Synthesis, Formulation, and Adjuvanticity of Monodesmosidic Saponins with Olenanolic Acid, Hederagenin and Gypsogenin Aglycones, and some C-28 Ester Derivatives.

In an attempt to discover a new synthetic vaccine adjuvant, the glycosylation of hederagenin, gypsogenin, and oleanolic acid acceptors with di- and trisaccharide donors to generate a range of mimics of natural product QS-21 was carried out. The saponins were formulated with phosphatidylcholine and cholesterol, and the structures analyzed by transmission electron microscopy. 3-O-(Manp(1→3)Glcp)hederagenin was found to produce numerous ring-like micelles when formulated, while C-28 choline ester derivatives preferred self-assembly and did not interact with the liposomes. When alone and in the presence of cholesterol and phospholipid, the choline ester derivatives produced nanocrystalline rods or helical micelles. The effects of modifying sugar stereochemistry and the aglycone on the immunostimulatory effects of the saponins was then evaluated using the activation markers MHC class II and CD86 in murine bone marrow dendritic cells. The most active saponin, 3-O-(Manp(1→3)Glcp)hederagenin, was stimulatory at high concentrations in cell culture, but this did not translate to strong responses in vivo.

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems.

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.

Comparison of chitosan nanoparticles and chitosan hydrogels for vaccine delivery.

In this work the potential of chitosan nanoparticles (CNP) and thermosensitive chitosan hydrogels as particulate and sustained release vaccine delivery systems was investigated. CNP and chitosan hydrogels were prepared, loaded with the model protein antigen ovalbumin (OVA) and characterised. The immunostimulatory capacity of these vaccine delivery systems was assessed in-vitro and in-vivo. Particle sizing measurements and SEM images showed that optimised OVA-loaded CNP had a size of approximately 200 nm, a polydispersity index < 0.2, and a positive zeta-potential of approximately 18 mV. The amount of OVA adsorbed onto CNP was high with an adsorption efficacy of greater than 96%. Raman spectroscopy indicated conformational changes of OVA when adsorbed onto the surface of CNP. Uptake of the dispersions and immunological activation of murine dendritic cells in-vitro could be demonstrated. Investigation of the release of fluorescently-labelled OVA (FITC-OVA) from CNP and chitosan hydrogels in-vitro showed that approximately 50% of the total protein was released from CNP within a period of ten days; release of antigen from chitosan gel occurred in a more sustained manner, with < 10% of total protein being released after 10 days. The slow release from gel formulations may be explained by the strong interactions of the protein with chitosan. While OVA-loaded CNP showed no significant immunogenicity, formulations of OVA in chitosan gel were able to stimulate both cell-mediated and humoral immunity in-vivo.

Immunostimulatory lipid implants containing Quil-A and DC-cholesterol.

Biocompatible lipid implants which promote the sustained release of antigen have potential as novel vaccine delivery systems for subunit antigen as they may reduce or remove the requirement for multiple administrations. Of particular interest are sustained release systems that release antigen incorporated into particles. Previous work has demonstrated that lipid implants prepared from phosphatidylcholine, cholesterol, the adjuvant Quil-A, and ovalbumin as the model antigen could stimulate an immune response equivalent to that induced by a prime and boost with a comparable injectable vaccine. However, entrapment of antigen into particles released from the implant was low. Therefore the aim of this study was to firstly determine if the inclusion of a cationic derivative of cholesterol, DC-cholesterol, into the implants increased antigen entrapment and immunogenicity, and secondly, if a cationic implant could induce at least a comparable immune response as compared to a prime and boost with an injectable vaccine. The inclusion of DC-cholesterol had only a minor effect on antigen entrapment into particles released from the implants and the implants did not stimulate cellular responses as effectively as the comparable injectable vaccine or the unmodified implant containing Quil-A and cholesterol, although the vaccine did induce stronger responses than either soluble protein alone, or protein co-delivered in alum.

In vivo activity of cationic immune stimulating complexes (PLUSCOMs).

A particulate vaccine delivery system consisting of cationic ISCOM derivatives (PLUSCOMs) was compared to classic anionic ISCOMs with regard to antigen attachment and ability to elicit in vivo T cell responses against a model protein antigen (ovalbumin [OVA]). ISCOMs did not incorporate hydrophilic OVA whilst OVA readily adsorbed onto PLUSCOMs with increasing adsorption at higher protein concentrations. The zeta-potential of PLUSCOMs significantly decreased with increasing protein load, suggesting neutralization of the cationic charge upon absorption of the anionic OVA. Antigen-specific CD8 T cell responses were demonstrated in mice vaccinated with either PLUSCOMs or ISCOMs. Ex vivo restimulation of harvested T cells demonstrated that cells isolated from PLUSCOM and ISCOM vaccinated mice responded to the secondary OVA challenge more efficiently than mice vaccinated with OVA in solution. Restimulated cells from the mice vaccinated with particulate vaccines produced significantly more INF-gamma. Therefore PLUSCOMs are as effective as classic ISCOMs in inducing antigen-specific CD8 T cell responses and have advantages with regard to the incorporation of purified anionic antigens.

Immunostimulatory biodegradable implants containing the adjuvant Quil-A--Part II: In vivo evaluation.

Sustained-release formulations have drawn the attention of formulation scientists working in the area of vaccine research because these systems may reduce the need for booster immunisations. This would be of great advantage especially for the administration of subunit vaccines. The aim of this study was to illustrate the performance of liposome-forming, sustained-release lipid implants containing 2% of the adjuvant Quil-A (QA) (w/w of total lipids) and ovalbumin (OVA) as a model antigen, in an in vivo study using C57Bl/6 mice. QA/OVA-containing lipid implants were administered subcutaneously and stimulated a similar magnitude of immune response when compared with an immediate-release formulation that contained an equivalent amount of adjuvant and antigen but was administered twice. The novel implant system presented here combines the advantages of both sustained release and particulate delivery in one formulation.

On the preparation, microscopic investigation and application of ISCOMs.

ISCOM matrices constitute colloidal structures formed from Quillaja saponins, cholesterol and phospholipid. Addition of protein antigens to these matrices leads to the formation of ISCOMs. In this review we report on microscopic investigations of ISCOM matrices and ISCOMs as well as related colloidal structures, such as helices, worm-like micelles, ring-like micelles, and lamellae structures. We briefly outline the immunologic basis for the use of ISCOMs as vaccine delivery systems, and describe the various methods to form ISCOMs. Negative staining transmission electron micrographs of all colloidal structures are presented and described. On the basis of our microscopic investigations, different formation mechanisms of ISCOMS are discussed.

Immunostimulatory colloidal delivery systems for cancer vaccines.

Cancer vaccine delivery is a multidisciplinary scientific field that is currently undergoing rapid development. An important component of cancer vaccines is the development of novel vaccine delivery strategies, such as colloidal immunostimulatory delivery systems. The importance of formulation strategies for cancer vaccines can be explained by the poor immunogenicity of tumour antigens. Colloidal vaccine delivery systems modify the kinetics, body distribution, uptake and release of the vaccine. This review explores recent research that is directed towards more targeted treatments of cancer through to colloidal vaccine delivery systems. Widely investigated carrier systems include polymeric micro- and nanoparticles, liposomes, archaeal lipid liposomes (archaeosomes), immune-stimulating complexes and virus-like particles. These systems are evaluated in terms of their formulation techniques, immunological mechanisms of action as well as the potential and limitations of such colloidal systems in the field of cancer vaccines.