Sulfated archaeol glycolipids: Comparison with other immunological adjuvants in mice.

Archaeosomes are liposomes traditionally comprised of total polar lipids (TPL) or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glyco- and amino-head groups. As adjuvants, they induce robust, long-lasting humoral and cell-mediated immune responses and enhance protection in murine models of infectious disease and cancer. Traditional total polar lipid (TPL) archaeosome formulations are relatively complex and first generation semi-synthetic archaeosomes involve many synthetic steps to arrive at the final desired glycolipid composition. We have developed a novel archaeosome formulation comprising a sulfated disaccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA) that can be more readily synthesized yet retains strong immunostimulatory activity for induction of cell-mediated immunity following systemic immunization. Herein, we have evaluated the immunostimulatory effects of SLA archaeosomes when used as adjuvant with ovalbumin (OVA) and hepatitis B surface antigen (HBsAg) and compared this to various other adjuvants including TLR3/4/9 agonists, oil-in-water and water-in-oil emulsions and aluminum hydroxide. Overall, we found that semi-synthetic sulfated glycolipid archaeosomes induce strong Ag-specific IgG titers and CD8 T cells to both antigens. In addition, they induce the expression of a number of cytokines/chemokines including IL-6, G-CSF, KC & MIP-2. SLA archaeosome formulations demonstrated strong adjuvant activity, superior to many of the other tested adjuvants.

Adjuvant potential of archaeal synthetic glycolipid mimetics critically depends on the glyco head group structure.

Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8(+) T cell activity, with highest responses to the antigen entrapped in archaeosomes containing disaccharides of glucose in beta- or alpha1-6 linkage (beta-gentiobiose, beta-isomaltose), or of beta-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive, and potent glyco-adjuvant.

The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo.

The unique glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with potent adjuvant activity. We studied the effect of archaeosomes on APCs to elucidate the mechanism(s) of adjuvant action. Exposure of J774A.1 macrophages to archaeosomes in vitro resulted in up-regulation of B7.1, B7.2, and MHC class II molecules to an extent comparable to that achieved with LPS. Similarly, incubation of bone marrow-derived DCs with archaeosomes resulted in enhanced expression of MHC class II and B7.2 molecules. In contrast, conventional liposomes made from ester phospholipids failed to modulate the expression of these activation markers. APCs treated with archaeosomes exhibited increased TNF production and functional ability to stimulate allogenic T cell proliferation. More interestingly, archaeosomes enhanced APC recruitment and activation in vivo. Intraperitoneal injection of archaeosomes into mice led to recruitment of Mac1alpha(+), F4/80(+) and CD11c(+) cells. The expression of MHC class II on the surface of peritoneal cells was also enhanced. Furthermore, peritoneal cells from archaeosome-injected mice strongly enhanced allo-T cell proliferation and cytokine production. The ability of archaeosome-treated APCs to stimulate T cells was restricted to Mac1alpha(high), B220(-) cells in the peritoneum. These Mac1alpha(high) cells in the presence of GM-CSF gave rise to both F4/80(+) (macrophage) and CD11c(+) (dendritic) populations. Overall, the activation of APCs correlated to the ability of archaeosomes to induce strong humoral, T helper, and CTL responses to entrapped Ag. Thus, the recruitment and activation of professional APCs by archaeosomes constitutes an efficient self-adjuvanting process for induction of Ag-specific responses to encapsulated Ags.

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help.

The unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.