TY  - JOUR
AB  - Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.
AU  - Michler, T.
AU  - Zillinger, J.
AU  - Hagen, P.
AU  - Cheng, F.
AU  - Festag, J.
AU  - Kosinska, A.
AU  - Protzer, U.
C1  - 70595
C2  - 55589
TI  - The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice.
JO  - Antiviral Res.
VL  - 226
PY  - 2024
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Bispecific antibodies (bsAbs) are engineered immunoglobulins that combine two different antigen-binding sites in one molecule. BsAbs can be divided into two molecular formats: IgG-like and non-IgG-like antibodies. Structural elements of each format have implications for engaging the immune system. T cell engager antibodies (TCEs) are bsAbs designed to engage T cells with target cells. TCEs can be applied not only in cancer but also in infectious disease therapy to activate T-cell responses. In this review, we focus on current literature on the design and use of bsAbs as an innovative strategy to enhance adaptive antiviral immune responses. We summarized the novel T cell-related immunotherapies with a focus on TCEs, that are developed for the treatment of chronic hepatitis B. Chronic infection with the hepatitis B virus (HBV) had a death toll of 1.1 million humans in 2022, mainly due to liver cirrhosis and hepatocellular carcinoma developing in the more than 250 million humans chronically infected. A curative treatment approach for chronic hepatitis B is lacking. Combining antiviral therapy with immune therapies activating T-cell responses is regarded as the most promising therapeutic approach to curing HBV and preventing the sequelae of chronic infection. Attracting functionally intact T cells that are not HBV-specific and, therefore, have not yet been exposed to regulatory mechanisms and activating those at the target site in the liver is a very interesting therapeutic approach that could be achieved by TCEs. Thus, TCEs redirecting T cells toward HBV-positive cells represent a promising strategy for treating chronic hepatitis B and HBV-associated hepatocellular carcinoma.
AU  - Xie, Z.
AU  - Protzer, U.
C1  - 71385
C2  - 56088
TI  - Activating adaptive immunity by bispecific, T-cell engager antibodies bridging infected and immune-effector cells is a promising novel therapy for chronic hepatitis B.
JO  - Antiviral Res.
VL  - 229
PY  - 2024
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - 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.
AU  - Ko, C.
AU  - Su, J.
AU  - Festag, J.
AU  - Bester, R.
AU  - Kosinska, A.
AU  - Protzer, U.
C1  - 62553
C2  - 50909
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
SP  - 05140
TI  - Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors.
JO  - Antiviral Res.
VL  - 194
PB  - Elsevier
PY  - 2021
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of Fc-receptor activation and antibody-dependent cellular cytotoxicity. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 alpha, beta and delta variants of concern. Importantly, these variants of concern are inhibited at picomolar concentrations proving that ACE2-IgG4 maintains – in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics.
AU  - Svilenov, H.L.*
AU  - Sacherl, J.
AU  - Reiter, A.*
AU  - Wolff, L.S.
AU  - Cheng, C.-C.
AU  - Stern, M.*
AU  - Grass, V.
AU  - Feuerherd, M.
AU  - Wachs, F.P.*
AU  - Simonavicius, N.*
AU  - Pippig, S.*
AU  - Wolschin, F.*
AU  - Keppler, O.T.*
AU  - Buchner, J.*
AU  - Brockmeyer, C.*
AU  - Protzer, U.
C1  - 63605
C2  - 51599
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
TI  - Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.
JO  - Antiviral Res.
VL  - 196
PB  - Elsevier
PY  - 2021
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Chronic hepatitis B virus (HBV) infection remains a major global concern due to its high prevalence and the increased probability of progressing toward cirrhosis and hepatocellular carcinoma (HCC). While currently available therapies are effective in controlling HBV replication, they rarely achieve functional cure. Similarly, effective treatment options for HBV-related HCC (HBV-HCC) are limited and primarily applicable only for early stages of the disease. With the general success of chimeric antigen receptor T-cell immunotherapy against B-cell leukemia, adoptively transferring engineered autologous T cells specific for HBV or HCC antigens might represent a promising therapeutic approach for both chronic HBV infection and HBV-HCC. This review will describe the novel T cell-related immunotherapies being developed for both indications and discuss the approach of each strategy, their considerations and limitations when applied for treatment of chronic HBV infection (CHB) and HBV-HCC.
AU  - Tan, A.T.*
AU  - Schreiber, S.
C1  - 58496
C2  - 48139
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
TI  - Adoptive T-cell therapy for HBV-associated HCC and HBV infection.
JO  - Antiviral Res.
VL  - 176
PB  - Elsevier
PY  - 2020
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.
AU  - Xia, Y.
AU  - Schlapschy, M.*
AU  - Morath, V.*
AU  - Röder, N.
AU  - Vogt, E.I.*
AU  - Stadler, D.
AU  - Cheng, X.
AU  - Dittmer, U.*
AU  - Sutter, K.*
AU  - Heikenwälder, M.
AU  - Skerra, A.*
AU  - Protzer, U.
C1  - 54756
C2  - 45808
SP  - 134-143
TI  - PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.
JO  - Antiviral Res.
VL  - 161
PY  - 2019
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Hepatitis B Virus (HBV) is a strictly hepatotropic pathogen which is very efficiently targeted to the liver and into its host cell, the hepatocyte. The sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a key virus entry receptor, but the early steps in the virus life cycle are still only barely understood. Here, we investigated the effect of lipase inhibition and lipoprotein uptake on HBV infection using differentiated HepaRG cells and primary human hepatocytes. We found that an excess of triglyceride rich lipoprotein particles in vitro diminished HBV infection and a reduced hepatic virus uptake in vivo if apolipoprotein E is lacking indicating virus transport along with lipoproteins to target hepatocytes. Moreover, we showed that HBV infection of hepatocytes was inhibited by the broadly active lipase inhibitor orlistat, approved as a therapeutic agent which blocks neutral lipid hydrolysis activity. Orlistat treatment targets HBV infection at a post-entry step and inhibited HBV infection during virus inoculation strongly in a dose-dependent manner. In contrast, orlistat had no effect on HBV gene expression or replication or when added after HBV infection. Taken together, our data indicate that HBV connects to the hepatotropic lipoprotein metabolism and that inhibition of cellular hepatic lipase(s) may allow to target early steps of HBV infection.
AU  - Esser, K.
AU  - Lucifora, J.
AU  - Wettengel, J.M.
AU  - Singethan, K.
AU  - Glinzer, A.*
AU  - Zernecke, A.*
AU  - Protzer, U.
C1  - 52688
C2  - 44230
CY  - Amsterdam
SP  - 4-7
TI  - Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting an early step in the virus life cycle.
JO  - Antiviral Res.
VL  - 151
PB  - Elsevier Science Bv
PY  - 2018
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection.
AU  - Golsaz-Shirazi, F.*
AU  - Amiri, M.M.*
AU  - Farid, S.*
AU  - Bahadori, M.*
AU  - Bohne, F.
AU  - Altstetter, S.
AU  - Wolff, L.
AU  - Kazemi, T.*
AU  - Khoshnoodi, J.*
AU  - Hojjat-Farsangi, M.*
AU  - Chudy, M.*
AU  - Jeddi-Tehrani, M.*
AU  - Protzer, U.
AU  - Shokri, F.*
C1  - 51386
C2  - 43195
CY  - Amsterdam
SP  - 153-163
TI  - Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).
JO  - Antiviral Res.
VL  - 144
PB  - Elsevier Science Bv
PY  - 2017
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Newcastle disease (ND) is still one of the major plagues of birds worldwide. Combat actions are limited to vaccines, highlighting the urgent need for new and amply available antiviral drugs. Previous results have shown that Newcastle disease virus (NDV) downregulates the intracellular Raf kinase inhibitor protein (RKIP) expression for efficient replication, suggesting that this molecular may be a suitable target for antiviral intervention. In the present work, we investigated whether or not the Raf kinase inhibitor V (RKIV), which functions in the same way as RKIP by targeting the intracellular Raf kinase, is able to suppress the propagation of enzootic virulent NDV in vitro and in vivo. In vitro antiviral activity of RKIV was assessed by cell-based assay, and in vivo activity was determined in the chicken model. Our results clearly showed that RKIV treatment protected the cells from NDV-induced CPE with the effective concentrations on nM level, and inhibited virus replication in the lungs of infected chickens in a dose-dependent manner and protected chickens from the lethal infection by NDV. Thus, we conclude that the Raf kinase inhibitor compound RKIV, by inhibiting the host cellular target Raf kinase, might be very promising as a new class of antivirals against the enzootic virulent NDV infection.
AU  - Yin, R.*
AU  - Liu, X.*
AU  - Zhang, P.*
AU  - Chen, Y.*
AU  - Xie, G.*
AU  - Ai, L.*
AU  - Xue, C.*
AU  - Qian, J.*
AU  - Bi, Y.*
AU  - Chen, J.*
AU  - Sun, Y.*
AU  - Stöger, T.
AU  - Ding, Z.*
C1  - 49237
C2  - 33769
CY  - Amsterdam
SP  - 140-144
TI  - A Raf kinase inhibitor demonstrates antiviral activities both in vitro and in vivo against different genotypes of virulent Newcastle disease virus.
JO  - Antiviral Res.
VL  - 133
PB  - Elsevier Science Bv
PY  - 2016
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Chronic hepatitis B (CHB) is currently treated with IFN-α and nucleos(t)ide analogues, which have many clinical benefits, but there is no ultimate cure. The major problem consists in the persistence of cccDNA in infected hepatocytes. Because no antiviral drug has been evaluated which significantly reduces copies of cccDNA, cytolytic and noncytolytic approaches are needed. Effective virus-specific T- and B-cell responses remain crucial in eliminating cccDNA-carrying hepatocytes and for the long-term control of HBV infection. Reduction of viremia by antiviral drugs provides a window for reconstitution of an HBV-specific immune response. Preclinical studies in mice and woodchucks have shown that immunostimulatory strategies, such as prime-boost vaccination and PD-1 blockade, can boost a weak virus-specific T cell response and lead to effective control of HBV infection. Based on data obtained in our preclinical studies, the combination of antiviral drugs and immunomodulators may control HBV viremia during a patient's drug-off period. In this article, we review current immune-modulatory approaches for the treatment of chronic hepatitis B and the elimination of cccDNA in preclinical models. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis".
AU  - Zhang, E.*
AU  - Kosinska, A.
AU  - Lu, M.*
AU  - Yan, H.*
AU  - Roggendorf, M.
C1  - 47243
C2  - 40612
SP  - 193-203
TI  - Current status of immunomodulatory therapy in chronic hepatitis B, fifty years after discovery of the virus: Search for the "magic bullet" to kill cccDNA.
JO  - Antiviral Res.
VL  - 123
PY  - 2015
SN  - 0166-3542
ER  - 

TY  - JOUR
AB  - Current treatment of chronic hepatitis B virus (HBV) infection mainly targets viral replication in hepatocytes and leads to curing only in exceptional cases. Despite their potential to improve therapeutic success, no drugs interfering with early infection steps of the hepatotropic pathogen HBV are available to date. Recently, entry of the hepatitis C virus (HCV) has been shown to occur along hepatic cholesterol uptake pathways and ezetimibe, a drug which blocks this lipid transport, has been shown to inhibit HCV infection. We here investigated the effect of ezetimibe on HBV infection using differentiated HepaRG cells as a cell-culture infection model. Treatment with ezetimibe inhibited establishment of intrahepatic cccDNA and expression of viral replication markers when cells were infected with HBV virions, while we observed no effect when the HBV viral genome was transduced via an adenoviral vector. Our data suggest that modulating hepatic cholesterol uptake by ezetimibe inhibits early HBV infection and that ezetimibe sensitive lipid transport pathways represent new targets for antiviral therapy in HBV infection.
AU  - Lucifora, J.
AU  - Esser, K.
AU  - Protzer, U.
C1  - 22443
C2  - 30861
SP  - 195-197
TI  - Ezetimibe blocks hepatitis B virus infection after virus uptake into hepatocytes.
JO  - Antiviral Res.
VL  - 97
IS  - 2
PB  - Elsevier Science
PY  - 2013
SN  - 0166-3542
ER  -