Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

Background

There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.

Results

The cucumarioside A₂-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.

Conclusions

We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.