Engineering immunosuppressive drug-resistant armored (IDRA) SARS-CoV-2 T cells for cell therapy.

Solid organ transplant (SOT) recipients receive immunosuppressive drugs (ISDs) and are susceptible to developing severe COVID-19. Here, we analyze the Spike-specific T-cell response after 3 doses of mRNA vaccine in a group of SOT patients (n = 136) treated with different ISDs. We demonstrate that a combination of a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and prednisone (Pred) treatment regimen strongly suppressed the mRNA vaccine-induced Spike-specific cellular response. Such defects have clinical consequences because the magnitude of vaccine-induced Spike-specific T cells was directly proportional to the ability of SOT patients to rapidly clear SARS-CoV-2 after breakthrough infection. To then compensate for the T-cell defects induced by immunosuppressive treatment and to develop an alternative therapeutic strategy for SOT patients, we describe production of 6 distinct SARS-CoV-2 epitope-specific ISD-resistant T-cell receptor (TCR)-T cells engineered using the mRNA electroporation method with reactivity minimally affected by mutations occurring in Beta, Delta, Gamma, and Omicron variants. This strategy with transient expression characteristics marks an improvement in the immunotherapeutic field and provides an attractive and novel therapeutic possibility for immunosuppressed COVID-19 patients.

Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

SARS-CoV-2 breakthrough infection in vaccinees induces virus-specific nasal-resident CD8+ and CD4+ T cells of broad specificity.

Rapid recognition of SARS-CoV-2-infected cells by resident T cells in the upper airway might provide an important layer of protection against COVID-19. Whether parenteral SARS-CoV-2 vaccination or infection induces nasal-resident T cells specific for distinct SARS-CoV-2 proteins is unknown. We isolated T cells from the nasal mucosa of COVID-19 vaccinees who either experienced SARS-CoV-2 infection after vaccination (n = 34) or not (n = 16) and analyzed their phenotype, SARS-CoV-2 specificity, function, and persistence. Nasal-resident SARS-CoV-2-specific CD8+ and CD4+ T cells were detected almost exclusively in vaccinees who experienced SARS-CoV-2 breakthrough infection. Importantly, the Spike-specific T cells primed by vaccination did not suppress the induction of T cells specific for other SARS-CoV-2 proteins. The nasal-resident T cell responses persisted for ≥140 d, with minimal sign of waning. These data highlight the importance of viral nasal challenge in the formation of SARS-CoV-2-specific antiviral immunity at the site of primary infection and further define the immunological features of SARS-CoV-2 hybrid immunity.

Immunological alterations after immunotherapy with short lived HBV-TCR T cells associates with long-term treatment response in HBV-HCC.

The application of hepatitis B virus (HBV)-T-cell receptor (TCR) T-cell immunotherapy in patients with HBV-related hepatocellular carcinoma (HBV-HCC) has been apathetic, as the expression of HBV antigens by both normal HBV-infected hepatocytes and HCC cells with HBV-DNA integration increases the risk of on-target off-tumor severe liver inflammatory events. To increase the safety of this immunotherapeutic approach, we developed messenger RNA (mRNA) HBV-TCR-redirected T cells that-due to the transient nature of mRNA-are functionally short lived and can be infused in escalating doses. The safety of this approach and its clinical potential against primary HBV-HCC have never been analyzed in human trials; thus, we studied the clinical and immunological parameters of 8 patients with chronic HBV infection and diffuse nonoperable HBV-HCC treated at weekly intervals with escalating doses (1 × 104 , 1 × 105 , 1 × 106 , and 5 × 106 TCR+ T cells/kg body weight) of T cells modified with HBV-TCR encoding mRNA. The treatment was well tolerated with no severe systemic inflammatory events, cytokine storm, or neurotoxicity observed in any of these patients throughout treatment. Instead, we observed a destruction of the tumor lesion or a prolonged stable disease in 3 of 8 patients. Importantly, the patients without clinically relevant reductions of HCC did not display any detectable peripheral blood immunological alterations. In contrast, signs of transient localized liver inflammation, activation of the T-cell compartment, and/or elevations of serum chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL10 levels were detected in patients with long-term clinical benefit. Conclusion: We show that despite the reduced in vivo half-life (3-4 days), adoptive transfer of mRNA HBV-TCR T cells into patients with HBV-HCC show long-term clinical benefit that was associated with transient immunological alterations.

Immunotherapy of HBV-related advanced hepatocellular carcinoma with short-term HBV-specific TCR expressed T cells: results of dose escalation, phase I trial.

BACKGROUND & AIMS: Immunotherapy with hepatitis B virus (HBV)-specific TCR redirected T (HBV-TCR-T) cells in HBV-related hepatocellular carcinoma (HBV-HCC) patients after liver transplantation was reported to be safe and had potential therapeutic efficacy. We aim to investigate the safety of HBV-TCR-T-cell immunotherapy in advanced HBV-HCC patients who had not met the criteria for liver transplantation. METHODS: We enrolled eight patients with advanced HBV-HCC and adoptively transferred short-lived autologous T cells expressing HBV-specific TCR to perform an open-label, phase 1 dose-escalation study (NCT03899415). The primary endpoint was to evaluate the safety of HBV-TCR-T-cell therapy according to National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03) during the dose-escalation process. The secondary endpoint was to assess the efficacy of HBV-TCR-T-cell therapy by evaluating the anti-tumor responses using RECIST criteria (version 1.1) and the overall survival. RESULTS: Adverse events were observed in two participants among the 8 patients enrolled. Only one patient experienced a Grade 3 liver-related adverse event after receiving a dose of 1 × 105 HBV-TCR-T cells/kg, then normalized without interventions with immunosuppressive agents. Among the patients, one achieved a partial response lasting for 27.7 months. Importantly, most of the patients exhibited a reduction or stabilization of circulating HBsAg and HBV DNA levels after HBV-TCR-T-cell infusion, indicating the on-target effects. CONCLUSIONS: The adoptive transfer of HBV-TCR-T cells into advanced HBV-HCC patients were generally safe and well-tolerated. Observations of clinical efficacy support the continued development and eventual application of this treatment strategy in patients with advanced HBV-related HCC. CLINICAL TRIALS REGISTRATION: This study was registered at ClinicalTrials.gov (NCT03899415).

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals.

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.

A Dynamic Immune Response Shapes COVID-19 Progression.

The inflammatory response to SARS-coronavirus-2 (SARS-CoV-2) infection is thought to underpin COVID-19 pathogenesis. We conducted daily transcriptomic profiling of three COVID-19 cases and found that the early immune response in COVID-19 patients is highly dynamic. Patient throat swabs were tested daily for SARS-CoV-2, with the virus persisting for 3 to 4 weeks in all three patients. Cytokine analyses of whole blood revealed increased cytokine expression in the single most severe case. However, most inflammatory gene expression peaked after respiratory function nadir, except expression in the IL1 pathway. Parallel analyses of CD4 and CD8 expression suggested that the pro-inflammatory response may be intertwined with T cell activation that could exacerbate disease or prolong the infection. Collectively, these findings hint at the possibility that IL1 and related pro-inflammatory pathways may be prognostic and serve as therapeutic targets for COVID-19. This work may also guide future studies to illuminate COVID-19 pathogenesis and develop host-directed therapies.

HBV as a target for CAR or TCR-T cell therapy.

Engineering HBV-specific T cells utilizing a chimeric antigen receptor (CAR) or a classical T cell receptor (TCR) provides a well characterized, sizeable and functionally intact population of HBV-specific T cells with identical in vitro functionality to the T cells isolated in patients who resolved acute HBV infection. In this review we present evidences of the virological and immunological features of chronic HBV infection, alone or in combination with Hepatitis Delta that might make it amenable for CAR/TCR-T cells therapy.

Challenges of CAR- and TCR-T cell-based therapy for chronic infections.

While therapy with T cells engineered with a chimeric antigen receptor (CAR) or a classical T cell receptor (TCR) is revolutionizing cancer treatment, its adoption in infectious diseases has been met with considerable resistance. Can we find its value for the cure of infections?

Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is often associated with hepatitis B virus (HBV) infection. Cells of most HBV-related HCCs contain HBV-DNA fragments that do not encode entire HBV antigens. We investigated whether these integrated HBV-DNA fragments encode epitopes that are recognized by T cells and whether their presence in HCCs can be used to select HBV-specific T-cell receptors (TCRs) for immunotherapy. METHODS: HCC cells negative for HBV antigens, based on immunohistochemistry, were analyzed for the presence of HBV messenger RNAs (mRNAs) by real-time polymerase chain reaction, sequencing, and Nanostring approaches. We tested the ability of HBV mRNA-positive HCC cells to generate epitopes that are recognized by T cells using HBV-specific T cells and TCR-like antibodies. We then analyzed HBV gene expression profiles of primary HCCs and metastases from 2 patients with HCC recurrence after liver transplantation. Using the HBV-transcript profiles, we selected, from a library of TCRs previously characterized from patients with self-limited HBV infection, the TCR specific for the HBV epitope encoded by the detected HBV mRNA. Autologous T cells were engineered to express the selected TCRs, through electroporation of mRNA into cells, and these TCR T cells were adoptively transferred to the patients in increasing numbers (1 × 104-10 × 106 TCR+ T cells/kg) weekly for 112 days or 1 year. We monitored patients' liver function, serum levels of cytokines, and standard blood parameters. Antitumor efficacy was assessed based on serum levels of alpha fetoprotein and computed tomography of metastases. RESULTS: HCC cells that did not express whole HBV antigens contained short HBV mRNAs, which encode epitopes that are recognized by and activate HBV-specific T cells. Autologous T cells engineered to express TCRs specific for epitopes expressed from HBV-DNA in patients' metastases were given to 2 patients without notable adverse events. The cells did not affect liver function over a 1-year period. In 1 patient, 5 of 6 pulmonary metastases decreased in volume during the 1-year period of T-cell administration. CONCLUSIONS: HCC cells contain short segments of integrated HBV-DNA that encodes epitopes that are recognized by and activate T cells. HBV transcriptomes of these cells could be used to engineer T cells for personalized immunotherapy. This approach might be used to treat a wider population of patients with HBV-associated HCC.

Corrigendum: Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

[This corrects the article DOI: 10.3389/fimmu.2018.00416.].

Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

In the hepatitis B virus (HBV)-related hepatocellular carcinoma tumor microenvironment (TME), monocytes reportedly impede natural T cell functions via PD-L1/PD-1 signaling. However, it remains unclear if T cell receptor-redirected T cells (TCR T cells) are similarly inhibited. Hence, we developed a 3D intrahepatic TME microfluidic model to investigate the immunosuppressive potential of monocytes toward HBV-specific TCR T cells and the role of PD-L1/PD-1 signaling. Interestingly, in our 3D static microfluidic model, we observed that monocytes suppressed only retrovirally transduced (Tdx) TCR T cell cytotoxicity toward cancer cells via PD-L1/PD-1, while mRNA electroporated (EP) TCR T cell cytotoxicity was not affected by the presence of monocytes. Importantly, when co-cultured in 2D, both Tdx and EP TCR T cell cytotoxicity toward cancer cells were not suppressed by monocytes, suggesting our 3D model as a superior tool compared to standard 2D assays for predicting TCR T cell efficacy in a preclinical setting, which can thus be used to improve current immunotherapy strategies.

T-cell therapy for chronic viral hepatitis.

Although therapy for chronic hepatitis C virus infection has delivered remarkable cure rates, curative therapies for hepatitis B virus (HBV) may only be available in the distant future. The possibility to eliminate or at least stably maintain low levels of HBV replication under the control of a functional anti-host response has stimulated the development of specific immunotherapies for HBV infection. We reviewed the development of T-cell therapy for HBV, highlighting its potential antiviral efficiency but also its potential toxicities in different groups of chronic HBV patients. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the only two communicable diseases in which there have been increases in related morbidity and mortality over the past 20 years [1]. Both viruses are chronically infecting about 500 million people (HBV ~350 million, HCV ~150 million) and represent the seventh most frequent cause of death worldwide [1]. HBV and HCV are hepatotropic, non-cytopathic viruses able to establish persistent infections that cause different degrees of hepatic inflammation (chronic hepatitis), leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). The two viruses are unrelated and virologically different. HCV remains prevalent in North America and Europe, whereas chronic hepatitis B is prevalent in Asia and sub-Saharan Africa [1,2]. HCV is an RNA virus belonging to the Flaviviridae family, and HBV is a DNA virus of the Hepadnaviridae family and uses reverse transcriptase to synthesize its DNA from a pre-genomic RNA form [3]. HCV is able to activate in the infected host a classical type I interferon (IFN)-mediated innate response [3], whereas HBV generally escapes innate immune recognition and does not activate type I IFN-mediated immunity. Chronic HBV and HCV infections are both characterized by quantitative and functional defects of virus-specific T-cell response [4,5]. The frequency of virus-specific T cells is extremely low, and virus-specific T cells show features of exhaustion in both chronic HBV and HCV patients [6]. However, the quantitative and functional defects are more pronounced in HBV infections, with T cells virtually undetectable in the blood of many chronic HBV patients by ex vivo analysis [7-9]. In addition, while frequency and impact of viral mutations in T cell epitopes are frequently detectable in HCV infections [10], viral mutations affecting CD8 T-cell epitopes are scarcer in chronic HBV patients [6,11,12]. Of extreme practical importance in relation to the potential impact of T-cell therapy for HBV and HCV are the efficacies of currently available treatments. New therapies for HCV have delivered remarkable cure rates, with more than 90% of patients achieving viral clearance with all oral direct-acting antivirals [13]. In contrast, curative therapies for HBV will not be available until the distant future (14). Thus, although it is difficult to see a possible therapeutic advantage of a new T-cell-based therapy in chronic HCV patients, the fact that current therapies for HBV only partially suppress but do not eliminate HBV from the infected host has encouraged research for new and more radical therapies designed to eliminate or at least stably maintain low levels of HBV replication under the control of a functional anti-host response. For these reasons, in this review, we concentrate on the development of T-cell therapy for HBV. T-cell therapy for HCV chronic infection is certainly important for understanding the mechanisms of T-cell antiviral control [15,16], but their use for therapy appears unlikely.

Targeted Therapy of Hepatitis B Virus-Related Hepatocellular Carcinoma: Present and Future.

Cancer immunotherapy using a patient's own T cells redirected to recognize and kill tumor cells has achieved promising results in metastatic melanoma and leukemia. This technique involves harnessing a patient's T cells and then delivering a gene that encodes a new T cell receptor (TCR) or a chimeric antigen receptor (CAR) that allow the cells to recognize specific cancer antigens. The prospect of using engineered T cell therapy for persistent viral infections like hepatitis B virus (HBV) and their associated malignancies is promising. We recently tested in a first-in-man clinical trial, the ability of HBV-specific TCR-redirected T cells to target HBsAg-productive hepatocellular carcinoma (HCC) and demonstrated that these redirected T cells recognized HCC cells with HBV-DNA integration [1] We discuss here the possibility to use HBV-specific TCR-redirected T cells targeting hepatitis B viral antigens as a tumor specific antigen in patients with HBV-related HCC, and the potential challenges facing the development of this new immunotherapeutic strategy.

Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire.

Repertoire composition, quantity, and qualitative functional ability are the parameters that define virus-specific T-cell responses and are linked with their potential to control infection. We took advantage of the segregation of different hepatitis B virus (HBV) genotypes in geographically and genetically distinct host populations to directly analyze the impact that host and virus variables exert on these virus-specific T-cell parameters. T-cell responses against the entire HBV proteome were analyzed in a total of 109 HBV-infected subjects of distinct ethnicities (47 of Chinese origin and 62 of Caucasian origin). We demonstrate that HBV-specific T-cell quantity is determined by the virological and clinical profiles of the patients, which outweigh any influence of race or viral diversity. In contrast, HBV-specific T-cell repertoires are divergent in the two ethnic groups, with T-cell epitopes frequently found in Caucasian patients seldom detected in Chinese patients. In conclusion, we provide a direct biological evaluation of the impact that host and virus variables exert on virus-specific T-cell responses. The discordance between HBV-specific CD8 T-cell repertoires present in Caucasian and Chinese subjects shows the ability of HLA micropolymorphisms to diversify T-cell responses and has implications for the rational development of therapeutic and prophylactic vaccines for worldwide use.

Understanding the immunopathogenesis of chronic hepatitis B virus: an Asian prospective.

The study of hepatitis B virus (HBV) immunity has been mainly focused on understanding the differences between subjects who are able to control HBV infection and patients with persistent infection. These studies have been instrumental in increasing our knowledge on the pathogenesis of the disease caused by HBV. However, it is possible that heterogeneity of host and virus factors which segregate in ethnically distinct HBV infected populations might modify important aspects of the immune response against HBV. In this review, we reexamine the kinetics and the pattern of HBV-specific immunity associated with control or persistence of infection. We then discuss how the epidemiological, genetic and viral characteristics peculiar to Asian patients can impact the profile of HBV-specific immunity.