Ultrasensitive HIV-1 p24 Assay Detects Single Infected Cells and Differences in Reservoir Induction by Latency Reversal Agents.

The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4+ T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4+ T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4+ T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals.IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it.

Monitoring the emergence of resistance mutations in patients infected with HIV-1 under salvage therapy with raltegravir in Rio de Janeiro, Brazil: a follow-up study.

The present study describes a follow-up of a prospective and observational cohort of patients infected with HIV-1 and treated with raltegravir for salvage therapy in Brazil. Two groups of patients were analyzed: switching from T20 to RAL (Group 1, n = 9) and salvage therapy containing RAL (Group 2, n = 10). Blood samples were drawn for CD4(+) T-cell counts and HIV-1 viral load determinations. Protease, reverse transcriptase, and integrase genotyping were performed at baseline and at the time of virologic failure. CD4(+) T-cells increased at 6 and 12 months in both groups; HIV-1 viral load was continuously suppressed for Group 1, and for Group 2 it significantly decreased after starting a RAL-containing regimen. Three out of 10 patients from Group 2 could not suppress HIV-1 viral load. The mutations Q148H + G140S were observed for two patients and for the third patient only mutations to PR/RT inhibitors were detected. The genotypic sensitivity score (GSS) was analyzed for all patients of Group 2 and both patients who developed resistance to raltegravir presented a GSS < 2.0 for the RAL-containing scheme, which could be associated to the lack of effectiveness of the proposed scheme. The present study describes, for the first time in Brazil, the close follow-up of a series of patients using a raltegravir-containing HAART, showing the safety of the enfuvirtide switch to RAL and the effectiveness of a therapeutic regimen with RAL in promoting immune reconstitution and suppressing HIV replication, as well as documenting the occurrence of resistance to integrase inhibitors in the country.

Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.

BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.

Association of single nucleotide polymorphisms in the lens epithelium-derived growth factor (LEDGF/p75) with HIV-1 infection outcomes in Brazilian HIV-1+ individuals.

The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

Near full-length genome characterization of HIV type 1 unique BC recombinant forms from Southern Brazil.

Abstract In southern Brazil, CRF31_BC is the major HIV-1 recombinant form and URFs_BC occurs very infrequently. The near full-length genomes of four samples displaying a similar recombination profile in the integrase region and different profiles in the protease/reverse transcriptase regions were analyzed to elucidate their mosaic structure and to describe for the first time the near full-length genomes of URFs_BC circulating in Brazil. For this purpose, overlapping PCR amplifications followed by direct sequencing were carried out. Despite the observation that these samples share a similar recombination profile in the integrase region, phylogenetic, bootscan, and informative site analyses revealed that the four samples displayed distinct mosaic structures. Additional recombination events occurred mainly in the gag, reverse transcriptase, and env regions. Our data provide the first description of the near full-length genomes of URFs_BC in Brazil and offer important insights about the complexity of HIV-1 recombinant strains currently in circulation in the south of Brazil.

Genetic characterization of HIV-1 BC recombinants and evolutionary history of the CRF31_BC in Southern Brazil.

To evaluate the recombination profiles and evolutionary history of HIV-1 BC recombinants in Southern Brazil, 81 isolates collected in the city of Porto Alegre (Rio Grande do Sul State) from 1998 to 2006 previously subtyped as C (env-gp120/C2V3) were screened in the protease-reverse transcriptase (pr/rt), integrase and gp41 genomic regions. Detailed phylogenetic, bootscan and informative site analyses were performed to trace the subtype classification. The evolutionary rate and divergence time of the Brazilian CRF31_BC epidemic were estimated using a Bayesian Markov Chain Monte Carlo framework. Analysis of the four target regions identified: 43 isolates as "pure" subtype C, 23 as CRF31_BC, and 15 as unique BC recombinant forms (URFs_BC). Recombination breakpoints were mainly localized in the rt gene and 100% of the recombinant samples could be detected analyzing only this region. Most URFs_BC (86.7%) contained small subtype B fragments (

Estudos sobre aspectos virais e genéticos relacionados à integrase e ao processo de integração do HIV-1 / Studies on viral and genetic aspects related integrase and the process of integration of HIV-1

A integrase (IN) é uma enzima chave para o ciclo de replicação do HIV, sendo responsável por catalisar a integração do genoma do HIV no cromossomo hospedeiro. Devido ao papel essencial desta enzima para a patogênese da infecção pelo HIV, a recente introdução dos inibidores de IN (INI) na prática clínica e em vista da escassez de informação sobre a diversidade genética da IN do HIV no Brasil, o presente estudo tem como objetivos a) investigar a diversidade genética da IN e os níveis de resistência primária nos subtipos B, C e F do HIV que são prevalentes no Brasil; b) acompanhar pacientes sob terapia antirretroviral em esquemas contendo raltegravir (RAL) a fim de monitorar a emergência de mutações de resistência aos INI; c) desenvolver um método de genotipagem da IN do HIV para ser usado na pratica clínica no Brasil; e d) investigar o envolvimento do processo de integração no controle da replicação do HIV. Não foram detectadas mutações principais associadas aos INI entre os indivíduos virgens de tratamento infectados com diferentes subtipos de HIV-1. O nível de mutações acessórias observadas foi bem baixo, e algumas posições foram polimórficas nas amostras brasileiras dos subtipos B, C e F. Esses resultados encorajam o uso de INI no Brasil. Analisando as coortes de pacientes que trocaram a enfuvirtida por RAL ou sob terapia de resgate com RAL, nós observamos um aumento nas contagens de células T CD4+ e uma rápida diminuição da carga viral no grupo sob terapia de resgate. Três pacientes não atingiram supressão virológica e as mutações Q148H+G140S foram detectadas em dois deles. A fim de monitorar o crescente número de pacientes sob terapia com RAL no Brasil, nós desenvolvemos um método de genotipagem in-house que está atualmente em teste pela rede de Genotipagem do Ministério da Saúde do Brasil para futura incorporação no monitoramento de pacientes falhando INI. Sobre o papel da IN no controle da infecção pelo HIV, não observamos mutações nos resíduos importantes para a atividade catalítica nas sequências de IN obtidas de pacientes controladores da infecção pelo HIV, nem acúmulo de DNA 2-LTR, sugerindo que não há um mecanismo bloqueando a integração nestes pacientes. Juntos, os resultados apresentados trazem informações importantes sobre a diversidade genética da IN, resistência aos INI e sobre o papel da IN na patogênese da infecção pelo HIV.

Caracterização molecular da região da integrase do gene pol de subtipos de HIV-1 prevalentes no Brasil: avaliação de marcadores de resistência e antigenicidade / Molecular characterization of the region of integrase of the gene pol of subtypes of HIV-1 in Brazil: evaluation of resistance markers and antigenicity

A diversidade genética do HIV-1 tem sido bem estudada tendo-se como alvo as proteínas estruturais. Porém, na região da integrase essa diversidade ainda não foi bem caracterizada. A integrase é uma enzima essencial para o ciclo de replicação do HIV-1 e é um alvo promissor para a terapia anti-retroviral. Atualmente, a terapia inclui inibidores de protease (PR), transcriptase reversa (TR) e de entrada viral. Entretanto, não está claro por quanto tempo os benefícios clínicos serão mantidos devido à emergência de variantes com resistência a múltiplas drogas. Neste contexto, o objetivo foi caracterizar a diversidade genética da integrase do HIV-1 em amostras dos subtipos B(B), C e F, obtidas de pacientes virgens de tratamento, e identificar a presença de mutações naturais relacionadas aos inibidores de integrase em amostras de indivíduos apresentando falha terapêutica, a fim de verificar se esta região ainda permaneceria como um alvo íntegro para o tratamento. O DNA proviral foi extraído de 111 amostras de sangue de indivíduos virgens de tratamento, infectados com os subtipos prevalentes no Brasil, previamente determinados com base na região C2-V3 do envelope. O RNA viral foi extraído de amostras de plasma de 30 indivíduos apresentando falha terapêutica às drogas correntes, infectados pelo subtipo B do HIV-1, previamente determinado com base no gene pol (PR/RT). As amostras foram amplificadas pela metodologia de nested PCR e seqüenciadas por sequenciamento automatizado. O pacote de programas DNASTAR, ClustalX e MEGA foram usados para edição, alinhamento, tradução, análise filogenética (neighbor-joining) e definição das seqüências consenso. Não verificamos a ocorrência de recombinação inter-gênica entre a região da integrase e os genes env e pr/rt, tipados previamente. Entretanto, identificamos genomas apresentando recombinação intra-gênica na região da integrase (3B/F, 3B/C) em amostras de indivíduos virgens de tratamento. Todos os recombinantes B/C apresentaram um...