MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 34, Number 2, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0079

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Monoclonal Antibodies Against Hepatitis B Viral Surface Antigens and Epitope Grouping Gregory Lee and Suefay Liu

Monoclonal antibodies (MAbs) were generated against subtypes (ad/ad/rw) of the human hepatitis B viral surface antigen (HBsAg). Among dozens of antibodies that were generated, the majority was shown to commonly react with various ad/ay subtypes of the S protein. Epitope(s) of these antibodies were grouped by various immunoassay methods, and at least four distinct epitope regions were identified. Some of these antibodies were selected to formulate sandwich enzyme immunoassays for quantitative determinations of HBsAg in reconstituted specimens. Epitope-defined monoclonal antibodies with high affinity and specificity might be suitable for formulations as vaccines (containing a mixture of humanized monoclonal antibodies) for passive immunization in humans for immunoprophylaxis of HBV infection.

therapy. In particular, monoclonal antibodies (MAbs) are specific for a single antigen, and therefore are target-oriented. Antibody-based therapy has become a common practice for disease treatments, especially in cancer, arthritis, and infectious diseases. Attempts have also already been made to utilize anti-HBsAg monoclonal antibodies to neutralize HBV in animal models.(5–8,10) Based on these considerations, MAbs of high affinity and specificity were generated against various components of HBsAg and characterized according to established methodology. We believe that some of the monoclonal antibodies reported in this study can be selected for humanization and used as anti-HBV drugs for prophylactic and therapeutic applications in humans.



epatitis B virus (HBV) infections are a major public health concern worldwide. Currently, the World Health Organization estimates that approximately 2 billion people worldwide are infected with HBV with 350 million of these individuals suffering from chronic HBV infections.(1) This results in at least 600,000 HBV-related deaths annually, most commonly due to cirrhosis, liver failure, or hepatocellular carcinoma.(2,3) A safe and effective vaccine against HBV has been available since 1982 and has drastically decreased the global incidence of HBV infection.(1) However, a variety of problems are associated with the vaccine, including non-response, adverse effects, and the emergence of vaccine escape mutants.(4) Approximately 5–10% of individuals vaccinated against HBV will develop no immune response against the virus and subsequently are at risk of acquiring HBV infections.(5) This is especially the case for seniors and immunosuppressed patients, in which the vaccine efficacy has been reported to be less than 70%.(4) Therefore, it is imperative that a more effective means is identified to prevent and to treat HBV infections. HBV is a hepa-associated DNA virus with a genome size of 3.2 kb. The hepatitis B surface antigen (HBsAg) is composed of three proteins, large (L), middle (M), and small (S). These proteins are translated from a single open reading frame and can be divided into three distinctive regions: preS1, preS2, and S.(6) L, M, and S exist in the HBV virion envelope with a proportion of 2/1/7, respectively.(6–9) The HBsAg is the antigen against which protective immunity, following primary infection or vaccination, is generated. Antibodies have been known to neutralize micro-organisms or eliminate cancer cells in current clinical immuno-

Materials and Methods Generation of monoclonal antibodies against various components of human hepatitis B viral surface antigen

Various preparations of HBsAg were used as immunogens for the generation of monoclonal antibodies.(6) Notably, the HBsAg preparations for human vaccine formulations, which contain mixtures of different HBsAg subtypes (ad/ay/rw), were used for immunization in BALB/c mice. Initially, 100 mg of HBsAg in 100 mL phosphate-buffered saline (PBS) was homogenized with an equal volume of complete Freund’s adjuvant, and injected subcutaneously into each mouse. Two and four weeks later, another immunization was performed with the same amount of antigens, except in incomplete Freund’s adjuvant. The mice with the highest antibody titers against HBsAg (as determined by enzyme linked immunosorbent assay [ELISA], as described below) were

UBC Center for Reproductive Health, Vancouver, British Columbia, Canada.


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boosted through intraperitoneal injections with 100 mg of immunogen in PBS. Three days after the boosted injections, spleen cells were removed and fused with Sp-2 myeloma cells. The hybrid cells were cultured in a semi-solid medium containing 1.25% methylcellulose in Iscove’s Modified Dulbecco’s Medium (IMDM) with 2.5% bovine calf serum and hypoxanthine, aminopterin, and thymidine (HAT) selection reagents. They were cultured in a 37C 5% CO2 incubator with saturated humidity. Seven to 10 days later, the hybrid clones were removed and cultured in 96-well microtiter plates in the presence of RPMI1640 medium containing 10% bovine calf serum, HT supplement, and antibodies. After 3 days, microwells with individual hybrid cell colonies were screened for reactivity by ELISA with HBsAg-coated microwells. Antibody-positive hybridomas were further screened by ELISA with microwells coated with different subtypes and/or components of HBsAg. Only those antibodies capable of crossreacting with different subtypes were retained for further evaluations. The isotypes of the established monoclonal antibodies were determined as described previously.(11) All the established hybrid cell lines were used to produce ascitic fluid in mice according to the established procedure.(9) Monoclonal antibodies were purified separately from ascitic fluid by protein A affinity column, as reported previously.(9,11) The subclass and purity of the purified MAbs were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% acrylamide gel). Because HBS-9 is of the immunoglobulin M (IgM) class, it was purified by different procedures.(12) Preparation of enzyme conjugates of anti-HBsAg monoclonal antibodies

For the pairing analysis via sandwich enzyme immunoassays, HBS-8, HBS-18, and HBS-19 were selected and conjugated with horseradish peroxidase (HRP) by lab using standard protocol.(13) Bindings, pairings, and epitope analysis of anti-HBsAg monoclonal antibodies

In order to establish the epitope relationships among the generated anti-HBsAg MAbs, two enzyme immunoassay procedures were used. Initially, microwells coated with different subtypes and components (ad/ay; preS1, preS2, and S) were coated according to standard procedures.(14) Briefly, following the antigen coatings of 1 mg/mL in 40 mM Trishydrochloric acid (HCl, pH 8.0), coated wells were stored overnight at 4C. The microwells were blocked with a blocking solution containing PBS, 0.1% bovine serum albumin (BSA), 1% sucrose, and 0.1% thiomersal (TMS). Enzyme-linked immunosorbent assay were performed by first incubating a serial dilution of MAbs, starting from a dilution of 1 mg/mL, for 1 h at 37C. This was followed by fives washes with PBS and 0.1% Tween-20 (PBS-T), and the addition of alkaline phosphatase (ALP)-labeled goat anti-mouse immunoglobulin G (IgG) for an additional 1 h incubation. The color reaction with p-nitrophenylphosphate as the substrate was determined spectrophotometrically at 405nm. ELISA was used to estimate the dissociation constant (Kd) of each MAb generated against HBsAg with the established methods. To establish the pairing relationship among different MAbs, sandwich enzyme immunoassays (SEIA) were per-


formed. Three MAbs were labeled separately with HRP, including HBS-8, HBS-18, and HBS-19. SEIA was performed by the established two-step assays. Briefly, microtiter wells were coated with 1 mg/mL of the different MAbs in 40 mM Tris-HCl (pH 8.0). 100 mL of the MAb, diluted in 400 mM Tris-HCl (pH 8.0), were dispensed into each well. The wells were left at 4C overnight. The content of the wells was removed and wells were washed with deionized water, followed by blocking of each well with 200 mL of EIA blocking solution (0.1% BSA, 0.1% sucrose, 0.1% TMS in PBS) to minimize any non-specific binding. The two-step sandwich enzyme immunoassays were performed with three separate HRP-conjugates (HBS-8, HBS18, and HBS-19). In the first step, the microtiter wells coated with 1 mg/mL of the different MAbs were first incubated with 100 mL of HBsAg standard at appropriate dilutions for 1 h at 37C. After incubation, the plates were washed five times with PBS and 0.05% Tween (PBS-T). In the second step, 100 mL of horseradish peroxidase (HRP) conjugated HBS-8, HBS-18, or HBS-19 were added to the wells at the appropriate dilutions, and wells incubated again for 1 h at 37C. The same washing procedure was repeated. Microwells were incubated with 100 mL of 3,3¢,5,5¢-tetramethylbenzidine (TMB) reagent (Moss, Inc., Pasadena, MD) and left to stand for 20 min. The color reaction was stopped by adding 50 mL of 2 M HCl in each well and optical density read at 450nm spectrophotometrically with a microplate reader. Alternatively, one-step SEIA was also performed with preparations of HBsAg standards for the co-incubation of antibody-coated wells with HRP-labeled MAbs for 1–3 h incubation at 37C. This was followed by washes and substrate TMB color development with TMB substrate, as described above. To establish epitope relationships with anti-HBsAg MAbs, inhibition studies with anti-HBsAg MAbs were performed with various established two-step SEIA. Initially, 1 mg/mL of different anti-HBsAg MAbs was added in each well to coincubate with HBsAg for 1 h incubation at 37C. This was followed by the same washing procedure mentioned above to remove the excess, unbound MAb and antigen. HRP-labeled HBS-8, HBS-18, and HBS-19 were added separately to wells for an additional hour of incubation at 37C, followed by color development. For one-step SEIA, the HBsAg standards were co-incubated with HRP conjugate of HBS-19 in MAb-coated wells for 1 h incubation at 37C. Results of SEIA were determined by the color development at 450nm, following the addition of onestep TMB reagent for the color development, as described above. The degree of inhibition by a given free MAb can be assessed based on the results of these SEIA assays. Western blot assays

Serum purified HBsAg (General Biologicals, Taiwan), which was used to generate the anti-HBsAg monoclonal antibodies, was subjected to SDS-PAGE on a 10% gel using a discontinuous buffer system with a concentration of about 80 mg/mL of HBsAg. Proteins were transferred to a polyvinyl difluoride (PVDF) membrane for 90 min at 50V using a wet transfer system (BioRad, Hercules, CA). The membrane was then blocked for 1 h in Tris-based saline (TBS) containing 5% non-fat milk and cut into strips. Western blot assays were performed with anti-HBsAg MAbs at 10 mg/mL for 2 h at

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FIG. 1. Relative dose-dependent bindings of anti-HBsAg monoclonal antibodies to microwell-coated standard preparation of HBsAg (subtype ay). room temperature on a shaker. TBS containing 5% non-fat milk was used as the dilution buffer. Normal mouse IgG was used as the negative control. After primary antibody incubation, strips were washed with TBS containing 0.1% Tween-20 (TBST). Strips were then incubated in alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) diluted 1:10,000 in TBS containing 5% non-fat milk for 1 h at room temperature on a shaker. Following washing with TBST, color development was performed with nitroblue tetrazolium in aqueous dimethyl formamide (DMF) containing MgCl2 and 5-bromo-4-chloro-3-indonyl in DMF (BioRad). Results Generation of anti-HBsAg monoclonal antibodies

Several HBsAg preparations were used as immunogens to immunize mice, followed by cell fusions and hybridoma screenings.(15–18) The yield of the hybridomas secreting the desirable monoclonal antibodies varied significantly. For example, the subtype ad preparation of HBsAg yielded a number of MAbs against HBsAg, as determined by ELISA, successfully. On the other hand, the subtype ay preparation yielded few useful MAbs. Following the generation of MAbs, secondary screening was performed to select those crossreacting with all different subtypes of HBsAg. Based on the immunobinding ELISA, only those with sufficiently high affinity and specificity were retained for further studies. Further characterizations of these MAbs involved the use of different immunoassay methods to identify their relative affinity to HBsAg and their mutual pairing and epitope relationships. Among these MAbs, all were shown to be of the IgG subclass, except for HBS-9, which was found to be of the IgM class by use of the subclass determination enzyme immunoassay kit (BioRad). The purity of the affinity-purified MAbs was analyzed by SDS-PAGE and their specificity was confirmed by Western blot assay.

Characterizations and epitope mappings of anti-HBsAg monoclonal antibodies

The relative dose-dependent bindings between the wellcoated HBsAg and different anti-HBsAg MAbs are presented in Figure 1. By using dose-dependent binding ELISA with well-coated HBsAg, the Kd between these MAbs and HBsAg were estimated in the range of nanomoles (nM), as shown in Table 1. Among these MAbs, HBS-L, HBS-2, HBS-18, and HBS-19 were shown to have the highest affinity to HBsAg. Monoclonal antibody-coated wells and HRP conjugations

Table 1. Determination of Dissociation Constants of Anti-HBsAg MAbs to HBsAg Epitope mapping group

Dissociation Pairinga constant (nM) Antibodyb HBS-8 HBS-18 HBS-19

HBS-2* HBS-1* HBS-3* HBS-22* Group 1 HBS-23* HBS-8* Group 2 HBS-19* HBS-18* HBS-28* HBS-9 Group 3 HBS-20* Group 4 HBS-C (not yet determined) HBS-L

1.7 4.2 4.2 6.3 2.5 3.6 2.1 2.1 4.2 N/Ac 7.1 4.0 1.5

++ ++ +/+ /– ++ ++ +/+/+ +/-

+ + + +/++

+ + +/+++ +/+ + +/+++

Classification of epitope-distinct groups of these MAbs are based on results from pairing and competition studies through SEIA. a Signals of SEIA are based on pairing among different MAbs. The signal intensities follow the order + + + , + + , + , + / - , and - . b Antibodies followed by asterisks represent antibodies that have demonstrated positive results also seen with Western blot assay. HBS-9 did not demonstrate positive results due to assay design. HBS-C and HBS-L have not undergone Western blot testing yet. c The dissociation constant of HBS-9 could not be accurately obtained due to experimental design.

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FIG. 2. SEIA results of pairing among different anti-HBsAg MAbs. HBS-8, HBS-18, or HBS-19 were employed separately in a two-step SEIA under the same assay conditions with the same HBsAg standards. The signals from the SEIA were obtained with different well-coated MAbs to pair with either one of the three HRP-labeled MAb conjugates. They are presented in Figure 2 for comparisons. The relationships of mutual pairing among different MAbs can therefore be obtained. Results of such SEIAs are presented qualitatively in Table 1 for comparative purposes. To map the epitope relationships, inhibition studies by use of SEIAs was performed in the presence of free MAbs. The presence and absence of inhibition by a given free MAb in the SEIA would indicate the relative epitope locations of different MAbs due to the mutual competitions. Based on the results of such analysis, these MAbs were generally grouped into at least four different epitope regions. HBS-20 MAb and two others could not be properly mapped with the other MAbs under our current assay conditions.

the fact that HBsAg samples exist in polymeric form or as an aggregate in vaccine formulations, only broad high molecular weight protein bands were observed (Mwt ‡ 50–100 kDa) for all of the monoclonal antibodies employed in our studies (examples are presented in Fig. 4). Discussion

In this study, efforts were made to generate and screen dozens of MAbs against HBsAg. Out of dozens of antiHBsAg MAbs, 11 were found to have sufficiently high affinity and desirable specificity for further studies. Epitope relationships were determined through the established enzyme immunoassay methods. In this study, only the MAbs against the a determinant of HBsAg (S protein) were recovered. Antibodies against the a determinant are generally believed to be essential for HBV clearance and protection against major HBV subtypes as the human humoral immunity is generally directed against the a determinant.(19–21)

Sandwich enzyme immunoassays

Based on the results of the pairing experiments, epitope mappings, and relative affinity of these antibodies to HBsAg, sandwich enzyme immunoassays (SEIA) were formulated. The typical results are presented in Figure 2 for various antibody-coated wells in combinations with HRP-conjugated HBS-8, HBS-18, and HBS-19 in SEIA. From the results of this assay, it can be demonstrated that an SEIA assay with a combination of HBS-9 coated wells and HBS-19-HRP yield the best signal-to-noise ratios and sensitivity with respect to detection of HBsAg in solution. The sensitivity of this assay is in the range of 0.5–1 ng/mL depending on assay conditions. The standard curve for the SEIA is presented in Figure 3. Western blot assays

All of the selected monoclonal antibodies were shown to react with human HBsAg by Western blot assays. In view of

FIG. 3. Standard curve of HBsAg using a SEIA with HBS-9 coated wells and HBS-19-HRP.

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FIG. 4.

Western blot assay examples demonstrating reactivity of anti-HBsAg MAbs.

However, other studies have suggested that MAbs against the a determinant may or may not be sufficient for the formulation of passively immunized anti-HBS vaccines and for immunodiagnostic purposes.(2,6) For example, in cases where the a determinant demonstrates an altered structure, HBV infection may be poorly detected due to a lack of recognition by MAbs against the a determinant.(6) Therefore, experiments are in progress to generate additional MAbs against the preS1 and preS2 regions of HBsAg, which can then serve as the repertoire for the testing of HBV immunodetection and neutralizing activity in the future.(5,7,8,22–29) Through our efforts, it was possible to identify a dozen MAbs against HBsAg for testing of anti-viral activity. Affinity and inhibition studies using binding ELISA and SEIA assays, respectively, were then performed to determine MAb affinity to HBsAg and to group these MAbs into four distinct epitope regions for further analysis (see Table 1). For detailed fine mapping of these MAbs, peptide microarrays of HBsAg would be a desirable choice in the future.(30) By targeting different epitope regions, these MAbs may prove beneficial in the immunoprophylaxis of HBV infection both for the wild-type virus and for vaccine escape mutants.(19) For example, in the study by Shirazi and colleagues, a repertoire of HBV-neutralizing MAbs against multiple epitopes of the a determinant was found to be capable of recognizing and neutralizing virus variants.(19) In this study, efforts were also made to identify the generated anti-HBsAg MAbs for optimal formulations of SEIA to detect HBsAg in solution with a high degree of sensitivity. From this, it was determined that HBS-9 coated wells with HRP-labeled HBS-19 yielded the best SEIA results in terms of detection sensitivity of HBsAg, with a minimal sensitivity range of 0.5–1 ng/mL (see Fig. 3). This result is believed to be due to the multivalent interaction of an IgM subclass monoclonal antibody and the polydeterminant HBsAg.(31) Wells coated with combinations of HBS-9 with other IgG subclass anti-HBsAg MAbs may also be useful and further increase assay sensitivity. In addition, the HBS-8 and HBS-8 pair demonstrated a strong signal, indicating epitope duplications in aggregates of HBsAg. The main objective of this study was to generate murine monoclonal antibodies against human HBsAg-based vaccines. Currently, more studies are required to demonstrate that these MAbs exhibit viral neutralizing activity in vivo and can be humanized for use as vaccines or for passive immunization in humans for immunoprophylaxis of HBV infection.(2) The efficacy of these MAbs to neutralize HBV or

inhibit the attachment of human hepatocytes as target cells remains to be investigated in the future. Nevertheless, it is reasonable to assume that both the antibody affinity and their respective epitope locations are critical factors during the efficacy evaluation of these anti-HBsAg MAbs.(6,22,32) In principle, a mixture of these monoclonal antibodies should mimic the compositions of human anti-HBs antigen antibodies obtained from vaccinated individuals with a positive response. Following standard humanization procedures, these humanized anti-HBsAg monoclonal antibodies can then be used for passive immunization of those non-responsive individuals for temporary protection (2–4 weeks) of HBV infection or as prolonged therapy for those suffering from adverse side effects of traditional HBV medication. A repertoire of these humanized anti-HBsAg MAbs may also be useful in prophylaxis or immunotherapy of virus mutants. Author Disclosure Statement

The authors have no financial interests to disclose. References

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Address all correspondence to: Gregory Lee UBC Center for Reproductive Health 9117 Shaughnessy Street Vancouver, BC V6P 6R9 Canada E-mail: [email protected] Received: October 8, 2014 Accepted: December 23, 2014

Monoclonal antibodies against hepatitis B viral surface antigens and epitope grouping.

Monoclonal antibodies (MAbs) were generated against subtypes (ad/ad/rw) of the human hepatitis B viral surface antigen (HBsAg). Among dozens of antibo...
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