The aim of this study is the gain new insights into HIV latency before and after cure intervention studies through extensive blood and tissue sampling (lymph node and colon biopsies) from 30 individuals.
Study Type
OBSERVATIONAL
Ghent University Hospital
Ghent, Belgium
Quantification of total and intact HIV DNA and HIV RNA
Rainbow assay: multiplex digital PCR approach that combines five different HIV-1 regions to quantify total HIV-1 DNA and intact HIV-1 DNA simultaneously (Qiacuity dPCR platform, Qiagen). mutliplex digital PCR approach to quantify HIV RNA
Time frame: 5 years
Integration site analysis
HIV/host DNA junctions will be amplified using the Integration Site Loop Amplification (ISLA) assay, and resulting chimeric amplicons will be sequenced by Sanger.
Time frame: 5 years
Full-length HIV genome analysis
Full-Length Individual Proviral Sequencing (FLIPS) assay: nested PCR with Illumina MiSeq.
Time frame: 5 years
Epigenetic analysis
Methylation (bisulfite conversion) and chromatin accessibility (Assay for Transposase-Accessible Chromatin using sequencing)
Time frame: 5 years
Matched integration site and proviral sequencing
MIP-seq: captures full-length viral genome sequences in conjunction with its associated viral integration site
Time frame: 5 years
Proviral UMI-mediated Long-read Sequencing
HIV-PULSE: characterize the composition of the viral reservoir using long-read sequencing. Involves pre-amplifying individual proviral genomes using PCR and tagging them with dual UMIs, followed by long-range PCR amplification and long-read sequencing on the Oxford Nanopore MinION platform
Time frame: 5 years
Transcriptome analysis
* Bulk RNA sequencing on extracted RNA (Illumina Hiseq 2500 with 10-100 ng input of ribodepleted RNA) * Single cell RNA sequencing (10x genomics technology )
This platform is for informational purposes only and does not constitute medical advice. Always consult a qualified healthcare professional.
Time frame: 5 years
High dimensional phenotyping
CyTOF (mass cytometry, Fluidigm) combined with bioinformatics approach to extensively characterize the phenotype of latently infected cells
Time frame: 5 years
Immunohistochemistry, RNA- and DNA In Situ Hybridization
Immunochemistry will be used to study the expression of activation and exhaustion markers on tissues samples , while viral expression will be assessed through DNAScope and RNAScope technologies
Time frame: 5 years
Immunometabolic profile analysis
Mass spectrometry metabolomics will be used to study the immunometabolic profile of latently infected cells
Time frame: 5 years
Detection of translation-competent reservoirs
HIV-Flow assay: flow cytometry based assay using a combination of 2 antibodies targeting the p24 protein and allowing the detection of cells containing translation-competent viruses. p24+ cells detected by this assay can be sorted for downstream applications and further characterization of translation-competent reservoirs. The Simultaneous TCR Integration site and Provirus sequencing (STIP-seq) assay will be performed to sequence the proviral genome and matched integration sites of the translation-competent viruses, as well as phenotypic characterization and TCR sequencing of the host cell. characterization of translation-competent reservoirs.
Time frame: 5 years
Immunological analysis-FACS
Immunophenotyping by flow cytometric assays will be performed of different cells to assess the phenotype of innate immune cells, using FACS analysis.
Time frame: 5 years
Immunological analysis-ELISA
Immunophenotyping by flow cytometric assays will be performed of different cells to assess the phenotype of innate immune cells, using ELISA.
Time frame: 5 years
Microbiome monitoring
Gut microbiome will be analyzed in stool and colon biopsies using next-generation sequencing (NGS) of rRNA gene amplicons to identify bacteria at genus/species level
Time frame: 5 years