Title:  Submicron emulsions as vaccine adjuvants

United States Patent:   5,961,970

Assignee:  Pharmos Corporation (New York, NY); The United States of America as represented by the Secretary of the Army (Washington, DC)

Filed: April 29, 1996

A vaccine adjuvant composition of an oil-in-water submicron emulsion that has about 0.5 to 50% of a first component of an oil, about 0.1 to 10% of a second component of an emulsifier, about 0.05 to 5% of a nonionic surfactant, about 0.00001 to 1% of an immunogen, and an aqueous continuous phase. This submicron emulsion has a mean droplet size in the range of between about 0.03 and 0.5 .mu.m, and preferably 0.05 and 0.2 .mu.m.

SUMMARY OF INVENTION

The present invention provides vaccine adjuvant compositions in the form of an emulsion of a plurality of submicron oil-in-water droplets having a particle size in the range of between about 30 nm to about 500 nm to effect enhanced immunogenicity of antigens incorporated intrinsically or extrinsically into the droplets. These droplets or particles form a submicron emulsion ("SME") for use as the vaccine adjuvant.

In marked contrast to the aforementioned disclosures, as will be described, the present invention does not require use of any immunostimulatory mycobacteria or muramyl peptide-like additives for its submicron emulsion to be effective. Moreover, as will be seen, a preferred embodiment of the present invention consists of intrinsically incorporating the antigen into the emulsion at the time of formation of the emulsion; this is in distinct contrast to mixing the antigen with the emulsion after the emulsion has been independently extrinsically formed. It will be appreciated that intrinsic formulation will be effective even in situations and conditions and species where extrinsic formulation is not. In this regard as well, the present invention is uniquely different and not at all implied by the previously mentioned applications which indeed teaches away from the present invention in stating that it is sufficient to simply mix the antigen with the extrinsically previously formed emulsion.

The vaccine compositions of this invention also do not include any polyoxypropylene-polyoxyethylene block polymer, trehalose dimycolate, or cell wall skeleton, as are found in prior art compositions.

Another aspect of this invention is to provide compositions and methods for the preparation of submicron emulsions containing antigens, incorporated either intrinsically (emulsified together with the oil and surfactant) or extrinsically (added externally to a prepared SME).

In some cases, the submicron emulsion of the present invention can be administered in combination with other adjuvant systems, such as proteosomes, as indicated in the examples.

The size, concentration and specific formulation of SMEs may be varied to suit the particular antigen used. Moreover, such adjuvant preparations may enhance both humoral and cell-mediated immunity (CMI) as do Freund's adjuvants. The SMEs here described have been developed for human use and since the oily droplets of the emulsions are of submicron size and contain no added pyrogenic moieties such as mycobacteria or MDP derivatives they have, unlike Freund's adjuvants, great safety potential. They may be especially applicable to antigens that are vaccine candidates to protect against biologic toxins or infectious agents, which have natural hydrophobic moieties as a component including transmembrane viral, bacterial or parasite proteins, membrane proteins such as proteosomes, lipopolysaccharides, glycolipids such as gangliosides, or a variety of proteins or peptides to which hydrophobic anchors have been chemically or genetically added.

Another aspect of the invention provides compositions and methods to achieve mucosal immunity by using an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, immunogenic peptide or antigen, and an aqueous continuous phase, which induces mucosal immunity by achieving mucoadhesion of the emulsion particles to mucosal surfaces. Mucous surfaces suitable for application of the emulsions of the present invention may include-ocular (corneal, conjunctival), oral (buccal, sublingual), nasal, vaginal and rectal routes of administration.

The emulsion particles have a hydrophobic core comprising a lipid or lipid-like composition and are stabilized with amphiphilic and/or non-ionic surfactants.

A wide variety of immunogens, including both water-soluble and water-insoluble peptides or polysaccharides, may be accommodated in the present emulsions. The hydrophobic core and surfactant provide a microenvironment which accommodates lipophilic immunogens such as lipid A or lipopolysaccharides as well as membrane-associated peptide antigen domains, while the aqueous continuous phase accommodates water-soluble peptide domains, or oligosaccharides.

The term "peptide" herein includes both oligopeptides and proteins. To facilitate intestinal uptake, the emulsions may be encapsulated in gelatin capsules or otherwise enterocoated to prevent their exposure to gastric fluids when the oral route of administration is selected. Furthermore, the emulsions may be lyophilized as disclosed previously in Pharmos Corp. International Application Publication WO 93/15736 prior to their encapsulation in order to achieve added stability of the antigen.

Claim 1 of 25 Claims

1. A vaccine adjuvant composition of an oil-in-water submicron emulsion consisting essentially of about 0.5 to 50% of a first component of an oil, about 0.1 to 10% of a second component of an emulsifier, wherein the emulsifier is a phospholipid compound or a mixture of phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin, about 0.05 to 5% of a non-ionic surfactant, about 0.00001, to 1% of an immunogen, and an aqueous continuous phase, said submicron emulsion having a mean droplet size in the range of between about 0.03 and 0.5 .mu.m, which composition is substantially free of added amounts of muramyl peptides or their lipophilic derivative

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