WO2000076476A1

WO2000076476A1 - Adjuvant-containing polymerized liposomes for oral, mucosal or intranasal vaccination

Info

Publication number: WO2000076476A1
Application number: PCT/US2000/015914
Authority: WO
Inventors: Hansi J. Dean; Robert N. Brey; Elya Bolotin; Denise Bucher; Patrick J. Frenchick
Original assignee: Endorex Corp
Current assignee: Soligenix Inc
Priority date: 1999-06-11
Filing date: 2000-06-09
Publication date: 2000-12-21
Anticipated expiration: 2001-12-11
Also published as: AU5731200A

Abstract

The present invention encompasses novel liposomal compositions, particularly comprising polymerizable liposomes, which are useful for the oral, intranasal and/or mucosal delivery of vaccines. In particular, the present invention relates to pharmaceutical compositions comprising polymerizable liposomes; antigens for inducing an immune response; adjuvants for enhancing an immune response to antigens; and stabilizing compounds for preserving the primary, secondary and tertiary structure of peptide and protein antigens during preparation and storage. These compositions may optionally comprise a targeting ligand. In addition, the invention relates to methods for forming liposomes by controlling the content of polymers in the lipid bilayer membrane. The invention still further relates to the use of the liposomal composition utilizing polymerized liposomes as, or in, pharmaceutical compositions for oral delivery of a variety of diganostic or therapeutic agents, including drugs and vaccines. The liposomes of the present invention provide increased stability in the gastrointestinal (G-I) tract, and provide for more effective vaccines that can be administered to humans and animals by the oral route. Further, the liposomal composition provide for more effective vaccines that can be administered by the intranasal route.

Description

ADJUVANT-CONTAINING POLYMERIZED LIPOSOMES FOR ORAL. MUCOSAL OR INTRANASAL VACCINATION

1 INTRODUCTION

The present invention relates to novel hposomal compositions which are useful as orally, mtranasally and/or mucosaliy delivered vaccines and complex molecular drugs In particular, the present mvention relates to vaccines composed of polymerizable hposomes that contain antigens for inducing an immune response, adjuvants for enhancing an immune response to antigens, "targeting" ligands that bind to specific cellular receptors, and stabilizing compounds for preserving the pπmary and tertiary structure of peptide and protem antigens duπng preparation and storage The vaccines of the present invention can be used to induce active immunity to antigens or, m some embodiments, induce tolerance useful in therapeutic treatment of allergies or autoimmune diseases The invention still further relates to the use of polymerized hposomes as, or in, pharmaceutical compositions for oral delivery of a vaπety of diagnostic or therapeutic agents, including drugs and vaccmes The hposomes of the present invention provide increased stability in the gastrointestinal (G-I) tract and shelf-life stability, and provide for efficacious vaccmes that can be administered to humans and animals by the oral route Further, the invention provides for more effective vaccmes that can be administered by the mtranasal route

2 BACKGROUND OF THE INVENTION

2 1 VACCINE DELIVERY

Drug and vaccine delivery takes a vaπety of forms, depending on the agent to be delivered and the administration route Drug and vaccine delivery systems are often designed to administer drugs to specific areas of the body In the gastrointestinal tract it is important that the drug not be degraded or eliminated before it has had a chance to exert a localized effect or to pass into the bloodstream or to interact with lymphoid tissue in the local environment In the nasopharyngeal tract, it is important that the drug or vaccine remains in proximity to the absorptive cells, m the case of a vaccine, it is important that antigens remain in contact with lymphoid cells before the antigen is washed through the nasal tract or is swallowed Further, it is important that antigen, vehicle, and immune stimulator be co-delivered in a single complex

Traditionally, immunization of humans and animals to modulate a disease state or to prevent acquisition of disease caused by infectious agents has been accomplished by admmisteπng inactivated whole organisms or cells, extracts of microorganisms or cells, or isolated components of those such as protem or peptide antigens Immunization has been achieved by oral or intramuscular administration of attenuated live organisms Typically, humans and animals are injected with such compositions in the presence of preservatives, adjuvants, and other excipients via intramuscular or subcutaneous routes to elicit protective immunity m normal pediatπc or adult vaccines in common use Parenteral immunization rarely is capable of eliciting an effective mucosal immune response that results in antibody production, particularly production of immunoglobulin A (IgA), which is important as a first defense barrier to invading microorganisms For this reason, it would be beneficial to provide oral vaccination, if the problems of low absorbability and instability associated with oral vaccination could be overcome Moreover, oral administration of vaccmes is the most convenient way to administer vaccines for reasons of increasing compliance m patient populations, avoidance of pam and needlestick injury

Currently, only live attenuated viruses or bacteπa are capable of inducing protective immune response m humans or animals when administered orally To date, very few vaccines have been developed that can be administered by the oral or intranasal route and these vaccines are mvaπably live organisms such as the oral polio virus vaccmes and a commonly used oral salmonella typhoid fever vaccine For example, live attenuated influenza virus prepared by cold-adaptation can be used to vaccinate humans against influenza by mtranasal administration

Typically, vaccines m common use are prepared from cellular extracts or produced by recombinant production methodology, these vaccmes are usually composed of proteins, microbial toxoids, inactivated whole viruses, or polysacchaπdes of microbial oπgm These types of mateπals are usually effective m induction of disease protective immune responses when administered by injection However, such mateπals are poorly lmmunogenic or non- lmmunogenic when administered through oral or mtranasal administration, even though the intestinal and nasopharyngeal tract of humans and most animals is πchly endowed with cells and tissues capable of induction of immune response to non-host antigens One of the mam failures of orally or mtranasally administered vaccmes is that antigens are poorly absorbed and unstable duπng passage Proteins and peptides that are administered orally are invaπably degraded in the GI tract by action of proteases and other hydrolytic enzymes, and stomach acid Thus, vaccines composed of subcellular immunogens are ineffective when administered by the oral or mtranasal routes Therefore, compositions that induce effective immune responses after oral or mtranasal administration would provide a more useful and convenient means to vaccinate animals and humans

Enteric coated formulations have been widely used for many years to protect drugs administered orally through the stomach, as well as to delay release of drug or vaccine antigens Several microsphere formulations have been proposed as a means for oral drug or vaccine delivery For example, PCT US90/06433 by Enzytech discloses the use of a hydrophobic protein, such as zem, to form microparticles, U S Patent No 4,976,968 to Sterner et al discloses the use of "protemoids" to form microparticles, US patent 5,075,109 by the UAB Research Foundation and Southern Research Institute discloses the use of synthetic polymers such as polylactic acid-glycohc acid to form microspheres, and PCT US95/04711 discloses the use of microcapsules composed of anionic polymers such as sodium algmate encasing inner aqueous cores formed by instantaneous contact with solution of cations such as spermine

Particles less than ten microns in diameter, such as the microparticles of EPA 0,333,523, can be taken up by cells in specialized areas, such as Peyer's patches and other intestinal mucosal lymphoid aggregates, located in the intestine, especially in the lleurn, into the lymphatic circulation Entrapping a drug or antigen m a microparticulate system can protect the drug or antigen from acidic and enzymatic degradation, yet still allow the drug or antigen to be administered orally The entrapped drugs or vaccmes are taken up by the specialized mucosal tissue and cells, and release the entrapped mateπal m a sustained manner or are processed by phagocytic cells such as macrophages

2 2 LIPOSOMES In addition to polymeπc microcapsules, liposomes can piovide a convenient and efficient means to encapsulate proteins, peptides, and sacchaπde antigens, and DNA or nucleic acid that encodes antigemc mateπal Liposomes have been used to develop several drugs useful in the treatment of cancers and fungal disease, these drugs include hposome and hpid-complex amphoteπcm B and doxorubicm Because of their relative ease of preparation and compatibility with a vaπety of complex molecules, liposomes are ideal vehicles for the construction of mucosal vaccines Similar vehicles have been described, for example, immune stimulatory complexes (ISCOMS, composed of cholesterol, saponms, and viral surface glycoproteins) provide for vaccines that contain adjuvant (sapomn, Quil A), vehicle and antigen together in a single complex ISCOMS can be administered for induction of immune response by both parenteral and mucosal routes, but are difficult to prepare and suitable only for a narrow set of antigemc mateπals Liposomes are structures consisting of a membrane bilayer composed of phosphohpids of biological or synthetic oπgin, usually spheπcal in shape Liposomes form naturally when phosphohpids or hpids contact water The structure of liposomes can be manipulated by methods to form them in the laboratory, including the input of energy m the form of heat, sonic energy, freeze-thaw cycles, or shear forces Because liposomes have features of biological membranes, they can be engineered in the laboratory to contain a vaπety of biologically and therapeutic relevant complex molecules, including proteins The phospholipid bilayer membrane of liposomes separates and protects entrapped materials in the inner aqueous core from the outside. Both water-soluble and -insoluble substances can be entrapped in different compartments, the aqueous core and bilayer membrane, respectively, of the same liposome. Chemical and physical interaction of these substances can be eliminated because the substances are in these different compartments. However, liposomes are physically and chemically unstable, entrapped material leaks during storage and, more important, leaks rapidly during transit through the GI tract or through the nasopharynx.

Conventional liposomes have been proposed for use as an oral drug delivery system, for example, by Patel and Rvman. FEBS Letters 62(1), 60-63 (1976). Liposomes are typically less than 10 microns in diameter, and may be absorbed through Peyer's patches. However, the efficiency of transport is compromised by instability in the intestine and in the stomach. Liposomes also have some features that should be advantageous for a particulate system for oral drug or antigen delivery. Without fortifying the liposomes, they are not good candidates for oral drug or antigen delivery. Thus, despite the early proposal for use of conventional liposomes in oral drug delivery, their use has still not been accepted. Several methods have been tried to fortify liposomes. Some methods involve intercalating cholesterol into the bilayer membrane or generating the liposomes in the presence of polysaccharides. Alternatively, synthetic or natural lipids that are thought to form more stable interactions in the bilayer have been used in certain experimental vaccines. Examples of phosphohpids that are thought to form more stable membranes include liposomes composed of distearoyl phosphotidyl choline (DSPC). DSPC-containing liposomes have been used in several experimental vaccines for dental caries. Preparation of such liposomes is limited to higher temperatures, above DSPC transition temperature (-55 °C), a condition unfavorable to most complex proteins and polypeptides. Generally, these methods are not believed to be sufficient in making liposomes for oral delivery since during oral delivery liposomes are exposed to an acidic pH in the stomach and bile salts and phospholipases in the intestine. These conditions break down the cholesterol and polysaccharide in the liposomes, and degrade the contents. Polymerized liposomes have been developed in attempts to improve oral delivery of encapsulated drugs (Chen et al., WO 9503035) and vaccines (Okada et al, US 5,762,904). A number of compounds have been reported to form polymerized liposomes. For example, U.S. Patent No. 4,485,045 discloses polymerizable phosphatidyl choline derivatives containing an unsaturated lower aliphatic acyloxy longer chain alkanoyloxy moiety; U.S. Patent No. 4,808,480 discloses heterocyclic compounds containing disulfide bonds that are used to form polymerizable phosphohpids; U.S. Patent No. 4,594,193 discloses polymerizable Hpid compounds containing mercaptan groups; U.S. Patent No. 5,160,740 discloses polymerization of a polymerizable 2,4-diene phospholipid, cholesterol, and a polymerizable 2,4-diene fatty acid to form a polymerized macromolecular endoplasmic reticulum; and Regen, in Liposomes: from Biophysics to Therapeutics (Ostro, ed., 1987), Marcel Dekker, N.Y, describes additional polymerizable phosphohpids. The ability of polymerized liposomes to survive the G-I tract has also been investigated (Chen et al., 1995, Proceed. Internat. Symp. Control. Rel. Bioact. Mater. 22; Chen et al., 1995 Proc. 3rd U.S. Japan Symposium on Drug Delivery Systems). Although polymerized liposomes, generally, are more stable than their unpolymerized counterparts, it is not clear that the o improved stability thus far achieved is by itself sufficient to enable these liposomes to deliver effective doses of drugs administered orally. Therefore, despite the advances in liposome technology and drug delivery, there remains a need for stable and efficacious polymerized liposomes, and new polymerizable compounds that can be incorporated into polymerizable liposomes to improve stability, binding selectivity, and efficiency of drug 5 delivery. There additionally remains a need for new processes to manufacture polymerizable liposomes incorporating targeting molecules or ligands, and to manufacture polymerizable liposomes which encapsulate drugs. Further, there remains a need to preserve the conformational structure of antigens incorporated in polymerized liposomes, especially if the antigen is composed of protein or is peptidic in nature. 0

3. SUMMARY OF THE INVENTION

The present invention encompasses novel Hposomal compositions, particularly comprising polymerizable liposomes, which are useful for the oral, intranasal and/or mucosal delivery of vaccines. In particular, the present invention relates to pharmaceutical 5 compositions comprising polymerizable liposomes; antigens for inducing an immune response; adjuvants for enhancing an immune response to antigens; and stabilizing compounds for preserving the primary, secondary and tertiary structure of peptide and protein antigens during preparation and storage. These compositions may optionally comprise a targeting ligand. In addition, the invention relates to methods for forming 0 Hposomes by controlling the content of polymers in the Hpid bilayer membrane. The invention still further relates to the use of the Hposomal composition utilizing polymerized liposomes as, or in, pharmaceutical compositions for oral delivery of a variety of diagnostic or therapeutic agents, including drugs and vaccines. The liposomes of the present invention provide increased stability in the gastrointestinal (G-I) tract, and provide for more effective 5 vaccmes that can be administered to humans and animals by the oral route. Further, the Hposomal compositions provide for more effective vaccines that can be administered by the intranasal route.

The present invention provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes and antigen, wherein the antigen is in the interior space of the polymerized liposome and external to the polymerized liposomes. The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes and an antigen, wherein the antigen is in the interior space of the polymerized liposome and/or the leaflet of the polymerized liposomes. The present invention provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized Hposomes, a stabilizer, and an antigen, wherein the stabilizer is in the interior of the polymerized Hposomes and the antigen is in the interior space of the polymerized liposome and external to the polymerized liposomes. The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized Hposomes, a stabilizer, and an antigen, wherein the stabilizer is in the interior of the polymerized Hposomes and the antigen is in the interior space of the polymerized liposome and/or the leaflet of the polymerized liposomes. The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes, an adjuvant, and an antigen, wherein the adjuvant in the interior space of the polymerized liposomes and/or the leaflet of the polymerized liposomes, and the antigen is in the interior space of the polymerized liposome and external to the polymerized Hposomes. The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes, an adjuvant, and an antigen, wherein the adjuvant in the interior space of the polymerized liposomes and/or the leaflet of the polymerized liposomes, and the antigen is in the interior space of the polymerized liposome and/or the leaflet of the polymerized liposomes.

The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes, a stabilizer, an adjuvant, and an antigen, wherein the stabilizer is in the interior of the polymerized Hposomes, the adjuvant in the interior space of the polymerized liposomes and/or the leaflet of the polymerized Hposomes, and the antigen is in the interior space of the polymerized liposome and external to the polymerized Hposomes. The present invention also provides methods and compositions for orally or intranasally administering an antigen to a mammal, comprising polymerized liposomes, a stabilizer, an adjuvant, and an antigen, wherein the stabilizer is in the interior of the polymerized liposomes, the adjuvant m the mteπor space of the polymeπzed liposomes and/or the leaflet of the polymeπzed liposomes, and the antigen is in the mteπor space of the polymerized liposome and/or the leaflet of the polymenzed liposomes

The present invention also provides methods and compositions for orally or intranasally admimstenng an antigen to a mammal, comprising polymeπzed liposomes, a stabilizer, an adjuvant, a targeting molecule, and an antigen, wherein the stabilizer is in the inteπor of the polymeπzed liposomes, the adjuvant in the mteπor space of the polymerized Hposomes and/or the leaflet of the polymeπzed liposomes, the targeting molecule is on the surface of the polymerized liposomes, and the antigen is m the mtenor space of the polymeπzed liposome and external to the polymerized liposomes The present invention further provides methods and compositions for orally or intranasally administeπng an antigen to a mammal, compπsmg polymeπzed liposomes, a stabilizer, an adjuvant, a targeting molecule, and an antigen, wherein the stabilizer is in the interior of the polymerized liposomes, the adjuvant in the interior space of the polymeπzed liposomes and/or the leaflet of the polymeπzed liposomes, the targeting molecule is on the surface of the polymeπzed Hposomes, and the antigen is in the interior space of the polymeπzed liposome and/or the leaflet of the polymenzed liposomes

The present invention is based on Applicants' discovery that vaccine compositions compπsmg an adjuvant, an antigen, and a stabilizing agent delivered with, or preferably encapsulated in a polymeπzed liposome, provide enhanced vaccine efficacy

3 1 DEFINITIONS

As used herein, the term "liposome" is defined as an aqueous compartment enclosed by a Hpid bilayer (Stryer, Biochemistry, 2d Edition, W H Freeman & Co , p 213 (1981)) The liposomes can be prepared by a thin film hydration technique followed by a few freeze- thaw cycles Liposomal suspensions can also be prepared accordmg to methods known to those skilled in the art, for example, as descπbed in U S. Patent No 4,522,811, which is incorporated herein by reference m its entirety

As used herein, the term "polymenzed liposome" is defined as a liposome in which some, most or all of the constituent phosphohpids are covalently bonded to each other by inter and mtra molecular interactions The phosphohpids can be bound together withm a single layer of the phospholipid bilayer (the leaflets) and/or bound together between the two layers of the bilayer

The degree of crosslinkmg in the polymeπzed liposomes can range from approximately 5 percent to 100 percent (i e , up to 100 percent of the available bonds are formed), 35 percent to 90 percent, or 40 percent to 60 percent Preferably, the degree of crosslinking in the polymerized liposomes ranges from 5 percent to 100 percent. The size range of polymerized liposomes is between approximately 15 nm to 10 μm. The polymerized liposomes can be loaded with up to 100% of the material to be delivered, when the material is hydrophobic and attracted by the phospholipid layers. In general, about 5 to about 40 percent of the material is encapsulated when the material is hydrophilic, although under certain conditions, up to 100% of material can be loaded.

As used herein, the term "conventional liposome" refers to an unpolymerized liposome, not having crosslinked polymers as components of the Hpid bilayer membrane.

As used herein, the term "trap ratio" is defined as the ratio of inner aqueous phase volume to total aqueous phase volume used.

As used herein, the term "entrapment efficiency" is defined as the ratio of material contained within the liposome structure to the amount of material initially available before the formation of liposome membranes.

As used herein, the term "external to polymerized Hpsomes" is defined as material that is located on the surface or in the milieu surrounding a liposome vehicle.

As used herein, the term "radical initiator" is defined as a chemical which initiates free-radical polymerization.

As used herein, the term "reverse phase evaporation technique" is defined as a method involving dissolving a Hpid in an organic solvent, adding a buffer solution, and evaporating the organic solvent at reduced pressure, as described by Skoza, F. Jr., and

Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA. Volume 75, No. 9, pp. 4194-4198 (1978).

As used herein, the term "freeze-thaw technique," or "F-T," is defined as freezing a suspension in a cryogenic fluid such as liquid nitrogen, and subsequently thawing the suspension in a roughly 30 °C water bath. As used herein, the terms "mucosa" or "mucosal" refers to a mucous tissue such as epithelium, lamina propria, a layer of smooth muscle in the digestive tract. Mucosal delivery as used herein is meant to include delivery through bronchi, gingival, lingual, nasal, oral, vaginal, rectal, and intestinal mucosal tissue.

As used herein, the term "buffer solution" is defined as an aqueous solution or aqueous solution containing less than 25% of a miscible organic solvent, in which a buffer has been added to control the pH of the solution. Examples of suitable buffers include but are not limited to PBS (phosphate buffered saline), TRIS (tris-

(hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidine ethane sulfonic acid), and TES (2-[(tris-hydroxymethyl)methyl]amino-l-ethanesulfonic acid). As used herein, the term "leaflets" is defined as a single layer of phosphohpids in the bilayer forming the liposome. 4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Tetanus toxoid-specific serum IgG responses in mice vaccinated with polymerized liposome formulations and control formulations: Group # 11 - TT/polymerized Hposomes/MPLA; Group # 7 - TT/polymerized

Hposomes/MPLA/trehalose; 17 - 1 μg soluble TT (subcutaneous vaccination); Group # 22 - soluble TT/MPLA; Group # 23 - polymerized Hposomes/MPLA. Mice were orally vaccinated on days 0, 14 and 28 with the indicated formulation. For each group, geometric mean titer is indicated for blood samples taken on days 13, 27, and 41. Figure 2. A. Serum IgG titers, expressed as nanograms of IgG per ml, in individual animals. Group #7: polymerized liposomes TT + MPLA + trehalose; group #22: TT + MPL. B. Group #11 : polymerized Hposomes + TT + MPL; group # 23: polymerized liposomes + MPLA.

Figure 3. Tetanus toxoid-specific intestinal IgA responses in individual mice vaccinated with polymerized liposome formulations and control formulations: Group # 11 - TT/polymerized Hposomes/MPLA; Group # 7 - TT/polymerized Hposomes/MPLA trehalose; Group # 17 - 1 μg soluble TT (subcutaneous vaccination); Group # 22- soluble TT/MPLA; Group # 25 - polymerized Hposomes/MPLA/trehalose. Mice were orally vaccinated on days 0, 14 and 28 with the indicated formulation. Bars represent the value for 3 individual mice from the indicated groups, from samples taken 48 days following the first vaccination.

Figure 4. Tetanus toxoid-specific serum IgG responses in mice orally vaccinated with polymerized liposome formulations and control formulations (see Table 3).

Figure 5. Tetanus toxoid-specific serum IgG2a responses in mice orally vaccinated with polymerized liposome formulations and control formulations (see Table 3).

Figure 6. Ratio of tetanus toxoid-specific serum IgG2a to IgGl responses in mice orally vaccinated with polymerized liposome formulations and control formulations (see Table 3).

Figure 7. Intranasal immunization protocol. The figures indicates when mice where bleed (B) and when vaccine (V) was administered intranasally to mice.

Figure 8. Tetanus toxoid-specific serum IgG responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations: Group #1 - soluble TT/MPLA; Group #2 - TT/liposomes/MPLA/trehalose; Group #3 - TT/polymerized Hposomes/MPLA/trehalose; group #4 - polymerized Hposomes/MPLA/trehalose. Mice were intranasally vaccinated on day 0 with the indicated formulation. For each group, individual values are indicated for serum samples taken 14 days after vaccination.

Figure 9. Tetanus toxoid-specific serum IgG responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations: DODPC/TT/MPL (40%); native TT mixed with MPL; and empty polymerized Hposomes and MPL.

Figure 10. Tetanus toxoid-specific IgA in the nasal wash of mice intranasally vaccinated with polymerized liposome formulations and control formulations: Hposomal

TT-MPL; soluble TT-MPL; Hposomal MPL; and TT (administered subcutaneously). Figure 11. Tetanus toxoid-specific serum IgA responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations on day 41 after initial immunization: native TT/MPL; 40% polymer content Hposomes/TT/MPL; and

40% polymer content Hposomes/MPL.

Figure 12. Tetanus toxoid-specific serum IgG2a responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations on day 41 after initial immunization: native TT/MPL; 40% polymer content Hposomes/TT/MPL; and

40% polymer content Hposomes/MPL.

Figure 13. Tetanus toxoid-specific serum IgG2b responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations on day 41 after initial immunization: native TT/MPL; 40% polymer content Hposomes/TT/MPL; and

40% polymer content Hposomes/MPL.

Figure 14A-B. Tetanus toxoid-specific serum IgG responses in mice intranasally vaccinated with polymerized liposome formulations and control formulations: (■) soluble

TT/MPL; (A) Lip- TT/MPL 3 μg; (T) Lipo-TT/MPL 2 μg + TT 1 μg; (♦) Lip-TT/MPL 1 μg + TT 2 μg; (•) Lipo-MPL + TT 3 μg; (□) TT 1 μg (subcutaneously administered); and

( ) Lipo-MPL.

Figure 15. Tetanus toxoid-specific serum IgG2a responses in mice intranasally vaccinated four times with polymerized liposome formulations and control formulations: soluble TT/MPL; Lipo-TT/MPL 2 μg + TT 1 μg; TT 1 μg (subcutaneously administered); and Lipo-MPL.

Figure 16. Tetanus toxoid-specific IgA in nasal wash fluids from mice intranasally vaccinated four times with polymerized liposome formulations and control formulations: soluble TT/MPL; Lipo-TT/MPL 2 μg + TT 1 μg; TT 1 μg (subcutaneously administered); and Lipo-MPL. Figure 17. Tetanus toxoid-specific IgA in the serum from mice intranasally vaccinated four times with polymerized liposome formulations and control formulations: soluble TT/MPL; Lipo-TT/MPL 2 μg + TT 1 μg; TT 1 μg (subcutaneously administered); and Lipo-MPL.

5. DETAILED DESCRIPTION OF THE INVENTION

5 The invention relates to an oral, intranasal or mucosal drug delivery system to deliver drugs and vaccines to the mucosal tissue of the intestine, which utilizes polymerized liposomes as the drug carriers. In accordance with the present invention, polymerized liposomes are prepared from mixtures of polymerizable and non-polymerizable phosphohpids, fatty acids and substituted derivatives and contain between approximately

10 10% and 90% cross-linked groups, and other lipid compounds that have immune stimulatory properties, such as monophosphoryl Lipid A (MPLA) or stabilizing agents such as cholesterol. In a prefeπed embodiment, Hposomal compositions of the present invention contain a high content of polymers for the enhanced stability, which is in turn is related to a more efficient delivery mechanism for drugs or vaccines.

15 In another embodiment, the invention is directed to Hposomal compositions that contain immunostimulatory compounds, (adjuvants) designed to enhance the efficacy of vaccines. The presence of adjuvants in the liposomes decreases the effective amount of antigen required to elicit an immune response to orally or mucosally administered vaccines. Further, the invention relates to inclusion of stabilizing agents in the aqueous interior space 0 of liposomes along with an aqueous soluble biologically active material to preserve function of the material during formation, during storage, and to alter other biological effects of the vaccine, such as the nature, onset, and duration of the immune response, and GI transit.

The present invention is based on Applicants' discovery that vaccine compositions comprising an adjuvant, an antigen, and a stabilizing agent delivered with, or preferably 5 encapsulated in a polymerized liposome, provide enhanced vaccine efficacy. In particular, these vaccine compositions when administered orally or intranasally result in an enhanced immune response. In addition, vaccine compositions containing stabilizer provided enhanced stability of the antigen. Further, the present invention is based on Applicants' discovery that the polymerized liposome compositions have enhanced stability against the 0 harsh environment of the gastrointestinal tract particularly when compared to unpolymerized liposomes. Thus, the polymerized Hposomal compositions of the present invention provide an optimal method of immunizing animals, particularly, human, by mucosal vaccination regimes.

In one embodiment, pharmaceutical compositions of the present invention comprise 5 polymerized liposomes (e.g., polymerized phosphohpids or a mixture of polymerized phosphohpids and polymerized fatty acids), an antigen, and a stabilizing agent. In a preferred embodiment, pharmaceutical compositions of the present invention comprise polymerized liposomes (e.g., polymerized phosphohpids or a mixture of polymerized phosphohpids and polymerized fatty acids), an antigen, an adjuvant, and a stabilizing agent. In a more preferred embodiment, pharmaceutical compositions of the present invention comprise polymerized liposomes (e.g., polymerized phosphohpids or a mixture of polymerized phosphohpids and polymerized fatty acids), an antigen, an adjuvant, a targeting molecule, and a stabilizing agent.

In one embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more antigens and an adjuvant in the interior space of the polymerized Hposomes. In another embodiment, polymerized

Hposomal compositions for drug or vaccine delivery comprise polymerized Hposomes with one or more antigens and an adjuvant contained in the membrane bilayer of the polymerized liposomes. In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more antigens and an adjuvant contained in the membrane bilayer of the polymerized liposomes and in the interior space of the polymerized liposomes. In yet another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more antigens in the interior space of the polymerized liposomes and external to the polymerized liposomes, and an adjuvant contained in the membrane bilayer of the polymerized liposomes or the interior space of the polymerized Hposomes. In accordance with these embodiments, polymerized Hposomal compositions for drug or vaccine discovery may further comprise a targeting molecule.

In a specific embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes and one or more antigens, wherein the ratio of antigens encapsidated in the liposomes to antigens external to the liposomes is 1 :100, 1 :75, 1 :50, 1 :25, 1 :20: 1 : 15, 1 :10, 1 :8, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1, 2: 1, 3:1, 4:1, 5: 1, 6:1, 8: 1, 10:1, 15: 1, 20:1, 25: 1, 50:1, 75:1, or 100:1. In a prefeπed embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes and one or more antigens, wherein the ratio of antigens encapsidated in the liposomes to antigens external to the liposomes 2:1. In accordance with these embodiments, the polymerized Hposomal compositions may further comprise a adjuvant, a stabilizer, a targeting molecule, or any combination thereof. Polymerized Hposomal compositions of the invention may comprise more than one antigen, adjuvant, stabilizer or targeting molecule.

The polymerized liposomes of the present invention comprise polymerizable phosphohpids and have about 10 to about 90% polymer content. Alternatively, polymerized liposomes of the present invention comprise a mixture of polymerizable phosphohpids and non-polymerizable phosphohpids, and have about 10 to about 90% polymer content. Alternatively, polymerized liposomes of the present invention comprise a mixture of polymerizable phosphohpids and polymerizable fatty acids, and have about 10 to about 90% polymer content. In a prefeπed embodiment, antigens or immunogens encompass any agent that induces an immune response in an animal. Antigens or immunogens include, but are not limited to, glycoproteins, peptides, and carbohydrates. For example, antigens include diphtheria toxoid, tetanus toxoid, influenza hemagglutinin, ospA antigen from Lyme disease bacterium, and HTLV envelope protein. In a prefeπed embodiment, adjuvants encompass any agent that enhances an immune response to antigens. Adjuvants encompass any compound capable of enhancing an immune response to an antigen. Examples of adjuvants which may be effective, include, but are not limited to: aluminum hydroxide, monophosphoryl Hpid A (MPLA) -acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, simple immunostimulatory oligonucleotides, cytokines such as IL-12, IL-2 or IL-1, saponins, and microbial toxins such as cholera toxin, heat labile toxin and genetically altered derivatives of them.

In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more therapeutics and an adjuvant in the interior space of the polymerized liposomes. Therapeutics include, but are not limited to, antiviral agents, antibacterial agents, attenuated viruses, antifungal agents, cytokines, hormones, insulin, calcitonin, fertility drugs, antibiotics, and chemotherapy agents.

In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more hydrophobic antigens and an adjuvant in the leaflet of the polymerized liposomes. In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery, comprise polymerized liposomes with one or more antigens, an adjuvant, and a stabilizing agent in the interior space of the polymerized liposomes. Stabilizing agents are compounds that protect or preserve conformational structure of an antigen. Examples of stabilizing agents include, but are not limited to, polyols with multiple hydroxyl groups, such as trehalose, marmitol, sorbitol, sucrose, and surfactants, such as pluronic F-68 and polyethylene-polypropylene block polymers. Other stabilizing agent include gelatin, glycine, EDTA, polyethylene glycols, polyvinyl pyπolidone, and ZnCl₂. In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more hydrophobic antigens and an adjuvant in the leaflet of the polymerized liposomes, and a stabilizing agent in the interior space of the polymerized liposome. In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more antigens in the interior space of the polymerized liposome and external to the polymerized liposomes, an adjuvant in the leaflet of the polymerized liposomes, and a stabilizing agent in the interior space of the polymerized liposome. In yet another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes with one or more antigens in the interior space of the polymerized liposome and external to the polymerized liposomes, an adjuvant in the interior space of the polymerized liposomes, and a stabilizing agent in the interior space of the polymerized liposome. In accordance with these embodiments, the polymerized Hposomal compositions for drug or vaccine delivery may further comprise a targeting molecule.

In another embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes, a targeting ligand, one or more antigens and adjuvant. In accordance with this embodiment, the targeting ligand may be on the surface of the polymerized liposome, and the antigen and adjuvant either in the interior of the polymerized liposome or in the leaflet of the polymerized liposome. Alternatively, the targeting ligand may be on the surface of the polymerized liposome, the adjuvant either in the interior of the polymerized liposome or in the leaflet of the polymerized liposome, and the antigen in the interior space of the polymerized liposome and external to the polymerized liposome. In a preferred embodiment, polymerized Hposomal compositions for drug or vaccine delivery comprise polymerized liposomes, a targeting ligand, one or more antigen, an adjuvant, and a stabilizing agent. In accordance with this embodiment, the targeting ligand may be on the surface of the polymerized liposome, the antigen and adjuvant in the interior of the polymerized liposome or in the leaflet of the polymerized liposome, and the stabilizer in the interior space of the polymerized liposome. Alternatively, the targeting ligand may be on the surface of the polymerized liposome, the adjuvant in the interior of the polymerized liposome or in the leaflet of the polymerized liposome, the stabilizer in the interior space of the polymerized liposome, and the antigen in the interior space of the polymerized liposome and external to the polymerized liposome.

In accordance with the invention, an antigen, an adjuvant or targeting molecule may be covalently linked, ionic linked, intercalated or complexed to the polymerized liposomes of the invention. 5.1. POLYMERIZED LIPOSOMES

5.1.1. PREPARATION OF POLYMERIZED LIPOSOMES The polymerized liposomes of the present invention may be prepared by a variety of techniques as described infra. For example, and not by way of limitation, polymerized liposomes are prepared by polymerizing double and triple bond-containing olefmic and acetylenic phosphohpids. In addition, polymerized liposomes can also be prepared by chemical oxidation of thiol groups in the phosphohpids to disulfide linkages. The polymerization can take place in a solution containing a biologically active substance, such as a drug, antigen or adjuvant, in which case the substance is encapsulated during the polymerization. Alternatively, the liposomes can be polymerized first, and the biologically active substance can be added later by resuspending the polymerized liposomes in a solution of a biologically active substance, and entrapping the substance by sonication of the suspension. Another method of entrapping a biologically active substance in polymerized liposomes is to dry the polymerized liposomes to form a film, and hydrate the film in a solution of the biologically active substance. The above conditions are typically mild enough to entrap biologically active substances without denaturing them.

The polymerized liposomes are generally prepared by polymerization of double bond-containing monomeric phosphohpids. These phosphohpids may contain any unsaturated functional group, including polymerizable functional group double or triple bonds, any may contain more than one polymerizable functional group double or triple bonded. Suitable monomeric phosphohpids are known to those skilled in the art, and include, but are not limited to, phosphatidylaholines DODPC (l,2-di(2,4-Octadecadienoyl)- 3-phosphatidylcholine), 2,4-diene phosphohpids, di-yne phosphohpids, see e.g., U.S. Patent No. 4,485,045, U.S. Patent No. 4,861,521. In addition, polymerized liposomes can be prepared by polymerization of phosphohpids with negatively charged groups as described infra. If the liposome is polymerized by oxidation of thiol groups, it is prefeπed not to encapsulate thiol-containing biologically active substances, as they could be oxidized during the polymerization step.

The liposomes of the present invention may be polymerized by free radical initiation. The monomeric double bond-containing phosphohpids can be polymerized using a hydrophobic free radical initiator, such as AIBN (azo-bis-isobutyronitrile), or a hydrophilic free radical initiator such as AAPD (azo-bis-amidinopropane dihydrochloride). The latter is particularly useful for initiating polymerization between layers of the bilayer. The present invention also encompasses the use of other mild redox initiators, such as Na₂S₂O₅ and K₂S₂O₈. Alternatively, polymerization can be initiated by using a radiation source, such as ultraviolet or gamma radiation. Use of the free radical initiators is prefeπed if the biologically active substance to be entrapped is denatured when exposed to radiation. The ratio between the phospholipid and crosslinker and aqueous phase all affect the percent of crosslinking. In general, the percent crosslinking increases as the amount of crosslinker or time or temperature of reaction are increased. As the percent crosslinking increases, the release rate of the materials from the liposomes decreases and the stability increases.

The Hposomes of the present invention may be polymerized by radiation including, polymerization with ultraviolet and/or gamma radiation, provided the biologically active substance can survive exposure to the radiation. Typical conditions for initiating the polymerization with ultraviolet radiation include but are not limited to iπadiating the solution at 254 nm, 100 W, for 3 hours at room temperature. Typical conditions for initiating the polymerization with gamma radiation include but are not limited to iπadiating the solution at 0.3 mRad per hour for 3 hours at room temperature.

5.1.2. PREPARATION OF POLYMERIZED LIPOSOMES COMPRISING POLYMERIZABLE FATTY ACIDS

The present invention encompasses polymerized liposomes which incorporate polymerizable fatty acids, and the use of these polymerized liposomes as vehicles for the delivery of drugs and vaccines to the mucosal tissue of the intestine or other body mucosa, including the nasopharynx. The fatty acids can be used either in their non-derivatized form, to enhance the stability of the polymerized liposomes, or coupled to a ligand which targets particular cells in the G-I tract, as described infra. The polymerized Hposomes are obtained by polymerizing a mixture of a polymerizable lipid and a polymerizable fatty acid or polymerizable polymer-coupled fatty acid or polymerizable targeted fatty acid of the present invention, using conventional liposome polymerization techniques, such as irradiation, redox initiation, radical initiation, and the like.

The polymerizable lipids used in conjunction with the polymerizable fatty acids and targeted polymerizable fatty acids of the present invention are not limited to any particular lipids. Any lipid can be used which is polymerizable and is capable of forming polymerized Hposomes. A wide variety of polymerizable lipids have been described in the literature; see, e.g., Regen, in Liposomes: From Biophysics to Therapeutics (Ostro, ed., 1987), Marcel Dekker, N.Y., and Singh and Schnur, Polymerizable Phosphohpids, in Phosphohpids Handbook, 1993, Marcel Dekker, New York which are incorporated herein by reference. Preferred polymerizable lipids include diene containing phosphohpids with uncharged head groups, such as glycerol, inositol, or serine, or charged head groups such as choline or ethanolamine. A particularly prefeπed polymerizable lipid is 1 ,2-di(2,4- octadecadienoyl)-3-phosphatidylcholine (DODPC).

The polymerizable fatty acids and targeted polymerized fatty acids can be any of the species described herein. Without being bound by any particular theory, it is believed that the 2,4-ODPEGSu fatty acid co-polymerizes with the polymerizable lipid, and that the hydrophilic tail of 2,4-ODPEGSu incorporated into the liposome extends away from the liposome surface and into any suπounding aqueous phase. The PEG chain thus enhances the stability of the liposome by creating a sterically stabilized liposome, in which the liposome body is somewhat protected by the protruding and entangled copolymerized ODPEGSu chains. Higher molecular weight polyethylene glycols (i.e., average molecular weight above about 2000) may destabilize the liposome, while lower molecular weight PEGs (i.e., average molecular weights from about 200 to 2000) will have a net stabilizing effect. The resulting polymerized liposomes thus have increased stability in the G-I tract and ability to pass through mucus layer, and additionally can be targeted to particular cells of the intestine when targeted polymerizable fatty acids are used.

When targeted fatty acids are used, the polymer chain of the surfactant group additionally serves as a spacer between the liposome and the targeting ligand attached to the fatty acid. Accordingly, the molecular weight of the polyethylene glycol should be chosen in order to achieve the desired spacing, while still allowing the fatty acid to copolymerize with the lipids. The lower molecular weight polyethylene glycols are thus believed to be more suitable; i.e., those with average molecular weights of about 200 to about 2000, preferably about 200 to about 1500, and most preferably about 400.

The polymerized liposomes of the present invention can additionally contain non- polymerizable compounds, so long as the amounts of polymerizable lipids and polymerizable fatty acids or targeted polymerizable fatty acids are sufficient to give the resulting polymerized liposomes adequate stability. For example, non-polymerizable fatty acids or non-polymerizable phosphohpids known in the art and used for conventional liposome formation may be used. In addition, cholesterol can be used for added stability. A preferred non-polymerizable compound is cholesterol which can be included in molar ratios of up to 50% with the polymerizable components.

5.1.3. NEGATIVELY CHARGED POLYMERIZABLE PHOSPHLIPIDS

For still greater flexibility and utility in creating oral and/or intranasal drug delivery systems, the present invention also encompasses polymerizable phosphohpids with negatively charged groups. Incorporating negatively charged groups into a polymerized liposome greatly expands the use of the liposomes by taking advantage of the desirable properties of Hposomes while additionally utilizing the electrostatic charge to improve and enhance the ability of the liposomes to entrap therapeutic agents. The resulting negatively charged polymerizable liposomes have superior trap ratios, and thus are especially effective in delivering the entrapped therapeutic agents. The negatively charged polymerizable lipids of the present invention include polymerizable lipids which have phosphatidyl inositol (PI), phosphatidyl glycerol (PG) or phosphatidyl serine (PS) groups on a polymerizable backbone. These polymerizable lipids can be used to create polymerizable liposomes incorporating the negatively-charged PI, PG or PS groups, using conventional techniques. Because of capacity to interact with divalent cations, such negatively charged polymerizable phosphohpids can assume alternate configurations in aqueous suspensions. In the presence of metal ions, for example, Ca²" or Mg²⁺ ions, jelly-roll or cochelate structures can be formed; these structures consist of tightly packed bilayer membranes in which water has been squeezed out of the internal spaces. By harvesting such structures by centrifugation, followed optionally by lyophilization, and exposure to divalent metal-ion chelating agents, such as EGTA or EDTA, in the presence of drug or protein to be encaptured, a high and reproducible degree of loading of typically configured spherical bilayer liposomes can be obtained.

The negatively charged polymerizable lipids have the structure:

CH,-O-R

CH-O-R'

CH₂-O-R" wherein at least one of R, R' or R" is independently phosphoryl inositol, phosphoryl glycerol or phosphoryl serine and at least one of the remaining two groups is a polymerizable group, consisting of acyl chains containing dienoic acids, diacetylenic acids, methacrylate side groups, acrylates, or thiol or disulfide containing acids. For example, the negatively charged polymerizable lipids may also have the following structure: CH₂-O-CO-R,

CH-O-CO-R CH₂-O-PO₃X where X is glycerol, inositol or serine; and R, and R₂ are independently a polymerizable group selected from the group consisting of a diene group, a diacetylene group, a methacrylate group, and a thiol group. The polymerizable group is preferably a hydrocarbon chain containing one or more of the above-mentioned polymerizable moieties. The hydrocarbon chain can be from C₄ to C₃₀ and higher if desired. Although any polymerizable backbone can be used with these negatively charged polymerizable lipids, a particularly prefeπed backbone is 2,4-DODPC. Thus, a prefeπed negatively charged polymerizable lipid is

CH₂-O-C(O)-CH=CH-CH=CH-(CH₂)₁₂-CH₃

CH-O-C(O)-CH=CH-CH=CH-(CH₂)₁₂-CH₃

CH₂-O-R in which R is phosphoryl inositol, phosphoryl glycerol phosphoryl or serine.

These novel negatively charged polymerizable lipids can be synthesized according to the methods described in Confurius and Zwaal, Biochimin Biophysica Acta. 488:36-42 (1977) wherein polymerizable PG, PI or PS may be synthesized by a transphosphatidylation catalyzed by phospholipase D in the presence of protected glycerol, inositol or serine followed by a deprotection step. For example, DODPC is dissolved in diethyl ether (distilled from P₂O₅ to remove trace of alcohol) at a concentration of 20 mg/ml. L-serine is first lyophilized from a 10% (w/v) aqueous solution to remove trace of methanol and is subsequently dissolved at 45 °C at different concentrations up to saturation (46% w/v) in 100 mM acetate buffer (Ph 5.6) containing 100 mM CaCl₂. Phospholipase D is added to the serine solution at 45 °C to a final concentration of 1 IU/ml. An equal volume of the DODPC solution in ether is added and the incubation flask is immediately closed, in order to avoid ether evaporation. Incubation is carried out at 45 °C with stiπing to complete mixing of both phases. Usually, two additional portions of phospholipase D equal to the starting amount are added after 30 minutes and 60 minutes respectively. Incubation is stopped after 90 minutes by addition of 100 mM EDTA (equivalent to two volumes of acetate buffer). Ether is evaporated at room temperature under a stream of nitrogen gas and the aqueous layer is mixed with 4.3 vol. of chloroform/methanol (5.8 v/v) and is stiπed for 30 min. The single phase mixture is filtered through a glass filter G-2 and the filtrate is stiπed for 10 min. with 1 volume of water and 3.7 volumes of chloroform. After centrifugation (10 min, 3000 x g) the lower chloroform layer is collected and mixed with an equal volume of absolute ethanol, followed by evaporation to dryness under reduced pressure. The residue is dissolved in chloroform. Similar incubation is carried out at 37 °C in which serine is replaced by ethanolamine, glycerol, methanol, or ethanol in order to establish optimal conditions leading to the highest yields of DODPS.

The negatively charged polymerizable phosphohpids, chelated with metal ions such as Ca²", can be formed into water-free liposomes and converted into spherical bilayer liposomes by exposure to chelating agents. Additionally, negatively charged polymerizable phosphohpids can be mixed with the novel fatty acids and targeted fatty acids described above, and water- free composite structures can be formed in the presence of divalent cations. Following conversion of water- free Hposomes to spherical liposomes with internal aqueous space in the presence of chelating agents, resulting liposomes can be cross-linked for stabilization by polymerization initiators in the same manner as for the non-charged liposomes described above.

5.2 POLYMERIZABLE FATTY ACIDS

The present invention encompasses, in one embodiment, novel polymerizable fatty acids which can be used both to increase the stability of polymerized liposomes incorporating them, and to provide a functional acid linking group to conveniently, efficiently and effectively attach targeting ligands to polymerized liposomes. The o polymerizable fatty acids comprise at one end a polymerizable group, at the other end an acid functional group, and a surfactant group, between the polymerizable and functional groups, forming the central portion of the fatty acid, and optionally chemically stable linking moieties between these groups. It is prefeπed that the functional group be an acid functional group. 5 For example, the structure of these novel fatty acids in one embodiment is:

R₄-X-PEG-Y-B wherein R₄, the polymerizable group, is a lipophilic chain (fatty acid chain) with at least one polymerizable functional group that will enable polymerization; X or Y are independently a functional linkage such as an ester bond, an ether bond, an amide bond or a carbamate; B is 0 an acid functionality, -NH₂, or an aldehyde; and PEG is the preferred surfactant group which can vary in molecular weight as described below.

The structure of these novel fatty acids gives them unique functionality and particular utility when used in conjunction with polymerizable liposomes. The polymerizable group allows the novel fatty acid molecules to co-polymerize with 5 polymerizable phosphohpids in a polymerizable liposome, so that the molecules are covalently bound to the polymerized liposome, rather than attached in a less-stable fashion, such as by intercalation or steric entanglement. The functional acid group provides a convenient reaction site which can be derivatized using known techniques to attach any targeting ligand capable of bonding to the acid or derivatized acid moiety. The surfactant 0 group is disposed between the polymerizable group and the functional acid group, and comprises a polymeric chain with hydrophilic and hydrophobic regions.

The surfactant group serves several functional purposes. The length of the polymeric chain of the surfactant group can be chosen to be short or long, and the relative hydrophilicity/hydrophobicity of the chain can be altered, depending on the desired 5 properties of the liposome. The polymeric chain should not be long enough to affect the ability of the lipophilic moiety to participate in the lipid packing. A long-chain surfactant group with significant hydrophilicity, for example, can extend away from the liposome into the suπounding solution, providing the liposome with numerous hydrophilic "hairs" protecting the liposome body and effectively "disguising" it to aid its passage through the G-I tract. A short-chain surfactant group with less hydrophilicity will stay closer to the body of the liposome, and will tend to coil and tangle, to give the liposome numerous hydrophilic coils or tangles close to the liposome surface. It will be appreciated that several configurations can be achieved, by varying the length and hydrophilicity of the polymer chain. When the fatty acid is coupled to a targeting ligand and incorporated into a polymerized liposome, the polymer chain of the surfactant group additionally serves as a "spacer" between the liposome and the targeting group, allowing the targeting group to be held closer or farther from the body of the liposome, as desired.

The polymerizable group can be any group capable of coupling to the surfactant group and co-polymerizing with polymerizable phosphohpids. A wide variety of polymerizable groups are suitable, and it will be appreciated that the particular choice of polymerizable groups will depend upon the polymerizable phospholipid and surfactant groups chosen. For example, it is convenient to use a mono-, di- or poly-unsaturated aliphatic carboxylic acid, which can polymerize with a polymerizable phospholipid through the double or triple bond or bonds, and can couple to hydroxy-terminated surfactant groups through the acid moiety. Specific examples of polymerizable groups include, but are not limited to, unsaturated aliphatic acid groups such as CH₃(CH₂)_mCH=CH-CH=CHCOOH where the number of methylene groups (m) can vary from 4 to 12. The double bonds or polymerizable functionalities can be anywhere in the chain so long as they provide an environment suitable for polymerization and packing. One or more of such functionalities can be present in a molecule. The surfactant group comprises a polymeric chain with hydrophilic and hydrophobic regions, capable of coupling to both the polymerizable group and the functional acid group. Polyethers such as polyethylene glycol, polypropylene glycol, and their copolymers, for example, are suitable surfactant groups. Poly(lactic acid) may also be used. A prefeπed surfactant group is polyethylene glycol, as it is readily coupled to the prefeπed enoic polymerizable groups and the prefeπed dioic functional acid groups discussed below. The functional group can be an acid capable of coupling to the surfactant group. Diacids are prefeπed, as they are easily attached to the prefeπed polyether surfactant groups. Particularly preferred are saturated, aliphatic diacids of the formula: HO-C(O)-(CH₂)_b-C(O)-OH where b is an integer from 0 (i.e., oxalic acid) to 12, preferably 0 to 4. Unsaturated diacids having from 2 to 14 carbon atoms are also suitable. For convenient coupling to the surfactant group, diacids which can be used in their anhydride form are especially prefeπed, such as succinic acid (succinic anhydride). Alternatively, the functional group can be an amine, an amide or diamine.

In a prefeπed embodiment, the polymerizable group is a 2,4-dienoyl, the surfactant is a polyethylene glycol group (PEG), and the functional group is a short-chain diacid acid group. In this embodiment, the fatty acids have the formula:

CH₃-(CH₂)_a-CH=CH-CH=CH-C(O)-(OCH₂CH₂)_n-O-C(O)-(CH₂)_b-CO₂H where a is an integer from 0 to 18, preferably 4 to 12, b is an integer from 0 to 12, preferably 0 to 4 and the value of n depends on the average molecular weight of the polyethylene glycol reagent used to synthesize the fatty acids; n can range from about 4 (PEG-200) to about 45 (PEG-2000). It will be appreciated that n is an average value, not generally integral, which characterizes a mixture of chain lengths present in commercially available polyethylene glycols of a particular molecular weight average.

In a more preferred embodiment, the polymerizable group is an 2,4 octadecadienoyl group (2,4OD), the surfactant is a polyethylene glycol group (PEG), and the functional acid group is a succinic acid group (Su). In this particularly prefeπed embodiment, the fatty acids have the formula

CH₃-(CH₂)₁₂-CH=CH-CH=CH-C(O)-(OCH₂CH₂)_n-O-C(O)-CH₂-CH₂-CO₂H where n is about 8.7, coπesponding to the average n in PEG-400. A polyethylene glycol of any desired molecular weight can be incorporated into the 2,4-ODPEGSu fatty acid. For use with the targeting ligands described below, however, polyethylene glycols with average molecular weights from about 200 to about 2000 are prefeπed, and a molecular weight average of about 400 or about 1900 is most prefeπed. These prefeπed ODPEGSu polymerizable fatty acids can be formed by first reacting 2,4-octadecadienoic acid with a desired molecular weight polyethylene glycol to form 2,4-ODPEG, then derivatizing the ODPEG product with succinic anhydride to form ODPEGSu. For a detailed synthesis, see the example section of U.S. Patent Application Serial No. 09/002,145, filed on December 31, 1997, which is incorporated herein in its entirety.

5.3. TARGETED MOLECULES

A variety of molecules and ligands may be used to modify the polymerized liposomes of the present invention in order to target them to a specific site cell type, including, but not limited to, glycoproteins, carbohydrates, lectins, monoclonal antibodies, antibody fragments, mimetic peptides, natural or synthetic organic or inorganic molecules, specific cell surface receptors of ligands, viral proteins, bacterial proteins, i.e., cholera toxin B subunit, phage display hybrid peptides and magnetic particles. The present invention encompasses the fatty acids described supra, which have been further derivatized (if necessary) and coupled to a ligand or molecule capable of targeting particular cells in the gastrointestinal tract. It is expected that Hposomes prepared with these targeted ligands will selectively bind to the targeted cells, thereby increasing the effectiveness of delivery of encapsulated drugs. The targeting ligand can be, for example, a glycoprotein, a carbohydrate, a lectin, a monoclonal antibody, an antibody fragment, a mimetic peptide, a natural or synthetic organic or inorganic molecule, a specific cell surface receptor ligand, a viral protein or a bacterial protein with affinity for human and mammalian intestinal M cells. The polymerized liposomes of the invention comprising polymerizable fatty acids are easily derivatized and covalently linked to ligands capable of targeting particular cells in the G-I tract. The fatty acids are first coupled to a targeting ligand, then copolymerized with a polymerizable liposome, to form a stable, polymerized liposome with the desired targeting ligand covalently attached. The polymerization can be carried out in the presence of a desired therapeutic agent, such as a vaccine or antigen, or the polymerized liposome can be loaded with a therapeutic agent after polymerization, using known techniques. The resulting stable, targeted liposome can be used to effectively and selectively deliver therapeutic agents to M cells in the G-I tract. The targeted fatty acids of the present invention are thus especially useful for the oral, intranasal and/or mucosal delivery of therapeutic agents, such as vaccines.

In a prefeπed embodiment of the present invention carbohydrates or lectins are used to target the polymerized liposomes of the present invention to M cells and Peyer's Patch cells of the small intestine. In another prefeπed embodiment of the present invention, lectins which bind to fucosyl sugars are used to modify the polymerized liposomes. Lectins are a heterogenous group of proteins or glycoproteins that recognize carbohydrate residues on cell surface glycoconjugates with a high degree of specificity. Examples of lectins that may be used to modify the polymerized Hposomes of the present invention, include but are not limited to, lectins specific for fucosyl glycoconjugates, such as Ulex Europeas Agglutinin I (UEA); lectins specific for galactose/N-acetylgalactoseamine, such as Phaseolus vulgaris haemagglutinin (PHA), tomato lectin (Lycopersicon esculentum) (TL), wheat germ agglutinin (WGA); lectins specific for mannose, such as, Galanthus nivalis agglutinin (GNA); lectins specific for mannose/glucose, such as, con A/concavalan A. (See e.g., Lehr et al., 1995, in Lectins Biomedical Perspectives, pp. 117-140). These targeting molecules can be derivatized if desired. See e.g., Chen et al., 1995, Proceed. Internat. Symp. Control. Rel. Bioact. Mater. 22 and Cohen WO 9503035. In another embodiment of the invention, polymerized liposomes may be modified with viral proteins or bacterial proteins that have an affinity for a particular residue expressed on a cell surface or that have an affinity for a cell surface protein or receptor. Examples of such proteins include, but are not limited to, cholera toxin B subunit, bacterial adhesotopes.

In yet another embodiment of the present invention, polymerized liposomes may be modified with monoclonal antibodies or fragments of antibodies which target the polymerized liposome to a particular cell type. The polymerized liposomes of the present invention may be modified with ligands for specific mucosal cell surface receptors and proteins. As used herein, the term "ligand" refers to a ligand attached to the polymerized liposomes which adheres to the mucosa in the intestine or can be used to target the liposomes to a specific cell type in the G-I tract or following absorption. These can range from ligands for specific cell surface proteins and antibodies or antibody fragments immunoreactive with specific surface molecules, to less specific targeting such as coatings of materials which are bioadhesive, such as alginate and polyacrylate. In general, ligands are bound to or inserted within the polymerized phosphohpids; adhesive polymers are applied as a coating to the particles.

As noted above, the liposomes can be modified, for example, by attaching to the surface of the particle specific ligands for given cells in a mixture of cells. When magnetic particles are also incorporated, the particles can be targeted using the ligands, such as tissue specific surface proteins, then maintained as the targeted cells using a magnetic field while the particles are imaged or a compound to be delivered is released. Such magnetic particles are known in the art and include aqueous-based feπo fluid EMB 807 (Feπofluids, NH). The targeting ligand can be, for example, a lectin with an affinity for human and mammalian intestinal M cells. Prefeπed ligands are lectins such as EEA (Euonymus

Europaeus Agglutinin), fluorescently labeled EEA, FITC-EEA (fluorescein isothiocyanate- EEA), UEA-I (Ulex Europaeus Agglutinin I), and WGA (Wheat Germ Agglutinin). Many other ligands are potentially useful for mucosal application; for example, class II framework Mab, ICAM-1, CTB (cholera toxin B subunit), LT-B (heat labile toxin subunit), reovirus sigma 1 proteins, antibodies to sialyl Lewis A antigen, microbial outer membrane proteins, fimbrial adhesin molecules from salmonella such as lpf gene product, or any protein or peptide Hgand that binds to M cells using the fatty acid substrate.

In a prefeπed embodiment, the targeting ligand is a lectin such as EEA, and the fatty acid is a dienoyl-polyethylene glycol-diacid derivative, such as ODPEGSu. The coupling reaction to covalently attach a lectin to a fatty acid such as ODPEGSu can be carried out using the techniques known in the art such as that described in Chen et al., 1996, Pharmaceutical Research 13: 1378-1383. See Example 2 of U.S. Patent Application Serial No. 09/002,145, filed on December 31, 1997 for more details regarding the coupling of 2,4- ODPEGSu with EEA.

The amount of targeting ligand used in the polymerizable liposome will depend on the specific target.

5.4. MATERIALS TO BE ENCAPSULATED

The polymerized liposomes and targeted polymerized liposomes of the present invention are used as the carriers in a drug delivery system, especially an oral, intranasal and intranasal drug delivery system. The polymerized Hposomes of the present invention may be utilized for the delivery of a wide variety of compounds, including vaccines, antigens, allergens and other therapeutic agents or diagnostics. In one embodiment, polymerized Hposomes of the present invention are utilized for the delivery of a biologically active substance. As used herein, the term "biologically active substance" refers to eukaryotic and procaryotic cells, viruses, vectors, proteins, peptides, nucleic acids, polysaccharides and carbohydrates, lipids, glycoproteins, and combinations thereof, and synthetic organic and inorganic drugs exerting a biological effect when administered to an animal. For ease of reference, the term is also used to include detectable compounds such as radiopaque compounds including air and barium, magnetic compounds, fluorescent compounds, and radioactive compounds. The active substance can be soluble or insoluble water. Examples of biologically active substances include anti-angiogenesis factors, antibodies, antigens, growth factors, hormones, enzymes, and drugs such as steroids, anticancer drugs or antibiotics, as well as materials for use as insecticides or insect repellents, fertilizers and vitamins, or any other material having a biological effect where controlled release is desirable.

In one embodiment, polymerized Hposomes of the present invention can be used for the oral, intranasal and/or mucosal delivery of a wide variety of therapeutics, including but limited to, chemotherapy agents, antibiotics, insulin, cytokines, interferon, hormones, calcitonin, hormones, fertility drugs, antiviral agents (ddl, AZT, ddC, acyclovir and the like), antibacterial agents, antifungal agents, DNA and RNA nucleotides, i.e., useful for gene therapy. In another embodiment, polymerized liposome of the present invention can be used diagnostically. In particular, a pharmaceutically acceptable gamma- emitting moiety, including but not limited to, indium and technetium, magnetic particles, radiopaque materials such as air or barium and fluorescent compounds, can be incorporated into the polymerized liposomes of the present invention. 5.4.1 ANTIGENS In a prefeπed embodiment, antigens or immunogens encompass any agent that induces an immune response in an animal. In another prefeπed embodiment, the polymerized liposomes of the present invention have utility in the oral, intranasal and/or mucosal delivery of vaccines and antigens. For example, the polymerized Hposomes of the present invention may be designed to carry a wide variety of antigens including, but not limited to, diphtheria toxoid, tetanus toxoid, influenza hemagglutinin, ospA antigen from Lyme disease bacterium, and HTLV envelope protein antigen. Antigens to poliovirus, rhinovirus, rabies, vaccinia, Epstein-Ban virus, hepatitis, HIV-1 and HIV-2, herpes virus and human immunodeficiency virus are just examples of the many types of antigens which may be encapsulated into the liposomes of the present invention. They may also be utilized for the oral delivery of a wide variety of therapeutics, including but not limited to, chemotherapy agents, antibiotics, insulin, cytokines, interferon, hormones, calcitonin, hormones, fertility drugs, antiviral agents (ddi, AZT, ddc, acyclovir and the like), antibacterial agents, antifungal agents, DNA and RNA nucleotides. The polymerized liposomes of the present invention have utility for the oral, intransal and/or mucosal delivery of vaccines, antigens, allergens, diagnostic agents therapeutic agents and drugs. The polymerized liposomes of the present invention may be designed to carry a wide variety of antigens including, but not limited to diphtheria toxoid, tetanus toxoid, influenza hemagglutinin, ospA antigen from Lyme disease bacterium, and HIV envelope protein antigen. Antigens to poliovirus, rhinovirus, rabies, vaccinia, Epstein-Ban virus, hepatitis, HTLV, herpes virus and human immunodeficiency virus are just examples of the many types of antigens which may be encapsulated into the Hposomes of the present invention. In one embodiment, the polymerized Hposomal compositions of the present invention comprise polymerized liposomes, a stabilizer, and an antigen, wherein the antigen is hydrophobic. In another embodiment, the polymerized Hposomal compositions of the present invention comprise polymerized liposomes and antigen, wherein the antigen is amphopathic. In accordance with these embodiments, the polymerized Hposomal compositions of the present invention may further comprise an adjuvant and/or a targeting molecule.

5.4.2. ADJUVANTS

A convenient means to enhance the efficacy of vaccines to include materials that function as adjuvants. In a prefeπed embodiment, adjuvants encompass any agent that enhances an immune response to antigens. There are a variety of compounds that can act as adjuvants. Examples of adjuvants which may be effective, include, but are not limited to: aluminum hydroxide, monophosphoryl lipid A (MPLA) N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine, simple immunostimulatory oligonucleotides, cytokines such as IL-12, IL-2 or IL-1, saponins, and microbial toxins such as cholera toxin, heat labile toxin and genetically altered derivatives of them.

The effectiveness of an adjuvant may be determined by measuring the induction of antibodies directed against an immunogenic polypeptide containing an antigenic epitope, the antibodies resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants. The effectiveness of an adjuvant may also be determined by measuring the induction of specific killer cells (e.g., cytotoxic T cells) that recognize an antigen. For example, the induction of cytotoxic T cells in response to the administration of a vaccine containing an antigen can be measured utilizing techniques known to one of skill in the art.

5.5. STABILIZERS

In one embodiment, the polymerized liposome compositions of the present invention contain stabilizing agents. In a prefeπed embodiment, stabilizing agents are included in the aqueous interior space of liposomes along with an aqueous soluble biologically active material. The term "stabilizer" or "stabilizing agent" as used herein refers to any agent which protects, preserves or stabilizes the conformational structure of a biologically active material, e.g., antigen, incorporated into polymerized lipsomes during production, storage and/or uptake by an animal. In particular, stabilizing agents include, but are not limited to, polyols with multiple hydroxyl groups, such as trehalose, mannitol, sorbitol, sucrose, and surfactants, such as pluronic F-68 and polyethylene- polypropylene block polymers. Other stabilizing agent include gelatin, glycine, EDTA, polyethylene glycols, polyvinyl pyπolidone, and ZnCl₂.

In one embodiment, stabilizers protect biologically active material incorporated into the polymerized liposome compositions of the present invention from degradation. In a prefeπed embodiment, stabilizers protect antigen incorporated into polymerized liposome compositions of the invention from degradation and preserve the conformational structure (i.e., the primary, secondary and tertiary structure) of the antigen. In particular, stabilizers protect antigen from nucleophilic attack by free radical ions and preserve the conformation of the antigen. Conformation of structure is important in eliciting appropriate immune responses to a variety of vaccines, including viral subunit vaccines such as HIV gpl20, herpes virus glycoprotein D antigen, and other viral glycoproteins. Conformational properties are also important for preservation of bacterial antigen structures. Preserving these conformational epitopes in formulation and during storage influences the quality of host immune response and will differentiate an effective vaccine composition from an ineffective one.

5.6. ENCAPSULATION OF MATERIAL INTO LIPOSOMES

Materials are generally incorporated into the liposomes at the time of formation, following polymerization using sonication of a solution of the material which contains the liposomes, and following polymerization by rehydration of a thin film of the liposomes.

The following is a general method for the preparation of polymerized liposomes wherein a biologically active substance is entrapped prior to the polymerization of the monomeric polymerizable liposome. First, the monomeric liposome is prepared by the thin film hydration of a monomeric double bond-containing phospholipid. The monomeric phospholipid and, optionally, a polymerizable fatty acid, non-polymerizable lipids or adjuvants, targeted polymerizable fatty acid, is dissolved, and the solution is then dried to form a thin film of phospholipid. A solution containing substance to be entrapped is added, preferably with a catalytic amount (1-3 percent by weight) of free radical initiator. At this stage, it is preferable to establish an inert atmosphere. The lipid is then hydrated by gently shaking the mixture at a temperature of from about 20 to 50 °C, usually around 25 °C, for between five minutes and two hours, preferably around five minutes. Once the Hpid film is hydrated, the trap ratio of the liposome can be increased by performing one or more freeze- thaw cycles on the liposome solution. This is particularly useful when the material being incorporated is hydrophilic in nature. Next, the polymerization is carried out at room temperature, or from about 20 - 37°C, preferably at around 25 °C, for 30 minutes to 20 hours, preferably about 5 hours, or until the polymerization is essentially complete. The desired degree of crosslinking is from 10 to 100 percent.

Unentrapped biologically active substance can be removed by several means, including repeated centrifugation, decantation, gel filtration, and dialysis. The polymerized liposomes are then suspended in a buffer solution. The buffer solution has a pH preferably between pH 4.5 and pH 9.5, more preferably at physiological pH.

This method of entrapping biologically active substances is prefeπed because it does not involve the use of organic solvents. Use of organic solvents can denature biologically active substances. Further, the temperature requirements are mild, with the temperature typically not exceeding 25 °C. If the biologically active substance cannot tolerate the temperature conditions or exposure to radiation, a third method of entrapping the substance in a polymenzed liposome is prefeπed In this method, the liposomes are polymenzed, m the presence of a polymerizable fatty acid or targeted polymenzable fatty acid, if desired, before adding the mateπal to be encapsulated After the polymerization is complete, the polymerized Hposomes are added to an aqueous solution of the mateπal The solution should be aqueous, although it can include small amounts of organic solvent The solution is sonicated, and the somcation entraps the substance mside the polymenzed liposomes Another method for entrapping biologically active substances in polymenzed liposomes is to dissolve the partially polymerized liposomes or mixture of partially polymeπzed hposomes with a polymenzable fatty acid and/or a targeted polymenzable fatty acid, m a suitable organic solvent, such as tetrahydrofuran, acetone, ether, chloroform, methylene dichloπde, and ethyl acetate, and evaporate the solvent to form a thin film of partially polymeπzed liposome Following encapsulation, polymenzation is completed Hydrophobic matenals are preferably encapsulated in the liposomes by dissolving the matenals in an organic solvent with the phospholipid, before forming the liposomes Hydrophilic materials are more preferably incorporated by hydratmg a thin film of polymerized liposomes in the presence of an aqueous solution of the substance

Mateπals can be entrapped withm the liposomes, as well as or alternatively in one or more of the hpid layers of the phospholipid bilayer This is typically determined by the hydrophobicity/hydrophilicity of the mateπal to be incorporated as well as the method of preparation

5 7 MODES OF ADMINISTERING THE

POLYMERIZED LIPOSOMES TO A PATIENT

The polymeπzed hposomes of the present invention are administered by those routes which optimize uptake by mucosa For example, oral, sublmgual, buccal, rectal and intranasal are prefeπed routes of administration However, topical, transdermal and parenteral delivery may also be used The most prefened route is oral Further, the polymeπzed liposomes are particularly suitable for delivery through mucosal tissue or epithelia The polymeπzed liposomes of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, creams, ointments, suppositoπes and the like When the dosage unit form is a capsule, it can contain, m addition to the matenal of the above type, a liquid earner or adjuvant, when the liposomes contain an antigen Liposomes can also be administered with unentrapped antigen If administered topically the liposomes will typically be administered in the form of an ointment or transdermal patch If administered intranasally the liposomes will typically be administered in an aerosol form, spray, mist or in the form of drops. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, PA (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.

The polymerized liposomes of the present invention are suitable for administration to mammals, including humans, as well as other animals and birds. For example, domestic animals such as dogs and cats, as well as domesticated herds, cattle, sheep, pigs and the like may be treated or vaccinated with the polymerized liposomes of the present invention. The polymerized liposomes of the present invention have use in vaccine preparations. The preparation of vaccines containing an immunogenic polypeptide as the active ingredient is known to one of skill in the art.

5.8. VACCINE FORMULATIONS Suitable preparations of vaccines include liquid solutions or suspensions; solid forms such as capsules and tablets, liquids for injections, may also be prepared. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combmations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.

Immunogenic polypeptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

The vaccines of the invention may be multivalent (contain more than one antigen) or univalent. For example, vaccines that comprise recombinant viruses that direct the expression of more than one viral antigen are multivalent. Many methods may be used to introduce the vaccine formulations of the invention; these include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, rectal, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle).

The patient to which the vaccine is administered is preferably a mammal, most preferably a human, but can also be a non-human animal including but not limited to cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats. The vaccine formulations of the invention comprise an effective immunizing amount of the antigenic protein and a pharmaceutically acceptable caπier or excipient. Vaccine preparations comprise an effective immunizing amount of one or more antigens and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable caπiers are well known in the art and include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combmations thereof. One example of such an acceptable carrier is a physiologically balanced salt solution containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc. The caπier is preferably sterile. The formulation should suit the mode of administration. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is administered by injection, an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.

The precise dose of vaccine preparation to be employed in the formulation will also depend on the route of administration, and the nature of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to produce an immune response to the antigen in the host to which the vaccine preparation is administered.

Use of purified antigens as vaccine preparations can be carried out by standard methods. For example, the purified protein(s) should be adjusted to an appropriate concentration, formulated with any suitable vaccine adjuvant and encapsulated within the polymerized liposome. Suitable adjuvants may include, but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin or pluronic polyols; polyanions; peptides; oil emulsions; alum, Lipid A and derivatives of Lipid A, cytokines, and MDP. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. In instances where the recombinant antigen is a hapten, i.e., a molecule that is antigenic in that it can react selectively with cognate antibodies, but not immunogenic in that it cannot elicit an immune response, the hapten may be covalently bound to a carrier or immunogenic molecule; for instance, a large protein such as serum albumin will confer immunogenicity to the hapten coupled to it. The hapten-carrier may be formulated for use as a vaccine.

Effective doses (immunizing amounts) of the vaccines of the invention may also be extrapolated from dose-response curves derived from animal model test systems.

The present invention thus provides a method of immunizing an animal, or treating or preventing various diseases or disorders in an animal, comprising administering to the animal an effective immunizing dose of a vaccine encapsulated within a polymerized liposomes of the present invention.

5.9. USE OF ANTIBODIES GENERATED

BY THE VACCINES OF THE INVENTION

The antibodies generated against the antigen by immunization with the polymerized liposome delivered antigenic protein also have potential uses in diagnostic immunoassays, passive immunotherapy, and generation of antiidiotypic antibodies.

The generated antibodies may be isolated by standard techniques known in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays. The antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art, such as those listed supra, may be used for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays, to name but a few.

The polymerized liposome encapsulated vaccines of the present mvention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of pre-formed antibody directed against a heterologous organism.

The antibodies generated by the vaccines of the present invention can also be used in the production of antiidiotypic antibody. The antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Arm. Immunol. (Paris) 125c:373; Jerne et al, 1982, EMBO J. 1:234).

In immunization procedures, the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be 5 administered by reference to the immune response and antibody titers of the subject.

5.10. PACKAGING

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may 10 for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical earner may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.

15

6. EXAMPLE 1. PREPARATION AND ANALYSIS OF POLYMERIZED

LIPOSOMES FOR ORAL ADMINISTRATION

A mixture of lipids containing polymerizable phosphohpids, non-polymerizable phosphohpids, polymerizable and non-polymerizable fatty acids and other lipids and adjuvants can be lyophilized to form a powdery material, then rehydrated with a solution

20 containing the desired peptides or antigens to be encapsulated in the liposome or to be incorporated into the Hpid bilayer. The rehydrating takes place at a temperature above the melting point of the lipid mixture. The resulting liposomes are shown to be composed of mostly multi-lamellar vesicles of wide-size distribution. To reduce the size of liposomes and to control the lamellarity, vesicles are extruded through specific pore sized

25 polycarbonate membranes. To achieve naπow size distribution, the liposome preparation is extruded up to 20 cycles and can be subjected to freeze-thaw one to four times during the process.

The Hposomes are then polymerized using any known technique. Polymerization is initiated with water soluble free radical initiators, lipophilic free radical initiators or by

30 exposure of the liposomes to short wave radiation such as uv or gamma iπadiation. The unincorporated peptides or antigens can be separated from the liposomes by centrifugation at approximately 100,000 x g. or the liposome solutions can be passed through an ultrafiltration column to purify and concentrate the liposomes containing peptides or

„ . antigens.

35 ^s

A prefeπed method uses a mixture of 2,4-DODPC as the polymerizable lipid mixture, dissolved in tert-butanol. After rehydration with a solution of the desired peptide or antigen, the liposomes are extruded and polymerized using a sodium bisulfite (580 mM) potassium persulfate (127 mM) redox couple initiator.

Preparation of polymerized liposomes containing a lipophilic adjuvant, tetanus antigen and trehalose as the stabilizing agent

Liposomes were designed to be used as mucosally administered vaccines. In addition to application of vaccines at bodily mucosal surfaces, prefeπed routes of administration are via the oral route or the intranasal route. To accomplish this, liposomes were constructed to contain in the interior aqueous compartment an antigen, an adjuvant, and a saccharide stabilizer to enhance stability of the proteinaceous antigen during formulation, handling, and subsequent storage. Further, cross-linked or polymerized phosphohpids were used to construct the membrane bilayers, to withstand the degradative environment of the GI tracts and other mucosal surfaces. Liposomes were constructed using 2,4 DODPC lipid (NOF Lipidure PC 8082 ND-P9A). 2,4 DODPC was dissolved in t- butanol and co-lyophilized with monophosphoryl Lipid A (MPLA: Ribi Immunochem Lot

# 67039-E0896B) in t-butanol for 24 hours ( Ratio 1500 μmoles of 2,4 DODPC: 6 mg

MPLA). This sample was then rehydrated with an isotonic saline solution containing 21 mgs tetanus toxoid (TT) per ml (Massachusetts Biological Laboratories Lot # CTP-996) in equal volume to 2 M Tris pH 8.5 containing 500 mM trehalose. For every 100 μmole of 2,4

DODPC, 0.5 ml of tetanus solution and 0.5 ml of Tris/trehalose buffer were added. This suspension was then mixed vigorously and extruded through 100 nm polycarbonate filters ten cycles, freeze/thawed (liquid nitrogen/30°C) three times, and re-extruded 10 cycles through 100 nm filters. Sodium bisulfite and potassium persulfate were added to initiate the polymerization reaction in the following ratios: 47.4 mg and 26.8 mg respectively per 100 μmoles of DODPC. When the polymerization reaction reached > 36% at 25 °C the sample was diluted with cold saline and immediately spun on the ultracentrifuge. The sample was washed two times with isotonic saline and reconstituted in its original volume. The washing was to remove the initiators and any free tetanus from the polymerized liposomes.

This sample was labeled # 7.

Preparation of polymerized liposomes containing a lipophilic adjuvant and tetanus antigen

As above, DODPC (NOF Lipidure PC 8082 ND-P9A) was dissolved in t-butanol and co-lyophilized with MPLA (Ribi Immunochem Lot # 67039-E0896B) in t-butanol for

48 hours (Ratio 1500 μmoles of DODPC: 4.5 mg MPLA). This sample was then rehydrated with a 1.8 mg/ml TT isotonic saline solution (Massachusetts Biological

Laboratories Lot # TP-1001) in equal volume to 2 M Tris pH 8.5, without a stabilizing agent. For every 100 μmole of DODPC, 0.5 ml of tetanus solution and 0.5 ml of buffer were added. This suspension was then vigorously mixed, subjected to three cycles of freeze/thaw (liquid nitrogen 30 °C) and extruded through 100 nm filters eleven cycles. The size after extrusion was 137 nm. Sodium bisulfite and potassium persulfate were added to

5 initiate the polymerization reaction at the following proportions: 23.7 mg and 13.4 mg respectively per 100 μmoles of DODPC. When the polymerization reaction reached > 25% at 25 °C the sample was diluted with cold saline and immediately washed two times with isotonic saline and reconstituted in its original volume. The washing was to remove the initiators and any unincorporated tetanus toxoid in the liposome preparation. This

10 preparation was labeled #11.

Preparation of polymerized liposomes containing tetanus toxoid and adjuvant for intranasal immunization

A third tetanus vaccine was constructed using polymerized Hposomes. This vaccine differed from the two prior examples in the quantity of tetanus antigen contained within the liposomes and in the content of the adjuvant MPLA and in the content of polymers in the liposome bilayer membrane. As above, 2,4 DODPC (NOF Lipidure PC 8082 ND-P9C,D,E) was dissolved in t-butanol and co-lyophilized with MPLA (Ribi Immunochem Lot # 67039-

E0896B) in t-butanol for 24 hours ( Ratio 4000 umoles of DODPC: 14.4 mg MPLA). The samples were then rehydrated with a 3.6 mg/ml Tetanus toxoid (TT) isotonic saline solution

(Massachusetts Biological Laboratories Lot # CTP-996) in equal volume to 2 M Tris pH 8.5 with 500 mM Trehalose. For every 100 umole of DODPC, 0.5 ml of TT solution and 0.5 ml of buffer were added. This suspension was then mixed vigorously, subjected to three cycles of freeze/thaw (liquid nitrogen/30C) and extruded 5 cycles through 200 nm polycarbonate membrane followed by 4 cycles of extrusion through 100 nm polycarbonate membranes. Approximately 88% of the resulting liposomes after extrusion and before polymerization of the lipid bilayer were 147 nm in diameter. Sodium bisulfite and potassium persulfate were added to initiate the polymerization reaction in the following ratios: 14.22 mg and 8.844 mg respectively per 100 umoles of DODPC. When the

_„ polymerization reaction reached 27% at 25° C, the sample was passed over a gel filtration to remove the initiators and any remaining unincorporated tetanus toxoid. Samples were then filtered with an 800 nm filter to remove gel packing from formulations. Mean particle diameter was 188 nm and content of cross-linked polymers was approximately 40%. Most of the liposomes were small unilamellar vesicles, as observed by negative staining electron

-._<- microscopy. Analysis of polymerized liposomes

Liposomes prepared by the above means were analyzed for several properties before examining capability of inducing biological activity. These properties include size and size distribution, surface charge, morphology, content of biologically active material, polymer content, content of adjuvant and other formulation materials, and stability properties. Size of the liposome preparations was determined by laser light scattering techniques using a Coulter N4B particle size analyzer. For all liposome preparations the major peak of liposomes comprising no less than 85% of the liposomes, was between 140 and 160 nanometers. Further analysis of the preparations was done with negative stain electron micrography; the vast majority of liposomes were typical unilamellar structures. A few isolate structures with intermediate morphologies were observed, thought to be due to freezing of intermediates by polymerization. To analyze the encapsulation of tetanus toxoid, Hposomes were subjected to SDS-PAGE gel electrophoresis. Using this technique, the presence of tetanus was estimated by scanning densitometry of the stained gel. Because of the relatively insensitive densitometry assay, a sandwich ELISA assay was developed to assess the presence of tetanus toxoid on the surface of liposomes and within the inner aqueous space. This technique was adequate to measure surface associated tetanus toxoid, which was for all preparations less than 10% of the total liposome-associated tetanus toxoid. Exposure of polymerized liposomes to triton X-100 in excess of the amount to which they are normally stable (>1 mM) allowed for release of tetanus. Total protein was determined by dye absorption techniques. Results from all these techniques coπelated to yield a determination of 15% entrapment ratio of tetanus toxoid. The lipid concentration was determined in the samples by phosphorous analysis, by the Bartlett method. The polymer content of the membranes at the final steps of the process was 40-60%, as determined by relative ratio of absorbance at 205 nm/254 nm.

The parameters for particular formulations used for oral administration of vaccines are given in Table 1 and Table 3 (infra). The equivalent parameters for formulations administered intranasally is presented in Table 4 and Table 7 (infra). The results characterizing polymerized liposomes for intranasal administration is in Table 5 and Table 6 (infra).

7. EXAMPLE 2. ORAL IMMUNIZATION OF ANIMALS

7.1 MATERIALS & METHODS To examine the effect of the stabilized liposome formulations on inducing a relevant immune response in animals, the various preparations were used to immunize inbred mice. The parameters for the particular formulations for oral administration of the vaccines are given in Table 1. Several control groups were also included: 1 μg/ TT administered (group #17), free unformulated tetanus toxoid at 50 μg/dose with MPLA (group # 22), and polymerized liposomes containing MPLA and trehalose but no tetanus.

Table 1. Parameters of polymerized liposome formulations for oral immunization

Female Balb/C mice, 7- 9 weeks old, were inoculated by gastric intubation techniques at 0, 14, and 28 days with a 500 microliters of samples or control preparations. At appropriate time after each immunization blood was withdrawn from each animal and serum samples were analyzed for tetanus specific total IgG, by a direct ELISA. Proliferation in response to TT was measured by reduction of the tetrazolium dye WST-1 (Roche Molecular Biochemicals) as described. To assess the presence of secretory antibodies in the intestine (slgA), a length of small intestine from the distal side of the stomach to the proximal end of the colon was removed and washed with PBS. The clarified gut wash was assayed for the presence of TT specific IgA. Samples were standardized for total IgA content and TT specific IgA tested by ELISA as described below.

ELISA Assay for Tetanus Antibodies

The wells of a Nunc Maxi-Sorp plate were coated with 50 μl of tetanus toxoid (Mass. Biol. Lab.) diluted to 2 μg/ml in 50mM bicarbonate buffer, pH 9.5. The plate was incubated overnight at room temperature After washing once on a BioTek ELx50 strip washer with PBS-Tween 20 (0 002%), unbound protem binding sites were blocked with PBS-BSA The plate was incubated for 1-2 hour at room temperature Serum samples were diluted (typically 1/25, 1/75, and 1/225) in blocking solution and added in duplicate to TT coated wells The plate was incubated for 2 hours at room temperature After washing twice, goat anti-mouse IgG-horseradish peroxidase conjugate (Southern Biotechnology Associates, Birmingham, AL) (1/5000) was added and the plate is incubated for 1 hour Antibody subclass specific conjugates were used to differentiate between IgGl, IgG2a, IgG2b and IgA antibodies To develop the color, 100 μl TMB peroxidase substrate (KPL, Gaithersburg, MD) was added Color development was stopped by the addition of 1 N H₂SO₄ The OD 450 - 620 was read on a Perkin-Elmer HTS 7000 plate reader Anti-TT IgG m each sample was determined by companson of sample OD against the standard curve generated with a standard curve of mouse IgG absorbed to the plate using the non-linear regression function on Prism Graphpad software

Lymphoprohferation of spleen and mesentenc lymph node cells Isolated spleens from certain animals were collected at the termination of the experiment to assess the ability for spleen cell cultures to respond to tetanus toxoid Spleens and mesentenc lymph nodes were dissected from selected animals Single cell suspensions were made using standard techniques Red blood cells in the spleen cell preps were removed by lysis with ammonium chlonde For proliferation assays, cells were plated at 2 X 10⁵ cells/well in Falcon 96 well plates in RPMI-1% mouse serum The cells were stimulated with antι-CD3 (1 μg/ml) or TT and 1 and 10 μg/ml The plates were cultured at 37 ° for 5 days On day 5, 20 μl of WST-1 proliferation reagent was added to each well and the plates were incubated an additional 3 hours The OD 450-620 was read on the HTS 7000 plate reader Data were analyzed by determining the stimulation index for each sample Proliferation is considered positive if the stimulation index is > 2

Table 2 lists the oral immunizations study groups Group #7 was diluted 10 times before dosing whereas group # 11 was dosed without dilution Two negative control formulations were prepared (1) soluble TT mixed with MPLA adjuvant and (2) polymenzed DODPC without tetanus but with MPLA and trehalose

' Subcutaneous

7.2. RESULTS Immune Response to Orally administered polymerized liposomes Oral administration of polymerized liposome formulations containing tetanus toxoid and MPLA, both with and without trehalose stabilizer induced the production of tetanus specific serum IgG (groups 7 and 11, Figure 1). However, a higher, more consistent response was observed in all of the animals that were immunized with polymerized liposomes that contained trehalose as the stabilizing agent (Figure 2). Seroconversion was induced after 1 dose, and a boosting effect was observed after doses 2 and 3. In comparison, unformulated tetanus toxoid admixed with MPLA did not induce seroconversion. Further analysis of serum samples from individual mice revealed that all mice that were immunized with MPLA/TT/liposomes seroconverted after the first dose. In contrast, no mice seroconverted at any point when immunized with the same dose of non-liposome associated TT/MPL (Figures 1 and. 2). At the terminal point of the experiment, mice were sacrificed and intestinal lavage samples were obtained from three mice in each group, (normalized for total IgA content).

Increased slgA levels response were observed in each individal mouse immunized and boosted with the polymerized Hposome/MPLA compositions, whereas a statistically greater response was observed in individual animals immunized with the composition stabilized with trehalose. Little secretory IgA was observed in intestinal lavage samples from mice that had been parenterally immunized with 1 μg TT, although those animals responded vigorously with high titer serum IgG. No slgA response was detected in mice immunized with unencapsulated TT with MPLA (Figure 3).

Splenic lymphocytes were tested for the ability to proliferate in response to TT stimulation in vitro. Spleen cells were collected by standard methods and stimulated with TT at 10 μg/ml. Spleen cells from two of the 3 mice in group 7 proliferated in response to TT. The results demonstrate that tetanus-containing polymerized liposomes induce an potent immune response after oral administration that is increased in the presence of a polylol (trehalose) stabilizer. The humoral response was observed after a single dose of vaccine. This is in contrast to many studies conducted with alternative delivery systems published with TT containing PLGA particles that showed no IgG response or a response only after three immunizations. After one booster immunization mice receiving TT containing polymerized liposomes demonstrated an anamnestic response which was even greater after a second booster immunization.

Example 3. IgG SUBTYPES INDUCED BY

ORAL IMMUNIZATION OF ANIMALS

10

To examine the ability of stabilized polymerized liposome formulations to induce various IgG subtypes as a consequence of oral administration, various preparations were used to immunize inbred mice. The parameters for the particular formulations for oral administration of the vaccines are given in Table 3.

15

Table 3. Parameters of polymerized liposome formulations for oral immunization

20

25

30

* Each vaccine contained approximately 50 μg of tetanus toxoid for oral immunization.

** If MPLA was present in the vaccine, approximately 20 μg was present.

These liposome and control preparations were administered orally in a volume of , , 500 μl to Balb/c mice in groups of 5. The vaccines were administered to mice two weeks and four weeks after the initial immunization. Serum samples were obtained from each animal two weeks following each immunization procedure. Total anti-tetanus antibody IgG was determined in individual serum samples using the ELISA assay described in Example 2 supra and means were plotted (Figure 4). The highest titers of anti-tetanus antibodies were observed in groups immunized via the subcutaneous route. High response rates were observed in groups that included MPLA. The serum obtained after the third immunization (day 41) was also analyzed for the presence of IgG2a and IgGl (Figures 5) and the ratios were compared (Figure 6). The highest quantity of IgG2a was observed in same from group 7 that contained both the adjuvant and the stabilizer. IgG2a functions in complement fixation and virus neutralization.

9. Example 4. INTRANASAL IMMUNIZATION

To examine the capacity of polymerized liposomes to induce an immune response as a consequence of intranasal administration, groups of 5 female Balb/C mice were immunized by intranasal instillation 20 μl of either (1) unencapsulated TT including MPLA as an adjuvant or (2) TT-MPLA entrapped polymerized liposomes or (3) control Hposomes with MPLA but lacking tetanus antigen. The parameters of the liposome formulations is presented in Table 4 (infra). The polymerized liposomes were characterized as described in Example 1 supra and the results are presented in Tables 5 and 6. Polymerization resulted in increased detergerent-resistance of liposomes (Table 6).

Table 4. Parameters of polymerized liposome formulation used for intranasal immunization

Table 5. Characterization of polymerized liposome formulation

Table 6. Detergent Stability, As Determined By Particule Size

A total of 3 μg of TT were administered at each dose according to the protocol in Figure 7. No elevation of serum IgG was observed in mice immunized with liposome without tetanus and only a small amount was observed with admixtures of TT and MPLA after the third dose (Figure 8). These antibodies were detected 2 weeks following a single intranasal dose of vaccine and were further boosted by a second and a third intranasal dose of vaccine (Figure 9). Only a small amount of anti-tetanus toxoid antibodies was observed in animals after receiving three doses of native tetanus toxoid admixed with the adjuvant MPLA and none of those animals seroconverted after one or two doses of the adjuvant/antigen mixture. In addition, IgA, IgG2a, and IgG2b were measurable in sera of animals that were nasally vaccinated with polymerized liposomes containing tetanus toxoid and MPLA (Figures 10-13).

10. Example 5. INTRANASAL IMMUNIZATION WITH

POLYMERIZED LIPOSOMES & ANTIGEN

The influence of antigen localization was investigated using a single preparation of polymerized liposomes with encapsulated tetanus toxoid and MPLA. The vaccines were used to immunize mice intranasally. A total of 20 μl of Hposomal vaccine was administered to anesthetized mice up to four times two weeks apart. To some of the vaccines, native tetanus toxoid was added externally to the Hposomes, keeping the total dose of tetanus constant at 3 μg and by compensating for lipid concentration with empty polymerized liposomes, keeping the dose of MPL constant at approximately 20 μg.

Table 7. Parameters of polymerized liposome formulation for intranasal immunization

As controls, empty MPL/liposomes mixed with tetanus and empty MPL/liposomes without tetanus and native tetanus toxoid/MPL were used. Before each immunization, serum samples were obtained from each Balb/c mouse. Total anti-tetanus IgG was measured at these intervals. Two weeks following the last immunization, serum samples were obtained and monitored for the presence of IgA and IgG. The presence of IgA and IgG in the serum was determined as described in Example 2 supra. Figures 14A, 14B and 15 depicts the concentration of anti-tetanus toxoid IgG detected in the serum samples of the mice. The mice immunized intranasally with polymerized Hposomal vaccine with a ratio of 2:1 of tetanus toxoid encapsulated to external resulted in the highest induction of anti- tetanus toxoid IgG. Nasal lavage samples were also obtained and analyzed for the presence of anti- tetanus IgA. All of the animals that were immunized with the Hposomal MPL vaccine with a ratio of 2:1 seroconverted after the second dose of vaccine and had the highest levels of nasal wash IgA after four immunizations in comparison with native TT/MPL and other controls (Figures 16 and 17). In comparison with the identical liposomes where the ratio was modified to contain either 100% encapsulated antigen or 33% antigen, the highest titers of antibodies were observed in animals that had received the vaccine containing 66% internal encapsulated antigen. The vaccines that contained encapsulated antigen and added antigen yielded higher antibody titers than vaccines where the antigen was totally encapsulated. The vaccine where antigen was added to the exterior of empty liposomes failed to seroconvert any animal after two doses of vaccine.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. Certain embodiments of the invention are illustrated, and not limited, by the following working examples.

Claims

What is Claimed Is:

1 A method of dehvenng antigen to a mammal which compnses orally administenng to said mammal a composition compnsmg polymeπzed liposomes, antigen and stabilizer

2 A method of dehvenng antigen to a mammal which comprises intranasally administenng to said mammal a composition compnsmg polymerized liposomes, antigen and stabilizer

3 The method of Claim 1 or 2, wherein the composition further compπses adjuvant

4 The method of Claim 3 in which the antigen and the adjuvant are in the mtenor space of the polymenzed liposome

5 The method of Claim 3 m which the antigen is m the mtenor space of the polymenzed liposome and the adjuvant is in the leaflet of the polymenzed liposome

6 The method of Claim 1 or 2 in which the antigen is hydrophobic

7 The method of Claim 3 in which the antigen is hydrophobic or amphopathic

8 The method of Claim 3 m which the antigen and the adjuvant are m the leaflet of the polymenzed liposome

9 The method of Claim 3 in which the antigen is m the leaflet of the polymerized liposome, and the adjuvant is in the mtenor space of the polymerized liposome

10 The method of Claim 1 or 2 in which the polymenzed liposomes compnse a phospholipid bilayer having covalently bonded phosphohpids therein

11 The method of Claim 1 or 2 in which the polymeπzed liposome comprises one or more polymenzable lipids and polymerizable fatty acids

12. The method of Claim 1 or 2 in which the composition further comprises at least one targeting molecule selected from the group consisting of glycoproteins, antibodies, antibody fragments, mimetic peptides, natural or synthetic organic or inorganic molecules, and specific cell surface receptors and ligands.

13. The method of Claim 3 in which the composition further comprises at least one targeting molecule selected from the group consisting of glycoproteins, antibodies, antibody fragments, mimetic peptides, natural or synthetic organic or inorganic molecules, and specific cell surface receptors and ligands.

14. The method of Claim 1 or 2 in which the stabilizer selected from the group consisting of polycols with multiple hydroxyl groups, surfactants, gelatins, EDTA, polyethylene glycols, polyvinyl pyπolidone or ZnCl₂.

15. The method of Claim 1 or 2 in which the phospholipid is DODPC.

16. The method of Claim 3 in which the phospholipid is DODPC.

17. The method of Claim 10 in which the fatty acid is has the formula: CH₃-(CH₂),₂-CH=CH-CH=CH-C(O)-(OCH₂CH₂)_n-O-C(O)-CH₂-CH₂-CO₂H wherein n is an average number of -OCH₂CH₂- units from about 4 to about 45, or a pharmaceutically acceptable salt thereof.

18. The method of Claim 12 in which the targeting molecule is covalently bound to a polymerizable fatty acid.

19. The method of Claim 13 in which the targeting molecule is covalently bound to a polymerizable fatty acid.

20. The method of Claim 12 in which the targeting molecule is a lectin.

21. The method of Claim 13 in which the targeting molecule is a lectin.

22. The method of Claim 20 in which the lectin is UEA, EEA or FITC-EEA.

23. The method of Claim 21 in which the lectin is UEA, EEA or FITC-EEA.

24. The method of Claim 1 or 2 in which the liposomes are 5 to 100% polymerized.

25. The method of Claim 24 in which the Hposomes are 35 to 90% polymerized.

26. The method of Claim 25 in which the liposomes are 40to 60%> polymerized.

27. The method of Claim 26 in which the liposomes are 5 to 100% polymerized.

28. A method of delivering antigen to a mammal which comprises orally administering to said mammal a composition comprising polymerized liposomes, antigen, adjuvant, stabilizer, and a targeting molecule.

29. A method of delivering antigen to a mammal which comprises intranasally administering to said mammal a composition comprising polymerized liposomes, antigen, adjuvant, stabilizer, and a targeting molecule.

30. A method of delivering tetanus toxoid to a mammal which comprises orally administering to said mammal a composition comprising polymerized liposomes and tetanus toxoid.

31. A method of delivering tetanus toxoid to a mammal which comprises intranasally administering to said mammal a composition comprising polymerized liposomes and tetanus toxoid.

32. The method of Claim 30 or 31 in which the composition further comprises trehalose as a stabilizing agent.

33. The method of Claim 30 or 31, wherein the polymerized liposomes comprise 2, 4 DODPC lipid.

34. The method of Claim 30 or 31, wherein the polymerized Hposomes comprise 2, 4 DODPC lipid and monophosphoryl Lipid A.

35. A composition which comprises polymerized liposomes, an antigen, a stabilizer.

36. The composition of Claim 35 further comprising an adjuvant.

37. The composition of Claim 35 or 36 further comprising a targeting molecule.

38. The composition of Claim 37 in which the targeting molecule is a lectin.

39. The composition of Claim 35 or 36 in which the stabilizer is trehalose.

40. The composition of Claim 37 in which the stabilizer is trehalose.

41. A composition comprising polymerized liposomes and tetanus toxoid.

42. The composition of Claim 41 further comprising a stabilizer.

43. The composition of Claim 41 or 42 further comprising an adjuvant.

44. The composition of Claim 41 or 42 further comprising a targeting molecule.

45. The composition of Claim 43 further comprising a targeting molecule.

46. A method of delivering antigen to a mammal which comprises orally administering to said mammal a composition comprising polymerized liposomes and antigen, wherein the antigen is in the interior space of the polymerized liposome and external to the polymerized liposome.

47. A method of delivering antigen to a mammal which comprises intranasally administering to said mammal a composition comprising polymerized Hposomes and antigen, wherein the antigen is in the interior space of the polymerized liposome and external to the polymerized liposome.

48. The method of Claim 46 or 47 in which the composition further comprises an adjuvant.

49. The method of Claim 46 or 47 in which the composition further comprises a targeting molecule.

50. The method of Claim 48 in which the composition further comprises a targeting molecule.

51. The method of Claim 46 or 47 in which the composition further comprises a stabilizer.

52. The method of Claim 48 in which the composition further comprises a stabilizer.

53. The method of Claim 49 in which the composition further comprises a stabilizer.

54. A composition comprising polymerized Hposomes and antigen, wherein the antigen is in the interior space of the polymerized liposome and external to the polymerized liposome.

55. The composition of Claim 54 further comprising an adjuvant.

56. The composition of Claim 54 or 55 further comprising a targeting molecule.

57. The composition of Claim 54 or 55 further comprising a stabilizer.

58. The composition of Claim 56 further comprising a stabilizer.

59. The method of Claim 1 or 2 wherein the liposomes have primarily a negative charge.