| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | June 25, 1996 |
| PATENT TITLE |
Hydrogel microencapsulated vaccines |
| PATENT 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 adminstered 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 |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 4, 1993 |
| PATENT REFERENCES CITED |
Espraza et al. 1992. Parameters affecting the immunogenicity of microencapsulated tetanustoxoid. Vaccine. 10:714-720. Eldridge et al. 1991. Biodegradable microspheres as a vaccine delivery system. Mol. Immunol. 28(3):287-294. Eldridge et al. 1990. Controlled vaccine release in the gut-associated lymphoid tissues . . . J. Controlled Release. 11:205-214. Langer. 1990. New Methods of Drug Delivery. Science 249:1527-1533. Letvin 1993. Vaccines against human immunodeficiency virus-progress and prospects. NEJ Med. 329(19):1400-1405. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method of treating an animal to elicit an immune response comprising: treating an animal to elicit an immune response by mucosally administering to the animal a vaccine composition comprising hydrogel microparticles, wherein said microparticles comprise a polyphosphazene polymer and said microparticles contain an effective amount of an antigen to elicit an immune response, wherein the microparticles are 200 microns or less in diameter. 2. The method of claim 1 wherein the microspheres are administered to mucosal surfaces. 3. The method of claim 2 wherein the route to the mucosal surfaces is intratracheal. 4. The method of claim 2 wherein the route to the mucosal surfaces is intranasal. 5. The method of claim 2 wherein the mucosal surfaces is selected from the group consisting of rectal and vaginal. 6. The method of claim 2 wherein the route to the mucosal surfaces is orally. 7. The method of claim 1 wherein the microparticles have a diameter of between one micron and fifteen microns. 8. The method of claim 1 wherein the antigen is selected from the group consisting of compounds derived from cells, bacteria, and virus particles, wherein the compound is selected from the group consisting of proteins, peptides, polysaccharides, glycoproteins, glycolipids, and nucleic acids. 9. The method of claim 8 wherein the antigen is derived from an organism selected from the group consisting of rotavirus, measles, mumps, rubella, polio, hepatitis A and B, herpes viruses, Haemophilus influenza, Clostridium tetani, influenza, Corynebacterius diphtheria, and Neisseria gonorrhea. 10. The method of claim 1 wherein the polymer is covalently conjugated with the antigen. 11. The method of claim 1 wherein the microparticles are administered in combination with a material protecting the microparticles from the acid pH of the stomach. 12. The method of claim 1 wherein the microparticles have different release rates. 13. The method of claim 1 wherein the polyphosphazene polymer is a crosslinked polyphosphazene. 14. The method of claim 13 wherein the polymer is ionically crosslinked. 15. The method of claim 1 wherein the polyphosphazene is biodegradable. 16. The method of claim 1 wherein the microparticles comprise polyphosphazene and alginate. 17. The method of claim 1 wherein the polyphosphazene contains carboxylatophenoxy pendant groups. 18. The method of claim 1 wherein the polyphosphazene is a copolymer which comprises poly [di(carboxylatophenoxy)] phosphazene. 19. The process of claim 1 wherein the polyphosphazene polymer is poly [di (carboxylatophenoxy) phosphazene-co-di (chloro) phosphazene-co-(carboxylatophenoxy) (chloro)phosphazene]. 20. The method of claim 1 wherein the polyphosphazene is poly[dicarboxylatophenoxy phosphazene]. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
Induction of an Immune Response via Mucosal Surfaces The majority of viruses 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. Examples of synthetic polymers are the non-ionic block co-polymer surfactants as disclosed in Hunter, R. L. Topics in Vaccine Adjuvant Research, D. R. Spriggs and W. C. Koff (eds.) pp. 89-98 (CRC Press, 1991), which have molecular weights below approximately 10,000 and have a simple structure composed of two blocks of hydrophilic polyoxyethylene (POE) which flank 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 1982). 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(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 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 FIG. 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. FIG. 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). FIGS. 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 (FIG. 3a), and Mw 400 KDa (FIG. 3b). FIG. 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. FIG. 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. FIGS. 6a, 6b, and 6c are graphs of the flu-specific responses in the sera of animals immunized with flu virus in suspension (FIG. 6a), encapsulated flu virus in combination with cholera toxin (CT) in alginate microspheres (FIG. 6b), and flu virus encapsulated in alginate microspheres (FIG. 6c), measured as antibody titer (reading left to right: IgM, dark bars; IgG, hatched bars; IgA, stipled bars) at 7, 14, 21, and 28 days. FIG. 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. FIG. 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 others 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, N.H., 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.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 C.sub.2 to C.sub.20 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, 1,2,4-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-(car boxylatophenoxy) (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: ##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)amino- including alkylamino-, heteroaralkyl, di(aliphatic)amino- including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, -oxyaryl including but not limited to -oxyphennyCO.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).sub.x O].sub.y --CH.sub.2).sub.x NH.sub.2, --O--[(CH.sub.2).sub.x O].sub.y CH.sub.2).sub.x NH (CH2).sub.x SO.sub.3 H, and --O--[(CH.sub.2).sub.x O].sub.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: ##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)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, 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: ##STR3## 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.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)phosphazene-co(carb oxylatophenoxy)(glycinato)phosphazene], and poly[di(carboxylatophenoxy)phosphazene-co-di(chloro)phosphazene-co(carboxy latophenoxy)(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-GlPP) is prepared by the nucleophilic substitution reaction of the chlorine atoms of the poly(dichlorophosphazene) with propyl p-hydroxybenzoate and ethyl glycinate hydrochloride (PC-GlPP 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. 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 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. Pat. No. 5,149,543 to Cohen, et al., or U.S. Pat. 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 1 part 3% Na.sub.2 CO.sub.3 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 .mu.l/min to pump the polymer and antigen solution into an atomization nozzle (Turibotak, Ottawa Canada), or an ultrasonic spray nozzle (Medsonic, Inc., Farmingdale, N.Y.), 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% CaCl.sub.2 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% CaCl.sub.2 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 CaCl.sub.2 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.degree. 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.degree. 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 parentorally. 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 parentoral 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, as 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 brand mineral oil, Markol brand mineral oil, 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.degree. 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. |
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