Bacterial vesicles for vaccine application

Abstract

Klebsiella Pneumoniae (K. pneumoniae) is a major cause of high-mortality hospital-associated infections such as pneumonia and bacteraemia. Over the years, K. pneumoniae has been rapidly gaining hypervirulence and antimicrobial resistance, making treatment increasingly challenging. Despite the significant public health impact, no commercial vaccine against K. pneumoniae is currently available. This project aims to develop a novel vaccine strategy using bacterial mimetic extracellular vesicles (BMEVs) with encapsulated bacterial capsule. BMEVs retain the same pathogen-associated molecular patterns (PAMPs) as bacterial natural extracellular vesicles enabling effective immune stimulation while offering better safety, scalability and stability. BMEVs can be engineered to remove components such as lipopolysaccharide (LPS) which can trigger harmful immune responses. The bacterial capsule, the most crucial virulence factor that aids in immune evasion, was targeted for encapsulation to enhance immune recognition. Encapsulation success was confirmed through polysaccharide quantification using the phenol-sulfuric assay and uronic acid assay. BMEV showed significantly higher polysaccharide content (69.45 nmol/well total carbohydrates and 170.9 µg/ml uronic acid) compared to BEV (21.533 nmol/well total carbohydrates and 41.8 µg/ml uronic acid) pointing to successful incorporation of capsule. Vaccine efficacy was assessed through macrophage activation studies, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) analysis of pro- and anti-inflammatory markers expressed and Dot-Enzyme-Linked Immunosorbent Assay (Dot-ELISA). BMEVs induced higher expression of pro-inflammatory markers, and lower expression of anti-inflammatory markers relative to BEVs and successfully activated adaptive immune memory (positive Dot-ELISA results). In conclusion, BMEV encapsulating K. pneumoniae show promise as an effective vaccine strategy. Their ability to stimulate both innate and adaptive immunity highlights their potential for further development in combating the increasingly virulent and drug-resistant K. pneumoniae.