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Abstract: RNA interference is a powerful tool used to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. Almost all HIV-1 genes have been targets for small interfering RNA (siRNA) molecules, and HIV-1 replication can be specifically and successfully inhibited by this technique. RNA interference has been proposed as an alternative strategy to inhibit replication of drug-resistant viruses that emerge during suboptimal antiretroviral therapy for HIV-1. To investigate specific inhibition of drug-resistant HIV-1 by RNA interference, we designed siRNA molecules that recognize codons 181–188 of the reverse transcriptase (RT) gene of wild-type HIV-1 and HIV-1 carrying the M184V mutation, which confers high-level resistance to the RT inhibitor lamivudine. Using viral variants with single point mutations at codon 184, we measured the impact of these mutations on virus replication. We have demonstrated that siRNA targeting either wild-type HIV-1 or M184V variants inhibits replication of the corresponding virus, but does not influence replication of virus with a mismatch in the targeted region. Combining two effective siRNAs did not show synergistic inhibitory effect on HIV-1 replication. However, a combination of lamivudine and siRNA–M184V was very effective in inhibiting replication of both wild-type and variant M184V viruses in mixed infection experiments. Taken together, these results demonstrate that RNA interference might be useful in the treatment of drug-resistant HIV-1 infection. [Copyright &y& Elsevier]

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSFWT and HIV-1 JR-CSFK65R_M184V. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells. [ABSTRACT FROM AUTHOR]

In people with HIV-1 (PWH), Mycobacterium tuberculosis (MTB) infection poses a significant threat. While active tuberculosis (TB) accelerates immunodeficiency, the interaction between MTB and HIV-1 during asymptomatic phases remains unclear. Analysis of peripheral blood mononuclear cells (PBMC) transcriptomic profiles in PWH, with and without controlled viral loads, revealed distinct clustering in MTB-infected individuals. Functional annotation identified alterations in IL-6, TNF, and KRAS pathways. Notably, MTB-related genes displayed an inverse correlation with HIV-1 viremia, at both individual and signature score levels. These findings suggest that MTB infection in PWH induces a shift in immune system activation, inversely related to HIV-1 viral load. These results may explain the observed enhanced antiretroviral control in MTB-infected PWH. This study highlights the complex interplay between MTB and HIV-1, emphasizing the importance of understanding their interaction for managing co-infections in this population., Competing Interests: A.C. received grants from Merck Sharp & Dohme (MSD), ViiV Healthcare, and Gilead Sciences for unrelated research. R.D.K. received grants from Gilead Sciences and National Institutes of Health (NIH) for unrelated research. D.L.B. received honoraria for working on the advisory board of Gilead Sciences, Merck, ViiV, Pfizer, and AstraZeneca. D.L.B. received honoraria for presentations from Gilead Sciences and Merck. E.B. received grants from MSD for unrelated research. E.B. received payments for travel reimbursement from ViiV, MSD, Gilead Sciences, Pfizer, and Abbvie. E.B. received honoraria for working on the advisory board of ViiV, MSD, Pfizer, Gilead Sciences, AstraZeneca, and Ely Lilly. H.H.H. received honoraria for working on the advisory board of AiCuris, Merck, Vera Dx, and Molecular Partners. H.H.H. received honoraria for presentations from Merck, Gilead Sciences, Biotest, and Vera Dx. J.N. received honoraria for presentations from Oxford Immunotec, Gilead and ViiV. H.FG. received honoraria for working on the advisory board of Gilead Sciences, Merck, ViiV, Janssen, Johnson and Johnson, Novartis, and GlaxoSmithKline (GSK). H.F.G. received payments for travel reimbursements from Gilead Sciences. H.F.G. received grants from NIH, Yvonne Jacob Foundation, and Gilead Sciences., (© 2024 The Author(s).)

HIV-1 RNA genetic diversity predicts time since infection, which is important for clinical care and research. It is unclear, however, whether proviral DNA genetic diversity sampled under suppressive antiretroviral therapy can be used for this purpose. We tested whether proviral genetic diversity from next-generation sequencing predicts time since infection and recency in 221 people with HIV-1 with known infection time. Proviral diversity was significantly associated with time since infection (P < 5×10-7, R2 up to 25%) and predictive of treatment initiation during recent infection (area under the curve-receiver operating characteristic up to 0.85). This shows the utility of proviral genetic diversity as a proxy for time since infection., Competing Interests: Potential conflicts of interest. A. R. has received research grants from Gilead; travel expenses from Gilead and Pfizer; and honoraria for data safety monitoring board or advisory board consultations from MSD and Moderna, all paid to his institution. D. L. B. has received personal consulting fees from Gilead, MSD, and ViiV; personal honoraria for presentations from Gilead, Pfizer, MSD, and ViiV; and travel expenses from Gilead and ViiV, paid to his institution. E. B. has received research grants from MSD; consulting fees from Moderna; honoraria for presentations from Pfizer; travel expenses from ViiV, MSD, Gilead, and Pfizer; and honoraria for data safety monitoring board or advisory board consultations from ViiV, MSD, Pfizer, Gilead, Moderna, AstraZeneca, AbbVie, and Ely Lilly, all paid to his institution. H. F. G. has received research grants from the Swiss National Science Foundation, Swiss HIV Cohort Study, Yvonne Jacob Foundation, Gilead, ViiV, and Bill and Melinda Gates foundation, paid to his institution; personal honoraria for data safety monitoring board or advisory board consultations from Merck, ViiV Healthcare, Gilead Sciences, Janssen, Johnson and Johnson, Novartis, and GSK; and personal travel expenses from Gilead. J. N. has received research grants from the Swiss HIV Cohort Study and the cantonal hospital St Gallen, paid to her institution. K. J. M. has received unrestricted research grants from Gilead and Novartis, paid to her institution; and personal honoraria for advisory board consultations from ViiV. M. C. has received research grants from Gilead, ViiV, and MSD; payment for expert testimony from Gilead, ViiV, and MSD; and travel expenses from Gilead, all paid to his institution. M. S. has received honoraria for data safety monitoring board advisory board consultations from Gilead, ViiV, Moderna, Pfizer, and MSD; and travel expenses for conferences from Gilead, all paid to his institution. P. F. has received personal travel expenses from the University Zurich; payment for equipment from the University Zurich; and personal honoraria for presentations from the University Zurich. R. D. K. has received research grants from Gilead and NIH, paid to his institution. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Even during extended periods of effective immunological control, a substantial dynamic of the viral genome can be observed in different cellular compartments in HIV-1 positive individuals, indicating the persistence of active viral reservoirs. To obtain further insights, we studied changes in the proviral as well as in the viral HIV-1 envelope (Env) sequence along with transcriptional, translational and viral outgrowth activity as indicators for viral dynamics and genomic intactness. Our study identified distinct reservoir patterns that either represented highly sequence-diverse HIV-1 populations or only a single / few persisting virus variants. The single dominating variants were more often found in individuals starting ART during early infection phases, indicating that early treatment might limit reservoir diversification. At the same time, more sequence-diverse HIV reservoirs correlated with a poorer immune status, indicated by lower CD4 count, a higher number of regimen changes and more co-morbidities. Furthermore, we noted that in T-cell populations in the peripheral blood, replication-competent HIV-1 is predominantly present in Lymph node homing TN (naïve) and TCM (central memory) T cells. Provirus genomes archived in TTM (transitional memory) and TEM (effector memory) T cells more frequently tended to carry inactivating mutations and, population-wise, possess changes in the genetic diversity. These discriminating properties of the viral reservoir in T-cell subsets may have important implications for new early therapy strategies, underscoring the critical role of early therapy in preserving robust immune surveillance and constraining the viral reservoir., Competing Interests: K.J.M. has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV and Abbott; and the University of Zurich received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies that Dr Metzner serves as principal investigator, and advisory board honoraria from Gilead Sciences and ViiV., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

People with HIV may report neurocognitive complaints, with or without associated neurocognitive impairment, varying between individuals and populations. While the HIV genome could play a major role, large systematic viral genome-wide screens to date are lacking. The Swiss HIV Cohort Study biannually enquires neurocognitive complaints. We quantified broad-sense heritability estimates using partial 'pol' sequences from the Swiss HIV Cohort Study resistance database and performed a viral near full-length genome-wide association study for the longitudinal area under the curve of neurocognitive complaints. We performed all analysis (i) restricted to HIV Subtype B and (ii) including all HIV subtypes. From 8547 people with HIV with neurocognitive complaints, we obtained 6966 partial 'pol' sequences and 2334 near full-length HIV sequences. Broad-sense heritability estimates for presence of memory loss complaints ranged between 1% and 17% (Subtype B restricted 1-22%) and increased with the stringency of the phylogenetic distance thresholds. The genome-wide association study revealed one amino acid (Env L641E), after adjusting for multiple testing, positively associated with memory loss complaints ( P = 4.3 * 10 -6 ). Other identified mutations, while insignificant after adjusting for multiple testing, were reported in other smaller studies (Tat T64N, Env *291S). We present the first HIV genome-wide association study analysis of neurocognitive complaints and report a first estimate for the heritability of neurocognitive complaints through HIV. Moreover, we could identify one mutation significantly associated with the presence of memory loss complaints. Our findings indicate that neurocognitive complaints are polygenetic and highlight advantages of a whole genome approach for pathogenicity determination., Competing Interests: An.R. received research grants from Gilead, paid to his institution; travel expenses from Gilead and Pfizer, paid to his institution; and honoraria for data safety monitoring board or advisory board consultations from MSD and Moderna, paid to his institution. C.P. received personal research grants from the Swiss HIV Cohort Study, Collegium Helveticum and the University of Zurich. E.B. received research grants from MSD, paid to his institution; consulting fees from Moderna, paid to his institution; honoraria for presentations from Pfizer, paid to his institution; travel expenses from ViiV, MSD, Gilead and Pfizer, paid to his institution; and honoraria for data safety monitoring board or advisory board consultations from ViiV, MSD, Pfizer, Gilead, Moderna, AstraZeneca, AbbVie and Ely Lilly, paid to his institution. H.F.G. has received research grants from the Swiss National Science Foundation, Swiss HIV Cohort Study, Yvonne Jacob Foundation, NIH, Gilead, ViiV and Bill and Melinda Gates foundation, paid to his institution; personal honoraria for data safety monitoring board or advisory board consultations from Merck, ViiV healthcare, Gilead Sciences, Janssen, Johnson and Johnson, Novartis and GSK; and personal travel expenses from Gilead. J.N. received research grants from the Swiss HIV Cohort Study and the cantonal hospital St. Gallen, paid to her institution, and travel expenses from Gilead. K.J.M. received unrestricted research grants from Gilead and Novartis, paid to her institution, and personal honoraria for advisory board consultations from ViiV. M.C. received research grants from Gilead, ViiV and MSD, paid to his institution; payment for expert testimony from Gilead, ViiV and MSD, paid to his institution; and travel expenses from Gilead, paid to his institution. M.S. received honoraria for data safety monitoring board advisory board consultations from Gilead, ViiV, Moderna, Pfizer and MSD, paid to his institution, and travel expenses for conferences from Gilead, paid to his institution. P.F. received personal travel expenses from the University Zurich, payment for equipment from the University Zurich and personal honoraria for presentations from the University of Zurich. R.D.K. received research grants from Gilead and NIH, paid to his institution. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: Clinical and laboratory monitoring of patients on antiretroviral therapy is an integral part of HIV care and determines whether treatment needs enhanced adherence or modification of the drug regimen. However, different monitoring and treatment strategies carry different costs and health consequences., Materials and Methods: The SIMPL'HIV study was a randomised trial that assessed the non-inferiority of dual maintenance therapy. The co-primary outcome was a comparison of costs over 48 weeks of dual therapy with standard antiretroviral therapy and the costs associated with a simplified HIV care approach (patient-centred monitoring [PCM]) versus standard, tri-monthly routine monitoring. Costs included outpatient medical consultations (HIV/non-HIV consultations), non-medical consultations, antiretroviral therapy, laboratory tests and hospitalisation costs. PCM participants had restricted immunological and blood safety monitoring at weeks 0 and 48, and they were offered the choice to complete their remaining study visits via a telephone call, have medications delivered to a specified address, and to have blood tests performed at a location of their choice. We analysed the costs of both strategies using invoices for medical consultations issued by the hospital where the patient was followed, as well as those obtained from health insurance companies. Secondary outcomes included differences between monitoring arms for renal function, lipids and glucose values, and weight over 48 weeks. Patient satisfaction with treatment and monitoring was also assessed using visual analogue scales., Results: Of 93 participants randomised to dolutegravir plus emtricitabine and 94 individuals to combination antiretroviral therapy (median nadir CD4 count, 246 cells/mm3; median age, 48 years; female, 17%),patient-centred monitoring generated no substantial reductions or increases in total costs (US$ -421 per year [95% CI -2292 to 1451]; p = 0.658). However, dual therapy was significantly less expensive (US$ -2620.4 [95% CI -2864.3 to -2331.4]) compared to standard triple-drug antiretroviral therapy costs. Approximately 50% of participants selected one monitoring option, one-third chose two, and a few opted for three. The preferred option was telephone calls, followed by drug delivery. The number of additional visits outside the study schedule did not differ by type of monitoring. Patient satisfaction related to treatment and monitoring was high at baseline, with no significant increase at week 48., Conclusions: Patient-centred monitoring did not reduce costs compared to standard monitoring in individuals switching to dual therapy or those continuing combined antiretroviral therapy. In this representative sample of patients with suppressed HIV, antiretroviral therapy was the primary factor driving costs, which may be reduced by using generic drugs to mitigate the high cost of lifelong HIV treatment., Trial Registration: ClinicalTrials.gov NCT03160105.

Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner., (© 2023. The Author(s).)

Background: T-cell responses during chronic viral infections become exhausted, which is reflected by upregulation of inhibitory receptors (iRs) and increased interleukin 10 (IL-10). We assessed 2 iRs-PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3)-and IL-10 mRNAs in peripheral blood mononuclear cells (PBMCs) and their soluble analogs (sPD-1, sTim-3, and IL-10) in plasma in chronic HIV-1/hepatitis C virus (HCV) coinfection and explored the effect of HCV treatment on these markers. We also aimed to establish whether iR expression may be determined by the HCV CD8 + T-cell immunodominant epitope sequence., Methods: Plasma and PBMCs from 31 persons with chronic HIV-1/HCV coinfection from the Swiss HIV Cohort Study were collected before and after HCV treatment. As controls, 45 persons who were HIV-1 negative with chronic HCV infection were recruited. Exhaustion markers were assessed by enzyme-linked immunosorbent assay in plasma and by quantitative reverse transcription polymerase chain reaction in PBMCs. Analysis of an HCV epitope sequence was conducted by next-generation sequencing: HLA-A*02-restricted NS3 1073-1081 and NS3 1406-1415 and HLA-A*01-restricted NS3 1436-1444 ., Results: The study revealed higher plasma sPD-1 ( P = .0235) and IL-10 ( P = .002) levels and higher IL-10 mRNA in PBMCs ( P = .0149) in HIV-1/HCV coinfection. A decrease in plasma sPD-1 ( P = .0006), sTim-3 ( P = .0136), and IL-10 ( P = .0003) and Tim-3 mRNA in PBMCs ( P = .0210) was observed following successful HCV treatment. Infection with the HLA-A*01-restricted NS3 1436-1444 ATDALMTGY prototype variant was related to higher sTim-3 levels than infection with the ATDALMTGF escape variant ( P = .0326)., Conclusions: The results underscore the synergistic effect of coinfection on expression of exhaustion markers, their reduction following successful HCV treatment and imply that iR levels may operate on an epitope-specific manner., Competing Interests: Potential conflicts of interest. H. F. G. reports having received honoraria from Gilead Sciences, Merck, ViiV, GSK, Janssen, Johnson & Johnson, and Novartis for serving on data and safety monitoring and/or advisory boards and has received a travel grant from Gilead Sciences. In addition, he has received grants from the Swiss National Science Foundation, the SHCS, the Yvonne Jacob Foundation, and the National Institutes of Health and unrestricted research grants from Gilead Sciences, all paid to the institution. K. J. M. has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV, and Abbott and advisory board honoraria from Gilead Sciences and ViiV. The University of Zurich has received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies for which K. J. M. serves as principal investigator. All other authors report no potential conflicts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4 + T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory T cells (T CM s) as main players, critical for HIV-1 reservoir eradication., Competing Interests: K.J.M. has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV, and Abbott, and the University of Zurich received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies of K.J.M. T.K. has received honoraria outside of the present study from Gilead Sciences, ViiV Healthcare, and Roche Diagnostics., (© 2023 The Authors.)

Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing (SS) as the primary method for HIV genotypic resistance testing. However, there are limited systematic data on comparability of these methods in a clinical setting for the presence of low-abundance drug resistance mutations (DRMs) and their dependency on the variant-calling thresholds., Methods: To compare the HIV-DRMs detected by SS and NGS, we included participants enrolled in the Swiss HIV Cohort Study (SHCS) with SS and NGS sequences available with sample collection dates ≤7 days apart. We tested for the presence of HIV-DRMs and compared the agreement between SS and NGS at different variant-calling thresholds., Results: We included 594 pairs of SS and NGS from 527 SHCS participants. Males accounted for 80.5% of the participants, 76.3% were ART naive at sample collection and 78.1% of the sequences were subtype B. Overall, we observed a good agreement (Cohen's kappa >0.80) for HIV-DRMs for variant-calling thresholds ≥5%. We observed an increase in low-abundance HIV-DRMs detected at lower thresholds [28/417 (6.7%) at 10%-25% to 293/812 (36.1%) at 1%-2% threshold]. However, such low-abundance HIV-DRMs were overrepresented in ART-naive participants and were in most cases not detected in previously sampled sequences suggesting high sequencing error for thresholds <3%., Conclusions: We found high concordance between SS and NGS but also a substantial number of low-abundance HIV-DRMs detected only by NGS at lower variant-calling thresholds. Our findings suggest that a substantial fraction of the low-abundance HIV-DRMs detected at thresholds <3% may represent sequencing errors and hence should not be overinterpreted in clinical practice., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose of Review: HIV-1 drug resistance (HIV DR) testing is routinely performed by genotyping plasma viruses using Sanger population sequencing. Next-generation sequencing (NGS) is increasingly replacing standardized Sanger sequencing. This opens up new opportunities, but also brings challenges., Recent Findings: The number of NGS applications and protocols for HIV DR testing is increasing. All of them are noninferior to Sanger sequencing when comparing NGS-derived consensus sequences to Sanger sequencing-derived sequences. In addition, NGS enables high-throughput sequencing of near full-length HIV-1 genomes and detection of low-abundance drug-resistant HIV-1 variants, although their clinical implications need further investigation. Several groups have defined remaining challenges in implementing NGS protocols for HIV-1 resistance testing. Some of them are already being addressed. One of the most important needs is quality management and consequently, if possible, standardization., Summary: The use of NGS technologies on HIV DR testing will allow unprecedented insights into genomic structures of virus populations that may be of immediate relevance to both clinical and research areas such as personalized antiretroviral treatment. Efforts continue to tackle the remaining challenges in NGS-based HIV DR testing., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Genomic safe harbors (GSH) are defined as sites in the host genome that allow stable expression of inserted transgenes while having no adverse effects on the host cell, making them ideal for use in basic research and therapeutic applications. Silencing and fluctuations in transgene expression would be highly undesirable effects. We have previously shown that transgene expression in Jurkat T cells is not silenced for up to 160 days after CRISPR-Cas9-mediated insertion of reporter genes into the adeno-associated virus site 1 (AAVS1), a commonly used GSH. Here, we studied fluctuations in transgene expression upon targeted insertion into the GSH AAVS1. We have developed an efficient method to generate and validate highly complex barcoded plasmid libraries to study transgene expression on the single-cell level. Its applicability is demonstrated by inserting the barcoded transgene Cerulean into the AAVS1 locus in Jurkat T cells via the CRISPR-Cas9 technology followed by next-generation sequencing of the transcribed barcodes. We observed large transcriptional variations over two logs for transgene expression in the GSH AAVS1. This barcoded transgene insertion model is a powerful tool to investigate fluctuations in transgene expression at any GSH site., Competing Interests: K.J.M. has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV and Abbott; the University of Zurich received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies wherein K.J.M. serves as principal investigator, and advisory board honoraria from Gilead Sciences. All other authors declare no conflict of interest., (© 2022 The Author(s).)

CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the 'shock and kill' cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5'LTR promoter, making them a promising 'shock' strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5'LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3' half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located -165 to -106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the 'shock and kill' cure approach.

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1-infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1-infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells.

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts., (© 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Background: The primary hurdle for the eradication of HIV-1 is the establishment of a latent viral reservoir early after primary infection. Here, we investigated the potential influence of human genetic variation on the HIV-1 reservoir size and its decay rate during suppressive antiretroviral treatment., Setting: Genome-wide association study and exome sequencing study to look for host genetic determinants of HIV-1 reservoir measurements in patients enrolled in the Swiss HIV Cohort Study, a nation-wide prospective observational study., Methods: We measured total HIV-1 DNA in peripheral blood mononuclear cells from study participants, as a proxy for the reservoir size at 3 time points over a median of 5.4 years, and searched for associations between human genetic variation and 2 phenotypic readouts: the reservoir size at the first time point and its decay rate over the study period. We assessed the contribution of common genetic variants using genome-wide genotyping data from 797 patients with European ancestry enrolled in the Swiss HIV Cohort Study and searched for a potential impact of rare variants and exonic copy number variants using exome sequencing data generated in a subset of 194 study participants., Results: Genome-wide and exome-wide analyses did not reveal any significant association with the size of the HIV-1 reservoir or its decay rate on suppressive antiretroviral treatment., Conclusions: Our results point to a limited influence of human genetics on the size of the HIV-1 reservoir and its long-term dynamics in successfully treated individuals.

Background: The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2) ., Methods: We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2 , and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean + /mCherry + ) and inactive (Cerulean - /mCherry + ) HIV-1 promoters were characterised., Results: Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2 , the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]., Conclusion: Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity., Competing Interests: Karin J. Metzner has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, Tibotec, Bristol-Myers Squibb, and Abbott; the University of Zurich has received research grants from Gilead, Roche and Merck Sharp & Dohme for studies that Karin J. Metzner serves as principal investigator, and advisory board honoraria from Gilead Sciences. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All other authors declare no competing interests relevant to this study., (© 2020 Inderbitzin et al.)

The HIV-1 reservoir is the major hurdle to curing HIV-1. However, the impact of the viral genome on the HIV-1 reservoir, i.e. its heritability, remains unknown. We investigate the heritability of the HIV-1 reservoir size and its long-term decay by analyzing the distribution of those traits on viral phylogenies from both partial-pol and viral near full-length genome sequences. We use a unique nationwide cohort of 610 well-characterized HIV-1 subtype-B infected individuals on suppressive ART for a median of 5.4 years. We find that a moderate but significant fraction of the HIV-1 reservoir size 1.5 years after the initiation of ART is explained by genetic factors. At the same time, we find more tentative evidence for the heritability of the long-term HIV-1 reservoir decay. Our findings indicate that viral genetic factors contribute to the HIV-1 reservoir size and hence the infecting HIV-1 strain may affect individual patients' hurdle towards a cure.

The HIV-1 reservoir is the major hurdle to a cure. We here evaluate viral and host characteristics associated with reservoir size and long-term dynamics in 1,057 individuals on suppressive antiretroviral therapy for a median of 5.4 years. At the population level, the reservoir decreases with diminishing differences over time, but increases in 26.6% of individuals. Viral blips and low-level viremia are significantly associated with slower reservoir decay. Initiation of ART within the first year of infection, pretreatment viral load, and ethnicity affect reservoir size, but less so long-term dynamics. Viral blips and low-level viremia are thus relevant for reservoir and cure studies.

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.

Host-directed therapies against HIV-1 are thought to be critical for long term containment of the HIV-1 pandemic but remain elusive. Because HIV-1 infects and manipulates important effectors of both the innate and adaptive immune system, identifying modulations of the host cell systems in humans during HIV-1 infection may be crucial for the development of immune based therapies. Here, we quantified the changes of the proteome in human CD4 + T cells upon HIV-1 infection, both in vitro and in vivo A SWATH-MS approach was used to measure the proteome of human primary CD4 + T cells infected with HIV-1 in vitro as well as CD4 + T cells from HIV-1-infected patients with paired samples on and off antiretroviral treatment. In the in vitro experiment, the proteome of CD4 + T cells was quantified over a time course following HIV-1 infection. 1,725 host cell proteins and 4 HIV-1 proteins were quantified, with 145 proteins changing significantly during the time course. Changes in the proteome peaked 24 h after infection, concomitantly with significant HIV-1 protein production. In the in vivo branch of the study, CD4 + T cells from viremic patients and those with no detectable viral load after treatment were sorted, and the proteomes were quantified. We consistently detected 895 proteins, 172 of which were considered to be significantly different between the viremic patients and patients undergoing successful treatment. The proteome of the in vitro -infected CD4 + T cells was modulated on multiple functional levels, including TLR-4 signaling and the type 1 interferon signaling pathway. Perturbations in the type 1 interferon signaling pathway were recapitulated in CD4 + T cells from patients. The study shows that proteome maps generated by SWATH-MS indicate a range of functionally significant changes in the proteome of HIV-infected human CD4 + T cells. Exploring these perturbations in more detail may help identify new targets for immune based interventions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Genotypic monitoring of drug-resistance mutations (DRMs) in HIV-1 infected individuals is strongly recommended to guide selection of the initial antiretroviral therapy (ART) and changes of drug regimens. Traditionally, mutations conferring drug resistance are detected by population sequencing of the reverse transcribed viral RNA encoding the HIV-1 enzymes target by ART, followed by manual analysis and interpretation of Sanger sequencing traces. This process is labor intensive, relies on subjective interpretation from the operator, and offers limited sensitivity as only mutations above 20% frequency can be reliably detected. Here we present MinVar, a pipeline for the analysis of deep sequencing data, which allows reliable and automated detection of DRMs down to 5%. We evaluated MinVar with data from amplicon sequencing of defined mixtures of molecular virus clones with known DRM and plasma samples of viremic HIV-1 infected individuals and we compared it to VirVarSeq, another virus variant detection tool exclusively working on Illumina deep sequencing data. MinVar was designed to be compatible with a diverse range of sequencing platforms and allows the detection of DRMs and insertions/deletions from deep sequencing data without the need to perform additional bioinformatics analysis, a prerequisite to a widespread implementation of HIV-1 genotyping using deep sequencing in routine diagnostic settings., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Diversity, evolution, and epidemiology of HIV are directly relevant to HIV transmission and pathogenesis; hence, they play a key role in antiretroviral treatment and vaccine design. Global HIV whole-genome sequencing would provide a treasure chest of data to answer many questions still open in these fields. An article by Berg et al. in this issue of theJournal of Clinical Microbiologydescribes a universal strategy to amplify and sequence heterogeneous HIV whole genomes (M. G. Berg, J. Yamaguchi, E. Alessandri-Gradt, R. W. Tell, J.-C. Plantier, and C. A. Brennan, J Clin Microbiol 54:868-882, 2016,http://dx.doi.org/10.1128/JCM.02479-15)., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background.  The hepatitis C virus (HCV) epidemic is evolving rapidly in patients infected with human immunodeficiency virus (HIV). We aimed to describe changes in treatment uptake and outcomes of incident HCV infections before and after 2006, the time-point at which major changes in HCV epidemic became apparent. Methods.  We included all adults with an incident HCV infection before June 2012 in the Swiss HIV Cohort Study, a prospective nationwide representative cohort of individuals infected with HIV. We assessed the following outcomes by time period: the proportion of patients starting an HCV therapy, the proportion of treated patients achieving a sustained virological response (SVR), and the proportion of patients with persistent HCV infection during follow-up. Results.  Of 193 patients with an HCV seroconversion, 106 were diagnosed before and 87 after January 2006. The proportion of men who have sex with men increased from 24% before to 85% after 2006 (P < .001). Hepatitis C virus treatment uptake increased from 33% before 2006 to 77% after 2006 (P < .001). Treatment was started during early infection in 22% of patients before and 91% after 2006 (P < .001). An SVR was achieved in 78% and 29% (P = .01) of patients treated during early and chronic HCV infection. The probability of having a detectable viral load 5 years after diagnosis was 0.67 (95% confidence interval [CI], 0.58-0.77) in the group diagnosed before 2006 and 0.24 (95% CI, 0.16-0.35) in the other group (P < .001). Conclusions.  In recent years, increased uptake and earlier initiation of HCV therapy among patients with incident infections significantly reduced the proportion of patients with replicating HCV.

Objectives: It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART., Methods: This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression., Results: Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found., Conclusions: Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objective: The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients., Design: We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing., Methods: Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients., Results: Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant., Conclusion: As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

The human immunodeficiency virus type 1 (HIV-1) establishes a chronic, lifelong infection finally leading to the acquired immunodeficiency syndrome (AIDS), which is characterized by the onset of opportunistic infections and tumors. The natural course of HIV-1 infections is very variable and depends on the virus and its replication competence as well as the host. This review summarizes our current knowledge on HIV-1 and host factors influencing the pathogenesis of HIV-1.

Background: Human milk and infant gut microbiota are essential for the immune system maturation and protection against infections. There is scarce information on the microbiological composition of breast milk in general, and none from developing countries. The objective of the study was to characterize the breast milk and gut microbiota from mothers and infants from southern Mozambique, where infections and breastfeeding are prevalent., Methods: A community-based study was undertaken among 121 pairs of women and infants. Breast milk and infant's faeces were analyzed by bacterial culture and molecular methods. Breast milk samples were screened for HIV RNA by RT-PCR., Results: The most frequent bacterial groups isolated by culture media in breast milk were Staphylococci (96.4%), Streptococci (92.7%) and Lactobacilli (56.4%). HIV RNA was detected in 24% of the samples. Staphylococcus hominis, S. aureus, and S.parasanguis were more frequently isolated in infants ≤14 days of life. Women on exclusive breastfeeding presented higher proportion of S. parasanguis in breast milk than those on mixed infant feeding (36.4% versus 11.1%, p = 0.035). Bacterial diversity (mean number of bacterial species isolated by sample: 10.4 versus 8.5; p = 0.004) and the frequency of Lactobacillus spp (75.9% versus 36%, p = 0.003) were higher in the specimens with HIV RNA than in those without it. The main bacterial groups found in infant's faeces were Bifidobacterium, Streptococci and Enterococci., Conclusions: Women with HIV RNA in breast milk had a different pattern of microbiological composition, suggesting specific immunopathological phenomena in HIV-infected women. Both breast milk and faecal microbiota composition varied with lactation period, which might be related to changes in the type of feeding over time and/or in the milk's biochemical characteristics. These findings provide insights into interactions between commensal bacteria and HIV infection in human milk and the role of these bacteria in mucosal protection against infections in breastfed infants.

Background: Selection of preexisting minority variants of drug-resistant human immunodeficiency virus type 1 (HIV-1) can lead to virological failure in patients who receive antiretroviral therapy (ART) with low genetic resistance barriers. We studied treatment response and dynamics of minority variants during the first weeks of ART containing a ritonavir-boosted protease inhibitor (PI) and 2 nucleoside reverse-transcriptase inhibitors (NRTIs), which is a regimen with a high genetic resistance barrier., Methods: Plasma samples obtained prior to initiation of ART from 109 patients with primary HIV infection and samples obtained during viral decay during early ART from 17 of these 109 patients were tested by allele-specific polymerase chain reaction for K103N and M184V variants., Results: K103N and/or M184V mutations were detected in 15 (13.8%) of 109 patients prior to ART as minority variants. No selection of these variants was observed within the first weeks of ART in 7 of 15 patients with preexisting drug resistance mutations, nor was any selection observed in 10 patients without preexisting drug resistance mutations. Most patients received ART immediately after diagnosis of HIV-1 infection, showed a rapid decrease in viral load, and experienced sufficient suppression of viremia for 48 months., Conclusions: Minority variants, in particular viruses harboring the M184V mutation, were efficiently suppressed in patients with acute infection who received a ritonavir-boosted PI and 2 NRTIs (most regimens included lamivudine). Under this high genetic resistance barrier regimen, the M184V was not further selected.

Background: Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure., Methods: We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure., Results: Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07%-2.0%. A range of 1-4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times., Conclusions: Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrier.