Quantification of HIV-RNA from dried blood spots using the Siemens VERSANT(R) HIV-1 RNA (kPCR) assay

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Viruses

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

Plos One

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)

Recent HIV-1 Infection Contributes to the Viral Diffusion over the French Territory with a Recent Increasing Frequency

To analyse the contribution of primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) to the French viral epidemic. sequences included 987 PHI from the French ANRS PRIMO cohort between 1999 and 2010 and were analysed using a population-based phylogenetic approach. Clinical features, risk factors, sexual behaviour and drug resistance for clustered and nonclustered transmission events were ascertained.Viruses from 125 (12.7%) of PHI cosegregated into 56 transmission chains, with increasing frequency during the last years (10.2% before 2006 versus 15.2% of clusters in 2006–2010, p = 0.02). The mean number of patients per cluster was 2.44. Compared to unique PHI, clusters involved more often men, infected through homosexual intercourse, of young age, with a high number of casual sexual partnerships and frequent previous HIV serological tests. Resistant strains were found in 16.0% and 11.1% of clusters and unique PHI, respectively (p = 0.11). Overall, 34% (n = 19) clusters included patients followed in French regions far apart, involving 13 clusters with at least one Parisian patient.PHIs are a significant source of onward transmission, especially in the MSM population. Recently infected people contribute to the spread of the viral epidemic throughout the French territory. Survey of transmitted drug resistance and behavioural characteristics of patients involved into clustered PHI may help to guide prevention and treatment interventions

Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping

BACKGROUND: Trofile is the prospectively validated HIV-1 tropism assay. Its use is limited by high costs, long turn-around time, and inability to test patients with very low or undetectable viremia. We aimed at assessing the efficiency of population genotypic assays based on gp120 V3-loop sequencing for the determination of tropism in plasma viral RNA and in whole-blood viral DNA. Contemporary and follow-up plasma and whole-blood samples from patients undergoing tropism testing via the enhanced sensitivity Trofile (ESTA) were collected. Clinical and clonal geno2pheno[coreceptor] (G2P) models at 10% and at optimised 5.7% false positive rate cutoff were evaluated using viral DNA and RNA samples, compared against each other and ESTA, using Cohen's kappa, phylogenetic analysis, and area under the receiver operating characteristic (AUROC). RESULTS: Both clinical and clonal G2P (with different false positive rates) showed good performances in predicting the ESTA outcome (for V3 RNA-based clinical G2P at 10% false positive rate AUROC = 0.83, sensitivity = 90%, specificity = 75%). The rate of agreement between DNA- and RNA-based clinical G2P was fair (kappa = 0.74, p < 0.0001), and DNA-based clinical G2P accurately predicted the plasma ESTA (AUROC = 0.86). Significant differences in the viral populations were detected when comparing inter/intra patient diversity of viral DNA with RNA sequences. CONCLUSIONS: Plasma HIV RNA or whole-blood HIV DNA V3-loop sequencing interpreted with clinical G2P is cheap and can be a good surrogate for ESTA. Although there may be differences among viral RNA and DNA populations in the same host, DNA-based G2P may be used as an indication of viral tropism in patients with undetectable plasma viremia

Antiretroviral-naive and -treated HIV-1 patients can harbour more resistant viruses in CSF than in plasma

Objectives The neurological disorders in HIV-1-infected patients remain prevalent. The HIV-1 resistance in plasma and CSF was compared in patients with neurological disorders in a multicentre study. Methods Blood and CSF samples were collected at time of neurological disorders for 244 patients. The viral loads were >50 copies/mL in both compartments and bulk genotypic tests were realized. Results On 244 patients, 89 and 155 were antiretroviral (ARV) naive and ARV treated, respectively. In ARV-naive patients, detection of mutations in CSF and not in plasma were reported for the reverse transcriptase (RT) gene in 2/89 patients (2.2%) and for the protease gene in 1/89 patients (1.1%). In ARV-treated patients, 19/152 (12.5%) patients had HIV-1 mutations only in the CSF for the RT gene and 30/151 (19.8%) for the protease gene. Two mutations appeared statistically more prevalent in the CSF than in plasma: M41L (P = 0.0455) and T215Y (P = 0.0455). Conclusions In most cases, resistance mutations were present and similar in both studied compartments. However, in 3.4% of ARV-naive and 8.8% of ARV-treated patients, the virus was more resistant in CSF than in plasma. These results support the need for genotypic resistance testing when lumbar puncture is performe

SIVagm Infection in Wild African Green Monkeys from South Africa: Epidemiology, Natural History, and Evolutionary Considerations

Pathogenesis studies of SIV infection have not been performed to date in wild monkeys due to difficulty in collecting and storing samples on site and the lack of analytical reagents covering the extensive SIV diversity. We performed a large scale study of molecular epidemiology and natural history of SIVagm infection in 225 free-ranging AGMs from multiple locations in South Africa. SIV prevalence (established by sequencing pol, env, and gag) varied dramatically between infant/juvenile (7%) and adult animals (68%) (p<0.0001), and between adult females (78%) and males (57%). Phylogenetic analyses revealed an extensive genetic diversity, including frequent recombination events. Some AGMs harbored epidemiologically linked viruses. Viruses infecting AGMs in the Free State, which are separated from those on the coastal side by the Drakensberg Mountains, formed a separate cluster in the phylogenetic trees; this observation supports a long standing presence of SIV in AGMs, at least from the time of their speciation to their Plio-Pleistocene migration. Specific primers/probes were synthesized based on the pol sequence data and viral loads (VLs) were quantified. VLs were of 104-106 RNA copies/ml, in the range of those observed in experimentally-infected monkeys, validating the experimental approaches in natural hosts. VLs were significantly higher (107-108 RNA copies/ml) in 10 AGMs diagnosed as acutely infected based on SIV seronegativity (Fiebig II), which suggests a very active transmission of SIVagm in the wild. Neither cytokine levels (as biomarkers of immune activation) nor sCD14 levels (a biomarker of microbial translocation) were different between SIV-infected and SIV-uninfected monkeys. This complex algorithm combining sequencing and phylogeny, VL quantification, serology, and testing of surrogate markers of microbial translocation and immune activation permits a systematic investigation of the epidemiology, viral diversity and natural history of SIV infection in wild African natural hosts. © 2013 Ma et al

Phylodynamic and Phylogeographic Profiles of Subtype B HIV-1 Epidemics in South Spain

Since 1982, HIV-1 epidemics have evolved to different scenarios in terms of transmission routes, subtype distribution and characteristics of transmission clusters. We investigated the evolutionary history of HIV-1 subtype B in south Spain.We studied all newly diagnosed HIV-1 subtype B patients in East Andalusia during the 2005-2012 period. For the analysis, we used the reverse transcriptase and protease sequences from baseline resistance, and the Trugene® HIV Genotyping kit (Siemens, Barcelona, Spain). Subtyping was done with REGA v3.0. The maximum likelihood trees constructed with RAxML were used to study HIV-1 clustering. Phylogeographic and phylodynamic profiles were studied by Bayesian inference methods with BEAST v1.7.5 and SPREAD v1.0.6.Of the 493 patients infected with HIV-1 subtype B, 234 grouped into 55 clusters, most of which were small (44 clusters ≤ 5 patients, 31 with 2 patients, 13 with 3). The rest (133/234) were grouped into 11 clusters with ≥ 5 patients, and most (82%, 109/133) were men who have sex with men (MSM) grouped into 8 clusters. The association with clusters was more frequent in Spanish (p = 0.02) men (p< 0.001), MSM (p<0.001) younger than 35 years (p = 0.001) and with a CD4+ T-cell count above 350 cells/ul (p<0.001). We estimated the date of HIV-1 subtype B regional epidemic diversification around 1970 (95% CI: 1965-1987), with an evolutionary rate of 2.4 (95%CI: 1.7-3.1) x 10-3 substitutions/site/year. Most clusters originated in the 1990s in MSMs. We observed exponential subtype B HIV-1 growth in 1980-1990 and 2005-2008. The most significant migration routes for subtype B went from inland cities to seaside locations.We provide the first data on the phylodynamic and phylogeographic profiles of HIV-1 subtype B in south Spain. Our findings of transmission clustering among MSMs should alert healthcare managers to enhance preventive measures in this risk group in order to prevent future outbreaks

Multiclass primary antiretroviral drug resistance in a patient presenting HIV-1/2 dual infection.

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