Synthetic archaeosome vaccines containing triglycosylarchaeols can provide additive and long-lasting immune responses that are enhanced by archaetidylserine.

The relation between archaeal lipid structures and their activity as adjuvants may be defined and explored by synthesizing novel head groups covalently linked to archaeol (2,3-diphytanyl-sn-glycerol). Saturated archaeol, that is suitably stable as a precursor for chemical synthesis, was obtained in high yield from Halobacterium salinarum. Archaeosomes consisting of the various combinations of synthesized lipids, with antigen entrapped, were used to immunize mice and subsequently determine CD8(+) and CD4(+)-T cell immune responses. Addition of 45 mol% of the glycolipids gentiotriosylarchaeol, mannotriosylarchaeol or maltotriosylarchaeol to an archaetidylglycerophosphate-O-methyl archaeosome, significantly enhanced the CD8(+) T cell response to antigen, but diminished the antibody titres in peripheral blood. Archaeosomes consisting of all three triglycosyl archaeols combined with archaetidylglycerophosphate-O-methyl (15/15/15/55 mol%) resulted in approximately additive CD8(+) T cell responses and also an antibody response not significantly different from the archaetidylglycerophosphate-O-methyl alone. Synthetic archaetidylserine played a role to further enhance the CD8(+) T cell response where the optimum content was 20-30 mol%. Vaccines giving best protection against solid tumor growth corresponded to the archaeosome adjuvant composition that gave highest immune activity in immunized mice.

Isopranoid- and dipalmitoyl-aminophospholipid adjuvants impact differently on longevity of CTL immune responses.

The success of lipid membranes as cytotoxic T-cell (CTL) adjuvants requires targeted uptake by antigen-presenting cells (APCs) and delivery of the antigen cargo to the cytosol for processing. To target the phosphatidylserine (PS) receptor of APCs, we prepared antigen-loaded liposomes containing dipalmitoylphosphatidylserine and archaeal lipid liposomes (archaeosomes), containing an equivalent amount of archaetidylserine, and compared their ability to promote short and long-term CTL activity in animals. CTL responses were enhanced by the incorporation of PS into phosphatidylcholine/cholesterol liposomes and, to a lesser extent, into phosphatidylglycerol/cholesterol liposomes, that correlated to the amount of surface amino groups reactive with trinitrobenzoyl sulfonate. Archaeosomes contrasted to the liposome adjuvants by exhibiting higher amounts of surface amino groups and inducing superior shorter and, especially, longer-term CTL responses. The incorporation of dipalmitoyl lipids into archaeosomes induced instability and prevented long-term, but not short-term, CTL responses in mice. The importance of glycero-lipid cores (isopranoid versus dipalmitoyl) to the longevity of the CTL response achieved was shown further by incorporating dipalmitoyl phosphatidylethanolamine (DPPE) or equivalent amounts of synthetic archaetidylethanolamine (AE) into archaeosome adjuvants. Both DPPE and AE at equivalent (5 mol%) concentrations enhanced the rapidity of CTL responses in mice, indicating the importance of the head group in the short term. In the longer term, 5% of DPPE (but not 5% of AE) was detrimental. In addition to head-group effects critical to the potency of short-term CTL responses, the longer term CTL adjuvant properties of archaeosomes may be ascribed to stability imparted by the archaeal isopranoid core lipids.

Development of new glycosylation methodologies for the synthesis of archaeal-derived glycolipid adjuvants.

To commercialize the production of glycolipid adjuvants, their synthesis needs to be both robust and inexpensive. Herein we describe a semi-synthetic approach where the lipid acceptor is derived from the biomass of the archaeon Halobacterium salinarum, and the glycosyl donors are chemically synthesized. This work presents some preliminary results using the promoter system N-iodosuccinimide (NIS) and a stable 0.25 M solution of boron trifluoride-trifluoroethanol (BF(3) x TFE(2)) in dichloromethane. This promoter system allows for the use of peracetyl alkyl(aryl)thioglycosides that are available in high yield from inexpensive disaccharide starting materials by promoting clean glycosylation reactions from which pure product can be easily isolated. Conventional glycosylation using NIS-silver trifluoromethanesulfonate (AgOTf) leads to extensive acetyl transfer to the archaeol acceptor and numerous byproducts that make purification complicated. As part of preliminary structure-adjuvant activity studies, we describe the one-pot synthesis of a gentiobiose beta-Glcp-(1-->6)-Glcp-SEt donor with an O-2 benzoyl group, which can be used to prepare a disaccharide attached to archaeol in 85% overall yield, and the related glycolipid trisaccharide beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-Glcp-(1-->O)-archaeol. The synthesis of the isomeric beta-Glcp-(1-->6)-alpha-Glcp-(1-->O)-archaeol featuring a >10:1 alpha/beta alpha-selective glycosylation using the promoter system N-phenylselenylphthalimide-trifluoromethanesulfonic acid (TfOH) is also presented, along with the adjuvant properties of the corresponding archaeosomes (liposomes comprised entirely of combinations of isoprenoid archaeal-like lipids). These new vaccine formulations extend previous observations that glycolipids are integral to the activation of MHC type I pathways via CD8(+) antigen-specific T-cells. The beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-Glcp-(1-->O)-archaeol trisaccharide is shown to be more active than the Glcp-(1-->6)-beta-Glcp-(1-->O)-archaeol disaccharide.