Epitope Mapping of Tetanus Toxin by Monoclonal Antibodies: Implication for Immunotherapy and Vaccine Design

Tetanus as a life-threatening disease is characterized by muscle spasm. The disease is caused by the neurotoxin of Clostridium tetani. Active form of tetanus neurotoxin is composed of the light chain (fragment A) and the heavy chain. Fragment A is a zinc metalloprotease, which cleaves the neuronal soluble N-ethylmaleimide-sensitive attachment receptor (SNARE) protein, leading to the blockade of inhibitory neurotransmitter release and subsequent generalized muscular spasm. Two functional domains of the heavy chain are fragment C, which is required for neuronal cell binding of the toxin and subsequent endocytosis into the vesicles, and fragment B, which is important for fragment A translocation across the vesicular membrane into the neuronal cytosol. Currently, polyclonal immunoglobulins against tetanus neurotoxin obtained from human plasma of hyper-immunized donors are utilized for passive immunotherapy of tetanus; however, these preparations have many disadvantages including high lot-to-lot heterogeneity, possibility of transmitting microbial agents, and the adverse reactions to the other proteins in the plasma. Neutralizing anti-tetanus neurotoxin monoclonal antibodies (MAbs) lack these drawbacks and could be considered as a suitable alternative for passive immunotherapy of tetanus. In this review, we provide an overview of the literature discussing epitope mapping of the published neutralizing MAbs against tetanus toxin. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

Inhibitory Effect of Polyclonal Antibodies Against HER3 Extracellular Subdomains on Breast Cancer Cell Lines

OBJECTIVE: Human epidermal growth factor receptor 3 (HER3) is a unique member of the tyrosine kinase receptors with an inactive kinase domain and is the preferable dimerization partner for HER2 which lead to potent tumorigenic signaling. METHODS: In this study, the expression plasmids coding for the human HER3 subdomains were transfected into CHO-K1 cells. Produced proteins were characterized by ELISA and SDS-PAGE. Rabbits were immunized and produced polyclonal antibodies (pAbs) that were characterized by ELISA, Immunoblotting and flowcytometry and their inhibitory effects were assessed by XTT on BT-474 and JIMT-1 breast cancer cell lines. RESULT: The recombinant subdomains were highly immunogenic in rabbits. The pAbs reacted with the recombinant subdomains as well as commercial HER3 and the native receptor on tumor cell membranes and could significantly inhibit growth of Trastuzumab sensitive (BT-474) and resistant (JIMT-1) breast cancer cell lines in vitro. CONCLUSION: It seems that HER3 extra cellular domains (ECD) induce a strong anti-tumor antibody response and may prove to be potentially useful for immunotherapeutic applications.

Human Leukocyte Antigens Influence the Antibody Response to Hepatitis B Vaccine

Hepatitis B virus (HBV) infection and its sequelae such as cirrhosis and hepatocellular carcinoma has remained a serious public health problem throughout the world. The WHO strategy for effective control of HBV infection and its complications is mass vaccination of neonates and children within the framework of Expanded Programme on Immunization (EPI). Vaccination with hepatitis B surface antigen (HBsAg) induces protective antibody response (anti-HBs ≥ 10 IU/L) in 90-99% of vaccinees. The lack of response to HBsAg has been attributed to a variety of immunological mechanisms, including defect in antigen presentation, defect in HBsAg-specific T and/or B cell repertoires, T-cell suppression, increase in the regulatory T cell count, lack of necessary help of T-cells for production of anti-HBs by B cells, defect in Th1 and/or Th2 cytokine production and selective killing of HBsAg-specific B-cells by human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes. The HLA complex plays an important role in many of these immunological processes.A variety of HLA class I, II, and III alleles and antigens have been reported to beassociated with antibody response to HBsAg vaccination in different ethnic populations. Moreover, some HLA haplotypes were also associated with responsiveness to HBsAg.In this review the association of the HLA specificities with antibody response to hepatitis B (HB) vaccine is discusse

Optimization of an efficient cell culture hepatitis B infection system for assessment of hepatitis B virus neutralizing monoclonal antibodies.

Background: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. Objective: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. Methods: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. Results: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. Conclusion: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics

Monoclonal antibodies to various epitopes of HBs antigen inhibit HBV infection.

BACKGROUND AND AIM: Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating HBV particles and to prevent HBV infection. It has been proposed, that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity. METHODS: Nine murine HBsAg specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro. RESULTS: All MAbs were able to inhibit the establishment of HBV infection in a dose dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into 3 subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (<40%) to variants with mutations within the first loop of "a" determinant, five MAbs displayed negligible binding to variants with mutations within the second loop and one MAb lost its binding to variants having mutations in both loops of the "a" determinant. CONCLUSIONS: Our results indicate that antibodies against different epitopes of the "a" determinant of HBsAg are able to neutralize HBV. It seems that mutations within a single or a limited number of amino acids within this determinant can hardly result in viral escape. These results have important implications for the development of antibody-based therapies against HBV

Molecular Characterization of Murine Monoclonal Antibody Variable Regions Specific for Hepatitis B Surface Antigen

Background: Hepatitis B virus (HBV) surface antigen (HBsAg) induces a vigorous neutralizing antibody response, which causes effective protection against HBV infection. Little is known about the profile of variable region genes of immunoglobuline heavy (VH) and light (VL) chains rearranged in anti-HBs antibodies, and also the possible association of this profile with specificity pattern of these antibodies to mutant forms of HBsAg. Aims: The present study determined the nucleotide sequence of VH and VL genes of mouse monoclonal antibodies (MAbs) generated against HBsAg. Methods: Hybridoma clones secreting anti-HBsAg MAbs were developed from hyperimmunized Balb/c mice. VH and VL gene sequences of all MAbs were determined by amplifying the genes using a panel of VH and VL family specific primers by reverse transcription polymerase chain reaction. The reactivity pattern of anti-HBs MAbs with different mutant forms of HBsAg was evaluated by enzyme-linked immunosorbent assay, and then the profile of antigen specificity and its association to VH/VL family expression was analyzed. Results: Twenty-three murine hybridomas producing anti-HBs MAbs were generated. Nucleotide sequence analysis revealed that heavy chains of these MAbs were encoded by IGHV genes from the HV1 (52), HV6 (22), HV5 (17), and HV3 (9) families in combination with IGHJ2 (57), HJ1 (26), and HJ4 (17). Besides, 56 of MAbs used IGHD1 genes in their VDJ rearrangements. Concerning the IGKV gene, 26 and 22 of clones used KV4 and KV10 gene families, while the rest of the clones used KV8, KV6, KV1, KV12, and KV14 gene families. Besides, the IGKJ2 gene was the most represented KJ gene (43). No association was found between the specificity pattern of MAbs to mutant forms of HBsAg with their preferential V, D, and J genes usage for most of MAbs. Conclusion: The data suggest that heavy chains of anti-HBs MAbs preferentially use genes derived from the IGHV1, IGHV6, IGHJ2, and IGHD1 families. In contrast to heavy chains, which predominantly use four families of IGHV genes, light chains use more diverse IGKV gene families. © Copyright 2015, Mary Ann Liebert, Inc. 2015

Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).

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

T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is a global health threat responsible for 880,000 deaths per year. Current antiviral therapies control but rarely eliminate the virus, and leave chronic HBV carriers at risk to develop hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring anti-viral T-cell immunity to achieve HBV elimination in chronically infected patients will help to cure HBV. METHODS: We constructed bispecific T-cell engager antibodies that are designed to induce anti-viral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and cluster of differentiation 3 or 28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of T-cell response was determined by detection of pro-inflammatory cytokines, effector function by cytotoxicity and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBV envelope-positive and -negative hepatoma cells. RESULTS: The two T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent anti-viral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. CONCLUSION: This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. LAY SUMMARY: T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies they on the hand bind HBV envelope proteins displayed on the surface of HBV-infected cells or HBV-positive hepatoma and on the other hand attract and stimulate T cells by binding CD3 or CD28 on the T cell. Hereby, they activate a potent antiviral and cytotoxic response resulting in the elimination of HBV-positive cells. Their potential to activate T cells to resolve HBV infection renders T-cell engagers interesting candidates for the therapy of chronic hepatitis B and HBV-associated hepatocellular carcinoma