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Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4 + T cells and macrophages. IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4 + T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis., (Copyright © 2021 Zhong et al.)

HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain-containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4 + T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for the clearance of persistent HIV-1 infection., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.

Granuloma formation is the pathologic hallmark of tuberculosis (TB). Few studies have detailed the exact production of cytokines in human granulomatous inflammation and little is known about accessory molecule expressions in tuberculous granulomas. We aimed to identify some of the components of the immune response in granulomas in HIV-positive and -negative lymph nodes. We investigated the immunohistochemical profiles of CD4+, CD8+, CD68+, Th-17, Forkhead box P3 (FOXP3) cells, accessory molecule expression (human leukocyte antigen [HLA] classes I and II), and selected cytokines (interleukins 2, 4, and 6 and interferon-γ) of various cells, in granulomas within lymph nodes from 10 HIV-negative (-) and 10 HIV-positive (+) cases. CD4+ lymphocyte numbers were retained in HIV- granulomas, whereas CD4+:CD8 + cell were reversed in HIV+ TB granulomas. CD68 stained all histiocytes. Granulomas from the HIV+ group demonstrated a significant increase in FOXP3 cells. Interleukin-2 cytoplasmic expression was similar in both groups. Interferon-gamma (IFN-γ) expression was moderately increased, IL-6 was statistically increased and IL-4 expression was marginally lower in cells from HIV- than HIV+ TB granulomas. Greater numbers of cells expressed IFN-γ and IL-6 than IL-2 and IL-4 in HIV- TB granulomas. This study highlights the varied cytokine production in HIV-positive and -negative TB granulomas and indicates the need to identify localized tissue factors that play a role in mounting an adequate immune response required to halt infection. Although TB mono-infection causes variation in cell marker expression and cytokines in granulomas, alterations in TB and HIV coinfection are greater, pointing toward evolution of microorganism synergism.

Understanding the causes of the diverse outcome of COVID-19 pandemic in different geographical locations is important for the worldwide vaccine implementation and pandemic control responses. We analyzed 42 unexposed healthy donors and 28 mild COVID-19 subjects up to 5 months from the recovery for SARS-CoV-2 specific immunological memory. Using HLA class II predicted peptide megapools, we identified SARS-CoV-2 cross-reactive CD4 + T cells in around 66% of the unexposed individuals. Moreover, we found detectable immune memory in mild COVID-19 patients several months after recovery in the crucial arms of protective adaptive immunity; CD4 + T cells and B cells, with a minimal contribution from CD8 + T cells. Interestingly, the persistent immune memory in COVID-19 patients is predominantly targeted towards the Spike glycoprotein of the SARS-CoV-2. This study provides the evidence of both high magnitude pre-existing and persistent immune memory in Indian population. By providing the knowledge on cellular immune responses to SARS-CoV-2, our work has implication for the development and implementation of vaccines against COVID-19., Competing Interests: AlS is listed as inventor on patent application no. 63/012,902, submitted by La Jolla Institute for Immunology, that covers the use of the megapools and peptides thereof for therapeutic and diagnostic purposes. AlS is a consultant for Gritstone, Flow Pharma, Merck, Epitogenesis, Gilead and Avalia. 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 © 2021 Ansari, Arya, Sachan, Jha, Kalia, Lall, Sette, Grifoni, Weiskopf, Coshic, Sharma and Gupta.)

Heterologous prime-boost immunization regimens are a common strategy for many vaccines. DNA prime rAd5-GP boost immunization has been demonstrated to protect non-human primates against a lethal challenge of Ebola virus, a pathogen that causes fatal hemorrhagic disease in humans. This protection correlates with antibody responses and is also associated with IFNγ + TNFα + double positive CD8 + T-cells. In this study, we compared single DNA vs. multiple DNA prime immunizations, and short vs. long time intervals between the DNA prime and the rAd5 boost to evaluate the impact of these different prime-boost strategies on vaccine-induced humoral and cellular responses in non-human primates. We demonstrated that DNA/rAd5 prime-boost strategies can be tailored to induce either CD4 + T-cell or CD8 + T-cell dominant responses while maintaining a high magnitude antibody response. Additionally, a single DNA prime immunization generated a stable memory response that could be boosted by rAd5 3 years later. These results suggest DNA/rAd5 prime-boost provides a flexible platform that can be fine-tuned to generate desirable T-cell memory responses., Competing Interests: 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., (Copyright © 2021 Marcus, Thompson, Zhou, Bailey, Donaldson, Stanley, Asiedu, Foulds, Roederer, Moliva and Sullivan.)

Despite the advances in Human Immunodeficiency Virus (HIV) treatment, the cure for all HIV patients still poses a major challenge, which needs to be surpassed in the coming years. Among the strategies pursuing this aim, the 'kick-and-kill' approach, which involves the reactivation and elimination of a latent HIV reservoir that resides in some CD4 + T cells, appears promising. The first step of this approach requires the use of latency reversal agents (LRAs) that induce the reactivation of the latent virus. Although several classes of LRAs have been reported so far, some limitations of these compounds still need to be overcome before their clinical use. The complete exhaustion of the reservoir of latent virus will contribute to promote the second step of this approach, facilitating the elimination of the reactivated HIV. Therefore, potent, safe, and non-toxic LRAs are necessary to promote efficient elimination of the HIV-1 virus from its reservoir. In this review article, we focus on the promising LRAs that have been described in the literature over the past few years, highlighting the advantages and disadvantages of their use in the 'kick and kill' approach, thus opening a new avenue in the development of a potential cure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The prophylactic vaccines available to protect against infections by HPV are well tolerated and highly immunogenic. People with HIV have a higher risk of developing HPV infection and HPV-associated cancers due to a lower immune response, and due to viral interactions. We performed a systematic review of RCTs to assess HPV vaccines efficacy and safety on HIV-infected people compared to placebo or no intervention in terms of seroconversion, infections, neoplasms, adverse events, CD4+ T-cell count and HIV viral load. The vaccine-group showed a seroconversion rate close to 100% for each vaccine and a significantly higher level of antibodies against HPV vaccine types, as compared to the placebo group (MD = 4333.3, 95% CI 2701.4; 5965.1 GMT EL.U./ml for HPV type 16 and MD = 1408.8, 95% CI 414.8; 2394.7 GMT EL.U./ml for HPV type 18). There were also no differences in terms of severe adverse events (RR = 0.6, 95% CI 0.2; 1.6) and no severe adverse events (RR = 0.6, 95% CI 0.9; 1.2) between vaccine and placebo groups. Secondary outcomes, such as CD4 + T-cell count and HIV viral load, did not differ between groups (MD = 14.8, 95% CI - 35.1; 64.6 cells/µl and MD = 0.0, 95% CI - 0.3; 0.3 log10 RNA copies/ml, respectively). Information on the remaining outcomes was scarce and that did not allow us to combine the data. The results support the use of the HPV vaccine in HIV-infected patients and highlight the need of further RCTs assessing the effectiveness of the HPV vaccine on infections and neoplasms.

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies., Competing Interests: The authors declare no competing interest.

We identified types of immune cells that contribute to clearing COVID-19 during the acute phase of the infection in mouse model and human. Our results suggest that both innate and adaptive immune responses are essential for controlling COVID-19 infection. Mild infection report of children by COVID-19 comparing adults' infection causes conclusion of higher resistance of immune system of children comparing adults. Our results show innate immune system including phagocytes contribute severely to the elimination of COVID-19 in both mouse model and human. Our results also show the elimination of COVID-19 required the activation of B cells by CD4+ T cells. CD4+ T cells play an important role in elimination of COVID-19 in primary effection. We measured IgM and IgG in all patients including adults and kids (human) and found IgM and IgG in kids patients are much higher than other adults patients. It causes production of much more natural antibodies in kids' bodies to protect them against COVID-19 that shows reason of mild effection of kids comparing adults. Our observations have important ramifications for the development of novel vaccination and medicine strategies to alleviate COVID-19. The most important result is for producing any vaccine for COVID-19, increasing and producing these factors must be included: (a) Phagocytes (IgM and IgG), (b) T Cells, and (c) White Cells., (© 2020 John Wiley & Sons Ltd.)

Background: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 ( 64 Cu) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH)., Methods: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and 64 Cu ( 64 Cu-3BNC117) in vitro to assess binding and neutralization. In a clinical trial 64 Cu-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL <20 c/mL; Group 3) participants using two dosing strategies: high protein (3mg/kg unlabeled 3BNC117 combined with <5mg 64 Cu-3BNC117) and trace (<5mg 64 Cu-3BNC117 only). All participants were screened for 3BNC117 sensitivity from virus obtained from viral outgrowth. Magnetic resonance imaging (MRI)/PET and pharmacokinetic assessments (ELISA for serum 3BNC117 concentrations and gamma counting for 64 Cu) were performed 1, 24- and 48-hours post dosing. The trial (clincialtrials.gov NCT03063788) primary endpoint was comparison of PET standard uptake values (SUVs) in regions of interest (e.g lymph node groups and gastrointestinal tract)., Findings: Comparison of unmodified and modified 3BNC117 in vitro demonstrated no difference in HIV binding or neutralisation. 17 individuals were enrolled of which 12 were dosed including Group 1 (n=4, 2 high protein, 2 trace dose), Group 2 (n=6, 2 high protein, 4 trace) and Group 3 (n=2, trace only). HIV+ participants had a mean CD4 of 574 cells/microL and mean age 43 years. There were no drug related adverse effects and no differences in tissue uptake in regions of interest (e.g lymph node gut, pharynx) between the 3 groups. In the high protein dosing group, serum concentrations of 3BNC117 and gamma counts were highly correlated demonstrating that 64 Cu-3BNC117 remained intact in vivo., Interpretation: In PLWH on or off ART, the intervention of infusing 64 Cu-3BNC117 and MRI/PET imaging over 48 hours, was unable to detect HIV-1 env expression in vivo. Future studies should investigate alternative radiolabels such as zirconium which have a longer half-life in vivo., Funding: Funded by the Alfred Foundation, The Australian Centre for HIV and Hepatitis Virology Research with additional support from the Division of AIDS, National Institute of Allergy and Infectious Disease, US National Institutes of Health (USAI126611). JHM and SRL are supported by the Australian National Health and Medical Research Council., Competing Interests: Declaration of Competing Interest PD holds intellectual property in, this area of research, which has been licensed from the University of Melbourne to Clarity Pharmaceuticals and has a financial interest in Clarity Pharmaceuticals. PD serves on the Scientific Advisory Board of Clarity Pharmaceuticals. MCN has a patent for 3BNC117 licensed to Gilead and is a Member of the Scientific Advisory Board of Celldex Pharmaceuticals and Frontier Bioscience. SRL reports grants from National Health and Medical Research Council of Australia (NHMRC), grants from National Institutes of Health (NIH), during the conduct of the study; grants from American Foundation for AIDS Research (amfAR), grants from Gilead Sciences, grants from Merck, grants from ViiV, grants from Leidos, grants from Wellcome Trust, grants from Australian Centre for HIV and Hepatitis Virology Research (ACH2), grants from Melbourne HIV Cure Consortium, grants from Victorian Department of Health and Human Services (DHHS), grants from Medical Research Future Fund (MRFF), Other authors have nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: One strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed., Methods: We quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat., Findings: In total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA., Interpretation: Following administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal., Funding: NHMRC, NIH DARE collaboratory., Competing Interests: Declaration of Interests SRL's institution has received funding from the National Health and Medical Research Council (NHMRC) of Australia, National Institutes for Health, American Foundation for AIDS Research; Merck, ViiV and Gilead for investigator-initiated research; Merck, ViiV and Gilead for educational activities. She is on the advisory board of Abivax and Innivirax., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step., Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs., Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-β, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit., Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1., Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency., Competing Interests: The authors have declared that no competing interests exist.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials., (Copyright © 2021 Gaebler et al.)

The human immunodeficiency virus (HIV) reservoir is responsible for persistent viral infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon antiretroviral therapy interruption, which is the major obstacle to a cure. However, markers that determine effective therapy and viral rebound posttreatment interruption remain unclear. In this study, we comprehensively and longitudinally tracked dynamic decay of cell-associated viral RNA/DNA in systemic and lymphoid tissues in simian immunodeficiency virus (SIV)-infected rhesus macaques on prolonged combined antiretroviral therapy (cART) and evaluated predictors of viral rebound after treatment cessation. The results showed that suppressive ART substantially reduced plasma SIV RNA, cell-associated unspliced, and multiply spliced SIV RNA to undetectable levels, yet viral DNA remained detectable in systemic tissues and lymphoid compartments throughout cART. Intriguingly, a rapid increase of integrated proviral DNA in peripheral mononuclear cells was detected once treatment was withdrawn, accompanied by the emergence of detectable plasma viral load. Notably, the increase of peripheral proviral DNA after treatment interruption correlated with the emergence and degree of viral rebound. These findings suggest that measuring total viral DNA in SIV infection may be a relatively simple surrogate marker of reservoir size and may predict viral rebound after treatment interruption and inform treatment strategies. IMPORTANCE Viral reservoirs are involved in persistent HIV infection, and a small number of mosaic latent cellular reservoirs promote viral rebound upon analytical treatment interruption, which is the major obstacle to a cure. However, early indicators that can predict resurgence of viremia after treatment interruption may aid treatment decisions in people living with HIV. Utilizing the rhesus macaque model, we demonstrated that increased proviral DNA in peripheral cells after treatment interruption, rather than levels of proviral DNA, was a useful marker to predict the emergence and degree of viral rebound after treatment interruption, providing a rapid approach for monitoring HIV rebound and informing decisions., (Copyright © 2021 Ziani et al.)

Lipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from plasma samples from 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load set points post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the proinflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral set points upon ART cessation. Furthermore, higher pre-ATI levels of the proinflammatory by-product of intestinal lysophosphatidylcholine metabolism, trimethylamine- N -oxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4 + T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible proinflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during and after ART warrants further investigation. IMPORTANCE The likelihood of HIV rebound after stopping antiretroviral therapy (ART) is a combination of the size of HIV reservoirs that persist despite ART and the host immunological and inflammatory factors that control these reservoirs. Therefore, there is a need to comprehensively understand these host factors to develop a strategy to cure HIV infection and prevent viral rebound post-ART. Lipids are important biologically active molecules that are known to mediate several cellular functions, including reactivating latent tumor cells; however, their role in HIV latency, persistence, and post-ART rebound has never been investigated. We observed significant links between higher levels of the proinflammatory lysophosphatidylcholine and its intestinal metabolic by-product, trimethylamine- N -oxide, and both faster time-to-viral-rebound and higher viral load set point post-ART. These data highlight the need for further studies to understand the potential contribution of phosphatidylcholine and lysophosphatidylcholine metabolism in shaping host immunological and inflammatory milieu during and after ART., (Copyright © 2021 Giron et al.)

Alveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24 + ) cells and bystander (p24 - ) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24 + cells had longer telomeres compared to p24 - cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24 + cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection., Competing Interests: 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., (Copyright © 2021 Cao, Khanal, Wang, Li, Zhao, Nguyen, Nguyen, Dang, Schank, Thakuri, Zhang, Lu, Wu, Morrison, El Gazzar, Ning, Moorman and Yao.)

Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.

As the immune system develops with age, children combat infections better. HIV-1, however, targets an activated immune system, potentially rendering children increasingly permissive to HIV-1 infection as they grow. How HIV-1 fitness changes with age in children is unknown. Here, we estimated the within-host basic reproductive ratio, R 0 , a marker of viral fitness, in HIV-1 subtype C-infected children in India, aged between 84 days and 17 years. We measured serial viral load and CD4 T cell counts in 171 children who initiated first-line ART. For 25 children, regular and frequent measurements provided adequate data points for analysis using a mathematical model of viral dynamics to estimate R 0 . For the rest, we used CD4 counts for approximate estimation of R 0 . The viral load decline during therapy was biphasic. The mean lifespans of productively and long-lived infected cells were 1.4 and 27.8 days, respectively. The mean R 0 was 1.5 in children aged < 5 years, increased with age, and approached 6.0 at 18 years, close to 5.8 estimated previously for adults. The tolerogenic immune environment thus compromises HIV-1 fitness in young children. Early treatment initiation, when the R 0 is small, will likely improve viral control, in addition to suppressing the latent reservoir.

HIV-1 infection elicits a complex dynamic of the expression various host genes. High throughput sequencing added an expressive amount of information regarding HIV-1 infections and pathogenesis. RNA sequencing (RNA-Seq) is currently the tool of choice to investigate gene expression in a several range of experimental setting. This study aims at performing a meta-analysis of RNA-Seq expression profiles in samples of HIV-1 infected CD4+ T cells compared to uninfected cells to assess consistently differentially expressed genes in the context of HIV-1 infection. We selected two studies (22 samples: 15 experimentally infected and 7 mock-infected). We found 208 differentially expressed genes in infected cells when compared to uninfected/mock-infected cells. This result had moderate overlap when compared to previous studies of HIV-1 infection transcriptomics, but we identified 64 genes already known to interact with HIV-1 according to the HIV-1 Human Interaction Database. A gene ontology (GO) analysis revealed enrichment of several pathways involved in immune response, cell adhesion, cell migration, inflammation, apoptosis, Wnt, Notch and ERK/MAPK signaling.

Background: HIV-1 proviruses persist in people on antiretroviral therapy (ART) but most are defective and do not constitute a replication-competent reservoir. The decay of infected cells carrying intact compared with defective HIV-1 proviruses has not been well defined in people on ART., Methods: We separately quantified intact and defective proviruses, residual plasma viremia, and markers of inflammation and activation in people on long-term ART., Results: Among 40 participants tested longitudinally from a median of 7.1 years to 12 years after ART initiation, intact provirus levels declined significantly over time (median half-life, 7.1 years; 95% confidence interval [CI], 3.9-18), whereas defective provirus levels did not decrease. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6-75). The proportion of all proviruses that were intact diminished over time on ART, from about 10% at the first on-ART time point to about 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was no evidence for associations between intact provirus levels and inflammation or immune activation., Conclusions: Cells containing intact, replication-competent proviruses are selectively lost during suppressive ART. Defining the mechanisms involved should inform strategies to accelerate HIV-1 reservoir depletion., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Kaposi's sarcoma (KS) has been the most common HHV-8 virus-induced neoplasm associated with HIV-1 infection. Although the standard KS therapy has not changed in 20 years, not all cases of KS will respond to the same therapy. The goal of current AIDS-KS treatment modalities is to reconstitute the immune system and suppress HIV-1 replication, but newer treatment modalities are on horizon. There are very few mathematical models that have included HIV-1 viral load (VL) measures, despite VL being a key determinant of treatment outcome. Here we introduce a mathematical model that consolidates the effect of both HIV-1 and HHV-8 VL on KS tumor progression by incorporating low or high VLs into the proliferation terms of the immune cell populations. Regulation of HIV-1/HHV-8 VL and viral reservoir cells is crucial for restoring a patient to an asymptomatic stage. Therefore, an optimal control strategy given by a combined antiretroviral therapy (cART) is derived. The results indicate that the drug treatment strategies are capable of removing the viral reservoirs faster and consequently, the HIV-1 and KS tumor burden is reduced. The predictions of the mathematical model have the potential to offer more effective therapeutic interventions based on viral and virus-infected cell load and support new studies addressing the superiority of VL over CD4 + T-cell count in HIV-1 pathogenesis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Since its first appearance in Wuhan, China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread throughout the world and has become a global pandemic. Several medical comorbidities have been identified as risk factors for coronavirus disease 2019 (COVID-19). However, it remains unclear whether people living with human immunodefeciency virus (PLWH) are at an increased risk of COVID-19 and severe disease manifestation, with controversial suggestion that HIV-infected individuals could be protected from severe COVID-19 by means of antiretroviral therapy or HIV-related immunosuppression. Several cases of coinfection with HIV and SARS-CoV-2 have been reported from different parts of the globe. This review seeks to provide a holistic overview of SARS-CoV-2 infection in PLWH., (© 2020 Wiley Periodicals LLC.)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 emerged in China in December 2019 and then rapidly spread worldwide. Why COVID-19 patients with the same clinical condition have different outcomes remains unclear. This study aimed to examine the differences in the phenotype and functions of major populations of immune cells between COVID-19 patients with same severity but different outcomes. Four common type adult inpatients with laboratory confirmed COVID-19 from Beijing YouAn Hospital, Capital Medical University were included in this study. The patients were divided into two groups based on whether or not COVID-19 polymerase chain reaction (PCR)-negative conversion occurred within 3 weeks. Peripheral blood samples were collected to compare the differences in the phenotype and functions of major populations of immune cells between the two groups of patients. The result shows that the proportions of CD3 + CD8 + CD38 + HLA-DR + CD27 - effector T killer cells generally declined, whereas that of CD3 + CD4 + CD8 + double-positive T cells (DPTs) increased in the persistently PCR-positive patients. In summary, considering the imbalance between effector T killer cells/CD3+CD4+CD8+ DPTs was a possible key factor for PCR-negative conversion in patients with COVID-19., (© 2020 Wiley Periodicals LLC.)

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication., Competing Interests: No authors have competing interests.

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies., Competing Interests: The authors have declared that no competing interests exist.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates., Competing Interests: The authors have declared that no competing interests exist.

Prolonged shedding of viral RNA occurs in some individuals following SARS-CoV-2 infection. We perform comprehensive immunologic evaluation of one individual with prolonged shedding. The case subject recovered from severe COVID-19 and tested positive for SARS-CoV-2 viral RNA repeatedly as many as 87 days after the first positive test, 97 days after symptom onset. The subject did not have any associated rise in anti-Spike protein antibody titers or plasma neutralization activity, arguing against re-infection. This index subject exhibited a profoundly diminished circulating CD8+ T cell population and correspondingly low SARS-CoV-2-specific CD8+ T cell responses when compared with a cohort of other recovering COVID-19 subjects. CD4+ T cell responses and neutralizing antibody responses developed as expected in this individual. Our results demonstrate that detectable viral RNA shedding in the upper airway can occur more than 3 months following infection in some individuals with COVID-19 and suggest that impaired CD8+ T cells may play a role in prolonged viral RNA shedding., Competing Interests: Author BP is employed by the company IncellDX. 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 © 2021 Turner, Day, Alsoussi, Liu, O’Halloran, Presti, Patterson, Whelan, Ellebedy and Mudd.)

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

The COVID-19 pandemic has led to a major setback in both the health and economic sectors across the globe. The scale of the problem is enormous because we still do not have any specific anti-SARS-CoV-2 antiviral agent or vaccine. The human immune system has never been exposed to this novel virus, so the viral interactions with the human immune system are completely naive. New approaches are being studied at various levels, including animal in vitro models and human-based studies, to contain the COVID-19 pandemic as soon as possible. Many drugs are being tested for repurposing, but so far only remdesivir has shown some positive benefits based on preliminary reports, but these results also need further confirmation via ongoing trials. Otherwise, no other agents have shown an impactful response against COVID-19. Recently, research exploring the therapeutic application of mesenchymal stem cells (MSCs) in critically ill patients suffering from COVID-19 has gained momentum. The patients belonging to this subset are most likely beyond the point where they could benefit from an antiviral therapy because most of their illness at this stage of disease is driven by inflammatory (over)response of the immune system. In this review, we discuss the potential of MSCs as a therapeutic option for patients with COVID-19, based on the encouraging results from the preliminary data showing improved outcomes in the progression of COVID-19 disease., (© American Society for Clinical Pathology 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The induction of inflammation and cytokine storm was proposed to play a critical role in COVID-19. This study is aimed at investigating the relationship between glucose metabolism and the inflammatory state of inpatients with COVID-19. 71 inpatients with COVID-19 were classified into nondiabetes mellitus (NDM) group, impaired fasting glucose (IFG) group, and diabetes mellitus (DM) group. The average hospitalization days were significantly shorter in DM patients when compared with patients in the IFG group and NDM group. CD4 + T cell percentage was higher while CD8 + T cells percentage was lower in the DM group than those in the NDM group. The serum levels of IL-6, IL-2, IL-10, and INF- γ in the DM group were upregulated when compared with those in the NDM group. The serum levels of TNF- α , IL-4, IL-2, IL-10, and INF- γ were significantly higher in the DM group than those in the IFG group. A significant difference was observed in CD4 + T cell, CD4 + /CD8 + ratio percentage, IL-6, and IL-10 between the NDM group and DM group with adjusted BMI. In conclusion, COVID-19 patients with elevated glucose levels have promoted cytokine profiles and immune response., Competing Interests: All authors declare no competing interests., (Copyright © 2021 Mao Zheng et al.)

HIV cure is thwarted by the presence of quiescent yet replication competent HIV-1 (HIV). Antiretroviral therapy (ART) is unable to eradicate reservoirs, and upon cessation of ART, HIV will rebound. This review encompasses the curative strategies of HIV in the context of NF-κB sub-pathways that are currently exploited and demonstrate promise in the disruption of latent HIV. Canonical NF-κB signaling has long been established to drive HIV proviral expression while noncanonical NF-κB signaling, a novel and perhaps more desirable mechanism of latency reversal due to its unique characteristics, has recently been shown to also promote HIV expression from latency. Furthermore, we discuss the previously unrecognized upstream signaling of NF-κB as a new avenue for exploration of a functional cure of HIV., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interests., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF- κ B p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF- κ B activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P =0.034) and ACH-2 cells (3.9±1.4; P =0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

Antiretroviral therapies efficiently block HIV-1 replication but need to be maintained for life. Moreover, chronic inflammation is a hallmark of HIV-1 infection that persists despite treatment. There is, therefore, an urgent need to better understand the mechanisms driving HIV-1 pathogenesis and to identify new targets for therapeutic intervention. In the past few years, the decisive role of cellular metabolism in the fate and activity of immune cells has been uncovered, as well as its impact on the outcome of infectious diseases. Emerging evidence suggests that immunometabolism has a key role in HIV-1 pathogenesis. The metabolic pathways of CD4 + T cells and macrophages determine their susceptibility to infection, the persistence of infected cells and the establishment of latency. Immunometabolism also shapes immune responses against HIV-1, and cell metabolic products are key drivers of inflammation during infection. In this Review, we summarize current knowledge of the links between HIV-1 infection and immunometabolism, and we discuss the potential opportunities and challenges for therapeutic interventions.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4 + T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.

HIV infects its target cell and integrates into its genome as an essential step in its replication cycle. Proviral DNA is also subjected to the same transcriptional regulation as the host cell genome by its own transcriptional factors, with activating or repressive activity. There is a clear interaction between the presence of transcriptional repressors and a decrease in the rate of HIV replication, promoting gene silencing in infected cells, which serve as viral reservoirs. This represents a major obstacle for HIV eradication. The ZBTB gene family comprises 49 genes that encode transcription factors that have a repressor function in differentiation and development of cells of the lymphopoietic lineage, including the main target cells of HIV, CD4 + T cells. In this cross-sectional study, we evaluated the expression profile of ZBTB genes in CD4 + T cells of HIV-positive individuals with different levels of infection control. We found upregulation of gene expression of ZBTB4 (p < 0.01), ZBTB7B (p < 0.001), and ZBTB38 (p < 0.05) and downregulation of ZBTB16 (p < 0.01) in HIV-positive patients compared to HIV-negative individuals. Interestingly, in a deeper analysis, we observed that elite controllers had the highest levels of expression of the ZBTB38, ZBTB2, HIC1, ZBTB7A, ZBTB7B (ThPOK) and ZBTB4 genes, showing 2.56- to 7.60-fold upregulation compare to the ART-naïve group. These results suggest a possible contribution of these ZBTB transcriptional repressors in HIV-positive patients and a possible new molecular mechanism of viral control.

Wild-type HBV infection is followed by the blood expression of its widely known serological markers of infection, and designated as, hepatitis B virus surface antigen (HBsAg) and its antibody (anti-HBs), anti-HBc antibodies (IgM/IgG), and hepatitis B virus 'e' antigen (HBeAg) and its antibody (anti-HBe). These markers are detected as the infection develops and its kinetic behavior serves as a basis for monitoring the disorder and for diagnosing the clinical form or infection phase. Among these, the HBeAg-anti-HBe system markers demonstrate a dynamic profile whose interpretation, both in the acute or chronic HBV infection context, can offer greater difficulty to the health professionals, due to its particularities. This review offers a revisit to the markers dynamics of this system in the acute and chronic HBV infection and to the clinical and laboratory significance of its expression in these two clinical contexts.

Human immunodeficiency virus (HIV) causes acquired immune deficiency syndrome (AIDS) and enters the host cell via CD4 and either CC-chemokine receptor 5 (CCR) or CXC-chemokine receptor 4 (CXCR4). HIV is directly recognized by toll-like receptor 4 (TLR4) and affects downstream immune-related signal pathways. In addition, stimulated TLR4 inhibits HIV-1 invasion, and the rs4986790 single nucleotide polymorphism (SNP) (D299G) of the TLR4 gene contributes to the risk of HIV-1 infection in an Indian population. To evaluate whether the rs4986790 SNP of the TLR4 gene is related to vulnerability to HIV-1 infection, we collected genetic information from HIV-1 patients in previous studies and performed an association analysis with a matched control population obtained from the 1000 Genomes Project. In addition, to strengthen the results of association analysis, we performed a meta-analysis. We identified a strong association between the rs4986791 SNP and susceptibility to HIV infection in HIV-infected patients in previous studies and a matched control population obtained from the 1000 Genomes Project. In addition, we found that the G allele of the rs4986791 SNP in the TLR4 gene is strongly related to susceptibility to HIV infection in three Caucasian populations (odd ratio = 2.29, 95% confidence interval: 1.72-3.07, p = 1.438 × 10 -7 ) and all four populations (odd ratio = 2.22, 95% confidence interval: 1.74-2.84, p = 2 × 10 -10 ) in a meta-analysis. To the best our knowledge, this was the first meta-analysis on the association between the rs4986791 SNP of the TLR4 gene and susceptibility to HIV infection.

It is known that a subpopulation of T cells expresses two T cell receptor (TCR) clonotypes, though the extent and functional significance of this is not established. To definitively evaluate dual TCRα cells, we generated mice with green fluorescent protein and red fluorescent protein reporters linked to TCRα, revealing that ∼16% of T cells express dual TCRs, notably higher than prior estimates. Importantly, dual TCR expression has functional consequences, as dual TCR cells predominated response to lymphocytic choriomeningitis virus infection, comprising up to 60% of virus-specific CD4 + and CD8 + T cells during acute responses. Dual receptor expression selectively influenced immune memory, as postinfection memory CD4 + populations contained significantly increased frequencies of dual TCR cells. These data reveal a previously unappreciated contribution of dual TCR cells to the immune repertoire and highlight their potential effects on immune responses., Competing Interests: The authors declare no competing interest.

Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4 + T cells. Trafficking of α 4 β 7 -expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α 4 β 7 that is expressed on gut endothelial cells. MAdCAM signaling through α 4 β 7 costimulates CD4 + T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α 4 β 7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α 4 β 7 resulting in CD4 + T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α 4 β 7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α 4 β 7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis., Competing Interests: The authors declare no competing interest.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103 + and CD103 - CD4 T cells from the blood and rectum of HIV-negative (HIV - ) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV + ) individuals. Like gut CD4 + T cells, circulating CD103 + cells harbored more HIV DNA than did CD103 - cells but transcribed less HIV RNA per provirus. Circulating CD103 + cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103 - cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103 + CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency. IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103 + CD4 + T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103 + T cells share a cellular transcriptome that more closely resembles that of CD4 + T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4 + or circulating CD103 + T cells from circulating CD103 - T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV., (Copyright © 2020 American Society for Microbiology.)

IFI16 is an important player of the host intrinsic immune response. Among others, it has been reported to sense intermediate products of HIV-1 reverse transcription in the cytosol and to sequester the transcription factor Sp1 in the nucleus to attenuate viral gene expression. Here, we present three different methods to reduce IFI16 protein expression levels in HIV-1 primary target cells. These techniques can be adapted for the investigation of other cellular factors in primary macrophages and CD4 + T lymphocytes. For complete details on the use and execution of this protocol, please refer to Hotter et al. (2019) and Bosso et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Human immunodeficiency virus (HIV) remains incurable due to latent viral reservoirs established in non-activated CD4 T cells that cannot be eliminated via antiretroviral therapy. Current efforts to cure HIV are focused on identifying drugs that will induce viral gene expression in latently infected cells, commonly known as latency reversing agents (LRAs). Some drugs have been shown to reactivate latent HIV but do not cause a reduction in reservoir size. Therefore, finding new LRAs or new combinations or increasing the round of stimulations is needed to cure HIV. However, the effects of these drugs on viral rebound after prolonged treatment have not been evaluated. In a previous clinical trial, antiretroviral therapy intensification with maraviroc for 48 weeks caused an increase in residual viremia and episomal two LTR-DNA circles suggesting that maraviroc could reactivate latent HIV. We amended the initial clinical trial to explore additional virologic parameters in stored samples and to evaluate the time to viral rebound during analytical treatment interruption in three patients. Maraviroc induced an increase in cell-associated HIV RNA during the administration of the drug. However, there was a rapid rebound of viremia after antiretroviral therapy discontinuation. HIV-specific T cell response was slightly enhanced. These results show that maraviroc can reactivate latent HIV in vivo but further studies are required to efficiently reduce the reservoir size.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4 + T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure., Competing Interests: The authors declare no competing interest.

The molecular mechanisms underlying HIV-induced inflammation, which persists even during effective long-term treatment, remain incompletely defined. Here, we studied pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections in macaques and African green monkeys, respectively. We longitudinally analyzed genome-wide DNA methylation changes in CD4 + T cells from lymph node and blood, using arrays. DNA methylation changes after SIV infection were more pronounced in lymph nodes than blood and already detected in primary infection. Differentially methylated genes in pathogenic SIV infection were enriched for Th1-signaling (e.g., RUNX3, STAT4, NFKB1) and metabolic pathways (e.g., PRKCZ). In contrast, nonpathogenic SIVagm infection induced DNA methylation in genes coding for regulatory proteins such as LAG-3, arginase-2, interleukin-21 and interleukin-31. Between 15 and 18% of genes with DNA methylation changes were differentially expressed in CD4 + T cells in vivo. Selected identified sites were validated using bisulfite pyrosequencing in an independent cohort of uninfected, viremic and SIV controller macaques. Altered DNA methylation was confirmed in blood and lymph node CD4 + T cells in viremic macaques but was notably absent from SIV controller macaques. Our study identified key genes differentially methylated already in primary infection and in tissues that could contribute to the persisting metabolic disorders and inflammation in HIV-infected individuals despite effective treatment.

Despite the introduction of combinatory antiretroviral therapy (cART), HIV-1 infection cannot be cured and is still one of the major health issues worldwide. Indeed, as soon as cART is interrupted, a rapid rebound of viremia is observed. The establishment of viral latency and the persistence of the virus in cellular reservoirs constitute the main barrier to HIV eradication. For this reason, new therapeutic approaches have emerged to purge or restrain the HIV-1 reservoirs in order to cure infected patients. However, the viral latency is a multifactorial process that depends on various cellular mechanisms. Since these new therapies mainly target viral transcription, their development requires a detailed and precise understanding of the regulatory mechanism underlying HIV-1 transcription. In this review, we discuss the complex molecular transcriptional network regulating HIV-1 gene expression by focusing on the involvement of host cell factors that could be used as potential drug targets to design new therapeutic strategies and, to a larger extent, to reach an HIV-1 functional cure.

To counter HIV latency, it is important to develop a better understanding of the full range of host factors promoting latency. Their identification could suggest new strategies to reactivate latent proviruses and subsequently kill the host cells ("shock and kill"), or to permanently silence these latent proviruses ("block and lock"). We recently developed a screening strategy termed "Reiterative Enrichment and Authentication of CRISPRi Targets" (REACT) that can unambiguously identify host genes promoting HIV latency, even in the presence of high background "noise" produced by the stochastic nature of HIV reactivation. After applying this strategy in four cell lines displaying different levels of HIV inducibility, we identified FTSJ3, TMEM178A, NICN1 and the Integrator Complex as host genes promoting HIV latency. shRNA knockdown of these four repressive factors significantly enhances HIV expression in primary CD4 T cells, and active HIV infection is preferentially found in cells expressing lower levels of these four factors. Mechanistically, we found that downregulation of these newly identified host inhibitors stimulates different stages of RNA Polymerase II-mediated transcription of HIV-1. The identification and validation of these new host inhibitors provide insight into the novel mechanisms that maintain HIV latency even when cells are activated and undergo cell division., Competing Interests: The authors have declared that no competing interests exist.