European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

The analytical performance of the Veris HIV-1 assay for use on the new, fully automated Beckman Coulter DxN Veris molecular diagnostics system was evaluated at 10 European virology laboratories. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.).

European Multicenter Study on Analytical Performance of DxN Veris System HCV Assay.

The analytical performance of the Veris HCV Assay for use on the new and fully automated Beckman Coulter DxN Veris Molecular Diagnostics System (DxN Veris System) was evaluated at 10 European virology laboratories. Precision, analytical sensitivity, specificity, and performance with negative samples, linearity, and performance with hepatitis C virus (HCV) genotypes were evaluated. Precision for all sites showed a standard deviation (SD) of 0.22 log10 IU/ml or lower for each level tested. Analytical sensitivity determined by probit analysis was between 6.2 and 9.0 IU/ml. Specificity on 94 unique patient samples was 100%, and performance with 1,089 negative samples demonstrated 100% not-detected results. Linearity using patient samples was shown from 1.34 to 6.94 log10 IU/ml. The assay demonstrated linearity upon dilution with all HCV genotypes. The Veris HCV Assay demonstrated an analytical performance comparable to that of currently marketed HCV assays when tested across multiple European sites.

Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

BACKGROUND: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. METHODS AND FINDINGS: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions­a proxy for recent infection­yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs­K101E, K103N, Y181C, and G190A­accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. CONCLUSIONS: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin.

The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.

Prevalence of hepatitis C virus (HCV) variants resistant to NS5A inhibitors in naïve patients infected with HCV genotype 1 in Tunisia.

BACKGROUND: Hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibitors have been recently developed to inhibit NS5A activities and have been approved for the treatment of HCV infection. However the drawback of these direct acting antivirals (DAAs) is the emergence of resistance mutations. The prevalence of such mutations conferring resistance to HCV-NS5A inhibitors before treatment has not been investigated so far in the Tunisian population. The aim of this study was to detect HCV variants resistant to HCV-NS5A inhibitors in hepatitis C patients infected with HCV genotype 1 before any treatment with NS5A inhibitors. METHODS: Amplification and direct sequencing of the HCV NS5A region was carried out on 112 samples from 149 untreated patients. RESULTS: In genotype 1a strains, amino acid substitutions conferring resistance to NS5A inhibitors (M28V) were detected in 1/7 (14.2 %) HCV NS5A sequences analyzed. In genotype 1b, resistance mutations in the NS5A region (R30Q; L31M; P58S and Y93H) were observed in 17/105 (16.2 %) HCV NS5A sequences analyzed. R30Q and Y93H (n = 6; 5.7 %) predominated over P58S (n = 4; 3.8 %) and L31M (n = 3; 2.8 %). CONCLUSIONS: Mutations conferring resistance to HCV NS5A inhibitors are frequent in treatment-naïve Tunisian patients infected with HCV genotype 1b. Their influence in the context of DAA therapies has not been fully investigated and should be taken into consideration.

Natural prevalence of hepatitis C virus (HCV) variants resistant to protease and polymerase inhibitors in patients infected with HCV genotype 1 in Tunisia.

Hepatitis C virus (HCV) protease inhibitors (PIs) and polymerase inhibitors: nucleos(t)ide inhibitors (NS5B-NIs) and non-nucleos(t)ide inhibitors (NS5B-NNIs) have been recently developed to inhibit protease (NS3) or polymerase (NS5B) activities. The drawback of antiviral treatment is the emergence of resistance mutations to the drugs. The prevalence of such mutations conferring resistance to PIs, NS5B-NIs, and NS5B-NNIs before treatment has not been investigated so far in the Tunisian population. The aim of this study was to investigate the prevalence of known substitutions conferring resistance to HCV-PIs, NS5B-NIs, and NS5B-NNIs in 149 untreated patients naïve of any novel or investigational anti-HCV drugs and infected with HCV genotype 1 (genotype 1a = 7; genotype 1b = 142). Twelve sequences (9.2%) of the 131/149 HCV NS3 sequences analyzed showed amino-acid substitutions associated with HCV PIs resistance mutations (T54S, n = 4 (3%); V55A, n = 2 (1.5%); Q80K, n = 4 (3%); R155K, n = 1 (0.7%); A156V, n = 1 (0.7%)). One (1%) of the 95/149 HCV NS5B sequences analyzed showed the substitution V321I conferring resistance to NS5B-NIs, while 34 of 95 (35.8%) showed substitutions conferring resistance to NS5B-NNIs (C316N, n = 2 (2%); M414L, n = 1 (1%); A421V, n = 8 (8.5%); M423A, n = 1 (1%); M423T, n = 2 (2%); I424V, n = 5 (5.2%); C445F, n = 1 (1%); I482T, n = 2 (2%); V494A, n = 1 (1%); P496A, n = 1 (1%); V499A, n = 15 (16%); S556G, n = 5 (5.2%)). Naturally occurring substitutions conferring resistance to NS3 or NS5B inhibitors exist in a substantial proportion of Tunisian treatment-naïve patients infected with HCV genotype 1. Their influence on treatment outcome should be assessed.

Activation of GCN2 upon HIV-1 infection and inhibition of translation.

Higher eukaryotic organisms have a variety of specific and nonspecific defense mechanisms against viral invaders. In animal cells, viral replication may be limited through the decrease in translation. Some viruses, however, have evolved mechanisms that counteract the response of the host. We report that infection by HIV-1 triggers acute decrease in translation. The human protein kinase GCN2 (eIF2AK4) is activated by phosphorylation upon HIV-1 infection in the hours following infection. Thus, infection by HIV-1 constitutes a stress that leads to the activation of GCN2 with a resulting decrease in protein synthesis. We have shown that GCN2 interacts with HIV-1 integrase (IN). Transfection of IN in amino acid-starved cells, where GCN2 is activated, increases the protein synthesis level. These results point to an as yet unknown role of GCN2 as an early mediator in the cellular response to HIV-1 infection, and suggest that the virus is able to overcome the involvement of GCN2 in the cellular response by eliciting methods to maintain protein synthesis.

Nucleoside reverse transcriptase inhibitor resistance mutations associated with first-line stavudine-containing antiretroviral therapy: programmatic implications for countries phasing out stavudine.

BACKGROUND: The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure. METHODS: We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance. RESULTS: Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ≥ two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M. CONCLUSIONS: Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.

Dried cervical spots for human papillomaviruses identification.

Financial and operational constraints limit low-resource countries in the screening of high-risk genital human papillomaviruses (HR-HPV), the etiological agents of cervical cancer. With its simple storage, conservation and shipping, dried cervical sample (DCS) could represent an efficient tool. The aim of the study was to evaluate the reliability of HPV genotyping from DCS. Cervical samples were obtained from 50 women infected with HIV-1 in Côte d'Ivoire. After DNA extraction from both DCS and matched liquid cervical samples (LCS), HPV genotyping was performed and the concordance of genotyping results was evaluated. HPV prevalence was 88% in LCS and 78% in DCS. Kappa statistic was 0.51 for the presence of any genotype (95% confidence interval, 0.25-0.77) and 0.73 for HR-HPV (0.45-0.99). Out of 50 samples, 45 were HPV-positive for DCS and/or LCS, and HR-HPV were detected in 37 samples (74%) with 36 HR-HPV multiple infections. Any genotype and HR genotype identification was concordant/compatible in 86% (43/50) and 88% (44/50) of samples, respectively. In most instances, kappa statistics for detection of type-specific HPV was over 0.6 (including HPV-16, -18, -31, -33). An excellent agreement (kappa statistic ≥ 0.81) was found for eight genotypes (HPV-6, -31, -35, -40, -56, -58, -66, and -82). In spite of interfering factors (multiple infections, different HPV loads, amplification competition, different inputs), DCS and LCS led to concordant/compatible results in most cases. DCS could represent an efficient tool for epidemiological field studies in resource-limited settings, and more importantly for improving the screening coverage and care management in women infected with HPV.

Phylogenetic analysis of HIV-1 archived DNA in blood and gut-associated lymphoid tissue in two patients under antiretroviral therapy.

One of the approaches to cure human immunodeficiency virus (HIV) is the use of therapeutic vaccination. We have launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles in aviremic patients under antiretroviral therapy (ART). A HIV-1 polypeptidic therapeutic vaccine based on viral sequence data obtained from circulating blood was proposed; here, our aim was to compare the proviral DNA in blood and gut-associated lymphoid tissue (GALT). Peripheral blood mononuclear cells and gut biopsies were obtained from two HIV-1 infected patients under successful antiretroviral therapy. Total DNA was extracted including the proviral DNA. The HIV-1 reverse transcriptase was sequenced in both compartments using next generation sequencing followed by single genome sequencing; phylogenetic trees were established and compared. The proviral sequences of both compartments intra-patient exhibited a very low genetic divergence while it was possible to differentiate the sequences inter-patients; single genome sequencing analysis of two couples of samples confirmed that there was no compartmentalization of the sequences intra-patient. We conclude that, considering these two cases, the proviral DNA sequences in blood and GALT are similar and that the epitope analysis of HIV-1 provirus in blood should be considered as relevant to that observed in the GALT, a hard-to-reach major compartment, and can therefore be used for therapeutic vaccine approaches.

Evolution of 2-long terminal repeat (2-LTR) episomal HIV-1 DNA in raltegravir-treated patients and in in vitro infected cells.

OBJECTIVES: Our aim was to analyse the evolution of HIV-1 2-long terminal repeat (2-LTR) circular DNA in vitro and ex vivo in the presence of raltegravir. PATIENTS AND METHODS: Twenty-five patients starting a raltegravir-based regimen were included. Total HIV-1 DNA and 2-LTR DNA were quantified at baseline and in follow-up samples up to month 12. The effect of raltegravir on the formation of 2-LTR circles was evaluated in HeLa P4 cells. The effect of raltegravir was also investigated by sequence analysis of the 2-LTR circle junctions. RESULTS: Among 21 patients with undetectable 2-LTR DNA at baseline, 7 had detectable 2-LTR DNA during the follow-up. Three of four patients with detectable 2-LTR DNA at baseline had undetectable 2-LTR DNA during the follow-up (P = 0.27). The mean 2-LTR level increased significantly (+0.07 log(10)/month, P = 0.02) in raltegravir-treated patients, and a 2-LTR increase was also observed in raltegravir-treated HeLa P4 cells, with a peak at 3 days post-infection. 2-LTR DNA showed a high prevalence of deletions ex vivo (64.5%) and in vitro (50%) in the presence of raltegravir, which was not statistically different from the prevalence in untreated patients or cells. CONCLUSIONS: In antiretroviral-experienced patients receiving raltegravir, 2-LTR DNA increased while total HIV-1 DNA decreased over time. The frequent rearrangements found in 2-LTR sequences warrant further investigations to determine the dynamics of evolution of unintegrated HIV-1 DNA.

HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens.

BACKGROUND: Our aim was to study the in vivo viral genetic pathways for resistance to raltegravir, in antiretroviral-experienced patients with virological failure (VF) on raltegravir-containing regimens. METHODS: We set up a prospective study including antiretroviral-experienced patients receiving raltegravir-based regimens. Integrase (IN) genotypic resistance analysis was performed at baseline. IN was also sequenced at follow-up points in the case of VF, i.e. plasma HIV-1 RNA>400 copies/mL at month 3 and/or >50 copies/mL at month 6. For phenotyping, the IN region was recombined with an IN-deleted HXB2-based HIV-1 backbone. A titrated amount of IN recombinant viruses was used for antiviral testing against raltegravir and elvitegravir. RESULTS: Among 51 patients, 11 (21.6%) had VF. Four different patterns of IN mutations were observed: (i) emergence of Q148H/R with secondary mutations (n=5 patients); (ii) emergence of N155H, then replaced by a pattern including Y143C/H/R (n=3); (iii) selection of S230N (n=1); and (iv) no evidence of selection of IN mutations (n=2). The median raltegravir and elvitegravir fold changes (FCs) were 244 (154-647) and 793 (339-892), respectively, for the Q148H/R pattern, while the median raltegravir and elvitegravir FCs were 21 (6-52) and 3 (2-3), respectively, with Y143C/H/R. The median plasma raltegravir Cmin was lower in patients with selection of the N155H mutation followed by Y143C/H/R compared with patients with Q148H/R and with patients without emerging mutations or without VF. CONCLUSIONS: Diverse genetic profiles can be associated with VF on raltegravir-containing regimens, including the dynamics of replacement of mutational profiles. Pharmacokinetic parameters could be involved in this genetic evolution.

ART-Treated Patients Exhibit an Adaptive Immune Response against the HFVAC Peptides, a Potential HIV-1 Therapeutic Vaccine (Provir/Latitude45 Study).

We proposed a new HIV-1 therapeutic vaccine based on conserved cytotoxic T lymphocyte (CTL) epitopes of archived HIV-1 DNA according to their affinity to the dominant HLA-A and -B alleles of the population investigated. Our proposal (Hla Fitted VAC, HFVAC) was composed of 15 peptides originating from the RT, gag and nef parts of proviral DNA. Our aim was to investigate baseline immune reactivity to the vaccine in HIV-1 chronically infected patients at success of antiretroviral therapy (ART) who would be eligible for a therapeutic vaccine. Forty-one patients were tested. Most of them had been infected with HIV-1 subtype B and all had been receiving successful ART for 2 to 20 years. The predominant HLA-A and -B alleles were those of a Caucasian population. ELISPOT was carried out using the HFVAC peptides. In 22 patients, the PD-1 marker was investigated on CD4+ and CD8+ T cells by flow cytometry in order to evaluate global T cell exhaustion. ELISPOT positivity was 65% overall and 69% in patients exhibiting at least one HLA allele fitting with HFVAC. The percentages of CD4+ and CD8+ T cells expressing PD-1 were high (median values 23.70 and 32.60, respectively), but did not seem to be associated with an impairment of the immune response investigated in vitro. In conclusion, reactivity to HFVAC was high in this ART-treated population with dominant HLA alleles, despite potential cellular exhaustion associated with the PD-1 marker.

HIV type-1 transmission dynamics in recent seroconverters: relationship with transmission of drug resistance and viral diversity.

BACKGROUND: HIV type-1 (HIV-1) has been shown to be frequently transmitted by acutely infected patients. We investigated the relationship between the dynamics of HIV-1 transmission within recently infected patients, the HIV-1 variability and the transmission of antiretroviral drug resistance. METHODS: We included patients infected between 1996 and 2006, with a plasma sample obtained <18 months after seroconversion and prior to antiretroviral therapy initiation. Reverse transcriptase (RT) and protease sequences were determined by direct population sequencing from plasma samples. Genotypic resistance was interpreted with the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales 2006 algorithm and International AIDS Society-USA list. Phylogenetic analysis (neighbour-joining and maximum likelihood methods) of RT sequences was used to determine the HIV-1 subtype and the interrelationship between sequences. RESULTS: Genotypic resistance was detected in 37/263 (14.1%) patients. Patients were infected by HIV-1 clade B in 222 (84%) cases and with non-B subtypes in 41 (16%). A total of 80 (30.4%) RT sequences were segregated in 24 clusters with bootstrap values >98% for 22 clusters. The frequency of grouping in clusters was higher within B sequences compared with non-B sequences (35.1% versus 4.9%; P<2.10(-4)). Drug-resistant isolates were retrieved in only 3 clusters, but the prevalence of resistance in clustering viruses (10/80, 12.5%) was not different than in isolated sequences. CONCLUSIONS: The segregation into clusters suggested frequent forward transmission events in patients infected with HIV-1 subtype B, including the possibility of transmission of drug-resistant isolates. These findings warrant increasing prevention efforts and serological screening in the at-risk populations.

Virological and immunological response in HIV-1-infected patients with multiple treatment failures receiving raltegravir and optimized background therapy, ANRS CO3 Aquitaine Cohort.

BACKGROUND: The efficacy of raltegravir plus optimized background therapy (OBT) has been demonstrated for antiretroviral (ARV)-experienced HIV-1-infected patients in randomized clinical trials. We studied viro-immunological response, pharmacokinetic parameters and genotypic test results in an observational cohort of multiple ARV class-experienced patients starting a raltegravir-based regimen. METHODS: Already enrolled ANRS CO3 Aquitaine Cohort patients with virological failure were included in this study after starting a raltegravir-based regimen (400 mg twice a day, week 0). Virological success was defined by the plasma HIV-1 RNA level [viral load (VL)] <2.7 log(10) copies/mL at week 12 and <1.7 log(10) copies/mL at week 24. One patient was excluded from further analysis (no follow-up after week 4). RESULTS: Fifty-one patients [male/female = 43/8, median age = 48 (interquartile range = 43, 55) years] were included. At week 0, median CD4 count was 244 (110; 310)/mm(3) and median VL was 4.2 (3.6, 4.7) log(10) copies/mL. At week 24, 39 (78%) patients experienced virological success: 4 (44%), 14 (82%) and 21 (87%) patients with a genotypic sensitivity score <1, > or =1 and <2 and > or =2 (P = 0.02), respectively. Raltegravir-related mutations emerged in 9 of 11 failing patients (82%): Q148H/R (n = 5), N155S/H (n = 3) and S230N (n = 1). Median CD4 increases from week 0 to week 4 and week 24 were 28 (-4, 85) and 57 (0, 156) cells/mm(3), respectively. A poor immune response was independently associated with a lower VL decline (week 0 to week 12) [odds ratio (OR): 3.5, 95% confidence interval (CI): 1.4, 8.4, for 1 log(10) less] and CD4+% at baseline (OR: 2.6, 95% CI: 0.97, 8.3, for 10% lower). CONCLUSIONS: Raltegravir plus OBT provided a good virological success rate in highly pre-treated patients under clinical routine conditions.

Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing.

Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Provir/Latitude 45 study: A step towards a multi-epitopic CTL vaccine designed on archived HIV-1 DNA and according to dominant HLA I alleles.

One of the approaches by which the scientific community is seeking to cure HIV is the use of therapeutic vaccination. Previous studies have highlighted the importance of the virus-specific CD8+ T cell cytotoxic responses for the immune control of HIV and have oriented research on vaccine constructs based on CTL epitopes from circulating HIV-1 strains. The clinical trials with therapeutic vaccines to date have had limited success likely due to (i) a discrepancy between archived CTL epitopes in the viral reservoir and those in circulating viruses before antiretroviral therapy (ART) initiation and (ii) the lack of strong affinity between the selected CTL epitopes and the HLA grooves for presentation to CD8+ cells. To overcome these limitations, we launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles of aviremic patients, most of whom are under ART. The near full-length genomes or Gag, Pol and Nef regions of proviral DNA were sequenced by Sanger and/or Next Generation Sequencing (NGS). The HLA-A and B alleles were defined by NGS or molecular analysis. The TuTuGenetics software, which moves a sliding window of 8 to 10 amino acids through the amino acid alignment, was combined with the Immune Epitope Data Base (IEDB) to automatically calculate the theoretical binding affinity of identified epitopes to the HLA alleles for each individual. We identified 15 conserved epitopes in Pol (11), Gag (3), and Nef (1) according to their potential presentation by the dominant HLA-A and B alleles and now propose to use the corresponding conserved peptides in a multi-epitopic vaccine (HLA-fitted VAC, HFVAC).