**3. Significance of capsular polysaccharide A**

Previous studies on the CPSA molecule have revealed it to be effective as a therapeutic molecule, the tetrasaccharide repeat needs to be a polymer of ten repeat units or longer. If shorter than that, it fails to activate the immune system [64, 65]. CPSA operon encodes for a flippase wzx, which takes the repeat unit and flips it from the cytoplasmic space to the periplasmic space, where the polymerase wzy, utilizes the repeat unit and polymerizes it till it reaches a length of approximately 130 repeat units [65, 66].

Recent successes in cancer vaccines and in monoclonal antibody cancer immunotherapy have given the impetus towards development of vaccines targeting cancer-associated carbohydrates. The Andreana group have been developing carbohydrate immunogens to elicit a T-cell dependent immune response. CPSA is known to stimulate a strong T-cell mediated response. They have successfully linked CPSA to the tumor-associated carbohydrate antigen (TACA), Sialyl Thomsen-nouveau (STn) and were able to obtain a robust immune response to the antigen [67–71]. They have further reported total synthesis of the CPSA unit in 19 steps with a final yield of 5% [67]. Chemoenzymatic assembly is a faster and scalable approach, that can be used as an alternative or in combination with chemical synthesis. CPSA

obtained in this way, can then be linked to the antigen. The chemoenzymatic method has also been used to create capsule polysaccharide based glycoconjugates for *Neisseria meningitidis* serotypes A, C and X [72–74]. In some cases, recombinant glycosyltransferases can be used to assemble non-native carbohydrate antigens in compliant host organisms like *Escherichia coli*. This method has been successfully used by the Brendan W. Wren lab for the in vivo assembly of capsular polysaccharide from several serotypes of *Streptococcus pneumoniae*. A similar approach is also currently being applied with respect to CPSA, wherein the whole CPSA biosynthesis and assembly will be done inside *E. coli*. This will allow to have access to longer oligomers of CPSA, which can be helpful in studies towards size requirement in eliciting immune response. So far there have been no reports of CPSA unit being polymerized synthetically.
