JPH1025250A - Method for improving yield of immune antibody in preventive vaccination of animal and man - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the enhancement of immune response to vaccine for domestic animal and man by using a specific adjuvant. SOLUTION: The adjuvant to be used in this process contains a glycopeptide GMDP (N-acetyl-glucosaminyl-N-acetylmuramyl-L-alanyl-Disoglutamine) and a colloidal solution of a solid lipid substance. The solid lipid substance is a biodecomposable substance containing a lipid having a melting point of >=37 deg.C. Examples of the substance are triglceride, lecithin, D,L-α-tocopheryl acetate, etc. A colloidal solution stable in the presence of emulsifier and dispersing agent is formed from the substance. The colloidal solution contains solid liquid particles having diameter of <0.2μm and is asepticized by filtration. The administration dose of glycopeptide is 10μg/kg-body weight. The adjuvant is effective for suppressing the formation of granuloma of gout, man, etc., by the synergistic interaction of GMDP and lipid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to immunological techniques, and more particularly to techniques for enhancing the natural response of livestock and humans to vaccines with adjuvants.

[0002]

BACKGROUND OF THE INVENTION Vaccination against infectious diseases results in the formation of antibodies in the host organism that are sufficient for sustained or lifetime protection. In the past, this has been satisfactorily achieved simply by conferring the antigen in the form of an inactivated pathogen, and it has also been relatively difficult to produce commercial quantities of pathogenic bacteria or viruses for vaccines. It was easy. However, in the last decade, the emergence of viral infections such as hepatitis B and HIV, where mass production of antigens is not possible, has been seen. One must resort to genetically engineered viral subunits that are much less immunogenic than whole pathogens and require repetitive applications. They are expensive and do not allow for the extremely necessary mass vaccination. Adjuvant
t), once a laboratory item, has become a goal of great medical and economic importance. Despite great efforts, truly efficient adjuvants that are recognized as modern medical and veterinary preventives have not become available.

[0003] Jules Freund, 1948
(J. Immunol. 60, 383-98) introduced the idea of an adjuvant and described its efficient realization. There is Freund's Incomplete Adjuvant (FIA), a mixture of mineral oil and commercial emulsifiers. To use it, add the same amount of aqueous antigen solution and repeatedly pass the mixture through the injection needle until a cream-like product that floats on the surface of the water behaves like an aqueous emulsion in oil Need to be done. Injection with the emulsion results in a dramatically stronger immune response than the antigen alone. M. tuberculosis or M. tuberculosis The addition of killed Mycobacterium, such as M. butyricum, to the emulsion further increases immunogenicity. This composition is called Freund's Complete Adjuvant (FCA) and is considered ideal in view of its remarkable and general efficiency. However, Freund's emulsion causes non-neoplastic and inflammatory nodules (granulomas) at the site of injection, which tends to rupture into purulent ulcers, and their use in prophylactic vaccines is a modern standard. Is not a problem at all. The only adjuvant currently approved for the human body is a fine suspension of aluminum hydroxide, the source of which is much older than Freund's adjuvant (AT Glenny).
et al. , J. et al. Pathol. 29, 31-40
(1926)). It is based on the hypothesis that when adsorbed on aluminum hydroxide, the antigen is recognized by the immune system somewhat effectively. Aluminum hydroxide is much less effective than Freund's adjuvant and causes granulomas.

The present inventor has reported that the biodegradable GMDP glycopeptide (N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-glutamine) is superior in mice even in the absence of any oil emulsion. Revealed that it was an adjuvant. The dose is 1 μg G
MDP / mouse only (US patent application Ser. No. 08/54)
2,643).

[0005] Surprisingly, in large animals such as rabbits, GMDP alone does not perform as well as FCA. In this case, obviously mineral oil is required, and the inventor has shown that dimethyldioctadecyl ammonium chloride (DDA) is a very effective alternative. Completely insoluble DDA provides zinc in the vial giving visible:
When present in the form of an intimate solid mixture with the L-proline complex, it could be dispersed and zinc was considered to be a cofactor. DDA with a size of 1-10 μm
Particles formed.

This formulation is at least as efficient as FCA and does not cause any side effects (NG
rubfer, Immunology Lette
rs, 44, 19-24 (1995). U.S. patent application Ser. No. 08 / 504,409). The lipid dose for rabbits is 100 μg, very low when compared to 100 mg oil per rabbit in Freund's adjuvant.

However, in goats, attainable antibody titers could not be measured up to FCA.

[0008]

It is an object of the present invention to propose an adjuvant formulation suitable for large animals and humans. This type of improved formulation must have the potential to be approved as an animal drug and injection, in addition to enhancing the immunogenic effect of the antigen (vaccine) to the level obtained with Freund's adjuvant.

[0009]

[Means for Solving the Problems]

Best Practices for Practicing the Invention Good results with Freund's adjuvant in large animals are most likely to be related to oversized doses of oil emulsions. 2.5 ml of Freund's standard emulsion
Considering the case of injection, goats receive more than 1 g of mineral oil. Freund himself has already shown that eliminating the fixed deposition of the oil emulsion after 2 hours does not significantly impair the immunostimulatory effect (1948, cited above). This means that the emulsion plugs are useless in practice, and the effect is that oils that can leak and penetrate quite quickly from the host organism's lymphatic system, possibly in the form of microdroplets, This is due to a small part. At the same time, more antigen becomes available that would otherwise be buried in the bulk of the emulsion.

[0010] This can in fact indicate that it is very possible. That is, Freund's emulsion in contact with a medium such as bovine serum, upon gentle agitation, rapidly breaks down into ultrafilterable droplets, and the presence of such droplets or small particles indicates the effect of Freund's adjuvant. (See Example 1).

[0011] Evolving research has therefore focused on improving the lipid portion of the adjuvant. The experiments were performed with sheep and goats and the results obtained at each stage of the study are summarized in Table 1.

Table 1 shows that the adjuvant effect and antibody titer of various formulations containing a lipid colloid solution containing no GMDP are set in relation to the titer of Freund's incomplete adjuvant (FIA). Antigen: 100 μg BSA. Animals: G: goat, S: sheep.

[0013]

[Table 1]

A _rel : Antibody titer on a given day divided by antigen titer using FIA in # 15. Materials and methods are described in Examples 1-3. The liquid contains 30 g dextran and 10 g L-proline per liter as solubilizers. The lipid is present as a colloidal solution containing particles or droplets smaller than 0.2 μm obtained by mechanical microhomogenization according to Example 3. Lipid dose: 50 mg / animal. Antigen: 100 μg BSA. Immunization: Example 6.

The data presented in Table 1 is summarized and interpreted as follows. 1. Microparticles or microdroplets generally work with an adjuvant, regardless of chemical structure. You are therefore free to choose the most appropriate one from various other perspectives.

2. Lipids are best prepared at about 50-
It is administered at 100 mg (〜1 mg / kg). This has to do with the fact that the dose must be immediately available to the host due to rapid clearance from the injection site. Particles (nanoparticles) with a size of less than 0.2 μm ensure that mobility is not impaired, with the added advantage that they can be sterilized by ultrafiltration.

3. Oils that are easily metabolized are much less effective than solid fats (# 6 vs. # 7). This can mean that they are hydrolyzed too quickly.

4. Solid lipids are more effective (# 9 vs. # 8 or # 10) because of their longevity in the host, thereby reaching their target, possibly macrophages, and staying there for a period of time Opportunities are given. It is important that the stability of the solution is significantly improved (Example 5).

[0019]

DETAILED DESCRIPTION OF THE INVENTION These data establish guidelines for further developmental research and represent the essence of the claims of the present invention. Subsequent immunization studies with goats yielded the results shown in Table 2. They are the object of the present invention, namely adjuvant formulations, which are as effective as FCA in large animals, but without side effects and are qualified as adjuvants for human vaccines,
Has been achieved.

Table 2 shows the adjuvant effect of various formulations containing a lipid colloid solution in combination with GMDP.
Antibody titers were determined using Freund's incomplete adjuvant (FIA).
It is set in relation to the titer. Animal: Goat. Antigen: Hepatitis B surface antigen: 20 μg / goat.

[0021]

[Table 2]

A _rel : antibody titer on day 63 divided by antigen titer using FCA in # 33. * D-T80 = Twe
en80 / sodium deoxycholate 1/1. Na
D = sodium deoxycholate. Hoe S403
9: di (stearoylhydroxyethyl) hydroxyethylmethylammonium chloride prepared according to Example 4. Materials and Methods: Example 3. Immunization: See Example 7.

The data is also shown in FIG. In FIG. 1, the horizontal axis corresponds to the vertical axis in Table 2, and the vertical axis is the antibody titer% of FCA.

The contents of Table 2 are summarized and interpreted as follows. 1. The same efficiency level as Freund's complete adjuvant is
This was achieved by an adjuvant formulation containing GMDP and a lipid compound present in the form of a colloidal solution capable of ultrafiltration.

2. A synergistic interaction between GMDP and lipid was further confirmed.

3. Lipids are triglycerides, lecithin,
Biodegradable natural compounds or mixtures thereof that are solid at body temperature, such as cholesterol or tocopheryl acetate.

4. Lipids are dispersed with a water-blast microhomogenizer to form a pharmaceutically acceptable emulsifier such as sodium deoxycholate or Tween 80 and dextran, polyvinylpyrrolidone and L
-Forming a stable colloidal solution in the presence of a dispersant such as proline. The resulting colloid solution contains solid lipid particles with a diameter of less than 0.2 μm and can be sterilized by filtration, ie, sterilized by filtration.

5. Quaternary ammonium salts, especially DDA
Hoe S4039, a biodegradable analog of DDA called (dimethyldioctadecyl ammonium chloride) or esterquat, also has value as an adjuvant, and both compounds are of great industrial importance as soft rinses for cleaners. And has non-toxic and abundant toxicological data.

6. A number of alternative formulations can be utilized for further optimization. esterqua
Because t is non-toxic and biodegradable, it is particularly promising as a very potent adjuvant lipid for veterinary vaccines.

[7] All goats were physically controlled and showed no signs of illness or granuloma formation at the injection site.

8. Human studies show no erythema, sensitivity or granuloma at the injection site.

[0032]

【Example】

Example 1 Demonstration of Microdroplets Obtained from Freund's Adjuvant Emulsion A regular FIA emulsion was mixed with 2 ml of FIA (Difc
o Laboratories, Detroit M
i. # 0639-60) and 2 ml of saline, double-hub needle (# 7979 fine emulsification needle 18xl)
-7/8, Popper & Sons New Hy
de Park NY 11040) for 20 double strokes. The obtained plug was subjected to ultrafiltration until clear to 100 m at 37 ° C.
1 fetal bovine serum. Upon slow magnetic stirring, most of the floating plugs disappeared into the aqueous phase and the aqueous phase became cloudy. This turbidity easily passed through the 0.2 μm filtration membrane.

Example 2 Preparation of a lipid colloid solution using a water spray homogenizer 20 recrystallized twice from acetone (20 g / liter)
gDA (Genamin SC Hoechst)
AG, D-65290 Frankfurt), 60
C. into a solution of 30 g dextran 40000 and 10 g L-proline warmed to <RTIgt; C </ RTI> and stirred with a household blender until homogeneous. The obtained emulsion was used for APV Gaulin Micron Lab.
40 Homogenizer (APV Gauli
n, D-23519 Lubeck, Ger. ) And pass through a 0.2 μm filtration membrane until 1000 μm
The cycle was repeated at bar (16000 psi).

Example 3 Preparation of a Colloidal Solution Used at Home 3.1 Using the colloidal solution of DDA containing 20 mg / ml prepared in Example 2, 2.5 m / goat
1 was injected.

3.2 Hoechst S4039,
[Di (stearoylhydroxyethyl) hydroxyethylmethylammonium chloride] (Hoechst A
G, D-69520 Frankfurt) in Example 4.
Purified as described in and used in the form of a thick suspension.
The dextran-L-proline solution was replenished at high concentration to reach the standard concentration after adding the required amount of esterquat suspension. The lipid was processed as in Example 2. Only a few cycles with an APV machine at 1000 bar were required to pass through a 0.2 μm membrane.

3.3 Lecithin from eggs, DAB 8
(Lipoid E 89, Lipoid GmbH
85 parts of Ludwigshafen) and Tween
n 80, 15 parts were processed as in Example 2.

3.4 DL-α-tocopheryl acetate, 85 parts of DAB 10 and 1 of Tween 80
Five parts were processed as in Example 2.

3.5 Lecithin / DL-α-tocopheryl acetate 1/1 was dissolved in absolute ethanol,
The ethanol was evaporated. The waxy mass obtained is about 4
Processed as in Example 2 after beginning to melt at 5 ° C. and adding 15 parts Tween 80.

3.6 Trioctanoin 99%, (S
Sigma T 9001 SigmaChem. C
o. , St. Louis Mo. 20 g) and Tw
3 g of een80 were processed as in Example 2. In this case, it was necessary to cool the emulsion immediately after leaving the homogenizer in order to prevent creaming.

3.7 Tridodecaninoin, "hard fat" (Witepsol E 85, Huls AG.
US-P XX and D for D-45764, Marl)
20 g of commercially available cocoglyceride and 2 g of sodium deoxycholate according to the AB 10 monograph were processed as in Example 2. Cooling was required as in Example 3.6.

3.8 Paraffin oil, "dunplus"
sig "DAB 10 (E. Merck D-6427)
1 Darmstadt) 20g and Tween
3 g of 80 were processed as in Example 2.

3.9 paraffin (curing point 54-56,
E. FIG. 20g of Merck DAB 10) and Twe
3 g of en 80 were processed as in Example 2. Cooling was required as in Example 3.6.

3.10 Silicone oil DC200 (5
Centi-stroke, Mr 770, Fluka # 85
411) was processed as in sub 3.8 and Example 2.

3.11 Melt 90 parts of trilaurin, in which 10 parts of cholesterol (Fluka # 26740 from lanolin) are dissolved by magnetic stirring and processed as in Example 2 to give a 5% solution Obtained.

3.12 To 1 ml of lecithin / D, L-α-tocopheryl acetate colloid solution prepared according to Example 3.7, add 0.05 ml of Alhydrogel.
l (registered trademark) (Superphos Biosect)
or A / S) was added. 1.05 ml of this mixture
Is used in a single injection with 100 μg of BSA.

3.13 Blockpolymer L
121, (Syperonic L121 C.H.
Erbsloh Dusseldorf, German
y) was processed as in Example 2.

3.14 Polysciences I
Polystyrene, microspheres 0.1 μm obtained from nc Warrington PaUSA was dispersed and processed as in Example 2.

3.15 Incomplete Freund's adjuvant was prepared according to Example 1 using 2.5 ml of FIA and 2.5 ml of phosphate buffered saline containing 100 μg BSA for one sheep or goat. did.

3.16 FCA # 0639 (Difc
o Labs. Complete Freund's adjuvant prepared according to Example 1 was processed as in Example 3.15 using Detroit Mi).

3.17 Aluminum hydroxide, Alhy
drogel (registered trademark) Superphos Bio
sector A / S was used. 10 ml of a commercial 2% suspension for associating the alumina with the protein
With 2 mg of BSA (# 3265-00, Interg
en Comp. Purchase, NY 10577
1) and incubated at room temperature (Erik
B, Lindblad, Aluminum Adjuvant (Alumini) in Adjuvant Theory and Practical Applications
um Adjuvants in the Theor
y and Practical Applicati
on of Adjuvants) pp. 21-35, 1
995, John Willer & Sons Ltd
d D. E. FIG. S. , Studard-Tull). For injection, aluminum hydroxide containing 1 mg aluminum hydroxide and 100 μg BSA-
0.05 ml of the BSA suspension was diluted to 5 ml.

3.28 Lecithin and tocopheryl acetate were melted as in Example 3.5 and processed as in Example 2 to a concentration of 50 mg lipid / ml. GMD
P was added to 0.3 μg / ml.

3.29 Same as # 3.28, but G
No MDP was added.

3.31 As in Example 3.2. 4
0.12 μg in a colloid solution adjusted to 0 mg / ml
Of GMDP / ml and used 2.5 ml per goat immunization.

3.32 As per 3.28: 50 m
Add 2 ml of colloid solution containing g lipid / ml
0.05 ml of aluminum hydroxide suspension was added per reaction.

3.33 FCA was processed as in Example 3.16.

3.35 90 parts of trilaurin + 10 parts of cholesterol were processed as in Example 3.11, and G
MDP was added to 0.12 μg / ml.

Example 4 Purification of esterquat Esterquat S 4039 [di (stearoylhydroxyethyl) hydroxyethylmethylammonium chloride] (Hoechst AG Frankfu)
rt) is available as a commercial viscous liquid containing metsulfate anion and 15% isopropanol. The crude material was purified by adding 230 g to 0.8 liter of hot saline (20% NaCl) in a household blender and gradually homogenizing. Lipids surface,
The lower liquid was siphoned off. Washing with 20% saline was repeated 5 times. The floating cake was transferred into a dialysis tube, crushed, and dialyzed until no trace of chloride was detected in the external solution. An aliquot is evaporated to give a soft yellow wax, mp 40-45 ° C., which is weighed to determine the lipid content in the main emulsion. Analysis of the chlorine, sulfur and nitrogen contents in the wax samples was performed and the results showed a minimum 98% exchange of the corresponding ion of metsulfate to chloride, the nitrogen content being reasonably consistent with the chlorine of the distearoyl compound. The bulk of the 12.5% emulsion was stored for use in adjuvant experiments.

Example 5 Stability of microemulsion compared to stability of microsuspension 3.8 ml sample of colloid solution containing liquid paraffin and 3.9 ml of solution containing solid paraffin
Were stored at 45 ° C. for 2 weeks. Another set of samples was frozen overnight and left at room temperature during the day for 12 repetition cycles. The solution of liquid paraffin began to cream after several days under both treatments and could no longer pass through the 0.2 μm membrane. The solution of solid paraffin did not change.

Example 6 Immunization of sheep 20 mg lipid and 150 μg GMDP per animal
And BS in water to give 100 μg BSA
A (# 3265-00, Intergen Comp.
Purchase, NY 10577) was supplemented with the colloid liquid solution and GMDP. Subcutaneous injections were made at sites 4-6. Booster injections of adjuvant and antigen were given 14 and 28 days later. Sheep were bled on day 63 and antibody titers were assayed by ELISA.

Example 7 Goat Immunization Instead of BSA, hepatitis B surface antigen (Intenat)
ionic Enzyme Inc. Fallbrook
k Calif. ) Was replaced. All goats are 50μ
g of antigen and 300 μg of GMDP were received and, except for experiment # 29, the total injection volume was 5 ml, which was dispersed by 6 subcutaneous injections. The immunization schedule is the same as in Experiment 6. Goat testing is available from Ramona, Cal.
ifnia 92065 USA HTI Bio
service Inc. I went in.

[0061]

EFFECT OF THE INVENTION A method for increasing the immune response of livestock and humans to the vaccine was established.

[Brief description of the drawings]

FIG. 1 shows anti-hepatitis titers in goats.

Claims (34)

[Claims] 1. The method of claim 1, wherein the injection of the glycopeptide G
Enhance animal and human immune responses by adding optimal doses of MDP (N-acetyl-glucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine) and solid lipid substances to the antigen solution to the antigen solution How to let. 2. The particle diameter of the colloidal solution is 0.2 μm.
2. The method of claim 1 wherein the method is less than. 3. The method according to claim 1, wherein the melting point of the lipid exceeds 37 ° C.
3. The method according to any one of 2 above. 4. The method according to claim 1, wherein the solid lipid is a biodegradable substance. 5. The solid lipid comprising lecithin and D, L-α-
The method according to any one of claims 1 to 3, wherein the method is tocopheryl acetate. 6. The method according to claim 1, wherein the solid lipid is a triglyceride. 7. The method according to claim 1, wherein the lipid is octadecane.
The method according to any one of the preceding claims. 8. The method according to claim 1, wherein the solid lipid is cholesterol. 9. The method according to claim 1, wherein the lipid is a quaternary ammonium compound. 10. The method according to claim 1, wherein the ammonium compound is dimethyldioctadecyl ammonium chloride. 11. The method according to claim 1, wherein the ammonium compound is dimethyl distearoyl hydroxyethyl ammonium chloride. 12. The process as claimed in claim 1, wherein sodium deoxycholate is used as the emulsifier. 13. The method according to claim 1, wherein sodium cholate is used as an emulsifier. 14. The method according to claim 1, wherein aluminum hydroxide is included as a synergist. 15. The method according to claim 1, wherein dextran is used to stabilize the lipid colloid solution. 16. The method according to claim 1, wherein polyvinylpyrrolidone is used to stabilize the lipid colloid solution. 17. The method according to any one of claims 1 to 16, wherein the glycopeptide is used at a dose of 10 μg / kg body weight, but may be reduced or omitted. 18. Glycopeptide GMDP (N-acetyl-glucosaminyl-N-acetylmuramyl-L-alanyl-) with a colloidal solution of a solid lipid substance in which the diameter of the particles in the colloidal solution is preferably less than 0.2 μm. D-
Isoglutamine), which enhances the immune response of animals and humans and is added to the antigen solution for injection into animals and humans. 19. The adjuvant of claim 18, wherein said solid lipid substance has a melting point above 37 ° C. 20. The adjuvant according to claim 18, wherein the solid lipid substance is a biodegradable substance. 21. The solid lipid substance comprising lecithin and D, L
The adjuvant according to any one of claims 18 to 20, which is a mixture with -α-tocopheryl acetate. 22. An adjuvant according to any one of claims 18 to 20, wherein the solid lipid substance is a triglyceride. 23. The adjuvant according to claim 18, wherein the solid lipid substance is octadecane. 24. An adjuvant according to any of claims 18 to 20, wherein said solid lipid substance is cholesterol. 25. The adjuvant according to claim 18, wherein the solid lipid substance is a quaternary ammonium compound. 26. The quaternary ammonium compound is dimethyl dioctadecyl ammonium chloride.
Adjuvant according to the above. 27. The adjuvant according to claim 25, wherein the ammonium compound is dimethyl distearoyl hydroxyethyl ammonium chloride. 28. The adjuvant according to claim 18, wherein sodium deoxycholate is used as emulsifier. 29. The adjuvant according to claim 18, wherein sodium cholate is used as an emulsifier. 30. The adjuvant according to claim 18, wherein aluminum hydroxide is included as a synergist. 31. An adjuvant according to any one of claims 18 to 30, wherein dextran is used to stabilize the lipid colloid solution. 32. An adjuvant according to claim 18, wherein polyvinylpyrrolidone is used to stabilize the lipid colloid solution. 33. The dose of the selected glycopeptide is 1
An adjuvant according to any one of claims 18 to 32, which reaches 0 μg / kg body weight. 34. The method of claim 1, wherein the glycopeptide is omitted.
The adjuvant according to any one of 8-32.