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Transplantation
A major roadblock to achieving a cure for human immunodeficiency virus type one (HIV-1) is the persistence of latent viral infections in the cells and tissue compartments of an infected human host. Latent HIV-1 proviral DNA persists in resting memory CD4+ T cells and mononuclear phagocytes (MPs; macrophages, microglia, and dendritic cells). Tissue viral reservoirs of both cell types reside in the gut, lymph nodes, bone marrow, spleen, liver, kidney, skin, adipose tissue, reproductive organs, and brain.
- HIV-1
- latency
- experimental cell systems
- animal models
- humanized mice
1. Introduction
Despite advances in antiretroviral therapy (ART) toward improving the quality and longevity of life for people infected with human immunodeficiency virus (HIV) (PLWH), the medicines must be taken for life. Adherence is fraught with personal, social, behavioral, drug resistance, and pharmacologic limitations [1,2]. While ART can prevent viral transmission and suppress viral transmission [1,3], it is not a cure. The virus persists as a latent infection in multiple cell and tissue compartments. This includes populations of resting memory CD4+ T and myeloid cells [4]. Indeed, immediately after infection, a stable integration of the viral genome into the genomic DNA of the host cell occurs with limited changes in viral gene expression [5]. A reservoir of latent virus is quickly established. This occurs within a few days after viral infection and even before the virus is detectable in systemic circulation [6,7]. Current ART regimens cannot affect latent viral infection, resulting in viral rebound once therapy is discontinued. While early ART administration can lower the number of infected cells via viral suppression and limiting spread, it cannot eliminate the persistent, latent HIV-1 proviral DNA. Studies of HIV latency necessitate a multifaceted approach, integrating the disciplines of virology, immunology, and cell biology. Studies of HIV latency are of immediate importance as viral elimination cannot be achieved without a thorough understanding of the mechanisms of viral persistence.
2. Pitfalls for Studies of Latent HIV Infection
Studies on HIV-1 latency are hindered by the difficulty of performing in vivo studies as the cells that carry proviral DNA are low in abundance. The resting CD4+ T cell reservoir with integrated provirus accounts for less than 0.05% of the entire resting cell population. This suggests that productively infected cells have higher rates of turnover. This may be partly due to apoptotic and pyroptic death caused by direct viral cytopathicity. Alternatively, host cytotoxic T-lymphocytes (CTLs) eliminate productively infected cells. Hence, most of the cells containing integrated provirus rarely persist to switch into a memory state [31]. It is also difficult to distinguish latently infected from uninfected cells without activation [32]. There is a lack of definitive biomarkers that differentiate latently infected from uninfected resting CD4+ T cells.
3. Model Systems for Studies of HIV Reservoirs
3.1. Cell-Based Model Systems
The selection of an appropriate model system is necessary when studying HIV latency and reactivation. Given the human host specificity of HIV-1, there are few animal models that can replicate long-term HIV infection and latency [33]. Consequently, in vitro models with primary and transformed cell lines are used to address the multifaceted challenges of virus–cell interactions. The HIV latencies observed in patients are affected by cell–tissue locations and environment and remain poorly understood [33]. Primary CD4+ T and myeloid cells recovered from peripheral blood mononuclear cells or lymphoid tissues [34,35] are the most commonly studied cell types when addressing latency [36]. A primary CD4+ T cell model was developed from PLWH memory CD4+ T cells that was subjected to two rounds of activation. Despite having similar reactivation patterns to latency-reversing agents (LRAs), CD4+ T cells can only survive for up to eight weeks [37]. Several groups have also attempted to mimic infection in activated CD4+ T cells by collecting and isolating CD4+ T cells from people without HIV (PWoH) and activating the cells [38,39]. The first reported model isolated CD4+ T cells from PWoH with combinations of a CD3 antibody and interleukin-2 (IL-2). The cells were then infected with replication-competent virus, and the infection progressed to quiescence (latency) [38]. Another study employed similar methods for the activation of CD4+ T cells, followed by their infection with HIV-1NL4-3 and HIV-1 NLENG1-IRES [39]. The system allowed the latent infection to be studied for up to two months. These approaches have also been used to investigate HIV vectors lacking multiple HIV genes—tat, env, gag, and vif—that are needed to infect activated cells [40,41]. An ex vivo primary cell system can mimic in vivo latency. Cells were transduced using a B cell lymphoma gene (Bcl2) to increase their lifespan [42]. Other studies demonstrated that ex vivo CD4+ T cells obtained from PLWH could be reactivated by various classes of LRAs; however, CD4+ T cells infected in vitro could be activated by protein kinase C (PKC) agonists [35]. HIV-1-specific CD8+ T cells fail to eliminate CD4+ T cells; however, they are able to eliminate CD4+ T cells infected with outgrown virus from the same individual [43]. Cell procurement from PLWH poses challenges [44] as the longevity of primary cells in culture is limited [42] and another hurdle is the necessity if manipulation of these replicate cells using transfection or electroporation to introduce the exogenous genes, proteins, or HIV-specific therapeutic agents into them. Primary cells can contain non-replication-competent viruses and have different integration sites [45] that could affect active viral transcription and large numbers of biological replicates are required to perform mechanistic studies and to reach statistical significance [33].
To generate meaningful data sets, researchers have employed immortalized cell lines that include CD4+ T cells (Jurkat, CEM, MOLT4, THP1, and SupT1) and the promonocytic U1 cell lines to study viral latency [46,47]. The advantage of using in vitro model systems is that large numbers of infected cells are used to evaluate gene expression and cell phenotype. Notably, the levels of involvement of transcription machinery in studying latency, specifically positive-transcription-elongation-factor-b-mediated pathways, are significantly elevated in the tested cell lines when compared to the primary resting CD4+ T cells and macrophages. Components of cellular transcription machinery are unique between cells, which can result in different experimental results when studies are performed to evaluate multiple stages of HIV transcription [48,49]. Also, resting primary CD4+ T cells carry latent HIV-1 in the G0 stage of the cell cycle [50,51,52]. Taking this into consideration, a major drawback of using transformed cell lines to study viral latency is that they may not accurately reflect what occurs in CD4+ T cells that carry latent HIV-1 in vivo.
Yet another complexity in assessing the latent viral reservoir are the inherent differences between CD4+ T and myeloid cells in their carrying capacities and transcriptional machineries that support infections. Both cell types are known reservoirs of HIV. Indeed, tissue macrophages and microglia, along with circulating monocytes, can play roles in bearing latent HIV. These cells can have longer lifespans and resist virus-induced cytopathology [53] and are resistant to CTL cell elimination [54]. Additionally, myeloid cell reservoirs in the brain and lymphoid organs may show differential responses to antiretroviral drugs [55,56]. The previous notion that macrophages lack the capacity for self-renewal has been challenged by recent findings that demonstrate a more prominent role of macrophages as HIV reservoirs [57,58,59]. Additionally, the lack of immune-mediated viral clearance of infected macrophages may make the replication-competent carriage of latent virus and ultimate viral rebound in these cells more likely during therapy interruption [58,59,60]. Additional studies on viral activation in tissue macrophages that include the broader use of newly minted viral outgrowth assays are needed [61]. This need is supported by recent studies that show monocyte-derived macrophages (MDMs) as sources of rebound virus [62,63,64]. Future studies must account for the tissue and cellular environments. This can affect, for example, macrophage polarization and ultimately, HIV reactivation [61]. Moreover, LRAs known to be effective in reversing HIV latency in CD4+ T cells have different effects in macrophages. These factors all require further attention.
Astrocytes, which represent some of the most abundant cell types in the brain, can harbor HIV [65,66]. The cells are involved in the secretion cellular and viral neurotoxic factors, including Tat [67]. They may affect the clinical signs and symptoms of HIV-associated neurocognitive disorders (HANDs) [68,69,70,71]. Despite prior studies demonstrating HIV infection in astrocytes [72,73,74], their role as viral reservoirs remains incompletely defined [75,76,77,78,79]. The presence of HIV-1p24 in astrocytes remains uncertain beyond 60 days after exposure to the virus [80,81]. Studies of HIV-1 infection in astrocytes were performed in cells with differential outcomes following viral exposures [82,83], or in cells isolated and differentiated from human fetal tissues [84]. The findings have not reliably allowed such virus-exposed cells to be used as accurate platforms for studies of HIV-1 latency. Newer approaches to studying viral latency in astrocytic cells have become available through HNSC-100, a human neural stem cell line. These cells may mimic the non-dividing nature of mature astrocytes in the adult human brain and are being used to analyze LRAs and inhibitors of latency reactivators [85]. However, suitable cell models remain in demand for studying HIV latency in CNS tissue compartments.
3.2. Nonhuman Primates (NHPs)
Over the last two decades, SIV/SHIV-infected NHP models of HIV-1 have been instrumental in studies of viral latency and cure research. This is based on the similarity at the cellular and molecular levels of the infection life cycle, disease progression, and immune responses observed [25,86,87,88]. In addition, when using NHPs, researchers can employ analytical treatment interruption to address questions relevant to HIV latency, persistent viral reservoirs, and cures which are risky and unethical to conduct in PLWH [89,90,91,92].
NHP models have allowed for the evaluation of novel therapeutic strategies that include immune regulators and boosters [93,94,95,96], LRAs [92,97,98], broad neutralizing antibodies (bnAbs) [89,90,99,100], HIV-specific chimeric antigen receptor T cell (CAR T) therapy [91,101,101,102], and CRISPR-based gene editing [103]. Prior studies tested antiviral efficacy against SIV/SHIV infection by measuring viral load, host antiretroviral immunity, and disease protection. However, few studies achieved control of SIV/SHIV latency [89,99,103,104]. Prior works have, nonetheless, demonstrated undetectable plasma SHIV after the administration of the bnAb PGT121. Here, a significant reduction in proviral DNA was demonstrated in the peripheral blood, gastrointestinal mucosa, and lymph nodes within weeks of treatment [104]. A delay in viral rebound after the antibody infusion was observed. Although the bnAb was not able to eliminate the virus from latent reservoir sites, it was able to reduce the reservoir size [104]. Similarly, in SHIV-infected macaques treated with five bnAbs, strong viral suppression was observed with a delay in viral rebound in less than five weeks compared with viral rebound seven days after single-bnAb infusions. The data indicate that combinations of bnAbs showed clearer advantages over single-bnAb therapy [99]. Regarding viral elimination studies in macaques, thus far, a single study reported a functional cure in a subset of macaques receiving a combination of a TLR agonist and antibody therapy [89]. Five out of eleven macaques in a dual-treated group had no viral rebound at 200 days after ART discontinuation, while the virus rebounded in most (10/11 and 9/11) macaques belonging to the single-treatment groups. AAV-mediated CRISPR-Cas9 targeting SIV LTR-Gag with daily ART in macaques was used to excise the latent provirus [103]. The authors reported an excision of SIV proviral DNA from the circulatory lymphocytes, spleen, lung, and lymph nodes of the treated animals at 6.5 months post infection, with differential efficiency among the animals. Interestingly, a substantial reduction (38–95%) in viral DNA was detected in the lymph nodes of all treated animals, suggesting the potential efficacy of CRISPR-mediated treatments.
Though NHP models have contributed to the characterization of latent reservoirs and the discovery/evaluation of novel therapeutic strategies, the dissimilarities in the structural and virological signatures of SIV and HIV-1 are major limitations to using SIV-infected macaque models in a translational approach [86,105]. For this reason, SIV-infected NHPs are presumed to respond differently to antiretroviral agents such as different classes of ART regimens, LRAs, and HIV-1-envelope-specific bnAbs. Though chimeric SHIV overcomes this barrier with the insertion of the HIV-1 envelope gene [106,107], several researchers have argued that the SHIV lacks naturally occurring broad viral diversity [108] and is thus less reflective of natural reservoir establishment in which a versatile founder virus is engaged. In fact, recent work revealed the existence of altered, persistent viral reservoirs between SIV, SHIV, and HIV1/2 infections following ART-mediated viral suppression. These results highlight the mechanistic complexity of strain-specific viral treatment responses [109].
In addition, the higher cost of animal maintenance, longer study period, and high laboratory and personnel requirements compared to rodent models are other practical limitations for latency and cure studies using NHP models. In summary, NHP viral latency studies have uncovered novel therapeutic pathways for viral suppression. However, there remains an immediate need for alternatives. These include small animal model systems that can accurately mimic natural HIV infection and disease progression while providing insights into established viral reservoirs. Such models can be employed to facilitate targeted therapeutic studies designed to improve viral suppression and elimination.
4. Rodent Animal Models
Rodents are the preferred small animal model systems for use in infectious disease research [110,111] because they mirror the human system in many ways and have well-characterized immune systems. However, due to the human host specificity of the HIV virus, it is very difficult to find an ideal mouse model for studying HIV latent reservoirs and the cure strategies targeting them. One earlier model, the EcoHIV infection model, in which parts of HIV-1 gp120 were replaced with gp80 from the ecotropic murine leukemia virus, allowed for permissive HIV infection in conventional mice [112]. Recent studies by different research groups have shown neurocognitive impairment in an EcoHIV model without any significant CD4+ cell depletion [113]. This model is also being used by researchers to investigate the roles of infected macrophages and monocytes to understand how the persistence of HIV affects cognitive function despite ART, using molecular and behavior tests [114]. However, the lack of a proper HIV disease progression pattern is a major limitation of this model for latency and cure studies. In addition, the considerable differences between murine and human immune systems in terms of immune cell profiles, innate and adaptive immune responses, and the production of HIV-1-associated antiviral cytokines make this model less ideal for studying long-term HIV latency and assessing therapeutic efficacy [115]. Thus, humanized mice that can recapitulate a functional human immune system and mimic a natural HIV infection process remain preferred for HIV persistence studies [116].
Currently, several humanized mouse models are being used by researchers for HIV-related studies and are mostly developed from severe immunodeficient backgrounds in which the mice are engrafted with human immune cells or tissues. The journey of the development and generation of humanized mice began with the discovery of an autosomal recessive mutation in a DNA-dependent protein kinase, Prkdcscid (severe combined immunodeficiency, SCID), on the CB17 mouse strain background [117]. SCID mice have severely impaired lymphopoiesis and differentiation; thus, they lack their own functional T and B lymphocytes [117]. In 1988, McCune et al reported the co-transplantation of human fetal liver hematopoietic cells (fetal thymus and lymph nodes) into SCID mice for the first time. Though this pioneering study showed reconstituted mature human T and B cells in mouse peripheral circulation 6–7 weeks post transplantation [118], the human T cell population and human IgG were found to be transiently present because of graft-versus-host disease (GVHD), and the model lacked a sustained hematopoietic supportive environment [118,119]. Shortly thereafter, Namikawa et al. found that the co-transplantation of human fetal thymus and intact fetal liver fragments into SCID mice could support human hematopoiesis for up to 11 months, marking their model as the first humanized mouse model for HIV studies [120].
In addition to SCID, further mutations targeting the common cytokine receptor gamma chain (γc or CD132) resulted in the generation of more immunodeficient mice strains: the NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG), NODShi.CgPrkdcscid Il2rgtm1Sug (NOG), and BALB/c-Rag2null IL2rgnull mice (BRG) strains [121,122,123,124], which took humanized mouse research progress one step forward. Since the common cytokine receptor gamma chain is shared by a family of cytokines, including IL-2, 4, 7, 9, 15, and 21, and is crucial for the development and survival of NK cells and lymphocytes, the knockout or truncated version of γc further reduced the risks of GVHD generation and enhanced the survival of the human cell engraftment in mice [125]. More advances have been made recently by knocking-in cytokine-encoding genes, for example, M-CSFh/h IL-3/GM-CSFh/h SIRPah/h TPOh/h RAG2−/− IL2Rg−/−, which led to support for the establishment of human cells in (MISTRG) mice [126]. Another mouse model with chimeric human–mouse class II transgenes, the NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg (DRAG) mouse, allowed for the engraftment of enhanced HLA-DR-matched hematopoietic stem cells (HSCs), which subsequently supported better human T and B cell development [127]. These most recent advances made in humanized mouse models further boost the planning and execution of infectious disease studies.
In spite of the advantages of rodent models, a few limitations do exist, including the shorter lifespan of human cell persistence in vivo, which limits long-term efficacy studies. Most of the humanized mouse models used for HIV latency and cure studies are developed on either well-characterized NSG, NOG-SCID, or BRG genetic backgrounds [128,129,130,131,132]. Depending on the different humanization methods, the three main mouse models are deployed to assess either short-term or long-term HIV persistence, virus–host interactions, and therapeutic developmental investigations that serve to best target the viral reservoirs (Figure 1).
Figure 1. Humanized mouse models for preclinical HIV therapeutics. (A) The hu-PBL model employs peripheral blood mononuclear cells (PBMC/PBLs) from human donors that are injected into immunocompromised mice. This model typically reaches functional maturity within 1–2 weeks but is highly susceptible to graft-versus-host disease (GVHD) within 4–5 weeks of cell implantation. The hu-PBL model is commonly used for drug discovery and quick screening of antiretroviral drug combinations as well as novel antiretroviral therapies. (B) The hu-BLT model involves the transplantation of human HSCs and fetal liver and thymus tissues into immunocompromised mice to generate a more complete human immune system. This model requires 13–15 weeks for human immune cells to reach full functional maturity, and it has been extensively used as a chronic HIV infection model for HIV latency and cure studies, enabling therapy testing for more than 10 weeks. (C) The hu-HSC model utilizes umbilical-cord-blood-isolated CD34+ hematopoietic stem cells (HSCs) that are transplanted into newborn pups to generate functional human immune systems. It necessitates 18–20 weeks for full maturation and allows for long-term testing of HIV reservoir establishment and therapeutic targeting studies for up to one year. The first report of an HIV cure in a humanized mouse model used sequential long-acting ART and CRISPR-based gene editing treatments. This model has better clinical translational potential than the other models.
5. Perspectives
Given the successful application of hu-HSC mice in long-term HIV studies due to the presence of a functional human immune system that can last up to one year (recent observations), this model is considered by far the closest small animal translational model system for HIV latency and cure studies. However, limitations do exist. First, the human T cells are produced and educated in the mouse thymus, which is of some concern for their development status [123,178]. A recent study, however, revealed that human T cells were preferentially developed in NRG-hu-BLT mice with more predominant and well-educated T cell populations than NRG-hu-HSC mice [183]. The same study also suggested that there are no significant differences in responses between hu-BLT and hu-HSC mice when used to study HIV-1 replication, pathogenesis, and therapeutics [183]. The primary advantages of hu-BLT mice over hu-HSC mice are in studies involving human HLA class I and class II restricted T cell responses. The second limitation is the underdeveloped lymph nodes and lower level of human cell reconstitution in the gut and mucosal tissues of the hu-HSC mouse. However, no significant differences were observed in HIV long-term studies compared to other animal model systems. Third, hu-HSC mice harbor fewer cells of human origin (microglia, macrophages, and astrocytes) responsible for carrying latent virus in CNS compartments [192,193]. Encouraging recent studies have described the development and successful use of another hu-HSC model with human microglia [194]. Human interleukin-34, a key macrophage–microglial differentiation factor, was introduced into the NOG strain, which led to the successful reconstitution of human microglia-like cells in the mouse brain, thus allowing for the study of principal CNS viral reservoirs. Additionally, this model was able to recapitulate neuroHIV. This was evidenced via transcriptomic analyses of antiviral, inflammatory, and human-specific molecular signatures, which offer insights into viral persistence in the brain.
CD34 mouse models are used in a variety of platforms and are employed as mainstream small animal models to study HIV pathobiology, drug safety and efficacy, latency, and reservoir targeting and in studies that can modulate the immune system. The optimization of preclinical small animal models is the key to testing the up-to-date clinical advances in HIV therapeutic developments, which can ensure the improved translation of various preclinical results into interventions and can ultimately benefit patients. In summary the hu-HSC background represents an ideal long-term model for studying HIV-1 latency and cure, with diverse cell types reconstituted in immunologically active compartments and well-defined human cells that can last for more than six months to one year, as described in Figure 2. Its advantages include the simplicity of the mouse generation procedure, abundant sources of CD34+ HSCs from umbilical cord blood, and the greatly reduced incidence of GVHD (less than 5%), making it the leading mouse model for studying and validating HIV cure strategies prior to assessments in NHP models or clinical translation.
Figure 2. HIV reservoir target cells in a suitable small animal model system to study HIV latency. Cells demonstrated to harbor latent HIV in different tissues are shown. We propose an ideal humanized rodent model system that depicts HIV reservoirs spread across various tissue types around the body. The majority of latent virus is found in CD4+ resting memory T cells (TRM), along with other CD4+ T cell subsets (TEM, TCM, and TH). MP reservoirs are in a range of tissue compartments. In addition to having a peripheral functional human immune system, the ideal humanized model system should have abundant cells of human origin which include T cells, B cells, dendritic cells, monocytes, and macrophages that can be maintained for a year for long-term HIV latency studies and subsequent targeting of those reservoirs for ultimate HIV elimination and for clinical translation. TEM = T effector memory; TCM = T central memory; TH = T helper; NK = natural killer.
This entry is adapted from the peer-reviewed paper 10.3390/pathogens12070879
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