Predictors of Cell-Associated Human Immunodeficiency Virus (HIV)-1 DNA Over 1 Year in Very Early Treated Infants.

BACKGROUND: Younger age of antiretroviral therapy (ART) initiation is associated with smaller viral reservoirs in perinatally acquired HIV-1 infection, but there is wide variability among early-treated infants. Predictors of this variability are not fully described. METHODS: Sixty-three neonates diagnosed with HIV-1 <48 hours after birth in Johannesburg, South Africa, were started on ART as soon as possible. Fifty-nine (94%) infants received nevirapine prophylaxis from birth until ART start. Viably preserved peripheral blood mononuclear cells (PBMCs) collected at regular intervals to 48 weeks, and from mothers at enrollment, were tested using integrase-targeted, semi-nested, real-time quantitative hydrolysis probe (TaqMan) PCR assays to quantify total HIV-1 subtype C viral DNA (vDNA). Predictors were investigated using generalized estimating equation regression. RESULTS: Thirty-one (49.2%) infants initiated ART <48 hours, 24 (38.1%) <14 days, and 8 (12.7%) >14 days of birth. Three-quarters were infected despite maternal antenatal ART (however, only 9.5% of women had undetectable viral load closest to delivery) and 86% were breastfed. Higher infant CD4+ T-cell percentage and viral load <100 000 copies/mL pre-ART were associated with lower vDNA in the first 48 weeks after ART start. No antenatal maternal ART and breastfeeding were also associated with lower vDNA. Older age at ART initiation had a discernible negative impact when initiated >14 days. CONCLUSIONS: Among very early treated infants, higher CD4+ T-cell percentage and viral load <100 000 copies/mL pre-ART, infection occurring in the absence of maternal antenatal ART, and breastfeeding were associated with lower levels of HIV-1 DNA in the first 48 weeks of treatment. Clinical Trials Registration. clinicaltrials.gov (NCT02431975).

Interleukin-8 genetic diversity, haplotype structure and production differ in two ethnically distinct South African populations.

A single nucleotide polymorphism (SNP), 251 bases upstream from the IL-8 transcription start (-251A>T, rs4073), has been extensively investigated in cancers and inflammatory and infectious diseases in predominantly European and Asian populations. We sequenced the IL-8 gene of 109 black and 32 white South African (SA) individuals and conducted detailed characterization of gene variation and haplotype structure. IL-8 production in phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) of a subset (black: N = 22; white: N = 32) of these individuals was measured using ELISA. Select variants were genotyped for additional black individuals (N = 141), and data from the 1000 Genomes Project were used for haplotype analysis and comparative purposes. In white individuals, the -251A>T SNP formed part of a prevalent six-variant haplotype [haplotype frequency (HF): 61%], Hap-1C, involving the following variants: -251A>T; +394T>G (rs2227307); +780C>T (rs2227306); +1240->A (rs2227541); +1635C>T (rs2227543) and +2770A>T (rs2227543). Hap-1C (-251T+394T+780C+1240+A+1635C+2770A) was composed of two three-variant sub-haplotypes [Hap-1Ca: -251T+394T+1240+A; Hap-1Cb: +780C+1635C+2770A) sharing similarities with haplotypes identified in the black population. Hap-1C was found to be present in European, East and South Asian populations. Four haplotypes were identified in the black population with the two prevalent haplotypes each comprised of two variants: Hap-1B [-251A>T and +1240->A; -251T+1240+A; HF: 14%] and Hap-2B [-743T>C (rs2227532) and +2452A>C (rs2227545); -743C+2452C; HF: 13%]. Populations did not differ in unstimulated PBMC IL-8 production. Upon PHA stimulation, PBMCs from white individuals produced more IL-8 (P = 0.04), suggesting the -251T allele is responsible for higher production, however further analysis revealed that Hap-1C (and constituent sub-haplotypes), did not associate with IL-8 production. Populations did however differ in monocyte number with the white population having significantly more monocytes compared to the black population (P = 0.025), and furthermore monocyte number strongly correlated with IL-8 production in both population groups (black: p = 0.0002, r = 0.71; white: P = 0.0005, r = 0.59). Hap-1B, Hap-2B, and a SNP located one base pair upstream of the IL-8 ATG start codon, +100C>T SNP (rs2227538), all associated with higher IL-8 production in the black population - individuals harbouring at least one of these haplotypes/variant associated with higher IL-8 production (P = 0.003) compared to individuals without. The black population was enriched for individuals harbouring Hap-1B and/or Hap-2B compared to the 1000 Genomes project sub-Saharan African population (P = 0.006), suggesting that SA black individuals may be high IL-8 producers. Given the paucity of IL-8-related studies that have been conducted in populations from sub-Saharan Africa, this study has significantly increased our understanding of this important chemokine in the South African population.

The FCGR2C allele that modulated the risk of HIV-1 infection in the Thai RV144 vaccine trial is implicated in HIV-1 disease progression.

In the HIV-1 Thai RV144 vaccine trial-the only trial to demonstrate any vaccine efficacy to date-a three-variant haplotype within the Fc gamma receptor 2C gene (FCGR2C) modified the risk of HIV-1 acquisition. A similar vaccine regimen is currently being evaluated in South Africa in the HVTN702 trial, where the predominant population is polymorphic for only a single variant in the haplotype, c.134-96C>T. To investigate the significance of c.134-96C>T in HIV-specific immunity in South Africans, this study assessed its role in HIV-1 disease progression. In a cohort of HIV-1-infected South African controllers (n = 71) and progressors (n = 73), the c.134-96C>T minor allele significantly associated with increased odds of HIV-1 disease progression (odds ratio 3.80, 95% confidence interval 1.90-7.62; P = 2.0 × 10-4, PBonf = 2.4 × 10-3). It is unlikely that the underlying mechanism involves wild-type FcγRIIc function, since only a single study participant was predicted to express wild-type FcγRIIc as determined by the FCGR2C c.798+1A>G splice-site variant. Conversely, in silico analysis revealed a potential role for c.134-96C> T in modulating mRNA transcription. In conclusion, these data provide additional evidence towards a role for FCGR2C c.134-96C>T in the context of HIV-1 and underscore the need to investigate its significance in the HVTN702 efficacy trial in South Africa.

Cis-regulatory genetic variants in the CCR5 gene and natural HIV-1 control in black South Africans.

Studies have investigated CCR5 haplotypes (HHA, HHB, HHC, HHD, HHE, HHF*1, HHF*2, HHG*1, HHG*2), defined by seven 5'UTR single nucleotide polymorphisms (SNPs), CCR2-V64I and CCR5Δ32, in HIV-1 disease. CCR5 cis-regulatory regions were sequenced, CCR2-V64I and CCR5Δ32 genotyped, and compared in HIV-1-infected black South Africans: 71 HIV-1 controllers (23 elite controllers, 37 viraemic controllers (VCs), 11 high viral load long-term non-progressors) and 74 progressors. The HHE haplotype and 3'UTR +2919 T > G SNP heterozygosity were underrepresented in total controllers and VCs vs. progressors (p = .004; p = .007 and p = .002, pbonferroni = 0.032; p = .004, respectively). Possession of the +2919 T > G SNP (dominant mode) was associated with HIV-1 progression (controllers vs. progressors: p = .001, pbonferroni = 0.016). The +2919 T > G SNP is in linkage disequilibrium (LD; r2 = 0.73) with two 5'UTR SNPs (-2459G > A and -2135 T > C; r2 = 1: 5'UTR-2SNP-hap). The 5'UTR-2SNP-hap was lower in total controllers and VCs vs. progressors (p = .003, pbonferroni = 0.048; p = .01, respectively). Results suggest -2459G > A, -2135 T > C, and + 2919 T > G as key CCR5 variants in HIV-1 control.

CCR5 expression, haplotype and immune activation in protection from infection in HIV-exposed uninfected individuals in HIV-serodiscordant relationships.

Several host factors have been implicated in resistance to HIV infection in individuals who remain HIV-seronegative despite exposure. In a cohort of HIV-serodiscordant heterosexual couples, we investigated interactions between systemic inflammation and T-cell activation in resistance to HIV infection. Males and females in stable long-term relationships with either HIV-infected or uninfected partners were recruited, blood T-cell activation (CD38, HLA-DR, CCR5 and Ki67) and plasma cytokine concentrations were evaluated. The HIV-negative exposed individuals had significantly lower frequencies of CCR5+ CD4+ and CD8+ T cells than unexposed individuals. Mean fluorescence intensity of CCR5 expression on CD4+ T cells was significantly lower in HIV-negative exposed than unexposed individuals. Protective CCR5 haplotypes (HHA/HHF*2, HHF*2/HHF*2, HHC/HHF*2, HHA/HHA, HHA/HHC and HHA/HHD) tended to be over-represented in exposed compared with unexposed individuals (38% versus 28%, P = 0·58) whereas deleterious genotypes (HHC/HHD, HHC/HHE, HHD/HHE, HHD/HHD and HHE/HHE) were under-represented (26% versus 44%; P = 0·16). Plasma concentrations of interleukin-2 (P = 0·02), interferon-γ (P = 0·05) and granulocyte-macrophage colony-stimulating factor (P = 0·006) were lower in exposed compared with unexposed individuals. Activation marker expression and systemic cytokine concentrations were not influenced by gender. We conclude that the dominant signature of resistance to HIV infection in this cohort of exposed but uninfected individuals was lower T-cell CCR5 expression and plasma cytokine concentrations.

CXCR6 gene characterization in two ethnically distinct South African populations and association with viraemic disease control in HIV-1-infected black South African individuals.

CXCR6 genetic variation was described for HIV-1-uninfected black (n=41) and Caucasian (n=40) South Africans. We also investigated the CXCR6 rs2234358 and rs2234355 single nucleotide polymorphisms in HIV-1 disease control in 124 HIV-1-infected drug-naïve black individuals [elite controllers (n=11), viraemic controllers (VCs, n=30), high viral load long-term nonprogressors (HVL LTNPs, n=11) and progressors (n=72)] compared to healthy controls (HCs; n=232). The rs2234358-T allele was underrepresented in VCs (40.0%) compared to HCs (59%, P=0.006), HVL LTNPs (72.7%, P=0.012) and progressors (59%, P=0.014). The rs2234358-TT genotype was underrepresented in VCs (7%) compared to progressors (32%; OR=6.57, P=0.006) and HCs (35%; OR=7.18, P=0.001, Pbonferroni=0.034). The rs2234355-GA genotype was overrepresented in VCs (80%) compared to HCs (50.4%; OR=0.25, P=0.003) and progressors (29.17%; OR=0.10, P=3.8×10-5, Pbonferroni=0.001). The combination of rs2234355-GA in the absence of rs2234358-TT was overrepresented in VCs (80%) compared to HCs (32.6%, OR=0.12, P=1×10-6, Pbonferroni=3.4×10-5) and to progressors (16.7%; OR=0.05, P<1×10-8, Pbonferroni<1×10-7).

Characterization of CYP2B6 and CYP2A6 Pharmacogenetic Variation in Sub-Saharan African Populations.

Genetic variation in CYP2B6 and CYP2A6 is known to impact interindividual response to antiretrovirals, nicotine, and bupropion, among other drugs. However, the full catalogue of clinically relevant pharmacogenetic variants in these genes is yet to be established, especially across African populations. This study therefore aimed to characterize the star allele (haplotype) distribution in CYP2B6 and CYP2A6 across diverse and understudied sub-Saharan African (SSA) populations. We called star alleles from 961 high-depth full genomes using StellarPGx, Aldy, and PyPGx. In addition, we performed CYP2B6 and CYP2A6 star allele frequency comparisons between SSA and other global biogeographical groups represented in the new 1000 Genomes Project high-coverage dataset (n = 2,000). This study presents frequency information for star alleles in CYP2B6 (e.g., *6 and *18; frequency of 21-47% and 2-19%, respectively) and CYP2A6 (e.g., *4, *9, and *17; frequency of 0-6%, 3-10%, and 6-20%, respectively), and predicted phenotypes (for CYP2B6), across various African populations. In addition, 50 potentially novel African-ancestry star alleles were computationally predicted by StellarPGx in CYP2B6 and CYP2A6 combined. For each of these genes, over 4% of the study participants had predicted novel star alleles. Three novel star alleles in CYP2A6 (*54, *55, and *56) and CYP2B6 apiece, and several suballeles were further validated via targeted Single-Molecule Real-Time resequencing. Our findings are important for informing the design of comprehensive pharmacogenetic testing platforms, and are highly relevant for personalized medicine strategies, especially relating to antiretroviral medication and smoking cessation treatment in Africa and the African diaspora. More broadly, this study highlights the importance of sampling diverse African ethnolinguistic groups for accurate characterization of the pharmacogene variation landscape across the continent.

KIR2DS4 allelic variants: Differential effects on in utero and intrapartum HIV-1 mother-to-child transmission.

KIR2DS4 is the only activating gene within the A haplotype, and alleles of KIR2DS4 can encode either functional (KIR2DS4-f) or non-functional (KIR2DS4-v) variants. To establish the role of KIR2DS4 in the context of HIV-1 mother-to-child transmission, we KIR genotyped 145 HIV-1 non-transmitting mothers (NT) and their exposed uninfected infants (EU), and 72 HIV-1 transmitting mothers (TR) and their infected infants [intrapartum (IP), in utero (IU) or IU2 (an IU-enriched infected group)]. The frequency of KIR2DS4-v was significantly higher in IU2 infants compared to EU infants (P=0.022, OR=2.88); this association was more significant amongst AA haplotypes (P=0.004, OR=18.4). Possession of KIR2DS4-f in the mother with absence in the infant (M+I- discordance) was associated with a higher risk of IP transmission (P=0.005, OR=3.84); whilst in M-I+ discordance, infant possession of KIR2DS4-v was associated with increased risk of IU acquisition (P=0.002; OR=6.40). This study highlights the importance of KIR2DS4 in HIV-1 transmission/acquisition.

Characterization of CYP2D6 Pharmacogenetic Variation in Sub-Saharan African Populations.

Cytochrome P450 2D6 (CYP2D6) is a key enzyme in drug response owing to its involvement in the metabolism of ~ 25% of clinically prescribed medications. The encoding CYP2D6 gene is highly polymorphic, and many pharmacogenetics studies have been performed worldwide to investigate the distribution of CYP2D6 star alleles (haplotypes); however, African populations have been relatively understudied to date. In this study, the distributions of CYP2D6 star alleles and predicted drug metabolizer phenotypes-derived from activity scores-were examined across multiple sub-Saharan African populations based on bioinformatics analysis of 961 high-depth whole genome sequences. This was followed by characterization of novel star alleles and suballeles in a subset of the participants via targeted high-fidelity Single-Molecule Real-Time resequencing (Pacific Biosciences). This study revealed varying frequencies of known CYP2D6 alleles and predicted phenotypes across different African ethnolinguistic groups. Twenty-seven novel CYP2D6 star alleles were predicted computationally and two of them were further validated. This study highlights the importance of studying variation in key pharmacogenes such as CYP2D6 in the African context to better understand population-specific allele frequencies. This will aid in the development of better genotyping panels and star allele detection approaches with a view toward supporting effective implementation of precision medicine strategies in Africa and across the African diaspora.

Marked differences in CCR5 expression and activation levels in two South African populations.

The chemokine receptor CCR5 is pivotal in determining an individual's susceptibility to HIV-1 infection and rate of disease progression. To establish whether population-based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5(+) cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) -cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5(+) CD8(+) T-cell subsets and CCR5(+) NK-cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA-DR expression, than SAC individuals in CD4(+) T-cell subsets (P = 0·002) and CD56(+) NK-cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both cell activation and CCR5 expression, which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression.

CCR5 promoter haplotypes differentially influence CCR5 expression on natural killer and T cell subsets in ethnically divergent HIV-1 uninfected South African populations.

CCR5 plays a critical and central role in HIV-1 infection and, to date, a number of genetic mutations and haplotypes within the gene have been found to positively or negatively influence an individual's susceptibility and rate of disease progression. In this study, we have evaluated the influence of CCR5 haplotypes, HHA, HHC, HHD, and HHE, on CCR5 expression in healthy HIV-1 uninfected individuals from two populations, South African Africans (SAA, n = 22) and South African Caucasians (SAC, n = 31). CCR5 haplotypes were determined through sequencing and real time polymerase chain reaction. Flow cytometry was used to quantitate CCR5 surface expression, as both CCR5 density and percentage of CCR5-expressing cells, on B, T, natural killer (NK) cells and monocytes. SAA individuals positive for the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells in comparison to individuals without HHA (P = 0.001). HHC+ SAC individuals had significantly higher CCR5 molecules per cell (density) on NK (CD56+) and CD16+ CD56+ NK cell subsets (P = 0.030 and P = 0.024, respectively) compared to HHC- SAC individuals. Haplotypes HHD and HHE had no impact on CCR5 expression. Overall, our data highlight that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals. Findings raise the question as to the role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression.

FCGR3A gene duplication, FcγRIIb-232TT and FcγRIIIb-HNA1a associate with an increased risk of vertical acquisition of HIV-1.

BACKGROUND: Some mother-to-child transmission (MTCT) studies suggest that allelic variations of Fc gamma receptors (FcγR) play a role in infant HIV-1 acquisition, but findings are inconsistent. To address the limitations of previous studies, the present study investigates the association between perinatal HIV-1 transmission and FcγR variability in three cohorts of South African infants born to women living with HIV-1. METHODS: This nested case-control study combines FCGR genotypic data from three perinatal cohorts at two hospitals in Johannesburg, South Africa. Children with perinatally-acquired HIV-1 (cases, n = 395) were compared to HIV-1-exposed uninfected children (controls, n = 312). All study participants were black South Africans and received nevirapine for prevention of MTCT. Functional variants were genotyped using a multiplex ligation-dependent probe amplification assay, and their representation compared between groups using logistic regression analyses. RESULTS: FCGR3A gene duplication associated with HIV-1 acquisition (OR = 10.27; 95% CI 2.00-52.65; P = 0.005) as did the FcγRIIb-232TT genotype even after adjusting for FCGR3A copy number and FCGR3B genotype (AOR = 1.72; 95%CI 1.07-2.76; P = 0.024). The association between FcγRIIb-232TT genotype and HIV-1 acquisition was further strengthened (AOR = 2.28; 95%CI 1.11-4.69; P = 0.024) if adjusted separately for FCGR2C c.134-96C>T. Homozygous FcγRIIIb-HNA1a did not significantly associate with HIV-1 acquisition in a univariate model (OR = 1.42; 95%CI 0.94-2.16; P = 0.098) but attained significance after adjustment for FCGR3A copy number and FCGR2B genotype (AOR = 1.55; 95%CI 1.01-2.38; P = 0.044). Both FcγRIIb-232TT (AOR = 1.83; 95%CI 1.13-2.97; P = 0.014) and homozygous FcγRIIIb-HNA1a (AOR = 1.66; 95%CI 1.07-2.57; P = 0.025) retained significance when birthweight and breastfeeding were added to the model. The common FCGR2A and FCGR3A polymorphisms did not associate with HIV-1 acquisition. CONCLUSIONS: Collectively, our findings suggest that the FcγRIIb-232TT genotype exerts a controlling influence on infant susceptibility to HIV-1 infection. We also show a role for less studied variants-FCGR3A duplication and homozygous HNA1a. These findings provide additional insight into a role for FcγRs in HIV-1 infection in children.

An HIV Vaccine Protective Allele in FCGR2C Associates With Increased Odds of Perinatal HIV Acquisition.

In the Thai RV144 HIV-1 vaccine trial, a three-variant haplotype within the Fc gamma receptor 2C gene (FCGR2C) reduced the risk of HIV-1 acquisition. A follow-on trial, HVTN702, of a similar vaccine candidate found no efficacy in South Africa, where the predominant population is polymorphic for only a single variant in the haplotype, c.134-96C>T (rs114945036). To investigate a role for this variant in HIV-1 acquisition in South Africans, we used the model of maternal-infant HIV-1 transmission. A nested case-control study was conducted of infants born to mothers living with HIV-1, comparing children with perinatally-acquired HIV-1 (cases, n = 176) to HIV-1-exposed uninfected children (controls, n = 349). All had received nevirapine for prevention of mother-to-child transmission. The FCGR2C copy number and expression variants (c.-386G>C, c.-120A>T c.169T>C, and c.798+1A>G) were determined using a multiplex ligation-dependent probe amplification assay and the c.134-96C>T genotype with Sanger sequencing. The copy number, genotype and allele carriage were compared between groups using univariate and multivariate logistic regression. The FCGR2C c.134-96C>T genotype distribution and copy number differed significantly between HIV-1 cases and exposed-uninfected controls (P = 0.002, PBonf = 0.032 and P = 0.010, PBonf = > 0.05, respectively). The FCGR2C c.134-96T allele was overrepresented in the cases compared to the controls (58% vs 42%; P = 0.001, PBonf = 0.016). Adjusting for birthweight and FCGR2C copy number, perinatal HIV-1 acquisition was associated with the c.134-96C>T (AOR = 1.89; 95% CI 1.25-2.87; P = 0.003, PBonf = 0.048) and c.169C>T (AOR = 2.39; 95% CI 1.45-3.95; P = 0.001, PBonf = 0.016) minor alleles but not the promoter variant at position c.-386G>C. The c.134-96C>T variant was in strong linkage disequilibrium with the c.169C>T variant, but remained significantly associated with perinatal acquisition when adjusted for c.169C>T in multivariate analysis. In contrast to the protective effect observed in the Thai RV144 trial, we found the FCGR2C variant c.134-96T-allele associated with increased odds of perinatal HIV-1 acquisition in South African children. These findings, taken together with a similar deleterious association found with HIV-1 disease progression in South African adults, highlight the importance of elucidating the functional relevance of this variant in different populations and vaccination/disease contexts.

Reduced CCR5 Expression and Immune Quiescence in Black South African HIV-1 Controllers.

Unique Individuals who exhibit either suppressive HIV-1 control, or the ability to maintain low viral load set-points and preserve their CD4+ T cell counts for extended time periods in the absence of antiretroviral therapy, are broadly termed HIV-1 controllers. We assessed the extent to which black South African controllers (n=9), differ from uninfected healthy controls (HCs, n=22) in terms of lymphocyte and monocyte CCR5 expression (density and frequency of CCR5-expressing cells), immune activation as well as peripheral blood mononuclear cell (PBMC) mitogen-induced chemokine/cytokine production. In addition, relative CD4+ T cell CCR5 mRNA expression was assessed in a larger group of controllers (n=20) compared to HCs (n=10) and HIV-1 progressors (n=12). Despite controllers having significantly higher frequencies of activated CD4+ and CD8+ T cells (HLA-DR+) compared to HCs, CCR5 density was significantly lower in these T cell populations (P=0.039 and P=0.064, respectively). This lower CCR5 density was largely attributable to controllers with higher VLs (>400 RNA copies/ml). Significantly lower CD4+ T cell CCR5 density in controllers was maintained (P=0.036) when HCs (n=12) and controllers (n=9) were matched for age. CD4+ T cell CCR5 mRNA expression was significantly less in controllers compared to HCs (P=0.007) and progressors (P=0.002), whereas HCs and progressors were similar (P=0.223). The levels of soluble CD14 in plasma did not differ between controllers and HCs, suggesting no demonstrable monocyte activation. While controllers had lower monocyte CCR5 density compared to the HCs (P=0.02), significance was lost when groups were age-matched (P=0.804). However, when groups were matched for both CCR5 promoter haplotype and age (n=6 for both) reduced CCR5 density on monocytes in controllers relative to HCs was highly significant (P=0.009). Phytohemagglutinin-stimulated PBMCs from the controllers produced significantly less CCL3 (P=0.029), CCL4 (P=0.008) and IL-10 (P=0.028) compared to the HCs, which was largely attributable to the controllers with lower VLs (<400 RNA copies/ml). Our findings support a hypothesis of an inherent (genetic) predisposition to lower CCR5 expression in individuals who naturally control HIV-1, as has been suggested for Caucasian controllers, and thus, likely involves a mechanism shared between ethnically divergent population groups.

Low Pretreatment Viral Loads in Infants With HIV in an Era of High-maternal Antiretroviral Therapy Coverage.

BACKGROUND: With expansion of antiretroviral therapy (ART) programs, transmission rates are low but new infant infections still occur. We investigated predictors of pre-ART viral load (VL) and CD4+ T-cell counts and percentages in infants diagnosed with HIV at birth in a setting with high coverage of maternal ART and infant prophylaxis. METHODS: As part of an early treatment study, 97 infants with confirmed HIV-infection were identified at a hospital in Johannesburg, South Africa. Infant VL and CD4+ T-cell parameters were measured before ART initiation. Data were collected on maternal characteristics, including VL, CD4+ T-cell counts and ART, and infant characteristics, including sex, birth weight, and mode of delivery. RESULTS: Pre-ART, median infant VL was 28,405 copies/mL [interquartile range (IQR): 2515-218,150], CD4+ T-cell count 1914 cells/mm (IQR: 1474-2639) and percentage 40.8% (IQR: 32.2-51.2). Most (80.4%) infants were born to mothers who received ART during pregnancy and 97.9% of infants received daily nevirapine prophylaxis until ART initiation at median of 2 days of age (IQR: 1-7). Infant pre-ART VL was more likely to be ≥1000 copies/mL when their mothers had VL ≥1000 copies/mL [Odds Ratio (OR): 6.88, 95% confidence interval (CI): 2.32-20.41] and was higher in boys than girls (OR: 3.29, 95% CI: 1.07-9.95). Lower maternal CD4+ T-cell count (<350 cells/mm) was associated with lower infant CD4+ T-cell count (<1500 cells/mm) (OR: 3.59, 95% CI: 1.24-10.43). CONCLUSIONS: Pre-ART VL and CD4+ T-cell parameters of intrauterine-infected infants were associated with VL and CD4+ T-cell counts of their mothers. Maternal ART during pregnancy may begin treatment of intrauterine infection and may mask the severity of disease in infected infants identified in the current era with high-maternal ART coverage.

Human leukocyte antigen class I (A, B, C) and class II (DPB1, DQB1, DRB1) allele and haplotype variation in Black South African individuals.

South Africa has a population of 58.78 million, of which 80.7% are Black African individuals, representing 9 predominant ethnic/linguistic groups (Zulu, Xhosa, Pedi, Tswana, South Sotho, Tsonga, Swati, Venda and Ndebele). HIV-1 and Mycobacterium tuberculosis infection are the leading causes of death (7.8% and 5.9%, respectively) in this population group. To provide reference HLA allele and haplotype data for studies of gene-associations with infectious/non-infectious diseases or vaccine development, we have updated previously published HLA class I (A, B, C) and class II DRB1 genotypes and determined high-resolution class II (DPB1, DQB1) genotypes for n = 142 healthy, unrelated Black South African individuals.

The impact of bone marrow stromal antigen-2 (BST2) gene variants on HIV-1 control in black South African individuals.

Bone marrow stromal cell antigen 2 (BST2 or tetherin) is a host-encoded, interferon-inducible antiviral restriction factor which blocks the release of enveloped viruses. Few studies have assessed the role of BST2 polymorphisms on HIV-1 acquisition or disease progression in sub-Saharan Africa. This study investigated the frequency of four HIV-1-associated BST2 variants rs3217318, rs12609479, rs10415893 and rs113189798 in uninfected and HIV-1 infected black South Africans. Homozygosity for the rs12609479-A minor allele, previously associated with decreased HIV-1 acquisition risk, was underrepresented in HIV-1 uninfected black South Africans (2%) compared to reference African (9%) and in particular European populations (61%) (p = .047 and p < .0001, respectively). To determine if any of these gene variants influenced HIV-1 control in the absence of antiretroviral treatment (ART), we compared HIV-1 infected ART-naïve progressors [n = 72] and controllers [n = 71], the latter includes elite controllers [EC: n = 23; VL < 50 RNA copies/ml]. Heterozygosity for the rs12609479 SNP (G/A) was enriched in progressors compared to ECs (47.2% vs 21.7%, OR = 3.50 [1.16-10.59], p = .03), while rs113189798 heterozygosity (A/G) showed a strong trend of overrepresentation in ECs compared to progressors (47.8% vs 26.4%, OR = 0.39 [0.14-1.04], p = .07). Heterozygosity for the promoter indel rs3217318 (i19/Δ19) was associated with a faster rate of CD4+ T-cell decline in progressors (p = .0134). Carriage of the rs3217318 (i19/Δ19), rs12609479 (G/G), rs10415893(G/A) and rs113189798 (A/G) combined genotype, denoted as i19Δ19 GG GA AG, was associated with significantly higher CD4+ T-cell counts in progressors (p = .03), a finding predominantly driven by the _GG_AG combination. Our data suggest that the possession of select BST2 genotype combinations may be implicated in HIV-1 disease progression and natural spontaneous control.

Quantifying the Dynamics of HIV Decline in Perinatally Infected Neonates on Antiretroviral Therapy.

BACKGROUND: Mathematical modeling has provided important insights into HIV infection dynamics in adults undergoing antiretroviral treatment (ART). However, much less is known about the corresponding dynamics in perinatally infected neonates initiating early ART. SETTING: From 2014 to 2017, HIV viral load (VL) was monitored in 122 perinatally infected infants identified at birth and initiating ART within a median of 2 days. Pretreatment infant and maternal covariates, including CD4 T cell counts and percentages, were also measured. METHODS: From the initial cohort, 53 infants demonstrated consistent decline and suppressed VL below the detection threshold (20 copies mL) within 1 year. For 43 of these infants with sufficient VL data, we fit a mathematical model describing the loss of short-lived and long-lived infected cells during ART. We then estimated the lifespans of infected cells and the time to viral suppression, and tested for correlations with pretreatment covariates. RESULTS: Most parameters governing the kinetics of VL decline were consistent with those obtained previously from adults and other infants. However, our estimates of the lifespan of short-lived infected cells were longer than published values. This difference may reflect sparse sampling during the early stages of VL decline, when the loss of short-lived cells is most apparent. In addition, infants with higher pretreatment CD4 percentage or lower pretreatment VL trended toward more rapid viral suppression. CONCLUSIONS: HIV dynamics in perinatally infected neonates initiating early ART are broadly similar to those observed in other age groups. Accelerated viral suppression is also associated with higher CD4 percentage and lower VL.

Genome-Wide Association Study Identifies Novel Colony Stimulating Factor 1 Locus Conferring Susceptibility to Cryptococcosis in Human Immunodeficiency Virus-Infected South Africans.

BACKGROUND: Cryptococcus is the most common cause of meningitis in human immunodeficiency virus (HIV)-infected Africans. Despite universal exposure, only 5%-10% of patients with HIV/acquired immune deficiency syndrome and profound CD4+ T-cell depletion develop disseminated cryptococcosis: host genetic factors may play a role. Prior targeted immunogenetic studies in cryptococcosis have comprised few Africans. METHODS: We analyzed genome-wide single-nucleotide polymorphism (SNP) genotype data from 524 patients of African descent: 243 cases (advanced HIV with cryptococcal antigenemia and/or cryptococcal meningitis) and 281 controls (advanced HIV, no history of cryptococcosis, negative serum cryptococcal antigen). RESULTS: Six loci upstream of the colony-stimulating factor 1 (CSF1) gene, encoding macrophage colony-stimulating factor (M-CSF) were associated with susceptibility to cryptococcosis at P < 10-6 and remained significantly associated in a second South African cohort (83 cases; 128 controls). Meta-analysis of the genotyped CSF1 SNP rs1999713 showed an odds ratio for cryptococcosis susceptibility of 0.53 (95% confidence interval, 0.42-0.66; P = 5.96 × 10-8). Ex vivo functional validation and transcriptomic studies confirmed the importance of macrophage activation by M-CSF in host defence against Cryptococcus in HIV-infected patients and healthy, ethnically matched controls. CONCLUSIONS: This first genome-wide association study of susceptibility to cryptococcosis has identified novel and immunologically relevant susceptibility loci, which may help define novel strategies for prevention or immunotherapy of HIV-associated cryptococcal meningitis.

HIV diagnostic challenges in breast-fed infants of mothers on antiretroviral therapy.

BACKGROUND: Prompt initiation of antiretroviral therapy (ART) for HIV-infected infants is strongly recommended but diagnostic confirmation is important as committing children to life-long ART carries serious health and social implications. METHODS: Two HIV-exposed infants in Johannesburg, South Africa were identified presenting with unusual trajectories of diagnostic nucleic acid amplification tests (NAAT) and viral load results. RESULTS: Case 1 had repeat indeterminate NAAT results during the first 3 weeks of life; repeat testing thereafter was negative with undetectable viral load including after daily nevirapine prophylaxis ended. ART was not initiated at this time. Case 2 had a single positive NAAT result at 1 month of age that prompted initiation of ART. Subsequent results were negative and ART was discontinued. Repeat negative NAAT with viral load below the limit of quantification or undetectable continued to be obtained. Shortly after and around weaning, positive NAAT results with high viral load (7.1 and 6.03 log10 copies/ml for Cases 1 and 2, respectively) were observed in both children. Both mothers were treated with tenofovir, emtricitabine and efavirenz during breastfeeding. Testing with ultrasensitive assays on early samples conclusively revealed HIV-1 proviral DNA in Case 1. Testing with ultrasensitive assays after the early period but prior to weaning did not detect HIV in either infant. CONCLUSION: We hypothesize that breast milk from the mothers of these two rare cases had HIV-specific or nonspecific factors that led to the undetectable results in already infected infants until breastfeeding ended. Our results raise the importance of repeat testing of HIV-exposed breast-fed infants after complete cessation of all breastfeeding.