HU176180B - Process for preparing antigenic compositions - Google Patents

Abstract

A novel antigenic preparation which comprises a plurality of microvesicles each formed of at least one lipid bilayer upon the exterior surface of which is bound an antigenic protein derived from a pathogenic organism, such as a virus, bacteria or protozoa.

Description

The present invention relates to an antigen composition comprising a plurality of microspheres, each of which has at least one lipoid bilayer on the outer surface to which an antigenic protein from a pathogenic organism is attached, such that

a) a vigorous mixing of a lipoid, preferably natural or synthetic lecithin, in an aqueous medium, followed by further vigorous mixing in the presence of an antigenic protein, or

b) subjecting the mixture of a lipoid, preferably natural or synthetic lecithin, to the aqueous medium and the antigenic protein to vigorous mixing.

While many inactivated viruses, such as influenza viruses, are known to have strong immunogenic effects, they also have a febrile effect. Successful attempts have been made to eliminate the feverish effect by purifying the immunogenic portion of the virus, but the immunogenic activity of the resulting virus subunits is greatly reduced.

Therefore, an appropriate additive is required which can be added to these subunits and other antigenic proteins to help the vaccine host maintain its immune response.

The use of liposomes as carriers for drug agents and enzymes is receiving increasing attention as they are effective immunological additives. Liposomes are aqueous dispersions of concentric spheres consisting of phospholipoid bilayers separated by aqueous moieties. They are essentially bulbous, a structure which has already been described in the literature. They may be prepared by shaking a dried film layer of a phospholipoid such as lecithin in the presence of a buffer.

Previous publications on the use of liposomes have shown that the intermediate aqueous portions between the multilamellar lipid moiety contain embedded drug substances or antigens.

It has been found that antigenic proteins can be attached to the outer surface of the outer lipoid phase of liposomes and to the outer surface of a single lipid bilayer having a similar unicellular body, and that these compositions exhibit superior antigenic properties over non-bound proteins. In the case where the antigenic protein is a viral subunit, the orientation of this subunit, according to the electron microscope image, is likely to be similar to that of the viral particle.

Accordingly, the present invention provides, as explained above, a method for producing an antigenic composition comprising a number of microspheres of Al and at least one lipoid bilayer of each microvessel to which an antigenic protein derived from a pathogenic organism is attached.

The microspheres themselves may be liposomes or unicellular bodies consisting of a single lipid bilayer encapsulating an aqueous portion. Antigenic proteins from the same strain type, different types of the same strain, or different strains may be linked to the same body or a composition may contain mixtures thereof. The preferred size of the microspheres is from 4 to 100 nm, although smaller or larger microvessels, such as 20 to 200 nm, are included within the scope of the invention.

The antigenic protein can be derived from any pathogenic microorganism, such as protozoa, metazoa, and bacteria, especially viruses. Other suitable protective surface antigens are those from mixoviruses, such as influenza A, B and C viruses, Newcastle disease viruses, parainfluenza viruses types 1, 2, 3 and 4, and other viruses such as measles virus, mumps virus, cytomegalovirus, and other herpes viruses, picorna viruses such as foot and mouth disease virus, corona viruses and capsomers, poliomyelitis viruses, rhinoviruses, warts.

To the best of our knowledge, protein antigens are hydrophobically bound to microvessels, and if the antigen protein itself does not contain a hydrophobic moiety capable of or capable of being attached, such a hydrophobic moiety may be formed, e.g., by a hydrophobic with a reagent containing a group such as palmityl, stearyl, lauryl, polyallyanyl or other long aliphatic chains.

The microspheres may be made of any lipid material, preferably one which is biodegradable in itself and has no antigenic properties. Common materials such as natural or synthetic lecithins or other phospholipoids are very suitable for this purpose. However, other lipoids may also be used, namely cholesterol as a booster, preferably in a volume of <30 moles based on the total lipoid composition. Optionally, a third substance may be added to provide a positive or negative charge to the formulation. Negative charge agents include phosphatidic acid, bovine ganglioside, dicetylphosphate, phosphatidylserine and phosphatidylinositol. Examples of positively charged materials are stearylamine and other primary amines.

The microspheres may further contain, when embedded, another known additive known in the art.

The antigenic protein may be added to the lipoid before or after the formation of the microvessels, but preferably afterwards to avoid the possibility of the protein being embedded in the body rather than the outer surface.

Multilamellar microspheres (liposomes) can be produced by any known method. Preferably, the starting lipid substance (s) is dissolved in a solvent and the solvent is evaporated. The lipid phase is then either in aqueous sodium chloride solution or in a buffer solution (if the protein is to be added after the formation of the bladder) or in an aqueous suspension of the protein (if the protein is intended to be added to the dispersion and vigorously stir the resulting mixture. If the protein has not yet been added, we can do so now and the microspheres are subjected to vigorous mixing.

If one-cell microspheres are to be prepared, the initial mixing time is extended to reduce the number of liposomes present.

Alternatively, the starting lipid substance (s) is added to an aqueous phase and the mixture is slowly heated. Next, vigorous mixing is started to form the liposomes. The aqueous phase may contain the antigenic protein or be added thereafter.

Another method of preparing microspheres (liposomes) is to rapidly inject an aqueous solution of phospholipoid in aqueous sodium chloride solution or in a pre-purged buffer solution with nitrogen gas. The resulting liposome composition is then concentrated by ultrafiltration under low pressure, under nitrogen, under high pressure to avoid the formation of larger, non-heterogeneous liposomes. Ethanol can be removed from the liposome fraction by dialysis or ultrafiltration washing. The antigenic protein to be attached to the liposome may be present in the aqueous solution or the liposome fraction obtained after ultrafiltration may contain a slightly sonicated form.

The antigen formulations obtained above are aqueous dispersions of microvessels and can be converted to vaccines by soldering in sterile form in unit dose or multidose container. Additionally, stabilizers, preservatives, and other conventional vaccine excipients may be added to the formulations.

The vaccines thus obtained may be administered by standard methods of administration of the antigenic protein or proteinaceous vaccine. This usually involves nasal administration, intramuscular or subcutaneous injection, both in humans and in animals. The dose administered will, of course, depend on the nature of the antigen, the animal being treated (human), the vaccination program and the amount or amount of additives in the preparation, and of course the opinion of the attending physician (veterinarian).

A single dose of the vaccine may generally be administered in a single unit or in divided doses several times over a fixed period.

Accordingly, the following is within the scope of the present invention;

a method for preparing an antigen composition comprising a plurality of microvessels having an antigenic protein attached to its outer surface.

The following examples illustrate the process of the invention without limiting it.

Table I

Antigen Dose Number of mice Virus Lung Titration (10g ¹⁰ ) PR8 subunits 5 Mg First Second ip Third -4.21 4th . 5th 6th PR8 subunits 50 gg 7th ip 8th -3.39 9th 10th liposomes 11th (PR8 subunits) 12th -3.4 the liposome below 5 Mg 13th 10-fold dilution ip 14th prepared suspension 15th liposomes 16th (PR8 subunits) 50 gg 17th -1.76 0.5% lipoid ip 18th (incremental virus increment 19th none except 1 mouse 20th 1/8 of your lungs)

Example 1

A. Production of Influenza Virus Subunits

Highly purified [JJ Skehcle and GC Schield (1971) Virology 44: 396-408] PR8 influenza virus (12 mg viral protein / ml) is mixed with enough nonionic detergent (Nonidet NP40) to make the final detergent 5% v / v and layered on 11 ml cesium chloride gradients containing 24-45% w / v cesium chloride (0.05 M sodium phosphate buffer, pH 7.0) and 0.3 ml sucrose supernatant . The gradients were centrifuged at 100,000 g for 3 hours or more, then fractionated and the fractions assayed for hemagglutination activity. The high activity fractions were pooled and then subjected to vacuum dialysis against phosphate buffered saline (PBS), then layered on a second gradient of 20-60% w / v sucrose in PBS and centrifuged for 16 hours. The fractions were again assayed for hemagglutination activity, the high activity fractions were pooled again and vacuum dialyzed against PBS. The final solution has a protein concentration of 1.76 mg / ml and a hemagglutination activity of about 10 ⁶ HAU / ml (HAU = haemagglutination activity unit). The final protein concentration of the subunit mixture is adjusted to 200 µg / ml to produce liposomes.

The phosphate-containing sodium chloride solution (PBS) has the following composition: 10 g / l sodium chloride,

0.25 g / l potassium chloride, 0.25 g / l potassium dihydrogen phosphate and 1.4375 g / l disodium hydrogen phosphate.

B) Preparation of liposomes

Dicetyl phosphate (2.5 mg) and lecithin (22.5 mg) were dissolved in about 50 ml of chloroform. The solution was evaporated to dryness under a Buchi evaporator (40) under a slight vacuum. 5 ml of PBS solution of viral antigen preparation is added to the contents of the flask and the flask is manually and mechanically stirred until the lipoid is suspended in the liquid. The mixture was placed in a vial and subjected to ultrasonic treatment for 1.5 minutes using 1 cm samples and 8 μ amplitude.

C) Protective effect test in mice

Liposome formulations are tested for protection.

mice were divided into four groups of 5, two groups were injected intraperitoneally with the viral subunits of Example 1A (55), one 50 g / 0.25 ml and the other 5 g / 0.25 ml. The third and fourth groups were treated similarly with the liposomes of Example 1B, one with 50 µg / ml / 0.25 ml and the other

5 µg / 0.25 ml was added.

days later, mice were inoculated with live viruses of the same strain and their lungs were eviscerated two days later. The lungs were homogenized and sonicated to release existing virus and the resulting suspension was examined by an allan sage-on-shell culture according to the procedure of Fazekas de St. Groth and White (1958, Journal of Hygiene 56, 151).

The results are summarized in Table I below. Subunits bound to 50 µg liposome 5 provided adequate protection against homologous viral infection 10 days after immunization. In this group of mice, only one mouse exhibited a trace amount of virus growth in the lungs. A similar level of protection was provided by a 5 µg / 10 dose liposome as a 50 µg / dose subunit only, illustrating that the liposome material had ten times the protective effect.

Example 2

A) 2.5 mg of dicetyl phosphate and 22.5 mg of lecithin are dissolved in about 50 ml of chloroform. The solution was evaporated to dryness as in Example 1 and phosphate buffered saline was added as above to give a lipoid concentration of 16.6 Mmol / ml.

This mixture was sonicated in an ultrasonic bath for 1.5 hours at 50 KHz.

B) Influenza virus subunits of Example 1A) are added to the resulting composition to a concentration of 200 µg / ml. This mixture is then sonicated in an ultrasonic bath for an additional 15 minutes at 30 50 KHz.

C) Electron microscopic examination of the microscopic bodies of Example 2A showed a small unicellular structure, which is slightly contaminated with larger multilamellar structures (i.e., liposomes) by negative staining. Most lipoid disks are 50 to 100nm in size.

D) Examination of the subunits of Example 2B, identical to C, shows only the typical star and wheel forms attributable to the hemagglutinin and neuraminidase subunits. No trace of a viral membrane can be found.

E) Examination of the subunits of Example B above, identical to C, shows that most of the subunits are located on the surface of unicellular bodies, thereby becoming similar to the influenza virus.

In most microscopic bodies, hemagglutinin subunits first appeared, but in some areas the neuramidine subunits were also associated. Further immunoelectron microscopic examination of these microscopic bodies shows that the addition of hyperimmune influenza A antiserum results in aggregation of the bodies into large complexes.

Example 3

Preparation of ethanol liposomes A solution of mg of lecithin in chloroform was dried on a rotary evaporator under partial vacuum. 2 ml of ethanol are then added to dissolve the dry lipoid and the resulting solution is drawn into a 2 ml syringe fitted with a 27 gauge needle.

A stream of nitrogen (0.16 M potassium chloride) was bubbled through the stream for 1 hour and the needle was injected with ethanol solution of lecithin.

The resulting liposome preparation is concentrated to about 3 ml by ultrafiltration using 52 ultrafiltration cells of the Amicon model with PM 30 membrane [l.B.B. Acta, 298: 1015-1019 (1973)].

II. Spreadsheet

Antigen Dose Number of mice Virus lung titration (10 g ¹⁰ ) mean average 42 Mg X31 subunit 0.25 ml First -1.75 -1.85 alcohol Second -1.75 ± 0.22 liposomes Third -1.75 4th -1.75 5th -2.25 42 Mg X31 42 Mg 16th -3.31 -2.95 szubegység 0.25 ml 17th -2.63 ± 0.78 PBS 18th -3.88 19th -3.13 20th -1.81 not immunized 21st -4.06 -4.42 22nd -4.50 ± 0.21 23rd -4.50 24th -4.56 25th -4.50

Example 4

The ethanol liposomes of Example 3 are mixed with an equal volume of influenza virus X31 subunits (250 mg / ml) and the mixture is slightly sonicated.

The resulting antigen formulation was tested in mice for protection as described in Example 1C).

The results are shown in Table II. Table summarizes:

Example 5

Parenteral solution

Lecithin / dicetyl

(9: 1 by volume) 1 mg influenza virus X31 protein szubegységek 50 Mg nátriummertiolát 40 gg phosphate buffered sodium chloride solution (pH 7.2) 0.2 ml Phosphate-buffered saline together- making: NaCI 10 g / L KCI 0.25 g / L potassium dihydrogenphosphate 0.25 g / L disodium hydrogen phosphate 1.4375 g / utero

with variable doses of inactivated diphtheria toxin or liposomes containing a similar dose of inactivated diphtheria toxin at a lipid concentration of 1% v / v. In each experiment, the negative control is a group of non-injected mice. After 21 days, the animals are bled and the serum is absorbed to isolate antibodies to sheep red blood cells and α ₂ macroglobulin. The serum is then subjected to hemagglutination assays using sheep red blood cells sensitized with inactivated diphtheria toxin [1. J. Immunol. 100, No.2. 274 (1968)]. The hemagglutination titer is considered to be the serum dilution that exhibits a definite agglutinator. For each assay, standard equine anti-diphtheria was used as a positive control, and serum was used as a negative control and serum-free cells were used as a negative control. The non-injected control group always showed a negative result. The results for each group are expressed as the mean of the reciprocal of the haemagglutination titer in all animals and are shown in Table III. and IV. are summarized in Table.

III. Spreadsheet

Hemagglutination assay of CBA male mice serum 21 days after liposome-subcutaneous injection of inactivated diphtheria loxin or inactivated diphtheria toxin

Example 6

Liposomes containing inactivated diphtheria toxin

A. Preparation of Liposomes Dissolve mg dicetyl phosphate and 27 mg lecithin in 4 ml chloroform and mix, add 3 ml phosphate buffered saline (PBS) and connect the resulting flask to a Buchi rotary evaporator. The aqueous and non-aqueous phases were stirred for 1 hour under magnetic stirring and rotation under gentle vacuum (100 mm Hg) at 20 ° C. Chloroform was then removed by increasing the vacuum. The resulting liposome preparation is then allowed to equilibrate for 3 hours. 0.3 ml of inactivated diphtheria toxin (9 mg / ml) (Wellcome Laboratories) is added to the preparation and the resulting mixture is stirred again for half an hour, in which case the liposome absorbs the surface of the inactivated diphtheria toxin. Excess inactivated toxin was removed by centrifugation at 40,000 g for 2 hours at 5 ° C and the resulting liposomes were resuspended in 3 ml phosphate-buffered saline. [1st Biochim. Biophys. Acta, 266, 322 (1972).]

B) Effect of liposomes containing inactivated diphtheria toxin on the immune response of mice

Groups of 5-12 CBA and Porton albino male mice injected subcutaneously

Concentration of inactivated diphtheria toxin in Mg Reciprocal of hemagglutination titer Negative control Only inactivated diphtheria toxin Liposomes containing inactivated diphtheria toxin 0 <2 - - . ⁷ ' ⁵ - <2 12.8 5.0 - 8.0 32 10.0 - 20.8 64

ARC. Spreadsheet

Blood hemagglutination assay of a Porton albino male mouse serum, blood is collected 21 days after injection of inactivated diphtheria toxin or liposome subcutaneously containing inactivated diphtheria toxin

Reciprocal of inactivated hemagglutination titer

concentration of diphtheria toxin in Mg Negative control Only inactivated diphtheria toxin Liposomes containing inactivated diphtheria toxin 0 <2 - - 8 - <2 11.3

C) Effect of liposomes containing inactivated diphtheria toxin on the immune response of guinea pigs

Groups of male porcine guinea pigs were injected subcutaneously on day 0 with 0.4 µg of inactivated diphtheria toxin or liposomes containing inactivated diphtheria toxin at a 1% v / v lipoid concentration. Blood is collected from the animals 21 days later. Animals in both groups were injected on day 28 with 0.4 µg of solution containing inactivated diphtheria toxin only and blood was collected after 7 days. Sera were subjected to haemagglutination assays as described in Example 6b) and the results obtained in each group were expressed as the mean of the reciprocal of the haemagglutination titer in all animals. Guinea pig pre-injection serum serves as a negative control and does not show an inactivated diphtheria toxin reaction. The results are summarized in Table V.

Table V. Haemagglutination test in guinea pig serum

1. 21 days after the first injection of liposome containing inactivated diphtheria toxin or inactivated diphtheria toxin. 2. a solution containing only inactivated diphtheria toxin; 7 days after the date of injection inactivated hemagglutination diphtheria reciter of titer toxin concen- inactivated metabolize inactivated diphtheria pg-in diphtheria toxin toxin containing liposomes First Primary effect 0.4 <2 <2 Second secondary effect 0.4 16 150

Patent claims:

Claims (15)

CLAIMS 1. A method of preparing an antigen composition comprising a plurality of microspheres, each of which has at least one lipoid bilayer on its outer surface to which an antigenic protein derived from a pathogenic microorganism is attached, wherein: (a) vigorously mixing a lipoid, preferably natural or synthetic lecithin, after optionally dissolving in an organic solvent and optionally evaporating it in an aqueous medium, subjecting the resulting blisters to further vigorous mixing in the presence of an antigenic protein, or b) subjecting the mixture of the lipid, preferably natural or synthetic leticin, to the aqueous medium and the antigenic protein to vigorous mixing. A process according to process variant (a) of claim 1, wherein the starting lipoid material is dissolved in an organic solvent, the solvent is evaporated, The resulting lipid phase was dispersed in an aqueous solution and stirred vigorously, then the antigenic protein was added and vigorous stirring continued. 3. Process according to process variant a) of claim 1, wherein the starting lipoid is added to an aqueous solution and heated with stirring, then the antigenic protein is added and the resulting mixture is again subjected to vigorous mixing. A process according to process variant (a) of claim 1, wherein the alkanolic solution of the starting lipid material is rapidly injected into an aqueous solution to give The liposome preparation was concentrated by ultrafiltration and the antigen protein was added and the resulting mixture was vigorously stirred. 5. A process according to any one of claims 1 to 4, characterized in that the unicellular microvessels formed during lipid mixing are mixed with antigenic proteins. 35 6. A method according to any one of claims 1 to 5, characterized in that the microvessels formed by mixing the lecithin are mixed with an antigenic protein. 40 7. A method according to any one of claims 1 to 3, wherein the antigenic protein is derived from pathogenic microorganisms. ⁴⁵ 8. A method according to any one of claims 1 to 4, wherein the antigenic protein is derived from a virus. 9. The method of claim 8, wherein the virus is influenza virus. 10. Figures 1-9. A method according to any one of claims 1 to 6, characterized in that the microvessels of the size range 20-200 nm obtained by mixing the lipoid are mixed with an antigenic protein. 11. A method according to any one of claims 1 to 3, wherein the vigorous mixing is performed by sonication. 12. A process according to any one of claims 1 to 4, wherein the aqueous solution is phosphate buffered solution. 13. The process of claim 2, wherein the solvent is chloroform. 14. A process according to claim 4, wherein the alkanolic solution is an ethanolic solution. 15. The method of claim 4 embodiment, wherein the ultrafiltration is carried out in a nitrogen atmosphere for ⁵ Christmas ala pressure.