Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice.
ABSTRACT: The capacity of toll-like receptor (TLR) 7/8 agonist-conjugated hepatitis B virus (HBV) proteins (HBV-Ag) to overcome established hepatitis B surface antigen (HBsAg)-specific immune tolerance was explored.A TLR7/8 agonist, CL097, was conjugated with alum-absorbed HBsAg and hepatitis B core antigen (HBcAg), as confirmed by ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). Mice from two independently generated HBV-transgenic (HBV-Tg) colonies, C57BL/6J-TgN (AlblHBV) 44Bri/J mice and C57BL/6-HBV-1.3 genome-eq mice, were immunized with CL097-conjugated HBV-Ag every 2 weeks, four times.After immunization, 8/11 (72.7%) of the AlblHBV mice and 10/13 (76.9%) of the HBV-1.3 genome-eq mice generated serum detectable antibodies against HBsAg (anti-HBs). HBsAg-specific interferon gamma (IFN-?)-producing CD4(+) and CD8(+) T-cells were detected in splenocytes from these mice. Naïve normal mice receiving splenocytes from the mice immunized with CL097-conjugated HBV-Ag generated immediate recall immune responses, e.g., the mice that received CD4(+)CD25(+)-depleted splenocytes generated anti-HBs on day 3 after HBsAg challenge while those receiving cells from sham-immunized mice did not.Immunization with CL097-conjugated HBV-Ag reversed immune tolerance in HBV-Tg mice and induced antigen-specific immune responses. TLR7/8 agonists appear to be potent adjuvants for the induction of antigen-specific Th1 responses in an immune tolerant state.
Project description:The conjugation of polysaccharides with an effective carrier protein is critical for the development of effective bacterial polysaccharide vaccines. Therefore, the identification and optimization of carrier proteins to induce an effective immune response is necessary for developing a combined vaccine. In the current study, we utilized hepatitis B virus surface antigen (HBsAg) as a novel carrier protein combined with a capsular polysaccharide molecule to develop a new pneumococcal conjugated vaccine. The specific antibodies and T cell immune response against the capsular polysaccharide and HBsAg in the mice immunized with this conjugated vaccine were evaluated. In addition, the unique gene profiles of immune cells induced by this conjugated vaccine in the immunized mice were analyzed. Our results demonstrated that the vaccine consisting of pneumonia type 33?F capsular polysaccharide (Pn33Fps) conjugated with HBsAg can induce strong specific immune responses against both antigens in vivo in immunized mice. Furthermore, the conjugated vaccine induced higher expression of genes related to the activation of immunity and higher antibody titers against Pn33Fps and HBsAg in mice than those obtained via vaccination with a single antigen. Analyses of the dynamic expression changes in immunity-related genes in mice immunized with Pn33Fps_HBs, Pn33Fps, or HBsAg indicated the potent immunogenicity of the conjugated vaccine. In addition, a pathological evaluation of the organs from immunized mice further suggested that the conjugated vaccine is safe. Together, these results indicate that a conjugated vaccine consisting of Pn33Fps with HBsAg is a novel and effective vaccine.
Project description:<h4>Background</h4>Glypican-3 (GPC3) is one of the key tissue markers that could discriminate malignant precancerous lesions from benign hepatic lesions in cirrhotic patients. We aimed to develop a GPC3 cancer vaccine to induce specific T cells to intervene in hepatocellular carcinoma (HCC) development.<h4>Methods</h4>Synthesizing mannosylated liposomes (LPMan) as vaccine delivery system, incorporating one Toll-like receptor (TLR)-7/8 agonist CL097 as adjuvant, we prepared a GPC3 nanovaccine, LPMan-GPC3/CL097. We injected 25 mg/kg diethylnitrosamine intraperitoneally to induce autochthonous HCC in HBV-transgenic mice, which persistently express hepatitis B surface antigen in hepatocytes. Starting from week 8 after diethylnitrosamine injection when malignant hepatocytes generated, we immunized the mice subcutaneously every 2 weeks 4 times with LPMan-GPC3/CL097 containing 5 µg of GPC3 plus 5 µg of CL097.<h4>Results</h4>The vaccine efficiently targeted draining lymph nodes where naïve T cells reside and enhanced the expression of molecules involved in antigen presentation in migratory dendritic cells (DCs). Antigen was professionally processed in endoplasmic reticulum-Golgi system of DCs, subsequently priming both CD4<sup>+</sup> and CD8<sup>+</sup> T cells. The LPMan-GPC3/CL097 immunization generated significantly more GPC3-specific CD4<sup>+</sup> IFN?- and CD8<sup>+</sup> IFN?-producing T cells in mice spleens and livers, which specifically eliminated GPC3-expressing tumor cells. One week after last immunization (week 15 after diethylnitrosamine), 5/5 un-immunized, 5/5 sham (LPMan-CL097) and 1/5 LPMan-GPC3/CL097-immunized mice developed HCC. By week 20 after diethylnitrosamine, significantly less HCC developed in LPMan-GPC3/CL097-immunized mice than in sham-immunized mice (<i>P</i><0.01).<h4>Conclusions</h4>LPMan-GPC3/CL097 immunization induced <i>de novo</i> generation of specific T cells against tumor-associated antigen GPC3 that could prevent HCC development in cirrhotic liver.
Project description:BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.
Project description:Development of therapeutic vaccines/strategies to control chronic hepatitis B virus (HBV) infection has been challenging because of HBV-induced tolerance. In this study, we explored strategies for breaking tolerance and restoring the immune response to the HBV surface Ag in tolerant mice. We demonstrated that immune tolerance status is attributed to the level and duration of circulating HBsAg in HBV carrier models. Removal of circulating HBsAg by a monoclonal anti-HBsAg Ab in tolerant mice could gradually reduce tolerance and reestablish B cell and CD4(+) T cell responses to subsequent Engerix-B vaccination, producing protective IgG. Furthermore, HBsAg-specific CD8(+) T cells induced by the addition of a TLR agonist resulted in clearance of HBV in both serum and liver. Thus, generation of protective immunity can be achieved by clearing extracellular viral Ag with neutralizing Abs followed by vaccination.
Project description:The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.
Project description:Hepatitis B virus (HBV) acute and chronic infections remain a major worldwide health problem. Towards developing an anti-HBV vaccine with single-dose scheme potential, we engineered infectious measles virus (MV) genomic cDNAs with a vaccine strain background and expression vector properties. Hepatitis B surface antigen (HBsAg) expression cassettes were inserted into this cDNA and three MVs expressing HBsAg at different levels generated. All vectored MVs, which secrete HBsAg as subviral particles, elicited humoral responses in MV-susceptible genetically modified mice. However, small differences in HBsAg expression elicited vastly different HBsAg antibody levels. The two vectors inducing the highest HBsAg antibody levels were inoculated into rhesus monkeys (Macaca mulatta). After challenge with a pathogenic MV strain (Davis87), control naive monkeys showed a classic measles rash and high viral loads. In contrast, all monkeys immunized with vaccine or a control nonvectored recombinant vaccine or HBsAg-expressing vectored MV remained healthy, with low or undetectable viral loads. After a single vaccine dose, only the vector expressing HBsAg at the highest levels elicited protective levels of HBsAg antibodies in two of four animals. These observations reveal an expression threshold for efficient induction of HBsAg humoral immune responses. This threshold is lower in mice than in macaques. Implications for the development of divalent vaccines based on live attenuated viruses are discussed.
Project description:Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.
Project description:Strong tolerance to hepatitis B virus (HBV) surface antigens limits the therapeutic effect of the conventional hepatitis B surface antigen (HBsAg) vaccination in both preclinical animal models and patients with chronic hepatitis B (CHB) infection. In contrast, we observed that clinical CHB patients presented less immune tolerance to the preS1 domain of HBV large surface antigen. To study whether targeting the weak tolerance of the preS1 region could improve therapy gain, we explored vaccination with the long peptide of preS1 domain for HBV virions clearance. Our study showed that this preS1-polypeptide rather than HBsAg vaccination induced robust immune responses in HBV carrier mice. The anti-preS1 rapidly cleared HBV virions in vivo and blocked HBV infection to hepatocytes in vitro. Intriguingly, vaccination of preS1-polypeptide even reduced the tolerized status of HBsAg, opening a therapeutic window for the host to respond to the HBsAg vaccine. A sequential administration of antigenically distinct preS1-polypeptide and HBsAg vaccines in HBV carrier mice could finally induce HBsAg/hepatitis B surface antibody serological conversion and clear chronic HBV infection in carrier mice. CONCLUSION:These results suggest that preS1 can function as a therapeutic vaccine for the control of CHB. (Hepatology 2017;66:1067-1082).
Project description:AIM: To investigate the immunomodulatory effects of andrographolide on both innate and adaptive immune responses. METHODS: Andrographolide (10 microg/mL in vitro or 1 mg/kg in vivo) was used to modulate LPS-induced classical activated (M1) or IL-4-induced alternative activated (M2) macrophages in vitro and humor immune response to HBsAg in vivo. Cytokine gene expression profile (M1 vs M2) was measured by real-time PCR, IL-12/IL-10 level was detected by ELISA, and surface antigen expression was evaluated by flow cytometry, whereas phosphorylation level of ERK 1/2 and AKT was determined by Western blot. The level of anti-HBs antibodies in HBsAg immunized mice was detected by ELISA, and the number of HBsAg specific IL-4-producing splenocyte was enumerated by ELISPOT. RESULTS: Andrographolide treatment in vitro attenuated either LPS or IL-4 induced macrophage activation, inhibited both M1 and M2 cytokines expression and decreased IL-12/IL-10 ratio (the ratio of M1/M2 polarization). Andrographolide down-regulated the expression of mannose receptor (CD206) in IL-4 induced macrophages and major histocompability complex/costimulatory molecules (MHC I, CD40, CD80, CD86) in LPS-induced macrophages. Correspondingly, anti-HBs antibody production and the number of IL-4-producing splenocytes were reduced by in vivo administration of andrographolide. Reduced phosphorylation levels of ERK1/2 and AKT were observed in macrophages treated with andrographolide. CONCLUSION: Andrographolide can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and Ag-specific antibody production. MAPK and PI3K signaling pathways may participate in the mechanisms of andrographolide regulating macrophage activation and polarization.
Project description:Previous studies have paid more attention to hepatitis B e antigen (HBeAg) intrauterine exposure, however the effect of solely hepatitis B surface antigen (HBsAg) exposure on the immune response of offspring against hepatitis B virus (HBV) is still unclear. In this study, we investigated whether HBsAg intrauterine exposure affected the offspring's immune response against HBV and the relevant mechanism, which is important for the prevention of HBV mother-to-infant transmission. Overall design: HBs-specific CD8+ T cells were sorted from C57BL/6 mice and C57BL/6-Tg (HBV Alb-1) Bri44 HBV transgenic mice liver, and the Smart-seq was used to compare the difference between two groups.