Mouse Model of Hepatitis B Virus Carrier
Total Page：16
File Type：pdf, Size：1020Kb
The Journal of Immunology IL-12–Based Vaccination Therapy Reverses Liver-Induced Systemic Tolerance in a Mouse Model of Hepatitis B Virus Carrier Zhutian Zeng,* Xiaohui Kong,* Fenglei Li,* Haiming Wei,*,† Rui Sun,*,† and Zhigang Tian*,† Liver-induced systemic immune tolerance that occurs during chronic hepadnavirus infection is the biggest obstacle for effective viral clearance. Immunotherapeutic reversal of this tolerance is a promising strategy in the clinic but remains to be explored. In this study, using a hepatitis B virus (HBV)-carrier mouse model, we report that IL-12–based vaccination therapy can efficiently reverse systemic tolerance toward HBV. HBV-carrier mice lost responsiveness to hepatitis B surface Ag (HBsAg) vaccination, and IL-12 alone could not reverse this liver-induced immune tolerance. However, after IL-12–based vaccination therapy, the majority of treated mice became HBsAg2 in serum; hepatitis B core Ag was also undetectable in hepatocytes. HBV clearance was dependent on HBsAg vaccine-induced anti-HBV immunity. Further results showed that IL-12–based vaccination therapy strongly enhanced hepatic HBV-specific CD8+ T cell responses, including proliferation and IFN-g secretion. Systemic HBV-specific CD4+ T cell responses were also restored in HBV-carrier mice, leading to the arousal of HBsAg-specific follicular Th–germinal center B cell responses and anti–hepatitis B surface Ag Ab production. Recovery of HBsAg-specific responses also correlated with both reduced CD4+Foxp3+ regulatory T cell frequency and an enhanced capacity of effector T cells to overcome inhibition by regulatory T cells. In conclusion, IL-12–based vaccination therapy may reverse liver-induced immune tolerance toward HBV by restoring systemic HBV-specific CD4+ T cell responses, eliciting robust hepatic HBV-specific CD8+ T cell responses, and facilitating the generation of HBsAg-specific humoral immunity; thus, this therapy may become a viable approach to treating patients with chronic hepatitis B. The Journal of Immunology, 2013, 191: 4184–4193. he liver favors induction of immune tolerance rather than Therapeutic vaccination against HBV is a promising approach immunity during chronic infection with hepatotropic patho- to reverse HBV tolerance in chronically infected individuals (6, 7). T gens (1), such as hepatitis B virus (HBV). HBV infection Previous animal studies revealed that immunization with a hepa- by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. is one of the most serious health concerns worldwide, because titis B surface Ag (HBsAg) vaccine resulted in decreased HBV chronically infected individuals are predisposed to development of titers and the generation of anti-HBs in HBV-transgenic (tg) mice severe liver cirrhosis and hepatocellular carcinoma (2, 3). Lifelong (8, 9). However, clinical studies using these strategies revealed viral persistence in chronic hepatitis B (CHB) carriers is due to poor therapeutic effects in CHB patients in an immunotolerant liver-induced immune tolerance toward HBV, which is character- phase (10, 11). The divergence in therapeutic outcome may be ized by defective HBV-specific T cell responses and undetectable due, in part, to differences in the type of tolerance induced be- anti–hepatitis B surface Ag Ab (anti-HBs) levels (4). Reversing this tween HBV-tg mice and CHB patients (12, 13). Therefore, a immune tolerance will therefore be a critical step toward completely mouse model that more accurately mimics HBV-induced “ac- eliminating HBV from the host (5, 6). quired” tolerance during CHB infection could aid in developing http://classic.jimmunol.org immunotherapeutic strategies against HBV. Recently, an immu- nocompetent HBV-carrier mouse model was established using *Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China; and †Hefei National Laboratory hepatic gene transfer of an HBV-containing plasmid by hydro- for Physical Sciences at Microscale, Hefei, Anhui 230027, China dynamic injection. Because these mice are completely unrespon- Received for publication December 19, 2012. Accepted for publication August 11, sive to peripheral HBsAg vaccination because of liver-induced 2013. systemic tolerance, they provide a better mouse model with which Downloaded from This work was supported by the Ministry of Science and Technology of China of to study liver tolerance during HBV persistence (12). the People’s Republic of China (973 Basic Science Project 2012CB519004, The transient and insufficient effects of vaccine therapy alone in 2009CB522403), the National Natural Science Foundation of China (91029303, 31021061), and National Science and Technology Major Projects (2012ZX10002- clinical studies also suggest the need for combination therapy (5, 014, 2012ZX10002006). 6). On one hand, combining HBsAg vaccine with an immunosti- Address correspondence and reprint requests to Dr. Zhigang Tian, School of Life mulator, such as CpG, exhibited improved efficacy to break HBV Sciences, University of Science and Technology of China, 443 Huang-Shan Road, tolerance via Th1-biased mechanisms (14). On the other hand, Hefei, Anhui 230027, China. E-mail address: [email protected] combining HBsAg vaccine with an antiviral agent, including lami- The online version of this article contains supplemental material. vudine, was favored in other trials, because HBV-specific T cell Abbreviations used in this article: alum, aluminum hydroxide; anti-HBs, anti–hepatitis B surface Ag Ab; CHB, chronic hepatitis B; dLN, draining lymph node; GC, germinal hyporesponsiveness during CHB infection always correlated with center; HBcAg, hepatitis B core protein; HBeAg, hepatitis B e Ag; HBsAg, hepatitis B high viral load (15). However, neither approach was completely surface Ag; HBV, hepatitis B virus; HBV-tg, HBV-transgenic; SI, stimulation index; effective in the clinic, suggesting the possibility that an agent pos- TdR, thymidine; Treg, regulatory T cell. sessing both immunostimulatory and antiviral effects could optimize Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 vaccination therapy in combination with a vaccine. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203449 The Journal of Immunology 4185 IL-12 is a typical proinflammatory cytokine that plays a critical (University of Science and Technology of China). All mice were main- role in host defense against pathogens, including HBV (16). Im- tained under specific pathogen-free conditions and used in accordance with paired IL-12 secretion by dendritic cells is implicated in con- the Guide for the Care and Use of Laboratory Animals. tributing to the chronicity of HBV infection (17). Importantly, IL- HBV-carrier mouse model 12 can also inhibit HBV replication in HBV-tg mice by inducing The pAAV-HBV1.2 plasmid, containing the full-length HBV DNA, was IFN-g production (18). However, the antiviral effect of IL-12 kindly provided by Dr. Ding-Shinn Chen (National Taiwan University alone does not appear to be more effective than other traditional College of Medicine, Taipei, Taiwan). To establish the HBV-carrier mouse drugs in clinical trials (19). In this study, we used an HBV-carrier model, we injected C57BL/6 mice (5-wk-old male mice) with 5 mg pAAV- mouse model to study the effect of IL-12 on overcoming liver- HBV1.2 plasmid using a hydrodynamic tail-vein injection approach. Six induced systemic tolerance. Although neither IL-12 nor HBV weeks later, mice with serum HBsAg levels of at least 200 ng/ml were considered to be HBV-carrier mice. vaccine alone could efficiently eliminate HBV, a combination treat- ment using both HBsAg vaccine and IL-12 led to effective HBV IL-12 treatment and immunization clearance. HBV-carrier mice were screened out 6 wk after HBV plasmid injection and s.c. injected with 100 ng IL-12 (in 200 ml total volume of PBS; Peprotech, Materials and Methods Rocky Hill, NJ) daily for 3 consecutive days, followed by s.c. immuni- Mice zation with 2 mg recombinant HBsAg vaccine (adw subtype; Biokangtai Company, Shenzhen, China) in the presence of 100 ng IL-12 as a coad- Five-week-old male C57BL/6 mice were purchased from SLAC Laboratory juvant. This treatment regimen was repeated weekly for 3 wk. In some Animal Corporation (Shanghai, China). HBV-tg mice were purchased from experiments, HBsAg vaccine was replaced by recombinant HBsAg protein the Infectious Disease Center of the No. 458 Hospital (Guangzhou, China). (HyTest, Turku, Finland) emulsified in aluminum hydroxide (alum) (Imject 2 2 Rag1 / mice were purchased from the Model Animal Research Center Alum, Pierce, IL). Blood samples were obtained weekly by tail bleeding, 2 2 (Nanjing, China). CD4 / mice were a kind gift from Dr. Zhexiong Lian and sera were kept at 220˚C until use. by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from FIGURE 1. IL-12–based vaccination therapy efficiently eliminated HBV in HBV-carrier mice. (A) HBV-carrier mice were screened out 6 wk after HBV plasmid injection and were treated with IL-12–based vaccination therapy (as shown), HBsAg vaccination alone, or no treatment. (B) HBsAg levels were monitored in PBS+HBsAg vaccine–treated, IL-12+HBsAg vaccine–treated, and untreated control mice. Data represent the pooled results from three separate experiments (each point represents one mouse). (C) The ratio of HBsAg-seropositive mice was analyzed. Mice exhibiting HBsAg levels ,2 ng/ml were considered HBsAg2.(D) Serum HBeAg levels and (E) HBV DNA copies were measured in each group 3 wk after treatment initiation. The cutoff value was 1 national clinical unit per milliliter (NCU/ml). (F) Immunohistochemical staining of HBcAg in liver sections from HBV-carrier mice treated with HBsAg vaccination alone or IL-12–based vaccination therapy. Micrographs (original magnification 3200) are representative liver sections from each group at week 4 after treatment initiation. A liver section from an HBV-carrier mouse that was screened out 6 wk after HBV injection and before treatment initiation was used as the control. (G) Serum ALT levels were monitored during treatment.