Stem cell-derived CAR T cells show greater persistence, trafficking, and viral control compared to ex vivo transduced CAR T cells.

Adoptive cell therapy (ACT) using T cells expressing chimeric antigen receptors (CARs) is an area of intense investigation in the treatment of malignancies and chronic viral infections. One of the limitations of ACT-based CAR therapy is the lack of in vivo persistence and maintenance of optimal cell function. Therefore, alternative strategies that increase the function and maintenance of CAR-expressing T cells are needed. In our studies using the humanized bone marrow/liver/thymus (BLT) mouse model and nonhuman primate (NHP) model of HIV infection, we evaluated two CAR-based gene therapy approaches. In the ACT approach, we used cytokine enhancement and preconditioning to generate greater persistence of anti-HIV CAR+ T cells. We observed limited persistence and expansion of anti-HIV CAR T cells, which led to minimal control of the virus. In our stem cell-based approach, we modified hematopoietic stem/progenitor cells (HSPCs) with anti-HIV CAR to generate anti-HIV CAR T cells in vivo. We observed CAR-expressing T cell expansion, which led to better plasma viral load suppression. HSPC-derived CAR cells in infected NHPs showed superior trafficking and persistence in multiple tissues. Our results suggest that a stem cell-based CAR T cell approach may be superior in generating long-term persistence and functional antiviral responses against HIV infection.

Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates.

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative in vivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Autophagy inducer rapamycin treatment reduces IFN-I-mediated Inflammation and improves anti-HIV-1 T cell response in vivo.

A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4+ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1-infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I-mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV.

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.

Correction: Robust CAR-T memory formation and function via hematopoietic stem cell delivery.

[This corrects the article DOI: 10.1371/journal.ppat.1009404.].

Primary, Recall, and Decay Kinetics of SARS-CoV-2 Vaccine Antibody Responses.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naïve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naïve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naïve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naïve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

Robust CAR-T memory formation and function via hematopoietic stem cell delivery.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques.

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.

Apolipoprotein A-I mimetics attenuate macrophage activation in chronic treated HIV.

OBJECTIVES: Despite antiretroviral therapy (ART), there is an unmet need for therapies to mitigate immune activation in HIV infection. The goal of this study is to determine whether the apoA-I mimetics 6F and 4F attenuate macrophage activation in chronic HIV. DESIGN: Preclinical assessment of the in-vivo impact of Tg6F and the ex-vivo impact of apoA-I mimetics on biomarkers of immune activation and gut barrier dysfunction in treated HIV. METHODS: We used two humanized murine models of HIV infection to determine the impact of oral Tg6F with ART (HIV+ART+Tg6F+) on innate immune activation (plasma human sCD14, sCD163) and gut barrier dysfunction [murine I-FABP, endotoxin (LPS), LPS-binding protein (LBP), murine sCD14]. We also used gut explants from 10 uninfected and 10 HIV-infected men on potent ART and no morbidity, to determine the impact of ex-vivo treatment with 4F for 72 h on secretion of sCD14, sCD163, and I-FABP from gut explants. RESULTS: When compared with mice treated with ART alone (HIV+ART+), HIV+ART+Tg6F+ mice attenuated macrophage activation (h-sCD14, h-sCD163), gut barrier dysfunction (m-IFABP, LPS, LBP, and m-sCD14), plasma and gut tissue oxidized lipoproteins. The results were consistent with independent mouse models and ART regimens. Both 4F and 6F attenuated shedding of I-FABP and sCD14 from gut explants from HIV-infected and uninfected participants. CONCLUSION: Given that gut barrier dysfunction and macrophage activation are contributors to comorbidities like cardiovascular disease in HIV, apoA-I mimetics should be tested as therapy for morbidity in chronic treated HIV.

Engineering CAR T Cells to Target the HIV Reservoir.

The HIV reservoir remains to be a difficult barrier to overcome in order to achieve a therapeutic cure for HIV. Several strategies have been developed to purge the reservoir, including the "kick and kill" approach, which is based on the notion that reactivating the latent reservoir will allow subsequent elimination by the host anti-HIV immune cells. However, clinical trials testing certain classes of latency reactivating agents (LRAs) have so far revealed the minimal impact on reducing the viral reservoir. A robust immune response to reactivated HIV expressing cells is critical for this strategy to work. A current focus to enhance anti-HIV immunity is through the use of chimeric antigen receptors (CARs). Currently, HIV-specific CARs are being applied to peripheral T cells, NK cells, and stem cells to boost recognition and killing of HIV infected cells. In this review, we summarize current developments in engineering HIV directed CAR-expressing cells to facilitate HIV elimination. We also summarize current LRAs that enhance the "kick" strategy and how new generation and combinations of LRAs with HIV specific CAR T cell therapies could provide an optimal strategy to target the viral reservoir and achieve HIV clearance from the body.

Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.

The Use of the Humanized Mouse Model in Gene Therapy and Immunotherapy for HIV and Cancer.

HIV and cancer remain prevailing sources of morbidity and mortality worldwide. There are current efforts to discover novel therapeutic strategies for the treatment or cure of these diseases. Humanized mouse models provide the investigative tool to study the interaction between HIV or cancer and the human immune system in vivo. These humanized models consist of immunodeficient mice transplanted with human cells, tissues, or hematopoietic stem cells that result in reconstitution with a nearly full human immune system. In this review, we discuss preclinical studies evaluating therapeutic approaches in stem cell-based gene therapy and T cell-based immunotherapies for HIV and cancer using a humanized mouse model and some recent advances in using checkpoint inhibitors to improve antiviral or antitumor responses.

Correction: Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

[This corrects the article DOI: 10.1371/journal.ppat.1006753.].

Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

New approaches for the enhancement of chimeric antigen receptors for the treatment of HIV.

HIV infection continues to be a life-long chronic disease in spite of the success of antiretroviral therapy (ART) in controlling viral replication and preventing disease progression. However, because of the high cost of treatment, severe side effects, and inefficiency in curing the disease with ART, there is a call for alternative therapies that will provide a functional cure for HIV. Cytotoxic T lymphocytes (CTLs) are vital in the control and clearance of viral infections and therefore immune-based therapies have attempted to engineer HIV-specific CTLs that would be able to clear the infection from the body. The development of chimeric antigen receptors (CARs) provides an opportunity to engineer superior HIV-specific CTLs that will be independent of the major histocompatibility complex for target recognition. A CD4-based CAR has been previously tested in clinical trials to test the antiviral efficacy of peripheral T cells armed with this CD4-based CAR. The results from these clinical trials showed the safety and feasibility of CAR T cell therapy for HIV infection; however, minimal antiviral efficacy was seen. In this review, we will discuss the various strategies being developed to enhance the therapeutic potency of anti-HIV CARs with the goal of generating superior antiviral responses that will lead to life-long HIV immunity and clearance of the virus from the body.

Chimeric antigen receptor engineered stem cells: a novel HIV therapy.

Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

Targeting type I interferon-mediated activation restores immune function in chronic HIV infection.

Chronic immune activation, immunosuppression, and T cell exhaustion are hallmarks of HIV infection, yet the mechanisms driving these processes are unclear. Chronic activation can be a driving force in immune exhaustion, and type I interferons (IFN-I) are emerging as critical components underlying ongoing activation in HIV infection. Here, we have tested the effect of blocking IFN-I signaling on T cell responses and virus replication in a murine model of chronic HIV infection. Using HIV-infected humanized mice, we demonstrated that in vivo blockade of IFN-I signaling during chronic HIV infection diminished HIV-driven immune activation, decreased T cell exhaustion marker expression, restored HIV-specific CD8 T cell function, and led to decreased viral replication. Antiretroviral therapy (ART) in combination with IFN-I blockade accelerated viral suppression, further decreased viral loads, and reduced the persistently infected HIV reservoir compared with ART treatment alone. Our data suggest that blocking IFN-I signaling in conjunction with ART treatment can restore immune function and may reduce viral reservoirs during chronic HIV infection, providing validation for IFN-I blockade as a potential therapy for HIV infection.

Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.

HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy.

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.