HRP930517A2 - Complexes having adjuvant activity - Google Patents

Description

The present invention relates to complexes that have auxiliary activity, to complexes that include a lipid and at least one glycoside, and to new glycosides for inclusion in said complexes.

Protective immunization of animals against microbes involves the administration of an antigen derived from the microbe together with a material that increases the antibody and/or cell-mediated immune response to the antigen in the animal, this material being known as an adjuvant. The only additives currently authorized for use on humans or pigs in many countries are aluminum hydroxide and aluminum phosphate. Although these adjuvants are satisfactory for many vaccines, studies have shown that Freund's complete adjuvant (FCA) or quill a (also known as saponin) are often more effective in eliciting antibody responses and cell-mediated immunity but, unfortunately, these adjuvants can cause animals to react adversely to vaccination.

EP-A-0 109 942 and EP-A-0 180 564 describe immunogenic complexes which are formed between glycosides, such as tritropenoid saponins (especially Quil A), and antigens containing a hydrophobic region. Immuno-stimulating complexes are given the name "iscomi". The amount of Quil-A in a single iscom can be about 10 to 100 times lower than when Quil A is mixed with antigen to produce the same antigenic effect. Iscom does not produce the adverse reactions associated with Quil A.

EP-A-231 indicates that the presence of antigen is not necessary for the formation of the basic iscom structure, hereafter called the iscom "matrix", because it is possible that the matrix is formed from some sterol, such as cholesterol, some phospholipid, such as phosphati- di1-ethanolamine, and a glycoside such as Quil A. However, there is no description of such matrices being useful as excipients; are described only in the section to show that iscoma-like structures can form in the absence of antigen. We have found that such empty matrices can be used to provide an auxiliary function by helping to add antigens to bacteria and microorganisms that do not form part of the iscoma matrix. Yes, in all three cited prior art documents, when an antigen is used that is insoluble in water, it is dissolved with a detergent and, then, after mixing with a glycoside, etc., the detergent is separated, for example by dialysis, so that the formation iscoma. We have found that iscomas will form in the presence of detergent and that this separation step is not necessary. Iscom matrices can be formed in a similar way.

One aspect of the invention provides a vaccine for use in a human or animal comprising (1) an antigen associated with a bacterium or a bacterium and (2) an iscoma matrix, wherein said antigen is not incorporated into the iscoma.

The invention also includes a process for making a vaccine for human or animal use, which comprises (1) forming a complex of a gylcoside, a sterol and, optionally, a phospholipid, and (2) mixing said complex with an antigen associated with a bacterium or a mycoplasma.

By animal we mean a non-human vertebrate, preferably a mammal such as a cow, pig, sheep, goat, horse, dog or cat.

The vaccine may comprise one or more diluents and carriers of a conventional nature. These will normally be added at or after stage (2) of the process.

By the term "antigen associated with a bacterium or a mycoplasma" we mean any unit that can produce a protective antibody or cell-mediated immune response against a bacterium or a mycoplasma in a cell exposed to the antigen. The antigen can be all or part of a protein, glycoprotein, glycolipid, polysaccharide. or lipopolysaccharide associated with a bacterium or mycoplasma, or may be a polypeptide or other unit that mimics all or part of such a protein, glycoprotein, glycolipid, polysaccharide or lipopolysaccharide. Because the antigen is not incorporated into the iscoma matrix during the formation of the latter, it is not necessary that it has a hydrophobic region or to be attached to a hydrophobic group, as in prior art methods.The bacterium or mycoplasma itself need not be present in the vaccine, but it can reduce the cost of the vaccine if whole cells are used, for example Haemophi1us p1europneumoniae instead of extracts.

By "mycoplasmas" we include the closely related organisms known as ureaplasmas and aholeplasmas.

Bacterial diseases include those caused by Mycobacterium (eg, M. tubercu1osis & M. bovis), C1ostridium (eg, C .. we1wchii), Rickettsia , Spirochaetes (eg, Treponema pal1idum or Leptospira pomona), Eschericia (eg ., E. coli), Staphylococci (eg, S. aureus), Hemophillius (eg, H. inf1uenzae and H. p1europneumoniae ), Bordetella (eg, B. pertussis), Vibrio (eg, V. cholerae) , Salmonella (eg, S. typhi and S. paratyphi), Streptococci (eg, S.aga1actie, Neisseria (eg, N. gonorrhoea) Pasteurella (eg, P. multocida), Legionella (eg, L. pneumoniae), Ch1amidia (eg, C. pistacci), Pseudomonas (eg, P. malliae), Actinobacillus (eg, A. pleuropneumafilae), Campylobacter (eg, C. jejuni), Listeria (eg, L. monocytogenes), Brucella (eg, B. abortus), Corynebacterium (eg, C. diphteriae), Yersinia (eg, Y. pseudotuberculosis), Pneumococci (eg, Streptococcus pneumoniae), Bacillus (eg, B. anthracis ), Klebsiella (eg, K. pneumoniae), Shigella (eg, S. dysenteriae) and Actinomycetes (eg, Nocardia asteroides).

Preferred bacteria are Mycobacterium (eg, M. tubercu1osisi), C1ostridium (eg, C. we1chii), Rickettsia, Spirochaetes (eg, Treponema pallidium), Escherichia (eg, E. coli), Staphy1ococci (eg, S . aureus), Hemophilus (eg, H. influenzae and H. pleuropneumoniae), Bordetella (eg, B. pertussis), Vibrio (eg, V. cholerae), Salmonella (eg, S. Typhi and S. paratyphi) , Streptococci (eg, S. aga1actie), Neisseria (eg, N. gonorrhoea), Pasteurella (eg, P. mu1tocida), Legionella (eg, L. pneumoniae), Pseudomonas (eg, P. malliae), Actinobacillus (eg, A. pleuropneumoniae), Campylobacter (eg, C. jejuni), Listeria (eg, L. monocytogenes). Certain antigens include the adhesion factor in Colla, eg, pili K 88, the proin protein in eg. Salmonella and other membrane proteins from B. pertussis and Neisseria meningitidis.

Preferably, the antigen is one that does not form a conventional iscom, ie as described in EP-A-109 and EP-A-180 564.

Vaccination of pigs against H. pleuropneumoniae is particularly desirable.

Mycoplasmas of veterinary or medical interest include Mycoplasma bovis, M. gallisepticum, M. agalactiae, M. hyopopneumoniae, M. synoviae, M. Arthritis, M. capricolium, M. dispar, M. hominis. M. mycoides subs. capri, M.. ora1e, M. Oripneumoniae, M. pu1monis, M. cynos, M. hyorhinis, H. mycoides, M. salvarium, M. fermentas.

Although the antigen is not incorporated into the matrix in an earlier way, namely as part of the iscoma structure itself, it may still be present on the outer part of the iscoma matrix or may later be integrated into the iscoma matrix. Alternatively, it may be present completely separately in solution or dispersion.

The iscoma matrix can be made in the manner described in EP-A-0 231 0'39, namely by mixing sterols, glycosides and (optionally) phospholipids together and then separating the solubilizing agents. Alternatively, the separation of the solubilizers can be omitted. An electron micrograph of iscoma obtained from Quil A, cholesterol and phosphatidylinosamine is described in Figure 1 of EP-A-0 231 039 (magnification 116,000x). If phospholipids are not used, dimensional structures are formed. An example of such structures is described in Figure 2 of EP-A-0 231 039. This image is an electron and micrograph (magnification 146,000 x ) of structures obtained from Quil A and ho1estero1a. The term ``iscoma matrix'' is also used for 3- dimensional and 2-dimensional structures.

The glycosides to be used in the process according to the invention can be the same as those mentioned in EP-A 0 109 942 and EP-A--0 231 039. They are mainly glycosides, glycosides that show amphipathic properties and include hydrophobic and hydrophilic regions in the molecule . It is preferable to use saponins, especially the saponin extract from Quillaja saponaria M-olina in the first place DQ-extract made according to K. Dalsgard: Saponin Adjuvants III Archiv fur die gesamete Virusforschung 44. 243-254 (1974). Other preferred saponins are aescin from Aesculus hippocastanum (T.Patt and W Winkler."Das terapeutische Wirsame Prinzip der Rosskatanie (Aesculus hippocastanum) Arzneimittelforschung 10 (4) 273-275 (1960) and sapoalbin from Gypsophilla struthium (R. Vochten. P. Joos and R. Ruyssen. Physicochemical properties of sapoalbin and their relation to the foam stability, J. Pharm. Belg. 42 213.266 (1968). The use of Quil A is particularly preferred.

In the process according to the invention, glycosides are used in the least critical concentration for the formation of micelles. In the case of Quli A, this concentration is about 0.03% by weight. Generally, the molar ratio of glycoside (especially when it is Quil A) to sterol (especially when it is cholesterol) to phospholipid is 1:1:0 ± 20% (preferably no more than ± 10%) for each digit. This is equivalent to a mass ratio of about 5:1 for Qui1 A : ho1estero1).

The sterols used in the process according to the invention can be known sterols of animal or vegetable origin, such as cholesterol, lanosterol, 1umisterol, stigmasterol and sitosterol. Cholesterol is preferably a sterol used in the process of the invention.

Preferably, a phospholipid is used so that it forms into 3-dimensional iscomas. Suitable phospholipids include phosphatidylcholine and phosphatidylethanolamine.

Another aspect of the invention provides a method for preparing an immunogenic complex comprising a water-insoluble antigen, wherein the method comprises dissolving the antigen with a solubilizing agent, mixing the dissolved antigen, glycosides, sterols and optionally, phospholipids and forming an iscome essentially without separating the solubilizing agents.

Thus, there is no need for the solvent to be separated by ultrafiltration, dialysis, ultracentrifugation or chromatographic procedures to form the iscoma.

The antigen is as defined above, except that it may be associated with fungi, parasites such as protozoa and g1ists, or viruses instead of neoplasms or bacteria.

Fungi associated with the disease include: Candida spp., Cryptococcus spp., Aspergilus spp., Microsporum spp., Trichophyton spp., Epidermophyton spp., and Dermatophilus spp.

Parasites associated with the disease include those listed in EP-A-109 942.

Viruses that have been associated with the disease include those listed in EP-A-109 942. Many of these have envelopes (capsules) from which antigens can be extracted. Certain viruses include equine influenza virus, equine herpes virus, bovine viral diarrhea virus, coronavirus, paravovirus, feline leukemia virus, feline insufficiency virus, and equine viral arteritis virus.

The solubilizing agent can be any of those mentioned in EP-A-0 109 942 or EP-A-0 231 039, for example, a detergent, carbamide or guanidine.

In general, a non-ionic, ionic or zwitterionic detergent or a detergent based on hydrochloric acid, such as sodium deoxycholate, can be used for this purpose. It is preferred to use the detergent octylglycoid, nonine-N-methylglucamide or decanoyl-N-methylglucamide, but alkylphenyl-polyoxyethylene ethers are also suitable, especially polyethylene glycol p-isooctylphenylether having 9 to 10 oxyethylene groups and commercialized under the trade name Triton X-100R.

The process according to the invention can be used to make immunogenic complexes from antigenic proteins or peptides showing amphipathic properties, for example, those described in EP-A-109 942.

In the process according to the second aspect of the invention, the described or dissolved antigen is mainly contacted with a solution containing a glycoside in the least critical concentration for the formation of micelles, some sterol and optionally some phospholipid.

If desired, the obtained solutions of immunogenic complexes can be lyophilized. Lyophilized preparations can then be reconstituted for use before adding water.

A third aspect of the invention provides a process for preparing an iscoma matrix, wherein the process comprises dissolving a sterol, and optionally a phospholipid, with a solubilizing agent and mixing with a glycoside solution, without separating the solubilizing agents.

The solubilizing agent, sterol, phospholipid and glycoside are as previously defined herein.

Further, the invention relates to a pharmaceutical preparation containing immunogenic complexes made using the first and second aspects of the invention. these properties can be obtained by preparing the immunogenic complex in a form suitable for parenteral administration, preferably for administration by injection and, in particular, for injection by subcutaneous or intramuscular routes. Generally, pharmaceutical preparations contain immunogenic complexes in an aqueous, physiologically acceptable medium, which, if desired, contains a buffer and/or such a salt as sodium chloride to adjust the osmotic pressure. These procedures are well known in the art.

The following non-limiting examples illustrate various preferred aspects of the invention.

Examples

Examples 1-6: Creating an empty iscom matrix

Required reagents

Phosphate buffered saline (Dulbecco's A, Oxoid)

Lipid mix (see below)

10% w/v solution of Quil A (superfos) in sterile distilled water.

Preparation of lipid membrane

Materials: Chloroform (analytical reagent), cholesterol (Quality I, from pig liver, Sigma). L-beta-phosphatidylcholine (Sigina), Mega 9 or 10 (Sigma) or Triton (Mega 9 is nonyl-N-methylglucamide, while Mega 10 is decanoyl-N-methylglucamide) and sterile distilled water.

Procedure

(a) Make 20% w/v Mega 10 in sterile distilled water, ie, measure 2g of Mega 10 and dissolve in 10 ml of water., (b) Measure 50 mg of cholesterol and add to 50 mg of phosphatidylcholine. (c) when cholesterol is dissolved in phosphatidylcholine, add 2 ml of chloroform (d) Add 10 ml of 20% w/v Mega 10. The mixture will appear milky-white. This will bring the solution to 12 ml. However, Chloromorph will evaporate after mixing and the final volume will be 10 ml. (e) Place on a magnetic stirrer in a hot room (37° C), with the lid of the container detached. After continued mixing, the mixture will become more mobile and clear. (f) Store at room temperature

Example 1

Protocol. Place 20 ml of PBSA in a sterile universal flask. Add 100 µl of lipid mix. Vortex the solution at full speed for 10 seconds. Sterilize the solution by passing it through a 0.2 µm filter (Millipore). Decant a small volume for electron microscopy study.

Example 2

The procedure from example 1 is followed, but with the following variants. Place 20 ml of PBSA in a sterile universal flask. Add 200 µl of 10% Quil A solution. Add 800 µl of lipid mix. Vortex the solution at full speed for 10 seconds. Sterilize through a 0.2 µm filter. Decant a small volume for electron microscopy study. Store at +4° C.

Example 3

Add 20 ml of PBSA to an Amicon mixed cell with a 30K Mwt (molecular weight) membrane cut off. To this add 800 µl of lipid mix plus 200 µl of Quil A (10% solution). The solution was concentrated to 5 ml and 100 ml of PBSA was added. The above phase is repeated x2. The content of 20 ml is filtered through a marked filter of 0.2 µm and stored at +4°C.

Example 4

20 ml of PBSA is added to the Amicon mixing cell with the cut-off 30K membrane in place. Add 400 µl lipid mix plus 100 µl 10% solution of Quil A. The above solution is concentrated to 5 ml and resuspended to 100 ml with PBSA. The above phase is repeated x2. The final contents of 20 ml of cells are sterilized by filtration through a 0.2 µm filter, labeled and stored at +4°C.

Example 5

20 ml. PBSA is added to the Amicon mixing cell, with the cut-off 30K Mwt membrane in place. Add 200 µl of lipid mix plus 50 µl of 10% Quil A solution. The above solution is concentrated to 5 ml and resuspended in 100 ml of PBSA. The upper phase is washed x2. The final preparation of 20 ml cells is then filtered through a 0.2 µm filter, labeled and stored at +4°C.

Example 6

Add 20 ml of PBSA to an Amicon mixed cell (30 K Mwt cut-off membrane). Add 100 µl of the lipid mix plus 25 µl of a 10% solution of Quil A. The above solution is concentrated to 5 ml and resuspended with 100 ml of PBSA. The above phase is repeated x2. The final contents of 20 ml cells are sterilized by filtration through a 0.2 µm filter, labeled and stored at +4°C.

In electron microscopy studies, iscoma matrices appear to be less numerous than in other examples.

Example 7: Study on mice: Efficacy

Female mice of the inbred species (Balb/c) of the same age (8 weeks) are used. Mice were obtained from the Harlan-Olac maintained behind partition (SPF) unit (Bicester). The Mycoplasma hyopneumoniae compound from the National Cell Type Collection was used in this study. The organism is cultivated in a modified Friis medium. Stored cultures are maintained at -70°C each suspension for incorporating the vaccine is made by inoculating 40 ml of broth with 4 ml of the stored culture at pH 7.4 and then incubating at 37°C for 3 days or until the pH drops to 6.8. By this time, the organism has reached the log phase of growth. Cultures are inactivated using binary ethyleneimine, concentrated using tangential flow ultrafiltration, and washed with phosphate buffered saline (PBS). The antigen concentration is adjusted so that all vaccines contain 10 µg of total protein per dose of 0.1 ml of mouse vaccine.

Iscom preparations from examples 3 to 6 were used as follows.

Eight groups of six mice are immunized on two occasions, and each mouse receives 0.1 ml. the following preparations.

Group to vaccinate

And only 10 µg of M. hyopneumoniae

B 10 µg M. hyopneumoniae supplemented with Freund's kit

C 10 µg M. hyopneumoniae with iscoma matrix containing 10 µg QA mouse in dose

D 10 µg M. hyopneumoniae with iscoma matrix containing 5 µg QA mouse in dose

E 10 µg M. Hyopnemoniae with iscoma matrix containing 2.5 µg QA mouse in dose

F 10 µg M. hyopneumoniae with iscoma matrix containing 1.25 µg QA mouse in dose

G 0 µg M. hyopneumoniae with iscoma matrix containing 5 µg QA mouse in dose

H 10 µg M. hyopneumoniae + 107 cfu H. pleurpneumoniae with iscoma matrix containing 5 µg QA mouse in dose

QA=Quil A

Groups of mice are prebled through the tail vein before primary immunization and then 14 days later. 28 days after the primary immunization, the animals received a further vaccination and were bled 7 days later and finally 28 days after the secondary vaccination. Serum was made from blood on the day of collection and was stored at -70°C for analysis by ELISA. Mice were observed after vaccination for any adverse local and systemic reactions.

the results

The results of the experiments planned to evaluate the adjuvant effect of Quil A in the iscomma preparation, where the antigen is external to the preparation, are shown below in Tables 1 and 2.

The results clearly demonstrate that the ISCOM preparation with the highest protein Quil A ratio of 10 µg:10 µg/mouse dose, produces the highest vaccine response, giving as good a response, if not better, than the Freund's adjuvant vaccine.

Groups of mice that received iscom vaccines containing less than 10 µg of Qull A per mouse dose were no better, in producing a response to the antigen, than the blank antigen alone. No local or systemic reactions to the vaccine were observed in any mice that received iscom vaccine or antigen alone. In mice immunized with Freund, small granulomas of approximately 3 mm in size were observed at the point of immunization, but as with all other mice, no signs of systemic disorders were observed.

Table 1: Anti M. hyopneumoniae in mice as measured by the indirect ELSA assay

[image]

Standard deviations of DP2°C - days after secondary vaccination are omitted.

Example 8: Studies in mice: Evaluation of blank iscom matrix as an adjuvant for Hemophilius pleuropneumoniae vaccines in mice.

Mice are immunized with vaccines containing whole inactivated H. pleuropneumoniae bacteria and an iscom matrix formulated with different levels of Quil A. Mice immunized twice with a vaccine containing 10 µg of Quil A/mouse gave a greater antibody response. from mice immunized with equivalent numbers of bacteria in Freund's adjuvant. Lower doses of Quil A had a visible auxiliary effect. No adverse local or systemic effects have been observed following administration of any vaccine containing iscoma matrix.

NCTC reference strain H. pleuropneumoniea serotype 3 (strain 1421) was cultured to log phase growth in yeast extract medium supplemented with B NAD. The culture is inactivated with 0.2% formalin and washed three times with phosphate buffered saline (PBS) so that a washed preparation of the inactivated antigen from the whole cell is obtained. Antigen is standardized by bacterial enumeration using a Neubaumer chamber with modified Thoma rules and stored in PBS at 4°C until use.

Groups of mice, six per group, are immunized with H. pleuropneumoniae alone, with H. pleuromoniae in FIA or H. pleuropneumoniae or H. pleuropneumoniae together with iscorna matrix containing different levels of Quil A, as detailed below. The final group received both H. pleuropneumoniae and H. hypopneumoniae antigens together with the iscoma matrix to investigate a possible interaction between the two antigens. Mice received two subcutaneous injections of 0.1 ml of the respective vaccine on two occasions with a 3-week interval between immunizations.

Immunization of mice

Group Vaccination

1 nothing

2 only Hpl

3 Hpl in FIA

4 Hpl with iscom matrix containing 10 µg QA/per mouse at vaccine dose

5 Hpl with iscom matrix containing 5 µg QA/per mouse at the vaccine dose

6 Hpl with isca matrix containing 2.5 µ gQA/per mouse in the vaccine dose

7 Hpl with iscom matrix containing 1.2 µg QA/per mouse at the vaccine dose

8 Hpl with iscom matrix containing 5 µg Q A / per mouse in the dose of vaccine and 10 µg / per mouse in the dose of Mycoplasma hyopneumoniae antigen.

After primary vaccination, there were few antibody reactions in any group. After the secondary vaccination, the rapid and pronounced increase in antibody levels disappeared in 2 of the 8 immunized groups. The largest response was seen in mice immunized with the bacteria together with isca matrix containing 10 µg/mouse Quil A which had an average peak of 1.24 (± 0.17). Unit of optical density; (UOD ) when measured 1 week after secondary vaccination. Mice immunized with bacteria and FIA had a similar but slightly lower antibody response with an average peak of 1.11 (± 0.08) UOD on the same occasion. Mice immunized with bacteria alone, bacteria with lower doses of Quil A, or bacteria together with M. hyopneumoniae antigen all had similar, lower, antibody responses with an average value between 0.43 and 0.63 UOD when measured one month after the secondary immunization .

Table 2: Anti-H. pleuropneumoniae reaction, as measured by the indirect ELSA test

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Example 9: Study on pigs

30 3-week-old landrace-cross piglets were obtained from a herd known to be free of all clinical and serological evidence of infection with H. pleuropneumoniae and M. Hyopneumoniae. Piglets are kept in isolation from the rest of the herd and are fed ad libitum and standard weanling feed.

A low passage (less than 10) field isoiate (SVA3) of M. hyopneumoniae is cultured to the log phase of growth in Friis medium.

400 µl of 10% Quil A solution and 1600 µl of lipid mix are added to 20 ml of sterile PBSA. The mixture is then vortexed for 20 seconds at maximum speed and then filtered through a 0.45 µm filter. To 10 ml of this ISCOM mix, 10 ml of 4 mg/ml M. hyopneumoniae antigen is added so that an ISCOM mix containing 2 mg/ml antigen and 1 mg/ml Quil A is obtained.

A group of 5 piglets is immunized with 1 ml of ISCOM matrix containing 2 mg/ml of M. Hyopneurnoniae whole broth antigen. Another 5 received 1 ml of 2 mg/ml M. hyopneumoniae with Freund's adjuvant. The final 5 piglets received only PBSA.

Piglets received their first vaccination at 3 weeks of age by deep intramuscular injection of 1 ml of the appropriate vaccine into the deep muscles of the left side of the neck. A second similar injection was given 6 weeks later in the right side of the neck. 2 weeks after the second immunization, when they were 11 weeks old, the piglets were experimentally infected. Piglets were killed approximately 2 weeks after infection for postmortem examination of lung pathology.

a) Clinical reactions to vaccination

No adverse clinical signs were observed in any piglets after primary or secondary vaccination. There were no significant increases in temperature after primary or secondary immunization in any group. The highest temperature increase of 1.1 ± 0.23°C was observed in piglets immunized with M. hyopneumoniae antigen in Freund's incomplete adjuvant after secondary immunization. Basically, the average temperature in the group of piglets rose by approximately 0.3 °C, which was insufficient to cause clinical signs of pyrexia. Vaccines that contained M. hyopneumoniae were not significantly more pyrogenic than those that did not.

No local reactions were visible nor could the injection sites of any vaccine be palpated at any time examined.

There is no difference between weight gain in piglets.

b) Clinical reactions to vaccines

Clinical reactions accompanying the injections were not visible. The experimental infection did not cause pyrexia, the piglets remained clinically normal 2 weeks after the infection. Weight gain during this period and food intake did not show significant differences between groups.

c) Measurement of antibodies in serum

After vaccination, only the group that received M. hyopneumoniae with Freund's adjuvant demonstrated an antibody response. However, after the second immunization, the pronounced increase in antibody levels disappeared in all animals that received the M. hyopneumoniae vaccine. Serum M. hyopneumoniae antibody responses were measured by ELSA OD (at 492 nm) before challenge and were as follows:

for animals that received ISCOM vaccine 0.6±0.156

for animals that received a vaccine with Freund's agent 1.7±0.089

An anti M. hyopneumoniae antibody reaction (OD at 492 nm= 0.6±0.156) was also observed in animals that received the ISCOM matrix vaccine.

There was no further significant increase in antibodies in any infection group.

d) Local reactions to vaccination

The strongest reactions were observed in piglets immunized with M. hyopneumoniae antigen in Freund's adjunct. The reactions consisted of multiple dispersed causative granulomas and were characteristic reactions to Freud's adjuvant in piglets. No damage was observed on the piglets that received the ISCOM vaccine.

Example 10: Study on sheep

Blackface x Swledale ewes were obtained from a farm known to have been free of OEA for at least 4 years. 5-6 year old animals were previously tested for antibodies to Chlamydia psittaci and Toxoplasma gondii before use. Sheep are kept in isolation from other herds and are fed hay and concentrates at appropriate levels for their physiological and nutritional status.

1. Vaccines

OEA strains A22 and S26/3 Chlamydia psittaci are cultured and later semi-purified. The initial culture volume for each 10L layer is concentrated to 1L in this procedure so that 10X Antigen is obtained.

Iscom vaccine was made as follows (IV): 50 m1 of 10X antigen is treated with 100 µl of stock (50%) glutaraldehyde for 15 min at 4°C. (b) 50 ml of 0.15 M glycerin sterilized on a PBS filter is added and the suspension is incubated for 5 min at 4°C. (c) The suspension is centrifuged at 17,000 g for 45 min at 4°C, then the granule is washed twice with sterilized PBS pH 7.2. (d) The pellet is resuspended in 50 ml of PBS. (e) Add thiomersal (1%; sterilized by filtration), 0.5 ml, so that a final concentration of 0.1% is obtained.

50 µl of 10% Quil A solution and 2 ml of lipid mix are added to 100 ml of sterilized PBSA. The mixture is mixed under a pressure of 62 kN.m2 and concentrated to a volume of 20 ml. Sterile PBSA is added to make the solution up to 100 ml and the volume is reduced to 20 ml. This is repeated twice. The final volume is filtered through a 0.45 µm filter.

The antigen preparation is added to the Iscom mix so that the antigen concentration is 0.25 X antigen/ml.

One oil-based vaccine (OBV) and one aqueous vaccine with adsorbed Alhydrogel (AAV) are prepared as controls so that 1X antigen/ml and 0.25 X antigen/ml are obtained.

The experiment included 5 groups and a total of 106 sheep. Groups 1, 2 and 3 were vaccinated with OBV, AAV and IV. Groups 4 and 5 were controls, and were infected/unvaccinated and uninfected/unvaccinated, respectively.

The OBV vaccine was given once daily as a dose of 1 m1 given 9 weeks before maturation. The other vaccines were given twice, 9 weeks and 3 weeks before maturation, and each injection comprised a dose of 2 ml (all vaccines delivered an amount of 4 µg of Chlamydial protein).

All vaccines were injected subcutaneously at an identified exposed site on the right side of the neck.

Sheep from groups 1, 2, 3 and 4 are infected with live chlamydia organisms at 85-90 d.g. Group 5 remained untreated.

During lambing or abortion, placentas were taken and, if accessible, for sampling. The state of the lamb at this time was recorded (alive, stillborn or dead). No attempt was made to keep sick lambs alive, other than providing colostrum.

The presence or other OEA damage in the placentas was noted and their content was evaluated. Placentas (or vaginal swabs) were examined for chlamydial bodies by staining using the Ziehl-Neelsen method, and were cultured in BHK21 cells treated with 5-iododeoxyridine.

the results

The results showed that the ISCOM vaccine provided protection against Chlamydial infection that was at least as old as that provided by the conventional vaccines used in the experiment.

Example 11: Iscom preparation for equine influenza virus

Protocol: Place 20 ml of PBSA in a sterile universal flask. Add 100 µl of 10% Quil A solution. Add 400 µg of lipid mix. Add ERIK-14 antigenic component of equine influenza at a final concentration of 5 µg/ml. Vortex at full speed for 20 seconds. Sterilize the solution by passing it through a 0.2 µm filter (Millipore). Decant a small volume for. electron microscopy studies (observed by iscom structures) and verification of bacterial contamination. Store at +4°C.

Claims (19)

1. A process for making a vaccine for human or animal use, characterized in that it comprises (1) formation of a matrix from glycosides and sterols and (2) mixing said matrix with an antigen accompanying a bacterium or mycoplasma. 2. The method according to Claim 1, characterized in that in phase (1) a phospholipid is included. 3. The method according to Claim 2, characterized in that the phospholipid is phosphatidylcholine. 4. The method according to any one of Claims 1 to 3, characterized in that the glycoside is Quil A. 5. The method according to any one of Claims 1 to 4, characterized in that the strol is cholestrol. 6. The method according to any of Claims 1 to 5, characterized in that the antigen follows one of the following types of genera or classes of organisms: Mycobacterium, Clostridium, Rickettsia, Spirochaetes, Escherichia, Staphylococci, Haemophilus, Bordetella, Vibrio, Salmonella, Streptococci, Pasteurella , Legionella, Chlamydia, Pseudomonas, Actinobacillus, Campylobacter, Listeria, Mycoplasma bovis, M. gallisepticum, M. agalactiae, M. hyopmneumoniae, M. synoviae, M. arthrilditis, M. capricolium, M. dispar, M. hominis, M. Mycoides subs. capri, M. orale, M. oripneumoniae, M. pulmonis, M. cynos, M„ hyorhinis, M. mycoides, M. salvarium, M. fermentas or the antigen comprises the inherence factor in Colli, porin protein or outer membrane proteins from B. pertussi or Neisseria meningitidis. 7. The method according to any of the preceding Claims, characterized in that phase (1) comprises the formation of a matrix from glycosides, sterols, phospholipids and solubilizing agents. 8. The method according to any of the preceding Claims, characterized in that phase (1) comprises the formation of a matrix from strol glycosides and solubilizing agents selected from octylglycosides, nonyl-N-methylglucamide, carbamide and guandine. 9. The method according to any of the preceding Claims, characterized in that the sterol used in phase (1) is different from cholesterol. 10. The method according to Claim 9, characterized in that the sterol is lanosterol, luminsterol, stigmasterol or sitosterol. 11. The method according to Claim 8, 9 or 10, characterized in that phase (1) comprises the formation of a matrix of glycosides, sterols and phospholipids. 12. The method according to any of the preceding claims, characterized in that the antigen is associated with Mycoplasma hyopneumoniae. 13. The method according to any of the preceding claims, characterized in that the solvent is used in phase (1) and is not separated before the formation of the iscorn matrix. 14. A method for making an immunogenic complex comprising a water-insoluble antigen, indicated by the fact that it comprises dissolving the antigen with a solubilizing agent, mixing the dissolved antigen, glycosides and sterols and forming an iscom essentially without separating the solubilizing agents. 15. The method according to Claim 17, characterized in that phospholipid is additionally used in the mentioned mixing phase. 16. The method according to Claim 15, characterized in that iscomas are formed differently than during ultrafiltration, dialysis, ultracentrifugation or chromatographic procedures. 17. The method according to any one of Claims 14 to 16, characterized in that the solvent is sodium deoxycholate, octylglycoside, nonyl-N-methylglucamide, decanolyl-N-methylglucamide or polyethyleneglycol p-isooctyl-phenylether having 9 to 10 oxyethylene group. 18. A process for making an iscoma matrix, characterized in that it comprises mixing glycosides, sterols and solubilizing agents and forming an iscoma matrix essentially without separating the solubilizing agents. 19. The method according to any of the preceding Claims, characterized in that the vaccine is for use in pigs or sheep.