RESUMO
BACKGROUND: Coinfection with two phylogenetically distinct Human Immunodeficiency Virus-1 (HIV-1) variants might provide an opportunity for rapid viral expansion and the emergence of fit variants that drive disease progression. However, autologous neutralising immune responses are known to drive Envelope (Env) diversity which can either enhance replicative capacity, have no effect, or reduce viral fitness. This study investigated whether in vivo outgrowth of coinfecting variants was linked to pseudovirus and infectious molecular clones' infectivity to determine whether diversification resulted in more fit virus with the potential to increase disease progression. RESULTS: For most participants, emergent recombinants displaced the co-transmitted variants and comprised the major population at 52 weeks postinfection with significantly higher entry efficiency than other co-circulating viruses. Our findings suggest that recombination within gp41 might have enhanced Env fusogenicity which contributed to the increase in pseudovirus entry efficiency. Finally, there was a significant correlation between pseudovirus entry efficiency and CD4 + T cell count, suggesting that the enhanced replicative capacity of recombinant variants could result in more virulent viruses. CONCLUSION: Coinfection provides variants with the opportunity to undergo rapid recombination that results in more infectious virus. This highlights the importance of monitoring the replicative fitness of emergent viruses.
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Coinfecção , Infecções por HIV , HIV-1 , Filogenia , Humanos , Infecções por HIV/virologia , Infecções por HIV/complicações , HIV-1/genética , HIV-1/fisiologia , Coinfecção/virologia , Evolução Molecular , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Proteína gp41 do Envelope de HIV/genética , Masculino , Feminino , Recombinação Genética , Internalização do Vírus , Adulto , Contagem de Linfócito CD4 , Replicação ViralRESUMO
BACKGROUND: HIV-1 has well-established mechanisms to disrupt essential pathways in people with HIV, such as inflammation and metabolism. Moreover, diversity of the amino acid sequences in fundamental HIV-1 proteins including Tat and Vif, have been linked to dysregulating these pathways, and subsequently influencing clinical outcomes in people with HIV. However, the relationship between Tat and Vif amino acid sequence variation and specific immune markers and metabolites of the tryptophan-kynurenine (Trp-Kyn) pathway remains unclear. Therefore, this study aimed to investigate the relationship between Tat/Vif amino acid sequence diversity and Trp-Kyn metabolites (quinolinic acid (QUIN), Trp, kynurenic acid (KA), Kyn and Trp/Kyn ratio), as well as specific immune markers (sCD163, suPAR, IL-6, NGAL and hsCRP) in n = 67 South African cART-naïve people with HIV. METHODS: Sanger sequencing was used to determine blood-derived Tat/Vif amino acid sequence diversity. To measure Trp-Kyn metabolites, a LC-MS/MS metabolomics platform was employed using a targeted approach. To measure immune markers, Enzyme-linked immunosorbent assays and the Particle-enhanced turbidimetric assay was used. RESULTS: After adjusting for covariates, sCD163 (p = 0.042) and KA (p = 0.031) were higher in participants with Tat signatures N24 and R57, respectively, and amino acid variation at position 24 (adj R2 = 0.048, ß = -0.416, p = 0.042) and 57 (adj R2 = 0.166, ß = 0.535, p = 0.031) of Tat were associated with sCD163 and KA, respectively. CONCLUSIONS: These preliminary findings suggest that amino acid variation in Tat may have an influence on underlying pathogenic HIV-1 mechanisms and therefore, this line of work merits further investigation.
Assuntos
Infecções por HIV , HIV-1 , Inflamação , Cinurenina , Triptofano , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Triptofano/metabolismo , Infecções por HIV/virologia , Infecções por HIV/genética , Masculino , HIV-1/genética , Adulto , Feminino , Cinurenina/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Pessoa de Meia-Idade , Biomarcadores/sangue , Receptores de Superfície Celular , Antígenos de Diferenciação Mielomonocítica , Antígenos CDRESUMO
INTRODUCTION: HIV drug resistance (HIVDR) is an important challenge in the fight against HIV/AIDS and can threaten progress toward achieving the target of HIV elimination by 2030. Genotyping pretreatment HIVDR testing (DRT) has been proposed as a potential solution. However, the cost-effectiveness of this intervention needs to be evaluated to determine its feasibility and potential impact on healthcare systems. This study aimed to assess the cost-effectiveness of DRT among people living with HIV (PLHIV) in Iran. METHODS: 1000 hypothetical PLHIV were simulated in terms of cost and effectiveness based on quality-adjusted life Years (QALY). The Markov Model was developed to calculate incremental cost-effectiveness ratio (ICER) using TreeAge Pro 2020. Deterministic and probabilistic analyses were performed for sensitivity analyses. RESULTS: Results showed that compared to not performing pretreatment HIVDR testing, this intervention gained 0.035999 QALY with an incremental cost of 1,695.32 USD. The ICER was calculated as 47,093.53 USD, indicating that pretreatment DRT was not cost-effective. The probability of opportunistic infection (OI) in people with viral failure, the effectiveness of Dolutegravir in people without drug resistance, and the quality of life (QoL) of people in the AIDS stage were found to be the most important variables affecting ICER. With an increasing willingness to pay more than 53,000 USD, pretreatment DRT testing will become cost-effective. CONCLUSION: Based on our findings, pretreatment HIVDR testing is not currently cost-effective in Iran as it imposes high costs on healthcare systems with few benefits for People living with HIV (PLHIV). However, if resources are available, drug resistance testing can be a valuable tool in generating HIV molecular data and molecular surveillance of HIV.
Assuntos
Análise Custo-Benefício , Farmacorresistência Viral , Infecções por HIV , Anos de Vida Ajustados por Qualidade de Vida , Humanos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Infecções por HIV/economia , Farmacorresistência Viral/genética , Irã (Geográfico)/epidemiologia , Masculino , Feminino , Cadeias de Markov , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/economia , HIV-1/efeitos dos fármacos , HIV-1/genética , Qualidade de Vida , AdultoRESUMO
HIV-1 CRF08_BC is a significant subtype in China, though its origin and spread remain incompletely understood. Previous studies using partial genomic data have provided insights but lack comprehensive analysis. Here, we investigate the early evolutionary and spatiotemporal dynamics of HIV-1 CRF08_BC in China and Myanmar using near-complete genome sequences. We analyzed 28 near-complete HIV-1 CRF08_BC genomes from China and Myanmar (1997-2013). Phylogenetic, molecular clock, and Bayesian discrete trait analyses were performed to infer the virus's origin, spread, and associated risk groups. Based on Bayesian time-scaled inference with the best-fitting combination of models determined by marginal likelihood estimation (MLE), we inferred the time to the most recent common ancestor (TMRCA) and evolutionary rate of HIV-1 CRF08_BC to be at 3 October 1991 (95% HPD: 22 February1989-27 November 1993) and 2.30 × 10-3 substitutions per site per year (95% HPD: 1.96 × 10-3-2.63 × 10-3), respectively. Our analysis suggests that HIV-1 CRF08_BC originated in Yunnan Province, China, among injecting drug users, and subsequently spread to other regions. This study provides valuable insights into the early dynamics of HIV-1 CRF08_BC through combined genomic and epidemiological data, which may inform effective prevention and mitigation efforts. However, the limited genomic data influenced the extent of our findings, and challenges in collecting accurate risk group information during surveillance were evident.
Assuntos
Teorema de Bayes , Evolução Molecular , Genoma Viral , Infecções por HIV , HIV-1 , Filogenia , HIV-1/genética , HIV-1/classificação , Humanos , Infecções por HIV/transmissão , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , China/epidemiologia , Mianmar/epidemiologia , MasculinoRESUMO
BACKGROUND: An increase in the prevalence of HIV drug resistance (HIVDR) has been reported in recent years, especially in persons on non-nucleoside reverse transcriptase inhibitors (NNRTIs) due to their low genetic barrier to mutations. However, there is a paucity of epidemiological data quantifying HIVDR in the era of new drugs like dolutegravir (DTG) in sub-Saharan Africa. We, therefore, sought to determine the prevalence and correlates of viral load (VL) suppression in adult people with HIV (PWH) on a fixed-dose combination of tenofovir disoproxil fumarate/lamivudine/dolutegravir (TLD) or tenofovir alafenamide/emtricitabine/dolutegravir (TAFED) and describe patterns of mutations in individuals failing treatment. METHODS: We conducted a cross-sectional study among 384 adults living with HIV aged ≥15 years between 5th June 2023 and 10th August 2023. Demographic, laboratory and clinical data were collected from electronic health records using a data collection form. Viral load suppression was defined as plasma HIV-1 RNA VL of <1000 copies/ml after being on ART for ≥ 6 months. SPSS version 22 to analyze the data. Descriptive statistics and logistic regression were the statistical methods used. RESULTS: The median (interquartile range (IQR)) age was 22 (IQR 18, 38) years, and 66.1% (n = 254) were females. VL suppression was 90.4% (n = 347); (95% confidence interval (CI) 87.6%-93.6%) after switching to TLD/TAFED. Among the virally suppressed, the majority (67.1%, n = 233) were female. Those who missed ≥2 doses in the last 30 days prior to the most recent review were less likely to attain viral suppression compared to those who did not miss any dose (adjusted odds ratio (AOR) 0.047; 95% CI 0.016-0.136; p<0.001). Four participants had resistance mutations to lamivudine and tenofovir. The most common NRTI mutations were M184MV and K65R while K101E was the most common NNRTI mutation. CONCLUSION: Our findings show that viral suppression was high after switching to TLD/TAFED; but lower than the last 95% target of the UNAIDS. Adherence to antiretroviral therapy was a significant correlate of VL suppression. We, therefore, recommend prompt switching of PWH to TLD/TAFED regimen and close monitoring to enhance adherence to therapy.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Infecções por HIV , HIV-1 , Lamivudina , Mutação , Piperazinas , Piridonas , Tenofovir , Carga Viral , Humanos , Feminino , Adulto , HIV-1/genética , HIV-1/efeitos dos fármacos , Masculino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Farmacorresistência Viral/genética , Carga Viral/efeitos dos fármacos , Zâmbia/epidemiologia , Estudos Transversais , Tenofovir/uso terapêutico , Tenofovir/farmacologia , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/farmacologia , Piperazinas/uso terapêutico , Lamivudina/uso terapêutico , Lamivudina/farmacologia , Piridonas/uso terapêutico , Oxazinas/uso terapêutico , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Emtricitabina/uso terapêutico , Pessoa de Meia-Idade , Adulto Jovem , Adolescente , Combinação de MedicamentosRESUMO
Efforts towards an effective HIV-1 vaccine have remained mainly unsuccessful. There is increasing evidence for a potential role of HLA-C-restricted CD8+ T cell responses in HIV-1 control, including our recent report of HLA-C*03:02 among African children. However, there are no documented optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C*03:02; additionally, the structural influence of HLA-C*03:02 on epitope binding is undetermined. Immunoinformatics approaches provide a fast and inexpensive method to discover HLA-restricted epitopes. Here, we employed immunopeptidomics to identify HLA-C*03:02 CD8+ T cell epitopes. We identified a clade-specific Gag-derived GY9 (GTEELRSLY) HIV-1 p17 matrix epitope potentially restricted to HLA-C*03:02. Residues E62, T142, and E151 in the HLA-C*03:02 binding groove and positions p3, p6, and p9 on the GY9 epitope are crucial in shaping and stabilizing the epitope binding. Our findings support the growing evidence of the contribution of HLA-C molecules to HIV-1 control and provide a prospect for vaccine strategies.
Assuntos
Epitopos de Linfócito T , HIV-1 , Antígenos HLA-C , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Humanos , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Antígenos HLA-C/imunologia , Antígenos HLA-C/metabolismo , Antígenos HLA-C/genética , HIV-1/imunologia , HIV-1/genética , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Ligação Proteica , Infecções por HIV/imunologia , Infecções por HIV/virologia , Antígenos HIVRESUMO
The HIV-1 capsid is composed of capsid (CA) protein hexamers and pentamers (capsomers) that contain a central pore hypothesised to regulate capsid assembly and facilitate nucleotide import early during post-infection. These pore functions are mediated by two positively charged rings created by CA Arg-18 (R18) and Lys-25 (K25). Here we describe the forced evolution of viruses containing mutations in R18 and K25. Whilst R18 mutants fail to replicate, K25A viruses acquire compensating mutations that restore nearly wild-type replication fitness. These compensating mutations, which rescue reverse transcription and infection without reintroducing lost pore charges, map to three adaptation hot-spots located within and between capsomers. The second-site suppressor mutations act by restoring the formation of pentamers lost upon K25 mutation, enabling closed conical capsid assembly both in vitro and inside virions. These results indicate that there is no intrinsic requirement for K25 in either nucleotide import or capsid assembly. We propose that whilst HIV-1 must maintain a precise hexamer:pentamer equilibrium for proper capsid assembly, compensatory mutations can tune this equilibrium to restore fitness lost by mutation of the central pore.
Assuntos
Proteínas do Capsídeo , Capsídeo , HIV-1 , Mutação , Montagem de Vírus , Replicação Viral , HIV-1/genética , HIV-1/fisiologia , Montagem de Vírus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Capsídeo/metabolismo , Humanos , Replicação Viral/genética , Vírion/metabolismo , Vírion/genética , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genéticaRESUMO
BackgroundIn the Netherlands, HIV pre-exposure prophylaxis (PrEP) has been available since 2019. However, the extent of PrEP use prior to HIV diagnosis and development of PrEP-resistance-associated mutations (RAMs) is not known.AimWe assessed prior PrEP use and potential transmission of PrEP RAMs among men who have sex with men (MSM) and transgender persons (TGP) with a new HIV diagnosis in the Netherlands.MethodsData on prior PrEP use between 1 January 2018 and 31 December 2022 were available from the Dutch national ATHENA cohort. We assessed proportion of prior PrEP use, detected PrEP associated RAMs and assessed potential onward transmission of RAMs between 2010 and 2022 using a maximum likelihood tree.ResultsData on prior PrEP use were available for 583/1,552 (36.3%) individuals, with 16% (94/583) reporting prior PrEP use. In 489 individuals reporting no prior PrEP use, 51.5% did not use PrEP due to: low HIV-risk perception (29%), no access (19.1%), personal preference (13.1%), and being unaware of PrEP (19.1%). For PrEP users, 13/94 (13.8%) harboured a M184V/I mutation, of whom two also harboured a K65R mutation. In people with a recent HIV infection, detection of PrEP RAMs increased from 0.23% (2/862) before 2019 to 4.11% (9/219) from 2019. We found no evidence of onward transmission of PrEP RAMs.ConclusionThe prevalence of PrEP-associated RAMs has increased since PrEP became available in the Netherlands. More widespread access to PrEP and retaining people in PrEP programmes when still at substantial risk is crucial to preventing new HIV infections.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Infecções por HIV , Homossexualidade Masculina , Mutação , Profilaxia Pré-Exposição , Pessoas Transgênero , Humanos , Masculino , Profilaxia Pré-Exposição/estatística & dados numéricos , Infecções por HIV/prevenção & controle , Infecções por HIV/epidemiologia , Países Baixos/epidemiologia , Homossexualidade Masculina/estatística & dados numéricos , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/administração & dosagem , Adulto , Farmacorresistência Viral/genética , Pessoas Transgênero/estatística & dados numéricos , HIV-1/genética , HIV-1/isolamento & purificação , Pessoa de Meia-Idade , Estudos de Coortes , FemininoRESUMO
This study investigates the prevalence and patterns of transmitted drug resistance mutations (TDRMs) and HIV-1 subtypes among antiretroviral therapy (ART) naïve individuals in Veneto, Italy, from 2017 to 2024. This research aims to understand the dynamic landscape of TDRMs and HIV-1 genetic diversity to inform treatment strategies effectively. We included all adult ART-naïve people with HIV (PWH) from seven infectious disease units in Veneto, Italy. We collected the genotypic resistance testing conducted to predict drug susceptibility and subtype distribution using the Stanford HIVdb algorithm. We included 762 PWH, showing a slight but statistically significant decline in the B subtype among Italian PWH (p = 0.045) and an increase in non-B subtypes among foreigners, though it was not statistically significant (p = 0.333). The most frequent mutations were in Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs), especially in non-B subtypes, with a notable rise from 10.7% in 2017-2019 to 15.5% in 2020-2024. Notably, TDRMs were consistently detected, highlighting an ongoing challenge despite the stable prevalence observed over the years. In addition, the data revealed a concerning rise in mutations against newer drug classes, such as integrase inhibitors. Conclusively, the study underscores the necessity of continuous surveillance of HIV subtypes and resistance patterns to adapt ART regimens optimally. Despite the stable levels of drug resistance, the emergence of resistance against newer drugs necessitates ongoing vigilance and possible adjustment in treatment protocols to enhance clinical outcomes and manage HIV drug resistance effectively.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Genótipo , Infecções por HIV , HIV-1 , Mutação , Humanos , HIV-1/genética , HIV-1/efeitos dos fármacos , HIV-1/classificação , Itália/epidemiologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Farmacorresistência Viral/genética , Masculino , Adulto , Feminino , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Prevalência , Pessoa de Meia-Idade , Adulto Jovem , Variação GenéticaRESUMO
M184V is a single-base mutation in the YMDD domain of reverse transcriptase (RT). The M184V resistance-associated mutation (RAM) is related to virological unresponsiveness to lamivudine (3TC) and emtricitabine (FTC) and induces high-level resistance to these two antiretroviral agents. M184V is rapidly selected in the setting of non-suppressive antiretroviral therapy (ART) and accumulates in the HIV reservoir. There were continuous efforts to evaluate the impact of the M184V mutation on the treatment outcomes in people living with HIV (PLWH). Since 3TC remains an extensively used part of recommended antiretroviral combinations, M184V is commonly detected in patients with virological failure (VF). ART guidelines do not recommend the use of drugs impacted by RAMs as they have been confirmed to comprise a risk factor for VF. However, there is evidence that 3TC/FTC can remain active even in the presence of M184V. Given the potential benefits of 3TC in ART combinations, the investigation of M184V remains of high interest to clinicians and researchers, especially in certain regions with limited resources, and especially for its unusual effects. This is a review of the literature on the challenges in treating both naïve and experienced individuals carrying the M184V mutation, including virological failure, virological suppression, and resistance to ART.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Infecções por HIV , HIV-1 , Mutação , Humanos , HIV-1/genética , HIV-1/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Farmacorresistência Viral/genética , Fármacos Anti-HIV/uso terapêutico , Transcriptase Reversa do HIV/genética , Lamivudina/uso terapêutico , Emtricitabina/uso terapêuticoRESUMO
BACKGROUND: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. METHODS: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. RESULTS: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools. CONCLUSION: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.
Assuntos
Farmacorresistência Viral , Infecções por HIV , HIV-1 , Sequenciamento de Nucleotídeos em Larga Escala , HIV-1/genética , HIV-1/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Itália , Farmacorresistência Viral/genética , Infecções por HIV/virologia , Mutação , Genótipo , RNA Viral/genética , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêuticoRESUMO
The human immunodeficiency virus type 1 (HIV-1) epidemic has been a major public health threat on a global scale since the early 1980s. Despite the introduction of combination antiretroviral therapy (cART), the incidence of new HIV-1 infections continues to rise in some regions around the world. Thus, with the continuous transmission of HIV-1 and the lack of a cure, it is imperative for molecular epidemiological studies to be performed, to monitor the infection and ultimately be able to control the spread of this virus. This work provides a comprehensive molecular epidemiological analysis of the HIV-1 infection in Cyprus, through examining 305 HIV-1 sequences collected between 9 March 2017 and 14 October 2021. Employing advanced statistical and bioinformatic techniques, the research delved deeply into understanding the transmission dynamics of the HIV-1 epidemic in Cyprus, as well as the monitoring of HIV-1's genetic diversity and the surveillance of transmitted drug resistance. The characterization of Cyprus's HIV-1 epidemic revealed a diverse landscape, comprising 21 HIV-1 group M pure subtypes and circulating recombinant forms (CRFs), alongside numerous uncharacterized recombinant strains. Subtypes A1 and B emerged as the most prevalent strains, followed by CRF02_AG. The findings of this study also revealed high levels of transmitted drug resistance (TDR) patterns, raising concerns for the efficacy of cART. The demographic profiles of individuals involved in HIV-1 transmission underscored the disproportionate burden borne by young to middle-aged Cypriot males, particularly those in the MSM community, who reported contracting the virus in Cyprus. An assessment of the spatiotemporal evolutionary dynamics illustrated the global interconnectedness of HIV-1 transmission networks, implicating five continents in the dissemination of strains within Cyprus: Europe, Africa, Asia, North America, and Oceania. Overall, this study advances the comprehension of the HIV-1 epidemic in Cyprus and highlights the importance of understanding HIV-1's transmission dynamics through continuous surveillance efforts. Furthermore, this work emphasizes the critical role of state-of-the-art bioinformatics analyses in addressing the challenges posed by HIV-1 transmission globally, laying the groundwork for public health interventions aimed at curbing its spread and improving patient outcomes.
Assuntos
Farmacorresistência Viral , Epidemias , Infecções por HIV , HIV-1 , Filogenia , Chipre/epidemiologia , Humanos , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/efeitos dos fármacos , HIV-1/classificação , Farmacorresistência Viral/genética , Masculino , Feminino , Adulto , Epidemiologia Molecular , Pessoa de Meia-Idade , Adulto Jovem , Variação Genética , Adolescente , Genótipo , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/farmacologiaRESUMO
We assessed the performance and clinical relevance of Illumina MiSeq next-generation sequencing (NGS) for HIV-1 genotyping compared with Sanger sequencing (SS). We analyzed 167 participants, 45 with virologic failure (VL ≥ 1000 copies/mL), i.e., cases, and 122 time-matched participants with virologic suppression (VL < 1000 copies/mL), i.e., controls, 12 months post-ART initiation. Major surveillance drug resistance mutations (SDRMs) detected by SS were all detectable by NGS. Among cases at 12 months, SS identified SDRMs in 32/45 (71.1%) while NGS identified SDRMs among 35/45 (77.8%), increasing the number of cases with SDRMs by 3/45 (6.7%). Participants identified with, and proportions of major SDRMs increased when NGS was used. NGS vs. SS at endpoint revealed for NNRTIs: 36/45 vs. 33/45; Y181C: 26/45 vs. 24/45; K103N: 9/45 vs. 6/45 participants with SDRMs, respectively. At baseline, NGS revealed major SDRMs in 9/45 (20%) cases without SDRMs by SS. Participant MBL/043, among the nine, the following major SDRMs existed: L90M to PIs, K65R and M184V to NRTIs, and Y181C and K103N to NNRTIs. The SDRMs among the nine increased SDRMs to NRTIs, NNRTIs, and PIs. Only 43/122 (25.7%) of participants had pre-treatment minority SDRMs. Also, 24.4% of the cases vs. 26.2 of controls had minority SDRMs (p = 0.802); minority SDRMs were not associated with virologic failure. NGS agreed with SS in HIV-1 genotyping but detected additional major SDRMs and identified more participants harboring major SDRMs, expanding the HIV DRM profile of this cohort. NGS could improve HIV genotyping to guide treatment decisions for enhancing ART efficacy, a cardinal pre-requisite in the pursuit of the UNAIDS 95-95-95 targets.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Genótipo , Infecções por HIV , HIV-1 , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Humanos , HIV-1/genética , HIV-1/efeitos dos fármacos , Farmacorresistência Viral/genética , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Masculino , Feminino , Adulto , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/farmacologia , Pessoa de Meia-Idade , Carga Viral , Terapia Antirretroviral de Alta AtividadeRESUMO
BACKGROUND: Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. MATERIALS AND METHODS: We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. RESULTS: The samples had a median 4.8 log [IQR 4.4-5.2] HIV-1 RNA copies/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2/28) compared with AD4SEQ (13/28) (p < 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13/28 and 9/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. CONCLUSIONS: Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage.
Assuntos
Farmacorresistência Viral , Infecções por HIV , HIV-1 , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , HIV-1/genética , HIV-1/efeitos dos fármacos , Farmacorresistência Viral/genética , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Masculino , Feminino , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Genótipo , Adulto , Pessoa de Meia-Idade , RNA Viral/genéticaRESUMO
Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.
Assuntos
HIV-1 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Transdução Genética , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , HIV-1/genética , HIV-1/fisiologia , Células HEK293 , Pseudotipagem Viral , Vetores Genéticos/genética , COVID-19/virologia , Testes de NeutralizaçãoRESUMO
Doravirine (DOR) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with efficacy against some NNRTI-resistant mutants. Although DOR resistance mutations are established for HIV-1 subtype B, it is less clear for non-B subtypes. This study investigated prevalent NNRTI resistance mutations on DOR susceptibility in HIV-1 subtype C. Prevalent drug resistance mutations were identified from a South African genotypic drug resistance testing database. Mutations, single or in combination, were introduced into replication-defective pseudoviruses and assessed for DOR susceptibility in vitro. The single V106M and Y188L mutations caused high-level resistance while others did not significantly impact DOR susceptibility. We observed an agreement between our in vitro and the Stanford HIVdb predicted susceptibilities. However, the F227L mutation was predicted to cause high-level DOR resistance but was susceptible in vitro. Combinations of mutations containing K103N, V106M or Y188L caused high-level resistance, in agreement with the predictions. These mutations are frequently observed in patients failing efavirenz- or nevirapine-based first-line regimens. However, they are also observed in those failing a protease inhibitor-based second-line regimen, as we have observed in our database. Genotypic drug resistance testing is therefore vital prior to the initiation of DOR-based treatment for those previously exposed to efavirenz or nevirapine.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Genótipo , Infecções por HIV , HIV-1 , Mutação , Piridonas , Triazóis , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Farmacorresistência Viral/genética , Piridonas/farmacologia , Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Triazóis/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fenótipo , Testes de Sensibilidade Microbiana , África do SulRESUMO
The purpose of this study was to determine the contribution of genetic factors, i.e., the level of expression and polymorphisms of Toll-like receptors (TLR), to the susceptibility of latent tuberculosis infection in a Russian cohort of individuals infected with HIV. The patients (n = 317) with confirmed HIV infection were divided into two groups according to the results of the STANDARD E TB-Feron test: 63 cases with a latent TB infection and 274 controls without LTBI. Total DNA and RNA were isolated from whole-blood samples. SNP genotyping and expression levels of five TLR genes (TLR1, TLR2, TLR4, TLR6, and TLR8) were determined by means of real-time PCR. There were no significant differences in the expression levels of the TLRs between the case and control groups. In addition, we did not observe any significant association between the analyzed SNPs and the susceptibility of Latent tuberculosis infection (LTBI) in patients with HIV. However, patients from an entire cohort with the rs4986790-GG (TLR4) and rs5743708-GG (TLR2) genotypes were characterized by lower CD4 T-cell counts compared to carriers of alternative alleles. Moreover, we found a significant risk of a hazardous drop in the CD4 T-cell count below 350 cells/mm3 associated with the rs4986790-G (TLR4) allele. Latent tuberculosis infection in individuals infected with HIV does not significantly modify the level of TLR gene expression.
Assuntos
Predisposição Genética para Doença , Infecções por HIV , HIV-1 , Tuberculose Latente , Polimorfismo de Nucleotídeo Único , Receptores Toll-Like , Humanos , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , Masculino , Tuberculose Latente/genética , Feminino , Adulto , Receptores Toll-Like/genética , Pessoa de Meia-Idade , HIV-1/genética , Genótipo , Alelos , Federação Russa/epidemiologia , Contagem de Linfócito CD4 , Estudos de CoortesRESUMO
Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.
Assuntos
Benzofuranos , HIV-1 , RNA Viral , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-1/genética , Humanos , Replicação Viral/efeitos dos fármacos , RNA Viral/metabolismo , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Benzofuranos/farmacologia , Vírion/efeitos dos fármacos , Vírion/metabolismo , Linfócitos T CD4-Positivos/virologia , Montagem de Vírus/efeitos dos fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Fator de Iniciação 4A em Eucariotos/metabolismo , Genoma Viral/efeitos dos fármacos , Ligação Proteica , Linhagem CelularRESUMO
BACKGROUND: The human immunodeficiency virus 1 (HIV-1) infections in Brazil are predominantly caused by two subtypes, B and C. OBJECTIVES: Here we present the characterisation of a novel HIV-1 recombinant form, indicating a new Brazilian CRF_BC, named CRF146_BC. METHODS: RDP, JphMM and Simplot recombination tools were used to evaluate the mosaic pattern. FINDINGS: In this work, we identified three HIV-1 nucleotide sequences previously classified as unique recombinant forms (URFs), plus one new partial genome sharing the same BC recombination pattern. The mosaic genome is almost entirely represented by the subtype C sequence, with a small subtype B recombination region in the pol gene, at the Integrase level. The phylogenetic analyses strongly indicate a common origin between the strains, which were isolated in Rio Grande do Sul, Rio de Janeiro and Bahia states. MAIN CONCLUSIONS: Thus, the new HIV-1 CRF146_BC is circulating in three different Brazilian regions: South, Southeast and Northeast.