Novel active and passive immunotherapy regimens against nosocomial infections caused by multidrug resistant enterococci

Enterococci are natural inhabitants of the gastrointestinal tract and they are the second most common Gram-positive pathogens responsible for nosocomial infections. Passive and active immunotherapies targeting capsular polysaccharides and surface-associated proteins have emerged as a potential prevention and/or treatment strategy against this opportunistically pathogenic bacterium. In the first part of this study we evaluated the potential use of two protein immunogens from Enterococcus faecium, the secreted antigen A (SagA) and the peptidyl-prolyl cis-trans isomerase (Ppic), as carrier proteins conjugated with the capsular polysaccharide diheteroglycan (DHG) from E. faecalis. These two glycoconjugates (DHG-SagA and DHG-PpiC) could serve as cross-species vaccines against enterococcal infections. For this purpose, rabbits were immunized with the two glycoconjugates, and increasing IgG titers against the immunogens and their components were reported. In addition, the cross-reactivity of the sera was confirmed by evaluation of their in vitro opsonophagocytic killing activity and their immunoreactivity in whole cell ELISA against several E. faecalis and E. faecium strains. The sera, also, conferred protection in mice challenged with the respective enterococcal strains. These results support the potential use of these proteins as carrier proteins and the ability of these glycoconjugate vaccines to provide broader coverage against enterococcal infections. (Publication I) In the second part of this study we developed two mAbs against two well characterized immunogens, DHG and SagA, from E. faecalis and E. faecium, respectively. For the development of these mAbs mice were immunized with the glycoconjugate DHG-SagA, also used in the previous study, and the hybridoma technology was used. The implementation of the opsonophagocytic assay for the selection of the hybridomas was investigated. The two mAbs developed through this process were further characterized for their immunoreactivity by in vitro immunological assays. Both the generated mAbs, DHG.01 and SagA.01, exhibited high affinity and specificity to their targets. In addition, the in vitro opsonophagocytic killing activity of these mAbs against different enterococcal strains was proven, confirming their cross-specificity. These mAbs could serve as a potential immunotherapy against enterococcal infections especially upon humanization. For this purpose, the sequencing of the mAbs and the evaluation of the resulting sequences by the same immunological assays were performed. Finally, the development of these two mAbs upon immunization with DHG-SagA, combined with the previous project, confirm that SagA is a good carrier protein and that the DHG-SagA glycoconjugate is a potential vaccine candidate against prevalent enterococcal species. (Publication II