Subtype CRF01 Research Articles

Prevalence of HIV-1 Integrase Strand Transfer Inhibitor Resistance in Treatment-Naïve Voluntary Counselling and Testing Clients by Population Sequencing and Illumina Next-Generation Sequencing in Taiwan.

PurposeIntegrase strand transfer inhibitors (INSTIs) are used as first-line therapy for HIV-1-infected patients. Next-generation sequencing (NGS) can detect low-frequency mutants; however, the clinical value of NGS to detect resistance variants is unknown. This study aimed to evaluate the prevalence of INSTI resistance in southern Taiwan and determine the clinical implications of using NGS to detect integrase region low-level resistant variants.Patients and MethodsThis retrospective cohort study included antiretroviral therapy-naïve HIV-1-infected individuals at Kaohsiung Veterans General Hospital, Taiwan, from 2013 to 2017. Drug-resistance mutations were determined, and an in-house polymerase chain reaction was used for genotyping INSTI resistance. NGS was used to assess INSTI resistance (≧1%), and the results were compared with those from population sequencing. Drug resistance-associated mutations were defined according to the 2019 IAS-USA HIV drug resistance-associated mutations list, and accessory mutations by a Stanford HIVdb score ≥10 to at least one INSTI.ResultsA total of 224 patients were included. Subtype B HIV-1 strains were found in 96% of the individuals and subtype CRF01_AE in 4%. The prevalence rates for nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and INSTI resistance were 4%, 5.8%, 0.4% and 0.9%, respectively. The most common INSTI resistance-associated mutations were G163K (0.4%) and E138A (0.4%). Of the 38 patients diagnosed in 2017 who had both NGS and population sequencing data, none had INSTI resistance-associated mutations by population sequencing; however, NGS detected four more INSTI resistance-associated mutations with low frequencies (G163R 3.25%, S153F 3.21%, S153Y 1.36% and Y143H 2.06%). Two patients with S153F and S153Y low frequencies mutations started INSTI-based highly active antiretroviral therapy, and none had virological failure by week 48.ConclusionOur findings showed a low rate of HIV drug resistance to INSTIs (0.9%) in treatment-naïve patients. NGS detected more INSTI resistance-associated mutations at a low frequency. Low-level drug resistance-associated mutations to INSTIs identified by NGS did not have an impact on the treatment response to INSTI-based first-line therapy.

Determination of reverse transcriptase inhibitor resistance mutations in HIV-1 infected children in Côte d'Ivoire.

Treatment scale-up is leading to a progressive increase in HIV resistance to antiretrovirals, especially in children. To assess resistance to reverse transcriptase inhibitors (RTIs) in HIV-1 infected children in Côte d'Ivoire, genotypic resistance tests were performed and interpreted using the ANRS algorithm (www.hivfrenchresistance.org). Phylogenetic trees were created using BioEdit v7 and Mega7 software. The frequency of resistance to at least one RTI was 79%. It was 88% for nucleoside reverse transcriptase inhibitors (NRTIs), 71% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 63% for both classes (NRTI + NNRTI). The frequency of resistance was 50% for the ZDV + 3TC + EFV combination, 42% for the ABC + 3TC + EFV combination, and 8% for the TDF + 3TC + EFV combination. Frequently encountered resistance mutations were for NRTIs: M184V (88%), TAMs (67%), T215F/I/V/Y (33%), and L74I/V (24%); for NNRTIs: K103N/S (74%), P225H (26%), and G190A/E/Q (24%). The synthesis of phylogenetic analyses showed the predominance of the viral subtype CRF02_AG (85%). These results show a high prevalence of resistance to RTIs in children infected with HIV-1. Hence the interest of a more accessible monitoring of viral load and genotypic resistance tests in HIV-1 infected children undergoing treatment in Côte d'Ivoire.

Prevalence of Antiretroviral Drug Resistance Mutations Among Pretreatment and Antiretroviral Therapy-Failure HIV Patients in Uzbekistan

To evaluate the national prevalence of antiretroviral therapy (ART)-resistant HIV-1 viruses among both ART-initiators (pretreatment drug resistance, PDR) and ART-failure HIV patients in Uzbekistan. A nation-wide, cross-sectional active HIV-1 PDR surveillance was conducted in Uzbekistan from 2015 to 2016. In total, 713 blood plasma samples from adults were collected, including samples from ART-naive patients initiating ART and ART-failure HIV patients. HIV-1 genome polregion viral sequences were obtained from 309 patients, of those 106 on ART and 203 on ART-initiators. Analysis of HIV-1 subtypes and drug resistance mutations (DRMs) to HIV protease and reverse transcriptase inhibitors was performed. Among all the viruses studied, HIV-1 CRF 02_AG recombinant was the most common-57% (176/309). The second major group was represented by A1-40.5% (125/309). Two viruses were found to be recombinants formed by subtypes A1 and CRF02_AG sequences. ART-naive cohort I (PDR) included six samples that contained at least one surveillance drug resistance mutation (SDRM) (2.96%), with the most common being K103N mutation (4/6). In ART-experienced patients, cohort II, 77.4% (82/106) of viruses contained at least one mutation against PIs, NRTIs, or NNRTIs, with the most common mutations of M184V/I (49.1%; 52/106), K65R (18.9%; 20/106), K103N (23.6%; 25/106), and G190S (22.6%; 24/106). The significant difference in frequency of mutations was found between two dominant subtypes, A1 and CRF02_AG. The molecular epidemiological profile of HIV infection in Uzbekistan has changed toward a predominance of CRF02_AG viruses. In the first national-scale study of the PDR prevalence, it was found to be relatively low (2.96%). The DR mutations in failure patients correspond to the main therapy regimens (NRTI/NNRTI) adopted in the country. The observations provide new evidence for differences in ART efficacy and resistance profiles for different subtypes.

Exploring the energetic basis of binding of currently used drugs against HIV-1 subtype CRF01_AE protease via molecular dynamics simulations

Non-B strains cause nearly 90% of the worldwide human immunodeficiency virus (HIV) infections. At the same time, the protease inhibitors (PIs) were designed for subtype B. Therefore, the use of PIs in the non-B subtype context requires further investigation. Herein, we have investigated the effectiveness of currently used four PIs, namely atazanavir, darunavir, lopinavir, and tipranavir against subtype CRF01_AE (PRCRF) by employing the MD/MMPBSA (molecular dynamics/molecular mechanics Poisson-Boltzmann surface area) scheme. Our investigation reveals that tipranavir is the most potent inhibitor against PRCRF while the other three PIs display a similar binding affinity. The energetic penalty arises due to the desolvation of polar groups always disfavor the association between PRCRF and PI, and this contribution is the least in the case of tipranavir/PRCRF compared to the other three PI-PRCRF complexes resulting in a better binding affinity for tipranavir. Further, it is revealed that the primary interaction controlling the binding of inhibitors with PRCRF is the van der Waals forces. The dynamic cross-correlation map and principal component analysis show that the anti-correlated motion at the flap region of PRCRF is diminished after the ligand binding. Further, our studies indicate that D25' forms a stable H-bond with darunavir, lopinavir, and tipranavir, while D25 forms a stable H-bond with atazanavir. The per-residue based decomposition of free energy reveals the actual residual origin of the binding free energy and identify the hotspot residues. Overall, the data presented in this study can guide the computer-aided rational design of more potent drugs targetting HIV-1 PRCRF. Communicated by Ramaswamy H. Sarma

The Emerging of CRF01_AE: A Clinical Story and Future HIV/AIDS Situation in Thailand.

The HIV epidemic in Thailand in the 1980's compromised the country's socio-economic development. The epidemic first became evident in the community of men with male sexual partners (MSM), and subsequently spread to intravenous drug users (IVDU), female commercial sex workers (CSW) and their male clients, and, ultimately, to their partners and children. The HIV epidemic has devastated the country's working-age population. The extensive negative impact and social stigma associated with the disease do not only have an impact on the victims of HIV but also on their descendants and relatives. An epicenter of the HIV epidemic has been in the northern provinces of Thailand. An HIV-1 subtype CRF01_AE, a complex chimeric virus composed of both A and E subtypes, is prevalent in Northern Thailand. The virus has quickly become a predominant viral strain circulating in Thailand, other neighboring Southeast Asian countries, and China as well as some other countries throughout the world. The epidemiology, evolution, and biology of CRF01_AE offer a unique model for further scientific investigations which would advance the knowledge of and curative strategies against HIV. In addition, Thailand has developed suitable national guidelines on HIV/AIDS treatment and prevention in order to control the epidemic. Effective antiretroviral drugs are, therefore, able to be made available to those who live with HIV. The national surveillance system has also been effective. The great efforts and resources which Thailand has dedicated to the fight against the epidemic have eventually paid off. In 2010, a plan was proposed to eliminate mother-to-child HIV transmission and Thailand has become the first country to be effective in this objective. Thailand therefore has become recognized as being the global leader in HIV prevention and treatment. The experience which Thailand has gained from the past and the current research and management strategies of the HIV epidemic has prepared the country for emerging strains of HIV-1 in the future.

High rates of tenofovir failure in a CRF01_AE-predominant HIV epidemic in the Philippines

BackgroundThe Philippines has the fastest growing HIV epidemic in the Asia-Pacific. This increase was accompanied by a shift in the predominant HIV subtype from B to CRF01_AE. Increasing evidence points to a difference in treatment responses between subtypes. We examined treatment failure and acquired drug resistance (ADR) in people living with HIV (PLHIVs) after one year on antiretrovirals (ARVs). MethodsPLHIV maintained on ARVs for one year were recruited. Treatment failure was defined as a viral load of ≥1000 copies/mL. Sanger sequencing for genotyping and drug resistance mutation (DRM) detection was performed on patients failing treatment. Results513 PLHIV were enrolled. The most common antiretroviral regimens were TDF+3TC + EFV (269) and AZT+3TC + EFV (155). 53 (10.3%) subjects failed treatment. Among these, 48 (90.6%) had DRMs, 84.9% were subtype CRF01_AE. Tenofovir-based regimens performed worse than zidovudine-based regimens (OR 3.28, 95% CI 1.58–7.52 p < 0.001). Higher rates of NRTI, NNRTI, K65R tenofovir resistance, and multi-class resistance were found compared to those reported in literature. ConclusionsHIV treatment failure at one year of treatment in the Philippines is 10.3%. We found unusually high tenofovir and multiclass resistance, and optimal ARV regimens may need to be reevaluated for CRF01_AE-predominant epidemics.

High resistance to reverse transcriptase inhibitors among persons infected with human immunodeficiency virus type 1 subtype circulating recombinant form 02_AG in Ghana and on antiretroviral therapy.

This study sought to determine the dominant circulating human immunodeficiency virus type 1 (HIV-1) subtype and associated drug resistance mutations in Ghana.This cross-sectional study was conducted with archived samples collected from patients who received care at 2 hospitals in Ghana from 2014 to 2016. Blood samples were earlier processed into plasma and peripheral blood mononuclear cells and stored at -80 °C. Ribonucleic acid (RNA) was extracted from the archived plasma. Two HIV-1 genes; protease and reverse transcriptase, were amplified, sequenced using gene-specific primers and analyzed for subtype and drug resistance mutations using the Stanford HIV Database.Of 16 patient samples successfully sequenced, we identified the predominance of HIV-1 subtype CRF02_AG (11/16, 68%). Subtypes G (2/16, 13%), dual CRF02_AG/G (2/16, 13%), and CRF01_AE (1/16, 6%) were also observed. Major nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations, M184I/V, D67N, T215F, and K70R/E were found. Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, K103N, Y181C, V90I, F227L, and V106A were also prevalent. Additionally, and at a lower level, protease inhibitor (PI)-resistance mutations, M46I, I54 V, V82A, L90 M, and I471 V, were also present in the sequences from antiretroviral therapy (ART)-experienced individuals. Two NRTI-associated drug resistance mutations (DRMs) (D67N and T69N) were present in sequences from 1 ART-naive individual.HIV-1 subtype CRF02_AG was most frequently detected in this study thus confirming earlier reports of dominance of this subtype in the West-African sub-region and Ghana in particular. The detection of these drug resistance mutations in individuals on first-line regimen composed of NRTI and NNRTI is an indication of prolonged drug exposure without viral load monitoring. Routine viral load monitoring is necessary for early detection of virologic failure and drug resistance testing will inform appropriate choice of regimens for such patients.

Viral Tropism in Human Immunodeficiency Virus Type 1-Infected Children and Adolescents in Thailand.

Maraviroc, a C-C chemokine receptor 5 (CCR5) antagonist, has been used as an alternative antiretroviral drug in treatment-experienced adults and children infected by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. Prior to widespread use of this drug, rates of HIV-1 coreceptor tropism and factors associated with coreceptor tropism had to be determined. HIV-1-infected individuals aged <20 years with HIV-1 viral loads >1000 RNA copies/mL who were treatment-experienced or treatment-naive were enrolled. HIV-1 coreceptor tropism was determined using a genotypic test in which V3 sequences were analyzed with GENO2PHENO version 2.5 and a false discovery rate of 5%. Fifty-two HIV-1-infected patients were recruited. The median age of participants was 14.9 years (interquartile range [IQR], 8.9-16.8 years). The median CD4 cell count was 396.0 cells/µL (IQR, 72.0-630.3 cells/µL). The median HIV-1 viral load was 43 339 RNA copies/mL (IQR, 8874-197 055 copies/mL). Thirty-nine patients (75%) were treatment-experienced. The most prevalent HIV-1 subtype in this population was CRF01_AE (36 patients, 69.2%). Based on analyses of V3 loop sequences, 5 of 13 treatment-naive patients (38.5%) and 11 of 39 treatment-experienced patients (28.2%) were infected by R5 viruses, while 7 of 13 treatment-naive patients (53.8%) and 19 of 39 treatment-experienced patients (48.7%) were infected by X4 viruses. The only factor associated with the presence of X4 viruses was HIV-1 subtype CRF01_AE. X4-tropic viruses are associated with the CRF01_AE subtype. Hence, testing of HIV tropism should be performed before treatment with CCR5 inhibitors in children in areas where CRF01_AE predominates.

Prevalence of Non-B HIV-1 Subtypes in North Italy and Analysis of Transmission Clusters Based on Sequence Data Analysis.

HIV-1 diversity is increasing in European countries due to immigration flows, as well as travels and human mobility, leading to the circulation of both new viral subtypes and new recombinant forms, with important implications for public health. We analyzed 710 HIV-1 sequences comprising protease and reverse-transcriptase (PR/RT) coding regions, sampled from 2011 to 2017, from naive patients in Spedali Civili Hospital, Brescia, Italy. Subtyping was performed by using a combination of different tools; the phylogenetic analysis with a structured coalescence model and Makarov Chain Monte Carlo was used on the datasets, to determine clusters and evolution. We detected 304 (43%) patients infected with HIV-1 non-B variants, of which only 293 sequences were available, with four pure subtypes and five recombinant forms; subtype F1 (17%) and CRF02_AG (51.1%) were most common. Twenty-five transmission clusters were identified, three of which included >10 patients, belonging to subtype CRF02_AG and subtype F. Most cases of alleged transmission were between heterosexual couples. Probably due to strong migratory flows, we have identified different subtypes with particular patterns of recombination or, as in the case of the subtype G (18/293, 6.1%), to a complete lack of relationship between the sequenced strains, revealing that they are all singletons. Continued HIV molecular surveillance is most important to analyze the dynamics of the boost of transmission clusters in order to implement public health interventions aimed at controlling the HIV epidemic.

High rate of hepatitis C virus and human immunodeficiency virus false-positive results in serologic screening in sub-Saharan Africa: adverse impact on the blood supply.

False positivity in blood screening may cause unnecessary deferral of healthy donors and exacerbate blood shortages. An international multicenter study was conducted to estimate the frequency of HCV and HIV false seropositivity in seven African countries (Burundi, Cameroon, Democratic Republic of Congo, Madagascar, Mali, Mauritania, and Niger). Blood donations were tested for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) with rapid detection tests (RDTs), third-generation enzyme immunoassays (EIAs), or fourth-generation EIAs. HCV (456/16,613 [2.74%]) and HIV (249/16,675 [1.49%]) reactive samples were then confirmed with antigen/antibody assays, immunoblots, and nucleic acid testing. Partial viral sequences were analyzed when possible. The HCV reactivity rate with RDTs was significantly lower than with EIAs (0.55% vs. 3.52%; p < 0.0001). The HIV reactivity rate with RDTs was lower than with third-generation EIAs (1.02% vs. 2.38%; p < 0.0001) but similar to a fourth-generation assay (1.09%). Only 16.0% (57/357) and 21.5% (38/177) of HCV and HIV initial reactive samples, respectively, were repeatedly reactive. HCV and HIV infections were confirmed in 13.2% and 13.7%, respectively, of repeated reactive donations. The predominant HCV genotype 2 and 4 strains in West and Central Africa showed high genetic variability. HIV-1 subtype CRF02_AG was most prevalent. High rates (>80%) of unconfirmed anti-HCV and anti-HIV reactivity observed in several sub-Saharan countries highlights the need for better testing and confirmatory strategies for donors screening in Africa. Without confirmatory testing, HCV and HIV prevalence in African blood donors has probably been overestimated.

HIV transmission links between past and newly diagnosed infections: a molecular epidemiology study

BackgroundUnderstanding the driving forces of HIV infection is crucial for developing intervention strategies. We aimed to infer these forces from genetic linkages between past and newly diagnosed cases by a molecular approach. MethodsWe recruited newly diagnosed HIV patients from all three HIV specialist clinics in Hong Kong, China, between 2016 and 2018 to collect their HIV sequences with corresponding behavioural and clinical data. We retrieved archived HIV sequences from 1994 to 2012 separately. We pooled and analysed sequences by constructing a network with 1·5% TN93 distance threshold and measured node degree centrality. We did univariate analyses to identify factors associated with genetic links with archived sequences among newly diagnosed patients, and used a generalised linear model to differentiate archived sequences having links with new cases adjusting for diagnosis year. Findings2778 partial pol sequences were amalgamated for network construction, of which 426 were newly diagnosed. The network contained 1908 (69%) nodes and 23 305 edges. Some 703 (3%) edges linked 192 archived samples with 135 new cases. Of 239 newly diagnosed patients with genetic connections, those linked with past infections were more likely to be subtype B (odds ratio 3·31; p<0·0001) and be 30 years or younger (2·26; p=0·0065). Men who have sex with men (MSM) linked with past infections were less likely to be engaged in chemsex (0·52; p=0·039) or users of a mainland Chinese gay dating mobile application (0·39; p=0·035). Generalised linear model showed that people with past infections linked to new cases were younger (p=0·0002), had a higher degree centrality (p<0·0001), were less likely to be subtype CRF01_AE (p=0·0019), and infected through heterosexual contact (p<0·0001) or injection drug use (p=0·0002) compared with MSM contact. InterpretationHIV transmission chains linking new with past infections in Hong Kong were driven by younger MSM. Chemsex, emergence in recent years, only clustered in and contributed to some new infections. Formation of transmission linkages over time signified the importance of targeting new and established infections in developing prevention strategies. FundingResearch Grants Council of Hong Kong (14103315) and AIDS Trust Fund (MSS 229 R and 243 R).

HIV genetic subtypes and comparison of the first CD(4)(+)T cell counts in newly diagnosed HIV infected patients in Liuzhou, 1998-2012

Objective: To analyze the change trend of HIV genetic subtypes and compare the first CD(4)(+)T cell counts of newly diagnosed HIV infected patients in Liuzhou from 1998 to 2012, and provide a reference for AIDS prevention and control. Methods: Newly diagnosed HIV-infected patients from 1998 to 2012 in Liuzhou were selected through national HIV/ADIS comprehensive response information management system. Their plasma samples were used for RNA gene extraction, amplification, sequencing and genotyping. Coharan-Armitage trend test was used to analyze the ratio trend of genetic subtypes and phylogenetic clusters of HIV and Wilcoxon Rank Sum Test was used to compare the first CD(4)(+)T cell counts (CD(4)) of the different subtype HIV infected patients. Results: A total of 1 877 newly diagnosed HIV infected patients were included in the study. From 1998 to 2012, the proportions of CRF01_AE and CRF01_AE (Cluster 1) increased from 78.4% (76/97) to 91.5% (1 441/1 574), from 63.9% (62/97) to 74.0% (1 164/1 574), and the proportion of CRF07_BC decreased from 17.5% (17/97) to 4.6% (72/1 574), respectively (Z=4.632, P<0.001; Z=2.455, P=0.014; Z=-5.943, P<0.001). The median and interquartile range of the first CD(4) of the patients infected with subtype CRF01_AE (Cluster 1), CRF01_AE (Cluster 2), CRF07_BC and CRF08_BC were 230 (83-375), 215 (48-351), 365 (254-503) and 334 (206-479) cell/μl, respectively. The first CD(4) levels of the patients infected with subtype CRF01_AE (Cluster 1) or CRF01_AE (Cluster 2) were significantly lower than those of CRF07_BC (Z=-4.795, P<0.001; Z=-4.238, P<0.001). Conclusion: The genetic subtypes of HIV were mainly CRF01_AE in newly diagnosed HIV-infected patients and this subtype proportion was in increase and the first CD(4) levels of the patients were low in Liuzhou during 1998 to 2012.

Prevalence of drug resistance mutations among ART-naive and -experienced HIV-infected patients in Sierra Leone.

ObjectivesThe aim of this study was to assess the prevalence of HIV drug resistance (HIVDR) in HIV-infected ART-naive and -experienced patients in Sierra Leone.Patients and methodsWe conducted a cross-sectional study of HIV-positive adults aged ≥18 years at Connaught Hospital in Freetown, Sierra Leone in November 2017. Sequencing was performed in the reverse transcriptase, protease and integrase regions, and interpreted using the Stanford HIVDR database and WHO 2009 mutation list.ResultsTwo hundred and fifteen HIV-infected patients were included (64 ART naive and 151 ART experienced). The majority (66%) were female, the median age was 36 years and the median ART exposure was 48 months. The majority (83%) were infected with HIV-1 subtype CRF02_AG. In the ART-naive group, the pretreatment drug resistance (PDR) prevalence was 36.7% (14.2% to NRTIs and 22.4% to NNRTIs). The most prevalent PDR mutations were K103N (14.3%), M184V (8.2%) and Y181C (4.1%). In the ART-experienced group, 64.4% harboured resistance-associated mutations (RAMs) and the overall prevalence of RAMs to NRTIs and NNRTIs was 85.2% (52/61) and 96.7% (59/61), respectively. The most prevalent RAMs were K103N (40.7%), M184V (28.8%), D67N (15.3%) and T215I/F/Y (15.3%). Based on the genotypic susceptibility score estimates, 22.4% of ART-naive patients and 56% of ART-experienced patients were not susceptible to first-line ART used in Sierra Leone.ConclusionsA high prevalence of circulating NRTI- and NNRTI-resistant variants was observed in ART-naive and -experienced HIV-1-infected patients in Sierra Leone. This necessitates the implementation of HIVDR surveillance programmes to inform national ART guidelines for the treatment and monitoring of HIV-infected patients in Sierra Leone.

HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals.

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4+ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.

Baseline PI susceptibility by HIV-1 Gag-protease phenotyping and subsequent virological suppression with PI-based second-line ART in Nigeria.

ObjectivesPrevious work showed that gag-protease-derived phenotypic susceptibility to PIs differed between HIV-1 subtype CRF02_AG/subtype G-infected patients who went on to successfully suppress viral replication versus those who experienced virological failure of lopinavir/ritonavir monotherapy as first-line treatment in a clinical trial. We analysed the relationship between PI susceptibility and outcome of second-line ART in Nigeria, where subtypes CRF02_AG/G dominate the epidemic.MethodsIndividuals who experienced second-line failure with ritonavir-boosted PI-based ART were matched (by subtype, sex, age, viral load, duration of treatment and baseline CD4 count) to those who achieved virological response (‘successes’). Successes were defined by viral load <400 copies of HIV-1 RNA/mL by week 48. Full-length Gag-protease was amplified from patient samples for in vitro phenotypic susceptibility testing, with PI susceptibility expressed as IC50 fold change (FC) relative to a subtype B reference strain.ResultsThe median (IQR) lopinavir IC50 FC was 4.04 (2.49–7.89) for virological failures and 4.13 (3.14–8.17) for virological successes (P = 0.94). One patient had an FC >10 for lopinavir at baseline and experienced subsequent virological failure with ritonavir-boosted lopinavir as the PI. There was no statistically significant difference in single-round replication efficiency between the two groups (P = 0.93). There was a moderate correlation between single-round replication efficiency and FC for lopinavir (correlation coefficient 0.32).ConclusionsWe found no impact of baseline HIV-1 Gag-protease-derived phenotypic susceptibility on outcomes of PI-based second-line ART in Nigeria.

Human Leukocyte Antigen-Associated HIV-1 CRF02_AG gag and vif Polymorphisms in Ghana.

In human immunodeficiency virus type-1 (HIV-1) infections, cytotoxic T-lymphocyte (CTL) responses targeting human leukocyte antigen (HLA)-restricted viral epitopes exert strong suppressive pressure on viral replication and frequently select for mutations resulting in viral escape from CTL recognition. Numerous data on these HLA-associated mutations in HIV-1 subtypes B and C have been amassed with few reports described in other subtypes. In the present study, we investigated the HLA-associated mutations in HIV-1 subtype CRF02_AG prevailing in Ghana, Western Africa. We determined viral gag sequences in 246 out of 324 HIV-1-infected Ghanaians. Phylogeny analysis revealed that 200 (81.3%) individuals were infected with HIV-1 CRF02_AG. Full gag and vif sequences were obtained from 199 and 138, respectively, out of the 200 individuals infected with CRF02_AG and subjected to determination of HLA-associated mutations. The analysis found HLA-associated HIV-1 CRF02_AG non-synonymous polymorphisms at 19 sites; 13 in gag and six in vif, including those that were newly determined. Generation of this data is an important contribution to our understanding of HIV-1 CRF02_AG and host T cell interaction.

Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution.

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlowTM RNA Assay (RNAflow) and RNAscope® ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRF01_AE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF01_AE). RNAscope was applicable for the detection of HIV-1 in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence.

Human immunodeficiency virus type 1 drug resistance in a subset of mothers and their infants receiving antiretroviral treatment in Ouagadougou, Burkina Faso.

The emergence of HIV-1 drug resistance (HIVDR) is a public health problem that affects women and children. Local data of HIVDR is critical to improving their care and treatment. So, we investigated HIVDR in mothers and infants receiving antiretroviral therapy (ART) at Saint Camille Hospital of Ouagadougou, Burkina Faso. This study included 50 mothers and 50 infants on ART. CD4 and HIV-1 viral load were determined using FACSCount and Abbott m2000rt respectively. HIVDR was determined in patients with virologic failure using ViroSeq HIV-1 Genotyping System kit on the 3130 Genetic Analyzer. The median age was 37.28 years in mothers and 1.58 year in infants. Sequencing of samples showed subtypes CRF02_AG (55.56%), CRF06_cpx (33.33%) and G (11.11%). M184V was the most frequent and was associated with highlevel resistance to 3TC, FTC, and ABC. Other mutations such as T215F/Y, D67N/E, K70R, and K219Q were associated with intermediate resistance to TDF, AZT, and 3TC. No mutation to LPV/r was detected among mothers and infants. The findings of HIVDR in some mothers and infants suggested the change of treatment for these persons.

Transmitted drug resistance among HIV infected men who have sex with men in Tianjin, 2014-2017

Objective: To understand prevalence and transmission of transmitted drug resistance (TDR) among HIV infected men who have sex with men (MSM) in Tianjin from 2014 to 2017. Methods: A total of 225 blood samples were collected from HIV infected MSM in Tianjin from 2014 to 2017. Pol gene fragments were obtained by viral RNA extraction and nested PCR amplification. Phylogenetic and drug resistance analyses were conducted. Results: A total of 205 samples were successfully sequenced and analyzed. Based on pol sequences, 53.2% (109/205), 28.8% (59/205), 10.2% (21/205), 4.9% (10/205) and 2.9% (6/205) of the samples were positive for HIV subtypes CRF01_AE, CRF07_BC, B, CRF55_01B and unique recombinant forms (URFs). Twenty transmission clusters, including 75 sequences, were identified and 62.5% (10/16) of sequences with TDR were in 5 clusters. The prevalence of TDR was 7.8% between 2014 and 2017. The annual prevalence rate increased from 3.9% (2/51) in 2014, 5.7% (3/53) in 2015, 9.6% (5/52) in 2016 to 12.2%(6/49) in 2017, the difference was not significant (χ(2)=2.504, P=0.127). CRF01_AE and B strains had high TDR prevalence (3.4%, 7/205) and (2.9%, 6/205), respectively. The TDR mutation was mainly NNRTIs, the TDR prevalence was 6.3% (13/205). In contract, the TDR prevalence of NRTIs and PIs were 1.5% (3/205) and 1.0% (2/205) respectively. Conclusion: Results from this study suggested that the prevalence of HIV-1 TDR strains in MSM was serious in Tianjin. It is necessary to take effective prevention and control measures.

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant.

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.