IL30206A - Process for inactivating microorganisms and/or their antigens - Google Patents

Description

0"»Πσ·«3ΛΊ1Ν-Π *·η "701 flY?"»SS * *»Ν Ϊ\Ώ1ΊΧ7 Ι^ΓίίΙ PROCESS FOR INACTIVATING MICROOROANISMS AND / OR ANTIGENS.

Process for inactivating biological material (Fw 5299 A - Ma 87) The present invention relates to a process for inactivating biological material such as infectious microorganisms and/or their antigens .- There are already known processes which permit the inactivation of biological substances, in particular of infectious microorganisms and/or their antigens. For this purpose, either chemical or physical methods have been hitherto used. All these known processes have in common that the biological substances are inactivated in the liquid state.

The chemical methods have the disadvantage that for combining the biological material with the inactivating substance, the recipient must be opened and that thus the biological material may be contaminated by fungi and bacteria. In most cases, chemicals added do not permit exact discontinuation of the inactivation and affect desired properties, for example the antigenicity of virus and bacteria intended to be used for the manufacture of vaccines. The storability of such substances is limited for the same reason but just the storability of biological material is an important factor, for example during transportation or in tropical countries where higher temperatures are unavoidable and must be taken into account. Therefore, storage in a refrigerator is prescribed for such preparations. The. attempt was. also made, when formaldehyde had been used as the inactivating agent, to^add bisulfite for binding the unreacted ormaldehyde; but this again required opening of the recipient. Another disadvantage has also been the fact that the chemicals added, for example phenol, cause intolerance reactions in humans and animals.

The physical methods, too, have disadvantages. Thus, for example, for inactivating suspensions which contain the. mentioned biological substances,, these suspensions must be . subjected in a uniformly thick film to ultraviolet irradiation in a special apparatus. This may cause., under certain circumstances, formation of peroxides which may continue the inactivation for an undesirably long period of time.

The object of the present invention is a process for inactivating biological material such as infectious microorganisms and/or their antigens, wherein the aqueous suspension containing the mentioned biological material is combined with a stabilizer, freeze-dried and the dry substance obtained is heated for a period in the range of from 15 minutes up to 12 weeks to a temperature in the range of from JO to 110°C.

An object of the present invention is furthermore the inactivated biological material prepared according to the above process and serving for the manufacture of vaccines.

The process- of the present invention is novel, since no method has hitherto been known which would have permitted the inactivation of biological material such as infectious microorganisms and/or their antigens in dry form by the action of heat. As has already been pointed out above, the inactivation according to the known methods has always been carried out in the liquid phase, either with the aid o suitable chemical agents or physically, for example by means of ultraviolet irradiation. The inactivation of the mentioned biological material by the action of heat has generally been considered with a prejudice because earlier tests had—shown that the inactivation in the liquid phase generally caused denaturation of the material and therewith impairement of desired biological properties or even caused destruction of these properties.

V It was thus surprising that the inactivation according to the invention by the action of heat onto the dry biological material does not cause denaturation of the biological material.

Whereas chemical substances, for example phenol, formaldehyde, etc. are used as inactivating agents in the known methods of inactivation, no such chemical agents are added, in the process of the present invention, so that side effects such as intolerace reactions, which are due to the presence of the inactivation agents, do not occur when the preparations are administered medicinally. Furthermore, the method of the present invention has the advantage that no risk of bacterial contamination of the biological material during the inactivation process is implied, since the inactivation can be carried out in a closed vessel. In order to discontinue the inactivation,' it is only necessary to cool the material to a lower temperature, for example to room temperature. Another technical advantage resides in the fact that the preparations prepared according to the present invention have a stronger activity than the known preparations and that they may be exposed to such temperatures at which the vaccines of the state of the art are already inactivated.

For preparing a vaccine from the inactivated biological material prepared according to the present invention, the dry preparation is simply combined with . distilled water, up to the desired concentration, ' if desired with the addition of a suitable buffer, for example a phosphate solution which ma also contain an adjuvant, for example aluminium hydroxide.

The vaccine thus obtained permits the production in known manner of immunities against various infectious diseases, according to the starting material.

Furthermore, the vaccines prepared according to the present' invention have the advanta e over the vaccines obtained b known vaccines prepared ' according to known methods owing to thsir sensitivity to higher temperatures have to be stored and transported . at temperatures in proximity of the freezing point (0 - °C), which, for example under tropical conditions, is connected with difficulties, the vaccines prepared according to the process of the present invention are well storable also at elevated temperatures, for example at 60°C, even over prolonged periods of time.

The process of the present invention. is suitably carried out by combining an aqueous suspension prepared in known manner, of .the biological material such as infectious microorganisms, for example viruses and bacteria as well as their degradation and metabolism products that have antigenic action, with a stabilizing substance, then freeze-drying it and heating the dry preparation obtained for a period in the range of- rom 15 minutes up to 12 weeks, preferably for a period in the range of from 60 minutes to 10 weeks, to a temperature in the range of from JO to 110°C, preferably to 35-100°C. As stabilizers, there are-.advantageously used known preparations, for example a polymerisation product prepared according to German Patent 1 118792 from degraded gelatin, which is traded under the Registered Trade mark Haemaccel (R) or a Molico(R) -solution which is prepared from dry milk powder, furthermore a saccharose-glutamate-solution, a maltose-glutamate-solution or a glucose-gelatine-bouillon.

The duration of the action of heat is in inverse proportion to the temperature at which the inactivation according to the invention is carried out, i.e. the higher the ' inactivation temperature, the shorter the time required for complete inactivation. For example, the inactivation of rabies viruses in the presence of Haernaccel as the stabilizer at 37°C takes 60 days, whereas at 56°C it requires only 7 days. As viruses, products; which may be inactivated according to the present invention, there may be mentioned, for example: the viruses of rabies, swine fever : and horse influenza; the bacteria of swine erysipelas; hemagglutinin of measle viruses as well as botulin toxins and streptokinase.

After the treatment according to the present invention, the preparations are subjected to the usual tests in order to determine whether the pathogenicity is removed and whether the antigenicity is maintained.. For example, in the case of the rabies virus, the innocuousness is tested by intracerebral injection into mice and the antigenic activity is determined by the Habel test (WHO Monograph Series No. '23 1 0 (1966) . .

The following Examples illustrate the invention but they are not intended to limit it thereto: E x a m p l e 1 g of wet substance recovered from the brain and the spinal cord of a rabbit infected with fixed rabies virus were combined with l80 ml of a 3.5 ^ solution of a polymeric gelatin derivative, which is available in the commerce under the Registered Trade mark Haemaccel; the whole was treated in a homogenizer to a fine suspension, filled into flasks having a capacity of 7 ml and freeze-dried .

The virus concentration of the dried material, which had been reconstituted with 7 ml was controlled in mice having . a weight in the range of 11 - 15 g by intracerebral (i.e.) titration. It was found to be 10 4* 0LD U Per 0.05 ml.

For inactivation of the virus material, the flasks we're then heated for 7 days to 56°C. Macroscopically, the action · of heat did not change the dry material. After 7 days the material was diluted with distilled water to the original concentration (7 ml) and the innocuousness and the activity in the following manner: Test for innocuousness in a) Mice; Mice received 0.03 ml of the dissolved material by intracerebral injection. Within an observation period of 11- days, the animals showed no signs of rabies. The virus material was thus inactivated. The result was confirmed by several tests. b) Dogs: 2 Dogs received subcutaneously, on 6 consecutive days, ml of the vaccine prepared according to the present invention. During an observation period of 21 days, neither local nor general reactions were observed. c) Guinea pigs: Guinea pigs were administered intraperitoneally 2 ml each of the vaccine prepared according to the present ' invention. During an observation period of 10 days, neither local nor general reactions were observed. 6 Guinea pigs were administered subcutaneously 0.5 ml each of the vaccine to be tested. During an observation period of 10 days, no local reactions were observed. d) Rabbits : rabbits were administered intracerebrally O.J> ml each o During the observation period of l\2 days, no reactions were observed. The result was confirmed by repeated tests carried out under the same conditions and with the same number of animals . .

The above-described innocuousness tests -showed that the rabies vaccine prepared according to the present invention is innocuous and well tolerated.

Activity: a) Mouse The activity of the rabies vaccine was determined according to the so-called Habel test. To carry out this test'/ 60 mice were immunized by 6 intraperitoneal (i.p.) injections of Ο.25 ml each of a suspension, which was obtained by . diluting to a ratio of 1 : 20 the content of a flask (7 ml) with phosphate-buffered distilled water or physiological NaCl solution, and which thus contained the brain and spinal cord material in a concentration of 0.5 the injections were given each time on Monday, Wednesday and Friday of two consecutive weeks. 15 Days after the first vaccination, - /the mice were subjected to a challenge infection. For this purpose, 10 mice each were infected, intracerebrally (i.e.), . with O.O3 ml of the virulent rabies test virus CVS 27 I b 2 -1 -6 (Challenge virus strain) in dilutions from 10 up to 10 At the same time, 10 untreated mice were infected with -5 -7 dilutions from 10 J to 10 ' .

The 50 c/o end points of both test series were then , determined according to Reed and Munch (cf. Am. J. Hyg. 27, ^93 {1938)). The difference between both final points is the FDC-Q, i.e. the dose which is capable of protecting 50 of the animals against an infection of the indicated magnitude.

A vaccine is considered as having passed the Habel test if the difference between both 0 end points of the vaccinated group and of the control group, i.e. the reaches a value higher than 0 or more.

The. vaccine prepared according to the present invention, gave 4 0 ¾ ς in several tests a titer of 10 * to 10 mJ ?T^0 per 0.03 ml. b) D o g The vaccine prepared according to the present invention A 12 was administered subcutaneously, in doses o 1 X 10 ml at the beginning of the test were free from virus-neutr - lizing antibodies against rabies. The course of the immunity was controlled over a period of 52 w.eeks by examination of the mixed sera of the groups, of dogs by periodical withdrawal' of blood and determination of the antibodies by serum neutralization tests in a white mouse. 14 Days after the immunization, the maximum of immunity was already reached with titers between 1 : 12 and 1 : 150 ( 50 % neutralizing end point of the serum dilution, i.e. this dilution neutralizes 0 of a same volume of a virus suspension of the infectious rabies virus) and then receded slowly over a period of 50 weeks.

At .the end of the test, 2 weeks after the immunization, the 1 sera of the groups of dogs had the following values: Dog No. Vaccination Serum titer ( 50 % end point) 52 weeks after vaccination 6053 1 x 10 ml 1 : 12 .3 6083 6190 1 x 5 nil 1 : 7 . 2 6191 These dogs are immune against a lethal experimental- infection with rabies virus (Amer. J. Vet. Res. 26, 24 - 30 ( 1965 ) ) .

In the above-described immunization test in dogs, a rabies vaccine of the state of the art obtained by inactivation with phenol according to Hempt was administered subcutaneously in a dose of 1 x 10 ml to a group of two dogs, . at the same time as the vaccine of the present invention (serum titer at 50 % end point 1 : 12. 3 ) . .: ' 14.. Days after vaccination, serum titers of 1 : 12 were determined, at the end of the test ( 52 weeks after the vaccination) a--serum titer ( 50 end point) of 1 : 3 . This test, too, showed that the vaccine prepared according to the present invention had a Stability The vaccine prepared according to the present Example -was stored for 60 days at 56°C and compared with a rabies vaccine prepared by inactivation with phenol and stored at the lower temperature of 37°C.

The vaccine of the present invention exhibited after 60 days at 6°C no loss of activity, whereas the vaccine prepared in known manner showed already after 15 days at the lower temperature of 37°C, an activity of only 10 PD 50 per 0.03 ml..

As results from the above, the vaccine prepared according to the present invention withstands considerably better higher temperatures whereas the vaccine used for comparison had lost its antigenicity already at a lower temperature.

E x a m p l e 2 A vaccine prepared as described in Example 1 from rabies viruses, which had been stabilized with Haemaccel and Molico solution, was subjected to the Habel test as well as to the stability test. The test results with indication of the test conditions are shown in the following Table.

For comparison, two vaccines inactivated in known manner, with phenol were used.

Composition of Method of Result of activity the vaccine and inactivation test in the Habel stabilizer solution before after exposure exposure to to temperatempera-- ture · ture Rabies viruses 30 days + in 20 g of a rabbit' s brain with phenol 56°C l80 ml of Haemaccel (for comparison) 3.-7 = 1.2 Rabies viruses 60 days + in 20 g of a rabbit1 s brain according to • 56°C l80 ml of Haemaccel invention , = 3.8 Composition of Method of Result of a Vctivity the vaccine and inactivation test in the Habel stabilizer solution before after exposure exposure to to temperatemperature ture Rabies viruses with 7 days in 20 g of a rabbit's brain phenol 37¾C l8o ml of Molico solution (for comparison) = 0.8 Rabies viruses 60 days -f| in 20 g of a rabbit's brain according 180 ml of Molico solution invention 4.3 = 4.6 The test results show that the vaccines containing rabies viruses inactivated according. to the process of the present invention resist a considerably higher temperature strain than vaccines prepared according to known processes.

E x a m p l e 3 , Inactivation tests were carried out at various temperatures with samples of the material indicated in Example 1 and the following results were obtained: Temoerature Duration of inactivation 37 °C 60 days °C 35 days 56°C 7 days With further samples of the same virus material and Molico solution ( 9 strength) there were carried out the following inactivation experiments: Temperature Duration of inactivation 37°C 70 days 45°C 35 days 56°C 7 days Furthermore, the inactivation times were determined for Λ different stabilizers,, but at the same temperature: Stabilizers Temperature Inactivation time Haemaccel "5'6pC 7 days ° da s - - - V This Example shows that in the process of the present invention the inactivation time for a determined virus depends on the stabilizer and on the temperature.

All samples were tested for i.nnocuousness, activity and stability. The virus material w.as inactivated and active in the Habel test. After storage for βθ days at 56°C it showed no loss of activity.

E x a m p l e Rabies viruses were combined with various stabilizers and inactivated by heating to 98°C, as indicated in Examples 1 and 2. The following Table shows the test results.

Composition of the rabies Method of inactivation Test for inact vaccine Temperature i Time in a mouse i.e. injection g of a rabbit's brain 80 ml of physiol. NaCl÷solution 98°C 60 mill, no rabies viru 100 ml of sacoh.-glutamate- solution g of a rabbit's brain 40 ml of physiol.NaCl-solution 98°C 60 min. no rabies viru 50 ml of sacch.-glutamate- solution 90 ml of Haemaccel g of a rabbit's brain 40 ml of physiol. NaCl-solution 50 ml of sacch.-glutamate- 98°C 60 min, no rabies viru solution 90 ml Molico-solution It has been shown in this Example that even extreme temperatures may j without imparing the activity of the vaccine of the present invention thereby essentially reduced. \ Three vaccines, prepared in accordance with the present invention, i.e.

A 16 (inactivated for 5 days at +70° C) A 17 (inactivated for > days at +70° C) A 18 (inactivated for 7 days at +70° "C), · were tested and compared in the Habel test with two rabies vaecines prepared by inactivation with phenol according to Hempt (B^ and B^) .

As has already been explained in Example 1, the Habel test requires that 6 injections of 0.25 ml each of the vaccine, diluted to 0.5 of the nerve tissue proportion, protects against 1,000 LD^ (log 3) of the infectious virus. With the rabies vaccine according to Hempt, therefore, the 10 nerve tissue ■ suspension is normally diluted in the ratio of 1 : 20. , Vaccine Preparation Vaccine dilution ( of nerve tissue proportion 1 :20(0,5 ) 1 :40 (0,25 $) 1 :80 (0,125 $>) A 17 B 3 ' according to a 5, 7 5.7 not state of the b 2. 6, 3.6 tested art c 3. 1 2.1 It can be seen from the above. able that the vaccines prepared according to the present invention in the dilutions 1 : 20, 1 : 40 and even 1 : 80 comply with the requirements of the Habel test. The rabies vaccines prepared according to Hempt corresponding to the state of the art, in a dilution of 1 : 20, comply with the requirements of the Habel test and in the dilution of 1 : 40, only one vaccine passed the test, and in the dilution of 1 : 80 this vaccine no longer complied with the requirements of the Habel. test. Thus, the vaccines of the present invention have a far better antigenicity than the vaccines prepared according to the state of the art.

E x a m p l e 6 liters of a suspension of Erysipelothrix rhusiopatiae, prepared in known manner, were mixed 'with 2.5 liters of a stabilizer solution consisting of 50 of Haemaccel and 50 o of a saccharose -glutamate solution, and the whole was freeze-dried. The dry material was then kept for 52 days at 70°C. a) Proof of inactivation: For proving the inactivation, the living germs were counted. On the 30 th day of inactivation, no growing of germs could be observed. b) Determination of the activity: The dry material was dissolved in a 0.8 ^ Al (OH).-, solution and the vaccine so prepared was compared according to the official prescriptions for the Testing of Swine erysipelas vaccines (Hellich-St riko, Die deutsche Tierseuchengesetzgebung 1953, page 623), by immunization of ,/ groups of mice and following infection with a standardized number of germs, with the activity of the international Swine erysipelas standard vaccine, whose content of International determined by means of the three-point method* A Swine . erysipelas vaccine which complies with the official. - /•requirements must have at least 50 International Units per ml over a period of 2 years.

The vaccine prepared according to the present invention showed in this comparative test with the international Swine. erysipelas standard vaccine Rfg, to have a content of 175 IU per ml. This is about three times the amount o the required number of International Units.

This Example shows that also bacteria can be inactivated by the process of the present invention.

E x a m p l e 7 , lethal doses for mice (when administered intraperitoneally), per ml were combined, while stirring continuously, with 30 ml of Haemaccel as the stabilizer. After thorough mixing, the ■ toxin was filled in quantities of 2 ml each into flasks- and freeze-dried. Inactivation was effected by heating the dry material to +100° C for 4 hours.

Tests: a) Test for toxicity prior to inactivation; 2 Mice each were given intraperitoneally in a dilution of 1 : 10, 0.1 ml of the dry material dissolved in 2 ml of distilled water. The dried toxin had a toxicity of mice lethal doses per 1 ml (MLD/ml).

Proof of inactivation 3 Mice received intraperi oneally 0.1 ml each of the inactivated dry material, dissolved in 2 ml. During an observation time of 3 days, all animals remained healthy. c) Test for activity of the vaccine prepared according to the invention botulism absorbate vaccine are not yet established at present. The vaccine prepared according to the present invention was tested in a manner analogous to the method devised by Prof. Rislakki, Finland. According to this method, 80 of the animals which receive the vaccine in a dilution of 1 : 4 and 50 % of the animals which receive the vaccine in a dilution of 1 : must survive.

Two groups of 10 mice each were injected subcutaneously Ο.25 ml of dilutions 1 : 4 and 1 :: 8 of the dry material which had been inactivated according to the present invention and dissolved in a 0.4 ^ Al (OH)^ suspension. 3 Weeks after the vaccination, the two groups o.f mice and 5 control animals were intoxicated subcutaneously with 10 MLD.

Of group 1 (vaccine dilution 1 : 4), 7 out of 10 animals survived = 70 Of group 2 (vaccine dilution 1 : 8) 8 out of 10 animals survived = 80 %.

All animals of the control group died on 2nd to 3rd day after the intoxication.

Claims (12)

We claim:

1. ) Process for inactivating infectious microorganisms and/or their antigens, wherein the aqueous suspension containing the infectious microorganisms and/or their antigens is combined v/ith a stabilizer, freeze-dried and the dry material obtained is heated for a period in the range of from 15 minutes to 12 weeks to a temperature in the range of from 30 to 110°C.

2. ) A process as claimed in claim 1, v/herein the dry material is heated for a period in the range of from 60 minutes to 10 weeks.

3. ) A process as claimed in claims 1 and 2, v/herein the dry material is heated to a temperature in the range of from · 35 to 100°C.

4. ) A process as claimed in claims 1 to 3 , v/herein a polymer from degraded gelatin is used as the stabilizer.

5. ) A process as claimed in claims 1 to 3 > wherein a solution obtained from dry milk powder is used as the stabilizer.

6. ) A process as claimed in claims to 3 > v/herein a saccha- roseglutamate solution, a maltose-glutamate solution or a glucose-gelatin bouillon is used as the stabilizer.

7. ) A process as claimed in claims 1 to 6, wherein viruses or bacteria or their antigens are used as infectious microorganisms.

8. ) A process as claimed in claims 1 to 6, wherein the degradation or metabolism products of infectious microorganisms are used as antigens.

9. ) A process as claimed in claims 1 to 7, wherein rabies virus is inactivated.

10. ) Inactivated infectious microorganisms and/or their antigens, I prepared according to the processes claimed in claims · 1 to 9.

11. ) Inactivated viruses or bacteria, prepared according to the processes claimed in claims 1 to 9.

12. ) Inactivated rabies viruses', prepared according to the processes claimed in claims 1 to 9. COHEN ZEDEK & SPISBACH P. O. Box 1169, Tel Λ v i v Attorneys for Applicant