Elaboration of immune stimulating lipid-saponin subunit antigen carrier based on glycolipid monogalactosyldiacylglycerol from sea macrophytes and triterpene glycosides from Cucumaria japonica

Abstract

The ability of some triterpene glycosides of holothurians: holotoxin A1 from Apostichopus japonicus and a mixture of monosulphated triterpene glycosides from Cucumaria japonica called cucumarioside (CD) to form supramolecular complexes with cholesterol (Chol) and monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC) was studied. A transmission electron microscopy method was used to observe supramolecular lipid-saponin complexes formed by holotoxin A1 and CD with cholesterol in the presence of membrane lipids. The observed supramolecular complexes are tubular nanoparticles with a length of 100-300 nm, an external diameter of 10-16 nm and an internal diameter of 2-6 nm. The formation of tubular nanoparticles was more effective in the presence of MGDG than with PC. Nanoparticles forming in the presence of MGDG are shaped as a tubule, have a constant diameter and a strongly pronounced internal channel. In contrast, PC has no such properties; this lipid is unable to fully integrate in tubular nanoparticles. Based on electron-microscopy data the range of weight ratio of MGDG-Chol-CD was determined as a 1-10:2:3 that provided most effective formation of tubular nanoparticles. Different methods of incorporation of model antigens in complex MGDG-Chol-CD were studied. Influenza haemagglutinin and neuraminidase from commercial vaccine "Influvac" and pore forming protein YompF from Yersinia pseudotuberculosis were used as model antigens. From 54 to 72% of protein of "Influvac" vaccine and 88-92% of YompF were incorporated in supramolecular complexes depending on the method of incorporation. The loss of functional activity of haemagglutinin of vaccine "Influvac" was the result of applying ultrasonic disintegration for incorporation of this protein in complex MGDG-Chol-CD. YompF incorporation in MGDG-Chol-CD complex led to the increased diameter of tubular particles, in the same time incorporation of vaccine "Influvac" antigens produced the "cap" formation at the end of tubules. The possibility of a described supramolecular complex MGDG-Chol-CD to be a carrier for subunit bacterial and viral antigens is shown.