Thomas Michler1, Anna D Kosinska1, Julia Festag2, Till Bunse1, Jinpeng Su2, Marc Ringelhan3, Hortenzia Imhof2, Dirk Grimm4, Katja Steiger5, Carolin Mogler5, Mathias Heikenwalder6, Marie-Louise Michel7, Carlos A Guzman8, Stuart Milstein9, Laura Sepp-Lorenzino9, Percy Knolle10, Ulrike Protzer11. 1. Institute of Virology, Technical University of Münich, Helmholtz Zentrum München, Münich, Germany; German Center for Infection Research, Münich, Heidelberg, Germany. 2. Institute of Virology, Technical University of Münich, Helmholtz Zentrum München, Münich, Germany. 3. Institute of Virology, Technical University of Münich, Helmholtz Zentrum München, Münich, Germany; Department of Internal Medicine II, University Hospital rechts der Isar, Technical University of Munich, Münich, Germany. 4. German Center for Infection Research, Münich, Heidelberg, Germany; Department of Infectious Diseases/Virology, Heidelberg University Hospital, BioQuant, Heidelberg, Germany. 5. Institute of Pathology, Technical University of Munich, Münich, Germany. 6. Division of Chronic Inflammation and Cancer, German Cancer Research Center, Heidelberg, Germany. 7. Institut Pasteur, Paris, France. 8. German Center for Infection Research, Münich, Heidelberg, Germany; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. 9. Alnylam Pharmaceuticals, Cambridge, Massachusetts. 10. German Center for Infection Research, Münich, Heidelberg, Germany; Institute of Molecular Immunology, University Hospital rechts der Isar, Technical University of Munich, Münich, Germany. 11. Institute of Virology, Technical University of Münich, Helmholtz Zentrum München, Münich, Germany; German Center for Infection Research, Münich, Heidelberg, Germany. Electronic address: protzer@tum.de.
Abstract
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. 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.
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. 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.
Authors: Josep M Llovet; Florian Castet; Mathias Heikenwalder; Mala K Maini; Vincenzo Mazzaferro; David J Pinato; Eli Pikarsky; Andrew X Zhu; Richard S Finn Journal: Nat Rev Clin Oncol Date: 2021-11-11 Impact factor: 65.011
Authors: Peter Jianrui Liu; James M Harris; Emanuele Marchi; Valentina D'Arienzo; Thomas Michler; Peter A C Wing; Andrea Magri; Ana Maria Ortega-Prieto; Maarten van de Klundert; Jochen Wettengel; David Durantel; Marcus Dorner; Paul Klenerman; Ulrike Protzer; Efstathios S Giotis; Jane A McKeating Journal: Sci Rep Date: 2020-08-24 Impact factor: 4.379
Authors: Valeria Fumagalli; Pietro Di Lucia; Valentina Venzin; Elisa B Bono; Robert Jordan; Christian R Frey; William Delaney; Francis V Chisari; Luca G Guidotti; Matteo Iannacone Journal: J Exp Med Date: 2020-11-02 Impact factor: 14.307