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Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presenc

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found i

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

T cell activation by antigen/MHC induces the expression of several genes critical to the immune response, including interleukin-2. T cells from mice deficient for the NF-kappa B family member c-rel cannot activate IL-2 gene expression. However, mutating the NF-kappa B site in the IL-2 promoter has only moderate effects. To investigate additional ways c-Rel could regulate IL-2 gene expression, we determined whether c-rel overexpression could increase the activity of other transcription factors involved in IL-2 promoter regulation: NF-AT, Oct/OAP (ARRE-1), and AP-1. In Jurkat TAg cells, overexpression of c-Rel increased AP-1 activation approximately 17-fold. Moreover, AP-1 activity stimulated by anti-TCR Abs or PMA/ionomycin was further increased by c-Rel overexpression. c-Rel overexpression did not affect NF-AT or ARRE-1 activity. Additionally, overexpression of c-Rel activated the nonconsensus AP-1 site from the IL-2 promoter (NF-IL-2B), although to a lesser extent, approximately sixfold. AP-1 activation required both the DNA binding and transactivation domains of c-Rel. Our results may provide an explanation for the effect on IL-2 gene activation in c-rel-deficient mice.

Considerable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-1 nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef+ HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nef mutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef+ and Nef- viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef- viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact nef for subsequent viral growth. We now provide evidence that the Nef+ HIV is more infectious than Nef- HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef+ viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef+ virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef+ and Nef- viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef+ infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMC-derived Nef+ HIC.(ABSTRACT TRUNCATED AT 400 WORDS)

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL-2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low-affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.

Monoclonal antibodies J5, VIL-A1, and BA-3, known to react with the common acute lymphoblastic leukemia antigen (CALLA) were found to specifically stain normal human polymorphonuclear neutrophils (PMN). The antigen detected on PMN had a molecular weight (95,000-110,000 mol wt) close to that of CALLA (95,000-100,000 mol wt) and thus these surface membrane antigens are likely related, if not identical. The fluorescent staining intensity of PMN is comparable to that of CALLA-positive leukemic cells and the presence of PMN in patient samples could potentially produce false-positive results in diagnosis.

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection 1-4 . Despite the clinical evidence 1,5-7 , the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits 1,5,8-10 . Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID., (© 2024. The Author(s).)

Hybrid immunity against SARS-CoV-2 has not been well studied in pregnancy. We conducted a comprehensive analysis of neutralizing antibodies (nAb) and binding antibodies in pregnant individuals who received mRNA vaccination, natural infection, or both. A third vaccine dose augmented nAb levels compared to the two-dose regimen or natural infection alone; this effect was more pronounced in hybrid immunity. There was reduced anti-Omicron nAb, but the maternal-fetal transfer efficiency remained comparable to that of other variants. Vaccine-induced nAbs were transferred more efficiently than infection-induced nAbs. Anti-spike receptor binding domain (RBD) IgG was associated with nAb against wild-type (Wuhan-Hu-1) following breakthrough infection. Both vaccination and infection-induced anti-RBD IgA, which was more durable than anti-nucleocapsid IgA. IgA response was attenuated in pregnancy compared to non-pregnant controls. These data provide additional evidence of augmentation of humoral immune responses in hybrid immunity in pregnancy., (© 2024. The Author(s).)

Despite the success of combination antiretroviral therapy (ART) for individuals living with HIV, mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation, neuronal dysfunction, and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia, we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1, ISG15, ISG20, IFI27, IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART, consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However, S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells, mature and immature ChP cells, neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons, accompanied by increased expression of genes promoting cellular senescence and cell death. Together, these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Host repressors mediate HIV latency, but how they interactively silence the virus remains unclear. Here, we develop "reiterative enrichment and authentication of CRISPRi targets for synergies (REACTS)" to probe the genome for synergies between HIV transcription repressors. Using eight known host repressors as queries, we identify 32 synergies involving eleven repressors, including BCL7C, KANSL2, and SIRT2. Overexpression of these three proteins reduces HIV reactivation in Jurkat T cells and in CD4 T cells from people living with HIV on antiretroviral therapy (ART). We show that the BCL7C-containing BAF complex and the KANSL2-containing NSL complex form a "supercomplex" that increases inhibitory histone acetylation of the HIV long-terminal repeat (LTR) and its occupancy by the short variant of the acetyl-lysine reader Brd4. Collectively, we provide a validated platform for defining gene synergies genome wide, and the BAF-NSL "supercomplex" represents a potential target for overcoming HIV rebound after ART cessation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5 + cells and (ii) CCR2/5 + cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5 + central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5 + CD4 + T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath., (© 2022. The Author(s).)

The efficacy of HIV pre-exposure prophylaxis (PrEP) is high in men who have sex with men, but much more variable in women, in a manner largely attributed to low adherence. This reduced efficacy, however, could also reflect biological factors. Transmission to women is typically via the female reproductive tract (FRT), and vaginal dysbiosis, genital inflammation, and other factors specific to the FRT mucosa can all increase transmission risk. We have demonstrated that mucosal fibroblasts from the lower and upper FRT can markedly enhance HIV infection of CD4+ T cells. Given the current testing of tenofovir disoproxil fumarate, cabotegravir, and dapivirine regimens as candidate PrEP agents for women, we set out to determine using in vitro assays whether endometrial stromal fibroblasts (eSF) isolated from the FRT can affect the anti-HIV activity of these PrEP drugs. We found that PrEP drugs exhibit significantly reduced antiviral efficacy in the presence of eSFs, not because of decreased PrEP drug availability, but rather of eSF-mediated enhancement of HIV infection. These findings suggest that drug combinations that target both the virus and infection-promoting factors in the FRT-such as mucosal fibroblasts-may be more effective than PrEP alone at preventing sexual transmission of HIV to women.

The Infectious Diseases Institute (IDI), established in 2001, was the first autonomous institution of Makerere University set up as an example of what self-governing institutes can do in transforming the academic environment to become a rapidly progressive University addressing the needs of society This paper describes the success factors and lessons learned in development of sustainable centers of excellence to prepare academic institutions to respond appropriately to current and future challenges to global health. Key success factors included a) strong collaboration by local and international experts to combat the HIV pandemic, along with b) seed funding from Pfizer Inc., c) longstanding collaboration with Accordia Global Health Foundation to create and sustain institutional strengthening programs, d) development of a critical mass of multi-disciplinary research leaders and managers of the center, and e) a series of strong directors who built strong governance structures to execute the vision of the institute, with subsequent transition to local leadership., Conclusion: Twenty years of sustained investment in infrastructure, human capital, leadership, and collaborations present Makerere University and the sub-Saharan Africa region with an agile center of excellence with preparedness to meet the current and future challenges to global health., (© 2022 Nakanjako D et al.)

Studies are needed to evaluate the safety and effectiveness of mRNA SARS-CoV-2 vaccination during pregnancy, and the levels of protection provided to their newborns through placental transfer of antibodies. Here, we evaluate the transplacental transfer of mRNA vaccine products and functional anti-SARS-CoV-2 antibodies during pregnancy and early infancy in a cohort of 20 individuals vaccinated during late pregnancy. We find no evidence of mRNA vaccine products in maternal blood, placenta tissue, or cord blood at delivery. However, we find time-dependent efficient transfer of IgG and neutralizing antibodies to the neonate that persists during early infancy. Additionally, using phage immunoprecipitation sequencing, we find a vaccine-specific signature of SARS-CoV-2 Spike protein epitope binding that is transplacentally transferred during pregnancy. Timing of vaccination during pregnancy is critical to ensure transplacental transfer of protective antibodies during early infancy., (© 2022. The Author(s).)

SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals 1,2 . The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals., (© 2022. The Author(s).)

Pregnancy confers unique immune responses to infection and vaccination across gestation. To date, there are limited data comparing vaccine- and infection-induced neutralizing Abs (nAbs) against COVID-19 variants in mothers during pregnancy. We analyzed paired maternal and cord plasma samples from 60 pregnant individuals. Thirty women vaccinated with mRNA vaccines (from December 2020 through August 2021) were matched with 30 naturally infected women (from March 2020 through January 2021) by gestational age of exposure. Neutralization activity against the 5 SARS-CoV-2 spike sequences was measured by a SARS-CoV-2-pseudotyped spike virion assay. Effective nAbs against SARS-CoV-2 were present in maternal and cord plasma after both infection and vaccination. Compared with WT spike protein, these nAbs were less effective against the Delta and Mu spike variants. Vaccination during the third trimester induced higher cord-nAb levels at delivery than did infection during the third trimester. In contrast, vaccine-induced nAb levels were lower at the time of delivery compared with infection during the first trimester. The transfer ratio (cord nAb level divided by maternal nAb level) was greatest in mothers vaccinated in the second trimester. SARS-CoV-2 vaccination or infection in pregnancy elicits effective nAbs with differing neutralization kinetics that are influenced by gestational time of exposure.

CD4 T lymphocytes belong to diverse cellular subsets whose sensitivity or resistance to HIV-associated killing remains to be defined. Working with lymphoid cells from human tonsils, we characterized the HIV-associated depletion of various CD4 T cell subsets using mass cytometry and single-cell RNA-seq. CD4 T cell subsets preferentially killed by HIV are phenotypically distinct from those resistant to HIV-associated cell death, in a manner not fully accounted for by their susceptibility to productive infection. Preferentially-killed subsets express CXCR5 and CXCR4 while preferentially-infected subsets exhibit an activated and exhausted effector memory cell phenotype. Single-cell RNA-seq analysis reveals that the subsets of preferentially-killed cells express genes favoring abortive infection and pyroptosis. These studies emphasize a complex interplay between HIV and distinct tissue-based CD4 T cell subsets, and the important contribution of abortive infection and inflammatory programmed cell death to the overall depletion of CD4 T cells that accompanies untreated 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 © 2022 Luo, Frouard, Zhang, Neidleman, Xie, Sheedy, Roan and Greene.)

T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56 bright TCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features., 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 © 2022 George, Luo, Neidleman, Hoh, Vohra, Thomas, Shin, Lee, Blish, Deeks, Greene, Lee and Roan.)

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28 in vitro stimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5 + T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increase ex vivo infection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12., 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 Pedersen, Egedal, Packard, Thavachelvam, Xie, van der Sluis, Greene, Roan and Jakobsen.)

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts., Competing Interests: JN, XL, MM, GX, VM, WG, SL, NR No competing interests declared, (© 2021, Neidleman et al.)

The progressive loss of CD4+ T cells during HIV infection of lymphoid tissues involves both the apoptotic death of activated and productively infected CD4 T cells and the pyroptotic death of large numbers of resting and abortively infected bystander CD4 T cells. HIV spreads both through cellular release of virions and cell-to-cell transmission involving the formation of virological synapses. Cell-to-cell transmission results in high-level transfer of large quantities of virions to the target cell exceeding that achieved with cell-free virions. Broadly neutralizing anti-HIV antibodies (bNAbs) binding to HIV envelope protein capably block cell-free virus spread, and when added at higher concentrations can also interdict cell-to-cell transmission. Exploiting these distinct dose-response differences, we now show that four different bNAbs block the pyroptotic death of bystander cells, but only when added at concentrations sufficient to block cell-to-cell transmission. These findings further support the conclusion that HIV killing of abortively infected bystander CD4 T cells requires cell-to-cell transfer of virions. As bNAbs attract more interest as potential therapeutics, it will be important to consider the higher concentrations of these antibodies required to block the inflammatory death of bystander CD4 T cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The majority of HIV infections are established through the genital or rectal mucosa. Fibroblasts are abundant in these tissues, and although not susceptible to infection, can potently enhance HIV infection of CD4+ T cells. Hyaluronic acid (HA) is a major component of the extracellular matrix of fibroblasts, and its levels are influenced by the inflammatory state of the tissue. Since inflammation is known to facilitate HIV sexual transmission, we investigated the role of HA in genital mucosal fibroblast-mediated enhancement of HIV infection. Depletion of HA by CRISPR-Cas9 in primary foreskin fibroblasts augmented the ability of the fibroblasts to increase HIV infection of CD4+ T cells. This amplified enhancement required direct contact between the fibroblasts and CD4+ T cells, and could be attributed to both increased rates of trans-infection and the increased ability of HA-deficient fibroblasts to push CD4+ T cells into a state of higher permissivity to infection. This HIV-permissive state was characterized by differential expression of genes associated with regulation of cell metabolism and death. Our results suggest that conditions resulting in diminished cell-surface HA on fibroblasts, such as genital inflammation, can promote HIV transmission by conditioning CD4+ T cells toward a state more vulnerable to infection by HIV., (© 2021. The Author(s).)

Although T cells are likely players in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe coronavirus disease 2019 (COVID-19). We analyze T cells from 34 individuals with COVID-19 with severity ranging from mild (outpatient) to critical, culminating in death. Relative to individuals who succumbed, individuals who recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 cases display elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4 + T cells, as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4 + T cells in the lungs of individuals with severe COVID-19, these results support a model where lung-homing T cells activated through bystander effects contribute to immunopathology, whereas a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Relatively little is known about features of T cells targeted by HIV in vivo. By applying bioinformatics analysis to mass cytometry (CyTOF)-phenotyped specimens from individuals with viremia and in-vitro-infected cells from uninfected donors, we provide an atlas of the phenotypes of in vivo and in vitro HIV-susceptible cells. T helper 17 (Th17) and α4β1 + cells are preferentially targeted in vivo, whereas T effector memory (Tem), T transitional memory (Ttm), Th1, and Th1/Th17 subsets are targeted in vitro. Multiple proteins-including chemokine and cytokine receptors-are remodeled by HIV in vivo, and these changes are mostly recapitulated in vitro. HIV remodels cells to a T follicular helper (Tfh) phenotype. Using clustering, we uncover a subset of CD29-expressing, Tem-like cells that are highly susceptible to infection in vivo and in vitro and experimentally confirm that susceptibility. These studies provide an in-depth look at features of HIV-susceptible cells in individuals with viremia and demonstrate that some-but not all-HIV-susceptible cells identified in vitro effectively model in vivo susceptibility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined. IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs., (Copyright © 2021 American Society for Microbiology.)

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.

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells., Competing Interests: The authors have declared that no competing interests exist.

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence., Competing Interests: JN, XL, JF, GX, FH, TM, VM, AL, ST, RT, WT, BW, RH, MS, PV, JM, KJ, NA, PH, SD, SY, SP, WG, NR No competing interests declared, (© 2020, Neidleman et al.)

Convalescing coronavirus disease 2019 (COVID-19) patients mount robust T cell responses against SARS-CoV-2, suggesting an important role of T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells and predominantly Tcm cells with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can proliferate homeostatically, and can persist for over 2 months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection and support an important role of SARS-CoV-2-specific T cells in host control of COVID-19., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

The female reproductive tract (FRT) is the most common site of infection during HIV transmission to women, but viral remodeling complicates characterization of cells targeted for infection. Here, we report extensive phenotypic analyses of HIV-infected endometrial cells by CyTOF, and use a 'nearest neighbor' bioinformatics approach to trace cells to their original pre-infection phenotypes. Like in blood, HIV preferentially targets memory CD4+ T cells in the endometrium, but these cells exhibit unique phenotypes and sustain much higher levels of infection. Genital cell remodeling by HIV includes downregulating TCR complex components and modulating chemokine receptor expression to promote dissemination of infected cells to lymphoid follicles. HIV also upregulates the anti-apoptotic protein BIRC5, which when blocked promotes death of infected endometrial cells. These results suggest that HIV remodels genital T cells to prolong viability and promote viral dissemination and that interfering with these processes might reduce the likelihood of systemic viral spread., Competing Interests: TM, XL, AG, GM, NS, TS, LG, WG, NR No competing interests declared

The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts., Competing Interests: The authors have declared that no competing interests exist.

Study Question: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?, Summary Answer: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization., What Is Known Already: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis., Study Design, Size, Duration: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization., Participants/materials, Setting, Methods: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs., Main Results and the Role of Chance: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization., Large Scale Data: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640., Limitations, Reasons for Caution: This study is limited to in vitro analyses., Wider Implications of the Findings: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation., Study Funding/competing Interest(s): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Human immunodeficiency virus (HIV-1) indefinitely persists, despite effective antiretroviral therapy (ART), within a small pool of latently infected cells. These cells often display markers of immunologic memory and harbor both replication-competent and -incompetent proviruses at approximately a 1:100 ratio. Although complete HIV eradication is a highly desirable goal, this likely represents a bridge too far for our current and foreseeable technologies. A more tractable goal involves engineering a sustained viral remission in the absence of ART--a "functional cure." In this setting, HIV remains detectable during remission, but the size of the reservoir is small and the residual virus is effectively controlled by an engineered immune response or other intervention. Biological precedence for such an approach is found in the post-treatment controllers (PTCs), a rare group of HIV-infected individuals who, following ART withdrawal, do not experience viral rebound. PTCs are characterized by a small reservoir, greatly reduced inflammation, and the presence of a poorly understood immune response that limits viral rebound. Our goal is to devise a safe and effective means for replicating durable post-treatment control on a global scale. This requires devising methods to reduce the size of the reservoir and to control replication of this residual virus. In the following sections, we will review many of the approaches and tools that likely will be important for implementing such a "reduce and control" strategy and for achieving a PTC-like sustained HIV remission in the absence of ART., Competing Interests: The authors declare no conflicts of interest.