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Mice infected with a recombinant adeno-associated virus carrying a replication-competent hepatitis B virus genome (rAAV-HBV) via the intravenous route establish a persistent HBV replication in hepatocytes and develop immune tolerance. They serve as models to evaluate antiviral immunity and to assess potential therapeutic approaches for chronic HBV infection. Combining selected HBV variants and different mouse genotypes allows for addressing a broad spectrum of research questions. This chapter describes the basic principles of the rAAV-HBV mouse model, rAAV-HBV production and purification methods, and finally, the in vivo application.
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Dependovirus , Modelos Animales de Enfermedad , Vectores Genéticos , Virus de la Hepatitis B , Replicación Viral , Animales , Dependovirus/genética , Dependovirus/aislamiento & purificación , Virus de la Hepatitis B/genética , Ratones , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Humanos , Hepatitis B Crónica/virología , Hepatitis B Crónica/inmunología , Hepatitis B/virología , Hepatitis B/inmunologíaRESUMEN
Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
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Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de LinfocitosRESUMEN
To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.
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Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Humanos , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Animales , Ratones , Antígenos de Superficie de la Hepatitis B/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos Monoclonales/inmunología , Inmunoterapia Adoptiva , Hepatitis B/inmunología , Hepatitis B/virología , Anticuerpos ampliamente neutralizantes/inmunología , Linfocitos B/inmunología , Linfocitos T/inmunologíaRESUMEN
Background & Aims: Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E. coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. Methods: bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. Results: While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Conclusion: Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic in vivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Impact and implications: Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.
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Here, we investigate the potential of CD70 co-expression during viral vector boost vaccination to improve an antigen-specific T cell response. To determine the chance of activating antigen-specific T cells by CD70, we used the HBV core antigen as a model antigen in a heterologous protein-prime, Modified Vaccinia virus Ankara (MVA) boost vaccination scheme. Both the HBV core and a CD70 expression cassette were co-expressed upon delivery by an MVA vector under the same promoter linked by a P2A site. To compare immunogenicity with and without CD70 co-expression, HBV-naïve, C57BL/6 (wt) mice and HBV-transgenic mice were prime-vaccinated using recombinant HBV core antigen followed by the MVA vector boost. Co-expression of CD70 increased the number of vaccine-induced HBV core-specific CD8 T cells by >2-fold and improved their effector functions in HBV-naïve mice. In vaccinated HBV1.3tg mice, the number and functionality of HBV core-specific CD8 T cells was slightly increased upon CD70 co-expression in low-viremic, but not in high-viremic animals. CD70 co-expression did not impact liver damage as indicated by ALT levels in the serum, but increased the number of vaccine-induced, proliferative T cell clusters in the liver. Overall, this study indicates that orchestrated co-expression of CD70 and a vaccine antigen may be an interesting and safe means of enhancing antigen-specific CD8 T cell responses using vector-based vaccines, although in our study it was not sufficient to break immune tolerance.
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BACKGROUND & AIMS: We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. METHODS: Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. RESULTS: Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2 T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. CONCLUSIONS: Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. IMPACT AND IMPLICATIONS: Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.
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Vacunas contra Hepatitis B , Hepatitis B Crónica , Ratones , Humanos , Animales , Virus de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Antígenos del Núcleo de la Hepatitis B , Linfocitos T CD4-Positivos , Inmunización , Vacunación/métodos , Anticuerpos contra la Hepatitis B , Linfocitos T CD8-positivos , Ratones Transgénicos , Adyuvantes Inmunológicos , AntiviralesRESUMEN
Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.
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In chronic hepatitis B virus (HBV) infection, virus-specific T cells are scarce and partially dysfunctional. Therapeutic vaccination is a promising strategy to induce and activate new virus-specific T cells. In long-term or high-level HBV carriers, however, therapeutic vaccination by itself may not suffice to cure HBV. One reason is the impairment of antiviral T cells by immune checkpoints. In this study, we used small-interfering RNA (siRNA) in combination with a heterologous prime-boost therapeutic vaccination scheme (TherVacB) to interfere with a major immune checkpoint, the interaction of programmed death protein-1 (PD-1) and its ligand (PDL-1). In mice persistently replicating HBV after infection with an adeno-associated virus harboring the HBV genome, siRNA targeting PD-L1 resulted in a higher functionality of HBV-specific CD8+ T cells after therapeutic vaccination, and allowed for a more sustained antiviral effect and control of HBV in peripheral blood and in the liver. The antiviral effect was more pronounced if PD-L1 was down-regulated during prime than during boost vaccination. Thus, targeting PD-L1 using siRNA is a promising approach to enhance the efficacy of therapeutic vaccination and finally cure HBV.
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Hepatitis B Crónica , Animales , Antivirales , Antígeno B7-H1/genética , Linfocitos T CD8-positivos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/terapia , Ratones , ARN Interferente Pequeño/genética , VacunaciónRESUMEN
T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo, induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity.
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Traslado Adoptivo , Caspasa 9/biosíntesis , Antígenos de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , Linfocitos T/trasplante , Traslado Adoptivo/efectos adversos , Animales , Caspasa 9/genética , Muerte Celular , Línea Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática , Femenino , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/virología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Simplexvirus/enzimología , Simplexvirus/genética , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/patología , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transducción GenéticaRESUMEN
During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(-) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(-) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with TherVacB, a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The TherVacB immunization of HBeAg(+) and HBeAg(-) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(-) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.
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The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5' but not in the 3' core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.
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ADN Circular/genética , Dependovirus/genética , Genoma Viral , Virus de la Hepatitis B/genética , Hígado/virología , Recombinación Genética , Replicación Viral/genética , Animales , Replicación del ADN , ADN Viral/genética , Vectores Genéticos , Genotipo , Células Hep G2 , Virus de la Hepatitis B/clasificación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. METHODS: We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. RESULTS: In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. CONCLUSIONS: In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.
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Antígenos de Superficie de la Hepatitis B/genética , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Linfocitos B/inmunología , Portador Sano/inmunología , Portador Sano/virología , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/virología , Humanos , Inmunización Secundaria , Inmunogenicidad Vacunal , Masculino , Ratones , Linfocitos T Citotóxicos/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
BACKGROUND: Chronic hepatitis B develops more frequently in countries with high prevalence of helminth infections. The crosstalk between these 2 major liver-residing pathogens, Schistosoma mansoni and hepatitis B virus (HBV), is barely understood. METHODS: We used state-of-the-art models for both acute and chronic HBV infection to study the pathogen-crosstalk during the different immune phases of schistosome infection. RESULTS: Although liver pathology caused by schistosome infection was not affected by either acute or chronic HBV infection, S mansoni infection influenced HBV infection outcomes in a phase-dependent manner. Interferon (IFN)-γ secreting, HBV- and schistosome-specific CD8 T cells acted in synergy to reduce HBV-induced pathology during the TH1 phase and chronic phase of schistosomiasis. Consequently, HBV was completely rescued in IFN-γ-deficient or in TH2 phase coinfected mice demonstrating the key role of this cytokine. It is interesting to note that secondary helminth infection on the basis of persistent (chronic) HBV infection increased HBV-specific T-cell frequency and resulted in suppression of virus replication but failed to fully restore T-cell function and eliminate HBV. CONCLUSIONS: Thus, schistosome-induced IFN-γ had a prominent antiviral effect that outcompeted immunosuppressive effects of TH2 cytokines, whereas HBV coinfection did not alter schistosome pathogenicity.
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Linfocitos T CD8-positivos/inmunología , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/inmunología , Esquistosomiasis mansoni/complicaciones , Esquistosomiasis mansoni/inmunología , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Virus de la Hepatitis B/fisiología , Interferón gamma/inmunología , Hígado/parasitología , Hígado/patología , Hígado/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Recuento de Huevos de Parásitos , Schistosoma mansoni , Células Th2/inmunología , Replicación ViralRESUMEN
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.
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Adyuvantes Inmunológicos/uso terapéutico , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/prevención & control , Oligodesoxirribonucleótidos/uso terapéutico , Vacunación/métodos , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Femenino , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/uso terapéutico , Hepatitis B Crónica/inmunología , Hígado/inmunología , Masculino , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Vacunas de ADN , Vacunas Virales/inmunologíaRESUMEN
Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-B/inmunología , Hepatitis D/inmunología , Virus de la Hepatitis Delta/inmunología , Antígenos de Hepatitis delta/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Linfocitos T CD8-positivos/metabolismo , Epítopos de Linfocito T/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Hepatitis D/genética , Hepatitis D/virología , Virus de la Hepatitis Delta/genética , Antígenos de Hepatitis delta/metabolismo , Humanos , Mutación , Homología de SecuenciaRESUMEN
A therapeutic vaccine is meant to activate the patient's immune system to fight and finally control or ideally eliminate an already established infectious pathogen. Whereas the success of prophylactic vaccination is based on rapid antibody-mediated neutralization of an invading pathogen, control and elimination of persistent viruses such as hepatitis, herpes or papilloma viruses requires multi-specific and polyfunctional effector T cell responses. These are ideally directed against continuously expressed viral antigens to keep the pathogen in check. Activation of a humoral immune response in order to lower viral antigen load and to limit virus spread, however, confers an additional benefit. Therapeutic vaccines are under development for a number of chronic infections and require an intelligent vaccine design. Hepatitis B virus (HBV) infection may serve as a prime example since a spontaneous, immune-mediated recovery of chronic hepatitis B and an elimination of the virus is possible even if it is observed only in very rare cases. In this review, we summarize the current knowledge and potential improvements of therapeutic vaccines for chronic hepatitis B.
Asunto(s)
Vacunas contra Hepatitis B/uso terapéutico , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , Inmunoterapia/métodos , Descubrimiento de Drogas/tendencias , HumanosRESUMEN
Hepatitis B virus (HBV) infection shows significant gender-related differences in pathogenesis, disease progression, and development of hepatocellular carcinoma. The gender-associated differences in HBV replication and viral protein levels may be associated with distinct HBV-specific immune responses in the host. In the present study, we examined the impact of gender on HBV-specific immune responses in two different mouse models representing transient and persistent hepadnaviral infection; hydrodynamic injection with the HBV genome mimicked acute HBV infection, whereas the efficacy of therapeutic vaccination was studied in the woodchuck hepatitis virus transgenic mouse model. Consistent with previous reports, significantly higher HBV DNA and protein levels were detected in male compared to female mice. Although hydrodynamic injection with the HBV genome resulted in similar numbers of intrahepatic HBV-specific cluster of differentiation 8-positive (CD8+ ) T cells, their functionality was significantly reduced in males and correlated with higher numbers of intrahepatic regulatory T cells (Tregs). Similar effects were observed in woodchuck hepatitis virus transgenic mice immunized with a DNA prime-recombinant adenovirus boost vaccination protocol. Male mice showed functionally suppressed woodchuck hepatitis virus-specific CD8+ T-cell responses in the liver and significantly higher numbers of intrahepatic Tregs compared to females. Blockade of Treg responses in male mice led to augmented effector functions of specific CD8+ T cells and subsequently improved virus control in both models of transient and persistent hepadnaviral infection. CONCLUSION: The functionality of virus-specific CD8+ T cells in male mice was suppressed by intrahepatic Tregs and inversely correlated with levels of hepadnaviral DNA and viral protein; the induction of intrahepatic Tregs by viral replication and/or protein levels may explain the gender-related differences in the outcomes of HBV infection and limit the success of immunotherapeutic strategies in male patients. (Hepatology 2017;66:69-83).
Asunto(s)
Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Inmunidad Celular/fisiología , Linfocitos T Reguladores/inmunología , Análisis de Varianza , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Hepatitis B/prevención & control , Vacunas contra Hepatitis B/administración & dosificación , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Distribución Aleatoria , Factores de Riesgo , Factores Sexuales , Estadísticas no Paramétricas , Vacunación/métodosRESUMEN
BACKGROUND: Therapeutic vaccination is a novel treatment approach for chronic hepatitis B, but only had limited success so far. We hypothesized that optimized vaccination schemes have increased immunogenicity, and aimed at increasing therapeutic hepatitis B vaccine efficacy. METHODS: Modified Vaccinia virus Ankara (MVA) expressing hepatitis B virus (HBV) antigens was used to boost protein-prime vaccinations in wildtype and HBV-transgenic (HBVtg) mice. RESULTS: Protein-prime/MVA-boost vaccination was able to overcome HBV-specific tolerance in HBVtg mice with low and medium but not with high antigenemia. HBV-specific antibody titers, CD8+ T-cell frequencies and polyfunctionality inversely correlated with HBV antigen levels. However, optimization of the adjuvant formulation, increasing the level of antigen expression and utilization of HBsAg of heterologous subtype induced HBV-specific CD8+ and CD4+ T-cells and neutralizing antibodies even in high-antigenemic HBVtg mice. CONCLUSIONS: Our results indicate that high HBV antigen levels limit the immunological responsiveness to therapeutic vaccination but optimization of the vaccine formulation can overcome tolerance even in the presence of high antigenemia. These findings have important implications for the development of future therapeutic hepatitis B vaccination strategies and potentially also for the stratification of chronic hepatitis B patients for therapeutic vaccination.
Asunto(s)
Vacunas contra Hepatitis B/inmunología , Hepatitis B/prevención & control , Tolerancia Inmunológica , Virus Vaccinia , Animales , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Anticuerpos contra la Hepatitis B/sangre , Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos e de la Hepatitis B/sangre , Inmunización Secundaria , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de NeutralizaciónRESUMEN
The woodchuck hepatitis virus (WHV) and its host, the eastern woodchuck, is a very valuable model system for hepatitis B virus infection. Many aspects of WHV replication and pathogenesis resemble acute and chronic hepatitis B infection in patients. Since the establishment of immunological tools, woodchucks were used to develop new therapeutic vaccines and immunomodulatory approaches to treat chronic hepadnaviral infections. Combination therapy of nucleos(t)ide analogs, with prime-boost vaccination and triple therapy, including immunomodulatory strategies by blocking the interaction of the programmed death-1 (PD-1) receptor with its ligand inducing a potent T-cell response in chronic WHV carrier woodchucks, suppression of viral replication, and complete elimination of the virus in 30% of the animals. Both strategies may be used for future therapies in patients with chronic hepatitis B.