Gastroenterology. 2020 Jan 28. pii: S0016-5085(20)30125-6. doi: 10.1053/j.gastro.2020.01.032. [Epub ahead of print]
Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice.
Michler T1, Kosinska AD1, Festag J2, Bunse T1, Su J2, Ringelhan M3, Imhof H2, Grimm D4, Steiger K5, Mogler C5, Heikenwalder M6, Michel ML7, Guzman CA8, Milstein S9, Sepp-Lorenzino L9, Knolle P10, Protzer U11.
Author information
1
Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
2
Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany.
3
Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; Department of Internal Medicine II, University Hospital rechts der Isar, Technical University of Munich, Ismaninger Str. 22, D-81675 Munich, Germany.
4
Department of Infectious Diseases/Virology, Heidelberg University Hospital, BioQuant, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
5
Institute of Pathology, Technical University of Munich, Trogerstrasse 18, D-81675 Munich, Germany.
6
Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany.
7
Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France.
8
Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
9
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
10
Institute of Molecular Immunology, University Hospital rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany.
11
Institute of Virology, Technical University of Munich / Helmholtz Zentrum München, Trogerstrasse 30, D-81675 Munich, Germany; German Center for Infection Research (DZIF), Munich, Heidelberg and Hannover/Braunschweig partner sites, Germany. Electronic address: [email protected].
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 (HBsAg) 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 twelve 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 following 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.