George, AF;Roan, NR;
PMID: 36689119 | DOI: 10.1007/s11904-023-00649-x
This review describes how advances in CyTOF and high-dimensional analysis methods have furthered our understanding of HIV transmission, pathogenesis, persistence, and immunity.CyTOF has generated important insight on several aspects of HIV biology: (1) the differences between cells permissive to productive vs. latent HIV infection, and the HIV-induced remodeling of infected cells; (2) factors that contribute to the persistence of the long-term HIV reservoir, in both blood and tissues; and (3) the impact of HIV on the immune system, in the context of both uncontrolled and controlled infection. CyTOF and high-dimensional analysis tools have enabled in-depth assessment of specific host antigens remodeled by HIV, and have revealed insights into the features of HIV-infected cells enabling them to survive and persist, and of the immune cells that can respond to and potentially control HIV replication. CyTOF and other related high-dimensional phenotyping approaches remain powerful tools for translational research, and applied HIV to cohort studies can inform on mechanisms of HIV pathogenesis and persistence, and potentially identify biomarkers for viral eradication or control.
Donoso, M;D'Amico, D;Valdebenito, S;Hernandez, CA;Prideaux, B;Eugenin, EA;
PMID: 35954221 | DOI: 10.3390/cells11152379
The major barrier to cure HIV infection is the early generation and extended survival of HIV reservoirs in the circulation and tissues. Currently, the techniques used to detect and quantify HIV reservoirs are mostly based on blood-based assays; however, it has become evident that viral reservoirs remain in tissues. Our study describes a novel multi-component imaging method (HIV DNA, mRNA, and viral proteins in the same assay) to identify, quantify, and characterize viral reservoirs in tissues and blood products obtained from HIV-infected individuals even when systemic replication is undetectable. In the human brains of HIV-infected individuals under ART, we identified that microglia/macrophages and a small population of astrocytes are the main cells with integrated HIV DNA. Only half of the cells with integrated HIV DNA expressed viral mRNA, and one-third expressed viral proteins. Surprisingly, we identified residual HIV-p24, gp120, nef, vpr, and tat protein expression and accumulation in uninfected cells around HIV-infected cells suggesting local synthesis, secretion, and bystander uptake. In conclusion, our data show that ART reduces the size of the brain's HIV reservoirs; however, local/chronic viral protein secretion still occurs, indicating that the brain is still a major anatomical target to cure HIV infection.
Journal of Virus Eradication
Pumtang-On, P;Sevcik, E;Davey, B;Goodarzi, N;Vezys, V;Casares, S;Rao, M;Skinner, P;
| DOI: 10.1016/j.jve.2022.100255
Background: HIV-specific chimeric antigen receptor T (CAR T) cells are being developed as a potential approach towards curing HIV infection. During infection, HIV replication is concentrated in B cell follicles, and viral reservoirs such as B cell follicles are a significant barrier to an HIV cure. We developed HIV-specific CAR T cells expressing the follicular homing receptor CXCR5 (CAR/CXCR5 T cells) to target follicular HIV reservoirs. We hypothesized after infusion of CAR/CXCR5 T cells in humanized HIV-infected DRAGA mice, CAR/CXCR5 T cells would accumulate in lymphoid follicles, make direct contact with HIV+ cells, lead to reductions in HIV viral loads, and preserve human CD4 T cells. Methods: Fourteen female humanized DRAGA mice were included in this study. Twelve mice were infected with 10 000 TCID50 of HIV-1 BaL. Levels of HIV-1 plasma viral loads and CD4 T cells were monitored using qRT-PCR and flow cytometry. Two spleens from uninfected mice were used to produce transduced CAR/CXCR5 T cells and transduced cell products (2×105 cells/gram) were infused in six HIV-infected mice. RNAscope combined with immunohistochemistry was used to visualize locations and quantities of CAR/CXCR5 T cells and HIV vRNA+ cells in lymphoid tissues. Results: All mice were HIV-1 detectable nbefore infusion of CAR/CXCR5 T cells. High levels of CAR/CXCR5 T cells and HIV vRNA+ cells were detected at 6 days post-infusion in lymphoid tissues. Many CAR/CXCR5 T cells were found in direct contact with HIV vRNA+ cells. However, many CAR/CXCR5 T cells, presumably CD4+ cells, were HIV vRNA+ and likely spreading infection. No differences in HIV plasma viral loads or CD4 T cell counts were observed between control and treated animals. Conclusions: These studies support the use of the HIV-infected DRAGA mouse model for HIV cure research studies. Using this model, we showed CAR/CXCR5 T cells accumulate in follicle-like structures with HIV vRNA+ cells and come in contact with vRNA+ cells. The simultaneous detection of CAR T cells with high levels of HIV vRNA+ cells indicates the need for HIV-resistant CAR T cells. These preliminary findings demonstrate the HIV-infected DRAGA mouse model is extremely valuable for evaluating HIV cure approaches.
Zerbato, JM;Avihingsanon, A;Singh, KP;Zhao, W;Deleage, C;Rosen, E;Cottrell, ML;Rhodes, A;Dantanarayana, A;Tumpach, C;Tennakoon, S;Crane, M;Price, DJ;Braat, S;Mason, H;Roche, M;Kashuba, ADM;Revill, PA;Audsley, J;Lewin, SR;
PMID: 36502576 | DOI: 10.1016/j.ebiom.2022.104391
HIV can infect multiple cells in the liver including hepatocytes, Kupffer cells and infiltrating T cells, but whether HIV can persist in the liver in people with HIV (PWH) on suppressive antiretroviral therapy (ART) remains unknown.In a prospective longitudinal cohort of PWH and hepatitis B virus (HBV) co-infection living in Bangkok, Thailand, we collected blood and liver biopsies from 18 participants prior to and following ART and quantified HIV and HBV persistence using quantitative (q)PCR and RNA/DNAscope. Antiretroviral (ARV) drug levels were quantified using mass spectroscopy.In liver biopsies taken prior to ART, HIV DNA and HIV RNA were detected by qPCR in 53% (9/17) and 47% (8/17) of participants respectively. Following a median ART duration of 3.4 years, HIV DNA was detected in liver in 61% (11/18) of participants by either qPCR, DNAscope or both, but only at very low and non-quantifiable levels. Using immunohistochemistry, HIV DNA was observed in both hepatocytes and liver infiltrating CD4+ T cells on ART. HIV RNA was not detected in liver biopsies collected on ART, by either qPCR or RNAscope. All ARVs were clearly detected in liver tissue.Persistence of HIV DNA in liver in PWH on ART represents an additional reservoir that warrants further investigation.National Health and Medical Research Council of Australia (Project Grant APP1101836, 1149990, and 1135851); This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024.
Baharlou, H;Canete, N;Vine, EE;Hu, K;Yuan, D;Sandgren, KJ;Bertram, KM;Nasr, N;Rhodes, JW;Gosselink, MP;Di Re, A;Reza, F;Ctercteko, G;Pathma-Nathan, N;Collins, G;Toh, J;Patrick, E;Haniffa, MA;Estes, JD;Byrne, SN;Cunningham, AL;Harman, AN;
PMID: 36130503 | DOI: 10.1016/j.celrep.2022.111385
The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4+ T cells, the primary targets of downstream infection. HIV+ DCs accumulate near and within lymphoid aggregates, which act as early sanctuaries of high viral titers while facilitating HIV passage to the submucosa. Finally, HIV entry induces recruitment and clustering of target cells, facilitating DC- and macrophage-mediated HIV transfer and enhanced infection of CD4+ T cells. These data demonstrate a rapid response to HIV structured to maximize the likelihood of mucosal infection and provide a framework for in situ studies of host-pathogen interactions and immune-mediated pathologies.
HIV Persistence in the Spleen: Opportunities for Pharmacologic Intervention
AIDS research and human retroviruses
Devanathan, AS;Kashuba, A;
PMID: 33499746 | DOI: 10.1089/AID.2020.0266
The persistence of HIV in the spleen, despite combination antiretroviral therapy (cART), is not well understood. Sustained immune dysregulation and delayed immune recovery, in addition to immune cell exhaustion, may contribute to persistence of infection in the spleen. Eliminating HIV from this secondary lymphoid organ will require a thorough understanding of antiretroviral (ARV) pharmacology in the spleen, which has been minimally investigated. Low ARV exposure within the spleen may hinder the achievement of a functional or sterilizing cure if cells are not protected from HIV infection. Here we provide an overview of the anatomy and physiology of the spleen, review the evidence of the spleen as a site for persistence of HIV, discuss the consequences of persistence of HIV in the spleen, address challenges to eradicating HIV in the spleen, and examine opportunities for future curative efforts.
Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells
Rhodes, JW;Botting, RA;Bertram, KM;Vine, EE;Rana, H;Baharlou, H;Vegh, P;O'Neil, TR;Ashhurst, AS;Fletcher, J;Parnell, GP;Graham, JD;Nasr, N;Lim, JJK;Barnouti, L;Haertsch, P;Gosselink, MP;Di Re, A;Reza, F;Ctercteko, G;Jenkins, GJ;Brooks, AJ;Patrick, E;Byrne, SN;Hunter, E;Haniffa, MA;Cunningham, AL;Harman, AN;
PMID: 33846309 | DOI: 10.1038/s41467-021-22375-x
Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).
AIDS research and human retroviruses
Sanders-Beer, BE;Archin, NM;Brumme, ZL;Busch, M;Deleage, C;O'Doherty, U;Hughes, SH;Jerome, K;Jones, RB;Karn, J;Kearney, MF;Keele, B;Kulpa, D;Laird, G;Li, JZ;Lichterfeld, M;Nussenzweig, MC;Persaud, D;Yukl, S;Siliciano, RF;Mellors, JW;
PMID: 37126090 | DOI: 10.1089/AID.2022.0188
Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Secondly, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.
Prolonged Posttreatment Virologic Control and Complete Seroreversion After Advanced Human Immunodeficiency Virus-1 Infection
Open forum infectious diseases
Uruena, A;Cassetti, I;Kashyap, N;Deleage, C;Estes, JD;Trindade, C;Hammoud, DA;Burbelo, PD;Natarajan, V;Dewar, R;Imamichi, H;Ward, AJ;Poole, A;Ober, A;Rehm, C;Jones, S;Liang, CJ;Chun, TW;Nath, A;Lane, HC;Smith, BR;Connors, M;Migueles, SA;
PMID: 33511235 | DOI: 10.1093/ofid/ofaa613
Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.
Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype
Tong, O;Duette, G;O'Neil, TR;Royle, CM;Rana, H;Johnson, B;Popovic, N;Dervish, S;Brouwer, MAE;Baharlou, H;Patrick, E;Ctercteko, G;Palmer, S;Lee, E;Hunter, E;Harman, AN;Cunningham, AL;Nasr, N;
PMID: 33872331 | DOI: 10.1371/journal.ppat.1009522
Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.
Khanal, S;Cao, D;Zhang, J;Zhang, Y;Schank, M;Dang, X;Nguyen, LNT;Wu, XY;Jiang, Y;Ning, S;Zhao, J;Wang, L;Gazzar, ME;Moorman, JP;Yao, ZQ;
PMID: 36146709 | DOI: 10.3390/v14091902
The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.
Rose R, Lamers SL, Nolan DJ, Maidji E, Faria NR, Pybus OG, Dollar JJ, Maruniak SA, McAvoy AC, Salemi M, Stoddart C, Singer E, McGrath MS.
PMID: 27466425 | DOI: 10.1128/JVI.00684-16
Abstract
While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious co-morbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies.We obtained DNA and RNA env, nef and pol sequences using single genome sequencing from post mortem tissues of three HIV+/cART+ individuals with undetectable viral load and metastatic cancer at death, and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient.Tissue-associated virus evolved at a similar rate in cART+ and cART- patients. Phylogenetic trees were characterized by two distinct features: 1) branching patterns consistent with constant viral evolution and dispersal amongst tissues; and 2) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Cancer diagnoses were temporally associated with diversification of viral lineages. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA+ cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV-expressing cells.Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread.
IMPORTANCE:
Combined anti-retroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. Here, we isolated and sequenced HIV from post mortem tissues from three HIV+/cART+ individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV diversity, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.
