WO1995002416A1 - Hydrogel microencapsulated vaccines - Google Patents

Abstract

Water soluble polymers or polymeric hydrogels are used to encapsulate antigen to form vaccines. The antigen is mixed with a polymer solution, microparticles are formed of the polymer and antigen, and, optionally, the polymer is crosslinked to form a stable microparticle. Preferred polymers are alginate and polyphosphazenes, and mixtures thereof. Microparticles can be administered parenterally or mucosally. For oral delivery, the microparticles are preferably fifteen microns or less in diameter, and adhere to the mucosal lining of the gastrointestinal tract, increasing uptake by the reticuloendothelium.

Description

HYDROGEL MICROENCAPSULATED VACCINES

Background of the Invention

The present invention is a microsphere configured vaccine vehicle based on a water soluble polymer or hydrogel. This is a continuation in part of U.S.S.N. 08/090,841 entitled "Phosphazene Polyelectrolytes as Im-munoadjuvants" filed July 12, 1993.

Induction of an Immune Response via Mucosal Surfaces

The majority of virus utilize mucosal surfaces as the primary site of infection. Depending on the virus, the infection either remains localized to the mucosal surface or disseminates to establish a systemic infection. Examples of viruses eliciting local infections are influenza, parainfluenza and common cold viruses which propagate in the respiratory mucosa and rotavirus and the Norwalk agent that replicate in the intestinal mucosa. Viruses that induce systemic viral infections that spread from the mucosa are exemplified by measles, mumps, rubella, polio, hepatitis A and B and herpes viruses.

During the last few years a great deal of information has accrued on the induction of mucosal immunity. In the gut, for example, the immune response is localized to the Peyer's patches embedded in the gut mucosa. Lymphoid tissue at these locations is exposed to the lumen of the gut (gut associated lymphoid tissue, GALT) , permitting a constant sampling of the luminal contents. Similar lymphoid tissue called the bronchiolar associated lymphoid tissue (BALT) is located in the respiratory mucosa. Currently, the majority of viral vaccines establish a state of systemic protective immunity following injection of live attenuated or inactivated virus preparations. The success of such vaccines is due to the induction of a cell mediated and/or humoral immune response in the vaccinee. This systemic immunity prevents the onset of disease by reducing viral replication at the mucosa and eliminating the spread of the virus to important target organs.

The use of injectable vaccines has dramatically reduced the incidence of many viral diseases. Nevertheless, their usage is associated with some undesirable effects. Live attenuated virus vaccines can cause systemic complications whereas inactivated vaccines can cause local reactions and even induce an allergic state. Two important consequences of these vaccine side effects are low compliance and litigation. The former leads to reduced immunity and increased rates of natural infection whereas the latter impedes the improvement of current vaccines and development of new vaccines.

An alternative to the use of injectable vaccines is the oral administration of antigen, especially of a live attenuated virus. Such a vaccine induces both a strong mucosal and systemic immunity mimicking the immune response induced by natural infection with the wild type virus. This constellation of immune responses eliminates not only the systemic spread of virus but also viral replication in the mucosa. Thus, the immune response elicited by a replicating oral vaccine is superior to that induced by injectable live or inactivated vaccines. The best example of this type of vaccine is the live attenuated oral polio virus vaccine (OPV) . Unfortunately, oral administration of live virus is limited to those viruses which survive passage through the stomach and which do not easily revert to virulence.

The most effective non-replicating antiviral vaccines thus far developed have been inactivated virus particles. The efficacy of peptide and subunit vaccines in animal models has had limited success and currently there are no human vaccines using these kinds of formulations. In the early years of recombinant DNA engineering, many groups fully expected not only the development of protective immunity but also resolution of safety issues by producing non-infectious viral antigens.

Unfortunately, it has become increasingly clear that there is no reason to assume that a viral protein produced in a laboratory expression vector, highly purified and injected into a vaccinee will assume a conformation in vivo which even remotely approximates the antigenic state found in natural infection. To date, the only successful recombinant derived vaccine has been the hepatitis B surface (HBS) antigen synthesized in an eucaryotic (yeast) expression system.

There is a growing body of evidence demonstrating that oral presentation of non-replicating antigens in the particulate state induces both a mucosal and systemic immunity that closely mimics the immunity induced by natural infection. This is in contrast to oral immunization with non-replicating soluble antigens which not only fail to induce systemic immunity but very often induce a state of systemic tolerance. Furthermore, the antigen doses required to elicit this immunity are far lower than that required for parenteral immunization with the same antigen. The major advantages inherent in such a vaccine formulation are the ease of administration and complete safety. Adjuvants The advent of modern molecular biology has provided a means of producing immunogens with unprecedented ease and precision. It is ironic that these new methodologies generate purified immunogens that do not generally induce a strong immune response in the absence of an effective adjuvant. The development of improved vaccine adjuvants for use in humans has therefore become a priority area of research. Nevertheless, research on adjuvants has lagged seriously behind the work done on immunogens. For decades the only adjuvant widely used in humans has been alum. Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. New chemically defined preparations such as muramyl dipeptide and monophosphoryl lipid A are being studied.

The traditional view on how adjuvants exert their effect is that adjuvants such as mineral oil emulsions or aluminum hydroxide form an antigen depot at the site of injection that slowly releases antigen. However, excision of the injection site after three days was found to have little effect on immune responses. Recent studies indicate that adjuvants enhance the immune response by stimulating specific and sometimes very narrow arms of the immune response by the release of cytokines, as reviewed by A.C. Allison and N.E. Byars, in: "Vaccines: New

Approaches to Immunological Problems", R.W. Ellis, ed. , p. 431 (Butterworth-Heinemann, Oxford 1992) . It is desirable to have an adjuvant that would act as a simple depot for the release of antigens over an extended period. An area of adjuvant research that has developed over the last few years is the utilization of synthetic polymers in the formulation of a vaccine. The non-ionic block co-polymer surfactants of Hunter, R.L. Topics in vaccine adjuvant research. D.R. Spriggs and .C. Koff (Eds), pp. 89-98 (CRC Press, 1991) with molecular weights below approximately 10,000, have a simple structure composed of two blocks of hydrophilic polyoxyethylene (POE) flanking a single block of hydrophobic polyoxypropylene (POP) . They are considered to be among the least toxic of surfactants and are widely used in foods, drugs and cosmetics.

Some of the large hydrophobic co-polymers are effective adjuvants while closely related preparations are not. There is a correlation between the adjuvant activity of these copolymers with differences in the chain links of the POE and POP. Currently, these adjuvants are used in an oil and water emulsion.

A wide range of polyelectrolytes of various molecular weights have also been shown by Petrov, et al. Sov. Med. Rev. Section D Immunology, 4:1-113 (1992), to have an adjuvant activity. Macromolecules bearing either positive or negative charges have displayed a similar immunostimulatory activity. The polyelectrolytes form complexes with antigens through electrostatic and hydrophobic bonds. On the other hand, neutral and uncharged polymers had no effect on the immune response. Controlled Release of Drugs and Antigens . There is currently considerable interest in the development of controlled release vaccines, since the major disadvantage of several currently available vaccines is the need for repeated administrations. Controlled release vaccines could obviate the need for booster immunizations, which would be particularly advantageous in developing countries, where repeated contact between the healthcare worker and the vaccine recipient is often difficult to achieve. There is a growing body of evidence showing that antigen persisting on the external membrane of follicular dendritic cells and lymph node organs is involved in the recruitment of B memory cells to form antibody secreting cells. The continual release of circulating antibodies suggests this recruitment happens continually. As the level of antigen decreases this allows the well established phenomena of affinity maturation of antibody to occur. Acceptance of the antigen persistence concept has an important implication in vaccine development. Ideally, it would be advantageous to be able to formulate vaccines in a way such that antigen is presented to the immune system and, in particular, the follicular dendritic cells, over an extended period of time. A number of polymers have been used to entrap antigens, as well as other proteins and compounds. An early example of this is the polymerization of influenza antigen within methyl methacrylate spheres having diameters less than one micron (1,000 nanometers) to form so-called nano particles, reported by Kreuter, J. Microcapsules and Nanoparticles in Medicine and

Pharmacology. M. Donbrow (Ed)., p. 125-148 (CRC Press). The antibody response as well as the protection against infection with influenza virus was significantly better than when antigen was administered in combination with aluminum hydroxide. Experiments with other particles demonstrated that the adjuvant effect of these polymers depends on particle size and hydrophobicity.

Several factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Biodegradable polymers may be designed around one of many types of labile bonds. Examples are polycarbonates, polyesters, polyurethanes, polyorthoesters and polyamides. One of the advantages of using a synthetic polymer for microencapsulation, rather than a naturally occurring polymer, is that the relative rates of hydrolysis of these bonds under neutral conditions can be influenced by the substituents to the polymer backbone. Substituent modification can also be used to alter the solubility and hydrophilicity/hydrophobicity of the polymer. A frequent choice of a carrier for pharmaceuticals and more recently for antigens, is poly (D,L-lactide-co-glycolide) (PLGA) . Acceptability by the regulatory authorities remains a significant obstacle for any antigen delivery system. PLGA polymers are biodegradable and biocompatible polyesters which have been used as resorbable sutures for many years, as reviewed by Eldridge, J.H., et al. Current Topics in Microbiology and Immunology. 1989, 146: 59-66. The entrapment of antigens in PLGA microspheres of 1 to 10 microns in diameter has been shown to have an adjuvant effect. A major disadvantage of the PLGA system is the use of organic solvents and long preparation times for the microencapsulation of the antigens. The process utilizes a phase separation of a water-in-oil emulsion. The compound of interest is prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvents such as methylene chloride and ethyl acetate. These two immiscible solutions are coemulsified by high-speed stirring. A nonsolvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules. The microcapsules are collected, and stabilized with a polyelectrolyte such as polyvinyl alcohol (PVA) , gelatin, alginate, polyvinylpyrrolidone (PVP) , or methyl cellulose, and the solvent removed by either drying in vacuo or solvent extraction. While these preparation conditions have been used successfully for microencapsulation of a variety of peptide drugs and hardy immunogens such as staphylococcal enterotoxin B and keyhole limpet cyanin, as demonstrated by J.H. Eldridge, et al., Infection and Immunity 9:2978 (1991), the high shear forces, the use of organic solvents and the long preparation times needed for microencapsulation using PLGA could be detrimental to important epitopes on complex labile immunogens such as enveloped viruses.

It is therefore an object of the present invention to provide materials for encapsulation and delivery by parenteral or mucosal administration of vaccines which do not require the use of organic solvents or long preparation times.

It is another object of the present invention to provide a system for delivery of antigen to mucosal surfaces, especially through oral delivery.

It is a further object of the present invention to provide a delivery system for delivery of antigens which elicits a broad spectrum of immunogenic responses.

It is a still further object of the present invention to provide a delivery system for delivery of vaccines which enhances the immunogenicity of the vaccines. It is yet another object of the present invention to provide a biodegradable delivery system providing controlled release of antigen.

Summary of the Invention

Water soluble polymers and polymeric hydrogels are used to microencapsulate antigen for delivery to mucosal surfaces and for the controlled release of antigen at the mucosal surface, or for injection (parenteral administration) . In the most preferred embodiment, the encapsulated antigen is administered orally or intranasally. The polymer can be any biocompatible, crosslinkable water-soluble polymer or polymeric hydrogel which can be used to form a microparticle having a diameter of two hundred microns or less, under conditions which are gentle and do not denature the antigen to be incorporated therein. Preferred natural water soluble polymers include alginate, gelatin, pectin, and collagen; preferred synthetic water soluble polymers include poly(aerylamide) , poly(methacrylamide) , poly(vinyl acetate) , poly(N-vinyl pyrrolidone) , poly(hydroxyethylmethacrylate) , poly(ethylene glycol) , polyvinylamines, poly(vinylpyridine) , phosphazene polyelectrolytes, and poly(vinyl alcohols) ; preferred polymers forming hydrogels by ionic crosslinking include salts of poly(acrylic acids) or poly(methacrylic acid) , sulfonated polystyrene, quaternary salts of either polyamines or poly(vinylpyridine) ; and mixtures and copolymers of the polymers or monomers thereof. The most preferred polymers are alginate, polyphosphazenes, and mixtures thereof.

To prepare the encapsulated antigen, the antigen is mixed with a polymer solution, microparticles are rapidly formed of the polymer and antigen without the use of significant quantities of organic solvents, and the polymer is crosslinked ionically or covalently to form a stable biodegradable microparticle. The microparticles adhere to mucosal surfaces such as the mucosal lining of the gastrointestinal tract, increasing takeup by the reticuloendothelium of antigen as it is released over time. The polymers are preferably alginate or a polyphosphazene, most preferably crosslinked ionically with a polyion or divalent cation, such as calcium chloride. Examples demonstrate the enhanced immunogenicity of polymer encapsulated antigen, alone or in combination with a mucosal stimulant such as cholera toxin, as well as how to manipulate the polymers to alter release rates and humoral response, when administered parenterally, orally, or intranasally.

Brief Description of the Drawings

Figure 1 is a graph of the permeability of polyphosphazene microspheres, measured as percent release as a function of encapsulated protein molecular weight and polymer concentration. Rainbow protein markers were microencapsulated in three concentrations of poly[di(carboxylatophenoxy)phosphazene- co-di(glycinato)phosphazene] (PP) : 3.3% (dotted bars)., 2.5% (hatched bars) , and 1.5% (dark bars) , and incubated in HEPES buffer pH 7.4 at room temperature for 24 hours before the amount of protein in the supernatant was spectrophotometrically measured.

Figure 2 is a graph of the effect of molecular weight on erosion profiles of polyphosphazene microspheres, measured as percent mass loss over time in days: PC-GIP, 130 KDa (squares) ; PCPP, 3900 KDa (diamonds) ; PC-GIP, 170 KDa (circles) ; and PCPP, 400 KDa (triangles) .

Figures 3a and 3b are molecular weight degradation profiles over time in days for PCPP hydrogels with different starting molecular weights of polyphosphazenes: Mw, molecular weight, Mn, number average molecular weight, initial Mw 3,900 KDa (Figure 3a) , and Mw 400 KDa (Figure 3b) .

Figure 4 is a molecular weight degradation profile over time in days for PC-GIPP hydrogel for Mw 170 KDa, comparing molecular weight of polymer in the matrix with molecular weight of polymers in solution.

Figure 5 is a graph of percent release of polystyrene beads from polyphosphazene microspheres coated with poly-L- lysines of different molecular weights: 12,000 mw (squares), 62,500 mw (diamonds), 140,800 mw (circles), and 295,000 mw (triangles) . Fluorescent polystyrene (PS) beads measuring 20 nm in diameter were encapsulated in polymer 1 and then coated with poly-L-lysines of different molecular weights. The coated beads were incubated in HEPES buffer pH 7.4 at room temperature.

Polystyrene beads released into the supernatant were measured by quantitative fluorimetry and expressed as a percent of the initially encapsulated beads.

Figures 6a, 6b, and 6c are graphs of the flu-specific responses in the sera of animals immunized with flu virus in suspension (Figure 6a) , encapsulated flu virus in combination with cholera toxin (CT) in alginate microspheres (Figure 6b) , and flu virus encapsulated in alginate microspheres (Figure 6c) , measured as antibody titer (reading left to right: IgM, dark bars; IgG, hatched bars; IgA, stipled bars) at 7, 14, 21, and 28 da Figure 7 is the flu specific antibody response in the sera following oral administration of influenza encapsulated in alginate in combination with CT, measured at seven, 14, 21, 28, and 35 days post immunization, for IgM, dark bars; IgG, hatched bars; IgA, stipled bars.

Figure 8 is a graph of the flu-specific antibody response in the fecal samples following administration orally of influenza in alginate microcapsules in combination with CT, following an oral boost, measured at seven, 14, 21, 28 and 35 days after the boost, for IgM, dark bars; IgG, hatched bars; IgA, stipled bars.

Detailed Description of the Invention

In general, microspheres for delivery of antigen are formed by covalent or ionic crosslinking of water soluble polymers or polymers that form hydrogels. In the preferred embodiment, the polymers are formed of water soluble polymers such as alginate or polyphosphazenes which are ionically crosslinked with divalent cations such as calcium ions to form a water-insoluble hydrogel encapsulating antigen. Antigen is mixed with the polymer solution prior to crosslinking to insure dispersion of the antigen throughout the microsphere. More stable microspheres can be formed by further crosslinking the microspheres with a polyelectrolyte such as a polyamino acid. Polymers useful for making Microspheres. The polymer can be almost any biocompatible, crosslinkable water-soluble polymer or polymeric hydrogel which can be used to form a microparticle having a diameter of ten microns or less, under conditions which are gentle and do not denature the antigen to be incorporated therein. As used herein, a hydrogel is defined as any water-swollen polymer. Water- soluble polymers are those that are at least partially soluble (typically to an extent of at least 0.001% by weight) in water, an aqueous buffered salt solution, or aqueous alcohol solution. Preferred natural water soluble polymers include alginate, gelatin, pectin, and collagen; preferred synthetic water soluble polymers include poly(acrylamide) , poly(methacrylamide) , poly(vinyl acetate), poly(N-vinyl pyrrolidone) , poly(hydroxyethylmethacrylate) , poly(ethylene glycol), polyvinylamines, poly(vinylpyridine) , phosphazene polyelectrolytes, and poly(vinyl alcohols) ; preferred polymers forming hydrogels by ionic crosslinking include poly(acrylic acids) or poly(methacrylic acid) , sulfonated polystyrene, quaternary salts of either polyamines or poly(vinylpyridine) ; and mixtures and copolymers of the polymers or monomers thereof. The most preferred polymers are alginate, polyphosphazenes, and mixtures thereof.

The polymers can be crosslinked either by ionic crosslinking, covalent crosslinking or physical crosslinking to render the water-soluble polymers water-insoluble. Gelation by ionic crosslinking of an aqueous based polymer solution at room temperature eliminates the long exposure to organic solvents, elevated temperatures and drying required by polymers dissolved in organic solvents. The polymers can be crosslinked in an aqueous solution containing multivalent ions of the opposite charge to those of the charged side groups, 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, or polycations such as poly(amino acid)s, poly(ethyleneimine) , poly(vinylamine) , poly(vinylpyridine) , polysaccharides, and other that can form polyelectrolyte complexes.

Alginates.

The best studied ion crosslinkable polymer is the naturally occurring alginate that is prepared from brown algae for use in foodstuffs, for example, Protanal LF 20/60 (Pronova, Inc., Portsmouth, NH, USA).

The polymer is cross-linked with a multivalent ion, preferably using calcium chloride or other divalent or multivalent cation.

Polyphosphazenes. The elucidation of a class of ion cross-linkable water soluble polyphosphazenes, described by H.R. Allcock and S. Kwon. , Macromolecules 22, 75-79 (1989), has made it possible to generate microspheres containing antigens that throughout preparation are exposed only to an aqueous environment. 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, to C^, 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) , -C0₂H, -S0₃H, -P0₃H, -C0₂alkyl, amide, amino, alkylamino and dialkylamino, and wherein the aryl group can have up to 3 substituents.

The term aliphatic refers to hydrocarbon, typically of Cj to C₂₀, 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, 1,2,4- thiadiazolyl, 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 ion" refers to an organic or inorganic moiety that carries a charge and that can be administered as a counterion in a phosphazene polyelectrolyte.

The term heteroalkyl, as used herein, refers to an 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.

The terms poly[ (carboxylatophenoxy) (glycinato) phosphazene] , poly[di(carboxylatophenoxy)phosphazene-co- di(glycinato)phosphazene-co-(carboxylatophenoxy) (glycinato)phosphazene] and poly[di(carboxylatophenoxy) phosphazene-co-di(glycinato)phosphazene] as used herein refer to the same polymer.

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 Phosphazene 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:

P - H - -

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)amino- including alkylamino-, heteroaralkyl, di(aliphatic)amino- including dialkylamino-, arylamino-', diarylamino-, alkylarylamino-, -oxyaryl including but.not limited to -oxypheny1C0₂H, -oxyphenylS0₃H, -oxyphenylhydroxyl and -oxypheny1P0₃H; -oxyaliphatic including - oxyalkyl, -oxy(aliphatic)C0₂H, -oxy(aliphatic)S0₃H, -oxy(aliphatic)P0₃H, and -oxy(aliphatic)hydroxyl, including -oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic including - thioalkyl, -thioalkaryl, -thioaralkyl, -NHC(0)0-(aryl or aliphatic), -0-[ (CH₂)_xO]_y-CH₂)_xNH₂, -O-[ (CH₂)_x0]_yCH₂)_XNH(CH2)_xS0₃H, and -0-[ (CH₂)_x0]_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 biodegradable polyphosphazene has the formula:

B 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)amino- including dialkylamino-, arylamino-, diarylamino- , alkylarylamino-, - oxyaryl including but not limited to -oxypheny1C0₂H, -oxyphenylS0₃H, -oxyphenylhydroxyl and -oxypheny1P0₃H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)C0₂H, -oxy(aliphatic)S0₃H,

-oxy(aliphatic)PO₃H, and -oxy(aliphatic)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.

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 polyphosphazene 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(ethylglycinato)-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 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:

wherein n is an integer, preferably an integer between 10 and 10,000. This polymer has the chemical name poly[di(carboxylatophenoxy)phosphazene] or, alternatively, poly[bis(carboxylatophenoxy)phosphazene] (PCPP).

The phosphazene polyelectrolyte is preferably biodegradable. 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°C - 37°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)phosphazene-co- (carboxylatophenoxy) (glycinato)phosphazene] , and poly[di(carboxylatophenoxy)phosphazene- co-di(chloro)phosphazene-co-

(carboxylatophenoxy) (chloro)phosphazene] .

The toxicity of the polyphosphazene can be 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 can be prepared by combining a phosphazene polyelectrolyte with a metal multivalent cation such 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(dichlorophosphazene) 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(dichlorophosphazene) 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.

For example, poly[ (carboxylatophenoxy)- (glycinato)phosphazene] (PC-G1PP) is prepared by the nucleophilic substitution reaction of the chlorine atoms of the poly(dichlorophosphazene) with propyl p-hydroxybenzoate 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), 515- 521 (1982); Allcock, H.R. ; et al., Macromolecules 22:75-79 (1989); U.S. Patent Nos. 4,440,921, 4,495,174, 4,880,622 to Allcock, H.R. ; et al.,; U.S. Patent No. 4,946,938 to Magill, et al., U.S. Patent 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 antigen can be derived from a cell, bacteria, or virus particle, or portion thereof. As defined herein, antigen 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 cells where the nucleic acid is expressed.

Examples of preferred antigens include viral proteins such as influenza proteins, human immunodeficiency virus (HIV) proteins, Haemophilus influenza , and hepatitis B proteins, and bacterial proteins and lipopolysaccharides such as gram negative bacterial cell walls and Neisseria gonorrhea proteins.

Virus infection of cells in culture generates two kinds of virus particles; mature infectious virus and some non- infectious virus-like particles devoid of nucleic acid. It is preferred to use inactivated mature virus particles in oral vaccines in those cases where the virus replicates to a high titer in cell culture. For virus that either cannot be grown in cell culture or that are tumorigenic, one can use recombinant DNA technology to produce non-replicating virus-like particles

(VLPs) . Using recombinant technology, one can construct virus¬ like particles that display on their surface protective antigens (pseudotyping) from virus that because of their inherent complexity do not lend themselves to either of the above two approaches. All of the antigens described above are virus particle structural components, however, not all antigens that elicit protective immunity are structural antigens. In those instances where the protective antigen is a non-structural component, one can genetically fuse such antigens to the surface of self-assembling virus-like particles.

Adjuvants

In some embodiments it may be desirable to include an adjuvant with the antigen which is encapsulated for mucosal or parenteral delivery. Adjuvants for oral administration.

It is known that oral administration of an admixture of trace amounts of cholera toxin (CT) (either cholera toxin subunit A, cholera toxin subunit B, or both) and a second antigen stimulate a mucosal immunity to the coadministered antigen. Furthermore, there is a dramatic humoral immune response to the second antigen instead of the immune tolerance that is elicited by oral delivery of the antigen alone. Thus, mucosally delivered CT functions as a powerful immunostimulant or adjuvant of both mucosal and humoral immunity. The mechanism for this adjuvant effect may be due to the ability of CT to specifically bind to the dome cells (or M cells) overlying the Peyer's patches and then to alter the lymphoid cells in a manner that favors immunoresponsiveness to antigens that may or may not normally bind to the dome cells. Recently, the binding function was localized to the non-toxic B subunit of the cholera toxin (CT-B) molecule. It has now been demonstrated that the addition of CT-B to antigens will mimic the immune response elicited by CT to the same antigens. It is therefore frequently preferred to enhance immunogenicity of the orally administered antigen by including CT in the microencapsulated vaccine.

Adjuvants for parenteral administration. Examples of adjuvants include muramyl dipeptides, muramyl tripeptide, cytokines, diphtheria toxin, and exotoxin A. Commercially available adjuvants include QS-21 from Cambridge Biosciences, Worcester, MA, and monophosphoryl lipid A (MPLA) from Ribi Immunochem.

It is also demonstrated herein that polyphosphazenes can also have an adjuvant effect when administered orally or parenterally. In particular, examples demonstrate the enhanced immunogenicity of microspheres formed of 95% alginate and 5% polyphosphazene (PCPP) .

Preparation of an Immunogenic Composition The polymer is used to encapsulate the antigen, for example, using the method of U.S. Patent 5,149,543 to Cohen, et al., or U.S. Patent No. 4,352,883 to Lim, et al., the teachings of which are incorporated 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.

As used herein, the term "microcapsule" encompasses microparticles, microspheres, and microcapsules unless otherwise stated. In general, those microcapsules which are useful will have a particle diameter of between one and 200 microns, preferably between one and 15 microns for oral administration, and preferably between one and 100 microns for injection, although the limiting factor for injection is the needle size. In the preferred embodiment, polyphosphazene/antigen solutions are prepared by first dissolving antigen in l part 3% Na₂C0₃ with stirring, followed by the addition of PCPP with stirring until dissolved and then slowly adding 3 parts phosphate buffer pH 7.4. The detergent Brij58 is added to the stirring polymer solution at a final concentration of 0.2%. The final concentration of PCPP is 2.5%. Sodium alginate/antigen solutions are prepared by dissolving the appropriate amount of antigen in deionized water. The alginate is then slowly added to the antigen solution so that the final concentration of alginate is 1.25%. Constant stirring, as well as the slow addition of the polymer to the antigen, is necessary in order to obtain a homogeneous solution.

In the most preferred embodiment for making microspheres for oral delivery, microspheres are generated using a syringe pump at a speed of 150 μl/min to pump the polymer and antigen solution into an atomization nozzle (Turbotak, Ottawa Canada) , or an ultrasonic spray nozzle (Medsonic, Inc. , Farmingdale, NY) , equipped with an 18 gauge blunt-end needle. The needle enables the solution to be delivered directly to the point of atomization in the nozzle. The polymer solution containing dispersed antigens is then forced through a 1.0 mm orifice in the nozzle under approximately 35 pounds per square inch of air pressure. For polyphosphazenes, the microdroplets cross-link when they impact a 7.5% CaCl₂ 0.5% Brij58 bath at a distance 35 cm from the nozzle. The Brij58 is added in order to prevent agglomeration of the microspheres. A 1.5% CaCl₂ bath (no Brij58) is used for gelation of alginate microspheres. The microspheres are then quickly transferred to a centrifuge tube and rocked gently for approximately 30 minutes to complete the cross-linking process and to avoid microsphere aggregation as they settle out of the CaCl₂ bath. Aggregation may be due to Ca++ crosslinking between exposed carboxylic groups on the microsphere surface and/or hydrophobic interactions between microspheres. After 30 minutes, the microspheres are collected by centrifugation at 4°C, 2800 rpm for 15 minutes. The supernatant is discarded, the pellet is both washed one time and resuspended in sterile deionized water. The microspheres are stored at 4°C until analysis. Approximately 90% of polyphosphazene microspheres generated under these conditions had diameters in the one to ten micron range.

Larger microspheres are made by using a larger orifice and lower air pressure.

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. Administration of Immunogenic Composition

Hydrogel microspheres containing antigen can be administered mucosally or parenterally. Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue) , respiratory, vaginal, and rectal. Nonlimiting examples of parenteral delivery include intradermal, subcutaneous, and intramuscular.

Antigens can be encapsulated in both naturally occurring alginate and synthetic polyphosphazenes. The level of antigen loading, release kinetics and the microsphere size distribution are used to vary the resulting immune response. 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, aε demonstrated by the following examples.

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 Drakeol™, Markol™, and squalene, to form an emulsion, or in combination with aqueous buffers, or encapsulated within a capsule or enteric coating to protect the microcapsules from degradation while passing through the stomach. Storage of Immunogenic Compositions Ionically cross-linked microspheres need to be stored in buffers that are conducive to the maintenance of their integrity. Conditions have been defined that maintain the integrity of the microspheres as well as antigens entrapped within the polymer matrix. Microspheres containing antigen are stable for seven days stored at 4°C in sterile deionized water. Standard buffers such as phosphate buffered saline (PBS) cannot be used because the replacement of calcium ions with sodium leads to the liquification of the matrix. Coating the microspheres with an amino acid polymer such as poly L-lysine or other crosslinking agent allows storage in PBS.

The present invention will be further understood by reference to the following non-limiting examples. Example l: Toxicity Studies.

Alginate is approved for human consumption. The polyphosphazenes can be tested to demonstrate non-toxicity using standard methodology. Polyphosphazenes have previously been demonstrated to be non-toxic to living cells. As reported by M.C. Bano, et al., Bio/Technology. 9:468 (1991), hybridoma cells were encapsulated in polyphosphazene microspheres having a diameter between 150 and 200 microns. The encapsulated hybridoma cells were able to undergo cell divisions, and by ten days after encapsulation the microspheres were essentially filled with living cells. Additional studies are described herein.

In the first study, cell culture dishes were coated with the polyphosphazene and then chicken embryo fibroblasts seeded onto the coated petri dish. Three days after seeding the chicken embryo fibroblasts, the cells had become flattened and spindle formed and under phase contrast microscopy one could see mitotic figures. This demonstrated the innocuous nature of the polyphosphazenes in cell culture.

In a second in vivo toxicity study, the in vivo acute toxicity of alginate and polyphosphazene was evaluated in 6-8 week old Sprague-Dawley rats. The study consisted of four groups of five male rats/group. Following an overnight fast, each animal in each group received a single oral dose of 5000 mg polymer/kg (in water) via gavage. The dose volume was 20 ml/kg. Group one rats received water and served as a control group. Group two animals received alginate microspheres. Group three rats received alginate microspheres coated with poly-L-lysine (M.W. 68,000). Group four animals received poly[di(carboxylatophenoxy)phosphazene] microspheres. The animals were clinically observed for 7 days. Body weights were recorded on day l prior to immunization and at euthanasia. Blood samples were obtained by puncture of the retro-orbital sinus after anesthetization with C0₂ at euthanasia. Animals were food fasted overnight prior to blood collection. Tissues were examined and saved at necropsy.

There were no significant differences in body weight gain between the ratε that received microsphereε and the rats in the control group. The results of hematology and clinical chemistry were normal for all rats in each group. There were no treatment related abnormalities observed in any organ at necropsy. This study demonstrated that at an oral dose of 5000 mg/kg, polyphosphazene and alginate microspheres are not acutely toxic.

Example 2: Incorporation of Proteins and Release

Characteristics of microspheres.

In order for the microencapsulated antigens to elicit an immune response, the antigen must be released from the microspheres. Antigen is released from a microsphere through the two different but not mutually exclusive procesεes of diffusion and erosion. If the hydrogel is permeable to the dispersed antigens, then the antigens can simply diffuεe out of the microspheres following the water phase that fills the matrix of the microsphere. Release of antigen is, therefore, an indication of the permeability of the microsphere matrix to the antigen. Conversely, adsorption of the antigens to the polymer matrix will serve to either reduce or eliminate the diffusion of the antigen out of the microsphere. Characterization of release kinetics. Protein molecular weight markers (Amersham) and FITC-labelled bovine serum albumin (Sigma) were microencapsulated to study release kinetics of soluble proteins. The release kinetics of 20 nm polystyrene beads (Duke Scientific) can be used for comparative purposes.

Quantitation of Protein in Microspheres. For immunogenicity studies, the protein content of microspheres is determined both directly after generation of the microspheres to assess the percent incorporation and also immediately before injection into animals to insure delivery of known antigen quantities.

The protein content of the microspheres can not be assessed by a standard assay such as the Bio-Rad protein assay. Although the protein can be released from the microspheres by chelating the Ca++ responsible for forming the hydrogel, the addition of the Bio-Rad reagent which contains divalent cations causes the polymer to re-cross-link, rendering the antigen unavailable to the dye reagent.

The quantitation of protein antigens encapsulated in ionically cross-linked microsphereε iε determined by electrophoresing a known quantity of intact microsphereε in SDS- PAGE. During electrophoresis, the proteinε migrate out of the microsphere matrix and into the polyacrylamide gel. The protein concentration is determined by comparison to known quantities of the encapsulated protein electrophoresed in parallel to the microsphere preparation.

Determination of Microsphere Size. One to fifteen micron microspheres are believed to have an adjuvant effect and are therefore preferred. The size of alginate and polyphosphazene microspheres is measured utilizing a Coulter

LS100 Particle sizer. The size is reported as % number in-the one to ten micron size range.

Modification of antigen release from polyphosphazene microspheres.

Effect of polymer concentration and molecular weight of the antigen .

The permeability of the poly[di(carboxylatophenoxy) phosphazenes] waε investigated by encapsulating protein molecular weight markers (Rainbow™ protein molecular markers (Amersham

Corporation), ranging in molecular weight from 14,000 to 200,000 daltonε, that are commonly used in polyacrylamide gel electrophoresis. Release of the proteins was assayed by spectrophotometric measurementε of the εupernatant. The reεultε are shown in Figure 1. The permeability of a particular protein such as the 14.3 KDa molecular weight lysozyme was affected by the concentration of the polymer in the gel. As the polymer concentration rises from 1.5% to 3.3% there is a marked decrease in the diffusion of the protein out of the microcapsule matrix. Similarly, as the molecular weight of the protein increaseε, diffusion of the protein out of the matrix is retarded. For example, the 200 KDa molecular weight myoglobin protein was unable to diffuse out of a 3.3% polyphosphazene matrix in a time period of 24 hours. Effect of polymer molecular weight and composition . The second mechanism by which the antigens can be released from microspheres is through the erosion of the polymer matrix making up the microsphere. Erosion can occur through the reversal of the gelation reaction, resulting in the solubilization of polymer molecules and their return to the surrounding aqueous environment. Degradation of polyphosphazene microspheres was studied in saline solution (pH 7.4) by monitoring mass loss, molecular weights of polymer matriceε and formation of soluble products. Erosion profiles for PCPP microsphereε of varied molecular weights are εhown in Figure 2.

No detectable mass losε waε obεerved during 20 days incubation of high molecular weight PCPP microspheres in solution, and for the period of time extended to 180 days. However GPC data show significant decrease in polymer molecular weight during the εame period of time (Figure 3a) . The mechanism of degradation apparently can involve intramolecular carboxylic group catalysis. Uεe of low molecular weight PCPP for microεphere preparation leadε to significant erosion of the hydrogel during the first 10 days and a decrease in molecular weight of polymer (Figure 3b) . Water-soluble polymeric products of practically the εame molecular weight aε in the matrix were detected.

These data indicate that there is a molecular weight threshold of approximately 200 KDa in the release of polyphosphazene from the matrix into the solution in this system. However, polymer solubility also depends on the amount of calcium ions (or other multivalent cationε or polymerε) held by the matrix and the ionization degree of macromolecules. The observed differences in the erosion of PCPP are of prime importance for the design of antigen delivery syεtems. Polyphoεphazeneε can be efficiently tailored by incorporating appropriate εide-groupε to provide a controllable set of properties, including hydrolytic degradability. Introduction of a hydrolysis-εenεitive pendant group, εuch as glycinato group, increaseε the degradation rate in an aqueouε environment. Cleavage of an external P-N bond occurring in neutral media in these aminophosphazenes to yield hydroxy derivatives confers hydrolytic instability in the polymer.

Poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato) phosphazene] (PC-G1PP) containing 10% of glycinato groups was used for the preparation of microspheres and degradation studieε. Eroεion rateε for theεe polymer hydrogelε alεo depend on the molecular weight of polyphosphazeneε. PC-GIPP with weight average molecular weight 130 KDa haε a 100% maεε loss within 3 days, as shown by Figure 2. The GPC analysiε of matrix and soluble products shows in Figure 4 that a 240 day incubation in an aqueous environment results in breakdown of the polymer backbone leading to fragments with molecular weightε lower than 1 KDa and inorganic phoεphate. Coating hydrogel microspheres with Poly-L-lysine (M.W. 62 KDa) to yield a polyelectrolyte complex membrane significantly decreaseε the eroεion rate by 2.5 timeε apparently because of steric hindrances, providing an additional approach to control the degradation and εtability of polyphoεphazene microspheres. Effect of Crosslinking agents .

The third means by which one can regulate the releaεe of antigen from microεpheres is by coating the polyphosphazene microspheres with poly-L-lysine or a similar polyion to form a semi-permeable membrane on the outside of the microspheres. The microsphere core can then be liquified by the addition of chelating agentε εuch as EDTA which reverse the gelation process and result in the solubilization of the polyphosphazene matrix. The degree of permeability can be regulated by the size of the polyion that is used in the coating process. The percent release from microεphereε croεεlinked with poly-L-lysines ranging in molecular weight from 12 to 295 KDa iε shown in Figure 5. Aε the molecular weight of the poly-L-lysine increases, the permeability of the coating increaseε, reεulting in an increaεed releaεe of 20 n polyεtyrene beads from the microsphere. The ability to vary the polyphosphazene concentration in the microspheres, alter the side chains on the polymer and coat microspheres with poly-L-lysine makeε it poεεible to formulate microεphereε that will releaεe antigenε with pulεatile and/or εuεtained release kinetics. The manipulability of this polymer εyεtem combined with the very gentle conditionε for gelation and microεphere formation make thiε polymer εyεtem particularly deεirable for developing εingle doεe vaccineε which may elicit both antibody and cellular immune reεponεeε.

Example 3: Efficacy of Influenza vaccine encapsulated in alginate administered orally to mice as measured by In vitro and in vivo immune response studies.

Microencapsulated antigens were used to immunize mice by the oral route. The kinetics of the immune responεe were first determined by in vitro assays for humoral immunity. The use of in vivo studieε allowε determination of the capacity to effect antibody class switching, the effect of doεe and route of immunization on the rapidity, amplitude and duration of the immune response, and the need for boosting the immune responεe. ELISA waε uεed to evaluate total antigen εpecific responses as well as subclasεes of IgG responεe, aε deεcribed below. CTL aεsayε could be performed to evaluate the cell mediated responseε.

As described in detail below, tetanus toxoid (Connaught Laboratories) and influenza virus were encapεulated for the immunogenicity studies. Microencapsulated antigens were prepared and quantitated as described above. The antigen concentration in alginate and polyphosphazene microspheres as determined by -SDS- PAGE was adjusted with sterile deionized water before adminiεtration. Female 7 to 8 week old BALB/c mice were randomized into groupε of five. Thirty microgramε of flu antigen were adminiεtered orally by intubation. Blood samples were taken from the retroorbital sinus of C0₂ anaesthetized mice. Mice were euthanized with C0₂ in an inhalation chamber. The influenza mouse disease model syεtem developed by

Novak at al., Vaccine, 11:55-60 (1992), could be uεed to εtudy the protection afforded by immunization with microencapεulated influenza. Mice are challenged at variouε timeε after immunization and the levelε of viruε replication in various organs determined. Although in previous studieε parenteral immunization did not completely protect the nose and trachea, it does completely protect against virus propagation in the lungs. Thus, vaccine efficacy can be evaluated on the basis of the level of virus replication in the lungs. Influenza waε grown in eggε according to εtandard methodε and quantitated by protein, hemagglutination and plaque aεsays. Influenza was formalin inactivated by the addition of a 38% formaldehyde solution at a final dilution of 1:4000. Virus infectivity was also inactivated by exposure to gamma irradiation from a '"Co source to 1.2 x 10⁶ rad. Anti-influenza specific antibodies in mouεe εerum were determined by ELISA in 96-well microtiter plateε coated with 10 μg/ml of influenza infected MDCK cell lyεate in εodium carbonate buffer pH 9.6. Sites available for non-specific binding of protein after coating and washing were blocked by adding 2.5% BSA in PBS solution. After blocking and washing, two-fold serial dilutions of sera in 1% BSA/PBS were added to the wells. Unbound serum was waεhed away and horseradish peroxidase-labelled goat anti-mouse IgG added. Unbound conjugate waε waεhed away and serum antibody detected by adding the subεtrate o- phenylenediamine dihydrochloride. The reaction was stopped by the addition of 2 M H₂S0₄ and the absorbance read at 490 nm. The endpoint titers are the reciprocal of the greatest sample dilution producing a signal significantly greater than that of an antibody negative sample at the same dilution.

The IgG isotypes of the ELISA reactive influenza specific antibodies were determined by the detection of murine antibodies bound to the antigenε. Horseradish peroxidase labelled sheep anti-mouse antibody specific for mouse IgG subclasεeε 1, 2a, 2b and 3 was reacted with the mouse antibodies bound to the antigen in the ELISA plates.

The influenza hemagglutination inhibition antibody asεay was done with heat-inactivated mouse serum that had been incubated for 30 minutes with 10% chicken red blood cells to remove non-specific inhibitors. Twofold dilutions of sera were added to a 96 well microtiter plate and 8 HA units of viruε suspension in an equal volume were added to each well and incubated at room temperature for 30 minutes. A 0.5% suεpenεion of chicken red blood cellε waε added to each well and incubated at room temperature for 45-60 minuteε. The HI titerε are expressed as the reciprocal of the highest dilution that completely inhibits hemagglutination of erythrocytes.

In the first group of studies, five groups of BALB/c mice, conεiεting of two mice per group, were immunized by oral intubation with εterile deionized water (Group I) , empty alginate microspheres (Group II) , alginate microspheres containing 30 μg Influenza (Group III) , alginate microεpheres containing 30 μg Influenza plus 10 μg cholera toxin (CT) admixed (Group IV) , or 30 μg soluble Influenza (Group V) . Blood and fecal samples were collected on days 7, 14, 21 and 28 post-immunization and the class specificity of influenza antibody reactivity was determined.

Animals were immunized as described above with influenza antigen encapsulated in alginate, alone or in combination with cholera toxin.

The results with alginate encapsulated influenza antigen are shown in Figures 6a, 6b, and 6c. Control mice that received no influenza antigen (groups I and II) showed no flu- specific serum IgM or IgG responses. Soluble influenza (Group V) induced a low IgM titer at day 7 that persisted at least through day 14 but there was no detectable IgG response, as shown in Figure 6a. Encapsulated flu together with CT induced high levels of flu-specific IgG at day 14 post-immunization, as εhown in Figure 6b. These levels were maintained up to day 28.. Alginate encapsulated flu alone induced flu-specific IgG titers that were equivalent to those seen in the animals that received the microsphere influenza-CT admixture, aε shown in Figure 6c. Good antibody titers were observed as early as 14 days, with high titers of IgG preεent through at leaεt 77 days. Animals immunized with alginate encapsulated influenza plus cholera toxin were boosted at 35 days post primary immunization. The results are shown in Figure 7. Boosting with influenza in combination with cholera toxin elicits production of IgA, as measured in the fecal samples.

In summary, the alginate encapsulated flu did not require the mucosal adjuvant CT for the induction of antigen specific IgM and IgG in the sera. The resultε obtained with alginate encapsulated influenza show that a single oral dose in the absence of CT elicits high flu specific serum IgG responseε.

Reεults in Figure 7 show that IgA antibodies are induced following a single oral boost with influenza encapsulated in alginate with CT.

Example 4: Production of Antibody by oral administration of Influenza vaccine encapsulated in polyphosphazene to mice as measured by In vitro and in vivo immune response studies.

The same protocol was followed for immunization of animals with influenza alone or in combination with cholera toxin, encapsulated in polyphosphazene microεphereε, aε deεcribed above.

The reεultε are εhown in Figure 8. In the abεence of cholera toxin there iε no production of anti-influenza antibodies measurable in either the serum or the feces. With the combination of influenza antigen and cholera toxin there iε production of IgM in a similar manner to that demonstrated with alginate encapsulated antigen (Figure 6b) , although slightly delayed in onset. Example 5: Intranasal immunization of mice with microencapsulated tetanus toxoid.

Mice were divided into four groups and inoculated intranasally with (1) tetanus toxoid in water (9 animals) ; (2) tetanus toxoid in alginate microsphereε (9 animals) ; (3) tetanus toxoid in PCPP microspheres (10 animals) ; and (4) tetanus toxoid in microsphereε consiεting of 95% alginate/5% PCPP (9 animalε) .

In each caεe 50 μg of antigen waε adminiεtered. Mice were aεεayed by ELISA for antibody production after two weekε (serum) and three weeks (bronchial and nasal washes) . The results are shown in Table 1.

These results clearly demonstrate that intranasal administration of antigen in a polyphosphazene or alginate/polyphosphazene microsphere induces a serum IgG responεe. Moreover, the results demonstrate that this method of administration can be used to elicit production of IgA moleculeε, when the antigen iε encapεulated within the combination of alginate and PCPP.

Table 1: Intranasal inoculation with microencapsulated tetanus toxoid. Group/Animal treatment anti-tetanus toxoid titer (log2) IgG IgA

1 tetanus toxoid <256 (<8) 2 tetanus toxoid <256(<8) 3 tetanus toxoid <256(<8) 4 tetanus toxoid <256(<8) 5 tetanus toxoid <256(<8) 6 tetanus toxoid 256( 8) 7 tetanus toxoid 256( 8) 8 tetanus toxoid <256(<8) 9 tetanus toxoid <256(<8)

10 tetanus toxoid in alginate microsphereε <256 (<8) 11 tetanus toxoid in alginate microεphereε <256(<8) 12 tetanus toxoid in alginate microεphereε <256(<8) 13 tetanus toxoid in alginate microspheres <256(<8) 14 tetanus toxoid in alginate microspheres <256(<8) 15 tetanus toxoid in alginate microsphereε <256(<8) 16 tetanus toxoid in alginate microεpheres <256(<8) 17 tetanus toxoid in alginate microsphereε <256(<8) 18 tetanus toxoid in alginate microεphereε <256(<8)

19 tetanus toxoid in PCPP microεphereε 512 9) <2(<1) 20 tetanus toxoid in PCPP microεpheres 2048 11) <2(<1) 21 tetanus toxoid in PCPP microsphereε 512 9) <2(<1) 22 tetanus toxoid in PCPP microspheres 512 9) 23 tetanus toxoid in PCPP microsphereε 2048 ID 24 tetanus toxoid in PCPP microspheres 512 9) 25 tetanus toxoid in PCPP microspheres 1024 10) 26 tetanus toxoid in PCPP microsphereε 1024 10) 27 tetanus toxoid in PCPP microεphereε 1024 10) 28 tetanus toxoid in PCPP microεpheres 512 9)

29 tetanus toxoid in Alginate/5% PCPP ms 4096 12) 8(3) 30 tetanus toxoid in Alginate/5% PCPP mε 4096 12) 32(5) 31 tetanus toxoid in Alginate/5% PCPP ms 512 9) 8(3) 32 tetanus toxoid in Alginate/5% PCPP ms 256 <8) 33 tetanus toxoid in Alginate/5% PCPP ms 2048 11) 34 tetanus toxoid in Alginate/5% PCPP ms 2048 ID 35 tetanus toxoid in Alginate/5% PCPP ms 2048 11) 36 tetanus toxoid in Alginate/5% PCPP mε 2048 11) 37 tetanus toxoid in Alginate/5% PCPP mε 1024 10) Example 6: Parenteral immunization of mice with tetanus toxoid encapsulated in microspheres and comparison with immunization with conventional adjuvants.

Traditionally, most injected non-replicating vaccines have required multiple doses to achieve sufficient serum antibody titers to be protective. For obvious reasons, it would be much more desirable to achieve protection with a single inoculation.

Therefore, the effect of polyphoεphazene on the immunogenicity of antigenε waε examined in mice that were immunized εubcutaneously with a single dose. Antigen formulated in water, alum and complete Freund's adjuvant was included in many experiments as a comparator.

The immunogenicity of tetanus toxoid antigen formulated in polymeric microspheres composed of alginate or polyphosphazene was compared to soluble tetanus toxoid and tetanus toxoid in the standard adjuvants, alum and complete Freund's adjuvant (CFA) . Groupε of five mice were immunized by the εubcutaneouε route with 20 μg of tetanus toxoid.

The results are shown in Table 2. The anti-tetanus toxoid serum immune responses were assayed by ELISA. Soluble tetanus toxoid antigen and alginate microencapsulated tetanuε toxoid induced a maximum titer of 512 by week 13. Polyphosphazene microεphereε containing tetanus toxoid induced higher antibody titers at earlier times poεt immunization than alum or complete Freund'ε adjuvanted tetanus toxoid.

Furthermore, polyphoεphazene microεphereε containing tetanus toxoid induced antibody tierε that were still rising at 13 weeks post immunization. At this late time point, tetanus toxoid in polyphosphazene microspheres had elicited a titer of 65,536, which was approximately 100 times aε strong a response aε seen for soluble tetanus toxoid and as good as or slightly better (two to four fold higher) than was seen for alum and complete Freund's adjuvant. Polyphosphazene microspheres were clearly superior to alginate microspheres in the induction of antibodies to tetanus toxoid.

Table 2: ELISA Titers in Mice Inoculated SC with Tetanus Toxoid anti TT ELISA titer week 3 week 5 week 7 week 9 week 13

TT in Water <256 256 256 256 512

TT in Alginate MS 256 512 512 512 512

TT in Alum 2048 8192 16384 32768 32768

TT in CFA 2048 16384 16384 32768 16384

TT in Poly¬ phosphazene MS 8192 16384 32768 32768 65536

The dose dependent effect of immunization with tetanus toxoid was examined by immunizing mice with varying amounts of tetanus toxoid formulated into polyphosphazene microspheres or complete Freund's adjuvant. The results are shown in Table 3. The immunogenicity of tetanus toxoid in polyphosphazene microspheres compared very favorably with complete Freund's adjuvant formulated tetanus toxoid. At all time points and tetanus toxoid doses, the ELISA titers for the two formulations were within a two-fold dilution of each other.

Table 3: ELISA Titers in Mice Inoculated SC with Tetanus Toxoid TT (μg) anti-TT ELISA titer

TT + polyphosphazene TT + complete Frends adjuvant week3 week5 week7 week9 week3 weeks week7 week9

25 32768 65536 131072 131072 16384 131072 262144 262144

5 8192 32768 65536 65536

2.5 4096 16384 32768 16384

1 4096 16384 65536 65536 16384 32768 32768 32768

0.2 2048 4096 8192 8192 1024 4096 4096 4096

0.04 <256 <256 256 256 <256 <256 <256 <256

Example 7: Parenteral Immunization of mice with influenza particles formulated in polymeric microspheres or with adjuvant.

Mice were also immunized with 5 μg of formalin inactivated influenza virus particleε formulated in polymeric microspheres, alum and complete Freund's adjuvant to determine if the relative efficiencieε of the formulationε would be the same for an enveloped virus aε they were for tetanuε toxoid.

The results are shown in Table 4. Again, polyphosphazene microspheres were as efficient aε complete

Freund'ε adjuvant but much more efficient than water, alum or alginate microεphereε at inducing a very high titer anti-flu immune response. In contrast to the tetanus toxoid results, alum adjuvanted influenza was no better than soluble influenza and alginate microencapsulated influenza in eliciting a rather low titer anti-flu responεe. Taken together, theεe reεultε demonstrate that polyphosphazene microsphereε containing an antigen provoke an antibody reεponse equal in magnitude to complete Freund's adjuvant formulated antigens.

Table 4: ELISA Titers in Mice Inoculated SC with x-31 Influenza anti- lu ELISA titer week 3 week 5 week 7 week 9 week 13

Flu in Water 256 1024 1024 512 512

Flu in Alginate

MS 512 1024 2048 2048 2048

Flu in Alum <256 512 1024 2048 2048

Flu in CFA 8192 16384 32768 32768 16384

Flu in Poly¬ phosphazene MS 8192 32768 32768 8192 16384

The mouse εera were teεted for the presence of functional antibodies by hemagglutination inhibition and neutralization assays. The reεults of the hemagglutination assay are shown in Table 5. As measured by the HAI assay, the polyphosphazene microspheres containing flu elicited an antibody titer of 1280 by week 7, while the Freund's adjuvanted flu, as well as the flu in alum and alginate microsphereε, elicited either no detectable or very low HAI titers.

Table 5: Hemagglutination Inhibition Assay titers in mice inoculated SC with x-31 Influenza

HAI titer week 3 week 5 week 7 week 9 week 13

Flu in Water neg neg neg 40 neg

Flu in

Alginate MS neg neg 40 40 40

Flu in Alum neg neg neg neg neg

Flu in CFA neg neg neg 40 neg

Flu in Poly¬ phosphazene MS 320 640 1280 1280 1280

Water* neg neg neg neg neg

♦Negative control had a titer of 20 due to non-specific serum hemagglutination inhibitors. Neg < 20.

Antibodies that neutralize influenza infectivity were assayed in a 50% plaque reduction assay. Flu in polyphosphazene microsphereε induced a detectable titer of 800 by week 13, whereas, flu in water and complete Freund's adjuvant did not elicit detectable neutralizing antibody titers. The HAI and neutralization assays are sensitive functional antibody assays for influenza. Thus, the immune reεponεe engendered by polyphoεphazene microεpheres is superior to complete Freund's adjuvant.

Table 6: Influenza Plaque Reduction Assay week 13

Flu in Polyphosphazene MS 800 Flu in Water <200

Flu in CFA <200

Normal mouse serum <200 The IgG isotypeε of the antibodies induced by theεe formulationε were determined by an ELISA aεsay. The resultε are shown in Table 7. Alum adjuvanted influenza elicited a purely IgGl response as expected. Flu formulated in Complete Freund's Adjuvant induced mostly an IgGl response that peaked by week 7 and was waning by week 13. Flu formulated in alginate and polyphosphazene microspheres also induced largely an IgGl response that by week 7 was higher than flu formulated in alum. Again, polyphosphazene microsphere formulated antigen induced titers that compared very favorably with those induced by complete Freund's adjuvant formulated antigen. Polyphosphazene microspheres like complete Freund's adjuvant was able to induce significant levels of IgG2a and IgG2b antibodies. A εignificant difference in the immune response was found in the level of activity detected in the IgG3 isotype. Polyphosphazene microsphereε were the only formulation able to induce a significant lgG3 antibody titer.

Table 8: Flu ELISA Isotyping Results

3 Weeks 7 Weeks 13 Weeks

IgGl IgG2A IgG2B IgG3 IgGl IgG2A IgG2B IgG3 IgGl IgG2A IgG2B

Flu in alginate MS 1024 <256 256 <256 65536 1024 512 <256 8192 512 <256 <2

Flu in PPP MS 8192 4096 512 512 131072 16384 1024 4096 16384 16384 2048 10 Flu in Alum 512 <256 <256 <256 16384 <256 <256 <256 8192 <256 <256 <2 Flu in CFA 8192 1024 4096 <256 >52428 8192 4096 <256 32768 2048 2048 <2

Flu in Water 256 512 256 <256 2048 1024 256 <256 1024 512 <256 <2

51

Modifications and variations of the present invention, polymer adjuvants and methods of synthesis and use in vaccine compositions, will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.

Claims

We claim:

1. A vaccine compoεition compriεing hydrogel microparticles formed of biocompatible polymers encapεulating an effective amount of an antigen to elicit an immunogenic effect, wherein the microparticleε have a diameter of 200 microns or less.

2. The composition of claim 1 wherein the microparticles have a diameter of between one micron and fifteen microns.

3. The composition of claim 1 wherein the polymer is a biocompatible polymer selected from the group conεisting of polymers and salts of polymers that can be crosslinked by physical crosslinking, covalent crosεlinking or ionic croεεlinking to form a hydrogel.

4. The composition of claim 3 wherein the polymer is selected from the group consisting of alginate, gelatin, pectin, collagen, phosphazene polyelectrolytes, poly(aerylamide) , poly(methacrylamide) , poly(vinyl acetate) , poly(N- vinyl pyrrolidone) , poly(hydroxyethylmethacrylate) , poly(ethylene glycol) , poly(vinyl alcohols) , poly(acrylic acidε) , poly(methacrylic acidε) , sulfonated polystyrene, polyamines, poly(vinylpyridine) , and mixtures and copolymers. of the polymerε and monomerε thereof.

5. The composition of claim 3 wherein the polymer is a phosphazene polyelectrolyte of the formula R p - N 4-

R

wherein A and B can vary independently in the polymer, and can be:

(i) a group that iε εuεceptible to hydrolysis under the conditions of use; or

(ii) a group that is not susceptible to hydrolysis under the conditions of use selected from the group consisting of aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, heteroalkyl, (aliphatic) mino-, heteroaralkyl, di(aliphatic)amino- arylamino-, diarylamino-, alkylarylamino-, -oxyaryl, -oxypheny1C0₂H, -oxypheny1S0₃H,

-oxyphenylhydroxy1, -oxypheny1P0₃H, -oxyaliphatic, -oxyalkyl, -oxy(aliphatic)C0₂H, -oxy(aliphatic)S0₃H, -oxy(aliphatic)P0₃H, -oxy(aliphatic)hydroxyl, -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic, -thioalkaryl, thioaralkyl, or -NHC(0)0-(aryl or aliphatic), -0-[ (CH₂)_xO]_y-CH₂)_xNH₂, -0-[ (CH₂)_xO]_yCH₂)_XNH(CH2)_xS0₃H, and

-0-[ (CH₂)_x0]_y-(aryl or aliphatic), wherein x is 1-8 and y is an integer of 1 to 20; and wherein n between 10 and 20,000.

6. The composition of claim 1 wherein the polymer is crosε-linked with a multivalent cation or polyelectrolyte.

7. The compoεition of claim 4 wherein the polymer iε a mixture of alginate and phoεphazene polyelectrolyte.

8. The composition of claim 1 wherein the antigen iε selected from the group consisting of compounds derived from cells, bacteria, virus particles, and portions thereof, wherein the compound is selected from the group consisting of proteins, peptides, polysaccharides, glycoproteins, glycolipidε, nucleic acid, or combinationε thereof.

9. The composition of claim 8 wherein the antigen is derived from an organism selected from the group consiεting of rotavirus, measles, umpε, rubella, polio, hepatitis A and B and herpes viruses, human immunodeficiency virus, Haemophilus influenza , Clostridium tetani , influenza, Cory-neJacteriujn diphtheria, and Neisseria gonorrhea .

10. The compoεition of claim 1 wherein the polymer iε covalently conjugated with the antigen.

11. The composition of claim 1 further comprising an adjuvant from the group consiεting of muramyl dipeptideε, muramyl tripeptide, cytokines, diphtheria toxin, exotoxin A, cholera toxin-A, cholera toxin-B, and soluble phosphazene.

12. The composition of claim 1 wherein the microparticles are within a coating protecting the microparticles from the acid pH of the stomach.

13. A method of causing an immune response in an animal comprising the steps of administering to the animal a vaccine composition comprising hydrogel microparticles formed of a biocompatible polymer encapsulating an effective amount of an antigen to elicit an immunogenic effect, wherein the microsphereε are 200 microns or less in diameter.

14. The method of claim 13 wherein the microspheres are administered to mucosal surfaces.

15. The method of claim 14 wherein the route to the mucoεal surfaces is intratracheal.

16. The method of claim 14 wherein the route to the mucosal surfaces is intranasal.

17. The method of claim 14 wherein the mucosal surfaces is selected from the group consisting of rectal and vaginal.

18. The method of claim 14 wherein the route to the mucosal εurfaceε iε orally.

19. The method of claim 14 wherein the route to the mucosal surfaces is parenterally.

20. The method of claim 13 wherein the microparticles have a diameter of between one micron and fifteen microns.

21. The method of claim 13 wherein the polymer is selected from the group consiεting of alginate, gelatin, pectin, collagen, phosphazene polyelectrolyte, poly(aerylamide) , poly(methacrylamide) , poly(vinyl acetate) , poly(N- vinyl pyrrolidone) , poly(hydroxyethylmethacrylate) , poly(ethylene glycol) , poly(vinyl alcohols) , poly(acrylic acids) , poly(methacrylic acids) , sulfonated polystyrene, polyamines, poly(vinylpyridine) , and mixtureε and copolymerε of the polymerε and monomerε thereof.

22. The method of claim 21 wherein the phoεphazene polyelectrolyte is of the formula

A

B wherein A and B can vary independently in the polymer, and can be:

(i) a group that is εuεceptible to hydrolyεiε under the conditionε of use; or

(ii) a group that is not susceptible to hydrolysiε under the conditions of use εelected from the group conεiεting of aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, heteroalkyl, (aliphatic)amino-, heteroaralkyl, di(aliphatic)amino- arylamino-, diarylamino-, alkylarylamino-, -oxyaryl, -oxypheny1C0₂H, -oxypheny1S0₃H,

-oxyphenylhydroxyl, -oxypheny1P0₃H, -oxyaliphatic, -oxyalkyl, -oxy(aliphatic)C0₂H, -oxy(aliphatic)S0₃H, -oxy(aliphatic)P0₃H, -oxy(aliphatic)hydroxyl, -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic, -thioalkaryl, thioaralkyl, -NHC(0)0-(aryl or aliphatic), -0-[ (CH₂)_xO]_y-CH₂)_xNH₂, -0-[ (CH₂)_x0]_yCH₂)_XNH(CH2)_xS0₃H, or

-0-[ (CH₂)_x0]_y-(aryl or aliphatic), wherein x iε 1-8 and y is an integer of 1 to 20; and wherein n is between 10 and 20,000.

23. The method of claim 21 wherein the polymer is a mixture of alginate and phosphazene.

24. The method of claim 13 wherein the antigen is selected from the group conεisting of compounds derived from cells, bacteria, virus particles, and portions thereof, wherein the compound is selected from the group consisting of proteins, peptides, polysaccharides, glycoproteinε, glycolipids, nucleic acid, or combinations thereof.

25. The method of claim 24 wherein the antigen is derived from an organism selected from the group consiεting of rotavirus, measles, mumps, rubella, polio, hepatitis A and B and herpeε viruses, human immunodeficiency virus, Haemophiluε influenza , Clostridium tetani , influenza, Corynebacterium diphtheria , and Neisseria gonorrhea .

26. The method of claim 13 wherein the polymer iε covalently conjugated with the antigen.

27. The method of claim 13 further comprising an adjuvant from the group consisting of muramyl dipeptides, muramyl tripeptide, cytokines, diphtheria toxin, exotoxin A, cholera toxin-A, cholera toxin-B, and soluble phosphazene.

28. The method of claim 13 wherein the microparticles are administered in combination with a material protecting the microparticles from the acid pH of the stomach.

29. The method of claim 13 wherein the microparticles have different release rateε.

30. A method for making a vaccine composition comprising forming microparticles having a diameter of 200 micronε or leεs by forming a hydrogel of biocompatible polymer in the presence of an antigen.

31. The method of claim 30 wherein the polymer iε formed into a hydrogel by physical crosslinking, covalent crosslinking or ionic crosεlinking.

32. The method of claim 31 wherein the polymer is selected from the group conεiεting of alginate, gelatin, pectin, collagen, phoεphazene polyelectrolyteε, poly(acrylamide) , poly(methacrylamide) , poly(vinyl acetate), poly(N- vinyl pyrrolidone) , poly(hydroxyethylmethacrylate) , poly(ethylene glycol) , poly(vinyl alcoholε) , poly(acrylic acids) , poly(methacrylic acids) , sulfonated polyεtyrene, polyamineε, poly(vinylpyridine) , and mixtureε and copolymerε of the polymerε and monomers thereof.

33. The method of claim 32 wherein the polymer is a phosphazene polyelectrolyte of the formula

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; or (ii) a group that iε not εuεceptible to hydrolyεiε under the conditions of use εelected from the group conεiεting of aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, heteroalkyl, (aliphatic)amino-, heteroaralkyl, di(aliphatic)amino- arylamino-, diarylamino-, alkylarylamino-, -oxyaryl, -oxypheny1C0₂H, -oxypheny1S0₃H,

-oxyphenylhydroxyl, -oxypheny1P0₃H, -oxyaliphatic, -oxyalkyl, -oxy(aliphatic)C0₂H, -oxy(aliphatic)S0₃H, -oxy(aliphatic)P0₃H, -oxy(aliphatic) ydroxyl, -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic, -thioalkaryl, thioaralkyl, or -NHC(0)0-(aryl or aliphatic), -0-[ (CH₂)_xO]_y-CH₂)_xNH₂, -0-[ (CH₂)_x0]_yCH₂)_XNH(CH2)_xS0₃H, and

-0-[ (CH₂)_x0]_y-(aryl or aliphatic), wherein x iε 1-8 and y is an integer of 1 to 20; and wherein n between 10 and 20,000.

34. The method of claim 30 further comprising εelecting polymers having a specific molecular weight to achieve a controlled rate of release of the antigen.

35. The method of claim 30 further comprising εelecting the croεεlinking cation or polymer molecule having a particular molecular weight to achieve a controlled rate of release of the antigen.

36. The method of claim 30 further comprising selecting the polymer with a hydrolysis-sensitive pendant group to increase the degradation rate in an aqueous environment.

37. The method of claim 30 further comprising mixing microparticles containing antigen which have different release rates together to form an immunogenic composition having pulsed release.

38. The method of claim 30 further comprising mixing microparticles containing antigen which have different release rates together to form an immunogenic composition having extended release over a period of time due to release of antigen from εome microparticleε occurring after a longer period of time than other microparticleε.