Vaccine composition

A vaccine composition which comprises an immnunologically active substance embedded in microparticles essentially consisting of starch having an amylopectin content exceeding 85% by weight, of which at least 80% by weight has an average molecular weight within the range of 10-10000 kDa, and without any covalent chemical cross-linking between the starch molecules. A process for preparing such vaccine composition.

EXAMPLES 
 Example 1 
 Preparation of OVA Starch Particles 40-100&mgr; (Batch D-018) Immobilization of OVA in starch microspheres produced from highly branched, sheared starch. All utensils were detoxified at 180° C. for 3 hours and autoclaved prior formulation. A starch solution (30%) of highly branched, shared starch with an av. mol. wt. 408 kDa, a PEG solution (37.5% av. mol. wt. 20 kDa) and an OVA solution (0.38 mg/ml, purified from polymeric residues by means of gel chromatography) were prepared in 10 mM sodium phosphate buffer pH&equals;7.4. The temperature of the starch solution was adjusted to 50° C. and the other solutions to approx. 23° C. The starch solution (14 g) was mixed with the OVA solution (4.69 ml) in an 250 ml IKA reactor equipped with an anchor propeller. The PEG solution (214 g) was added whilst stirring. The starch droplets were solidified at 20° C. for 7 h thereafter 37° C. for 17 hours. The starch microspheres containing OVA were washed with 10 mM sodium phosphate buffer pH&equals;6.4, and freeze-dried. The yield of dry particles in the range of 40-100&mgr; was 70%. The dried microspheres were dissolved by enzymatic action with a-amylase and amyloglucosidase for determining the protein and starch yield, and the protein loading. The loading of OVA was 0.40 &mgr;g/mg (analyzed by ELISA) giving a yield of 100%. The mean particle determined with a Malwern Mastersizer was 77 &mgr;m. ELISA Analyzing OVA Plates were coated with 50 &mgr;l/well with 20 &mgr;g/ml anti-OVA (Biodesign) in PBS at 4° C. over night. The wells were quenched with 100 &mgr;l 1% BSA for 1 h 37° C., Samples and standard were diluted PBS containing 0.2% BSA and subsequently diluted in a non-adsorbing plate 1&plus;1 before transferring 50 &mgr;l to the ELISA plate and incubating for 1 h at 37° C. 50 &mgr;l anti-OVA-HRPO (RDI) was diluted 6000 times in 0.05% Tween 20 and applied into the wells and left 1 h at 37° C. OPD was used as substrate. Between all the steps the plates were washed with 0.1% Tween 20 in PBS. 
 Example 2 
 Preparation of OVA Starch Particles Containing Alum, 40-100&mgr; (Batch D-004) Immobilization of OVA in starch microspheres produced from highly branched, sheared starch. All utensils were and autoclaved prior formulation. An OVA solution (1 mg/ml)was prepared in WFI of which 5.3 ml was mixed with 10.6 ml ALUM gel (Superfos Alhydrogel 2%) and left binding during 30 minutes. A starch solution (30%) of highly branched, shared starch with an av. mol. wt. 529 kDa and a PEG solution (42% av. mol. wt. 20 kDa) were prepared in a 10 mM sodium phosphate buffer pH&equals;6.4 The temperature of the starch solution was adjusted to 50° C. and the other solutions to approx. 38° C. The starch solution (12 g) was mixed with the OVA-ALUM solution (5.4 ml) and buffer (6.6 ml) in an 250 ml IKA reactor equipped with an anchor propeller. The PEG solution (175 g) was added whilst stirring. The starch droplets were solidified at 20° C. for 7 h thereafter 37° C. for 17 hours. The starch microspheres containing OVA were washed with 10 mM sodium phosphate buffer pH&equals;6.4, and freeze-dried. The yield of dry particles in the range of 40-100 &mgr;m was 58%. The dried microspheres were dissolved by enzymatic action with a-amylase and amyloglucosidase for determining the protein and starch yield, and the protein loading. The theoretic load of OVA was 0.54 &mgr;g/mg. The mean particle determined with a Malwern Mastersizer was 70 &mgr;m. 
 Example 3 
 Preparation of OVA Starch Particles <10&mgr; (Batch D-027) Immobilization of OVA in starch microspheres produced from highly branched, sheared starch. All utensils were detoxified at 180° C. for 3 hours and autoclaved. A starch solution (30%) of highly branched, shared starch with an av. mol. wt. 408 kDa, a PEG solution (37.5% av. mol. wt. 20 kDa) and an OVA solution (0.31 mg/ml, purified from polymeric residues by means of gel chromatography) were prepared in 10 mM sodium phosphate buffer pH&equals;7.4. The temperature of the starch solution was adjusted to 50° C. and the other solutions to approx, 23° C. The starch solution (1.6 g) was mixed with the OVA solution (0.50 ml) in an 50 ml cerbo beaker. The PEG solution (20 ml) was added. Microdroplets were formed by an ultra turrax (IKA T-25). The starch droplets were solidified at 4° C. for approximately 24 h thereafter 37° C. for approximately 24 hours. The starch microspheres containing OVA were washed with 10 mM sodium phosphate buffer pH&equals;6.4, and freeze-dried. The dried microspheres were dissolved by enzymatic action with a-amylase and amyloglucosidase for determining the protein and starch yield, and the protein loading. The loading of OVA was 0.13 &mgr;g/mg as determined by ELISA giving a yield of 43%. The mean particle size determined using light microscopy was 3.3 &mgr;m. 
 Example 4 
 Preparation of OVA Starch Particles <10 &mgr;m by Soaking (Batch D-015, Kla01001) Immobilization of OVA was done by soaking into preformed microspheres produced from highly branched, sheared starch. All utensils were detoxified at 180° C. for 3 hours and autoclaved. A starch solution (30%) of highly branched, sheared starch with an av. mol. wt. 408 kDa and a PEG solution (37.5% at. mol. wt. 20 kDa) were prepared in 10 mM sodium phosphate buffer pH&equals;7.4. The temperature of the starch solution was adjusted to 50° C. and the other solutions to approx. 23° C. The starch solution (1.6 g) was mixed with the OVA solution (0.50 ml) in an 50 ml cerbo beaker. The PEG solution (20 ml) was added. Microdroplets were formed by an ultra turrax (IKA T-25). The starch droplets were solidified at 4° C. for approximately 24 h thereafter 37° C. for approximately 24 hours. The placebo starch microspheres were washed with 10 mM sodium phosphate buffer pH&equals;6.4, and freeze-dried. An OVA solution (1.0 mg/ml, purified from polymeric residues by means of gel chromatography) was prepared in WFI. The OVA solution (300 &mgr;l) was mixed with WFI (500 &mgr;l) and subsequently 505 mg placebo starch microspheres. The mixture was incubated 37° C. for 30 minutes. The dried microspheres were dissolved by enzymatic action with a-amylase and amyloglucosidase for determining the protein and starch yield, and the protein loading. The loading of OVA was 0.23 &mgr;g/mg as determined by ELISA. The mean particle size as determined with light microscopy was 3.2 &mgr;m. 
 Example 5 
 Procedure for Coating of Starch Microspheres Containing OVA. (Coat 345, RG 502H) The OVA-containing starch microspheres obtained in Examples 2 and 3 were coated with a release-controlling shell made from PLGA by means of air suspension technology according to WO97/14408 with the RG502H (Boehringer Ingelheim). After the coating operation, the coating was dissolved with a mixture of methylene chloride and acetone in a ratio of 1:3 and, after these solvents have been washed away, for example by repeated centrifugation, the microspheres were dissolved with &agr;-amylase. The OVA content was determined by analysis with ELISA. The protein content was around 0.02 percent by weight. The, release kinetics for OVA from the coated microspheres was determined in vitro. With this process, parentally administrable microspheres can thus be produced so as to be suitable for vaccine delivery, The microparticles thus obtained were then subjected to an experiment concerning release in vitro in 30 mM sodium phosphate, pH 7.4, containing 82 mM sodium chloride, 3 mM sodium azide, 0.5 mM calcium chloride, 0.2% bovine serum albumin and 185 U/l &agr;-amylase, at 37° C. with intermittent agitation. The studies were performed by suspending 40 mg of microspheres in 1.5 mL of buffer. At specific times 1 mL aliquots of said buffer were removed and replaced by fresh buffer. 1 FIG. 1 . In vitro release profile for coat 345, RG502H 
 Example 6 
 In vivo Assessment of Immune-activity Experiment 1 Female BalbC mice 20-22 g (6-8 weeks old) were used in the study, 8 animals/group. The animals were primed s.c. on day 0 with 1 &mgr;g OVA 1 s.c. In the neck and subsequently boostered s.c. the neck at day 21 with 1 &mgr;g OVA. A CMC solution was used as diluent. Blood samples were taken on day 0, 21, 35, 49, 63, 77 and 91 from the tail vein. The blood samples were stored at 4° C. over night and centrifuged at 3000 rpm for 10 minutes. From the sera of each animal 5 &mgr;l sera were transferred to a pooled group serum. Before ELISA analysis the samples were stored at −20° C., ELISA Analysis of Total IgG Plates were coated with 50 &mgr;l/well with 5 &mgr;g/ml OVA (Sigma A-5503) in PBS at 4° C. over night. The wells were quenched with 100 &mgr;l 1% BSA for 1 h 37° C. Monoclonal IgG (Sigma A-6075 was used as a standard. Sera and standard were diluted PBS containing 0.2% BSA and subsequently diluted in a non-adsorbing plate 1&plus;1 before transferring 50 &mgr;l to the ELISA plate and incubating for 1 h at 37° C. 50 &mgr;l anti-mouse-IgG-HRPO was diluted 6000 times in 0.05% Tween 20 and applied into the wells and left 1 h at 37° C. OPD was used as substrate. Between all the steps the plates were washed with 0.1% Tween 20 in PBS. 1 TABLE 3 Overview of immunisations, Experiment 1 Group Prima Day 0: 1 &mgr;g OVA Booster Day 21: 1 &mgr;g OVA 1 ALUM ALUM 2 Coated starch microspheres Coated starch microspheres PLGA resomer RG 502H PLGA resomer RG 502H 3 Starch microspheres 40-100 &mgr; Starch microspheres 40-100 &mgr; 4 Starch microspheres <10 &mgr; Starch microspheres <10 &mgr; 5 Soaked starch microspheres Soaked starch microspheres <10 &mgr; <10 &mgr; 2 FIG. 2 . In vivo immunisation, experiment 1, using coated and un-coated starches microspheres. Experiment 2 Female BalbC mice 20-22 g (6-8weeks old) were used in the study, 8 animals/group. The animals were primed s.c. on day 0 with 2.5 &mgr;g OVA a.c. In the neck and subsequently boostered s.c. the neck at day 21 with 2.5 &mgr;g OVA. A starch solution was used as diluent. Blood samples were taken on day 0, 21, 35, 49 and 91 from the retroorbital point. From the sera of each animal 5 &mgr;l sera were transferred to a pooled group sera, Before ELISA analysis the samples were stored at −20° C. 2 TABLE 4 Overview of immunisations, Experiment 1 Group Prime Day 0: 1 &mgr;g OVA Booster Day 21: 1 &mgr;g OVA 1 ALUM ALUM 2 ALUM Coated ALUM containing starch microspheres PLGA resomer RG 502H 3 FIG. 2 . In vivo immunisation, experiment 1, using coated and un-coated starches microspheres. 
 Example 7 
 Preparation of DNA Plasmid Starch Particles 40-100&mgr; Immobilization of plasmid DNA (5 kb, pDNA-3, INVITROGEN) in starch microspheres produced from highly branched, sheared starch. All utensils were autoclaved prior formulation. A starch solution (31.5%) of highly branched, sheared starch with an av. mol. wt. 408 kDa prepared in distilled water, a PEG solution (40% av. mol. wt. 20 kDa) and a DNA-plasmid solution (1.4 mg/ml) were prepared in 10 mM sodium phosphate buffer with 1 mM EDTA pH&equals;7.0. The temperature of the starch solution was adjusted to 50° C. and the other solutions to 37° C. The starch solution (1.4 g) was mixed with 0.446 ml 10 mM sodium phosphate buffer containing 1 mM EDTA pH&equals;7.0 and DNA solution (0.154 ml) in an 50 ml beaker equipped with an anchor propeller. The PEG solution (22 g) was added whilst stirring. The starch droplets were solidified at 4° C. for 4 h thereafter 37° C. for 17 hours. The starch microspheres containing DNA were washed with 1 mM EDTA solution and subsequently with an ethanol solution, and dried in an LAUF-hood. The yield of dry particles in the range of 40-100&mgr; was 60%. The dried microspheres were dissolved by enzymatic action with &agr;-amylase for determining the DNA and starch yield, and the protein loading. The loading of DNA was 0.19 &mgr;g/mg (fluorometically analyzed by Pico Green method, Molecular Probes) giving a yield of 80%, the starch yield was 95%. The DNA-containing starch microspheres obtained in Example 7 were coated with a release-controlling shell made from PLGA by means of air suspension technology according to WO97/14408 with the a polymer composition consisting of 75% RG502H and 25% RG 756 (Boehringer Ingelheim). After the coating operation, the DNA load was assessed by dissolving the coating with a mixture of methylene chloride and acetone in a ratio of 1:3 and, after these solvents had been washed away, for example by repeated centrifugation, the microspheres were dissolved with &agr;-amylase. The DNA content was determined, by analysis with Pico Green Metod, Molecular Probes. The release kinetics for DNA from the coated microspheres were determined in vitro. With this process, parentally administrable microspheres can thus be produced so as to be suitable for vaccine delivery. The microparticles thus obtained were then subjected to an experiment concerning release in vitro in 30 mM sodium phosphate, pH 7.4, at 37° C. with intermittent agitation. The studies were performed by suspending 40 mg of microspheres in 1.5 mL of buffer. At specific times 1 mL aliquots of said buffer were removed and replaced by fresh buffer. 4 FIG. 3 . In vitro release profile for PLGA coated DNA microspheres.