| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | February 27, 1996 |
| PATENT TITLE |
Phosphazene polyelectrolytes as immunoadjuvants |
| PATENT ABSTRACT |
An immunoadjuvant soluble polyphosphazene polyelectrolyte is disclosed. In one embodiment, the polymeric adjuvant is an poly(organophosphazene) with (i) ionized or ionizable pendant groups that contain, for example, carboxylic acid, sulfonic acid, or hydroxyl moieties, and (ii) pendant groups that are susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 11, 1994 |
| PATENT REFERENCES CITED | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method for producing an immune response in an animal comprising: producing an immune response in an animal by administering to the animal an antigen and a polyphosphazene polyelectrolyte adjuvant in an amount effective to elicit an immune response in the animal against said antigen, said polyphosphazene being at least partially soluble in water. 2. The method of claim 1 wherein the antigen and polyphosphazene are first combined and the combination is administered to the animal. 3. The method of claim 1 wherein said administering is a parenteral administration. 4. The method of claim 1 wherein the polyphosphazene contains carboxylatophenoxy pendant groups. 5. The method of claim 1 wherein the polyphosphazene is a copolymer which comprises poly [di(carboxylatophenoxy)phosphazene]. 6. The method of claim 1 wherein the polyphosphazene polymer is poly [di(carboxylatophenoxy)phosphazene-co-di(chloro)phosphazene-co-(carboxylat ophenoxy)(chloro)phosphazene)]. 7. The method of claim 1 wherein the polyphosphazene polyelectrolyte contains hydrolyzable side chains selected from the group consisting of amino acid, amino acid ester, chlorine, imidazole, glycerol, and glucosyl. 8. The method of claim 1 wherein the polyphosphazene polyelectrolyte is cross-linked by a multivalent cation. 9. The method of claim 1 wherein the polyphosphazene polyelectrolyte is physically mixed with the antigen. 10. The method of claim 1 wherein the antigen is selected from the group consisting of proteins, peptides, polysaccharides, glycoproteins, and glycolipids. 11. The method of claim 1 wherein the antigen is selected from the group consisting of influenza proteins, hepatitis B proteins, bacterial proteins and bacterial lipopolysaccharides. 12. The method of claim 3 wherein the polyphosphazene contains carboxylatophenoxy pendant groups. 13. The method of claim 9 wherein said administering is a parenteral administration. 14. The method of claim 1 wherein the polyphosphazene has a molecular weight in excess of 1000. 15. The method of claim 1 wherein the ratio of antigen to polyphosphazene is from 0.5:1 to 0.0001:1. 16. The method of claim 15 wherein the polyphosphazene has a molecular weight in excess of 1000. 17. The method of claim 14 wherein the polyphosphazene is a copolymer which comprises poly [di(carboxylathophenoxy)phosphazene]. 18. The method of claim 15 wherein the polyphosphazene is a copolymer which comprises poly [di(carboxylathophenoxy)phosphazene]. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION This application is in the area of polymers for biomedical applications, and in particular describes polymers that can be used as immunoadjuvants. VACCINE DEVELOPMENT A wide variety of antigens stimulate the production of antibodies in animals and confer protection against subsequent infection. However, some antigens are unable to stimulate an effective immune response. The immunogenicity of a relatively weak antigan is often enhanced by the simultaneous administration of the antigan with an adjuvant, a substance that is not immunogenic when administered alone, but will induce a state of mucosal and/or systemic immunity when combined with the antigen. It has been traditionally thought that adjuvants, such as mineral oil emulsions or aluminum hydroxide, form an antigen depot at the site of injection that slowly releases antigen. Recent studies by Allison and Byars, in: "Vaccines: New Approaches to Immunological Problems:, R. W. Ellis, ed., p. 431, Butterworth-Heinemann, Oxford (1992) indicate that adjuvants enhance the immune response by stimulating specific and sometimes very narrow branches of the immune response by the release of cytokines. Unfortunately, many immunoadjuvants, such as Freund's Complete Adjuvant, are toxic and are therefore only useful for animal research purposes, not human vaccinations. Freund's Complete Adjuvant contains a suspension of heat-killed Mycobacterium tuberculosis in mineral oil containing a surfactant and causes granulomatous lesions in animals at the site of immunization. Freund's adjuvant may also cause the recipient of a vaccine to test positive for tuberculosis. Some synthetic polyelectrolytes have been found to provide immunostimulation when combined with an antigen. For example, the adjuvant activity of polyacrylic acid (PAA), copolymers of acrylic acid and N-vinylpyrrolidone (CP-AAVPD), poly-2-methyl-5-vinyl pyridine (PMVP), poly-4-vinylN-ethylpyridinium bromide (PVP-R.sub.2) and similar compounds, when conjugated to an antigen, has been studied by Petrov et. al., Jhurnal Vses. Khim. Ob-va im. D. I. Mendeleeva, 33:22-42 (1988). The immunomodulatory effect of polyelectrolyte complexes containing many of these same polyelectrolytes has also been more recently reviewed by Petrov, et al., Sov. Med. Rev. D. Immunol., 4:1-113 (1992). However, the toxicity and biodegradability of these polymers has not been studied and may prevent use of these polymers as adjuvants for use in humans. A non-toxic adjuvant or carrier having the ability to stimulate an immune response to non-antigenic or weakly antigenic molecules would be useful in the development and administration of vaccines. Therefore, it is an object of the present invention to provide an adjuvant that can be safely administered to humans and animals with minimal toxicity. It is a further object of the present invention to provide an adjuvant that is soluble and biodegradable. It is a further object of the present invention to provide a vaccine that confers protection against an organism such as the influenza virus or Clostridium tetani bacteria. It is a further object of the present invention to provide a rapid and efficient method of synthesizing a polymer, such as polyphosphazene, for use as an adjuvant. SUMMARY OF THE INVENTION A synthetic, water-soluble polyphosphazene is disclosed for use as an adjuvant. In a preferred embodiment, the phosphazene is a polyelectrolyte that is biodegradable and that exhibits minimal toxicity when administered to animals, such as humans. In one embodiment, the polymeric adjuvant is an poly(organophosphazene) with (i) ionized or ionizable pendant groups that contain, for example, carboxylic acid, sulfonic acid, or hydroxyl moieties, and (ii) pendant groups that are susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer. Suitable hydrolyzable groups include, for example, chlorine, amino acid, amino acid ester, imidazole, glycerol, and glucosyl. Two examples of polyphosphazenes that are useful as immunoadjuvants are poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato)phosphazene-co(carb oxylatophenoxy)(glycinato)phosphazene] and poly[di(carboxylatophenoxy) phosphazene-co-di(chloro)phosphaz ene-co-(carboxylatophenoxy)-(chloro)phosphazene]. A vaccine composition is prepared by either mixing or conjugating the polymer adjuvant with an antigen prior to administration. Alternatively, the polymer and antigen can be administered separately to the same site. When cross-linked with a multivalention, the polymer becomes less soluble, resulting in slower release of the polymer from the site of administration. DETAILED DESCRIPTION OF THE INVENTION The term amino acid, as used herein, refers to both natural and synthetic amino acids, and includes, but is not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, and histidinyl. The term amino acid ester refers to the aliphatic, aryl or heteroaromatic carboxylic acid ester of a natural or synthetic amino acid. The term alkyl, as used herein, refers to a saturated straight, branched, or cyclic hydrocarbon, or a combination thereof, typically of C.sub.1 to C.sub.20, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, octyl, nonyl, and decyl. The term (alkyl or dialkyl)amino refers to an amino group that has one or two alkyl substituents, respectively. The terms alkenyl and alkynyl, as used herein, refers to a C2 to C20 straight or branched hydrocarbon with at least one double or triple bond, respectively. The term aryl, as used herein, refers to phenyl or substituted phenyl, wherein the substituent is halo, alkyl, alkoxy, alkylthio, haloalkyl, hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(O)(lower alkyl), --CO.sub.2 H, --SO.sub.3 H, --PO.sub.3 H, --CO.sub.2 alkyl, amide, amino, alkylamino and dialkylamino, and wherein the aryl group can have up to 3 substituents. The term aliphatic refers to hydrocarbon, typically of C.sub.1 to C.sub.20, that can contain one or a combination of alkyl, alkenyl, or alkynyl moieties, and which can be straight, branched, or cyclic, or a combination thereof. The term halo, as used herein, includes fluoro, chloro, bromo, and iodo. The term aralkyl refers to an aryl group with an alkyl substituent. The term alkaryl refers to an alkyl group that has an aryl substituent, including benzyl, substituted benzyl, phenethyl or substituted phenethyl, wherein the substituents are as defined above for aryl groups. The term heteroaryl or heteroaromatic, as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, or nitrogen in the aromatic ring, and that can be optionally substituted as described above for aryl groups. Nonlimiting examples are furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbozolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. The term "pharmaceutically acceptable cation" refers to an organic or inorganic moiety that carries a positive charge and that can be administered as a countercation in a phosphazene polyelectrolyte. The term heteroalkyl, as used herein, refers to a alkyl group that includes a heteroatom such as oxygen, sulfur, or nitrogen (with valence completed by hydrogen or oxygen) in the carbon chain or terminating the carbon chain. A synthetic polymer is provided for use as an immunoadjuvant. The polymer adjuvant is a polyphosphazene that is at least partially soluble in water (typically to an extent of at least 0.001% by weight), an aqueous buffered salt solution, or aqueous alcohol solution. The polyphosphazene preferably contains charged side groups, either in the form of an acid or base that is in equilibrium with its counter ion, or in the form of an ionic salt thereof. The polymer is preferably biodegradable and exhibits minimal toxicity when administered to animals, including humans. SELECTION OF POLYPHOSPHAZENE POLYELECTROLYTES. Polyphosphazenes are polymers with backbones consisting of alternating phosphorus and nitrogen, separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two pendant groups ("R"). The repeat unit in polyphosphazenes has the following general formula: ##STR1## wherein n is an integer. The substituent ("R") can be any of a wide variety of moieties that can vary within the polymer, including but not limited to aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, carbohydrates, including glucose, heteroalkyl, halogen, (aliphatic)aminoincluding alkylamino-, heteroaralkyl, di(aliphatic)amino- including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, -oxyaryl including but not limited to -oxyphenylCO.sub.2 H, -oxyphenylSO.sub.3 H, -oxyphenylhydroxyl and -oxyphenylPO.sub.3 H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)CO.sub.2 H, -oxy(aliphatic)SO.sub.3 H, -oxy(aliphatic)PO.sub.3 H, and -oxy(aliphatic)hydroxyl, including oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, thioaliphatic including -thioalkyl, -thioalkaryl, thioaralkyl, --NHC(O)O-(aryl or aliphatic), --O--[(CH.sub.2)xO]y--CH.sub.2)--O--[(Ch.sub.2)xO]y(CH.sub.2)xNH(CH.sub.2) xSO.sub.3 H, and --O--[(CH.sub.2)xO]y-(aryl or aliphatic), wherein x is 1-8 and y is an integer of 1 to 20. The groups can be bonded to the phosphorous atom through, for example, an oxygen, sulfur, nitrogen, or carbon atom. In general, when the polyphosphazene has more than one type of pendant group, the groups will vary randomly throughout the polymer, and the polyphosphazene is thus a random copolymer. Phosphorous can be bound to two like groups, or two different groups. Polyphosphazenes with two or more types of pendant groups can be produced by reacting poly(dichlorophosphazene) with the desired nucleophile or nucleophiles in a desired ratio. The resulting ratio of pendant groups in the polyphosphazene will be determined by a number of factors, including the ratio of starting materials used to produce the polymer, the temperature at which the nucleophilic substitution reaction is carried out, and the solvent system used. While it is very difficult to determine the exact substitution pattern of the groups in the resulting polymer, the ratio of groups in the polymer can be easily determined by one skilled in the art. In one embodiment, the immunoadjuvant is a biodegradable polyphosphazene of the formula: ##STR2## wherein A and B can vary independently in the polymer, and can be: (i) a group that is susceptible to hydrolysis under the conditions of use, including but not limited to chlorine, amino acid, amino acid ester (bound through the amino group), imidazole, glycerol, or glucosyl; or (ii) a group that is not susceptible to hydrolysis under the conditions of use, including, but not limited to an aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, heteroalkyl, (aliphatic)amino- including alkylamino-, heteroaralkyl, di(aliphatic)aminoincluding dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, oxyaryl including but not limited to -oxyphenylCO.sub.2 H, -oxyphenyl SO.sub.3 H, -oxyphenylhydroxyl and -oxyphenylPO.sub.3 H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)CO.sub.2 H, -oxy(aliphatic)SO.sub.3 H, -oxy (aliphatic)PO.sub.3 H, and -oxy(aliphatics)hydroxyl, including -oxy(alkyl) hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic including -thioalkyl, -thioalkaryl, or thioaralkyl; wherein the polymer contains at least one percent or more, preferably 10 percent or more, and more preferably 80 to 90 percent or more, but less than 100%, of repeating units that are not susceptible to hydrolysis under the conditions of use, and wherein n is an integer of 4 or more, and preferably between 10 and 20,000 to 300,000. It should be understood that certain groups, such as heteroaromatic groups other than imidazole, hydrolyze at an extremely slow rate under neutral aqueous conditions, such as that found in the-blood, and therefore are typically considered nonhydrolyzable groups for purposes herein. However, under certain conditions, for example, low pH, as found, for example, in the stomach, the rate of hydrolysis of normally nonhydrolyzable groups (such as heteroaromatics other than imidazole) can increase to the point that the biodegradation properties of the polymer can be affected. One of ordinary skill in the art using well known techniques can easily determine whether pendant groups hydrolyze at a significant rate under the conditions of use. One of ordinary skill in the art can also determine the rate of hydrolysis of the polyphosphazenes of diverse structures as described herein, and will be able to select that polyphosphazene that provides the desired biodegradation profile for the targeted use. The degree of hydrolytic degradability of the polymer will be a function of the percentage of pendant groups susceptible to hydrolysis and the rate of hydrolysis of the hydrolyzable groups. The hydrolyzable groups are replaced by hydroxyl groups in aqueous environments to provide P--OH bonds that impart hydrolytic instability to the polymer. In other embodiments, the immunoadjuvant is: (i) a nonbiodegradable polyphosphazene wherein none, or virtually none, of the pendant groups in the polymer are susceptible to hydrolysis under the conditions of use, or (ii) a completely biodegradable polyphosphazene wherein all of the groups are susceptible to hydrolysis under the conditions of use (for example, poly[di(glycinato)phosphazene]). Phosphazene polyelectrolytes are defined herein as polyphosphazenes that contain ionized or ionizable pendant groups that render the polyphosphazene anionic, cationic or amphophilic. The ionic groups can be in the form of a salt, or, alternatively, an acid or base that is or can be at least partially dissociated. Any pharmaceutically acceptable monovalent cation can be used as counterion of the salt, including but not limited to sodium, potassium, and ammonium. The phosphazene polyelectrolytes can also contain non-ionic side groups. The phosphazene polyelectrolyte can be biodegradable or nonbiodegradable under the conditions of use. The ionized or ionizable pendant groups are preferably not susceptible to hydrolysis under the conditions of use. A preferred phosphazene polyelectrolyte immunoadjuvant contains pendant groups that include carboxylic acid, sulfonic acid, or hydroxyl moieties. While the acidic groups are usually on nonhydrolyzable pendant groups, they can alternatively, or in combination, also be positioned on hydrolyzable groups. An example of a phosphazene polyelectrolyte having carboxylic acid groups as side chains is shown in the following formula: ##STR3## wherein n is an integer, preferably an integer between 10 and 10,000 to 300,000. This polymer has the chemical name poly[di(carboxylatophenoxy)phosphazene] or, alternatively, poly[bis(carboxylatophenoxy)phosphazene] (PCPP) . The phosphazene polyelectrolyte is preferably biodegradable to prevent eventual deposition and accumulation of polymer molecules at distant sites in the body, such as the spleen. The term biodegradable, as used herein, means a polymer that degrades within a period that is acceptable in the desired application, typically less than about five years and most preferably less than about one year, once exposed to a physiological solution of pH 6-8 at a temperature of approximately 25.degree. C.-37.degree. C. Most preferably the polymer is a poly(organophosphazene) that includes pendant groups that include carboxylic acid moieties that do not hydrolyze under the conditions of use and pendant groups that are susceptible to hydrolysis under the conditions of use. Examples of preferred phosphazene polyelectrolytes with hydrolysis-sensitive groups are poly[di(carboxylatophenoxy)phosphazene-co-di(amino acid)phosphazene-co-(carboxylatophenoxy)(amino acid)phosphazene], specifically including poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato)phosp hazene-co-(carboxylatophenoxy)(glycinato)phosphazene], and poly[di(carboxylatophenoxy)phosphazene-co-di(chloro)phosphaz ene-co-(carboxylatophenoxy)(chloro)phosphazene]. The toxicity of the polyphosphazene determined using cell culture experiments well known to those skilled in the art. For example, toxicity of poly[di(carboxylatophenoxy) phosphazene] was determined in cell culture by coating cell culture dishes with the poly[di(carboxylatophenoxy) phosphazene]. Chicken embryo fibroblasts were then seeded onto the coated petri dishes. Three days after seeding the chicken embryo fibroblasts, the cells had become flattened and spindles formed. Under phase contrast microscopy, mitotic figures were observed. These observations provide evidence of the non-toxicity of poly[di(carboxylatophenoxy)phosphazene] to replicating cells. Crosslinked polyphosphazenes for use as immunoadjuvants can be prepared by combining a phosphazene polyelectrolyte with a metal multivalent cation s.about.uch as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium. SYNTHESIS OF PHOSPHAZENE POLYELECTROLYTES Polyphosphazenes, including phosphazene polyelectrolytes, can be prepared by a macromolecular nucleophilic substitution reaction of poly(dichloro phosphazene) with a wide range of chemical reagents or mixture of reagents in accordance with methods known to those skilled in the art. Preferably, the phosphazene polyelectrolytes are made by reacting the poly(dichloro phosphazene) with an appropriate nucleophile or nucleophiles that displace chlorine. Desired proportions of hydrolyzable to non-hydrolyzable side chains in the polymer can be obtained by adjusting the quantity of the corresponding nucleophiles that are reacted with poly(dichlorophosphazene) and the reaction conditions as necessary. Preferred polyphosphazenes for immunoadjuvant activity have a molecular weight of over 1,000. For example, poly[(carboxylatophenoxy)(glycinato) phosphazene] (PC-GIPP) is prepared by the nucleophilic substitution reaction of the chlorine atoms of the poly(dichlorophosphazene) with propyl phydroxybenzoate and ethyl glycinate hydrochloride (PC-G1PP synthesis). The poly[(aryloxy)(glycinato)phosphazene] ester thus obtained is then hydrolyzed to the corresponding poly(carboxylic acid). Other polyphosphazenes can be prepared as described by Allcock, H. R.; et al., Inorg. Chem. 11, 2584 (1972); Allcock, H. R.; et al., Macromolecules 16, 715 (1983);Allcock, H. R.; et al., Macromolecules 19,1508 (1986); Allcock, H. R.; et al., Biomaterials 19, 500 (1988); Allcock, H. R.; et al., Macromolecules 21, 1980 (1988); Allcock, H. R.; et al., Inorg. Chem. 21(2), 515521 (1982); Allcock, H.R.; et al., Macromolecules 22:7579 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174, 4,880,622 to Allcock, H.R.; et al.,; U.S. Pat. No. 4,946,938 to Magill, et al., U.S. Pat. No. 5,149,543 to Cohen et al., and the publication of Grolleman, et al., J. Controlled Release 3,143 (1986), the teachings of which, and polymers disclosed therein, are incorporated by reference herein. SELECTION OF AN ANTIGEN The antigan can be derived from a cell, bacteria, or virus particle, or portion thereof. As defined herein, antigan may be a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or combination thereof, which elicits an immunogenic response in an animal, for example, a mammal, bird, or fish. As defined herein, the immunogenic response can be humoral or cell mediated. In the event the material to which the immunogenic response is to be directed is poorly antigenic, it may be conjugated to a carrier such as albumin or to a hapten, using standard covalent binding techniques, for example, with one of the several commercially available reagent kits. In one embodiment, the polymer is used to deliver nucleic acid which encodes antigen to a mucosal surface where the nucleic acid is expressed. Examples of preferred antigens include viral proteins such as influenza proteins, human immunodeficiency virus (HIV) proteins, and hepatitis B proteins, and bacterial proteins and lipopolysaccharides such as gram negative bacterial cell walls and Neisseria gonorrhea proteins. PREPARATION OF AN IMMUNOGENIC COMPOSITION Combining Antigen with polymer for simultaneous administration. An immunogenic composition, or vaccine, is prepared by combining the polymer adjuvant with an antigan. Approximately 0.5-0.0001 parts of antigen is added to one part polymer, preferably by stirring a solution of polymer and antigan until a solution or suspension is obtained, preferably for 10 minutes or more at 25.degree. C. The polymer is preferably combined with the antigan using a method dispersing the antigan uniformly throughout the adjuvant. Methods for liquifying the polymer include dissolving the polymer in an aqueous-based solvent, preferably having a pH range of between 7.1 and 7.7, and melting the polymer. The latter is useful only when the antigan is stable at the polymer melting temperature. The antigan is then mixed with the polymer. The polymer and the antigen, in solid form, for example, when the antigen is lyophilized, can also be physically mixed together, for example, by compression molding. The polymer can also be used to encapsulate the antigen, for example, using the method of U.S. Pat. No. 5,149,543 to Cohen, et al., the teachings of which are incorporate herein, or by spray drying a solution of polymer and antigen. Alternatively, microspheres containing the antigen and adjuvant can be prepared by simply mixing the components in an aqueous solution, and then coagulating the polymer together with the substance by mechanical forces to form a microparticle. The microparticle can be stabilized, if necessary or desired, using electrolytes, pH changes, organic solvents, heat or frost to form polymer matrices encapsulating biological material. In a preferred embodiment, approximately one part of polymer is dissolved in 10 parts 3% Na.sub.2 CO.sub.3 aqueous solution while stirring, then 10 to 90 parts phosphate buffer pH 7.4 is slowly added. POLYMER--ANTIGEN CONJUGATES The polymer can also be covalently conjugated with the antigen to create a water-soluble conjugate in accordance with methods well-known to those skilled in the art, usually by covalent linkage between an amino or carboxyl group on the antigen and one of the ionizable side groups on the polymer. CROSS-LINKED POLYMER ADJUVANT In an alternative preferred embodiment, the polymer is cross-linked with a multivalent ion, preferably using an aqueous solution containing multivalent ions of the opposite charge to those of the charged side groups of the polyphosphazene, such as multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups. Preferably, the polymers are cross-linked by di and trivalent metal ions such as calcium, copper, aluminum, magnesium, strontium, barium, tin, zinc, and iron, organic cations such as poly(amino acid)s, or other polymers such as poly(ethyleneimine), poly(vinylamine) and polysaccharides. ADDITIVES TO THE POLYMER--ADJUVANT MIXTURE. It will be understood by those skilled in the art that the immunogenic vaccine composition can contain other physiologically acceptable ingredients such as water, saline or a mineral oil such as DrakeolTM, MarkolTM, and squalene, to form an emulsion. ADMINISTRATION OF POLYMER--AMTOGEN VACCINE The immunogenic composition can be administered as a vaccine by any method known to those skilled in the art that elicits an immune response, including parenterally, orally, or by transmembrane or transmucosal administration. Preferably, the vaccine is administered parenterally (intravenously, intramuscularly, subcutaneously, intraperitoneally, etc.), and preferably subcutaneously. Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), respiratory, vaginal, and rectal. The dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the polymer-antigen administration, as demonstrated by the following examples. Although in the preferred embodiment the polymerantigen mixture is administered simultaneously, in an alternative embodiment, the polymer and antigen are administered separately to the same or nearby site. The polymer serves to attract cells of the immune system to the site, where they process the antigen. |
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