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Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, BelgiumDepartment of Therapeutic Chemistry, Pharmaceutical and Drug Industries Research Institute, National Research Centre (NRC), Dokki, Cairo 12622, Egypt
Institute for Medical Virology, University Hospital, Goethe University, Frankfurt am Main, GermanyGerman Center for Infection Research, DZIF, External partner site, Frankfurt am Main, GermanyFraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Theodor Stern Kai 7, Frankfurt am Main, Germany
Corresponding author. Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University. Building MRBII, Corneel Heymanslaan 10, B-9000 Ghent, Belgium. Phone number: +32 (0)9 332 36 58
Laboratory of Liver Infectious Diseases (LLID), Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
A human-monoclonal antibody (hu-mAb) that targets HBsAg.
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The hu-mAb prevents in vitro HBV and HDV infection in permissive cell lines.
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The hu-mAb prevents HBV and HBV/HDV co-infection in human-liver chimeric mice.
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The hu-mAb prevents or at least attenuates HDV superinfection in vivo.
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The therapeutic potential of hu-mAb therapy is shown in vivo.
Abstract
Background and aims
Elimination of chronic hepatitis B and D virus (HBV/HDV) infection remains a major global health challenge. Targeting the excessive HBsAg release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals.
Methods
We evaluated a HBsAg-specific human monoclonal antibody as prophylactic and therapeutic strategy against HBV/HDV infection in cell culture models and in human-liver chimeric mice. In a preventative approach, mice were passively immunized prior to infection with HBV or HBV/HDV (co-infection and superinfection setting). The therapeutic efficacy was assessed in HBV and HBV/HDV (co-)infected mice during a 4-week therapy. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma.
Results
The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of ∼3.5 ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV (co-)infection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in HBsAg on-therapy, with a moderate viral rebound following therapy cessation.
Conclusion
We present a valuable antibody candidate to complement other antivirals to achieve a functional cure for chronic HBV and HDV infection.
Lay summary
Patients chronically infected with hepatitis B virus (HBV) may eventually develop liver cancer and are at great risk to acquire superinfection with hepatitis D virus (HDV), which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronic patients. In this study, we present a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development to potentially eliminate HBV and HDV infections.
Despite the availability of safe and efficacious hepatitis B virus (HBV) vaccines, worldwide approximately 300 million people are currently chronic carrier of hepatitis B surface antigen (HBsAg)
. Chronic HBV infection may eventually lead to liver cirrhosis and hepatocellular carcinoma (HCC) development. HBV is responsible for a heavy disease burden and an estimated 820,000 liver-related deaths annually
. Since HDV infection exacerbates disease progression tremendously by very prompt induction of cirrhosis, liver dysfunction and HCC, it is considered as the most severe type of all viral hepatitides
. Patients co-infected with HBV and HDV usually recover spontaneously through immune-mediated viral elimination. However, chronic HBV patients that become superinfected with HDV progress in 80% of the cases to a chronic disease state, resulting in rapid deterioration of the pre-existing HBV-related liver damage and very high fatality rates
Current lifelong HBV therapy with nucleos(t)ide analogues (NAs) suppresses viral replication, but only about 10% of all treated HBeAg-positive patients (and 1% in HBeAg-negative patients) may achieve a functional cure, i.e. complete HBsAg loss with/without seroconversion after a 5-year follow up
. Therefore, NAs will not eliminate the risk of cancer development. Regarding HDV therapy, this virus does not code for polymerases or proteases that could be therapeutically targeted as is the case for HBV or hepatitis C virus, but relies on both the host replicative machinery and on the helper function of HBV to complete its life cycle
. Specifically, clearance of serum HDV 6 months after cessation of a 48-week therapy was only achieved in 28% of subjects and 50% of the responders experienced late HDV RNA relapse during longer follow-up
. While phase III results are still pending, in 2020 the European Medicines Agency has conditionally approved the entry-inhibitor Myrcludex B (bulevirtide), a preS1-derived peptide able to block the HBV/HDV host receptor natrium taurocholate co-transporting polypeptide (NTCP), for the treatment of patients with compensated chronic HDV infection
Interim results of a multicentre, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of Myrcludex B in combination with Peg-Interferon alpha 2a in patients with chronic HBV/HDV co-infection.
Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV/HDV co-infection.
. It is however widely accepted that a combination of antiviral compounds targeting different steps in the viral life cycle is required to eliminate both viruses.
In this study, we evaluated the capacity of a previously generated HBsAg-specific human monoclonal antibody (hu-mAb) both in vitro and in vivo to prevent and treat HBV and HDV infection
. Targeting the entry-step in combination with diminishing circulating HBsAg would simultaneously block viral spread and potentially provide a window of opportunity for other antivirals to break immune tolerance in a chronic setting.
Material and methods
More details can be found in Supplemental Materials online.
Human monoclonal anti-HBsAg production and purification
The human monoclonal anti-HBsAg specific antibody was produced using classical hybridoma technology, more specifically by fusion of SCID-engrafted human peripheral blood lymphocytes isolated from a vaccinated individual (6,981 mIU/ml anti-HBsAg at time of blood donation) with K6H6/B5 heteromyeloma cells as previously described
. Fused cells were then seeded in culture medium supplemented with hybridoma growth factors and selection drugs hypoxanthine-aminopterin-thymidine and ouabain. Anti-HBs-positive cultures were sequentially cloned and several monoclonal hybridoma lines were isolated and confirmed via commercial anti-HBs ELISA (DiaSorin, Italy). The hu-mAb was then purified from collected supernatant using conventional Protein-G columns (Hitrap Protein G HP, Sigma Aldrich, USA). After evaluation by serum protein electrophoresis (SAS-MX Serum Protein-10, Sysmex, Japan), the antibody was concentrated (Amicon Ultra-15 50k, Sigma Aldrich, USA) and its concentration was determined using spectrophotometry at 280 nm.
In vitro prevention of HBV and HDV infection
For in vitro prevention of HBV infection, anti-HBsAg was applied to HepG2.hNTCP cells in duplicate at 5-fold serial dilutions ranging from 10 μg/ml to 0.128 ng/ml. Two hours later, cells were infected with HBV (4,990 IU/cell; genotype D) and after one week, infection was assessed using immunofluorescent (IF) staining of HBV-Core positive cells. Images were captured by automated spinning disk microscopy using a 40X objective (CSU-X1, Nikon). Per condition, a 20 x 10 field was captured (in duplicate) and positive cells were automatically counted using ImageJ software v1.53c. In vitro prevention of HDV infection (genotype 1) was examined in the same manner, but in 3 different cell lines (HepG2.hNTCP, Huh7.5.hNTCP and NEB2.7). Imaging was performed using the Leica TCS-SPE microscope with a 20X objective. Per condition, 3 random pictures were taken (in duplicate) and HDV-positive cells were automatically counted using ImageJ software v1.53c. More details are provided in Supplemental Materials.
Mice
All mice were bred under sterile conditions and all experiments were approved by the Animal Ethics Committee of the Faculty of Medicine and Health Sciences of Ghent University. Human-liver chimeric mice were generated by transplanting 106 primary human hepatocytes (donor C342, C399 and HH223 from Corning, the Netherlands; and donor L191501 from Lonza, Switzerland) into homozygous uPA+/+-SCID mice as previously described
. Human albumin was quantified in mouse plasma to evaluate successful humanization of the mouse liver using conventional ELISA (Bethyl Laboratories, USA). Mice with albumin levels ranging between 2-10 mg/ml were selected for this study and groups were randomized taking into consideration human albumin levels, infection levels and general condition (e.g. based on body weight). Mice were 8 to 9 weeks old at the start of infection.
In vivo prevention of HBV, HBV/HDV and HDV superinfection
For in vivo prevention of HBV, human-liver chimeric mice (n=6) repopulated with human hepatocytes from two different donors (n=3 for C342 and n=3 for HH223) were passively immunized through intraperitoneal (IP) administration of 1 mg hu-mAb 3 days before challenge with HBV (patient serum, 106 IU/mouse). As controls, 6 additional mice were infected without prior passive immunization. For prevention of HBV/HDV co-infection, humanized mice (n=4) engrafted with hepatocytes from donor C342 were passively immunized (1 mg hu-mAb, IP) 3 days prior to HBV/HDV co-infection with cell culture-derived virus (5 x 106 IU HBV DNA/mouse and 2.3 x 106 IU HDV RNA/mouse). As controls, 3 additional mice were HBV/HDV co-infected without prior passive immunization. Mice (co-)infected with HBV or HBV/HDV were monitored until week 12-13 post-inoculation. Finally, for prevention of HDV superinfection, humanized mice were first infected with either patient-derived HBV (106 IU/mouse, n=7, mice engrafted with donor L191501 hepatocytes) or cell-culture derived HBV (5 x 106 IU per mouse, n=14, i.e. 5 donor C342 and 9 C399 engrafted mice). Respectively 6 and 8 weeks later, mice were superinfected with HDV from either patient or cell culture origin (both 2.55 x 105 IU/mouse). Three days prior to superinfection, respectively 5 out of 7 and 9 out of 14 mice were passively immunized with 1 mg of hu-mAb (IP). Mice superinfected with HDV were monitored for 9-10 weeks following HDV inoculation. Blood plasma was collected every 1-2 weeks (depending on the condition of the mouse and the timing post-infection) and HBV DNA and HDV RNA levels were quantified via commercial HBV/HDV RealStar® (RT-)qPCR (Altona Diagnostics, Germany) or the RealTime m2000 HBV assay (Abbot, USA). Anti-HBsAg ELISA was performed using a commercially available quantitative kit (Beijing Wantai Biological, China).
Treatment of HBV and HBV/HDV (co-)infected mice
Human-liver chimeric mice (n=7) transplanted with donor C342 hepatocytes were chronically infected with cell-culture derived HBV (5 x 106 IU/mouse). Four out of 7 mice received 1 mg hu-mAb (IP) twice a week for 4 consecutive weeks (8 doses in total), starting at w11 post viral inoculation. The remaining 3 infected mice were left untreated as controls. In a second treatment experiment, 13 human-liver chimeric mice, transplanted with donor L191501 hepatocytes, were first infected with cell-culture derived HBV (5 x 106 IU/mouse) and 6 weeks later superinfected with cell-culture derived HDV (2.55 x 105 IU/mouse). At week 10, 7 out of 13 mice received 1 mg hu-mAb (IP) twice a week for 4 consecutive weeks (9 doses in total). The 6 remaining co-infected mice did not receive any treatment (control). Blood plasma was collected every 1 to 2 weeks (depending on the condition of the mouse and the timing post-infection) and the levels of HBV DNA, HDV RNA and anti-HBsAg mAb levels were determined as described above. Plasma samples were analyzed by SDS-PAGE and Western blot to assess HBsAg.
SDS-PAGE and Western blot analysis
Exactly 0.5μl of mouse plasma was used for sample preparation and denaturation (70°C, 10 minutes), and subjected to SDS-polyacrylamide gel (12%) electrophoresis and proteins were transferred to a PVDF membrane. Membranes were blocked (5% skim milk in TBS-T) and the expressed HBsAg was detected by a primary goat anti-HBsAg antibody (70-HG15; Fitzgerald industries; 1/1,000), followed by a HRP-conjugated rabbit anti-goat antibody (31402; Thermo Fisher Scientific; 1/20,000). Immunoblots were developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, USA) and exposed to the ImageQuant LAS4000 chemiluminescent imaging system (GE Healthcare, Diegem, Belgium). More details are provided in Supplemental Materials.
Statistics
GraphPad Prism v9.3.1 was used for graph visualization and statistical analyses. IC50 values were calculated using non-linear regression (curve fit). Normality tests and Mann-Whitney U-tests enabled comparisons between normalized log mean virus levels of treated versus non-treated mice. Data were considered statistically significant if p < 0.05.
Results
In vitro prevention of HBV infection
The HBV-permissive cell line HepG2.hNTCP was (pre-)incubated with 5-fold serial antibody dilutions before, during and after viral inoculation. Infected cells were visualized via immunofluorescent staining (HB-Core) after one week. Near complete abrogation of infection was shown when at least 80 ng/ml hu-mAb was applied, while an overall dose-dependent inhibitory effect was observed with an IC50 value of 3.531 ng/ml (Fig. 1).
Figure 1In vitro prevention of HBV infection. (A) A 5-fold serial dilution of hu-mAb was evaluated for the prevention of HBV infection in HepG2.hNTCP cells. Infectivity was assessed using HB-core IF staining one week after infection. Relative infection (%): total number of infected cells, relative to the control condition where no antibody was applied. (B) The IC50 value was calculated from the antibody dose-response curve by non-linear regression (curve fit). All conditions were performed in duplicate. All data represents mean ± standard deviation (error bars).
The HDV inoculum was pre-incubated with 5-fold serial hu-mAb dilutions and 3 cell lines were subjected to this virus-antibody mixture. Antibodies were maintained for one week until visualization of infected cells by IF. As shown in Fig. 2, infection was nearly completely abrogated using at least 16 ng/ml hu-mAb for all three cell lines tested. A clear dose-dependent inhibitory effect was observed with IC50 values of 3.275 ng/ml, 4.393 ng/ml and 3.080 ng/ml for respectively HepNB2.7, HepG2.hNTCP and Huh7.5.hNTCP cells.
Figure 2In vitro prevention of HDV infection. (A) A 5-fold serial dilution of hu-mAb was evaluated for the prevention of HDV infection in HepNB2.7 (red square), HepG2.hNTCP (blue triangle-up) and Huh7.5.hNTCP (green triangle-down) cells. The hu-mAb was pre-incubated with the HDV inoculum before challenge and the antibody was maintained for 7 days until HDAg IF staining. Relative infection (%): total number of infected cells relative to the control condition where no antibody was applied. (B) The IC50 values were calculated from the antibody dose-response curves by non-linear regression (curve fits). Baseline for background immunofluorescence is set on 0.04% (not depicted). All conditions were performed in duplicate. All data represents mean ± standard deviation (error bars).
Six human-liver chimeric mice (repopulated with hepatocytes from two distinct donors) were passively immunized (1 mg hu-mAb/mouse) 3 days prior to HBV inoculation. As controls, 6 animals were injected with the virus without prior passive immunization. In donor C342 engrafted mice, no virus was detected in the 3 passively immunized mice until at least week 6 (on which 2 were found dead) and one mouse experienced HBV DNA breakthrough at week 10, while the controls showed strongly increasing levels from week 4 onwards (Fig. 3A). This was also confirmed in mice repopulated with donor HH223 hepatocytes: 2 out of 3 passively immunized mice remained HBV DNA negative until at least week 12 post-infection (end of the observation period), one mouse only showed signs of infection at week 12 and all 3 control mice demonstrated increasing HBV DNA levels from week 3 onwards (Fig. 3B). In the two passively immunized mice that rebounded at week 10 and 12 post-infection, circulating plasma anti-HBsAg was no longer detectable as of week 6 (Suppl. Fig. 1).
Figure 3In vivo prevention of HBV infection. Mice transplanted with hepatocytes from donor C342 (A) or donor HH223 (B) were passively immunized with 1 mg of hu-mAb (IP) 3 days prior to HBV challenge (each n=3). Control mice (each n=3) were infected with the same viral inoculum (patient serum, 106 IU/mouse), but without prior passive immunization. HBV DNA was quantified using either the RealStar® qPCR (Altona Diagnostics, Germany; LOD = 3,750 IU/ml) (A) or the RealTime m2000 HBV assay (Abbot, US; LOD = 300 IU/ml) (B). LOD: limit of detection; †: mouse found dead.
Four human-liver chimeric mice were injected with 1 mg of hu-mAb (IP), 3 days prior to inoculation of a viral preparation containing 5 x 106 IU HBV and 2.28 x 106 IU HDV (both cell culture produced). Control mice (n=3), injected with the same virus prep without prior passive immunization, experienced increasing plasma levels of HBV DNA (Fig. 4A) and HDV RNA (Fig. 4B) from respectively week 4 and 8 onwards. Interestingly, all passively immunized mice did not show any signs of infection until 13 weeks post-infection when the experiment was terminated. Anti-HBs antibody levels exceeded 1,000 mIU/ml during the first 2-4 weeks following passive immunization, gradually declined over time and became undetectable in our assay at around 8-10 weeks post antibody injection (Suppl. Fig. 2).
Figure 4In vivo prevention of HBV/HDV co-infection. Passive immunization using 1 mg of hu-mAb (IP) was performed 3 days prior to HBV/HDV inoculation (n=4) in mice engrafted with donor C342 hepatocytes. Control mice (n=3) were accordingly infected with cell-culture derived virus (5 x 106 IU HBV and 2.28 x 106 IU HDV per mouse). HBV DNA (A) and HDV RNA (B) was analyzed in mouse plasma via RealStar® (RT-)qPCR (Altona Diagnostics, Germany; LOD HBV = 3,750 IU/ml; LOD HDV = 187.5 IU/ml). LOD: limit of detection; †: mouse found dead.
To evaluate whether a single antibody injection could avert an HDV infection in mice with pre-existing HBV infection, human-liver chimeric mice engrafted with donor L191501 hepatocytes (n=7) were first infected with HBV and 6 weeks later, 5 out of 7 mice were passively immunized with the antibody 3 days prior to HDV superinfection (Fig. 5A-B). At the moment of HDV inoculation (week 6), the HBV DNA plateau had been reached in all mice (Fig. 5A). In both control mice, HDV RNA levels could be detected already one week after HDV inoculation and these increased steeply to 108 IU/ml at week 10. In most passively immunized mice, HDV RNA was only detected at week 10 with a delayed peak at week 14 (Fig. 5B). Accordingly, circulating anti-HBs was lost between week 8 and 14 for all passively immunized mice (Suppl. Fig. 3A). This experimental set-up was repeated in 14 mice engrafted with either donor C342 or C399 hepatocytes, but here, passive immunization (in 9 out of 14 mice) and HDV superinfection was performed at week 8 (Fig. 5C-D). Interestingly, 8 mice were completely protected from HDV superinfection and the 5 animals that reached the endpoint of the observation period (week 16-17) were then still HDV RNA negative (Fig. 5D). The single passively immunized mouse that became HDV RNA positive at week 12 also lost its circulating anti-HBs at week 9 (Suppl. Fig. 3B).
Figure 5In vivo prevention of HDV superinfection. (A-B) Mice were engrafted with human hepatocytes from donor L191501 and infected at week 0 with patient-derived HBV (106 IU/mouse, n=7). Three days prior to HDV superinfection with patient-derived inoculum (2.55 x 105 IU/mouse) at week 6, passive immunization in 5 out of 7 mice was performed with 1 mg of hu-mAb (IP). (C-D) Mice were engrafted with human hepatocytes from donor C342 (n=5) or C399 (n=9) as depicted and infected at week 0 with cell culture-derived HBV (5 x 106 IU/mouse). Three days prior to HDV superinfection with either cell culture-derived inoculum or patient-derived inoculum (*) at week 8 (both using 2.55 x 105 IU/mouse), passive immunization in 9 (n=3 for C342 mice and n=6 for C399 mice) out of 14 mice was performed with 1 mg of hu-mAb (IP). HBV DNA (A, C) and HDV RNA (B, D) was quantified by RealStar® (RT-)qPCR (Altona Diagnostics, Germany; LOD HBV = 3,750 IU/ml; LOD HDV = 187.5 IU/ml). LOD: limit of detection; †: mouse found dead.
Humanized mice with an established HBV infection were treated twice a week for 4 consecutive weeks with 1 mg of hu-mAb (IP; n=4). While non-treated HBV-infected controls (n=3) demonstrated quite stable or moderately increasing plasma HBV DNA levels, all antibody-treated mice showed strongly decreasing viral levels on-therapy, although not statistically significant (Fig. 6A-B and Suppl. Table 1). Specifically, already after 1 week of therapy, mean plasma HBV DNA levels were reduced by 1.78 x log10 compared to non-treated mice. HBV DNA became even undetectable in our assay in one mouse (M240L). On week 2, 3 and 4 (end) of treatment, we respectively observed a 1.55 x log10, 2.09 x log10 and 2.02 x log10 fold reduction compared to the controls. Importantly, only 1 (M257RR) out of 4 mice rebounded after therapy cessation and this coincided with a complete loss of circulating anti-HBs antibodies (Fig. 6C). Three weeks post-therapy, a mean difference of 2.03 x log10 was still present between the two groups.
Fig. 6Treatment of chronic HBV infection. Human-liver chimeric mice (engrafted with donor C342 hepatocytes) were infected with HBV for 11 weeks and then treated with hu-mAb for 4 consecutive weeks: 2 IP injections of 1 mg per week (n=4). Control mice were accordingly infected with cell-culture derived virus (5 x 106 IU HBV/mouse), but were non-treated (n=3). (A) Plasma HBV DNA. LOD (HBV) = 3,750 IU/ml. (B) Log10 reduction of plasma HBV DNA, calculated towards levels observed one day prior to first antibody injection (w11). (C) Anti-HBsAg determined in mouse plasma via quantitative ELISA (Wantai, China). LLOQ (anti-HBsAg) = 100 mIU/ml and ULLOQ (anti-HBsAg) = 1,600 mIU/ml. LLOQ: lower limit of quantification; ULOQ: upper limit of quantification; LOD: limit of detection; †: mouse found dead.
Six weeks post HBV infection, 13 humanized mice were inoculated with HDV to obtain HBV/HDV co-infected mice. Four weeks later, the animals were divided in two groups: the first group (n=7) was given a 4-week hu-mAb therapy, while the second group (n=6) served as untreated control. At the start of therapy, plasma HBV DNA levels ranged between 106 and 109 IU/ml, while HDV RNA levels were more diverse, varying from just above the limit of detection (LOD) to almost 108 IU/ml and one mouse (M264RL) with undetectable HDV RNA in our assay (Fig. 7A,C). Two weeks following onset of therapy, significantly (p < 0.01 and p < 0.05) reduced mean HBV DNA and HDV RNA levels (resp. 1.96 x log10 and 2.82 x log10 difference) compared to control means were observed (Fig. 7A-D and Suppl. Table 2). Even more pronounced mean differences could be observed after 4 weeks (end of therapy): -2.82 x log10 and -3.15 x log10 (both p < 0.01) for resp. HBV DNA and HDV RNA, while stable or even increasing viral loads were measured in the controls. Two and 4 weeks after therapy cessation, mean differences of -1.97 x log10 and -1.69 x log10 for HBV DNA; and -3.20 x log10 and -3.10 x log10 for HDV RNA was shown with accompanying hu-mAb plasma levels exceeding 1,000 mIU/ml in treated animals. However, two mice (M241R and M298R) demonstrated HBV/HDV rebound at week 2-4 post-therapy, which coincided with hu-mAb loss at these time points (Fig. 7E). Remarkably, HBsAg was observed at the onset of therapy in the treated mice (w10), but was undetectable on-therapy using Western blot analysis (Fig. 7F). For the two mice showing strongly diminished or even absent viral loads post-therapy (M296 and M296R), HBsAg was still absent. On the other hand, the post-therapy viral rebound observed in M241R (at w16) and M298R (at w18) was accompanied with recurrence of HBsAg.
Fig. 7Treatment of chronic HBV/HDV infection. Human-liver chimeric mice engrafted with donor L191501 hepatocytes were infected with HBV and superinfected with HDV at week 6. From week 10 onwards, mice were treated with hu-mAb for 4 consecutive weeks: 2 IP injections of 1 mg per week (n=7). Control mice were accordingly infected with cell-culture derived virus (5 x 106 IU HBV and 2.55 x 105 IU HDV per mouse), but were non-treated (n=6). (A) Plasma HBV DNA. LOD (HBV) = 3,750 IU/ml. (B) Log10 reduction of plasma HBV DNA, calculated towards levels observed one day prior to first antibody injection (w10). (C) Plasma HDV RNA. LOD (HDV) = 187.5 IU/ml. (D) Log10 reduction of plasma HDV RNA, calculated towards levels observed one day prior to first antibody injection (w10). Statistics: ; * p-value < 0.05; ** p-value < 0.01. (E) Anti-HBsAg determined in mouse plasma via quantitative ELISA. LLOQ (anti-HBsAg) = 100 mIU/ml and ULLOQ (anti-HBsAg) = 1,600 mIU/ml. (F) HBsAg Western blot analysis. LLOQ: lower limit of quantification; ULOQ: upper limit of quantification; LOD: limit of detection; (g)p: (glycosylated) protein; †: mouse found dead.
The excessive HBsAg release in chronic HBV patients tolerizes antibody- and cell-mediated immune responses, and represent a major hurdle for viral eradication by current treatments
. Consequently, it is of utmost importance to find ways to break immune tolerance, enabling the host to mount effective immune responses to clear the virus and to prevent HDV superinfection. Here, we demonstrate the prophylactic and therapeutic efficacy of a hu-mAb targeting the HBV envelope in the context of HBV and HDV infection in vitro and in human-liver chimeric mice.
The hu-mAb neutralized HBV and HDV in vitro with respective IC50 values of 3.53 ng/ml (∼0.23 nM) and 3.58 ng/ml (∼0.24 nM) and can be considered as highly potent, since Hehle et al.
evaluated 170 different hu-mAbs isolated from natural HBV controllers or vaccinated individuals and showed that only 35% of antibodies harbored IC50 values <50 ng/ml.
Next, we demonstrated efficient prevention of HBV mono- and HBV/HDV co-infection in human-liver chimeric mice. Conceivably, once a chronic HBV infection is fully established, preventing HDV superinfection may be even more challenging due to massive HBsAg presence. However, depending on the hepatocyte donor, we showed either near complete prevention (donor C342 and C399) or at least delayed HDV RNA levels (donor L191501). Noteworthy, transplantation of mice with donor L191501 human hepatocytes rendered the animals more prone to hepatitis infection compared to other donors tested (data not shown), which may explain for the viral breakthrough. Overall, our HDV superinfection prevention data is quite impressive since only a single injection of 1mg hu-mAb was sufficient to overcome the high load of circulating HBsAg and to (partially) neutralize the high-titer HDV inoculum. Furthermore, since HDV can silently persist and replicate in human hepatocytes for up to 6 weeks, the hu-mAb effectively prohibits entry of HDV virions into hepatocytes
Notably, antibody half-life varies throughout our experiments, i.e. the antibody is still present in circulation 3-4 weeks or longer after a 1 mg injection. This may potentially be explained by variability in body weight (and hence distribution volume) since we injected 1 mg of antibody irrespective of mouse body weight. Furthermore, antibody clearance might be greatly accelerated by high replication of the virus (high circulating viral load). The source of viral inoculum also appears to influence the success of prevention and lack of viral rebound. For example, in the prophylactic HBV/HDV co-infection experiment, cell-culture derived virus was used and no viral rebound was observed, while in the prophylactic HBV mono-infection experiment, patient-derived virus was applied while we did observe HBV DNA rebound. Also more animals were protected from HDV superinfection when infected with cell-culture derived viruses. Therefore, it seems that primary patient-derived virus preparations are intrinsically less sensitive to antibody neutralization, or may contain variants that are.
The therapeutic potential of the hu-mAb was furthermore demonstrated by (significantly) reduced mean viral plasma levels at the end of a 4-week therapy in HBV-infected (2.02 x log10 drop) and HBV/HDV co-infected mice (2.82 x log10 and 3.50 x log10 drops for resp. HBV and HDV). Since the repeated injections of antibody result in a clear drop in HBV DNA (and HDV RNA levels) in this therpautic setup, we assume that the single dose regimen of the HDV superinfection prevention experiment was insufficient to show an antiviral effect on the already established HBV infection (i.e. no effect on HBV DNA). Interestingly, our 4-week therapy data are in line with the most potent hu-mAb (namely Bc1.187) tested by Hehle et al.
that treated HUHEP mice (weekly ∼1mg (50 mg/kg) hu-mAb IP for 3 weeks), inducing a 1.76 x log10 decrease in serum HBV DNA at the end of therapy. Interestingly, circulating HBsAg levels dropped and remained undetectable for 2 weeks following the last injection in 60% of treated animals. Importantly, these mice had initial lower HBV DNA levels (<106 IU/ml) compared to mice in our study. Another hu-mAb (HBC34) was evaluated at a very comparable schedule, but at lower dose (a 4-week treatment with 2 IP injections per week of 1mg/kg) in HBV/HDV co-infected USB/USG mice
Prevention of Viral Spread, Viremia Reduction and HBsAg Clearance in Hepatitis B and D Coinfected Humanized Mice by a Human Neutralizing Monoclonal Antibody.
Lempp FA, volz T, cameroni E, Benigni F, Zhou J, Rosen LE, et al. The potent broadly neutralizing antibody VIR-3434 controls Hepatitis B and D Virus infection and reduces HBsAg in humanized mice.; 2022.
. They revealed a similar reduction in HBV DNA and HDV RNA levels (mean 1 to 2.7 x log10 and 2.4 to 2.5 x log10 reduction, respectively, in both models) and a concomitant 2.7 to 2.8 x log10 reduction of HBsAg levels after 4 weeks. Finally, we also show HBsAg loss on-therapy with rebound after therapy cessation, a phenomenon that is frequently noted upon withdrawal of mAb therapy
. Interestingly, the HBsag loss might provide a therapeutic window for viral eradication using NAs, possibly in combination with other antivirals, that could take advantage of a (partially) reconstituted adaptive immune response
. Interestingly, although no effect on HBsAg was seen when the recently EU approved bulevirtide was used as mono-therapy, HBsAg decreases could be observed when combined with peg-IFNα
. Notably, decreasing HBsAg might be a valuable therapeutic strategy, but currently no direct evidence is available to support this hypothesis and therefore, additional studies addressing the relationship between circulating HBsAg levels and HBV-specific immune responses are required to provide further insights on the immunobiology of HBV, and presumably, additional immune activation would be necessary to control the virus
. Nowadays, polyclonal antibodies derived from pooled plasma from recovered or vaccinated individuals (HBIG) are successfully and routinely used in clinical practice as post-exposure prophylaxis, to avoid HBV recurrence after liver-transplantation or therapeutic immunosuppression, or to prevent perinatal transmission
. However, only a small antibody proportion in HBIG preparations is actually HBV-specific and neutralizing, and blood products always remain subject to bio-safety concerns, especially for immunosuppressed patients
. Consequently, monoclonal antibodies may be an attractive alternative since they represent a stable, reproducible source for prolonged immunotherapy. Hu-mAbs are already successfully explored in many therapeutic fields such as oncology
Efficacy and safety of two neutralising monoclonal antibody therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 (TICO): a randomised controlled trial.
Successful anti-scavenger receptor class B type I (SR-BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR-BI-targeting agents.
. Regarding other HBV-specific hu-mAbs, previous studies similarly have shown efficient prevention, delayed infection, or therapeutic efficacy in cell culture
Generation and Characterization of a Neutralizing Human Monoclonal Antibody to Hepatitis B Virus PreS1 from a Phage-Displayed Human Synthetic Fab Library.
Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.
Proceedings of the National Academy of Sciences of the United States of America.1993; 90: 3014-3018
Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.
Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.
demonstrated prevention in a mouse model harboring human NTCP-receptors, thus in absence of relevant HBV infection and moreover in a mouse background, and as mentioned above, antibody HBC34 revealed apotent decrease in HBV/HDV viremia in HBV/HDV co-infected USB/USG mice
Prevention of Viral Spread, Viremia Reduction and HBsAg Clearance in Hepatitis B and D Coinfected Humanized Mice by a Human Neutralizing Monoclonal Antibody.
Lempp FA, volz T, cameroni E, Benigni F, Zhou J, Rosen LE, et al. The potent broadly neutralizing antibody VIR-3434 controls Hepatitis B and D Virus infection and reduces HBsAg in humanized mice.; 2022.
Currently evaluated or approved antibody dosages vary tremendously in human applications, even beyond the field of infectious diseases. For example, omalizumab, a humanized recombinant monoclonal anti-IgE antibody approved for the treatment of allergic asthma is recommended at 75 to 600 mg every two weeks
. Rituximab, a genetically engineered chimeric mouse/human mAb approved for rheumatoid arthritis is recommended at 2 infusions of 1,000 mg IV, with 2 weeks of interval
. Eculizumab, a humanized mAb approved for myasthenia gravis, is administered at 600 mg IV weekly for 4 weeks, followed by 900 mg IV in the 5th week and then, 900 mg every 2 weeks as maintenance therapy
Casirivimab-Imdevimab treatment is associated with reduced rates of hospitalization among high-risk patients with mild to moderate coronavirus disease-19.
. These examples illustrate the high variability of applied doses in various applications, but also that much higher dosages are achievable and tolerated compared to our regimen. Assuming an average mouse body weight of 15 g, we applied about 66.7 mg/kg per injection. In the treatment regimen, we hence administered a total dose of 533 mg over a 4-week period; clearly in range of what is feasible in humans. However, dosages used in mice cannot simply be translated into human doses. More specifically, the common perception of scaling of dose based on body weight (mg/kg) alone is not entirely correct. This is primarily because the biochemical, functional systems in species vary which in turn alter pharmacokinetics
The main putative mechanism of action of the HBsAg-targeting antibody in the applied mouse model is functional neutralization of the viral envelope, which directly blocks entry of HBV and HDV, and consequently prevents spread of infection to naïve hepatocytes. Furthermore, the antibody also promotes HBsAg reduction (or clearance) in circulation. It has also been shown that HBsAg-specific antibodies could be internalized into infected cells and therefore partially inhibit HBsAg secretion from these cells, implicating intracellular blocking of virion and protein release as an additional possible mechanism of action
. Importantly, in the context of infected patients, additional intrinsic effector functions could be induced through the formation of immune complexes. More specifically, clearance of circulating virions, antigens or infected cells could be promoted via antibody-dependent cell-mediated cytotoxicity and/or other Fc-mediated effector responses such as complement lysis or phagocytosis
. Accordingly, many therapeutic advantages could be proposed in a clinical setting: 1) reduction of the hepatic accumulation of cccDNA, one of the main causes of HBV persistence
; 2) mediation of serum HBV/HDV and HBsAg clearance, which may ultimately counteract the challenging T-cell exhaustion mechanisms that are characteristic for patients with chronic HBV infection
In conclusion, we show the added value of an entry-inhibiting hu-mAb in the context of HBV and HDV (super)infection prophylaxis and its potential use as treatment for chronic HBV or HBV/HDV (co-)infection. For the here presented experiments we used either patient-derived virus or cell-culture derived virus (genotype D for HBV and genotype 1 for HDV) in human-liver chimeric mice that were transplanted with hepatocytes from in total 4 different donors. Further confirmation using additional viral genotypes and various hepatocyte donors having distinct genetic backgrounds is warranted. Overall, our data suggests that this hu-mAb is an interesting candidate to complement current therapies or antivirals (under development) to eradicate both HBV and HDV infections. Decreasing or even eliminating the high levels of circulating HBsAg in chronic patients may provide an opportunistic window for other antiviral therapies targeting later steps in the viral life cycle or the immune system to eventually cure chronic infection.
Data availability statement
The data that supports the findings of this study is available upon request. Please contact the corresponding author.
Conflict of interest
Nothing to declare related to the content of this manuscript. Please consult the specific ICMJE disclosure forms for more details.
Financial support
PM was supported by the Special Research Fund of Ghent University (BOFEXP2017001002), the Research Foundation-Flanders (FWO-Vlaanderen; projects G089515N, G047417N and Excellence of Science projects VirEOS and VirEOS2.0) and the European Union (FP7, HepaMab).
Author contributions
PM devised the study. General experimental set-up was designed by PM and RB. RB and FVH performed in vitro neutralization studies. Animal work was conducted by RB, LV, AAM and PM. Viral parameters were monitored in mouse plasma by RB and FV. GLR provided the antibody clone. SC, HW and PR delivered patient-derived viruses that were essential for the execution of the study. Data analyses and writing were performed by RB and PM. All authors approved the final version of the manuscript.
Acknowledgments
Cell lines HepNB2.7, HepG2.hNTCP and Huh7.5.hNTCP were kindly provided by Prof. Dr. Stephan Urban (Heidelberg University, Germany); HepAD38 cells by Dr. Christoph Seeger (Fox Chase Cancer Center, Philadelphia, USA), and Huh7.5 cells by Prof. Dr. Charles M. Rice (The Rockefeller University, USA). Dr. John Taylor and Prof. Dr. Camille Sureau provided plasmids pSVL(D)3 and pT7HB2.7. Recombinant HBsAg (HEF002) was kindly provided by GlaxoSmithKline Biochemicals (Rixensart, Belgium). We thank Sophie Vermaut for antibody purification and technical assistance. We acknowledge the Centre for Advanced Light Microscopy at Ghent University (Belgium) for the use and support on the Nikon spinning disk CSU-X1 microscope.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Interim results of a multicentre, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of Myrcludex B in combination with Peg-Interferon alpha 2a in patients with chronic HBV/HDV co-infection.
Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV/HDV co-infection.
Prevention of Viral Spread, Viremia Reduction and HBsAg Clearance in Hepatitis B and D Coinfected Humanized Mice by a Human Neutralizing Monoclonal Antibody.
Lempp FA, volz T, cameroni E, Benigni F, Zhou J, Rosen LE, et al. The potent broadly neutralizing antibody VIR-3434 controls Hepatitis B and D Virus infection and reduces HBsAg in humanized mice.; 2022.
Efficacy and safety of two neutralising monoclonal antibody therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 (TICO): a randomised controlled trial.
Successful anti-scavenger receptor class B type I (SR-BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR-BI-targeting agents.
Generation and Characterization of a Neutralizing Human Monoclonal Antibody to Hepatitis B Virus PreS1 from a Phage-Displayed Human Synthetic Fab Library.
Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.
Proceedings of the National Academy of Sciences of the United States of America.1993; 90: 3014-3018
Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.
Casirivimab-Imdevimab treatment is associated with reduced rates of hospitalization among high-risk patients with mild to moderate coronavirus disease-19.
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