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

Dendritic cells in cancer immunology and immunotherapy

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.S.K.W. is supported by a European Molecular Biology Organization Long- Term Fellowship (grant ALTF 438– 2016) and a CNIC–International Postdoctoral Program Fellowship (grant 17230–2016). F.J.C. is the recipient of a PhD ‘La Caixa’ fellowship. Work in the D.S. laboratory is funded by the CNIC, by the European Research Council (ERC Consolidator Grant 2016 725091), by the European Commission (635122-PROCROP H2020), by the Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R), by the Comunidad de Madrid (B2017/BMD-3733 Immunothercan- CM), by FIS- Instituto de Salud Carlos III, MCNU and FEDER (RD16/0015/0018-REEM), by Acteria Foundation, by Atresmedia (Constantes y Vitales prize) and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III, the MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015-0505).S

Migration of dendritic cells to the lymph nodes and its enhancement to drive anti-tumor responses.

Better prognoses associated with increased T cell infiltration of tumors, as seen with chimeric antigen receptor (CAR) T cell therapies and immune checkpoint inhibitors, portray the importance and potential of the immune system in controlling tumors. This has rejuvenated the field of cancer immunotherapy leading to an increasing number of immunotherapies developed for cancer patients. Dendritic Cells (DCs) vaccines represent an appealing option for cancer immunotherapy since DCs have the ability to circumvent tolerance to tumors by its adjuvant properties and to induce memory T cells that can become persistent after initial tumor clearance to engage potential metastatic tumors. In the past, DC-based cancer vaccines have elicited only poor clinical response in cancer patients, which can be attributed to complex and a multitude of issues associated with generation, implementing, delivery of DC vaccine and their potential interaction with effector cells. The current review mainly focuses on migration/trafficking of DCs, as one of the key issues that affect the success of DC-based cancer vaccines, and discusses strategies to enhance it for cancer immunotherapy. Additionally, impact of maturation, route of DC delivery and negative effects of tumor microenvironment (TME) on DC homing to LN are reviewed. Moreover, strategies to increase the expression of genes involved in Lymph node homing, preconditioning of the vaccination site, enhancing lymph node ability to attract and receive DCs, while limiting negative impact of TME on DC migration are discussed

Assessment of allergen-specific IgE by immunoblotting method in atopic dermatitis

Background and Objectives. Stimulating the immune system by exposure to various allergens to produce specific IgE has a significant role in the pathogenesis of atopic dermatitis. Identifying disease-causing allergens, prevention of exposure to those allergens, and immunotherapy will play an important role in the treatment of Atopic Disease. The purpose of this study was to determine the common allergens of northwest of Iran in patients with atopic dermatitis that are resistant to treatment. Materials and methods. In this descriptive-analytical study, serum levels of total IgE and frequency of specific IgE were measured by Immunoblotting against 20 common allergens in 150 cases of patients with atopic dermatitis, attending to dermatology and asthma and allergy clinics from 2010 to 2011. The control group consisted of individuals who had been clinically healthy. Results. In the 90% of patients that were included in this study, total IgE levels were higher than in healthy people with mean serum levels of total IgE 227.51 ± 103 IU/ml. 136 patients (90.6%) had specific IgE for at least one allergen. The frequency of positive allergens among the patients who were included in this study were 53.34%, 26.8%, and 19.56% respectively in plants and fungus allergens group, animal allergens group and food allergens group. After avoiding of the allergens (which they had been sensitized to), 60% of patients were cured with immune therapy, and total IgE serum levels in the control group were not increased. Conclusion. Identifying the abundant allergens such as cultivated rye, Timothy grass, house dust mite, birch, cat, horse, potato, dog, egg white, cow milk, in order to advise patients to avoid them or to do immunotherapy and desensitization, is useful in this area

The effect of date palm seed extract as a new potential radioprotector in gamma-irradiated mice

Objective: Date palm seed extract (DPSE) has various compounds revealing antioxidant features. This study aimed to evaluate the radioprotective effect of DPSE in total body gamma irradiation. Materials and Methods: At first, chemical characteristics of DPSE were analyzed by ultraviolet, visible and Fourier transform infrared spectroscopy. Then, the toxicity of DPSE was assessed. For this purpose, 60 mice were divided into five groups, and each of the groups were injected by the doses of 100, 200, 300, 400, and 500 mg/kg, respectively. At the termination of the experiment, mortality rate and weight loss of all mice were evaluated over a period of 30 days. Finally, the radioprotective effect of DPSE was evaluated by dividing 36 mice into three groups: control, test, and placebo and then were irradiated by Cobalt-60. Results: According to the findings, there was no mortality due to DPSE. Furthermore, for the maximum dose of 500 mg/kg, the number of mice surviving at the termination of the experiment with and without injection of DPSE was reported as 83 and 41, respectively. In addition, a significant difference was obtained between radiated mice with and without DPSE injection (P = 0.035). Conclusion: The findings showed that DPSE injected into mice before irradiation has no toxicity and could protect mice from lethal effects of total body irradiation. The use of DPSE as a new radioprotector agent in the human needs further studies, particularly clinical trials. © 2018 Journal of Cancer Research and Therapeutics | Published by Wolters Kluwer-Medknow