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HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

ABSTRACT: BACKGROUND:Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. METHODS:We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. RESULTS:HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. CONCLUSIONS:HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV.

SUBMITTER: Kruse RL

PROVIDER: S-EPMC6038120 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

Kruse Robert L RL Shum Thomas T Tashiro Haruko H Barzi Mercedes M Yi Zhongzhen Z Whitten-Bauer Christina C Legras Xavier X Bissig-Choisat Beatrice B Garaigorta Urtzi U Gottschalk Stephen S Bissig Karl-Dimiter KD

Cytotherapy 20180406 5

<h4>Background</h4>Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection.<h4>Methods</h4>We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human li ...[more]

PMID: 29631939

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Project description:BackgroundClinically, some patients whose HBsAg becomes negative owing to antiviral therapy or spontaneously still show a low level of HBV DNA persistence in serum. T-lymphocyte subsets, cytokine levels and HBV S gene sequences were analyzed in this study.MethodsA total of 52 HBsAg-negative and HBV DNA-positive patients(HBsAg-/HBV DNA+ patients), 52 persistently HBsAg-positive patients(HBsAg+/HBV DNA+ patients) and 16 healthy people were evaluated. T-lymphocyte subsets of these patients were detected by flow cytometry, serum cytokines and chemokines were detected by the Luminex technique, and the HBV S region was evaluated by Sanger sequencing. T%, T-lymphocyte, CD8+ and CD4+T lymphocyte were lower in the HBsAg-negative group than in the HC group. Compared with the HBsAg-positive group, the HBsAg-negative group had lower levels in T lymphocyte %, CD8+T lymphocyte %, CD8+T lymphocyte and CD4/CD8. These difference were statistically significant (P<0.05). Serum IFN-γ, IFN-α and FLT-3L levels were significantly higher in the HBsAg-negative group than in the HBsAg-positive group (P<0.05). However, levels of many cytokines related to inflammation (i.e., IL-6, IL-8, IL10, IL-12, IL-17A) were lower in the HBsAg-negative group. Fifty-two HBsAg-negative samples were sequenced, revealing high-frequency amino acid substitution sites in the HBV S protein, including immune escape mutations (i.e., Y100C, S114T, C124Y, P127L, G130R, T131N, M133T, C137S, G145A) and TMD region substitutions (i.e., E2K/R/D, G7D/R, G10D, A17R, F20L/S, L21V, L22V).ConclusionsAccording to the results of T-lymphocyte subsets and serum cytokines, it can be deduced that the cellular immune function of HBsAg-negative patients is superior to that of HBsAg-positive patients, with attenuation of liver inflammation. HBsAg-negative patients may show a variety of mutations and amino acid replacement sites at high frequency in the HBV S region, and these mutations may lead to undetectable HBsAg, HBsAg antigenic changes or secretion inhibition.

Project description:As alternative indexes of hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) transcriptional activity, hepatitis B surface antigen (HBsAg), hepatitis B core-related antigen (HBcrAg), and peripheral blood RNA known as pgRNA, have been advocated as novel serum markers for prediction of prognosis and treatment response in chronic hepatitis B (CHB). Since the availability of commercial quantitative assays of HBsAg in 2011, HBsAg has been widely used for predicting treatment response of patients with CHB. Patients who received interferon therapy have shown a sharper reduction of HBsAg level than those who received nucleoside drug (NAs) therapy. Upon peginterferon treatment, sustained responders have presented a larger reduction of HBsAg level than the non-responders. An absence of HBsAg decline, together with < 2log reduction in HBV DNA at week 12, can serve as a stopping rule in HBsAg-negative patients infected with genotype D HBV. A sharp reduction of HBsAg titer in the NAs therapy is a predictor of HBsAg clearance in long-term treatment. HBcrAg, which consists of three species of related proteins sharing an identical 149 amino acid sequence, including HbcAg, hepatitis B e antigen (HBeAg), and a truncated 22-kDa precore protein, is still detectable in situations where serum HBV DNA levels become undetectable or HBsAg loss is achieved. Therefore, HBcrAg remains a measurable serum marker to correlate with cccDNA in this situation. The decline in HBcrAg has been observed with NAs therapy and the pattern of decline might provide prognostic information on the risk of HBV post-treatment reactivation. Peripheral blood RNA, which is known as pgRNA, directly derives from cccDNA and reflects intrahepatic cccDNA level. Quantitative pgRNA has been suggested to be helpful in CHB management. However, commercial quantitative assays are lacking. Additionally, the use of simultaneous and continuous clearance of HBV RNA and HBV DNA in serum has been suggested to be a safe stopping rule of NAs therapy for patients with CHB. However, clinical studies of large sample sizes are needed to prove the feasibility and significance of using serum HBV RNA as the assessment standard of antiviral therapy in CHB and the safety of the stopping rule in clinics.

Project description:Chronic hepatitis B virus (HBV) infections remain a health burden affecting ~250 million people worldwide. Thus far, available interferon-alpha (IFNα)-based therapies have shown unsatisfactory cure rates, and alternative therapeutic molecules are still required. However, their development has been hampered because accessible cell models supporting relevant HBV replication and appropriate antiviral activity are lacking. Strategies that reverse epigenetic alterations offer a unique opportunity for cell reprogramming, which is valuable for restoring altered cellular functions in human cell lines. This work aimed to investigate the feasibility of converting HepG2 cells that stably overexpress the HBV entry receptor (sodium/taurocholate cotransporting polypeptide, NTCP) toward IFNα-responsive cells using epigenetic reprogramming. Herein, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine restored the anti-HBV action of IFNα in epigenetically reprogrammed HepG2-NTCP-C4 cells, named REP-HepG2-NTCP cells. Thus, a significant inhibition in HBV DNA levels was measured in REP-HepG2-NTCP cells after IFNα treatment. This inhibitory effect was associated with the enhancement of IFNα-mediated induction of critical interferon-stimulated genes (ISGs), which was limited in non-reprogrammed cells. In particular, our data indicated that re-expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) and interferon regulatory factor 9 (IRF9) was the result of an epigenetically driven unmasking of these genes in reprogrammed cells. At last, we evaluated the therapeutic potential of the IFN analog CDM-3008 in REP-HepG2-NTCP cells and demonstrated the efficiency of this chemical compound in triggering ISG induction and HBV inhibition. In summary, this study shows that epigenetic reprogramming promotes the IFNα response in HBV-infected cells and is potentially attractive for cell-based experimental screening of IFN-like compounds.

Dataset Information

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

Publications

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

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