Evolution of experimental design and research techniques in HIV-1 reservoir studies : a systematic review

Although HIV-1 has evolved from a deadly to a chronic disease over the past 20 years, an HIV-1 cure is still lacking due to the presence of persisting cellular viral reservoirs which are spread throughout the body in different anatomical compartments. Hence, the identification and characterization of these HIV-1 reservoirs were the focus of many studies during the past decades. In this review, a systematic literature screening and text mining approach were implemented to assess the evolution in experimental design of these HIV-1 reservoir studies. For this purpose. the online databases PubMed, Web of Science. and ClinicalTrials.gov were consulted and 1768 articles were identified, of which 106 are included in this review. We observed several evolutions that indicate a more structured approach of recent HIV-1 reservoir studies. This includes the use of well-characterized patient cohorts, tissue sampling at several time points and anatomical compartments, the inclusion of patients with different treatment status (on and off antiretroviral therapy), and the implementation of state-of-the-art research techniques such as single genome sequencing. In addition, there is an increased interest and sampling of lymphoid tissues and cerebrospinal fluid together with methods to investigate cellular subsets and HIV-1 sequences. Overall, this review describes an observed shift from detecting and quantifying HIV-1 toward a qualitative in-depth assessment of anatomical reservoirs and cellular subsets playing a role in H1V-1 persistence/latency. These trends coincide with the evolution in focus from controlling HIV-1 replication by currently available antiretroviral therapy toward HIV-1 curative strategies

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Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency

Tissue is the issue : when a second biopsy reveals the true diagnosis

We describe the case of a woman with minimal glomerular changes on initial kidney biopsy. On long-term follow-up, the patient developed nephrotic proteinuria and a second kidney biopsy was performed, which revealed focal segmental glomerulosclerosis (FSGS). Findings from electron microscopy (EM) examination suggested a genetic form of FSGS. Next-generation sequencing showed heterozygosity for a mutation in COL4A3. Collagen IV nephropathies can be linked to late-onset FSGS. By establishing a genetic cause of FSGS, immunosuppressive treatment can be avoided. This case emphasizes the importance of re-biopsy in cases of a non-explained rise in proteinuria. EM can be helpful in differentiating between primary and secondary FSGS and informing treatment strategies. In cases of adult-onset FSGS that cannot be categorized by clinical-pathological assessment, genetic testing should be considered

Motivations, barriers and experiences of participants in an HIV reservoir trial

Objectives: We aimed to investigate the motives, barriers and experiences of HIV-STAR study participants. The HIV-STAR study was an analytical HIV treatment interruption trial (ATI) aiming to evaluate the origin of viral rebound, conducted in Ghent, Belgium. Methods: A mixed-method study was performed among 11 participants of the HIV-STAR study. Two self-administered questionnaires with 32 and 23 items, respectively, assessed motives, barriers and experiences of the research participants. In-depth interviews were conducted to further explore and understand topics that had emerged from these surveys. Results: Motives of ATI study participants were primarily related to the improvement of their own health perspectives and to their contribution to find an HIV cure. Barriers for ATI participation mostly related to practical issues, such as difficulty in planning study visits. Ten out of 11 participants reported a very high overall satisfaction and were willing to participate in another ATI. This satisfaction was predominantly linked to clear communication and guidance. Invasive sampling during the ATI was less of a burden than anticipated by participants. However, most participants underestimated the emotional impact of HIV treatment interruption, which was associated with feelings of uncertainty and loss of control. Risk of HIV transmission because of viral rebound was also mentioned as burdensome during this phase. Conclusions: Involvement in an ATI was positively evaluated by HIV-STAR participants. Contributing to HIV cure research outweighed the burden of study participation for most participants. The latter aspects were attenuated by mutual decision making and the experience of empathy from the research team. Still, issues regarding privacy and the psychosocial impact of treatment interruption, including sexuality and HIV transmissibility, should be addressed in a better way

In-depth validation of total HIV-1 DNA assays for quantification of various HIV-1 subtypes

HIV-1 DNA quantification serves as an important reservoir biomarker in HIV cure trials. However, the high genetic diversity of HIV-1 represented by different subtypes may bring inaccuracy in quantifying HIV-1 DNA and a sensitive and validated assay covering diverse HIV-1 subtypes is lacking. Therefore, we cross-validated total HIV-1 DNA assays described in literature using a three-step comparative analysis. First, a bioinformatics tool was developed in-house to perform an in silico evaluation of 67 HIV-1 DNA assays. Secondly, these selected assays were in vitro validated using a panel of different HIV-1 subtypes and, finally, ex vivo assessed on selected patient samples with different HIV-1 subtypes. Our results show that quantification of HIV-1 DNA substantially differs between assays and we advise five best performing HIV-1 DNA assays for ddPCR and qPCR (Schvachsa_2007, Viard_2004, Heeregrave_2009, Van_der_Sluis_2013, Yu_2008 and Yun_2002). This in-depth analysis of published HIV-1 DNA assays indicates that not all assays guarantee an optimal measurement of HIV-1 DNA, especially when looking across subtypes. Using an in-depth cross-validation, we were able to validate HIV-1 DNA assays that are suitable for quantification of HIV-1 DNA in a wide variety of HIV-1 infected patients

Evaluating predictive markers for viral rebound and safety assessment in blood and lumbar fluid during HIV-1 treatment interruption

Background: Validated biomarkers to evaluate HIV-1 cure strategies are currently lacking, therefore requiring analytical treatment interruption (ATI) in study participants. Little is known about the safety of ATI and its long-term impact on patient health. Objectives: ATI safety was assessed and potential biomarkers predicting viral rebound were evaluated. Methods: PBMCs, plasma and CSF were collected from 11 HIV-1-positive individuals at four different timepoints during ATI (NCT02641756). Total and integrated HIV-1 DNA, cell-associated (CA) HIV-1 RNA transcripts and restriction factor (RF) expression were measured by PCR-based assays. Markers of neuroinflammation and neuronal injury [neurofilament light chain (NFL) and YKL-40 protein] were measured in CSF. Additionally, neopterin, tryptophan and kynurenine were measured, both in plasma and CSF, as markers of immune activation. Results: Total HIV-1 DNA, integrated HIV-1 DNA and CA viral RNA transcripts did not differ pre- and post-ATI. Similarly, no significant NFL or YKL-40 increases in CSF were observed between baseline and viral rebound. Furthermore, markers of immune activation did not increase during ATI. Interestingly, the RFs SLFN11 and APOBEC3G increased after ATI before viral rebound. Similarly, Tat-Rev transcripts were increased preceding viral rebound after interruption. Conclusions: ATI did not increase viral reservoir size and it did not reveal signs of increased neuronal injury or inflammation, suggesting that these well-monitored ATIs are safe. Elevation of Tat-Rev transcription and induced expression of the RFs SLFN11 and APOBEC3G after ATI, prior to viral rebound, indicates that these factors could be used as potential biomarkers predicting viral rebound

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

Rapid viral rebound after analytical treatment interruption in patients with very small HIV reservoir and minimal on-going viral transcription

Introduction: Viral remission after analytical treatment interruption (ATI), termed post-treatment control, has been described in a small proportion of HIV-positive patients. This phenomenon has been separately associated to both low levels of HIV-1 proviral DNA as well as cell-associated RNA. We investigated whether the combination of both parameters could help predict delayed viral rebound after treatment interruption (TI). Methods: We conducted an open single-arm ATI study in four Belgian HIV reference centres from January 2016 to July 2018. Eligible participants were adults who had fewer than 50 HIV-1 RNA copies/mL for more than two years, more than 500 CD4 cells/mu L for more than three months, and were in general good health. Consenting participants who had fewer than 66 copies total HIV-1 DNA (t-DNA) and fewer than 10 copies cell-associated HIV-1 unspliced RNA (US-RNA) per million peripheral blood mononuclear cells (PBMCs), interrupted therapy and were monitored closely. Antiretroviral therapy (ART) was resumed after two consecutive viral loads exceeding 1000 copies or one exceeding 10,000 copies/mL. The primary outcome was the proportion of participants with fewer than 50 HIV-1 RNA copies/mL 48 weeks after TI. Secondary outcomes were time to viral rebound, the frequency of serious adverse events (AEs) and evolution of t-DNA and US-RNA after TI. Results: All 16 consenting participants who interrupted therapy experienced rapid viral rebound two to eight weeks after TI. No serious AEs were observed. Levels of t-DNA and US-RNA increased after TI but returned to pre-ATI levels after treatment restart. None of the studied demographic, clinical and biological parameters were predictive of time of viral rebound. Conclusions: The combination of low levels of t-DNA and US-RNA in PBMCs, corresponding respectively to a small and transcriptionally silent viral reservoir, is not predictive of viral remission after TI in patients on ART