Development Of Outer Membrane Vesicles Vaccine Formulations And The Safety And Efficacy Evaluation In Vitro

Creative Commons License

Aral D., Varan G.

Extracellular Vesicles Conference 2026, Ankara, Turkey, 5 - 06 June 2026, pp.101, (Summary Text)

  • Publication Type: Conference Paper / Summary Text
  • City: Ankara
  • Country: Turkey
  • Page Numbers: pp.101
  • Hacettepe University Affiliated: Yes

Abstract

Antimicrobial resistance (AMR) causes approximately 700,000 deaths annually, and without necessary measures, it is projected that deaths could reach 10 million per year by 2050. In Turkey specifically, there are significantly higher cases of AMR compared to European countries, and difficulties are observed in treating infections caused by common bacteria such as Escherichia coli. The absence of an approved vaccine against this pathogen continues to pose challenges not only in treatment but also in prevention of the disease. In this study with the aim of developing a nanoparticle-based subunit vaccine formulation against AMR in Escherichia coli and evaluate its in vitro safety and effectiveness three different outer membrane vesicles (OMV) isolation methods were compared regarding to yield; protein amount, average size distribution and polydispersity index (PDI). Firstly E. coli was cultured in LB broth in shaking incubator. Then the bacteria were removed using centrifugation twice. The supernatant was filtered passing through syringe filters. The filtrate was divided into three groups for OMV isolation: ultracentrifugation, tangential flow filtration and ion exchange affinity column chromatography OMV Isolation Kit. The isolated OMVs were characterized regarding to their size and PDI by using DLS. The protein amounts of the OMVs were determined using BCA assay. Kit group was chosen for further experiments due to the highest yield in BCA in adding to optimum size and PDI values. The OMVs were imaged with electron microscopy. The effect of OMVs on cell viability was tested using L929 cells with MTT assay. The detected maximum non-toxic amount of OMV was used for vaccine formulations. As keeping the OMV amount stable, different kinds and amounts of adjuvants (MPLA, Alum) were added and the vaccine formulations were prepared. The prepared vaccine formulations were tested for stability for 30 days measuring average size distribution and zeta potential of the formulations at pre-determined time points. Also cell viability tests were performed using L929 cell line and CaCo-2:HT29 co-culture conditions. For co-culture studies CaCo-2 and HT29 cells were seeded onto the apical sides of thincerts with the ratio of 7:3 respectively. In order to evaluate in vitro efficacy of the vaccine formulations the cytokine amounts were measured using commercially available ELISA kits from the supernatants of THP-1 and CaCo-2:HT29 co-culture. It was shown that OMV isolated from E. coli can be used as a potential vaccine (less than 200 nm within PDI 0.2 and 1000 μg/mL protein amount) against AMR.

Acknowledgment:

This study was supported by TÜBİTAK BİDEB 2247-D National Early Stage Researchers Program and Dilara Aral is a recipient of a grant from TÜBİTAK BİDEB 2210/C National MSc/MA Scholarship Program in the Priority Fields in Science and Technology.