Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient-derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a human stroma-patient-derived xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrates greater resistance to targeted therapy and chemotherapy and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on recruitment of TAMs and tumor immune exclusion to impact to response to immunologic therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stroma-mediated immunosuppression. Combined, our data confirm a critical role for human stroma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing.

The persistence of human immunodeficiency virus (HIV) reservoirs in the lymphatic system remains a major obstacle to a complete cure, largely due to subtherapeutic concentrations of antiretrovirals (ARVs) in lymphatic tissues under current regimens. Developing innovative strategies to enhance ARV delivery to lymphatic tissues could therefore represent a paradigm shift in the pursuit of an HIV cure. Here, we describe a delivery strategy involving an Evans Blue (EB)-based prodrug conjugated with dolutegravir (DTG), a model ARV. This construct is designed to bind albumin with high affinity, facilitating transport to lymph nodes (LNs) via albumin-mediated trans-endothelial transcytosis. EB modification substantially improved the distribution of the conjugated molecule to LNs throughout the body, and the hEB-DTG prodrug increased DTG concentrations in LNs by up to 10-fold, suggesting a promising strategy for eradicating HIV reservoirs. STATEMENT OF SIGNIFICANCE: A major barrier to curing HIV infection is the persistence of viral reservoirs in lymphatic tissues, which current ARV therapies fail to eliminate due to poor drug penetration. To overcome this limitation, we developed a prodrug strategy that harnesses the natural trafficking of albumin to the lymphatic system. In this approach, ARVs are conjugated to EB, a high-affinity albumin-binding dye, to generate EB-ARV prodrugs that hijack albumin's endogenous transport pathways. After intravenous administration, these conjugates rapidly associate with circulating albumin, which mediates their transcytosis across endothelial barriers and directs their accumulation in lymph nodes and other lymphoid tissues. This strategy substantially enhances ARV exposure at the primary sites of viral persistence, representing a promising and potentially transformative approach for eradicating HIV reservoirs and advancing toward a functional cure.

In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.

Gamma-delta (γδ) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the Vδ2 cell subset compared to the Vδ1 cell subset of γδ T cells in the blood in most infected individuals, except for elite controllers. The capacity of Vδ2 T cells to kill HIV-infected targets has been demonstrated in vitro , albeit in vivo confirmatory studies are lacking. Here, we provide the first characterization of γδ T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of Vδ2 T cells in controlling HIV replication in vivo . We demonstrate a reduced proportion of Vδ2 T cells and an increased proportion of Vδ1 T cells in HIV-infected BLT humanized mice, like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of Vδ2 T cells, like in HIV-positive individuals. Adoptive transfer of activated Vδ2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia, suggesting that Vδ2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model γδ T cell-HIV interactions in vivo .

Gamma-delta (γδ) T cells recognize antigens in an MHC-independent manner, with demonstrable cytotoxicity against cancer and virally infected cells. Human immunodeficiency virus (HIV) infection severely depletes the Vγ9Vδ2 (Vδ2) subset of these T cells in most infected individuals, with the exception of elite controllers. The capacity of Vδ2 cells to kill HIV-infected targets has been demonstrated in vitro , but this has not been verified in vivo . Here, we examined the immunotherapeutic potential of Vδ2 cells in controlling HIV replication in vivo and provide the first characterization of reconstituted γδ T cell subsets in the peripheral blood and lymphoid tissue in a humanized mouse model. We demonstrate the depletion of Vδ2 cells and increase in Vδ1 cells in the blood following HIV infection, similar to that observed in HIV-infected humans. The functionality of human Vδ2 cells isolated from humanized mice was confirmed via ex vivo expansion in response to zoledronate and IL-2 treatment. The adoptive transfer of activated Vδ2 cells failed to control HIV infection in vivo but instead exacerbated viremia by serving as early targets for HIV infection. Our findings suggest that Vδ2 cells play a critical and unappreciated role as early HIV targets of infection to promote viral dissemination.

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The main advantage of animal models of infectious diseases over in vitro studies is the gain in the understanding of the complex dynamics between the immune system and the pathogen. While small animal models have practical advantages over large animal models, it is crucial to be aware of their limitations. Although the small animal model at least needs to be susceptible to the pathogen under study to obtain meaningful data, key elements of pathogenesis should also be reflected when compared to humans. Well-designed small animal models for HIV, hepatitis viruses and tuberculosis require, additionally, a thorough understanding of the similarities and differences in the immune responses between humans and small animals and should incorporate that knowledge into the goals of the study. To discuss these considerations, the NIAID hosted a workshop on 'Small Animal Models for HIV, Hepatitis B, and Tuberculosis' on May 30, 2019. Highlights of the workshop are outlined below.

The development of safe and effective combination antiretroviral therapies for human immunodeficiency virus (HIV) infection over the past several decades has significantly reduced HIV-associated morbidity and mortality. Additionally, antiretroviral drugs have provided an effective means of protection against HIV transmission. Despite these advances, significant limitations exist; namely, the inability to eliminate HIV reservoirs, the inability to reverse lymphoid tissues damage, and the lack of an effective vaccine for preventing HIV transmission. Evaluation of the safety and efficacy of therapeutics and vaccines for eliminating HIV reservoirs and preventing HIV transmission requires robust in vivo models. Since HIV is a human-specific pathogen, that targets hematopoietic lineage cells and lymphoid tissues, in vivo animal models for HIV-host interactions require incorporation of human hematopoietic lineage cells and lymphoid tissues. In this review, we will discuss the construction of mouse models with human lymphoid tissues and/or hematopoietic lineage cells, termed, human immune system (HIS)-humanized mice. These HIS-humanized mouse models can support the development of functional human innate and adaptive immune cells, along with primary (thymus) and secondary (spleen) lymphoid tissues. We will discuss applications of HIS-humanized mouse models in evaluating the safety and efficacy of therapeutics against HIV reservoirs and associated immunopathology, and delineate the human immune response elicited by candidate HIV vaccines. In addition to focusing on how these HIS-humanized mouse models have already furthered our understanding of HIV and contributed to HIV therapeutics development, we discuss how emerging HIS-humanized rat models could address the limitations of HIS-mouse models.

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family.

Abstract The development of Novel cancer immunotherapies has relied on the use of immune humanized mice. The field of study can be enhanced using an immune humanized rat, which will provide several advantages over the currently available humanized mouse models. The SRG OncoRat®; A Rag2/Il2rg double-knockout rat supports the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. Using this rat, we developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). We demonstrated the development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Furthermore, we demonstrated the presence of Human CD45+, CD3+, and CD20+ cells in peripheral blood, spleen, and bone marrow of the SRG OncoRat® engrafted with human PBMCs, hence humanizing the rat's immune system. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Bisoye Towobola Adedeji, Fallon K. Noto, Tseten Yeshi Jamling, Yash Argawal, Cole Jamison Beatty, Sara Grace Ho, Antu Das, Rajeev Kishore Salunke, Moses Turkle Bility. Humanized rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5621.

The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD- scid IL2Rγ null (NSG) mouse and Sprague–Dawley-Rag2 tm2hera Il2rγ tm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus . The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.

As a long-acting formulation of the nonnucleoside reverse transcriptase inhibitor rilpivirine (RPV LA) has been proposed for use as preexposure prophylaxis (PrEP) and the prevalence of transmitted RPV-resistant viruses can be relatively high, we evaluated the efficacy of RPV LA to inhibit vaginal transmission of RPV-resistant HIV-1 in humanized mice. Vaginal challenges of wild-type (WT), Y181C, and Y181V HIV-1 were performed in mice left untreated or after RPV PrEP. Plasma viremia was measured for 7 to 10 weeks, and single-genome sequencing was performed on plasma HIV-1 RNA in mice infected during PrEP. RPV LA significantly prevented vaginal transmission of WT HIV-1 and Y181C HIV-1, which is 3-fold resistant to RPV. However, it did not prevent transmission of Y181V HIV-1, which has 30-fold RPV resistance in the viruses used for this study. RPV LA did delay WT HIV-1 dissemination in infected animals until genital and plasma RPV concentrations waned. Animals that became infected despite RPV LA PrEP did not acquire new RPV-resistant mutations above frequencies in untreated mice or untreated people living with HIV-1, and the mutations detected conferred low-level resistance. These data suggest that high, sustained concentrations of RPV were required to inhibit vaginal transmission of HIV-1 with little or no resistance to RPV but could not inhibit virus with high resistance. HIV-1 did not develop high-level or high-frequency RPV resistance in the majority of mice infected after RPV LA treatment. However, the impact of low-frequency RPV resistance on virologic outcome during subsequent antiretroviral therapy still is unclear. IMPORTANCE The antiretroviral drug rilpivirine was developed into a long-acting formulation (RPV LA) to improve adherence for preexposure prophylaxis (PrEP) to prevent HIV-1 transmission. A concern is that RPV LA will not inhibit transmission of drug-resistant HIV-1 and may select for drug-resistant virus. In female humanized mice, we found that RPV LA inhibited vaginal transmission of WT or 3-fold RPV-resistant HIV-1 but not virus with 30-fold RPV resistance. In animals that became infected despite RPV LA PrEP, WT HIV-1 dissemination was delayed until genital and plasma RPV concentrations waned. RPV resistance was detected at similar low frequencies in untreated and PrEP-treated mice that became infected. These results indicate the importance of maintaining RPV at a sustained threshold after virus exposure to prevent dissemination of HIV-1 after vaginal infection and low-frequency resistance mutations conferred low-level resistance, suggesting that RPV resistance is difficult to develop after HIV-1 infection during RPV LA PrEP.

Immune humanized mice have been valuable in the development of novel cancer immunotherapies and have demonstrated stronger efficacy when combined with standard of care chemotherapy. An immune humanized rat could provide several advantages over the currently available humanized mouse models, including supporting the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. We developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). hSIS-humanized rat support development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Finally, we have demonstrated humanization of the rat immune system using human PBMCs. Human CD45+, CD3+, and CD20+ cells can be found in the peripheral blood, spleen, and bone marrow of engrafted rats. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Yash Agarwal, Cole Beatty, Sara Ho, Antu Das, Rajeev Salunke, Moses Bility, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat supports engraftment of human immune system for immunotherapy-based cancer efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A007. doi:10.1158/1535-7163.TARG-19-A007

Considerable progress has been made during the past 20 years towards elucidating the role of peroxisome proliferator-activated receptor-beta/delta (PPAR beta/delta) in skin cancer. In 1999, the original notion that PPAR beta/delta was involved with epithelial cell function was postulated based on a correlation between PPAR beta/delta expression and the induction of messenger RNAs encoding proteins that mediate terminal differentiation in keratinocytes. Subsequent studies definitively revealed that PPAR beta/delta could induce terminal differentiation and inhibit proliferation of keratinocytes. Molecular mechanisms have since been discovered to explain how this nuclear receptor can be targeted for preventing and treating skin cancer. This includes the regulation of terminal differentiation, mitotic signaling, endoplasmic reticulum stress, and cellular senescence. Interestingly, the effects of activating PPAR beta/delta can preferentially target keratinocytes with genetic mutations associated with skin cancer. This review provides the history and current understanding of how PPAR beta/delta can be targeted for both nonmelanoma skin cancer and melanoma and postulates how future approaches that modulate PPAR beta/delta signaling may be developed for the prevention and treatment of these diseases.

A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.

The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV-induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV-induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.

Despite the availability of a preventive vaccine, chronic hepatitis B virus (HBV) infection-induced liver diseases continue to be a major global public health problem. HBV naturally infects only humans and chimpanzees. This narrow host range has hindered our ability to study the characteristics of the virus and how it interacts with its host. It is thus important to establish small animal models to study HBV infection, persistence, clearance and the immunopathogenesis of chronic hepatitis B. In this review, we briefly summarize currently available animal models for HBV research, then focus on mouse models, especially the recently developed humanized mice that can support HBV infection and immunopathogenesis in vivo. This article is part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story."

Abstract HBV infection remains a leading cause of death worldwide. However, the lack of robust small animal model has hindered the progress in hepatitis B virus research and the development of new treatment options. Immunodeficient mice reconstituted with both human immune system and liver provide us a powerful in vivo model to study HBV pathogenesis and evaluate potential therapeutics. Cytokines targeting T cells and NK cells are attractive clinical interventions against viruses. Here we tested the therapeutic effect of human hyper-IL15(IL15 fused with IL15Rα sushi domain) on HBV replication in vivo in our humanized mice with both human liver and immune system. We showed that hydrodynamic injection of HBV1.3 expression plasmid led to persistent HBV replication for at least 4 months in humanized mice. Ectopic expression of hyper-IL15 in humanized mice one week after HBV plasmid injection significantly inhibited HBV replication. The HBV inhibition effect was correlated with increasing human T cell and NK cell numbers and activation in humanized mice. To further test the therapeutic potential of hyper-IL15 on chronic phase of hepatitis B, we treated the humanized mice with hyper-IL15 7.5 weeks after HBV persistence in vivo. Our data showed that the HBV genome was undetectable in 75% of hu-mice 3 weeks after hyper-IL15 treatment. Our encouraging results warrant future exploration of the use of hyper-IL15 as an immunotherapy reagent to treat chronic hepatitis B in humans.