Immunotherapy Development to Target Herpes Simplex Virus Infections

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

Interleukin-13 as an important cytokine: A review on its roles in some human diseases

Interleukin-13 (IL-13) as a pleiotropic cytokine acts through the IL-13Ra1/IL-4Ra complex to induce activation responses which contribute to the inflammatory diseases. Genetic polymorphisms in IL-13 and its receptor components have been proved to be associated with higher disease prevalence rates. Animal models such as in IL-13 deficient mice and transgenic animals also have been confirmed the critical role of this cytokine in the immune responses, mostly by IL-13 neutralization and IL-13/IL-4 dual neutralization strategies. This review highlights IL-13 structure as well as its pivotal roles in the normal physiologic and pathologic states. It is followed by a section on the recent findings on IL-13 receptors and signalling mechanisms to briefly summarize its functions in the immune systems. IL-13 roles in the human diseases such as asthma, systematic sclerosis, and some inflammatory diseases are described concisely. Finally some of the ongoing therapeutic applications are presented to comprehensively review IL-13 mediator roles

Development of a highly effective combination monoclonal antibody therapy against Herpes simplex virus.

BACKGROUND Infections with Herpes simplex virus (HSV)-1 or -2 usually present as mild chronic recurrent disease, however in rare cases can result in life-threatening conditions with a large spectrum of pathology. Monoclonal antibody therapy has great potential especially to treat infections with virus resistant to standard therapies. HDIT101, a humanized IgG targeting HSV-1/2 gB was previously investigated in phase 2 clinical trials. The aim of this study was to develop a next-generation therapy by combining different antiviral monoclonal antibodies. METHODS A lymph-node derived phage display library (LYNDAL) was screened against recombinant gB from Herpes simplex virus (HSV) -1 and HDIT102 scFv was selected for its binding characteristics using bio-layer interferometry. HDIT102 was further developed as fully human IgG and tested alone or in combination with HDIT101, a clinically tested humanized anti-HSV IgG, in vitro and in vivo. T-cell stimulating activities by antigen-presenting cells treated with IgG-HSV immune complexes were analyzed using primary human cells. To determine the epitopes, the cryo-EM structures of HDIT101 or HDIT102 Fab bound to HSV-1F as well as HSV-2G gB protein were solved at resolutions < 3.5 Å. RESULTS HDIT102 Fab showed strong binding to HSV-1F gB with Kd of 8.95 × 10-11 M and to HSV-2G gB with Kd of 3.29 × 10-11 M. Neutralization of cell-free virus and inhibition of cell-to-cell spread were comparable between HDIT101 and HDIT102. Both antibodies induced internalization of gB from the cell surface into acidic endosomes by binding distinct epitopes in domain I of gB and compete for binding. CryoEM analyses revealed the ability to form heterogenic immune complexes consisting of two HDIT102 and one HDIT101 Fab bound to one gB trimeric molecule. Both antibodies mediated antibody-dependent phagocytosis by antigen presenting cells which stimulated autologous T-cell activation. In vivo, the combination of HDIT101 and HDIT102 demonstrated synergistic effects on survival and clinical outcome in immunocompetent BALB/cOlaHsd mice. CONCLUSION This biochemical and immunological study showcases the potential of an effective combination therapy with two monoclonal anti-gB IgGs for the treatment of HSV-1/2 induced disease conditions