Highlights from the HIV Cure and Reservoir Symposium, 11-12 September 2017, Ghent, Belgium

For the second time, the HIV Cure Research Center (HCRC) at Ghent University organised the HIV Cure and Reservoir Symposium, in Ghent, Belgium, where in this two-day conference, virologists, molecular biologists, immunologists and clinicians presented the most recent achievements in the field of HIV cure, including data on therapeutic vaccines, HIV remission strategies such as 'shock and kill' or 'block and lock', benefits of early ART and potential of haematopoietic stem cell transplant in achieving cure. Furthermore, methods to characterise and quantify the HIV reservoir were discussed along with HIV reservoir characterisation in different body parts, including the central nervous system. An HIV activist and representative of a patients' agency also presented the patients' perspective on HIV cure. This report is a summary of all topics discussed during this symposium

Underestimated effect of intragenic HIV-1 DNA methylation on viral transcription in infected individuals

Background: The HIV-1 proviral genome harbors multiple CpG islands (CpGIs), both in the promoter and intragenic regions. DNA methylation in the promoter region has been shown to be heavily involved in HIV-1 latency regulation in cultured cells. However, its exact role in proviral transcriptional regulation in infected individuals is poorly understood or characterized. Moreover, methylation at intragenic CpGIs has never been studied in depth. Results: A large, well-characterized HIV-1 patient cohort (n = 72), consisting of 17 long-term non-progressors and 8 recent seroconverters (SRCV) without combination antiretroviral therapy (cART), 15 early cART-treated, and 32 late cART-treated patients, was analyzed using a next-generation bisulfite sequencing DNA methylation method. In general, we observed low level of promoter methylation and higher levels of intragenic methylation. Additionally, SRCV showed increased promoter methylation and decreased intragenic methylation compared with the other patient groups. This data indicates that increased intragenic methylation could be involved in proviral transcriptional regulation. Conclusions: Contrasting in vitro studies, our results indicate that intragenic hypermethylation of HIV-1 proviral DNA is an underestimated factor in viral control in HIV-1-infected individuals, showing the importance of analyzing the complete proviral genome in future DNA methylation studies

Single cell epigenetic visualization assay

Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.</jats:p

Single cell and spatial transcriptomics highlight the interaction of club-like cells with immunosuppressive myeloid cells in prostate cancer

Prostate cancer treatment resistance is a significant challenge facing the field. Genomic and transcriptomic profiling have partially elucidated the mechanisms through which cancer cells escape treatment, but their relation toward the tumor microenvironment (TME) remains elusive. Here we present a comprehensive transcriptomic landscape of the prostate TME at multiple points in the standard treatment timeline employing single-cell RNA-sequencing and spatial transcriptomics data from 120 patients. We identify club-like cells as a key epithelial cell subtype that acts as an interface between the prostate and the immune system. Tissue areas enriched with club-like cells have depleted androgen signaling and upregulated expression of luminal progenitor cell markers. Club-like cells display a senescence-associated secretory phenotype and their presence is linked to increased polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) activity. Our results indicate that club-like cells are associated with myeloid inflammation previously linked to androgen deprivation therapy resistance, providing a rationale for their therapeutic targeting

Evaluation of bisulfite kits for DNA methylation profiling in terms of DNA fragmentation and DNA recovery using digital PCR

<div><p>DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment kits provide good conversion efficiencies, DNA loss and especially DNA fragmentation remain troublesome. This hampers DNA methylation profiling of long DNA sequences. Here, we explored the performance of twelve commercial bisulfite kits by an in-depth comparison of DNA fragmentation using gel electrophoresis, qPCR and digital PCR, DNA recovery by spectroscopic measurements and digital PCR and conversion efficiency by next generation sequencing. The results show a clear performance difference between the bisulfite kits, and depending on the specific goal of the study, the most appropriate kit might differ. Moreover, we demonstrated that digital PCR is a valuable method to monitor both DNA fragmentation as well as DNA recovery after bisulfite treatment.</p></div

Cq values ± SD of the smallest and the largest amplicons.

<p>The data used are the geometric means of the average values from the five donor samples as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.s003" target="_blank">S3 Table</a>. The data labels refer to the kit number as provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.t001" target="_blank">Table 1</a>.</p

Effect of time and temperature.

<p>Effect of time and temperature of the conversion protocol on fragmentation between the different bisulfite conversion kits. Labels refer to the kit numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.t001" target="_blank">Table 1</a>. The upper panels (A-B) show the effect of different conversion temperatures between the kits measured by qPCR (A) and dPCR (B). The lower panels (C-D) show the effect of different conversion times between the kits measured by qPCR (C) and dPCR (D). The values on the x-axis are normalized to show the relative amount of fragmentation: 0 is least fragmenting and 1 is most fragmenting.</p