Current Pharmaceutical Design - Volume 24, Issue 11, 2018

Dengue is one of the most important arboviral infections worldwide, infecting up to 390 million people and causing 25,000 deaths annually. Although a licensed dengue vaccine is available, it is not efficacious against dengue serotypes that infect people living in South East Asia, where dengue is an endemic disease. Hence, there is an urgent need to develop an efficient dengue vaccine for this region. Data from different clinical trials indicate that a successful dengue vaccine must elicit both neutralizing antibodies and cell mediated immunity. This can be achieved by designing a multi-epitope peptide vaccine comprising B, CD8+ and CD4+ T cell epitopes. As recognition of T cell epitopes are restricted by human leukocyte antigens (HLA), T cell epitopes which are able to recognize several major HLAs will be preferentially included in the vaccine design. While peptide vaccines are safe, biocompatible and cost-effective, it is poorly immunogenic. Strategies to improve its immunogenicity by the use of long peptides, adjuvants and nanoparticle delivery mechanisms are discussed.

The prevalence of allergic diseases is increasing worldwide. It is estimated that more than 30% of the world population is now affected by one or more allergic conditions and a high proportion of this increase is in young people. The diagnosis of allergy is dependent on a history of symptoms on exposure to an allergen together with the detection of allergen-specific IgE. Accurate diagnosis of allergies opens up therapeutic options. Allergen specific immunotherapy is the only successful disease-modifying therapy for IgE-mediated allergic diseases. New therapeutic strategies have been developed or are currently under clinical trials. Besides new routes of administration, new types of allergens are being developed. The use of adjuvants may amplify the immune response towards tolerance to the antigens. In this review, we analyze different antigen-specific immunotherapies according to administration route, type of antigens and adjuvants, and we address the special case of food allergy.

Despite years of investigation, breast cancer remains a major cause of death worldwide. Phage display is a powerful molecular method in which peptide and protein libraries can be displayed via genetic fusions on the surface of phages. This approach has tremendous potential for biomedical applications and has already facilitated the discovery of specific antibodies, specific antigens, and peptides with potential roles in the diagnosis and treatment of malignancies including breast cancer. In this review, we discuss the new and the latest advancements in the applications of the phage display technique in the provision of immune therapeutics for breast cancer.

Background: Neisseria meningitidis is considered as a dangerous pathogen threatening human health. Nowadays, the new drug target is focused. Toxin antitoxin (TA) system is recently identified as an antimicrobial drug target. Also, in N. meningitidis, iron-uptake system could be an interesting target for drug discovery. Methods: In this study, fbpA and mazE genes were chosen as new antimicrobial targets and treated with antisense peptide nucleic acid (PNA). Firstly, they were evaluated by bioinformatics and then analyzed by experimental procedures. Secondly, the functionality was evaluated by stress conditions. Results: Our results interestingly demonstrated that when fbpA and mazE loci of N. meningitidis were targeted by antisense PNA, 8 μM concentration of fbpA-PNA as well as 30 μM concentration of mazE-PNA inhibited the growth of N. meningitides and were found to be bacteriostatic, whereas 10 μM concentration of fbpA-PNA showed bacteriocidal activity. Conclusion: Our findings demonstrated the bactriocidal activity of fbpA-PNA and bacteriostatic activity of mazEPNA. Therefore, mazE and fbpA genes should be potent antimicrobial targets but further analysis including in vivo analysis should be performed.

Background: The dimeric immunoglobulin (Ig) chimeras used for drug targeting and delivery are preferred biologics over their monomeric forms. Designing these Ig chimeras involves critical selection of a suitable Ig base that ensures dimer formation. In the present study, we systematically analyzed several factors that influence the formation of dimeric chimera. We designed and predicted 608 cytokine-Ig chimeras where we tested the contributions of (1) different domains of Ig constant heavy chain, (2) length of partner proteins, (3) amino acid (AA) composition and (4) position of cysteine in the formation of homodimer. Method: The sequences of various Ig and cytokines were procured from Uniprot database, fused and submitted to COTH (CO-THreader) server for the prediction of dimer formation. Contributions of different domains of Ig constant heavy chain, length of chimeric proteins, AA composition and position of cysteine to the homodimer formation of 608 cytokine-Ig chimeras were tested. Various in silico approaches were adopted for validating the in silico findings. Experimentally we also validated our approach by expressing the chimeric design of shorter cytokine with Ig domain in CHO cells and analyzing the protein by SDS-PAGE. Results: Our results advocate that while the CH1 region and the Hinge region of Ig heavy chain are critical, the length of partner proteins also crucially influences homodimer formation of the Ig-based chimera. We also report that the CH1 domain of Ig is not required for dimer formation of Ig based chimera in the presence of larger partner proteins. For shorter partner proteins fused to CH2-CH3, careful selection of partner sequence is critical, particularly the hydrophobic AA composition, cysteine content & their positions, disulphide bond formation property, and the linker sequences. We validated our in silico observation by various bioinformatics tools and checked the ability of chimeras to bind with the receptors of native protein by docking studies. As a proof of concept, we have expressed the chimeric proteins in CHO cells and found that our design favors the synthesis of dimeric proteins. Conclusion: Our structural prediction study suggests that extra amino acids in the range of 15-20 added to the CH2 domain of Ig is a critical requirement to make homodimer. This information from our study will have implication in designing efficacious homodimeric chimera.

Background: Unintentional passive diffusion of conventional small molecular weight pharmaceuticals across intact membranes of normal healthy cells in tissues and organ systems induces sequelae that limit therapeutic dosage and duration of administration. Selective “targeted” delivery of pharmaceuticals is a molecular strategy that can potentially provide heightened margins-of-safety with greater potency and improved efficacy. Materials and Methods: Monophosphate analogs of fludarabine, gemcitabine, and dexamethasone were combined with a carbodiimide reagent in the presence of imidazole to produce reactive intermediates that were subsequently covalently bound to monoclonal anti-IGF-1R or anti-EGFR IgG-immunoglobulin. The resulting covalent immunopharmaceutical end-products, fludarabine-(5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'- phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were evaluated by SDS-PAGE/chemiluminescent autoradiography (fragmentation/polymerization detection), UV spectrophotometric absorbance (purity; molar-incorporation-index), cell-ELISA (retained selective binding-avidity), and cell vitality-viability (selectively “targeted” anti-neoplastic cytotoxicity). Results: Maximum selectively “targeted” anti-neoplastic cytotoxicity of fludarabine-(5'-phosphoramidate)-[anti- IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was detected at the pharmaceutical-equivalent concentrations of 10-5 M (94.7%), 10-7 M (93.1%), and 10-7 M (64.9%) respectively. Discussion: Organic chemistry reactions were optimized in a template multi-stage synthesis regimen for fludarabine-( 5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-( C21-phosphoramidate)-[anti-EGFR]. Attributes of the synthesis regimen include; [-i-] covalent bonding of pharmaceutical moeities at high molar incorporation indexes, [-ii-] implementation of organic chemistry reactions in a non-dedicated synthesis regimen allowing component substitution and [-iii-] optional preservation of presynthesized amine-reactive pharmaceutical intermediates for on-demand immunopharmaceutical synthesis. Attributes of the covalent immunopharmaceuticals are; absence of any synthetically introduced chemical groups, retained IgG-immunoglobulin binding-avidity and potent selective “targeted” anti-neoplastic cytotoxic potency. Under in-vivo conditions, supplemental anti-neoplastic cytotoxicity is realized through trophic receptor inhibition and activation of multiple cytotoxic host immune responses.