Background Pretreatment HIV-1 drug resistance (PDR), including minority variants, may compromise antiretroviral therapy (ART) outcomes but remains underexplored in non-B HIV-1 subtypes. Methods This longitudinal study enrolled 106 ART-naïve Ugandan adults (2018–2020) to assess the impact of majority (≥ 20%) and minority (< 20%) pretreatment HIV-1 drug resistance mutations on virologic outcomes using next-generation sequencing of baseline HIV-1 RNA. Viral load was measured at 6, 12, and 24 months, and associations between PDR and cumulative viral load were evaluated among 38 participants with complete follow-up. Results Majority HIVDRMs were most frequent in NNRTIs and NRTIs, while minority variants were more common in PIs and INSTIs. Among participants with complete follow-up, 26% had detectable cumulative VL, of whom 91% harbored at least one HIVDRM. PDR was not significantly associated with detectable VL (OR = 5.29, 95% CI: 0.58–48.20; p = 0.224), although a trend toward poorer virologic outcomes was observed. Conclusion Pretreatment HIVDRMs, may adversely affect virologic outcomes, although no statistically significant association was observed. Expanding access to baseline resistance testing could improve ART selection and treatment outcomes in settings with diverse HIV-1 subtypes.

Background ODYSSEY showed superior efficacy for dolutegravir-based antiretroviral therapy (ART) versus standard of care (SOC) in children living with HIV starting first-line or second-line ART aged 4 weeks or older. Here, we aim to compare virological outcomes and resistance in the dolutegravir group versus SOC for first-line and second-line ART up to 96 weeks. Methods ODYSSEY was an open-label, multicentre, randomised, non-inferiority trial done in 29 centres in seven countries (Germany, Spain, South Africa, Thailand, the UK, Uganda, and Zimbabwe). ODYSSEY recruited children living with HIV aged at least 28 days and younger than 18 years, weighing at least 3 kg, starting first-line ART (ODYSSEY A), or switching to second-line therapy after treatment failure (ODYSSEY B). Children were randomly assigned (1:1) to dolutegravir plus two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs; dolutegravir group) versus the SOC group (non-nucleoside reverse transcriptase inhibitor [NNRTI], boosted protease inhibitor, or non-dolutegravir integrase strand-transfer inhibitor, plus two NRTIs). Two randomised cohorts were combined in this exploratory analysis: children weighing at least 14 kg were enrolled between Sept 20, 2016, and June 22, 2018, and children weighing less than 14 kg were enrolled between July 5, 2018, and Aug 26, 2019. Virological failure was defined as an inadequate virological response at week 24 with an ART switch or confirmed HIV-1 RNA viral load of at least 400 copies per mL after week 36. Virological suppression was defined as two consecutive viral loads of less than 400 copies per mL and was compared between groups, including an ART switch and death as competing risks. Children with virological failure were tested for post-failure genotypic resistance, with baseline results used to identify emergent resistance. Development of emergent resistance was a secondary trial outcome and all other outcomes are exploratory. ODYSSEY was registered with ClinicalTrials.gov (NCT02259127), EUDRACT (2014-002632-14), and ISRCTN (ISRCTN91737921). Findings In ODYSSEY at enrolment, 381 participants started first-line ART (ODYSSEY A: 189 in the dolutegravir group and 192 in the SOC group) and 407 participants started second-line ART (ODYSSEY B: 202 in the dolutegravir group and 205 in the SOC group). 72 participants in ODYSSEY A and 13 participants in ODYSSEY B weighed less than 14 kg. 401 (51%) of 788 participants were female and 387 (49%) were male. Virological suppression occurred significantly earlier in the dolutegravir group (adjusted [cause-specific] hazard ratio [HR] 157 [95% CI 135 to 183]; p<00001). Overall, 51 (13%) participants had virological failure by 96 weeks in the dolutegravir group versus 86 (22%) in the SOC group (including 18 [10%] vs 43 [22%] in ODYSSEY A and in 33 [16%] vs 43 [21%] in ODYSSEY B; adjusted HR 056 [040 to 079]; p=00011). Among ODYSSEY B participants starting dolutegravir, virological failure was higher in children starting zidovudine (HR 222 [101 to 488]; p=0048) and similar in those starting tenofovir disoproxil fumarate (119 [050 to 283]; p=070) compared with abacavir. Time to virological suppression was marginally faster in participants receiving second-line dolutegravir and abacavir with high-level abacavir resistance at baseline compared with those with no, low-level, intermediate- level resistance (cause-specific HR 170 [101 to 285]; p=0046); and failure rates by week 96 were similar (HR 090 [023 to 361]; p=088). An estimated 1% (95% CI 0 to 2) of participants in the dolutegravir group versus 20% (14 to 26) in the SOC group in ODYSSEY A had emergent resistance to at least one drug-class within their first-line regimen (risk difference -20% [-25 to -14]; p<00001); 4% (1 to 6) versus 5% (2 to 8) had resistance to drug within their initial second-line regimen (risk difference -1% [-5 to 3]; p=060). 3% (0 to 5) of participants in the dolutegravir group had emergent integrase strand-transfer inhibitors resistance compared with 3% (1 to 6) of participants in the SOC group who had emergent resistance to the anchor drug (risk difference 0% [-4 to 3]; p=078). Interpretation Dolutegravir led to faster virological suppression and lower risk of virological failure than NNRTIs and boosted protease inhibitor-based SOC. Participants starting second-line dolutegravir-based ART with an abacavir or tenofovir backbone were at lower risk of virological failure than those starting zidovudine. During first-line therapy, dolutegravir protected against emergent resistance; starting second-line therapy, the risk of emergent resistance to nucleoside reverse transcriptase inhibitor backbone, and anchor drugs, was similar among participants starting dolutegravir within their second-line regimen and those starting mainly boosted protease inhibitor-based SOC.

Mechanisms by which prior tuberculosis (TB) increases long-term risk for cancer, cardiovascular, and neurological disorders remain unclear, particularly in people with HIV (PWH). This study investigated DNA methylation (DNAm) patterns and associated pathways in PWH with and without prior TB infection. DNAm was analyzed in blood samples from 30 PWH (10 with prior latent TB infection [LTBI], 10 with previous successfully treated active TB, and 10 with no TB) using the Illumina MethylationEPIC BeadChip covering over 850,000 CpG sites. Epigenetic age was estimated, and age acceleration was calculated. Differentially methylated CpGs (dmCpGs) and regions (DMRs) were identified, and functional enrichment analyses for Gene Ontology, KEGG pathways, PANTHER database, and gene set enrichment analysis (DisGeNET, dbGaP) were performed. Statistical significance was set at a false discovery rate (FDR) of < 0.05. PWH exhibited significant epigenetic age acceleration, with a mean of 19.32 ± 10.82 years greater than chronological age. This accelerated aging was more pronounced in individuals with any prior TB infection (21.60 ± 12.03 years) compared to those without TB (17.42 ± 9.38 years). In the prior active TB vs. no TB comparison, 7461 dmCpGs were identified, corresponding to 150 DMRs (p < 0.05), with top associated genes including GRAMD1C (hypomethylation), DPP6 (hypermethylation), and HDAC4 (hypomethylation). In the LTBI vs. no TB comparison, 8598 dmCpGs were observed, corresponding to 39 DMRs (p < 0.05), associated with genes such as PLEKHG5 (hypermethylation), STK32C (hypermethylation), and SPATC1L. When comparing any prior TB (active or latent) to no TB, 71,774 dmCpGs and 14 DMRs were identified, including genes like PLEKHG5, KCNN3, and BRSK2. Pathway analyses of prior TB (active or latent) vs. no TB revealed enrichment in neurogenesis, neuron differentiation, axon guidance, and neuroactive ligand signaling. Additional enriched pathways included those related to platelet activation, vascular muscle contraction, and chemokine signaling. Cancer-related pathways such as proteoglycans in cancer, small cell lung cancer, prostate cancer, breast cancer, hepatocellular carcinoma, and thyroid cancer were also enriched. PANTHER analysis showed consistent enrichment in the Wnt signaling pathway and inflammation-mediated pathways across compared groups. DisGeNET analysis linked prior TB DNAm patterns to lymphoid leukemia, while dbGaP analysis identified associations with phenotypes like asthma, body mass index, tunica media, and lymphocyte count. Prior TB infection in PWH is associated with distinct DNAm changes in pathways related to neural function, cardiovascular health, and cancer risk, and is linked to more pronounced epigenetic age acceleration, suggesting epigenetic mechanisms for TB-related long-term complications.

With the current elimination of mother to child transmission (EMTCT) of HIV, the number of HIV-positive newborns has greatly reduced. Some countries have successfully eliminated HIV infections among newborn babies. This study was nested within the DRIBS (Drug Resistance testing among Infants at Baseline Study), which enrolled 100 infants at the time of treatment initiation between 2017 and 2023. Infants were followed for two years. Viral load (VL) was measured every six months and after completion of the three sessions of intensified adherence counseling (IAC). IAC and HIV drug resistance testing were performed for VL greater than 1000 copies/ml. The median age at diagnosis was 79 (IQR, 57.75;140.75) days, with 4

Background Several mechanisms including reduced CCR5 expression, protective HLA, viral restriction factors, broadly neutralizing antibodies, and more efficient T-cell responses, have been reported to account for HIV control among HIV controllers. However, no one mechanism universally accounts for HIV control among all controllers. In this study we determined whether reduced CCR5 expression accounts for HIV control among Ugandan HIV controllers. We determined CCR5 expression among Ugandan HIV controllers compared with treated HIV non-controllers through ex-vivo characterization of CD4 + T cells isolated from archived PBMCs collected from the two distinct groups. Results The percentage of CCR5 + CD4 + T cells was similar between HIV controllers and treated HIV non-controllers (ECs vs. NCs, P = 0.6010; VCs vs. NCs, P = 0.0702) but T cells from controllers had significantly reduced CCR5 expression on their cell surface (ECs vs. NCs, P = 0.0210; VCs vs. NCs, P = 0.0312). Furthermore, we identified rs1799987 SNP among a subset of HIV controllers, a mutation previously reported to reduce CCR5 expression. In stark contrast, we identified the rs41469351 SNP to be common among HIV non-controllers. This SNP has previously been shown to be associated with increased perinatal HIV transmission, vaginal shedding of HIV-infected cells and increased risk of death. Conclusion CCR5 has a non-redundant role in HIV control among Ugandan HIV controllers. HIV controllers maintain high CD4 + T cells despite being ART naïve partly because their CD4 + T cells have significantly reduced CCR5 densities.

Objective: In a multicountry prospective cohort of persons with HIV from six countries between 2007 and 2015, we evaluated long-term outcomes of first-line non-nucleoside reverse-transcriptase inhibitor-based antiretroviral therapy (ART), and risk factors for loss-to-follow-up, mortality, virological failure, and incomplete CD4+ T-cell recovery. Methods: We calculated cumulative incidence of lost-to-follow-up, death, virological failure (VL ≥ 1000 cps/ml) and incomplete CD4+ T-cell recovery (<500 cells/μl) at successive years, using Kaplan–Meier and Cox regression. Results: Of 2735 participants, 58.0% were female, median age was 37 (interquartile range [IQR] 32–43) years, and median pre-ART CD4+ T-cell count was 135 (IQR 63–205)/μl. Total follow-up time was 7208 person-years (median 24.3 months, IQR 18.7–58.3). Deaths by any cause and loss to follow-up occurred mostly during the first year of ART (84%, 201/240 and 56%, 199/353, respectively). During their first 6 years of ART, 71% (95% confidence interval [CI] 69.0–73.7) were retained on first-line, and among those 90–93% sustained viral suppression (<1000 cps/ml); CD4+ T-cell recovery was incomplete in 60% (220/363) of participants. The risk factors associated with poor outcomes during long-term ART were: for loss-to-follow-up, recent VL ≥1000 cps/ml, recent CD4+ T-cell count ≤50 cells/μl, age <30 years, being underweight; for mortality, recent CD4+ T-cell count ≤50 cells/μl; and, for virological failure, age <40 years, recent CD4+ T-cell count ≤200 cells/μl, poor adherence, male sex, and low-level viremia. Conclusion: To achieve long-term ART success towards the UNAIDS targets, early ART initiation is crucial, coupled with careful monitoring and retention support, particularly in the first year of ART. Male and youth-centred care delivery models are needed to improve outcomes for those vulnerable groups.

Background Our understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia. Results In this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4 + T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4 + counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001). Conclusions The smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

Abstract IntroductionBy Sept 2018, only 59.3% of all HIV positive children on cART under 5 years were achieving virological suppression compared to 88.4% in the general population in Uganda. CCR5 promoter genotype specifically single nucleotide polymorphisms have been linked to modulate patient virological status. However, the few studies that have studied the association in infants have utilized allele-specific PCR a genotypic method limited to detecting already known SNPs. By using Sanger sequencing, we explored the association taking into account novel mutations. Methods Following ethical approvals, a cross sectional study was conducted using archived buffy coat samples from a pediatric HIV drug resistance study between May 2019 and January 2022 at the Joint Clinical Research Centre in Kampala, Uganda. 100 HIV seropositive infant buffy coat samples were sequenced for CCR5 SNP genotype following nucleic acid extraction, polymerase chain reaction and sanger sequencing. Odds ratios were used to determine the association between CCR5 SNPs and infant HIV plasma loads. Results 10 SNPs were identified with frequencies as follows; 58227G/A (21.8%), 58636A/G (28.7%), 58934T/G (74.7%), 59029G/A (48.3%), 59353T/C (55.2%), 59356C/T (23.0%), 59402G/A (94.2%) 59653C/T (66.7%), 59802A/C (34.5%), and 60024A/G (34.5%). Notably, 58227G/A, 58636A/G, 59802A/C, and 60024A/G are novel SNPs. However, none of these SNPs was significantly associated with infant virological failure but SNPs 59029G/A and 59353T/C showed higher odds of occurring in non-suppressors whereas 59356C/T and 59402G/A appeared to correlate with reduced plasma HIV loads. Infant virological suppression remained at 59% and in agreement with the national data. Conclusion Our study augments previous studies that CCR5 promoter SNPs play a role in modulating patient virological status however, strong conclusions could be drawn from either utilizing in-vitro studies or large epidemiological studies.

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

OBJECTIVES:The second-generation integrase strand transfer inhibitor (INSTI) bictegravir is becoming accessible in low- and middle-income countries (LMICs), and another INSTI, cabotegravir, has recently been approved as a long-acting injectable. Data on bictegravir and cabotegravir susceptibility in raltegravir-experienced HIV-1 subtype A- and D-infected patients carrying drug resistance mutations (DRMs) remain very scarce in LMICs.PATIENTS AND METHODS:HIV-1 integrase (IN)-recombinant viruses from eight patients failing raltegravir-based third-line therapy in Uganda were genotypically and phenotypically tested for susceptibility to bictegravir and cabotegravir. Ability of these viruses to integrate into human genomes was assessed in MT-4 cells.RESULTS:HIV-1 IN-recombinant viruses harbouring single primary mutations (N155H or Y143R/S) or in combination with secondary INSTI mutations (T97A, M50I, L74IM, E157Q, G163R or V151I) were susceptible to both bictegravir and cabotegravir. However, combinations of primary INSTI-resistance mutations such as E138A/G140A/G163R/Q148R or E138K/G140A/S147G/Q148K led to decreased susceptibility to both cabotegravir (fold change in EC50 values from 429 to 1000×) and bictegravir (60 to 100×), exhibiting a high degree of cross-resistance. However, these same IN-recombinant viruses showed impaired integration capacity (14% to 48%) relative to the WT HIV-1 NL4-3 strain in the absence of drug.CONCLUSIONS:Though not currently widely accessible in most LMICs, bictegravir and cabotegravir offer a valid alternative to HIV-infected individuals harbouring subtype A and D HIV-1 variants with reduced susceptibility to first-generation INSTIs but previous exposure to raltegravir may reduce efficacy, more so with cabotegravir.

Background Mechanisms for HIV control among HIV-1 elite and viremic-controllers are not fully understood. In Uganda, Studies have reported individuals who without Antiretroviral therapy have the inherent ability to control HIV progression to AIDS for a period of greater than 5 years. However, reasons for this phenotype are not understood. The study objective was to determine the distribution of CCR5 co-receptor on CD4+ T-cells and its associated promoter variants among HIV-1 elite and viremic-controllers. Methods We isolated CD4+T-cells from PBMCs using EasySep CD4+ T-cell negative selection kit, and stimulated them with anti-CD3 and anti-CD28 for 48 hours. To quantify CCR5 expression, we performed immune-phenotyping using flow cytometry. CCR5 promoter polymorphisms were determined through sanger sequencing. The Kruskal–Wallis and the Mann-Whitney test were used to compare differences in the percentages of CCR5+ CD4+ T-cells and the differences in CCR5 densities on CD4+ T-cells respectively. p values < 0.05 were considered significant. Results The percentage of CCR5+CD4+ T-cells was higher among the non-controllers compared to the controllers although, the difference was not statistically significant; elite and viremic-controllers (p=0.9173), viremic and non-controllers (0.0702), elite and non-controllers (0.6010). Of significance was the CCR5 densities on CD4+ T-cells, which were significantly higher among non-controllers relative to the controllers; elite and viremic-controllers (p=3048), viremic and non-controllers (P=0.0312), elite and non-controllers (P=0.0210) From the sequence analysis, the rs1799987A>G mutation was found among elite (71%) and viremic-controllers (61%), while the -2459A/A and rs41469351C>T mutation were among the non-controllers (57%). This study also identified two novel mutations 1070T>G and 785A>G among the elite controllers (14.3%). Conclusion Rs1799987 SNP highly detected among the elite and viremic controllers may be associated with reduced CCR5 densities on CD4+ T-cells while higher frequency of -2459 A/A and rs41469351 SNP among non-controllers may be associated with increased CCR5 densities on CD4+ T-cells. Thus Rs1799987 SNP may be responsible for the delayed HIV progression among elite and viremic controllers, while -2459A/A and rs41469351 SNP may be responsible for the rapid progression of HIV among non-controllers. In vitro studies are needed to study the effect of the two novel mutations 1070T>G and 785A>G among elite-controllers.

Abstract 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 Resistance to antiretroviral drugs is a major challenge among Human Immunodeficiency Virus (HIV) positive patients receiving antiretroviral therapy (ART). Mutations that arise as a result of this are diverse across the various drugs, drug classes, drug regimens and subtypes. In Uganda, there is a paucity of information on how these mutations differ among the different drug regimens and the predominant HIV-1 subtypes. The purpose of this study was to determine mutation profile differences between first-line drug regimens: TDF/3TC/EFV and AZT/3TC/EFV and HIV-1 subtypes: A and D in Uganda. The study also investigated the potential usage of rilpivirine, doravirine and etravirine in patients who failed treatment on efavirenz. Methods A retrospective study was conducted on 182 archived plasma samples obtained from patients who were experiencing virological failure between 2006 and 2017 at five Joint Clinical Research Center (JCRC) sites in Uganda. Sanger sequencing of the Reverse Transcriptase (RT) gene from codons 1–300 was done. Mutation scores were generated using the Stanford University HIV Drug Resistance Database. A Chi-square test was used to determine the association between drug resistance mutations (DRMs) and drug regimens or HIV-1 subtypes. Results The prevalence of DRMs was 84.6% among patients failing a first-line efavirenz (EFV)-based regimen. The most prevalent Nucleoside Reverse Transcriptase Inhibitor (NRTI) mutations were M184V/I (67.3%), K219/Q/E (22.6%) and K65R (21.1%). While K103N (50.8%) and G190A/S/E/G (29.1%) were the most prevalent Non-Nucleoside Reverse Transcriptase Inhibitor (NNTRI) mutations. As expected, discriminatory DRMs such as K65R, L74I, and Y115F were noted in Tenofovir (TDF) containing regimens while the Thymidine Analogue Mutations (TAMs) L210W and T215 mutations were in Zidovudine (AZT)-based regimens. No significant difference (p = 0.336) was found for overall DRMs between HIV-1 subtypes A and D. Among the patients who had resistance to EFV, 37 (23.6%) were susceptible to newer NNRTIs such as Rilpivirine and Etravirine. Conclusion Accumulation of DRMs between AZT/3TC/EFV and TDF/3TC/EFV is comparable but individual mutations that confer resistance to particular drugs should be considered at virological failure. Having either HIV-1 subtype A or D is not associated with the acquisition of DRMs, therefore HIV diversity should not determine the choice of treatment. Rilpivirine, etravirine and doravirine had minimal benefits for patients who failed on efavirenz.

BACKGROUND Increasing first-line treatment failures in low- and middle-income countries (LMICs) have led to increased use of integrase strand transfer inhibitors (INSTIs) such as dolutegravir. However, HIV-1 susceptibility to INSTIs in LMICs, especially with previous raltegravir exposure, is poorly understood due to infrequent reporting of INSTI failures and testing for INSTI drug resistance mutations (DRMs). METHODS A total of 51 non-subtype B HIV-1 infected patients failing third-line (raltegravir-based) therapy in Uganda were initially selected for the study. DRMs were detected using Sanger and deep sequencing. HIV integrase genes of 13 patients were cloned and replication capacities (RCs) and phenotypic susceptibilities to dolutegravir, raltegravir and elvitegravir were determined with TZM-bl cells. Spearman's correlation coefficient was used to determine cross-resistance between INSTIs. RESULTS INSTI DRMs were detected in 47% of patients. HIV integrase-recombinant virus carrying one primary INSTI DRM (N155H or Y143R/S) was susceptible to dolutegravir but highly resistant to raltegravir and elvitegravir (>50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to raltegravir, elvitegravir and even dolutegravir. The former multi-DRM virus had WT RC whereas the latter had lower RCs than WT. CONCLUSIONS In HIV-1 subtype A- and D-infected patients failing raltegravir and harbouring INSTI DRMs, there is high-level resistance to elvitegravir and raltegravir. More routine monitoring of INSTI treatment may be advised in LMICs, considering that multiple INSTI DRMs may have accumulated during prolonged exposure to raltegravir during virological failure, leading to high-level INSTI resistance, including dolutegravir resistance.

BackgroundAlthough minority HIV-1 drug-resistant HIV-1 variants may be selected under antiretroviral pressure, leading to therapy failure, their clinical significance remains controversial. This is particularly relevant in the case of prevention of mother-to-child transmission (MTCT), where transmitted drug resistance can affect treatment outcomes.MethodsAn ultrasensitive HIV-1 genotyping assay based on deep sequencing (DEEPGENHIV) with a 1% mutation frequency sensitivity, was used to quantify MTCT drug-resistant variants in 38 prenatally HIV-infected children experiencing (Group I, n=27) or not (Group II, n=11) virologic failure 12 months after initiating first-line antiretroviral therapy (ART) as part of a paediatric cohort in Uganda.ResultsInfants were infected with subtype A(n=20), D(n=16) or C(n=2) HIV-1 strains, distributed equally between both patients’ groups. Similarly, no significant difference was observed in intra-patient HIV-1 diversity among viruses obtained from Group I or II individuals at baseline. DEEPGENHIV was able to detect all the mutations originally detected in samples obtained from four control patients in Group II, where drug resistance was identified at baseline using Sanger sequencing, e.g. K65R (78% mutation frequency), K103N (47%), or M184V (85%). More importantly, a series of low abundance (<20% detection limit of Sanger) primary and compensatory mutations associated with resistance to PIs (D30N, Q48V), NRTIs (D67N, K219Q), or NNRTIs (L100I, K103N) were identified in both groups of patients, although just a few seem to have been selected and became majority variants after 12 or 24 months of ART.ConclusionDEEPGENHIV improves the detection of minority viral variants in infants following MTCT; however, most of the emergent HIV-1 drug resistance mutations were not present at low frequency at baseline in subjects failing ART, most likely being generated and selected following exposure to treatment. Further studies, using this or other ultrasensitive assays, are needed to better understand the transmission, dynamics and overall evolution of minority drug-resistant viruses in MTCT.

Our understanding of HIV-1 and antiretroviral treatment (ART) is strongly biased towards subtype B, the predominant subtype in North America and western Europe. Efforts to characterize the response to first-line treatments in other HIV-1 subtypes have been hindered by the availability of large study cohorts in resource-limited settings. To maximize our statistical power, we combined HIV-1 sequence and clinical data from every available study population associated with the Joint Clinical Research Centre (JCRC) in Uganda. These records were combined with contemporaneous ART-naive records from Uganda in the Stanford HIVdb database.

The global HIV-1 pandemic comprises many genetically divergent subtypes. Most of our understanding of drug resistance in HIV-1 derives from subtype B, which predominates in North America and western Europe. However, about 90% of the pandemic represents non-subtype B infections. Here, we use deep sequencing to analyze HIV-1 from infected individuals in Uganda who were either treatment-naïve or who experienced virologic failure on ART without the expected patterns of drug resistance. Our objective was to detect potentially novel associations between mutations in HIV-1 integrase and treatment outcomes in Uganda, where most infections are subtypes A or D. We retrieved a total of 380 archived plasma samples from patients at the Joint Clinical Research Centre (Kampala), of which 328 were integrase inhibitor-naïve and 52 were raltegravir (RAL)-based treatment failures. Next, we developed a bioinformatic pipeline for alignment and variant calling of the deep sequence data obtained from these samples from a MiSeq platform (Illumina). To detect associations between within-patient polymorphisms and treatment outcomes, we used a support vector machine (SVM) for feature selection with multiple imputation to account for partial reads and low quality base calls. Candidate point mutations of interest were experimentally introduced into the HIV-1 subtype B NL4-3 backbone to determine susceptibility to RAL in U87.CD4.CXCR4 cells. Finally, we carried out replication capacity experiments with wild-type and mutant viruses in TZM-bl cells in the presence and absence of RAL. Our analyses not only identified the known major mutation N155H and accessory mutations G163R and V151I, but also novel mutations I203M and I208L as most highly associated with RAL failure. The I203M and I208L mutations resulted in significantly decreased susceptibility to RAL (44.0-fold and 54.9-fold, respectively) compared to wild-type virus (EC50=0.32 nM), and may represent novel pathways of HIV-1 resistance to modern treatments.Author summary There are many different types of HIV-1 around the world. Most of the research on how HIV-1 can become resistant to drug treatment has focused on the type (B) that is the most common in high-income countries. However, about 90% of infections around the world are caused by a type other than B. We used next-generation sequencing to analyze samples of HIV-1 from patients in Uganda (mostly infected by types A and D) for whom drug treatment failed to work, and whose infections did not fit the classic pattern of adaptation based on B. Next, we used machine learning to detect mutations in these virus populations that could explain the treatment outcomes. Finally, we experimentally added two candidate mutations identified by our analysis to a laboratory strain of HIV-1 and confirmed that they conferred drug resistance to the virus. Our study reveals new pathways that other types of HIV-1 may use to evolve resistance to drugs that make up the current recommended treatment for newly diagnosed individuals.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1-5 clones) than clones in the female genital tract (mean 5.7 clones, range 3-10 clones) (p<0.0001). The higher env diversity observed within the genital tract compared to plasma was independent of HIV-1 subtype (A, C and D). Our analysis of early mucosal infections in women revealed high HIV-1 diversity in the vaginal tract but few transmitted clones in the blood. These novel in vivo finding suggest a possible mucosal sieve effect, leading to the establishment of a homogenous systemic infection.