Author : Abhishek R. Vartak
Publisher :
Page : 194 pages
File Size : 13,61 MB
Release : 2019
Category : Antigens
ISBN :
Book Description
Over the last few decades, immuno-therapeutics have become an integral part of our health-care. We have come a long way since the pioneering work of the smallpox and anthrax vaccination. The current generic design of a vaccine platform includes isolated polysaccharide or peptide antigens, an immuno-adjuvant, and a carrier. The dissertation describes the development of an anti-cancer and anti-bacterial vaccine design and our efforts to improve its efficacy further. Use of the pattern recognition receptor (PRR) ligands to activate antigen presenting cells (APCs) is one of the pathway to augment the immunogenicity of peptide antigens. PRRs such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs) etc. bind to lipopolysaccharide, lipopeptide, and other pathogen-associated molecules to facilitate APC maturation. We utilized Pam3CysSK4, a synthetic lipopeptide related to the N-terminus of Escherichia coli lipoprotein and a well-known TLR-2 ligand. The peptide portion of the molecule, SK4 was synthesized using a solid phase peptide synthesis (SPPS) method with the Fmoc strategy. C-terminus of Pam3CysSK4 was coupled with azadibenzocyclooctyne-amine (DBCO-amine) in a presence of propylphosphonic anhydride (T3P) to afford conjugatable strained-alkyne terminated immuno-adjuvant. We have synthesized a putative anticancer immuno-therapeutic molecule consisting of a MUC-1 glyco-peptide bearing a tumor-associated carbohydrate antigen (TACA) along with a cytotoxic T-cell (CTL) epitope conjugated to Pam3CysSK4. A strain promoted alkyne-azide cycloaddition (SPAAC) was used to conjugate Pam3CysSK4 to the glycopeptide. The construct was incorporated into liposomes by the extrusion method. In addition, we have utilized L-rhamnose and mouse IgG3 Fc ligands for targeting antigen presenting cells (APC). Liposomal delivery of synthesized construct in C57BL6 mice showed increased priming of both CD4 and CD8 T-cells toward the MUC1-Tn cancer antigen. The primed CD8 T-cells were capable of tumor cell killing. Pseudomonas aeruginosa, a gram-negative bacterium is an opportunistic human pathogen with a leading cause of death among Cystic Fibrosis (CF) patients. With intrinsic antibiotic resistance, P. aeruginosa is also associated with hospital acquired pneumonia and blood infections. We have synthesized the valuable components required for immuno-therapeutics such as serotype independent outer core fragments of P. aeruginosa lipopolysaccharide (LPS) and the immunogenic domains of outer membrane protein F (OprF) of P. aeruginosa using chemical and biological methods.Pseudomonas aeruginosa, a gram-negative bacterium is an opportunistic human pathogen with a leading cause of death among Cystic Fibrosis (CF) patients. With intrinsic antibiotic resistance, P. aeruginosa is also associated with hospital acquired pneumonia and blood infections. We have synthesized the valuable components required for immuno-therapeutics such as serotype independent outer core fragments of P. aeruginosa lipopolysaccharide (LPS) and the immunogenic domains of outer membrane protein F (OprF) of P. aeruginosa using chemical and biological methods. The trisaccharide and tetrasaccharide fragments from the outer core domain of P. aeruginosa lipopolysaccharide (LPS) common to glycoform I and II were synthesized using TBDPS-protected hydroquinone (TPH), a novel reducing end capping group. We have developed a flexible synthetic strategy with Lev- and Fmoc- protected thioglycosides to assemble these complex oligosaccharide fragments. To gain access to oligosaccharide components in a short period of time, we demonstrated oligosaccharide synthesis on a commercially available, low cost, high molecular weight poly (2-hydroxyethylmethylacrylate) (pHEMA) using a very accessible, and novel photo-cleavable linker. This is a first report of pHEMA as a soluble platform for oligosaccharide synthesis. High polymer recoveries were achieved by precipitation with pHEMA. The soluble support afforded the synthesis of two trisaccharides in 48% and 39% overall yield respectively and was found to be compatible with a glycosylation and common de-protection conditions. The photo-cleavable linker was used to access 4-hydroxyphenyl glycosides which have a locked anomeric position to facilitate purification.
