Kostetsky EY et al. (2011), Tubular immunostimulating complex based on cucu...

ECB-ART-42167

J Nanobiotechnology 2011 Sep 02;9:35. doi: 10.1186/1477-3155-9-35.

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Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes.

Kostetsky EY , Sanina NM , Mazeika AN , Tsybulsky AV , Vorobyeva NS , Shnyrov VL .

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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.

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Genes referenced: LOC762715

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	Figure 1. Effect of different adjuvants on relative antibody response to porin YompF. Along the ordinate, percentages with respect to the value for an anti-porin reference serum (serum of mice immunized with pure porin). Along the horizontal axis, the experimental group of animals immunized with: FCA+porin - porin mixed with Freund's complete adjuvant (Sigma); ISCOM+porin - porin incorporated in ISCOMs, prepared as described [11]; TI(PC)+porin - porin incorporated in TI-complex consisting of a cucumarioside A2-2, cholesterol and PC from egg yolk at weight ratio of 3:2:6; TI+porin - porin incorporated in TI-complexes consisting of a cucumarioside A2-2, cholesterol and MGalDG from Ulva fenestrata at weight ratio of 3:2:6. Dose of antigen (denaturated monomeric porin YompF) - 10 μg/mouse.
	Figure 2. Effect of TI-complex on the content of anti-porin antibodies at different doses of porin YompF. Along the ordinate, optical density at 450 nm. Along the horizontal axis, experimental groups of animals immunized with: PBS - phosphate-buffered saline, Porin 0.1/1/10 - pure denaturated monomeric porin YompF at a dose of 0.1/1/10 μg/mouse, TI+porin 0.1/1/10 - denaturated monomeric porin YompF (a dose - 0.1/1/10 μg/mouse, respectively) incorporated in TI-complexes consisting of a cucumarioside A2-2, cholesterol and MGalDG from Ulva fenestrata at weight ratio of 3:2:6.
	Figure 3. Effect of TI-complexes on concentration of INF-γ (A) and IL-1β (B) at different doses of porin YompF. Experimental groups of animals immunized with: PBS - phosphate-buffered saline, Porin 0.1/1 - pure denaturated monomeric porin YompF at dose of 0.1/1 μg/mouse, TI+porin - denaturated monomeric porin YompF (a dose - 0.1 or 1 μg/mouse, respectively) incorporated in TI-complexes consisting of a cucumarioside A2-2, cholesterol and MGalDG from Ulva fenestrata at weight ratio of 3:2:6.
	Figure 4. Electron micrographs of the TI-complexes with different weight ratio of the components. The ratios of cucumarioside A2-2 - cholesterol - MGalDG: A, B - 3:2:6; C, D - 1.5:2:6; E, F - 6:2:6; G, H - 6:2:4; I, J - 6:2:10. I - TEM micrographs of negatively stained samples, bar is 100 nm. II - EFTEM micrographs of unstained samples, bar is 200 nm.
	Figure 5. Destruction of the TI-complex when diluted with phosphate buffer. The weight ratio of cucumarioside A2-2 - cholesterol - MGalDG was of 3:2:6. A - An initial complex, scale bar: 200 nm; B - 5-fold diluted, scale bar: 200 nm; C - 10-fold diluted, scale bar: 200 nm; D - 75-fold diluted, scale bar: 100 nm. Negative staining.
	Figure 6. Content of specific antibodies to the thermostable toxin (A) and YompF (B) from Yersinia pseudotuberculosis. Along the ordinate, optical density at 450 nm. Along the horizontal axis, the experimental group of animals immunized with: PBS - phosphate-buffered saline, TST - pure thermostable toxin, TST + TI - thermostable toxin in the TI-complex, TP - trimeric form of YompF, TP + TI - trimeric form of YompF in the TI-complex. TI-complexes comprised of a cucumarioside A2-2, cholesterol and MGalDG from Ulva fenestrata at weight ratio of 6:2:4.

References [+] :

Aguila, Induction of protective and mucosal immunity against diphtheria by a immune stimulating complex (ISCOMS) based vaccine. 2006, Pubmed