GB1567503A

GB1567503A - Vaccine compositions

Info

Publication number: GB1567503A
Application number: GB855677A
Authority: GB
Original assignee: Commonwealth Serum Laboratories
Current assignee: CSL Ltd
Priority date: 1976-03-04
Filing date: 1977-03-01
Publication date: 1980-05-14
Also published as: NZ183458A; AU513400B2; CA1084413A; JPS52125613A; AU2295877A; DE2709488A1; FR2342741A1

Description

(54) VACCINE COMPOSITIONS (71) We, COMMONWEALTH SERUM LABORATORIES COMMISSION, an Australian Body Corporate established under the Commonwealth Serum Laboratories Act, 1961, of 45 Poplar Road, Parkville, State of Victoria, Commonwealth of Australia, do herebydeclare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to vaccine compositions having improved performance for the control of prophylaxis of disease in animals.

The invention is specifically concerned with injectable vaccine compositions which on administration provide sustained and/or delayed and/or periodic release of antigenic material.

Vaccination against most diseases requires multiple dosage with antigen over an extended period of time in order to stimulate effective immunity. This presents substantial problems in the dosing, for example, of large groups of farm animals, where the animals have to be herded up for each dose. Clearly, if a vaccine composition could be provided which from a single dose would continuously or periodically release antigen over a period of time and thereby stimulate multiple vaccine dosing, the number of individual vaccinations required to produce immunity to infection would be minimized and thus offer considerable savings in labour and in convenience.

To date, however, there has been the problem of obtaining antigenic materials in a form capable of providing sustained or periodic release to the animal. Attempts have been made to produce "one dose" vaccines using oily adjuvants such as Freund's adjuvant, but while such vaccines do provide a measure of success, they also produce considerable necrosis and carcase damage and, for this reason, are not generally regarded as acceptable to the farming industry. They are also considered unacceptable for human use.

This invention is based upon the discovery that the performance of conventional liquid vaccines may be improved by the incorporation into such vaccines of a dispersed, solid, biodegradable matrix containing antigenic material which is available for subsequent release. With such combination vaccines, the primary immunizing dose is considered to be supplied by the conventional liquid vaccine with subsequent dose(s) provided by the release of antigen(s) from the incorporated matrix component. In this way, effective immunity may be stimulated by one vaccine dose whereas the conventional liquid vaccine alone requires multiple doses.

Thus, the present invention provides a vaccine composition comprising a vaccine in liquid form having dispersed therein a solid biodegradable matrix containing antigenic material, which matrix provides delayed, sustained or periodic release of the antigenic material upon injection into an animal.

The invention also provides a method for preparing a composition of the invention which method comprises mixing antigenic material with a matrix-forming material and subjecting the resulting mixture to cross-linking and/or polymerization to form a solid biodegradable matrix containing the antigenic material, and dispersing the thus-prepared matrix in a liquid vaccine.

Further provided by the invention is a method for improving the performance capability of a liquid vaccine, which method comprises dispersing in the vaccine a solid biodegradable matrix containing antigenic material, which matrix provides delayed, sustained or periodic release of the antigenic material upon injection into an animal.

In addition, the invention includes a method for the control of prophylaxis of disease in a non-human animal, which method comprises injecting into the animal a vaccine composition of the invention.

We have found that solid biodegradable matrices incorporating antigenic material can be produced by a simple cross-linking technique. Such matrices, after injection into animals, are capable of providing delayed, sustained or periodic release of the active ingredients, the type and time of release being dependent on the cross-linking conditions used in manufacture.

It is desirable that the base material used in the matrix is biologically and antigenically inactive and should be capable of being completely degraded and eliminated over a period of time. Generally, the matrix material will be polymeric in nature and, because of the requirements for low toxicity and biodegradability the preferred matrix materials will usually be derived from natural macromolecular substance, rather than synthetic polymers.

The antigenic material may be incorporated within the martrix by entrapment, linked to the matrix by chemical bonding or chemically bound in such a way as to become part of the matrix.

The preferred matrix material is gelatin cross-linked with glutaraldehyde but other proteins (or peptides) and other cross-linking agents can also be used.

When gelatin is reacted with the dialdehyde, glutaraldehyde, stable cross-links are formed by the reaction between the aldehyde groups of the glutaraldehyde and reactive groups (particularly amino groups) of the gelatin molecules. Several of the constituent amino acids of proteins or peptides are reactive with aldehydes and include lysine, histidine, arginine, cysteine, glutamine, asparagine, tryptophan and tyrosine. When an antigenic material for incorporation within the matrix contains such reactive groups then it will also be chemically linked by the glutaraldehyde and so may be either bound to the gelatin matrix or become a part of the matrix. Non-reactive antigenic materials are held within the matrix by physical entrapment.

To obtain a matrix which is capable of providing controlled antigen release, antigenic material is mixed with the major matrix-forming component, i.e., the polymer, monomer or prepolymer which is to be cross-linked into the matrix. The mixture is then subjected to cross-linking or polymerisation to form the matrix.

Control of the rate or time of release of antigenic material from the matrix is effected by controlling the extent of the cross-linking or polymerisation. In this way it is possible to produce matrices which will provide sustained release, i.e., the continuous release of antigenic material over an extended period following administration, and also matrices which will provide delayed release, i.e., release of all or part of the antigenic material at some time after administration. Obviously, by appropriate selection or combination of these two types of matrix it is possible to provide compositions which will effect extended dosage over long periods, or period dosages over extended periods.

The exact nature of the mechanism whereby antigenic materials are held in, and released by, the matrix is yet to be determined but it appears that both physical entrapment and chemical bonding of the antigen molecules may be involved. Whatever the mechanism involved, however, it is considered surprising that the antigen molecules can be bound in such matrices and subsequently released without any apparent effect on their antigenic properties.

The principles and practice of the invention will be further elucidated and illustrated by the following Examples. It should be clearly noted that Examples 1 and 2 do not exemplify the present invention as such but the cross-linked matrices described therein may be used in the invention and those Examples also provide useful description illustrative of the nature of sustained and delayed release of antigenic material.

Example 1 This Examples illustrates (a) that sustained antigen release may be attained from a matrix vaccine and (b) that the matrix used (gelatin cross-linked with glutaraldehyde) is capable of being completely degraded.

Spheres formed from a gelatin-antigen mixture were cross-linked for increasing periods of time by diffusing glutaraldehyde into the spheres. The outermost region of the spheres was therefore most cross-linked and the core least cross-linked. The spheres thus produced were implanted in guinea pigs and the cellular and humoral response of the guinea pigs to the spheres studied. At weekly intervals spheres and the surrounding tissues were excised, cryosectioned, stained and examined microscopically.

Five Clostridial antigens were used in the vaccines, namely: Activity units/dose of vaccine Clostridium welch it type D, epsilon toxoid 400 L+ Clostridium tetani toxide 50 Lf Clostridium oedematiens (novyi) toxoid 20 L+ Clostridium sepflctim toxoid 30 L+ Clostridium chauvoei cells Approx. 40 x l()" cells Vaccine spheres were formed as follows: Equal portions of 20 gelatin solution and antigen concentrate were mixed thoroughly at 37"C. The mixture was then dropped in 0.2 ml drops (of approx. 5 mm diameter) into paraffin oil which had been cooled to 4"C. As the mixture passed through the paraffin it formed spheres which set as the mixture cooled. After 30 min. at 40C the spheres were recovered from the paraffin washed in water to remove paraffin oil, and added to glutaraldehyde solution ( l Xo in 0.1 M phosphate buffer, pH 7.6 at 4"C). The penetration of glutaraldehyde into the sphere (and hence cross-linking) was assessed by noting the resistance of a bisected sphere to boiling (cross-linked gelatin is resistant to boiling, uncross-linked gelatin rapidly dissolves).

Batches of spheres were prepared in which cross-linking ranged from minimal (outer 1 mm of sphere resistant to boiling) to nearly complete (only centre [1 - 1.5 mm] not resistant to boiling). The spheres were implanted subcutaneously in unimmunized guinea pigs and thereafter at weekly intervals one guinea pig from each sphere batch was killed, its serum collected and the sphere and surrounding tissue excised, snap frozen, cryosectioned, stained and examined microscopically. The antibody levels in the sera were monitored and the cellular response to. and the clearance of. the implanted spheres was assessed from the cryosections.

The cryosections showed that after implantation an initial cellular response around the outside of the sphere was followed by a white blood cell invasion to the centre of the sphere from one or more points. The sphere was then progressively degraded from the centre to the outer skin, the outer skin being the most persistent. In every case the sphere was eventually totally degraded. For each batch of spheres, regardless of amount of cross linking, the pattern of cellular reponse and clearance of the spheres was similar. The amount of cross linking however determined the rate at which the breakdown occurred.

The least cross linked spheres were invaded in less than 6 days and totally removed by two weeks. The most cross linked spheres were slower to be invaded and degraded, and persisted over 6 weeks. The cellular response in each case was comprised mainly of polymorphonuclear leucocytes, macrophages and lymphocytes.

The sera of the guinea pigs showed antibody against each antigen. For example, the serum antibody levels against the flagella (or H) antigen of Cl. chauvoei were determined by an agglutination test and the results are shown in the following table: Cross Linking H agglutination titre of serum taken from of Sphere implanted guinea pigs at (weeks) 3 4 5 6 (a) Non-cross-linked control spheres 80 20 < 20 < 20 (b) Least cross-linked 1280 160 20 < 20 (c) [Intermediate 320 640 40 < 20 (d) [cross-linkage 160 640 640 .20 (e) Most cross-linked 160 640 320 640 Spheres of type (a) were controls, identical to the other spheres except that they were not cross linked. After one week they were not detectable in the animal.

The antibody responses shown in the table indicate that the time and duration of antigen release was dependent on the amount of cross linking within the matrix - the less cross linked matrices giving an earlier antigen release of shorter duration than the later and more sustained release from the more heavily cross linked matrices.

Example 2 This Example illustrates delayed release, c.f. the sustained release shown in Example 1.

Spheres were prepared as for example 1, but containing only tetanus and Clostridium chauvoei antigens. Glutaraldehyde penetration was observed to be rapid and spheres were prepared which were: (a) Non-cross-linked, i.e. antigen-gelatin only.

(b) Cross-linked for a period just sufficient to enable penetration of glutaraldehyde (and hence cross-linking) to the sphere centre.

(c) Cross linked for nine times the period allowed for (b).

Washed and dried spheres were implanted in guinea pigs and the guinea pigs humoral and cellular immune responses observed as in Example 1 over a period of 5 weeks.

Spheres of type (a) were undetectable after one week. Stained cryosections of tissue and spheres removed at weekly intervals from the guinea pigs implanted with spheres (b) and (c) revealed that a large white blood cell response was present around the spheres but that no penetration of the spheres occurred. During the period of observation a progressive clearing of bacterial cells from the outside to the inside of the spheres was noted, but no obvious dissolution of any of the spheres occurred until after week 4. By week 5 the spheres of type (b) were found to be much reduced in size and to have an increased white blood cell response around the outside. Cryosections showed the outer skin and a small central core of the sphere to be intact and apparently unaltered but with a large area of dissolution containing broken down matrix between the two. Spheres of type (c) remained unchanged, apart from bacterial clearing, over the 5 weeks of observation.

The serum antibody titres against tetanus toxin and Cl. chauvoei H antigen at 4 and 5 weeks were as shown.

Spheres Titres 4 weeks 5 weeks (a) Tetanus antitoxin, (International units, < 2 < 2 L+/10).

H antigen, Cl. chauvoei 40 40 (b) Tetanus 2 6 H antigen < 20 640 (c) Tetanus < 2 < 2 H antigen < 20 < 20 From the table it will be observed that release of the flagella and toxoid antigens was delayed for 4 weeks by using matrix of type (b).

Protective immunity against Cl. chauvoei is stimulated by the bacterial cells and not the flagella, and is assessed by measuring the resistance of vaccinated animals to challenge infection. Two guinea pigs, each of which receive spheres of types (a), (b) and (c) were challenged intramuscularly 5 weeks after implantation with a calcium chloride activated spore suspension of Cl. chauvoei. Guinea pigs vaccinated with implants (b) and (c) survived without symptoms whereas those given spheres (a) died within 48 hours. Four unvaccinated control guinea pigs died within 24 hours of challenge. These results, together with the observations of the cryosections, suggest that the bacterial cells were physically entrapped, but not chemically bound, within the matrix. This allowed sustained release of the cells, after breakage of their flagella, from the matrix. In contrast, the two protein antigens (flagella and tetanus toxoid) were chemically bound into the matrix and were only released after dissolution of the matrix had occurred (i.e. delayed release).

Example 3 This Example shows (a) that the number of vaccine doses required for effective immunity may be reduced by using antigen-containing cross linked matrix dispersed into conventional liquid vaccine, (b) that the immune responses stimulated by such vaccines may be earlier and of a greater magnitude than those stimulated by conventional vaccines and vaccination methods, and (c) that the time of release of antigen from the cross-linked matrix may be varied by altering the conditions of cross-linking. With such vaccines the conventional liquid vaccine provides the primary immunizing dose and secondary stimulation is provided by the release of antigen from the matrix.

A solid antigen-containing matrix was prepared containing the following antigens at the concentrations shown: Activity units of antigen/ dose of matrix (Y. welchii Type D epsilon toxoid 80 L+ (1. tetanus toxoid 10 I,f ('l. oedematiens toxoid 4 L+ ('l. septi(loul toxoid 6 L+ Cl. chauvoei cells Approx. 8 x 109 cells Two lots of equal quantities of 20% gelatin and antigen concentrate (at twice the above concentration) were mixed thoroughly and allowed to set at 40C. The set gels were then extruded into: A. 10 times the gel volume of 0.25% glutaraldehyde in 0.1M phosphate buffer, pH 7.6; or B. 40 times the gel volume of 0.25% glutaraldehyde in 0.1M phosphate buffer, pH 7.6.

In each case, the gels were allowed to cross-link at 4 C for 1 hour.

The cross-linked matrices were then washed free of glutaraldehyde and dispersed into a volume of liquid vaccine equal to 3 times that of the matrix before setting or cross linking (i.e. volume of gelatin and antigens). The liquid vaccine used was "C.S.L. 5-in-1" vaccine, an aluminium-adjuvanted vaccine containing the same 5 antigens as the matrices. Each matrix was finely dispersed into the liquid vaccine by using a tissue homogeniser. The liquid vaccines so formed were readily resuspended after settling and were found to be quite suitable for injection with automatic syringes.

For each of the two experimental vaccines (A and B), 4 sheep were vaccinated subcutaneously with one dose of 4 ml of vaccine. Twelve control sheep were vaccinated as follows: (a) Four sheep were given one dose of 4 ml of vaccine composed of one part of non-cross-linked gelatin-antigen dispersed in three parts of 5 in 1 vaccine. This vaccine was identical in composition to vaccines A and B except that the gelatin-antigen was not stabilized for delayed release by cross-linking with glutaraldehyde.

(b) Four sheep were given 2 doses of 2 ml of C.S.L. 5-in-1 vaccine, the second dose being given 4 weeks after the first (this is the recommended dosing procedure for immunizing sheep with this vaccine).

(c) Four sheep were each dosed intramuscularly with a vaccine containing the same quantity of antigens used in the experimental vaccines (A and B) emulsified in Freund's Complete Adjuvant.

One week after vaccination the sheep were checked for reactions to the vaccines and no abnormal lumps, swellings or abscesses were noted. At intervals after the first dose the sheep were bled and their sera pooled according to the date and type of vaccine they had received. The serum pools were assayed for antibodies to the four clostridial toxoids and their titres are shown in the following table in international units.

Antitoxic titres of serum pools at intervals (weeks shown) after first vaccine dose Vaccines Antitoxic titre against: Cl. welchii type D Tetanus toxin Cl. oedematiens toxin Cl. septicum toxin toxin 4 wks 6 wks 8 wks 12 wks 4 wks 6 wks 8 wks 12 wks 4 wks 6 wks 8 wks 12 wks 4 wks 6 wks 8 wks 12 wks Experimental Vaccine A 28 10 < 12 3 4 4 < 4 0.75 18 8 4 3 10 1 < 1 < 1 Experimental Vaccine B 81 26 19.6 7 16 12 5 1.5 60 24 10 6 10 5 3 < 1 Control Vaccine (a) < 12 < 10 < 12 < 1 < 6 2 2 < 0.25 10 6 8 1.5 < 2 < 2 < 1 < 1 (b) 14 15 < 12 3 7 12 10 3 < 8 < 24 22 3 2 7 5 < 1 (c) 18 18 19.6 8 10 12 12 7 10 10 10 6 < 2 2 < 1 < 1 Published Results* Commercial Vaccine A 6.4 7.4 14.8 2.4 Commercial Vaccine B 2.5 3.4 13.2 < 0.5 *G.M. titres from 8 sheep sera in 3 pools. Results from Frerichs, G.M., and Gray, A.K., Research in Veterinary Science, 1975, 18, 70.

Titres in International Units (I.U.) assayed according to methods prescribed by the British Veterinary Codex (B.V.C.).

The results of the foregoing table indicate: (i) that the inclusion of suitably cross linked antigen matrix into conventional vaccines is effective in causing a considerable improvement in antibody response to a single dose of vaccine. The titres stimulated by the single doses of experimental vaccine compare favourably with those produced in the above experiment by 2 doses of C.S.L. 5-in-1 vaccine and 1 dose of Freund's Complete Adjuvant vaccine, and are superior to those reported by Frerichs and Gray to be produced in sheep by two doses of commercial aluminium adjuvant clostridial vaccines.

(ii) that the conditions of cross linking affect the time and duration of antigen release from the matrix and hence the time and duration of peak antibody titre. The matrix of vaccine B was exposed to more glutaraldehyde than that of vaccine A and consequently, as a result of a more suitable mode of antigen release, stimulated a more durable antibody res onse than vaccine A.

(iii that the immune responses stimulated by one dose of the matrix containing vaccine may be earlier and of a greater magnitude than those stimulated by two doses of conventional aluminium adjuvant vaccine. This is illustrated in the accompanying drawing which also shows levels of immunity recommended by the British Veterinary Codex (2nd Edition) (B.V.C.) to be attained by approximately 6 weeks after the first vaccination dose.

Example 4 This illustrates the use of the matrix with a viral antigen - canine hepatitis virus.

Dogs are normally vaccinated against canine hepatitis with either two doses of a vaccine consisting of inactivated virus in a tissue culture medium (C.C.H. Vaccine) or one dose of aluminium adjuvanted C.C.H. Vaccine (K.C.H. Vaccine). The latter vaccine while offering the advantage of one dose vaccination may cause undesirable lumps to form on the dog at the site of injection.

This Example shows that a canine hepatitis virus matrix vaccine prepared in accordance with this invention gives a satisfactory antibody level with one dose and does not cause lumps or other undesirable side effects.

To prepare the virus containing matrix, equal quantities of C.C.H. Vaccine and 20% gelatin were mixed and allowed to set. The set gel was then macerated and dispersed into 33 times the gel volume of 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.6 at 4"C for 2 hours. The cross linked matrix was then washed and dispersed into a volume of C.C.H.

Vaccine equal to that of the gelatin antigen before setting.

Guinea Pigs were vaccinated as follows:1. Four guinea pigs were vaccinated subcutaneously with 2 ml of matrix vaccine.

2. Four guinea pigs were given two subcutaneous doses of 1 ml of C.C.H. Vaccine with a 2 week interval between doses.

3. Four guinea pigs were given 1 dose subcutaneously of 1 ml of aluminium adjuvanted C.C.H. Vaccine (K.C.H.).

In accordance with British Veterinary Codex requirements, sera were taken from the last guinea pig group (3), three weeks after the first dose, whereas the first two groups were bled 4 weeks after the first dose.

The sera were tested for virus neutralizing activity according to British Veterinary Codex requirements and gave the following results: Vaccine Group Pooled Serum Titre in International Units/ml 1. Experimental Matrix Vaccine > 57 (at 4 weeks) 2. 2 doses of 1 ml of C.C.H. Vaccine 57 (at 4 weeks) 3. 1 dose of 1 ml of K.C.H. Vaccine 25 (at 3 weeks) No lumps developed in guinea pigs given the matrix vaccine or 2 doses of C.C.H. Vaccine whereas 2 out of 4 guinea pigs developed small lumps at the side of injection after vaccination with K.C.H. vaccine.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within its spirit and scope.

Claims

WHAT WE CLAIM IS: l. A vaccine composition comprising a vaccine in liquid form having dispersed therein a solid biodegradable matrix containing antigenic material, which matrix provides delayed, sustained or periodic release of the antigenic material upon injection into an animal.
2. A composition as claimed in claim 1 wherein the base matrix material is biologically and antigenically inactive, and is capable of being completely degraded and elimated over a period of time.
3. A composition as claimed in claim 1 or claim 2 wherein the matrix is a natural macromolecular substance.
4. A composition as claimed in any one of the preceding claims wherein the antigenic material is incorporated within the matrix by entrapment, linked to the matrix by chemical bonding, or chemically bound in such a way as to become part of the matrix.
5. A composition as claimed in any one of the preceding claims wherein the matrix is a cross-linked protein or peptide.
6. A composition as claimed in claim 5 wherein the matrix is gelatin cross-linked with glutaraldehyde.
7. A composition as claimed in any one of the preceding claims wherein the antigenic material comprises or the vaccine incorporates one or more of the following antigens: Clostridium welchii type D, epsilon toxoid Clostridium tetani toxoid Clostridium oedematiens (novyi) toxoid Clostridium septicum toxoid Clostridium chau voci cells.
8. A composition as claimed in claim 7 in unit dosage form.
9. A vaccine composition as claimed in claim 1 and either substantially as hereinbefore described in Example 3 or Example 4 or which has been made using a solid cross-linked vaccine matrix substantially as hereinbefore described in Example 1 or Example 2.
10. A method for preparing a composition as claimed in claim 1, which method comprises mixing antigenic material with a matrix-forming material and subjecting the resulting mixture to cross-linking and/or polymerization to form a solid biodegradable matrix containing the antigenic material, and dispersing the thus-prepared matrix in a liquid vaccine.
11. A method as claimed in claim 10 wherein the extent of cross-linking and/or polymerization is accurately controlled in order to provide a composition with a specific release rate or time for the antigenic material.
12. A method as claimed in claim 10 or claim 11 wherein the matrix-forming material comprises a cross-linking agent and a polymer, monomer or prepolymer.
13. A method as claimed in claim 10 and either substantially as hereinbefore described in Example 3 or Example 4 or wherein a solid cross-linked vaccine matrix substantially as hereinbefore described in Example 1 or Example 2 is employed to provide the solid biodegradable matrix.
14. A composition as claimed in claim 1 which has been prepared by a method as claimed in any one of claims 10 to 13.
15. A method for the control or prophylaxis of disease in a non-human animal, which method comprises injecting into the animal a vaccine composition as claimed in any one of claims 1 to 9 or 14.
16. A method as claimed in claim 15 substantially as hereinbefore described in Example 3 or Example 4.
17. A method for improving the performance capability of a liquid vaccine, which method comprises dispersing in the vaccine a solid biodegradable matrix containing antigenic material, which matrix provides delayed, sustained or periodic release of the antigenic material upon injection into an animal.
18. A method as claimed in claim 17 when further defined by the specific features of any of claims 2 to 7.