutations have been previously reported to have clinical significance in ClinVar database (25,29).
In this cohort -2459 G/G is highly prevalent among ECs and VCs than NCs. It could be associated with the reduced CCR5 densities seen among ECs and VCs. These findings are in agreement with findings from other scholars who argued that this mutation was associated with reduced CCR5 expression (25,30). However, Janelle et al (2003), had controversial findings, she was unable to show any relationship between this mutation and CCR5 density on CD4 + T cells. This may be related to the recognized heterogeneity of CCR5 expression in different phenotypically defined CD4 + T-cell populations (29). As levels of viremia are closely associated with prognosis of HIV-1 infection, results from our study suggest that delay in progression to AIDS associated with the CCR5 -2459G allele may result from lower expression of CCR5 on susceptible cell populations (7,25,31,32).
At the same position, -2459 A/A, mutation was highly prevalent among the NCs. This mutation has been associated with increased CCR5 expression on CD4+ T cells and thus could explain the rapid progression to AIDS among the NCs. This mutation has been found to be associated with rapid progression in other studies (33)(34)(35). This mutation was significantly associated with disease acceleration, particularly an accelerated progression to death in Caucasians (16). It was also significantly associated with HIV-1 seroconversion, higher early HIV-1 RNA levels, and a shorter time to AIDS in diverse North American cohorts (17,36). Other studies in Rwanda, Spain, Thailand and Argentina have reported a strong association between this mutation and rapid HIV-1 disease progression (18,19,37) (18). Additionally, in a recent study investigating whether there is association between CCR5 haplotypes and HIV tropism in Estonian Caucasians found that -2459 A/A was associated with the presence of C-X-C chemokine receptor 4-tropic viruses (38). These findings consistently suggest that-2459 A/A leads to accelerated disease progression to AIDS in distinct genetic backgrounds.
The other identified mutation of clinical significance was -2132C/T, which was also found to be highly expressed amongst the non-controllers compared to the elite and viremic controllers. The finding of this study affirms those of Kostrikis LG et al. who reported that this mutation is associated with an increased rate of HIV-1 perinatal transmission (26). HIV-1-infected adults who were homozygotes for this mutation -2132C/T progressed rapidly to AIDS more rapidly (26).

Conclusions
Even though there was no statistical difference in the percentage of CD4+CCR5+ T cells observed among elite, viremic and non-controllers, there was significant reduction in the CCR5 densities on CD4+ T cells among the elite and viremic controllers compared to the non-controllers. We linked these variations to the polymorphisms within the promoter region of the CCR5 gene. -2459 G/G which is highly prevalent among ECs and VCs is associated with delayed progression to AIDS due to reduction in CCR5 densities on CD4 T cells, while -2459 A/A and -2132C/T which are highly prevalent among NCs are associated with rapid progression to AIDS due to increased expression of CCR5 on CD4 T cells. This study provides valuable information regarding the CCR5 promoter variants which are important to generate new concepts to further the understanding of the impact of CCR5 expression on host susceptibility to HIV-1.

Study design
This was a comparative cross-sectional study leveraging peripheral blood mononuclear cells (PBMC) samples collected by the Elite study that focused on the role of host genes in T cell resistance among elite and viremic controllers in Uganda. Cryopreserved PBMC samples collected from ART naïve HIV infected individuals followed for a duration greater than 5 years were used in this study.

Study Site Setting and Participants
Study participants were enrolled from Makerere University Joint AIDS Program (MJAP), Mulago ISS clinic. Patients who were ART naïve, maintained their CD4 count ≥ 500 cells/µl and their viral load of ≤ 2000 copies/ml for a period greater than 5 years were enrolled into this study. At enrollment, patients provided a peripheral blood sample, and HIV Viral Load was determined by qRT-PCR using Abbott Real Time HIV-1 assay (Abbott Molecular, USA). The time interval between initial viral load and enrollment viral load was determined and recorded in days.
To confirm EC/VC status, a follow-up VL was performed and the time interval between baseline and follow-up VL was also calculated. Any individuals with a hemoglobin of < 10mg/Dl and active opportunistic infection were excluded.

PBMC Thawing:
Cryopreserved PBMC samples were retrieved from liquid nitrogen at -196 0 C and immediately transferred to a preset 37 0 C water bath. Upon thawing, cells were washed with R10 media composed of RPMI 1640 medium (ThermoFisher Scientific, South America, catalogue no. 11875093), 1% Pen-Strep, 1% L-Glutamine, 1% Hepes buffer and 10% Fetal Bovine Serum (ThermoFisher Scientific, South America, catalogue no. 10270106) in a 15ml centrifuge tube. We then determined cell yield where viability testing was done using Trypan blue solution. Cells were stained using 0.4% trypan blue solution at 1:1 dilution ratio. Samples with at least 80% viability were considered for CD4+ T cell isolation. A portion of cells harvested off in R10 media were used for DNA extraction and the rest for CD4+ T cell isolation.

CD4+ T cells Isolation and Stimulation
Following thawing, CD4+ T cell were isolated using the EasySep TM Human Isolation Kit (Stem Cell Technologies, Catalogue no. 19052). The stem cell Isolation protocol was followed. But briefly, cells were centrifuged at 1500rpm for 10 minutes, decanted and the pellet re-suspended in 1ml of 2% FBS containing 0.5% EDTA. The samples were transferred into FACs tubes from where 50μl of the enrichment cocktail were added and then incubated at room temperature for 10 minutes. Thereafter, 100 μl of the magnetic beads were added and the sample incubated at room temperature for 5 minutes. The sample tube (lid removed) was then placed in the EasySep magnet and incubated at room temperature for 5 minutes. In one continuous motion, the sample (isolated CD4+ T cells) was poured into a second tube after the 5 minutes' incubation. The isolated CD4+ T cells were washed in 1ml PBS, centrifuged at 1500rpm for 10 minutes. These were re-suspended in 2ml R-10 media, stained for counting with trypan blue and then incubated at 37 0 C on a 24 well plate for 2 hours in a CO2 incubator. The cells were also stained for purity using anti-CD3, and anti-CD4 and ran on a BD FACS Canto II (BD Biosciences, Franklin lakes, New Jersey, USA). Samples with an average purity of 98% and above determined after staining for flow cytometry were considered for stimulation. Prior to stimulation, the cells were rested in a 24-well-plate at 37 0 C in a C0 2 incubator.

CD4+ T cell Stimulation
We prepared stimulatory antibodies; Anti-CD3 (eBioscience Clone CD28.2) and anti-CD28 (eBioscience clone OKT3) at a concentration of 5µg/ml each. A clearly mapped out 96-well plate was used. The plate was coated by adding 100 µl of anti-CD3 at a concentration of 5ug/ml and incubated for 2 hours. After incubation, the plate was blotted and 100,000 cells in 90µl per sample were added. Using a pipette, 110 µl anti-CD28 was added to each well to make a total final volume of 200µl at a concentration of 5ug/ml. For the negative control wells, 110µl of PBS was added to make volume of 200µl per well. The plate was incubated for a total of 48 hours at 37 0 C in a CO 2 incubator. After incubation, cells were washed with 200 µL staining buffer per well and then transferred to the 5 mm round bottomed polystyrene FACS tubes.

Cell Surface Staining
Subsequently, cells were surface stained and incubated for 30 minutes with the following monoclonal antibodies;

DNA Extraction
DNA was extracted using the QIAamp DNA mini Kit (Qiagen, Inc., Valencia, CA, USA) in accordance with the manufacturer's instructions as used in the previous studies (39). 200μl of sample containing 2x10 6 cells was added to micro-centrifuge tube together with 20μl of Qiagen protease. 200μl of buffer AL was added to the sample which was then mixed thoroughly to ensure efficient lysis and then incubated at 56 0 c for 10 minutes.
200μl of ethanol was added to the sample and then mixed by pulse vortexing. After vortexing, the mixture was added to span column (in a 2ml collection tube) and centrifuged at 8000rpm for 1 minute. The mini spin column was later placed into a clean 2 ml collection tube. The extracted DNA was washed using AW1 and AW2 and spun at 8000 rpm for 1 minute and 14000 for 3 minutes respectively. The empty column was span to prevent possible buffer AW2 carry over and later DNA was eluted using AE buffer into a new 1.5ml micro-centrifuge tube.

PCR and Sequencing
PCR amplification of CCR5 promoter region was carried out using the following cycling conditions; Initial denaturation at 95°C for 3 minutes; 31 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 2.40 minutes; followed by 68°C for 7 min. The PCR master mix contained High fidelity Super script III platinum Taq polymerase (Invitrogen, Carlsbad, CA, USA) in the presence of 2X reaction buffer, 5Mm MgCL2 with primers shown in table 2 developed using GenBank sequence with accession number U95626. as described in a similar study (40). The promote amplicon size was 2189 base pairs (40).

PCR clean up
From all samples that gave a single band after Gel electrophoresis, 10μl was aliquoted and added into a PCR tube followed by 2μl of ExoSAP IT. The samples were transferred into a thermocycler (Applied Biosystems, California, United States) and ran under the conditions: 37 0 C for 45 seconds, 800C for 45 seconds (inactivate ExoSAP-IT)2 and held at 4 0 C.

Cycle sequencing
Sequencing was performed using an ABI version 3.1 BigDye Kit (Applied Biosystems, Catalogue no. 4337456) and ABI3500xl Genetic Analyzer. Briefly, a master mix was prepared as follows; 0.5μl Big Dye terminator, 1.75μl 5X sequencing buffer, 2.5 μl primer as shown in the primer map ( Fig 5) and primer sequences are shown in

Data Analysis
Flow cytometry data were analyzed using FlowJo version 10.5.2 software. CD4+ T cells were distinguished by their surface expression of CD3 and CD4. Within these CD4 + T cells we identified CCR5+ T cells and determined both the percentage CD4+CCR5+ T cells and CCR5+ MFI (to ascertain the CCR5 density on CD4+ T cells). Statistical analysis was performed using GraphPad Prism 7. The Mann Whitney test for non-parametric variables facilitated comparison of differences among groups. P values < 0.05 indicated a significant difference.
Sanger Sequence data analysis was performed using mutation surveyor Mutation version 5.5 (SoftGenetics; Pennsylvania, USA). U95626 and NT_022517 reference sequences were used in assembly as used in other studies (18). A search of the GenBank NCBI SNP database (dbSNP) determined whether polymorphisms detected in this study had been previously reported. The CCR5 numbering system was used where the first nucleotide of the translational start site is designated as +1 and the nucleotide immediately upstream from that is −1 (41).   The CCR5 promoter primer map for the primers used in sanger sequencing.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. APPENDIX.docx