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Identifying HIV-1 envelope (Env) traits associated with neutralization cross-reactivity is crucial for vaccine design. Variable loops 1 and 2 (V1V2), positioned at the Env trimer apex, are key regions linked to neutralization. We describe non-canonical cysteine (Cys) residues in V1 that are enriched in individuals with elite neutralization breadth. Analyzing over 65,000 V1 sequences from the CATNAP database, AMP trials, and longitudinal HIV-1 cohorts (SHCS, ZPHI, and CAPRISA), we found that Env variants with extra V1 Cys are present at low levels and fluctuate over time. Extra V1 Cys associate with elite plasma neutralization, and two additional Cys are preferred, suggesting stabilization through disulfide bonds. Among 34 broadly neutralizing antibody (bnAb)-inducer Envs, 17.6% had elongated V1 regions with extra Cys. These extra Cys moderately increased neutralization resistance and altered bnAb epitope accessibility. Collectively, altering epitope exposure alongside Env stabilization renders the V1 twin Cys motif a promising feature for HIV-1 bnAb immunogens., Competing Interests: Declaration of interests All authors declare no direct competing interests related to this study, including financial interests, patents, board affiliations, and paid consultant activities., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

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).)

The development of broadly neutralizing antibody (bnAb)-based therapeutic HIV-1 vaccines and cure concepts depends on monitoring bnAb plasma activity in people with HIV (PWH) on suppressive antiretroviral therapy (ART). To enable this, analytical strategies must be defined to reliably distinguish antibody-based neutralization from drug inhibition. Here, we explore strategies that either utilize drug-resistant viruses or remove drugs from plasma. We develop ART-DEX (ART dissociation and size exclusion), an approach which quantitatively separates drugs from plasma proteins following pH-triggered release allowing accurate definition of antibody-based neutralization. We demonstrate that ART-DEX, alone or combined with ART-resistant viruses, provides a highly effective and scalable means of assessing antibody neutralization during ART. Implementation of ART-DEX in standard neutralization protocols should be considered to enhance the analytical capabilities of studies evaluating bnAb therapeutics and therapeutic vaccines, furthering the development of advanced ART and HIV-1 cure strategies., Competing Interests: Declaration of interests A.T. has received unrelated unrestricted research grants from the SNSF, Bill and Melinda Gates Foundation, Gilead Sciences, Novartis Biomedical Research Foundation, University of Zurich (UZH) Foundation, UZH Clinical Research Priority Program, the SHCS, honoraria from Roche Diagnostics and the Institute for biomedical research Bellinzona for consultant and scientific board activity, and directs the Swiss National Reference Center for Retroviruses together with M.H. H.F.G. has received unrelated unrestricted research grants from the SNSF, the SHCS, Yvonne Jacob Foundation, University of Zurich Clinical Research Priority Program, Systems.X, the National Institutes of Health, Gilead Sciences, and Roche. H.F.G. has further received personal fees from Merck, Gilead Sciences, ViiV, Janssen, GSK, Johnson and Johnson, and Novartis for consultancy or DSMB membership and a travel grant from Gilead. M.H. directs the Swiss National Reference Center for Retroviruses and has received unrelated unrestricted research grant from the UZH Clinical Research Priority Program, the SNSF, the SHCS, and the ETH PHRT. I.A.A. has received honoraria from MSD and Sanofi, a travel grant from Gilead Sciences, a grant from ProMedica Foundation, and she is member of the EKIF (Eidgenössische Kommission für Impffragen) of the Federal Office of Public Health. R.D.K. has received grants from SNSF, the National Institutes of Health, and Gilead Sciences., (Copyright © 2024 The Author(s). Published by Elsevier 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).)