Sustained release vaccine composition

Abstract

A non-liquid vaccine composition including one or more antigen component(s); a pharmaceutically acceptable non-liquid adjuvant therefor; and optionally a non-liquid vaccine protection agent.

Claims

1. A sustained release vaccine apparatus selected from the group consisting of an implant, a pellet, a tablet and a solid gel, the apparatus including: a non-liquid vaccine composition including: one or more antigen component(s); and a pharmaceutically acceptable adjuvant therefor; optionally, a non-liquid vaccine protection agent, wherein the vaccine apparatus comprises a pharmaceutically acceptable non-liquid matrix comprising at least a water-soluble substance which is a polysaccharide, and a sustained release support material which is biocompatible and biodegradable material; and wherein the vaccine apparatus is for oral administration to a non-human animal without liquid formulation, and the vaccine apparatus upon administration at least initiates and/or stimulates an immune response in the non-human animal.

2. The sustained release vaccine apparatus according to claim 1, wherein the apparatus provides, in use, approximately zero order release of antigen.

3. The sustained release vaccine apparatus according to claim 1, further including a water soluble or insoluble pharmaceutically active component, or mixtures thereof.

4. The sustained release apparatus according to claim 1, wherein the one or more antigen component(s) are contained in first dry milled powder particles dispersed in the matrix, and wherein the pharmaceutically acceptable adjuvant is a non-liquid mineral salt adjuvant consisting of second dry milled powder particles dispersed in the matrix.

5. The sustained release vaccine apparatus of claim 4, wherein the non-liquid mineral salt adjuvant is dispersed in the matrix in the form of dry powder particles consisting of Al(OH).sub.3.

6. The sustained release vaccine apparatus of claim 4, which is in the form of a biocompatible and biodegradable pellet or tablet adapted for oral administration.

7. The sustained release vaccine apparatus of claim 1, wherein the matrix further comprises an oligosaccharide.

8. The sustained release vaccine apparatus of claim 7, wherein the oligosaccharide is trehalose.

9. The sustained release vaccine apparatus of claim 1, wherein the matrix further comprises an amphipathic substance.

10. The sustained release vaccine apparatus of claim 9, wherein the amphipathic substance is in the form of a dry powder consisting of saponin.

11. The sustained release vaccine apparatus of claim 9, wherein the amphipathic substance is dispersed in the matrix in the form of dry milled particles consisting of the amphipathic substance dispersed in the matrix.

12. The sustained release vaccine apparatus of claim 11, wherein the amphipathic substance is in the form of a dry powder consisting of saponin.

Description

(1) In the Figures:

(2) FIG. 1 is a graph showing titres for ovalbumin (OVA) antibodies as described in Example 3.

(3) FIG. 2 is a graph showing titres for Salmonella antibodies as described in Example 4.

EXAMPLE 1

(4) A mixture of Tetanus Toxoid Antigen, Saponin, Human Gamma Globulin and NaCl in proportions specified in Table 1 below was produced. The obtained solid was milled and passed through a sieve. A portion of a powder thus obtained and Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 component B were mixed to give a drug dispersion component. Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 Component B were mixed to give a coating layer component. Thus obtained drug dispersion component and coating layer component were molded by extruding from a double extruder which enables them to be molded by extruding so that the drug dispersion is concentrically coated with the coating layer, and was allowed to stand at room temperature to cure, which was cut to obtain the cylindrical preparation 1 (the length of the preparation is 1.0 or 2 cm, the external diameter of the preparation is 1.5 mm) (See Table 2). Silicone comprised the other 50% of the formulation.

(5) TABLE-US-00002 TABLE 1 Outer Composition/Powder (%) Type Diameter (mm) Powder (%) TET SAP -Glob NaCl CR 1.5 50 2.68 2.68 34.64 10 Abbreviations CR = covered rod TET = Tetanus Toxoid Antigen 100 doses @ 384 ug/dose = 38.4 mg total weight Each dose contains 6Lf (flock units) of tetanus toxoid, which was freeze dried with 200 ug/dose of trehalose to provide some stability and bulk for handling. -Glob = Human Gamma Globulin SAP = Saponin adjuvant Outer diameter, mm = 1.5 mm Inner diameter, mm = 1.3 mm

(6) The cylindrical preparation 1 is then cut into various lengths as shown in Table 2 to provide the sustained release mini-pellets according to the present invention.

(7) TABLE-US-00003 TABLE 2 Design: Implant Length 1.0 cm 2.0 cm Total Length Number of implants 2 cm 2 2 cm 1 4 cm 4 4 cm 2 1 ml s.c day 0 and day 28
Groups Group 11 cm2 n=6 Group 22 cm1 n=3 Group 31 cm4 n=6 Group 42 cm2 n=2 Group 5ControlsGlanvac 6 in 1.
Examination 1

(8) Preparation 1 was subcutaneously administered to Merino and Merino cross sheep, whole blood was collected from the animal via the jugular vein, and then, the concentration of antibody in the plasma was determined by antibody titration. Animals: Merino/Merino Cross Sheep Identification: Ear tagsboth ears Implants: Single formulation Tetanus toxoidcontaining 384 microgram of antigen per cm of implant Cured at 37 C. for 3 days AdjuvantSaponin Type: Covered rod Dose: Two doses to be investigated2 cm and 4 cm Size: Implants used will be cut to 1.0 or 2.0 lengths Site: Neckat the base of the ear.

(9) Blood samples to be collected at 0, 7, 14, 21, 28, 42, 56, 84, 91 and 210 days

(10) Sheep were allocated to 5 treatment groups as outlined in Table 3.

(11) TABLE-US-00004 TABLE 3 Sample Numbers Group Number Sample Numbers 1 2, 4, 5, 16, 17, 19 2 6, 7, 21 3 1, 11, 13, 18, 20, 22 4 10, 23 5 3, 8, *9, 12, 14, 15, *Sheep euthanased 19/9/03 28 days - no more samples after this

(12) The results are provided in Table 4.

(13) TABLE-US-00005 TABLE 4 Merino/Merino Cross Sheep Sera, Trial - O-03-001 Technical Standard All titres are IU/ml Day 14 Day 42 Day 56 Day 91 Day 210 Sample ID - Group 1 2 7.72 4.48 5.68 N/S 4.26 4 28.24 1.22 0.79 5.4 7.44 5 6.22 24.2 17.06 1.49 N/S 16 4.23 7.77 5.26 4.48 4.47 17 N/S 8.46 8.47 16.75 5.54 19 5.8 8.56 3.65 3.82 3.38 GMT 8.02 6.49 4.81 4.71 4.84 Sample ID - Group 2 6 12.95 0.2 0.19 9.29 1.42 7 20.37 7.39 3.58 3.55 1.33 21 9.19 5.12 2.96 N/S 6.29 GMT 13.43 1.96 1.26 5.74 2.28 Sample ID - Group 3 1 7.8 6.97 5.53 4.41 5.32 11 18.72 12.86 6.86 3.48 3.47 13 1.01 2.24 3.29 1.43 5.36 18 9.14 7.6 3.34 5.05 1.63 20 9.7 12.38 6.69 6.3 7.3 22 28 15.8 8.55 5.88 9.08 GMT 8.46 9.64 5.36 4.00 4.69 Sample ID - Group 4 10 10.44 19.4 14.18 N/S 2.79 23 21.8 9.65 13.52 7.49 5.88 GMT 15.09 13.68 13.85 7.49 4.05 Sample ID - Group 5 3 1.82 25.55 0.71 2.86 4.11 8 0.14 N/S N/S N/S N/S 9 0.36 10.55 4.43 8.3 2.13 12 23.28 13.1 6.84 1.01 4.99 14 7.48 6.33 3.62 1.66 6.93 15 24.61 27.6 11.8 4.01 3.51 GMT 2.71 14.39 3.91 2.76 4.03 N/S = no sample
Groups Group 1cm2 n=6 Group 22 cm1 n=3 Group 31 cm4 n=6 Group 42 cm2 n=2 Group 5ControlsGlanvac 6 in 1.
Conclusions 1 The use of implants, the subject of the present invention (the division of a single larger implant into multiple small implants) results in a higher serum level of antibody in a more rapid time frame (see Table 4). 2 Optimum results were achieved with the Group 4, 2 cm2 implant combination. The results with the Group 3 and Group 1 combination showed some sustained immune response, but the Group 2 combination failed to provide sustained immunity.

EXAMPLE 2

(14) Example 1 was repeated utilising the vaccine composition formulations specified in Table 5 below.

(15) Mixtures of antigen and adjuvant (Saponin) or aluminium hydroxide in proportions specified in Table 5 below was produced.

(16) TABLE-US-00006 TABLE 5 1: Canine parvovirus 2a inactivated virus: antigen (freeze-dried) 30% adjuvant (saponin) 10% silicone formulation 60% 2: canine parvovirus 2a inactivated virus: antigen (freeze-dried) 30% adjuvant (powdered Al(OH).sub.3) 10% silicone formulation 60% 3: bovine rhinotracheiitis vaccine modified - live: antigen 15% adjuvant (saponin) 1% silicone formulation 84% 4: bovine rhinotracheiitis vaccine modified 0 live: antigen 15% adjuvant (powdered Al(OH).sub.3) 1% silicone formulation 84%

(17) The obtained solid was milled and passed through a sieve. A portion of a powder thus obtained and Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 component B were mixed to give a drug dispersion component. Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 Component B were mixed to give a coating layer component. Thus obtained drug dispersion component and coating layer component were molded by extruding from a double extruder which enables them to be molded by extruding so that the drug dispersion is concentrically coated with the coating layer, and was allowed to stand at room temperature to cure, which was cut to obtain the cylindrical preparation (the length of the preparation is 1.0 or 2 cm, the diameter of the preparation is 1.5 mm).

EXAMPLE 3

(18) A mixture of Ovalbumin, Gamma Globulin, NaCl and Aluminium hydroxide or Saponin in proportions specified in Table 6 below was produced. The obtained solid was milled and passed through a sieve. A portion of a powder thus obtained and Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 component B were mixed to give a drug dispersion component. Silastic Medical Grade ETR Elastomer Q7-4750 Component A and Silastic Medical Grade ETR Elastomer Q7-4750 Component B were mixed to give a coating layer component. Thus obtained drug dispersion component and coating layer component were molded by extruding from a double extruder which enables them to be molded by extruding so that the drug dispersion is concentrically coated with the coating layer, and was allowed to stand at room temperature to cure, which was cut to obtain the cylindrical preparation (the length of the preparation is 10 mm, the diameter of the preparation is 1.5 mm).

(19) TABLE-US-00007 TABLE 6 Vaccine M9 Ovalbumin 0.6% Porcine gamma globulin 38% Sodium chloride 10% Silicone 48.4% Aluminium hydroxide 3% Vaccine M11 Ovalbumin 0.6% Porcine gamma globulin 38% Sodium chloride 10% Silicone 48.4% Saponin 3%

EXPERIMENTAL PROCEDURE

(20) Mice injected intraperitoneally with implant measuring 1.5 mm (diameter)10 mm length.

(21) The implants fell into one of three categories: (a) blank implantnegative control (b) 100 g OVA/Al(OH).sub.3 (c) 100 g OVA/Saponin

(22) Mice bled at day 10 after vaccination.

(23) Titres measured for ovalbumin antibodies by ELISA. Animals: Balb/c mice Identification: Ear punch Implants: Single formulation

(24) The results are provided in FIG. 1.

(25) Conclusion

(26) Very high antibody titres were observed 10 days after vaccination for both preparations clearly showing that saponin and Al(OH).sub.3 are excellent adjuvants in the formulations.

EXAMPLE 4

Salmonella Vaccine in Horses

(27) Purpose of the Study:

(28) To evaluate the antibody response of horses to an experimental Samonella Newport inactivated vaccine comprising a bacterial extract of purified siderophore receptor and porin protein.

(29) Animals:

(30) 4 yearling horses (1 year of age) maintained in a barn and on pasture

(31) Vaccine:

(32) Silicone covered rod 1.5 mm outside diameter, 1.3 mm internal diameter. Each horse received 2 implants of 2 cm on day 0 of the experiment either subcutaneously in the neck (2 horses) or intramuscular in the ligamentum nuchae area (2 horses).

(33) Each 1 cm of implant contained 250 ug of Salmonella antigen adjuvanted with 3% aluminium hydroxide in powdered form (Sigma). The implants also contained 36% porcine globulin as a protection agent.

(34) Vaccine Formulation:

(35) TABLE-US-00008 % Formulation Inactivated Salmonella Antigens 1% Adjuvant: Powdered Al(OH).sub.3 3% NaCl 10% Protection Agent: Porcine -globulin 36% Silicone 50%
Samples:

(36) Blood samples were taken from the jugular vein on days 0, 8,16,24 and 32 of the experiment and analysed by ELISA.

(37) Site Reaction:

(38) No site reactions were observed at anytime over the 32 days the results were recorded.

(39) Results/Conclusions:

(40) The results are summarized in FIG. 2. ELISA SP refers to signal-to-positive. The value given is the test value divided by a positive control. The results from ELISA assays from positive horse samples (from unreported work) were used as positive controls. A result of near 1 or greater is thus indicative of an excellent immune response.

(41) All four horses showed a rapid onset of immunity to the antigen challenge with peak levels of antibody reached within 8 to 16 days. The results also show conclusively that Al(OH).sub.3 in a powdered form is an outstanding adjuvant. Both subcutaneous and intramuscular sites of implantation were successful with NO site reactions observed. This is an important finding as horses are extremely difficult to vaccinate being prone to adverse site reactions.

EXAMPLE 5

IBR Vaccine in Cattle

(42) Purpose of the Study:

(43) To evaluate the antibody response in healthy cattle against an inactivated infectious bovine rhinotracheitis (IBR) vaccine formulation as an aid in prevention of disease caused by Bovine Herpes Virus Types I & II.

(44) Animals:

(45) 18 Angus steers, aged 12-15 months maintained on pasture.

(46) Vaccine:

(47) Silicone covered rod 1.5 mm outside diameter, 1.3 mm internal diameter. One group of 5 cattle received 1 implant of 2 cm in length, another group of 6 cattle received 2 implants of 2 cm in length and a third group of 7 cattle served as negative controls and were not vaccinated. The implants were administered subcutaneously in the ear.

(48) Samples:

(49) Blood samples were collected at 0, 5, 7, 14, 21, 28, 56 and 84 days after implantation.

(50) Vaccine Formulation:

(51) TABLE-US-00009 % Formulation Inactivated IBR Antigen* 39.7% Adjuvant: Saponin 3.1% NaCl 10.2% Silicone 47% *includes high percentage of cell line debris and cell line culture media.
Site Reaction:

(52) No site reactions were observed during the 84 days over which the study was conducted.

(53) Results/Conclusions:

(54) High antibody titres at day 28 after implantation (measured by ELISA) were observed in 4/5 cattle implanted with 12 cm implants, 4/6 cattle implanted with 22 cm implants and 0/7 cattle in the negative control group.

EXAMPLE 6

Neonatal Calf Diarrhea Vaccine in Cattle

(55) Purpose of the Study:

(56) To evaluate the antibody response of healthy, pregnant cows to aid in passive maternal immunisation of their calves (through the consumption of colostrum) against neonatal calf diarrhea caused by bovine rotavirus, bovine coronavirus and entertoxigenic strains of Escherichia coli having the K.sub.99 pili adherence factor.

(57) Animals:

(58) 142 pregnant dairy cows vaccinated 6-8 weeks prior to calving.

(59) Vaccine:

(60) Silicone covered rod 3.0 mm outside diameter, 2.8 mm internal diameter. Each cow received 2 implants of 2 cm subcutaneously in the ear.

(61) Vaccine Formulation:

(62) TABLE-US-00010 Vaccine A % Formulation *Antigens: 37% Bovine Rotavirus, Bovine Coronavirus, K99 E. coli bacteria Adjuvant: Powdered Al(OH)3 3% Protection Agent: Human -globulin 11% Silicone 49% *includes high percentage of cell line debris, cell line culture media and bacterial culture media

(63) TABLE-US-00011 Vaccine S % Formulation *Antigens: 37% Bovine Rotuvirus, Bovine Coronavirus, K99 Ecoli bacteria Adjuvant: Saponin 3% Protection Agent: Human -globulin 11% Silicone 50% *includes high percentage of cell line debris, cell line culture media and bacterial culture media
Site Reaction:

(64) All 142 cows implanted had their implant sites inspected 7 days after implantation in the ear.

(65) TABLE-US-00012 All values expressed as percentages Vaccine A Vaccine B No swelling in ear 71 41 Swelling <2 mm 22 23 Swelling <4 mm 5 31 Implant lost from ear 2 4

(66) Vaccine A which included the adjuvant powdered Al(OH).sub.3 proved to be an extremely non-reactive vaccine formulation. Vaccine B which included Saponin as the adjuvant was slightly more reactive as a vaccine formulation.

(67) Results/Conclusions:

(68) Both vaccine preparations showed a rapid onset of immunity (in sera obtained 7 days after implantation) to the 3 antigens in the formulations with very high titres being observed in the colostrum of all the 138 cows that retained the implants in the ear at calving.

EXAMPLE 7

Extended Milk Life Vaccine in Cattle

(69) Purpose of the Study:

(70) To evaluate over the lactation cycle of dairy cows for up to 305 days the antibody response of healthy lactating cows to a vaccine formulation containing bacterial antigens to prevent bacterial decay of milk held at room temperature.

(71) Animals:

(72) 20 lactating cows vaccinated 4 weeks prior to calving.

(73) Vaccine:

(74) Silicone covered rod 1.5 mm outside diameter, 1.3 mm internal diameter. Each cow received 2 implants of 2 cm subcutaneously in the ear.

(75) Vaccine Formulation:

(76) TABLE-US-00013 Vaccine A % Formulation Purified Bacterial Antigens 1.1% Adjuvant: Powdered Al(OH)3 3% Protection Agent: Human -globulin 35.7% NaCl 10.2% Silicone 50%

(77) TABLE-US-00014 Vaccine S % Formulation Purified Bacterial Antigens 1.1% Adjuvant: Saponin 3% Protection Agent: Human -globulin 35.7% NaCl 10.2% Silicone 50%
Site Reaction:

(78) No site reactions were observed in the ear of the 20 vaccinated cows.

(79) Results/Conclusions:

(80) The milk of all 20 cows, observed up to 100 days after lactation, contained antibodies that showed significant inhibition of bacterial growth in milk at room temperature for 2 days and under refrigeration for up to 10 days.

(81) It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

(82) It will also be understood that the term comprises (or its grammatical variants) as used in this specification is equivalent to the term includes and should not be taken as excluding the presence of other elements or features.