In silico designing of vaccine candidate against Clostridium difficile.

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll-Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.

Optimization of a refolding step for a therapeutic fusion protein in the quality by design (QbD) paradigm.

Production of biotech therapeutics in Escherichia coli involves protein expression as insoluble inclusion bodies that need to be denatured and the resulting protein refolded into the native structure. In this paper, we apply a Quality by Design approach using Design of Experiments for optimization of the refolding process for a recombinant biotech therapeutic, granulocyte colony stimulating factor. First, risk analysis was performed to identify process parameters that require experimental examination. Next, the chosen parameters were examined using a fractional factorial screening design. Based on the results of this study, parameters that have significant effect on refold yield and product quality were identified and examined using a full factorial Design of Experiments for their interactions. The final model was statistically significant and delivered a refolding yield of 77%. Further, kinetics of refolding was evaluated under optimal conditions and was found to be of first order with a rate constant of 0.132/min. Design space was established for the three parameters for a given permissible range of yield, protein concentration, and purity. The primary objective of this paper is to provide a roadmap for implementing Quality by Design for development of a protein refolding step.