Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
ABSTRACT: Therapeutic vaccination against chronic hepatitis B must overcome high viral antigen load and local regulatory mechanisms that promote immune-tolerance in the liver and curtail hepatitis B virus (HBV)-specific CD8 T cell immunity. Here, we report that therapeutic heterologous HBcore-protein-prime/Modified-Vaccinia-Virus-Ankara (MVA-HBcore) boost vaccination followed by CpG-application augmented vaccine-induced HBcAg-specific CD8 T cell-function in the liver. In HBV-transgenic as well as AAV-HBV-transduced mice with persistent high-level HBV-replication, the combination of therapeutic vaccination with subsequent CpG-application was synergistic to generate more potent HBV-specific CD8 T cell immunity that improved control of hepatocytes replicating HBV.
Project description:An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4<sup>+</sup>/CD8<sup>+</sup> T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4<sup>+</sup>/CD8<sup>+</sup> T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4<sup>+</sup>/CD8<sup>+</sup> T cell level, CD4<sup>+</sup> T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4<sup>+</sup> Th and CD8<sup>+</sup> CTL responses.
Project description:Hepatitis B virus (HBV) is a global virus responsible for a universal disease burden for millions of people. Various vaccination strategies have been developed using viral vector, nucleic acid, protein, peptide, and virus-like particles (VLPs) to stimulate favorable immune responses against HBV. Given the pivotal role of specific immune responses of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in infection control, we designed a VLP-based vaccine by placing the antibody-binding fragments of HBsAg in the major immunodominant region (MIR) epitope of HBcAg to stimulate multilateral immunity. A computational approach was employed to predict and evaluate the conservation, antigenicity, allergenicity, and immunogenicity of the construct. Modeling and molecular dynamics (MD) demonstrated the folding stability of HBcAg as a carrier in inserting Myrcludex and "a" determinant of HBsAg. Regions 1-50 and 118-150 of HBsAg were considered to have the highest stability to be involved in the designed vaccine. Molecular docking revealed appropriate interactions between the B cell epitope of the designed vaccine and the antibodies. Totally, the final construct was promising for inducing humoral and cellular responses against HBV.
Project description:Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vaccination status in healthy individuals.Seventy two chronic Hepatitis B patients (CHB), 8 acute hepatitis B patients (AHB) and 80 healthy controls (HC) were tested by ELISA for IFN?- and IL2-secretion in whole blood after challenge with synthetic peptide libraries of hepatitis B core antigen (HBcAg) or hepatitis B surface antigen (HBsAg).The developed IGRA test reliably differentiated between Hepatitis B patients, vaccinees and unvaccinated healthy controls. Treatment naïve and treated CHB patients showed a weaker IFN? response to HBcAg (16 ± 5 and 35 ± 28 pg/ml, respectively) compared to the AHB group (82 ± 39 pg/ml), whereas HC remained unresponsive (6 ± 1 pg/ml). IL2 levels after HBcAg challenge were also higher in the AHB group compared to naive and treated CHB as well as HC (47 ± 21 vs. 12 ± 3, 15 ± 10 and 12 ± 9 pg/ml, respectively). HBsAg stimulation led to increased IFN? and IL2 levels in the AHB group (33 ± 12 and 22 ± 12 pg/ml) and even higher levels in HC due to a high hepatitis B vaccination rate (41 ± 10 and 167 ± 58 pg/ml). Naive and treated CHB patients developed no or only weaker IFN? or IL2 responses to HBsAg (5 ± 2 and 12 ± 7 pg/ml, for naive CHB, 12 ± 10 and 18 ± 15 pg/ml, for treated CHB). For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %.Our novel whole blood based cytokine release assay constitutes an easy and robust tool for screening HBV specific cellular immunity as alternative to flow cytometry or ELISPOT assays.
Project description:Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.
Project description:BACKGROUND: Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon ? and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators. METHODOLOGY/PRINCIPAL FINDINGS: To explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine. RESULTS: Functional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group. CONCLUSIONS/SIGNIFICANCE: For the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.
Project description:Current therapies for the hepatitis B virus (HBV), a major cause of severe liver disease, suppress viral replication but replication rebounds if therapy is withdrawn. It is widely accepted that immune activation is needed to control replication off-therapy. To specifically activate T cells crossreactive between the hepatitis B core and e antigens (HBcAg/HBeAg) in chronically infected patients, we developed a therapeutic vaccine candidate. The vaccine encompass codon-optimized HBcAg and IL-12 expressing plasmids delivered using targeted high-pressure injection combined with in vivo electroporation. One dose of the vaccine primed a B-cell-independent polyfunctional T-cell response, in wild-type, and in HBeAg-transgenic mice with an impaired ability to respond to HBc/eAg. The response peaked at 2 weeks and contracted at week 6 after vaccination. Coadministration of IL-12 improved antibody levels, and T-cell expansion and functionality. The vaccine primed T cells that, 2 weeks after a single dose, cleared hepatocytes transiently expressing HBcAg in vaccinated wild-type and HBeAg-transgenic mice. However, 4 weeks later, these functional responses were lost. Booster doses after 8-12 weeks effectively restored function and expansion of the rapidly contracting T cells. Thus, this vaccine strategy primes functional HBcAg-specific T cells in a host with dysfunctional response to HBV.
Project description:BACKGROUND:Although higher levels of hepatitis B virus (HBV) replication in HIV-HBV co-infection may relate to liver disease progression, this has not been completely elucidated. We used expression of hepatitis B core antigen (HBcAg) in liver biopsies from HIV-HBV co-infected and HBV mono-infected patients as a marker for HBV replication, and related these findings to clinical and histological parameters. METHODS:Data from 244 HBV patients were compared with 34 HIV-HBV patients. Liver biopsies were scored for inflammation, fibrosis, HBcAg, and hepatitis B surface antigen. Univariate and multivariate analyses were performed. RESULTS:HBcAg, but not hepatitis B surface antigen, staining was stronger in HIV co-infected than in HBV mono-infected. Co-infected and HBV mono-infected had similar alanine aminotransferase, inflammatory and fibrosis scores, and hepatitis B e antigen status. HBcAg staining correlated with HIV after correcting for HBV DNA and hepatitis B e antigen. CD4 counts and HIV RNA level did not correlate with intensity of HBcAg staining. HBV DNA levels were higher in HIV co-infected and correlated with HBcAg staining. CONCLUSIONS:By looking at HBcAg as a reflection of HBV replication in HIV-HBV co-infected with controlled HIV, our findings suggest that these patients may have subtle immune function defects, which could lead to adverse liver disease outcomes.
Project description:Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-?/? receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-?) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-? in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-? production induced by HBcAg. These results provide evidences for TNF-? mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.
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:Although several evidences suggesting the vital roles that innate immunity plays in the persistence and elimination of chronic hepatitis B virus (CHB) infection, the exact mechanism is still complicated. Here, we successfully polarized monocytes derived from healthy human peripheral blood mononuclear cells (PBMCs) into M1/M2 macrophages and detected the effects of hepatitis B core antigen (HBcAg) on the polarization and function of macrophages via the Toll-like receptor (TLR) 2 signaling pathway. The results showed that HBcAg had a negligible impact on M1 polarization, while it effectively impaired M2 polarization and promoted the production of pro-inflammatory cytokines such as IL-6 and TNF-?. Additionally, HBcAg treatment increased TLR2 expression on M2 macrophages and TLR2 blockade abolished the effects of HBcAg on the impaired phenotype and pro-inflammatory cytokine productions of M2 macrophages. Signaling pathway analysis revealed that the nuclear factor ?B (NF-?B) pathway, the downstream of TLR2, was upregulated upon HBcAg treatment in both M1 and M2 macrophages. Furthermore, a CD8+ T-macrophage coculture system implied that compared with PBS stimulation, HBcAg-stimulated M2 macrophages regained their ability to activate CD8+ T cells with higher secretion of IFN-?. Finally, we found impaired expression of M2-related molecules and increased levels of pro-inflammation cytokines in M2 macrophages from CHB patients upon HBcAg stimulation. In conclusion, these results imply a favorable role of HBcAg in the establishment of a pro-inflammatory microenvironment by macrophages, which may suggest a potential therapeutic strategy of HBcAg-induced macrophage activation in CHB infection.