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The glycoprotein gp350 of EBV - an in vivo antigen and a promising druggable target molecule
The glycoprotein gp350 of EBV - an in vivo antigen and a promising druggable target molecule
Epstein-Barr virus (EBV) infects more than 90% of the human population worldwide. The virus is directly responsible for acute infectious mononucleosis (IM), and is related to several malignancies like Hodgkin lymphoma (HL), Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and posttransplant lymphoproliferative diseases (PTLD). EBV is also associated with various autoimmune diseases like Multiple Sclerosis (MS). The present work aimed at investigating the biology of EBV in long-term infected healthy carriers, thereby focusing on the expression of the lytic envelope glycoprotein gp350. The protein is well known for its crucial role for the viral infection of human B cells and is therefore considered an attractive target for therapeutic approaches, and also a vaccine candidate. The first part of this thesis was dedicated to investigating the presence of gp350 and its potential role in vivo. Since gp350-specificic antibodies can be detected life-long in EBV seropositive individuals, there might be a constant trigger of the immune system by gp350. This hypothesis was corroborated by my observation that certain latently EBV infected cell lines express significant amounts of gp350 on the cell surface and/or release it via extracellular vesicles (EVs). Moreover, I could show for the first time that the gp350 protein is present on a subset of B cells from healthy EBV-seropositive donors, and that the protein is also present in sera. Intriguingly, I could observe a direct correlation between antibody and protein titers. Moreover, I could show that gp350 carrying vesicles isolated from donors reactivate autologous gp350-specific CD4+ T cells, implicating a potential immunological function, e.g., in maintaining viral latency and stable co-existence with the host. This is the first time that gp350 was detected in the blood of healthy donors. Additional studies are needed to elucidate its origin, exact status and biological function. Therapeutic monoclonal antibodies are a success story in modern medicine. Of particular potential are fully human IgG antibodies which combine the unprecedented specificities of antibodies with a very low immunogenicity. However, fully human antibodies targeting gp350 are not existent. In the second part of this thesis, we therefore developed a new technique to generate fully human IgG antibodies in humanized mice in collaboration with Prof. R. Stripecke (Med. Hochschule Hannover). These humanized mice were immunized with Epstein-Barr-virus like particles (EB-VLPs), and splenocytes were then immortalized with EBV in vitro. The supernatants of the EBV immortalized B cells were tested for human gp350-specific antibodies with neutralizing potential. The most promising antibody from the biological and neutralization assays was cloned. The immunoglobulin sequence was obtained, and classified the antibody as human IgG3, an Ig class strongly related with infectious diseases. The collaboration with the Stripecke lab resulted in the first fully human IgG antibodies generated in humanized mice. In summary, the present study showed for the first time that gp350 is present in sera from EBV-positive healthy carriers without previous viral reactivation. Moreover, this protein is able to reactivate specific cellular immune responses. Finally, a fully human IgG3 gp350-specific antibody was produced, that may be developed into a therapeutic tool for the treatment of EBV-associated diseases.
EBV, gp350, human antibody
Fiestas Cárcaba, Elena
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Fiestas Cárcaba, Elena (2021): The glycoprotein gp350 of EBV - an in vivo antigen and a promising druggable target molecule. Dissertation, LMU München: Faculty of Biology
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Abstract

Epstein-Barr virus (EBV) infects more than 90% of the human population worldwide. The virus is directly responsible for acute infectious mononucleosis (IM), and is related to several malignancies like Hodgkin lymphoma (HL), Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and posttransplant lymphoproliferative diseases (PTLD). EBV is also associated with various autoimmune diseases like Multiple Sclerosis (MS). The present work aimed at investigating the biology of EBV in long-term infected healthy carriers, thereby focusing on the expression of the lytic envelope glycoprotein gp350. The protein is well known for its crucial role for the viral infection of human B cells and is therefore considered an attractive target for therapeutic approaches, and also a vaccine candidate. The first part of this thesis was dedicated to investigating the presence of gp350 and its potential role in vivo. Since gp350-specificic antibodies can be detected life-long in EBV seropositive individuals, there might be a constant trigger of the immune system by gp350. This hypothesis was corroborated by my observation that certain latently EBV infected cell lines express significant amounts of gp350 on the cell surface and/or release it via extracellular vesicles (EVs). Moreover, I could show for the first time that the gp350 protein is present on a subset of B cells from healthy EBV-seropositive donors, and that the protein is also present in sera. Intriguingly, I could observe a direct correlation between antibody and protein titers. Moreover, I could show that gp350 carrying vesicles isolated from donors reactivate autologous gp350-specific CD4+ T cells, implicating a potential immunological function, e.g., in maintaining viral latency and stable co-existence with the host. This is the first time that gp350 was detected in the blood of healthy donors. Additional studies are needed to elucidate its origin, exact status and biological function. Therapeutic monoclonal antibodies are a success story in modern medicine. Of particular potential are fully human IgG antibodies which combine the unprecedented specificities of antibodies with a very low immunogenicity. However, fully human antibodies targeting gp350 are not existent. In the second part of this thesis, we therefore developed a new technique to generate fully human IgG antibodies in humanized mice in collaboration with Prof. R. Stripecke (Med. Hochschule Hannover). These humanized mice were immunized with Epstein-Barr-virus like particles (EB-VLPs), and splenocytes were then immortalized with EBV in vitro. The supernatants of the EBV immortalized B cells were tested for human gp350-specific antibodies with neutralizing potential. The most promising antibody from the biological and neutralization assays was cloned. The immunoglobulin sequence was obtained, and classified the antibody as human IgG3, an Ig class strongly related with infectious diseases. The collaboration with the Stripecke lab resulted in the first fully human IgG antibodies generated in humanized mice. In summary, the present study showed for the first time that gp350 is present in sera from EBV-positive healthy carriers without previous viral reactivation. Moreover, this protein is able to reactivate specific cellular immune responses. Finally, a fully human IgG3 gp350-specific antibody was produced, that may be developed into a therapeutic tool for the treatment of EBV-associated diseases.