Emerging PCR-based techniques to study HIV-1 reservoir persistence

While current antiretroviral therapies are able to halt HIV-1 progression, they are not curative, as an interruption of treatment usually leads to viral rebound. The persistence of this stable HIV-1 latent reservoir forms the major barrier in HIV-1 cure research. The need for a better understanding of the mechanisms behind reservoir persistence resulted in the development of several novel assays allowing to perform an extensive in-depth characterization. The objective of this review is to present an overview of the current state-of-the-art PCR-based technologies to study the replication-competent HIV-1 reservoir. Here, we outline the advantages, limitations, and clinical relevance of different approaches. Future HIV-1 eradication studies would benefit from information-rich, high-throughput assays as they provide a more efficient and standardized way of characterizing the persisting HIV-1 reservoir

The elusive source of HIV-1 rebound after treatment interruption

Identifying the source of viral rebound during a monitored analytical treatment interruption (ATI) would reveal potential targets for cure strategies. Therefore, we examined the genetic composition of proviral DNA in different subsets from participants on antiretroviral therapy and compared this to rebounding virus after an ATI. Eleven participants underwent a monitored ATI and were sampled from different anatomical sites prior to and after the ATI. From the peripheral blood, Naïve (TNA), central (TCM), transitional (TTM) and effector (TEM) memory CD4+ T cells were sorted as were CD45 cells from gut-associated lymphoid tissue (GALT). Using single-genome sequencing (SGS) the env region of HIV DNA and plasma-derived RNA was sequenced. In an ongoing study, Full-Length Individual Proviral Sequencing (FLIPS) and Integration Site Loop Amplification (ISLA) assays were performed on the T cell subsets from 2 participants. For participant STAR10, 87 integration sites (IS) and 113 proviral genomes were sequenced while only 3 unique intact proviruses (3%) were identified. A cluster of 17 identical defective proviruses were linked to an IS (9% of all IS) in STAT5B located in TCM, TNA, TEM and TTM. When comparing the FLIPS to SGS env sequences a 100% match was found between one defective provirus and one plasma HIV RNA sequence after rebound. For participant STAR11, 37 IS and 105 proviral genomes were sequenced yielding 14 intact proviruses (13%) with the highest proportion found predominantly in the TEM subset (n=13, 45%). Four different clusters of identical sequences could be identified of which 2 (n=3 and n=9) consisted of intact TEM sequences with the smaller cluster linked to an IS in ZNF274. A 99% match between 2 env from rebounding plasma RNA and this smaller cluster of intact proviral genomes was identified. Comparing proviral sequences and their IS to plasma-derived RNA sequences after an ATI reveals additional information in terms of the source of viral rebound. However, this comparison is complicated by multiple factors. For example, we found a plasma-derived RNA sequence obtained during viral rebound matched a defective proviral sequence which highlights the problem of using one HIV RNA subgenomic region for identifying replication-competent virus. In addition, ongoing viral replication during rebound may prevent a 100% match with genetically intact proviral sequences making it challenging to determine the absolute source of rebound

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers

Background: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). Methods: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. Results: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. Conclusion:GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.</p

The potential of long-read sequencing by MinION in the setting of a diverse genetic background of HIV-1

Recent advances in Nanopore sequencing have paved the way for new opportunities in the field of viral diagnostics in clinical settings (i.e. Ebola and Zika outbreaks). Therefore, we aimed to explore the clinical applicability of Nanopore sequencing (MinION) and its associated tools within an HIV setting, which is characterized by a diverse genetic background, as the generated long reads could lead to valuable information with a clear benefit to HIV cure research. This proof-of-concept study included several full-length HIV-1 amplicons which were previously sequenced via Illumina and were obtained via PCR amplification on a clinical blood sample from an HIV-1 patient on antiretroviral therapy. The sequencing library was constructed using the SQK-LSK109 kit from Oxford Nanopore Technologies, followed by sequencing on an R9.4 flow cell on a MinION device. Subsequent base calling was carried out with Albacore v2.3.4, while minimap2 was used to map the reads against the known HIV-1 amplicons. In a last step, consensus and de novo sequences were constructed using a range of tools like SAMtools or Canu in order to compare performance with the additional use of Nanopolish. The generated assemblies showed high accuracies (>95%) when aligned to their Illumina counterparts. When applying Nanopolish, a mild improvement in accuracy was noted. Overall, this proof-of-concept study showed that the MinION device is capable of generating accurate, long-read HIV-1 sequences while capturing the intrinsic viral diversity within an HIV-1 patient. Therefore, the MinION Nanopore sequencing technology can have a prominent role in clinical HIV-1 settings and is worth further research

Long-read sequencing assay allows accurate characterization of the HIV-1 reservoir

Background The advent of near full-length (NFL) HIV-1 proviral genome sequencing greatly expanded our understanding of the quality of the viral reservoir, revealing that only 2-5% of the persistent proviruses in ART-treated individuals can be considered genome-intact. However, current NFL assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. We developed a long-read sequencing assay to characterize many proviral genomes in parallel from bulk DNA. Methods The sensitivity of the long-read assay was determined on a DNA dilution series of J-Lat in uninfected Jurkat ranging from 80,000 to <8 HIV-1 copies. Next, the assay was performed on 15 chronic ART-suppressed individuals, using a fixed input of 500 ng DNA extracted from peripheral blood CD4 T cells (reservoir sizes ranging from 321 to 6581 total HIV-1 DNA copies/million CD4 T cells). Individual proviruses were tagged with a different unique molecular identifier (UMI) at each end during a single reaction, followed by NFL PCR amplification and long-read sequencing on an Oxford Nanopore MinION. UMI-based demultiplexing allowed for the construction of highly accurate consensus genomes, while excluding aberrant chimeric PCR artefacts. In addition, Full-Length Individual Provirus Sequencing (FLIPS) was performed on 2 individuals. Data from both assays were compared through phylogenetic analyses. Results The lower limit of the long-read assay was found to be <8 HIV-1 copies. The long-read assay yielded an average of 14 distinct HIV-1 proviruses per participant (range: 3-42). Across all participants, 213 distinct proviruses were retrieved of which 8% were considered putatively intact. In terms of reservoir composition, data obtained with FLIPS showed an overall agreement with data obtained with the long-read assay. In an individual with limited clonality (6% clonality of FLIPS data, n=1 clone) only 1 overlapping provirus was found, while an overlap of 3 proviruses was observed in an individual with higher clonality (91% clonality of FLIPS data, n=4 clones). Comparing the 4 overlapping proviral consensus genomes to their matching FLIPS counterparts showed an average sequence accuracy of 99,97%. Conclusion The long-read assay offers a high-throughput NFL sequencing method which enables an accurate characterization of the proviral landscape while retaining sequencing accuracy comparable to current gold standard NFL assays

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of residual viremia and rebound viruses

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HIV-PULSE: A long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1,308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11% vs 18%) and overall good concordance, though at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.</jats:p

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia

To provide in depth characterization of HIV reservoir cells, the authors here develop a single-cell approach to simultaneously sequence TCR, integration sites and proviral genomes, called STIP-Seq, and show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5’-end of the genome