Development, Optimization and Standardization of a method for the analysis of free polysaccharide content in the final multivalent pneumococcal vaccine

Abstract

Streptococcus pneumoniae, a gram-positive bacterium is the causative agent for community acquired pneumonia. The infection results in pus filled alveolar sacs in the lungs leading to chest pain and respiratory distress. To date, about 106 strains have been discovered. With the evolution of each new strain, an increasing resistance to antibiotics is seen. A glance at the morphology reveals the capsular polysaccharide layer as the main virulence factor aiding the survival of the organism. Infants (< 2 years), elderly (> 65 years) and immunocompromised individuals are most susceptible to the disease. The first vaccine developed against S. pneumoniae was a purified capsular polysaccharide-based vaccine. This vaccine generated T- cell independent immune responses- which provided short-term immunity and no memory B- cell formation. However, this vaccine failed to prove its immunogenicity in infants due to presence of immature B-cells. Thus, the capsular polysaccharides were conjugated in-vitro using carrier proteins- CRM197, Diphtheria Toxoid and Tetanus Toxoid, resulting in a polysaccharide-conjugate vaccine. This type of vaccine was able to generate T-cell dependent immune responses leading to formation of memory B-cells. Estimation of free polysaccharide in conjugate vaccine is necessary to evaluate the stability of vaccine that has direct impact on immunogenicity. Traditional methods like ELISA, are laborious and time consuming. Additionally, with the development of higher valent vaccines, it is necessary to look for alternate high throughput methods for estimation of free polysaccharide levels. Therefore, through this project, I aim to develop a high throughput method for detection and quantification of free polysaccharide content from the multivalent pneumococcal vaccine formulations using multiplexed sandwich bead-based assay.