title: Immunotherapy Development to Target Herpes Simplex Virus Infections creator: Seyfizadeh, Narges subject: ddc-570 subject: 570 Life sciences subject: ddc-610 subject: 610 Medical sciences Medicine description: The clinical manifestation and pathogenesis of Herpes Simplex infections depend on at least the site of primary infection, age, immune status of the host and the type of HSV. The frequency and severity of infections usually decrease in the immunocompetent population with standard antiviral therapies (e.g. acyclovir/valacyclovir or penciclovir/famciclovir). However, there is a growing concern to induce selection of therapy-resistant HSV strains in immunocompromised patients, stem cell transplant recipients and HIV/HSV co-infected patients, which requires the development of novel anti-HSV therapeutics with different mechanisms of action. In addition, the result of conventional standard therapies is often neither long-lasting nor very effective. Antibody immunotherapy has been demonstrated to be efficacious for the treatment and prevention of viral infections. There is a convincing number of evidence showing the protective role of antibodies against herpes simplex virus (HSV) and amelioration of the severity of HSV-related diseases through neutralizing antibodies in in-vivo models. This may portend a promising future for antibody therapy of HSV infection. Given the fact that there is always a risk of HSV resistance development against a specific therapy, there is still a need to develop new monoclonal antibodies with better efficacies and longer protection. This project’s aim has been to develop fully human therapeutic IgGs targeting glycoprotein B of HSV as a novel therapy. Fully human gB-specific single chain Fvs (scFvs) have been previously selected from patient-specific antibody library repertoires that were generated from lymph nodes of head and neck cancer patients. The project aimed to analyse the potential of selected antibodies for future therapeutic interventions in patients with HSV infections. For this purpose, selected scFvs were reformatted into IgG type molecules and produced, followed by functional in-vitro as well as in-vivo characterization. Antigen specificity of antibodies against HSV-1/2 was analysed by using cell associated HSV glycoproteins (infected Vero cells). The EC50 for binding of H4 (H28) to HSV-1 F- and HSV-2 G- infected Vero cells was 8.5(6.7) nM and 10.9 (9.95) nM, respectively. The neutralization capability of cell-free virus and cell to cell transmission of the antibodies was investigated. Analysis by immunofluorescence microscopy confirmed that cell-to-cell spread was completely inhibited in the presence of gB (HSV) specific mAb H4 and HDIT101 (75μg/ml) and the infection was limited to the initial infection of single cells via virions in the inoculum. In contrast, IgG H28 did not show efficient plaque-reducing effects at any used concentration up to 75μg/ml, which corroborates the limited neutralization efficiency of cell-free virus with this antibody. With the purpose of finding the epitopes targeted by the investigated antibodies, antibody-resistant mutants were propagated in-vitro and the capability of the antibodies to bind to resistant mutants, to neutralize cell-free virus infection and to block virus transmission via cell-to-cell spread were investigated. Escape mutations against antibodies H4 and HDIT101 in HSV-1 conferred amino acid substitutions including R304Q in HDIT101-resistant HSV-1F and R335Q in H4-resistant HSV-1F. According to the data from competitive binding assays and amino acid substitution found in generated viral escape mutants, H4 and H28 bind to different epitopes of gB than HDIT101. In addition, R304Q and R335Q are defining critical amino acids of HDIT101 and H4 epitopes respectively. In addition, Fc effector functions (ADCC, CMC and ADCP) were characterized using infected Vero cells as well as HEK293T stably expressing gB. According to the results, the anti-gB specific antibodies H4, H28 and HDIT101 are all capable of mediating ADCP, while an activity in ADCC or CMC was not detected. Both gB (HSV)-specific mAbs H4 and HDIT101 neutralize HSV-1F/2G with similar efficacies regardless of presence or absence of complement. Conclusively, high affinity, specificity and high neutralization capacity of gB(HSV) specific mAbs (HDIT101, H4) in-vitro and in-vivo independent to Fc functions of antibodies make them potentially promising anti HSV therapies. The results of this thesis suggest the possibility to translate the identified fully human antibodies into a clinical therapy and hence likely provide a novel way to combat HSV infection. date: 2023 type: Dissertation type: info:eu-repo/semantics/doctoralThesis type: NonPeerReviewed format: application/pdf identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/28894/1/Narges%20seyfizadeh.thesis%209.30.2019.pdf identifier: DOI:10.11588/heidok.00028894 identifier: urn:nbn:de:bsz:16-heidok-288946 identifier: Seyfizadeh, Narges (2023) Immunotherapy Development to Target Herpes Simplex Virus Infections. [Dissertation] relation: https://archiv.ub.uni-heidelberg.de/volltextserver/28894/ rights: info:eu-repo/semantics/openAccess rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html language: eng