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Identifying cellular and molecular mechanisms maintaining HIV-1 latency in the viral reservoir is crucial for devising effective cure strategies. Here we developed an innovative flow cytometry-fluorescent in situ hybridization (flow-FISH) approach for direct ex vivo reservoir detection without the need for reactivation using a combination of probes detecting abortive and elongated HIV-1 transcripts. Our flow-FISH assay distinguished between HIV-1-infected CD4+ T cells expressing abortive or elongated HIV-1 transcripts in PBMC from untreated and ART-treated PWH from the Amsterdam Cohort Studies. This flow-FISH method was employed to isolate CD4+ T cells expressing abortive or elongated HIV-1 transcripts from five ART-naïve PWH for transcriptomic analysis by 3' RNA sequencing. Supervised cluster analysis identified several differentially expressed mitochondrial genes in infected CD4+ T cells with abortive HIV-1 transcripts compared to cells containing elongated HIV-1 transcripts. Notably, enhancing mitochondrial function induced HIV-1 transcription in PBMC from PWH. Our data strongly suggests that cellular metabolism is involved in maintaining HIV-1 latency and show that improving mitochondrial functions induces HIV-1 transcriptional activity in PWH. These findings underline the relevance of metabolic regulation in HIV-1 infection, and support the development of strategies modulating immunometabolism to target viral latency., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Man 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.)

Dysbiosis of the vaginal microbiome poses a serious risk for sexual human immunodeficiency virus type 1 (HIV-1) transmission. Prevotella spp are abundant during vaginal dysbiosis and associated with enhanced HIV-1 susceptibility; however, underlying mechanisms remain unclear. Here, we investigated the direct effect of vaginal bacteria on HIV-1 susceptibility of vaginal CD4+ T cells. Notably, pre-exposure to Prevotella timonensis enhanced HIV-1 uptake by vaginal T cells, leading to increased viral fusion and enhanced virus production. Pre-exposure to antiretroviral inhibitors abolished P timonensis-enhanced infection. Our study shows that the vaginal microbiome directly affects mucosal CD4+ T-cell susceptibility, emphasizing importance of vaginal dysbiosis diagnosis and treatment., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. 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.)

Importance: HIV-1 continues to be a major global health challenge. Current HIV-1 treatments are effective but need lifelong adherence. An HIV-1 cure should eliminate the latent viral reservoir that persists in people living with HIV-1. Different methods have been investigated that focus on reactivation and subsequent elimination of the HIV-1 reservoir, and it is becoming clear that a combination of compounds with different mechanisms of actions might be more effective. Here, we target two host factors, inhibitor of apoptosis proteins that control apoptosis and the DEAD-box helicase DDX3, facilitating HIV mRNA transport/translation. We show that targeting of these host factors with SMAC mimetics and DDX3 inhibitors induce reversal of viral latency and eliminate HIV-1-infected cells in vitro and ex vivo ., Competing Interests: M.A., J.B., C.Z., and A.T. are employees of First Health Pharmaceuticals B.V.

Vaginal inflammation increases the risk for sexual HIV-1 transmission but underlying mechanisms remain unclear. In this study we assessed the impact of immune activation on HIV-1 susceptibility of primary human vaginal Langerhans cells (LCs). Vaginal LCs isolated from human vaginal tissue expressed a broad range of TLRs and became activated after exposure to both viral and bacterial TLR ligands. HIV-1 replication was restricted in immature vaginal LCs as only low levels of infection could be detected. Notably, activation of immature vaginal LCs by bacterial TLR ligands increased HIV-1 infection, whereas viral TLR ligands were unable to induce HIV-1 replication in vaginal LCs. Furthermore, mature vaginal LCs transmitted HIV-1 to CD4 T cells. This study emphasizes the role for vaginal LCs in protection against mucosal HIV-1 infection, which is abrogated upon activation. Moreover, our data suggest that bacterial STIs can increase the risk of HIV-1 acquisition in women., (© 2023. The Author(s).)

Pathogens trigger multiple pattern recognition receptors (PRRs) that together dictate innate and adaptive immune responses. Understanding the crosstalk between PRRs is important to enhance vaccine efficacy. Abortive HIV-1 RNA transcripts are produced during acute and chronic HIV-1 infection and are known ligands for different PRRs, leading to antiviral and proinflammatory responses. Here, we have investigated the crosstalk between responses induced by these 58 nucleotide-long HIV-1 RNA transcripts and different TLR ligands. Costimulation of dendritic cells (DCs) with abortive HIV-1 RNA and TLR7/8 agonist R848, but not other TLR agonists, resulted in enhanced antiviral type I IFN responses as well as adaptive immune responses via the induction of DC-mediated T helper 1 (T H 1) responses and IFNγ + CD8 + T cells. Our data underscore the importance of crosstalk between abortive HIV-1 RNA and R848-induced signaling for the induction of effective antiviral immunity., (© 2022 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

The most effective treatment for HIV-1, antiretroviral therapy, suppresses viral replication and averts the disease from progression. Nonetheless, there is a need for alternative treatments as it requires daily administration with the possibility of side effects and occurrence of drug resistance. Broadly neutralizing antibodies or nanobodies targeting the HIV-1 envelope glycoprotein are explored as alternative treatment, since they mediate viral suppression and contribute to the elimination of virus-infected cells. Besides neutralization potency and breadth, Fc-mediated effector functions of bNAbs also contribute to the in vivo efficacy. In this study multivalent J3, 2E7 and 1F10 anti-HIV-1 broadly neutralizing nanobodies were generated to improve neutralization potency and IgG1 Fc fusion was utilized to gain Fc-mediated effector functions. Bivalent and trivalent nanobodies, coupled using long glycine-serine linkers, showed increased binding to the HIV-1 Env and enhanced neutralization potency compared to the monovalent variant. Fusion of an IgG1 Fc domain to J3 improved neutralization potency compared to the J3-bihead and restored Fc-mediated effector functions such as antibody-dependent cellular phagocytosis and trogocytosis, and natural killer cell activation. Due to their neutralization breadth and potency and their ability to induce effector functions these nanobody-IgG1 constructs may prove to be valuable towards alternative HIV-1 therapies., Competing Interests: Authors GD and RH were employed by QVQ Holding BV. Author TV was employed by VerLin BV. The remaining 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., (Copyright © 2022 Schriek, van Haaren, Poniman, Dekkers, Bentlage, Grobben, Vidarsson, Sanders, Verrips, Geijtenbeek, Heukers, Kootstra, de Taeye and van Gils.)

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

The proinflammatory cytokine IL-1β mediates high levels of immune activation observed during acute and chronic human immunodeficiency virus 1 (HIV-1) infection. Little is known about the mechanisms that drive IL-1β activation during HIV-1 infection. Here, we have identified a crucial role for abortive HIV-1 RNAs in inducing IL-1β in humans. Abortive HIV-1 RNAs were sensed by protein kinase RNA-activated (PKR), which triggered activation of the canonical NLRP3 inflammasome and caspase-1, leading to pro-IL-1β processing and secretion. PKR activated the inflammasome via ROS generation and MAP kinases ERK1/2, JNK, and p38. Inhibition of PKR during HIV-1 infection blocked IL-1β production. As abortive HIV-1 RNAs are produced during productive infection and latency, our data strongly suggest that targeting PKR signaling might attenuate immune activation during acute and chronic HIV-1 infection., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

Current direct-acting antiviral therapies are highly effective in suppressing HIV-1 replication. However, mucosal inflammation undermines prophylactic treatment efficacy, and HIV-1 persists in long-lived tissue-derived dendritic cells (DCs) and CD4 + T cells of treated patients. Host-directed strategies are an emerging therapeutic approach to improve therapy outcomes in infectious diseases. Autophagy functions as an innate antiviral mechanism by degrading viruses in specialized vesicles. Here, we investigated the impact of pharmaceutically enhancing autophagy on HIV-1 acquisition and viral replication. To this end, we developed a human tissue infection model permitting concurrent analysis of HIV-1 cellular targets ex vivo. Prophylactic treatment with autophagy-enhancing drugs carbamazepine and everolimus promoted HIV-1 restriction in skin-derived CD11c + DCs and CD4 + T cells. Everolimus also decreased HIV-1 susceptibility to lab-adapted and transmitted/founder HIV-1 strains, and in vaginal Langerhans cells. Notably, we observed cell-specific effects of therapeutic treatment. Therapeutic rapamycin treatment suppressed HIV-1 replication in tissue-derived CD11c + DCs, while all selected drugs limited viral replication in CD4 + T cells. Strikingly, both prophylactic and therapeutic treatment with everolimus or rapamycin reduced intestinal HIV-1 productive infection. Our findings highlight host autophagy pathways as an emerging target for HIV-1 therapies, and underscore the relevancy of repurposing clinically-approved autophagy drugs to suppress mucosal HIV-1 replication.

Dendritic cell (DC) subsets are abundantly present in genital and intestinal mucosal tissue and are among the first innate immune cells that encounter human immunodeficiency virus type 1 (HIV-1) after sexual contact. Although DCs have specific characteristics that greatly enhance HIV-1 transmission, it is becoming evident that most DC subsets also have virus restriction mechanisms that exert selective pressure on the viruses during sexual transmission. In this review we discuss the current concepts of the immediate events following viral exposure at genital mucosal sites that lead to selection of specific HIV-1 variants called transmitted founder (TF) viruses. We highlight the importance of the TF HIV-1 phenotype and the role of different DC subsets in establishing infection. Understanding the biology of HIV-1 transmission will contribute to the design of novel treatment strategies preventing HIV-1 dissemination.

The mitochondrial antiviral protein MAVS is a key player in the induction of antiviral responses; however, human immunodeficiency virus 1 (HIV-1) is able to suppress these responses. Two linked single nucleotide polymorphisms (SNPs) in the MAVS gene render MAVS insensitive to HIV-1-dependent suppression, and have been shown to be associated with a lower viral load at set point and delayed increase of viral load during disease progression. Here, we studied the underlying mechanisms involved in the control of viral replication in individuals homozygous for this MAVS genotype. We observed that individuals with the MAVS minor genotype had more stable total CD4 + T cell counts during a 7-year follow up and had lower cell-associated proviral DNA loads. Genetic variation in MAVS did not affect immune activation levels; however, a significantly lower percentage of naïve CD4 + but not CD8 + T cells was observed in the MAVS minor genotype. In vitro HIV-1 infection of peripheral blood mononuclear cells (PBMCs) from healthy donors with the MAVS minor genotype resulted in decreased viral replication. Although the precise underlying mechanism remains unclear, our data suggest that the protective effect of the MAVS minor genotype may be exerted by the initiation of local innate responses affecting viral replication and CD4 + T cell susceptibility.

Sexually transmitted Hepatitis C virus (HCV) infections and high reinfections are a major concern amongst men who have sex with men (MSM) living with HIV-1 and HIV-negative MSM. Immune activation and/or HIV-1 coinfection enhance HCV susceptibility via sexual contact, suggesting that changes in immune cells or external factors are involved in increased susceptibility. Activation of anal mucosal Langerhans cells (LCs) has been implicated in increased HCV susceptibility as activated but not immature LCs efficiently retain and transmit HCV to other cells. However, the underlying molecular mechanism of transmission remains unclear. Here we identified the Heparan Sulfate Proteoglycan Syndecan 4 as the molecular switch, controlling HCV transmission by LCs. Syndecan 4 was highly upregulated upon activation of LCs and interference with Heparan Sulfate Proteoglycans or silencing of Syndecan 4 abrogated HCV transmission. These data strongly suggest that Syndecan 4 mediates HCV transmission by activated LCs. Notably, our data also identified the C-type lectin receptor langerin as a restriction factor for HCV infection and transmission. Langerin expression abrogated HCV infection in HCV permissive cells, whereas langerin expression on the Syndecan 4 expressing cell line strongly decreased HCV transmission to a target hepatoma cell line. These data suggest that the balanced interplay between langerin restriction and Syndecan 4 transmission determines HCV dissemination. Silencing of langerin enhanced HCV transmission whereas silencing Syndecan 4 on activated LCs decreased transmission. Blocking Heparan Sulfate Proteoglycans abrogated HCV transmission by LCs ex vivo identifying Heparan Sulfate Proteoglycans and Syndecan 4 as potential targets to prevent sexual transmission of HCV. Thus, our data strongly suggest that the interplay between receptors promotes or restricts transmission and further indicate that Syndecan 4 is the molecular switch controlling HCV susceptibility after sexual contact., (Copyright © 2020 Nijmeijer, Eder, Langedijk, Kaptein, Meeussen, Zimmermann, Ribeiro and Geijtenbeek.)

Strong innate and adaptive immune responses are paramount in combating viral infections. Dendritic cells (DCs) detect viral infections via cytosolic RIG-I like receptors (RLRs) RIG-I and MDA5 leading to MAVS-induced immunity. The DEAD-box RNA helicase DDX3 senses abortive human immunodeficiency virus 1 (HIV-1) transcripts and induces MAVS-dependent type I interferon (IFN) responses, suggesting that abortive HIV-1 RNA transcripts induce antiviral immunity. Little is known about the induction of antiviral immunity by DDX3-ligand abortive HIV-1 RNA. Here we synthesized a 58 nucleotide-long capped RNA (HIV-1 Cap-RNA 58 ) that mimics abortive HIV-1 RNA transcripts. HIV-1 Cap-RNA 58 induced potent type I IFN responses in monocyte-derived DCs, monocytes, macrophages and primary CD1c + DCs. Compared with RLR agonist poly-I:C, HIV-1 Cap-RNA 58 induced comparable levels of type I IFN responses, identifying HIV-1 Cap-RNA 58 as a potent trigger of antiviral immunity. In monocyte-derived DCs, HIV-1 Cap-RNA 58 activated the transcription factors IRF3 and NF-κB. Moreover, HIV-1 Cap-RNA 58 induced DC maturation and the expression of pro-inflammatory cytokines. HIV-1 Cap-RNA 58 -stimulated DCs induced proliferation of CD4 + and CD8 + T cells and differentiated naïve T helper (T H ) cells toward a T H 2 phenotype. Importantly, treatment of DCs with HIV-1 Cap-RNA 58 resulted in an efficient antiviral innate immune response that reduced ongoing HIV-1 replication in DCs. Our data strongly suggest that HIV-1 Cap-RNA 58 induces potent innate and adaptive immune responses, making it an interesting addition in vaccine design strategies., (Copyright © 2020 Stunnenberg, Sprokholt, van Hamme, Kaptein, Zijlstra-Willems, Gringhuis and Geijtenbeek.)

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission., Competing Interests: The authors have declared that no competing interests exist.

Sexual transmission of HIV-1 consists of processes that exert either positive or negative selection pressure on the virus. The sum of these selection pressures lead to the transmission of only one specific HIV-1 strain, termed the transmitted founder virus. Different dendritic cell subsets are abundantly present at mucosal sites and, interestingly, these DC subsets exert opposite pressure on viral selection during sexual transmission. In this review we describe receptors and cellular compartments in DCs that are involved in HIV-1 communication leading to either viral restriction by the host or further dissemination to establish a long-lived reservoir. We discuss the current understanding of host antiretroviral restriction factors against HIV-1 and specifically against the HIV-1 transmitted founder virus. We will also discuss potential clinical implications for exploiting these intrinsic restriction factors in developing novel therapeutic targets. A better understanding of these processes might help in developing strategies against HIV-1 infections by targeting dendritic cells.

HIV-1 sensors and their signaling features have been an ongoing topic of intense research over the last decade, as these mechanisms fail to establish protective immunity against HIV-1. Here, we discuss how HIV-1 infects dendritic cells (DCs) and which sensors play a role in recognizing viral DNA and RNA in these specialized immune cells. We will elaborate on the RNA helicase DDX3, which is crucial in translation initiation of HIV-1 mRNA, but also fulfills an important role as RNA sensor and inducer of antiviral immunity in DCs. As DDX3 is indispensable for HIV-1 replication, the virus cannot escape sensing by DDX3, which is an important aspect of its function. Last but not least, we will discuss how HIV-1 suppresses DDX3 sensing and how this impacts the viral load in HIV-1-infected individuals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Innate sensing of HIV-1 by dendritic cells (DCs) initiates cell-intrinsic signalling programs that direct virus restriction and antiviral defenses. These responses include the production of type I interferon (IFN) and a large number of IFN-stimulated genes (ISGs) with a broad spectrum of antiviral effector functions. Initial interactions of HIV-1 at the mucosal surfaces with DC-expressed innate immune factors including cGAS, TRIM5α and SAMHD1 are predictive of viraemia, inflammation and disease pathogenesis. Here, we review the molecular basis of HIV-1 sensing in the two major mucosal DC subsets, that is, epithelial Langerhans cells and subepithelial CD11c+ conventional DCs. We discuss the concerted actions of the host restriction factors and innate sensors as well as viral evasion mechanisms in determining HIV-1 susceptibility to infection and directing antiviral adaptive immune responses., (Copyright © 2017 Elsevier B.V. All rights reserved.)