During hepatitis B virus (HBV) infection, high numbers of non-infectious HBV surface antigen(HBsAg) particles are present in circulation. It is shown here that recombinant HBsAg (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. Attachment of rHBsAg to the THP-1 pre-monocytic cell line occurs upon 1,25-dihydroxyvitamin D3-induced differentiation. Binding to monocytes is enhanced by a heat-labile serum protein and is inhibited by Ca 2M /Mg 2M , low pH and an HBsAg-specific monoclonal antibody. Furthermore, it is shown that rHBsAg suppresses lipopolysaccharide-and IL-2-induced production of cytokines. These results suggest the existence of a monocyte-specific receptor, the engagement of which by HBsAg suppresses the activity of these cells.

Hepatitis B virus (HBV) infections cause 500,000 to 700,000 deaths per year as a consequence of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Efficient and safe antivirals to treat chronically infected patients and consequently to prevent development of hepatocellular carcinoma are still awaited. We isolated five single-domain antibodies (VHHs) that recognize the most abundant envelope protein (S) of HBV. VHHs, when expressed and retained in the endoplasmic reticulum as intrabodies, reduced levels of secreted hepatitis B surface antigen ( H epatitis B virus (HBV) imposes an important burden to human health worldwide. Approximately 350 to 400 million persons are chronically infected. Among these, 15% to 40% will develop cirrhosis, liver failure, and/or hepatocellular carcinoma, resulting in 500,000 to 700,000 deaths each year. 1,2 Potent and safe antivirals to succesfully treat chronically infected HBV patients and to prevent development of hepatocellular carcinoma are still awaited. To date, only interferon-2␤, pegylated interferon-2␣, and four nucleos-(t)ide analogues have been approved for the treatment of chronic HBV infections. However, interferons are only effective in a fraction (20%-40%) of patients and cause numerous side effects. The licensed synthetic nucleos-(t)ide analogues induce fewer side effects, but their therapeutic success is hampered by the appearance of drug-resistant mutants during long-term treatment. Discontinuation of treatment with nucleos(t)ide analogues generally leads to renewed viral replication that can be followed by acute exacerbation of liver disease. [3][4][5][6] Novel strategies to combat viral infections are being explored by several research groups. One approach is the ablation of viral messenger RNA expression by antisense oligonucleotides, peptide aptamers, ribozymes, and RNA interference-mediated gene silencing. 3 Another approach consists of the intrabody-mediated inhibition of viral protein functions or interactions. Intrabodies are antibody fragments that are expressed and function inside the cell. 7,8 Intrabodies can be targeted to a specific cell compartment such as the endoplasmic reticulum (ER), mitochondrium, nucleus, or cytoplasm. The most explored intrabody format is the single-chain variable fragment (scFv), which is derived from the variable antigen-binding domains of a conventional antibody. scFv intrabodies that interfere with viral replication have been developed against human immunodeficiency virus, 9 hepatitis C virus, 10,11 rotavirus, 12 herpesvirus, 13 flavivirus, 14 and HBV. 15,16 Single-domain antibodies (VHHs) are a new generation of recombinant antibody fragments. VHHs consist of the functional variable domain of heavy-chain-only antibodies of the family Camelidae, which are devoid of light chains. [17][18][19] VHHs are the smallest intact antigen-

End-stage liver disease (ESLD) caused by hepatitis C virus (HCV) infection is a major indication for liver transplantation. However, immediately after transplantation, the liver graft of viremic patients universally becomes infected by circulating virus, resulting in accelerated liver disease progression. Currently available direct-acting antiviral therapies have reduced efficacy in patients with ESLD and prophylactic strategies to prevent HCV recurrence are still highly needed. In this study, we compared the ability of two broadly reactive monoclonal antibodies (mAbs), designated 3/11 and AP33, recognizing a distinct, but overlapping, epitope in the viral E2 glycoprotein to protect humanized mice from a patient-derived HCV challenge. Their neutralizing activity was assessed using the HCV pseudoparticles and cell-culture-derived HCV systems expressing multiple patient-derived envelopes and a human-liver chimeric mouse model. HCV RNA was readily detected in all control mice challenged with a patient-derived HCV genotype 1b isolate, whereas 3 of 4 AP33-treated mice were completely protected. In contrast, only one of four 3/11-treated mice remained HCV-RNA negative throughout the observation period, whereas the other 3 had a viral load that was indistinguishable from that in the control group. The increased in vivo efficacy of AP33 was in line with its higher affinity and neutralizing capacity observed in vitro. Conclusions: Although mAbs AP33 and 3/11 target the same region in E2, only mAb AP33 can efficiently protect from challenge with a heterologous HCV population in vivo. Given that mAb AP33 efficiently neutralizes viral variants that escaped the humoral immune response and reinfected the liver graft of transplant patients, it may be a valuable candidate to prevent HCV recurrence. In addition, our data are valuable for the design of a prophylactic vaccine. (HEPATOLOGY 2016;63:1120-1134

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Hepatitis B virus (HBV) infections represent a global health problem, since these account for 350 million chronic infections worldwide that result in 500 000-700 000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes.

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

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The nucleocapsid of hepatitis B virus (HBV) allows insertions of heterologous peptides and even complete proteins. Because of its outstanding capacity to induce B-cell, T-helper and cytotoxic T-cell responses, this structure is considered to be an important instrument for future vaccine development. Most of the evidence for the unique immunogenic qualities of nucleocapsids has been generated in mice, which are not natural hosts of HBV. Moreover, most nucleocapsid preparations used in these studies were produced in a recombinant manner in Escherichia coli. Such preparations have been shown to contain lipopolysaccharide (LPS). Not unexpectedly, it isshown here that contaminating LPS, rather than the nucleocapsid structure itself, is responsible for the activation of human antigen-presenting cells. Careful examination of the literature dealing with the immunogenicity of HBV nucleocapsids suggests that the possible presence of LPS has been largely ignored or underestimated in several studies. This raises doubts on some of the underlying mechanisms that have been proposed to explain the unique immunogenicity of the HBV nucleocapsid. INTRODUCTIONThe initial step in the activation of a B cell is the crosslinking of the antigen receptor. A second signal is required to enhance the response and to induce a switch from IgM to IgG (Bachmann & Zinkernagel, 1997;Baumgarth, 2000). This signal is normally delivered by specific T-helper (Th) cells. Multivalent repetitive structures are often capable of triggering strong, T cell-independent IgM responses. Many viruses exhibit such repetitive structures and, indeed, have been shown to induce IgM responses that are completely T cell-independent (T-independent type I response) (Bachmann & Zinkernagel, 1996). If help is not provided, the switch to IgG does not occur unless very high antigen doses are used (Szomolanyi-Tsuda & Welsh, 1998). As always, there are exceptions to this paradigm: the nucleocapsid of the hepatitis B virus (HBV) was shown to induce the switch from IgM to IgG in nude mice or CD4-depleted xid mice (Milich & McLachlan, 1986;Milich et al., 1997;Fehr et al., 1998). In immune-competent mice, a single injection of capsids stimulates IgM and IgG antibodies and Th-cell function. Immunizations with as little as 6 ng capsid in saline resulted in detectable anti-HBV core antigen (HBcAg) production 2 weeks after a single injection. Furthermore, capsids elicited primarily IgG2a and IgG2b antibodies, with a low level of IgG3 and no IgG1 antibodies. Cytokine production by capsid-primed Th cells confirmed this Th1 phenotype: capsid-primed T cells efficiently produced interleukin 2 (IL2) and gamma interferon (IFN-c), but low levels of IL4 (Milich & McLachlan, 1986;Milich et al., 1997). It was reported by Riedl et al. (2002) that encapsidated RNA facilitates the priming of this Th1 immunity in mice. Recent work has indeed demonstrated that single-stranded RNA (ssRNA) has strong immunostimulatory potential, and Toll-like receptor (TLR) 7 and 8 have been shown to mediate recognit...