Abstract
Objectives
To further characterize HIV-1 viruses of patients experiencing unexplained virological failure (VF) on PI-containing regimens, ultradeep sequencing was performed on protease, gag ...and gp41 genes in patients failing a first-line treatment.
Methods
All naive patients initiating an antiretroviral treatment based on boosted darunavir, atazanavir or lopinavir and experiencing VF without any transmitted drug resistance mutation detected by Sanger sequencing on protease and reverse transcriptase genes were selected. Ultradeep sequencing (IlluminaTM Nextera®) was performed on protease, gag and gp41 genes in plasma before initiation of treatment and at VF to identify emergent mutations.
Results
Among the 32 patients included in the study, emergent and previously undescribed mutations in the viral protease gene were identified in five patients at VF: 64M (1 CRF02_AG), 64M/70R with mutation 15V (2 CRF02_AG), 79A (1 CRF06_cpx) and 79A with mutation 15V (1 CRF02_AG). Two patients showed the emergence of R286K in the gag region, outside of cleavage sites (2 CRF02_AG). In the gp41 region, the V321I mutation emerged inside the cytoplasmic tail (1 subtype A and 1 subtype B). All these patients were treated with a darunavir/ritonavir-based regimen.
Conclusions
In some cases of VF to PIs, we observed the emergence of protease, Gag or Gp41 mutations that had not previously been associated with VF or PI resistance. These mutations should be further studied, in particular the 15V/64M/70R pattern in the protease gene identified among CRF02_AG viruses.
The genetic barrier (defined as the number of genetic transitions/transversions needed to produce a resistance mutation) can differ between HIV-1 subtypes. The genetic barrier for the new attachment ...inhibitor BMS-626529 was evaluated in five HIV-1 subtypes.
Nine substitutions associated with BMS-626529 resistance at seven amino acid positions (116, 204, 375, 426, 434, 475 and 506) were analysed in 300 nucleotide sequences of the env gene encoding the gp120 protein from antiretroviral-naive patients (60 for each subtype and recombinant: B, C, D, CRF01_AE and CRF02_AG).
Differently from the B subtype, some resistance mutations were found as natural polymorphisms in the C and D subtypes and the CRF02_AG and CRF01_AE recombinants for four positions of the env gene encoding the gp120 protein (375, 426, 434 and 475). The majority (five out of seven) of amino acid positions studied (116, 426, 434, 475 and 506) were relatively conserved (>63%) between the five HIV-1 subtypes, leading to a similar genetic barrier to mutations associated with resistance to BMS-626529. However, at positions 116 and 506 a minority of C and CRF02_AG subtypes had codons leading to a higher genetic barrier. Different predominant codons were observed at two out of seven positions (204 and 375) between the subtypes, with no effect on the calculated genetic barrier. However, for position 375, a minority of CRF02_AG sequences showed a lower genetic barrier to S375M/T resistance mutations.
In non-B HIV-1 subtypes, four out of seven studied positions presented mutations implicated in BMS-626529 resistance. Despite great variability of the HIV-1 envelope, there was no major impact of polymorphisms on the genetic barrier to acquisition of BMS-626529 resistance.
A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood mononuclear cells (PBMCs). The HTLV-I copy number was ...referred to the actual amount of cellular DNA by means of the quantitation of the albumin gene. Ten copies of HTLV-I DNA could be detected with 100% sensitivity, and the assay had a wide range of at least 5
log
10. Intra- and inter-assay reproducibility was evaluated using independent extractions of PBMCs from an HTLV-I-infected patient (coefficients of variation, 24 and 7% respectively). The performance of this TaqMan PCR assay, coupled with its high throughput, thus allows reliable routine follow-up of HTLV-I proviral load in infected patients. Preliminary results using clinical samples indicate a higher proviral load in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers, and also suggest the usefulness of this quantitative measurement to assess the etiological link between HTLV-I and adult T-cell leukaemia/lymphoma-like syndromes.
Objectives: To examine viral diversity and resistance mutations in different brain areas in cases of HIV-encephalopathy.Design: Twelve postmortem brain areas from three cases of possible or certain ...HIV-encephalopathy were analyzed.Methods: After amplification of the reverse transcriptase and the V3 loop region of the gp120 protein, ultradeep sequencing was performed with Illumina technology. Phylogenetic analysis was performed with Fastree v2.1 using the generalized time-reversible (GTR) model. Identification of resistant viral variants was performed on Geneious software, according to HIV-1 genotypic drug resistance interpretation's algorithms, 2018 administered by the French Agency for Research on AIDS and Viral Hepatitis.Results: Phylogenetic analysis revealed significant inter-regional and intra-regional diversity reflecting persistent HIV-1 viral replication in the different brain areas. Although some cerebral regions shared HIV-variants, most of them harbored a specific HIV-subpopulation reflecting HIV compartmentalization in the central nervous system. Furthermore, proportion and distribution of resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors differed among different brain areas of the same case suggesting that penetration of antiretroviral treatment may differ from one compartment to another.Conclusion: This study, performed with a powerful sequencing technique, confirmed HIV compartmentalization in the central nervous system already shown by classical sequencing, suggesting that there are several reservoirs within the brain.
Determination of polyomavirus BK (BKV) load in urine and plasma has been advocated for monitoring adult renal transplant recipients suffering from BKV-related nephropathy. An “in-house” real-time ...quantitative PCR assay was developed using the BKV-1/BKV-3 primers set in the large tumor antigen (LT-ag) region to quantitate BK virus loads in plasma and urine in renal transplant patients. This assay was adapted to routine virology laboratory by evaluating two extraction procedures of nucleic acids from urine and plasma, one manual and the other using an automatic extractor, and by evaluating the Light Cycler versus Taqman apparatus. Both the manual and automatic extraction procedures and real-time PCR apparatus were equivalent. The Light Cycler and Taqman instruments allow similarly rapid, accurate, reproducible and specific quantitative detection of the three major BKV subtypes, with a detection limit of 10 BKV DNA copies/ml, and a range from 10
0 to 10
7
copies/ml. Of 855 renal transplant patients, 128 (15%) had BKV DNA in both plasma and urine samples with a mean viral load of 5.1
log/ml in plasma and 6.8
log/ml in urine and in 5 (4%) BKV-associated tubulo-interstitial nephropathy; 332 (39%) BKV DNA was found only in the urine, not in the plasma, without further development of nephropathy and 395 patients had no BKV in plasma and urine. These observations emphasize the usefulness of real-time PCR to assess the BKV load by routine testing, and confirm the need to combine both plasma and urine determinations of the BKV DNA load in order to identify renal transplant patient at high risk for BKV-associated nephropathy.
The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 ...(HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of Csubscript T values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of Csubscript T values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.
Non-hodgkin lymphomas (NHL) are among the most common and fatal cancers in HIV-infected patients and transplant recipients. They are frequently associated with the Epstein Barr Virus (EBV) and a ...central nervous system (CNS) localization. Cancers neoantigens derived from the somatic tumor variants are a key factor of anti-tumor activity and response to immunotherapies, but the immunogenomics and tumor microenvironment (TME) characteristics of NHL from immunodeficient (ID) patients are lacking. We hypothesized they might be influenced by the low immunological pressure, the viral stimulation and/or the immune-privileged site. We report the comprehensive and prospective study of the tumor mutational burden (TMB), numbers of tumor neoepitopes, neoepitopes specific T cell responses and TME in ID patients, according to the EBV status, the immune status and the disease localization.Consecutive HIV-infected patients or transplant recipients with treatment-naïve NHL were included and compared with immunocompetent patients with diffuse large B-cell lymphomas (DLBCL). A whole exome sequencing (WES) was carried out in parallel on tumor and on PBMC for TMB assessment and a whole genome RNA sequencing was performed on frozen tumor biopsies. The neoepitopes derived from the somatic tumor variants (called in WES and RNAseq data) were predicted in silico using a bioinformatic pipeline including pVACseq and NetMHC and filtered according to: VAF≥10%, gene expression≥1 TPM and affinity binding score ≤500 nM. Neoepitopes specific T cell responses were assessed by ELISPOT assays after a 10 days PBMC co-culture with peptides from the Top 50 best predicted autologous neoepitopes (positivity defined as spot forming cells >50/106 cells). For the TME study, the profiling of cell type abundance and the T-cell receptor (TCR) sequencing were performed with deconvolution tools (EPIC) and MiXCR respectively. All statistical tests used R.Sixty-seven patients were included so far: 33 post-transplant lymphoproliferative disorders (PTLD) and 16 HIV-positive NHL, 70% of whom having DLBCL, compared to 18 immunocompetent (IC)-DLBCL. Diseases were systemic in 73% and localized in CNS in 27% cases. The median age was 58 years (range 21-85) and 70% were male. Immuno-molecular data are available for 57 patients so far. The TMB was 3.7/Mb for all patients but was lower in EBV-positive NHL (2.7/Mb) compared to the negative ones (4/Mb) (p=0.012, t-test) (Fig 1) without difference according to the ID status. The overall median number of neoepitopes per tumor predicted from the non-immunoglobulin (Ig) variants was 113 (range 11-720) but was lower in EBV-positive NHL (49 vs 243, p= 0.04, t-test), correlating with the TMB (r=0.8, p<0.0001). Most neoepitopes were MHC-class II restricted (ratio MHC-class II/ MHC- class I= 4.1) independently of the EBV status. The median number of neoepitopes predicted from the Ig heavy chain (IgH) genes was 17.5 (range, 6-36) with a MHC-class II/MHC-class I ratio of 1.4. Neoepitopes specific T cell responses were detectable among 71% cases out of 14 patients tested so far, independently of ID status or CNS localization (Fig 2). All 3 positive responses against Ig-derived neoepitopes out of 5 tested cases were directed against the IgH, MHC-class II restricted and immunodominant compared to those against the non-Ig neoepitopes. There was no shared immunogenic neoepitopes between patients. The TME study showed a higher frequency of CD4+ and CD8+ T cells mean frequency in EBV-positive compared to EBV-negative NHL (11% vs 4% and 10% vs 2% respectively, p<0.03).The intra-tumoral TCR repertoire distribution was mostly polyclonal with a median frequency of dominant clones of 8% (range 1-61) and a lower repertoire diversity, assessed on the ratio of clonotypes number/ total TCR-β reads number, in EBV-positive NHL (29) than in EBV-negative ones (37) (p=0.02), without difference according to the immune status.Our data demonstrate that both immunogenomics and TME of NHL from immuno-compromized patients are preferentially influenced by the EBV status, with lower numbers of tumor neoepitopes correlating with lower TMB, and a lower intra-tumoral TCR diversity in EBV-positive versus EBV-negative NHL. We further demonstrated that anti-tumor immune responses can be mounted despite ID status or CNS localization, especially against MHC-class II presented Ig-variants, and might be targeted for therapeutic strategies.
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