Freya Van Houtte

Llama‐derived single‐domain intrabodies inhibit secretion of hepatitis B virions in mice

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 (HBsAg) particles in a cellular HBV model. In a hydrodynamics‐based HBV mouse model, these intrabodies caused a marked reduction in HBsAg concentrations and a 10‐ to >100‐fold reduction in the concentration of HBV virions in plasma. Conclusion: VHHs potently inhibited secretion of HBV virions in vivo, showing that this approach might be useful in the treatment of HBV. To our knowledge, this is the first report of intrabody‐mediated inhibition of viral secretion in mammals. (HEPATOLOGY 2009;49:39‐49.)

Production, characterization and in vitro testing of HBcAg-specific VHH intrabodies

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.

Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge

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)

Contamination of a Recombinant Hepatitis B Virus Nucleocapsid Preparation with a Human B-Cell Activator

The hepatitis B virus nucleocapsid is a very immunogenic structure (7) that activates mouse B cells in a T-cell-independent manner (3). We reported a similar observation with human B cells: nucleocapsids induced capsid-binding immunoglobulin M molecules when purified B cells of unprimed humans were transferred into the spleens of NOD/Scid mice (2). However, recent results which we report in this letter suggest that an immunostimulatory contaminant in the HBcAg preparation rather than the nucleocapsids themselves were likely responsible for the observed T-cell-independent B-cell activation. This emerged when four different nucleocapsid preparations were used to study the stimulatory potential of nucleocapsids for purified human B cells. HBcAg-c1 (Diasorin), HBcAg-c2 (Biodesign), and HBcAg-c3 (Biodesign) were produced in Escherichia coli. HBcAg-y was produced in Saccharomyces cerevisiae (GlaxoSmithKline Biologicals, Belgium). HBcAg-c1 nucleocapsids lack the first 2 amino acids and contain 11 foreign amino acids, 8 of which are derived from β-galactosidase (4). HBcAg-c1, HBcAg-y, and HBcAg-c2 contain encapsidated RNA, while HBcAg-c3 nucleocapsids do not, as these lack amino acids 145 to 183. The lipopolysaccharide (LPS) contents of HBcAg-c1, -y, -c2, and -c3 were 136, 0.422, 9.48, and 0.17 endotoxin units LPS/μg nucleocapsid, respectively. We first investigated whether nucleocapsids induced or enhanced the proliferation of purified B cells. Neither HBcAg-c1, HBcAg-y, nor HBcAg-c2 induced thymidine (TdR) incorporation (Fig. ​(Fig.1A).1A). The addition of HBcAg-c1 to B cells stimulated with interleukin-4 (IL-4), anti-CD40 plus IL-2, and anti-CD40 plus IL-4 had a clear synergistic effect on incorporation of TdR. This was not observed with HBcAg-y and HBcAg-c2. Next, we observed that HBcAg-c1 caused a clear up-regulation of CD86 and CD80 (Fig. ​(Fig.1B).1B). This was never observed with HBcAg-y, HBcAg-c2, and HBcAg-c3 (data not shown). A goat anti-human Fab-specific F(ab)2 fragment induced only the expression of CD86, indicating that the intracellular pathways triggered by HBcAg-c1 and the anti-Fab were different. Because only HBcAg-c1 stimulated B cells, we concluded that a contaminant was present. Total purified human circulating B cells do not respond to different toll-like receptor 2 (TLR2), TLR4, TLR2/TLR6, and TLR7/8 ligands (1), suggesting that HBcAg-c1-encapsidated RNA and contaminating LPS and TLR2 ligands (5, 6) were not responsible for the activation of B cells. The capacity of HBcAg-c1 to induce CD80 and CD86 was not destroyed by boiling for 30 min (data not shown), demonstrating indeed that HBcAg-c1 nucleocapsids themselves were not responsible for the stimulation of B cells. Unluckily, the use of HBcAg-c1 in our experiments suggested that nucleocapsids behaved as T-cell-independent B-cell antigens (2). We suggested previously that the stimulatory capacities of nucleocapsids for monocytes and dendritic cells might be attributed to contaminating TLR4 and TLR2 ligands (5, 6). Our data again highlight that caution is needed when studying the stimulatory capacities of nucleocapsids, especially when produced in a bacterium. Proteins produced in E. coli inherently contain products like LPS, bacterial DNA, porins, lipid A-associated proteins, fimbrial proteins, protein A, and lipoproteins. All these can activate cells (8). FIG. 1. (A) HBcAg-c1 enhances B-cell proliferation induced by IL-4 and anti-CD40 plus IL-2 or IL-4. Purified B cells were incubated for 3 days with 1 μg/ml of HBcAg-c1 (black), HBcAg-y (dark gray), or HBcAg-c2 (light gray) or without nucleocapsids (white), ...

Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

ABSTRACT 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. HighlightsDevelopment of a novel human monoclonal antibody (mAb), designated 2A5, that targets the HCV envelope glycoprotein E2.mAb 2A5 efficiently neutralizes HCVpp and HCVcc in a pan‐genotypic manner.mAb 2A5 protects human‐liver chimeric mice from HCV challenge.Our novel mAb could be used to protect liver transplant patients from HCV recurrence.The 2A5 epitope represents a valuable target for the development of HCV vaccines with broad‐spectrum activity.