Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

Objective: The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design: Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3 zeta domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results: In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon gamma, interleukin 2 and tumour necrosis factor a. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions: Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool

Secreted Interferon-Inducible Factors Restrict Hepatitis B and C Virus Entry In Vitro

Interferon-α (IFN-α) has been used for more than 20 years as the first-line therapy for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, because it has a number of antiviral effects. In this study, we describe a novel mode of its antiviral action. We demonstrate that the supernatant from IFN-α-treated cultured cells restricted HBV and HCV infection by inhibiting viral entry into hepatoma cells. The factors contained in the supernatant competed with the virus for binding to heparan glycosaminoglycans—the nonspecific attachment step shared by HBV and HCV. Secreted factors of high molecular mass that bind to heparin columns elicited the antiviral effect. In conclusion, IFN-α is able to induce soluble factors that can bind to heparan glycosaminoglycans thus leading to the inhibition of viral binding

Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use

<div><p>T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8⁺ as well as CD4⁺ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.</p></div

Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via non-cytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-gamma (IFN gamma) or tumor necrosis factor-a (TNF-alpha), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFN gamma and TNF-alpha were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFN gamma and TNF-alpha each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFN gamma and TNF-alpha, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFN gamma and TNF-alpha after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APO-BEC3B. CONCLUSIONS: IFN gamma and TNF-alpha, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay

Recognition of HBV-infected cells by TCR-transduced T cells.

<p>(A) Expression of HLA-A*02 on HepaRG target cells determined by flow cytometry. (B-D) HBV-infected HepaRG cells were co-cultured with decreasing numbers of TCR-transduced T cells for 44 hours. One TCR per peptide specificity was selected (5E: C18-specific, 4G: S20-specific, WL12: S172-specific). Mock = untransduced PBMC,— = medium control without PBMC. (B) T-cell activation measured by release of IFN-γ into the supernatant. (C) Alanine transaminase (ALT) released from dying hepatocytes. Supernatants from triplicate co-cultures per TCR were pooled for this analysis. + = Triton-X, positive control for cell lysis. (D) HBeAg in the supernatant was measured using a diagnostic ELISA. (E) Intracellular viral rcDNA and (F) nuclear cccDNA on day two were determined using qPCR. Values were normalized to the untreated medium control. Data are presented as mean values +/- SEM from triplicates.</p

Isolation of HBV-specific T cells.

<p>(A) Experimental procedure to isolate HBV-specific T cells. T cells from donor 1 with acute, donor 2 with protracted and donor 3 with resolving HBV infection were stimulated for two weeks with peptide-loaded, irradiated TAP-deficient T2 cells or by direct addition of HBV-derived peptides to PBMC. HBV-specific CD8⁺ T cells were isolated using reversible MHC multimers (MHC Streptamers) and cloned by limiting dilution. T-cell clones were expanded and subsequently analyzed for their specificity. (B) TCR β chains of all T-cell clones with specificity for HBV peptide C18, S20 or S172 were sequenced; n.d. = not determined because of unclear sequencing results. Each color represents identical TCR sequences. (C) Schematic representation of both TCR chains cloned as one transgene cassette into the retroviral vector MP71. Gene sequences were codon-optimized, constant regions were murinized introducing an additional disulfide bond, and TCR α and β chains were fused by a P2A element for polycystronic expression. (D) TCR-expressing T cells were generated by retroviral transduction: retroviral supernatant was obtained by transient transfection of 293T cells with gene-optimized constructs and packaging plasmids derived from the murine leukemia virus. Pre-stimulated PBMC were spinoculated two times on the plates coated with retrovirus supernatant. Successfully transduced T cells were identified by CD3, CD4 or CD8 and MHC Streptamer staining.</p

Characteristics of donors at the time of PBMC isolation.

<p>Characteristics of donors at the time of PBMC isolation.</p

Recognition of different HLA-A*02 subtypes.

<p>LCL with varying HLA-A*02 subtypes were loaded with HBV peptides (1 μM) and co-cultured with TCR-transduced C18-specific (A), S20-specific (B) or S172-specific (C) CD8⁺ T cells (E:T 1:1). T-cell activation was measured by IFN-γ ELISA. All donors, from which the TCRs originated, carried the HLA-A subtype *02:01 and therefore data were normalized to the recognition of the LCL with subtype *02:01 (set to 100%). The x-axis indicates the HLA-A*02 subtype of the target cells used (HLA-A*02:xx). Data are presented as mean values +/- SEM from triplicate co-cultures.</p

Recognition of endogenously processed HBV peptides by TCR-transduced T cells.

<p>After retroviral transduction with respective TCRs, CD8⁺ and CD4⁺ T cells were separated by MACS. Specific lysis of HBV-replicating HepG2.2.15 hepatoma cells or T-cell activation (indicated by IFN-γ secretion) by TCR-transduced CD8⁺ (A) or CD4⁺ (B) T cells was measured. The x-axis indicates the decreasing ratio of TCR⁺ effector to target cells. Each color represents one TCR. Data are presented as mean values +/- SEM from triplicate co-cultures.</p

Sensitivity of TCR-transduced T cells.

<p>TCR-transduced CD8⁺ (A) or CD4⁺ (B) T cells expressing C18-specific, S20-specific, or S172-specific TCRs were co-cultured with T2 cells pulsed with decreasing peptide concentrations (effector to target ratio, E:T = 1:1). Each color represents one TCR. Cytotoxicity of effector cells was assessed by chromium release assay. Data are presented as mean values +/- SEM from triplicate co-cultures.</p