The Journal of infectious diseases
Kroon, E;Chottanapund, S;Buranapraditkun, S;Sacdalan, C;Colby, DJ;Chomchey, N;Prueksakaew, P;Pinyakorn, S;Trichavaroj, R;Vasan, S;Manasnayakorn, S;Reilly, C;Helgeson, E;Anderson, J;David, C;Zulk, J;de Souza, M;Tovanabutra, S;Schuetz, A;Robb, ML;Douek, DC;Phanuphak, N;Haase, A;Ananworanich, J;Schacker, TW;
PMID: 35275599 | DOI: 10.1093/infdis/jiac089
Starting antiretroviral therapy (ART) in Fiebig 1 acute HIV infection limits the size of viral reservoirs in lymphoid tissues, but does not impact time to virus rebound during a treatment interruption. To better understand why the reduced reservoir size did not increase the time to rebound we measured the frequency and location of HIV RNA+ cells in lymph nodes from participants in the RV254 acute infection cohort. HIV vRNA+ cells were detected more frequently and in greater numbers when ART was initiated in Fiebig 1 compared to later Fiebig stages and were localized to the T cell zone compared to the B cell follicle with treatment in later Fiebig stages. Variability of virus production in people treated during acute infection suggests that the balance between virus producing cells and the immune response to clear infected cells rapidly evolves during the earliest stages of infection.
Journal of Virus Eradication
Hernandez, C;Eugenin, E;
| DOI: 10.1016/j.jve.2022.100212
Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.
Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques
Barber-Axthelm, IM;Barber-Axthelm, V;Sze, KY;Zhen, A;Suryawanshi, GW;Chen, IS;Zack, JA;Kitchen, SG;Kiem, HP;Peterson, CW;
PMID: 33427210 | DOI: 10.1172/jci.insight.141502
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.
Emerging microbes & infections
Gao, L;Jiao, YM;Ma, P;Sun, L;Zhao, H;Guo, AL;Fan, X;Zhang, C;Song, JW;Zhang, JY;Lu, F;Wang, FS;
PMID: 35253610 | DOI: 10.1080/22221751.2022.2049982
Semen is a known vector for both human immunodeficiency virus (HIV) infection and transmission. However, the distribution and characteristics of HIV-infected cells in semen remain unclear. Investigating the possibility of transmission through the spermatozoon in semen is of great clinical significance to improve the strategies for exposure prevention and assisted reproduction for HIV-infected partners. Twenty-six HIV-infected patients, including twelve treatment-naïve (TN) patients and fourteen antiretroviral treated (ART) patients, were enrolled in this study. HIV p24 protein in spermatozoa was detected using imaging flow cytometry and immunohistochemistry, and HIV RNA was identified using next-generation RNAscope in situ hybridization. Additionally, we described the rates of HIV-positive spermatozoon and CD4+ T lymphocytes in semen, and found that p24+ spermatozoon were mainly CD4 negative regardless of whether the patients received ART. Of note, p24-positive cells in semen are predominantly spermatozoa, and we confirmed that motile spermatozoa carried HIV into peripheral blood mononuclear cells of healthy men in vitro. Our findings provide evidence regarding the risk of HIV-infected spermatozoa.
Vasquez JJ, Aguilar-Rodriguez BL, Rodriguez L, Hogan LE, Somsouk M, McCune JM, Deeks SG, Laszik ZG, Hunt PW, Henrich TJ.
PMID: - | DOI: 10.20411/pai.v4i1.271
Abstract
Background: Identifying biomarkers for cells harboring replication-competent HIV is a major research priority. Recently, there have been mixed reports addressing the possibility that CD32-expressing T-cells are enriched for HIV. There is growing evidence that CD32 expression increases with cellular activation that may be related to, but not necessarily specific for, infection with HIV. However, the relationship of CD32 expression to HIV-infection in subtypes of tissue-resident leukocytes is unclear.
Methods: First, we used duplex chromogenic in situ hybridization to identify cells actively transcribing RNA for both CD32 and HIV on human gut tissues. Then we performed multiplexed immunofluorescence and in situ hybridization (mIFISH) on sections from the same tissues to determine the phenotype of individual cells co-expressing HIV-RNA and CD32-RNA.
Results: HIV-RNA+ cells were more abundant in tissues from viremic individuals compared to those on suppressive anti-retroviral therapy (ART). However, staining by both methods indicated that a higher proportion of HIV-RNA+ cells co-expressed CD32-RNA in ART-suppressed compared to viremic individuals. The majority of HIV-RNA+ cells were CD3+.
Conclusions: Our data suggest that the transcription of CD32-RNA is correlated with HIV transcriptional activity in CD3+ cells found within human gut tissue. Whether or not up-regulation of CD32-RNA is a direct result of HIV reactivation or more global T cell activation remains unclear.
Fletcher, CV;Kroon, E;Schacker, T;Pinyakorn, S;Chomont, N;Chottanapund, S;Prueksakaew, P;Benjapornpong, K;Buranapraditkun, S;Phanuphak, N;Ananworanich, J;Vasan, S;Hsu, D;RV254/SEARCH 010 study Group, ;
PMID: 35184069 | DOI: 10.1097/QAD.0000000000003201
The ability of antiretroviral drugs to penetrate and suppress viral replication in tissue reservoir sites is critical for HIV remission. We evaluated antiretroviral concentrations in lymph nodes and their impact on HIV transcription.Participants of the RV254/SEARCH010 Acute HIV Infection Cohort in Thailand were enrolled. Group 1 (n = 6) initiated and continued antiretrovirals with two nucleoside reverse transcriptase inhibitors (NRTIs), dolutegravir (DTG) and mar- aviroc (MVC). Group 2 (n = 12) initiated antiretrovirals with two NRTIs as well as efavirenz and were switched to two NRTIs as well as DTG. Antiretroviral concentrations were measured by mass spectroscopy. HIV RNA+ and DNA+ cells were measured by in-situ hybridization.All participants were MSM. At lymph node biopsy, all had plasma HIV RNA less than 20 copies/ml. Group 2 had longer durations of antiretroviral and DTG use (medians of 135 and 63 weeks, respectively) compared with Group 1 (median 44 weeks for both). TFV-DP, 3TC-TP, DTG and MVC were quantifiable in all lymph node samples from participants receiving those drugs versus carbovir-triphosphate (CBV-TP) in four out of 14. Median ratios of lymph node to peripheral blood concentrations were DTG, 0.014; MVC, 6.9; CBV-TP, 0.38; 3TC-TP, 0.32; and TFV-DP, 3.78. Median inhibitory quotients [ratios of lymph node concentrations to in-vitro inhibitory levels (IC50-or-90)] were DTG, 0.8; MVC, 38.8; CBV-TP, 0.5; 3TC- TP, 4.1; and TFV-DP, 1.8. Ongoing viral transcription was detected in lymph node of all participants. Median lymph node RNA+ cells were 71 350 versus 99 750 cells/g for Groups 1 and 2, respectively (P = 0.111).MVC has enhanced lymph node penetration and thereby may contribute to more complete viral suppression in the lymph node.
Flerin NC, Bardhi A, Zheng JH, Korom M, Folkvord J, Kovacs C, Benko E, Truong R, Mota T, Connick E, Jones RB, Lynch RM, Goldstein H.
PMID: PMID: 30626677 | DOI: DOI:10.1128/JVI.02051-18
Curing HIV infection has been thwarted by the persistent reservoir of latently-infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir-depletion strategies, we developed a novel pre-clinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, two weeks after PBMC injection, was 1,000-fold higher (mean=9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean=6,838 HIV copies/ml) or intravenously (mean=591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo reactivated primary latently infected cells. Time-to-viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, as compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in plasma of 10-1074-treated mice, while two new clusters emerged which were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV-cure strategies.IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time-to-viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV-cure strategies.
Astrocytes are HIV reservoirs in the brain: A cell type with poor HIV infectivity and replication but efficient cell-to-cell viral transfer
Journal of neurochemistry
Valdebenito, S;Castellano, P;Ajasin, D;Eugenin, EA;
PMID: 33655498 | DOI: 10.1111/jnc.15336
The major barrier to eradicating Human immunodeficiency virus-1 (HIV) infection is the generation of tissue-associated quiescent long-lasting viral reservoirs refractory to therapy. Upon interruption of anti-retroviral therapy (ART), HIV replication can be reactivated. Within the brain, microglia/macrophages and a small population of astrocytes are infected with HIV. However, the role of astrocytes as a potential viral reservoir is becoming more recognized because of the improved detection and quantification of HIV viral reservoirs. In this report, we examined the infectivity of human primary astrocytes in vivo and in vitro, and their capacity to maintain HIV infection, become latently infected, be reactivated, and transfer new HIV virions into neighboring cells. Analysis of human brain tissue sections obtained from HIV-infected individuals under effective and prolonged ART indicates that a small population of astrocytes has integrated HIV-DNA. In vitro experiments using HIV-infected human primary astrocyte cultures confirmed a low percentage of astrocytes had integrated HIV-DNA, with poor to undetectable replication. Even in the absence of ART, long-term culture results in latency that could be transiently reactivated with histone deacetylase inhibitor, tumor necrosis factor-alpha (TNF-α), or methamphetamine. Reactivation resulted in poor viral production but efficient cell-to-cell viral transfer into cells that support high viral replication. Together, our data provide a new understanding of astrocytes' role as viral reservoirs within the central nervous system (CNS).
Lamers SL, Rose R, Ndhlovu LC, Nolan DJ, Salemi M, Maidji E, Stoddart CA, McGrath MS.
PMID: 26572785 | DOI: -
Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4+ T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.
Cavrois M, Banerjee T, Mukherjee G, Raman N, Hussien R, Rodriguez BA, Vasquez J, Spitzer MH, Lazarus NH, Jones JJ, Ochsenbauer C, McCune JM, Butcher EC, Arvin AM, Sen N, Greene WC, Roan NR.
PMID: 28746881 | DOI: 10.1016/j.celrep.2017.06.087
To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.
Collins, DR;Hitschfel, J;Urbach, JM;Mylvaganam, GH;Ly, NL;Arshad, U;Racenet, ZJ;Yanez, AG;Diefenbach, TJ;Walker, BD;
PMID: 37205767 | DOI: 10.1126/sciimmunol.ade5872
Follicular CD8+ T cells (fCD8) mediate surveillance in lymph node (LN) germinal centers against lymphotropic infections and cancers, but the precise mechanisms by which these cells mediate immune control remain incompletely resolved. To address this, we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics, and transcriptional profiles of LN-resident virus-specific CD8+ T cells in persons who control HIV without medications. Antigen-induced proliferative and cytolytic potential consistently distinguished spontaneous controllers from noncontrollers. T cell receptor analysis revealed complete clonotypic overlap between peripheral and LN-resident HIV-specific CD8+ T cells. Transcriptional analysis of LN CD8+ T cells revealed gene signatures of inflammatory chemotaxis and antigen-induced effector function. In HIV controllers, the cytotoxic effectors perforin and granzyme B were elevated among virus-specific CXCR5+ fCD8s proximate to foci of HIV RNA within germinal centers. These results provide evidence consistent with cytolytic control of lymphotropic infection supported by inflammatory recruitment, antigen-specific proliferation, and cytotoxicity of fCD8s.
Vine, EE;Rhodes, JW;Warner van Dijk, FA;Byrne, SN;Bertram, KM;Cunningham, AL;Harman, AN;
PMID: 35173293 | DOI: 10.1038/s41385-022-00492-0
In tissue, mononuclear phagocytes (MNP) are comprised of Langerhans cells, dendritic cells, macrophages and monocyte-derived cells. They are the first immune cells to encounter HIV during transmission and transmit the virus to CD4 T cells as a consequence of their antigen presenting cell function. To understand the role these cells play in transmission, their phenotypic and functional characterisation is important. With advancements in high parameter single cell technologies, new MNPs subsets are continuously being discovered and their definition and classification is in a state of flux. This has important implications for our knowledge of HIV transmission, which requires a deeper understanding to design effective vaccines and better blocking strategies. Here we review the historical research of the role MNPs play in HIV transmission up to the present day and revaluate these studies in the context of our most recent understandings of the MNP system.
