Lyme disease, caused by Borrelia burgdorferi and transmitted by black-legged ticks, is a major public health concern in the United States, particularly in the Northeast, with no vaccine currently available. While antibiotics are effective post-infection if administered within 72 hours, no prophylactic treatments currently exist. Given the growing public health risk, this study aimed to develop a strategy for generating outer membrane vesicle (OMV)-based vaccine candidates with particle sizes that mimic those of B. burgdorferi (~1 µm) to enhance immunogenicity.
OMVs are spherical nanoparticles (20-250 nm) secreted by Gram-negative bacteria that can be engineered to express B. burgdorferi's antigens and elicit an immune response. Previous research suggests that particle geometry influences immune activation. Viruses, for example, have sizes ranging from 20-400 nm, similarly to OMVs, whereas bacteria such as B. burgdorferi are considered on the order of microscale particles (~1 µm). In order to use OMVs as a vaccine platform against Lyme disease, it is imperative to understand whether larger OMV particle sizes have an influence on the immune response. Thus, we investigated the efficiency of crosslinking in producing particle sizes larger than the native OMVs produced by bacteria, and how the ratio of reactants and molecular weight of homofunctional PEG di(carboxylic acid) linkers influences these results. OMVs were produced from genetically modified E. coli cultures, characterized for protein content and amine group availability, and subsequently crosslinked using linkers of varying molecular weights and geometry (20 kDa and 40 kDa, linear and branched) in the presence of DMTMM, a coupling agent. Multi-Angle Dynamic Light Scattering (MADLS) analysis confirmed successful crosslinking, with particle sizes ranging from 400 nm to over 1,700 nm. Reactions employing 40 kDa PEG produced the largest particles compared to other linkers for the same OMV-to-linker ratio, indicating that higher linker molecular weight leads to the formation of larger particles. Across all crosslinking reactions, no particle precipitation is observed, indicating that the OMVs remain stable. These findings demonstrate that PEG-based crosslinking is a viable approach for tuning OMV size while maintaining membrane and protein stability in solution.
Acknowledgements: This work was partially funded by NSF-ERI: "Evaluation of the Immune Response to Lyme Disease Antigens Using Bacterially-Derived Outer Membrane Vesicles" (CBET 2347479).