IL30168A - Antigen-free vaccine and process for preparing it - Google Patents

Description

ANTIGEN-FREE VACCINE AND PROCESS FOR PREPARING IT Antigen-free vaccine and process for preparing it (Fw 5407A - Ma 9?) The object of the present invention is an antigen-free vaccine containing an informationally active ribonucleic acid, and a process for preparing it.

For active immunization, either so-called killed vaccines, whioh contain killed virulent germs, or live vaccines, which contain living avirulent germs, are presently used. In addition thereto, toxid^s (detoxicated bacteria), toxines and subunits of viruses are used as vaccines. These conventional vaccines require from the manufacturer utmost oarefullness.

With killed vaccines, the inactivation must be carefully controlled, because on the one hand insufficient inactivation of the natural antigen may cause undesired side reactions in the vaccinated subject, whereas on the other hand, the natural antigen may be modified or damaged to a certain extent by the inactivation.

An insufficient attenuation of viruses in vaccines produced from proliferable viruses, too, may have severe consequences. Therefore, the manufacture of vaccines requires complicated, time-consuming and very costly controls. Despite these oontrols, however, intolerance reactions have been observed.

The disadvantages described above can be overcome when using for active immunization not the antigen itself but an informationally aotive ribonucleic acid (RNA) which contains the total building plan of the desired antibodies. The use of such an informationally aotive RNA as a vaccine would have considerable advantages over that of conventional vaccines. 1. The informationally active RNA is free from impurities. 2. Immunity after vaccination is rapidly reached, because the macroorganism directly obtains the building plan for the antibody synthesis and processing of the antigens by the macroorganism is not necessary. 2. The fact that the informationally active RNA can be synethesjLzed in vitro enables immunizations to be obtained which it has hitherto not been possible to obtain owing to the lack of suitable vaccines.

We have found that informationally active RNA, when injected to macroorganisms (human and animal), induces these macroorganisms to form antibodies after the injection and to produce a protection as proven by challenge.

Accordingly, the object of the present invention is a vaccine containing an informationally active ribonucleic acid, which is obtained by isolation in known manner, sterilisation and, if desired, combination with an adjuvant.

The informationally active RNA serving as the base for the vaccine is obtained by stimulating immunologically competent cells or, preferably, cell-free systems from immunologically oompetent cells, by the corresponding antigen.

Under "immunologically competent cells", there are to be understood such cells which are able to react, upon a primary stimulation by an antigen, with the formation of informationally active RNA. The competent cells are found in the lymphs, in lymph nodes, in the spleen, in exudates- for example in the peritoneal exudate - and especially in peripheral leukocytes (blood). The cells may be of human or animal origin depending on whether the vaccine should be used in humans or in animals. Thus, for preparing a vaocine for a determined species, suitably the immunologically competent oells of the species concerned are used. Human informationally active RNA may be obtained, for example, from peripheral manufacture of blood plasma.

For the stimulation of the immunologically competent oell material, there may be used antigens and toxins · from bacteria, viruses or their subunits, snake poison, etc.

As bacterial starting substances, there may be mentioned, for example tetanus toxin and diphtheria toxin, furthermore the products of metabolism or degradation of Bordetella pertussis, Mycobacterium tuberculosis as well as of salmonellae, shigellae, brucellae, and streptococci, viruses and their subunits, for example influenza viruses, influenza hemagglutinin, polio viruses or their protein envelope, measles, rabies, foot and mcruth disease, smallpox> swine fever, distemper, hepatitis contagiosa oanis and encephalitis viruses.

When, for producing the informationally active RNA and for preparing the vaccine, a cellular system is used, one may operate in general as follows: A cell culture of immunologically competent cells (for example human leukocytes) is stimulated with a previously determined optimal dose of the antigen (as a rule one antigen particle per 100 cells) and then incubated for 5 - 60 minutes, preferably for about 20 minutes, at about 37°C. The cell culture is then rapidly oooled to at least 0°C and washed.

The informationally active RNA is isolated from the washed cells acoording to known methods, for example by means of phenol, so that it is free from proteins and nucleases. For sterilization, the aqueous RNA suspension is filtered under sterile conditions or heated for about 5 minutes to about 100°C, if necessary after having been dialysed previously in order to remove traces of phenol. The product thus obtained constitutes the vaccine it may be combined with an adjuvant, for example aluminium hydroxide or aluminium phosphate.

When starting from a cell-free system, one may operate advantageously as follows t Washed, immunologically competent cells (for example leukocytes from the peripheral blood of human or animal origin) are taken up in a buffer solution having a molarity of 0.01 - 0.15, preferably about 0.1, and a pH-value of 7.6 -8.6, preferably about 7.8. A Tris/maleic acid or Tris/HCl buffer is the most suitable. The cells are destroyed by repeated freezing and thawing and the cell homogenate is centrifuged for about 30 minutes at 30000 g and at 0° C or below 0° C. The supernatant obtained after centrifugation is the cell-free system which contains all the soluble substances contained in the oells. It is stimulated with the optimal dose of a corresponding antigen and incubated for 2 - 60 minutes, preferably for about 10 minutes, at about 37° C. The further treatment is effected as described above.

To the manufacture of vaocines the process of the present invention offers many advantages. The inactivation step re-quired in the conventional manufacture of vaccines is no longer necessary. Side reactions (allergio reactions) and vaccination damages are not to be feared, since the vaccines are absolutely free from antigens. The starting materials for use in the process of the present invention are easy to obtain in large amounts. The oosts of the process are low.

The tolerance of the informationally active ribonucleic acid of the influenza virus was tested in mice. The mice were injected intraperitoneally 0.25 ml of an aqueous suspension of the informationally active ribonucleic acid.

No reaction of the mice was observed. Thus, the vacoine prepared according to the present invention was not toxic.

For determining the activity, a protection teat was carried out in mice, in this test the produot of the invention was compared with an influenza virus of the art,produced from viruses cultivated in eggs. For preparing the products to be compared (both without ad uvant)^ the same quantity was each time used. As results from the following table, the preparation of the present invention has greater aotivity than that prepared according to the state of the art.

Protection against k / Preparation Asia 1 6 Informationally active R A (antigen-free) 10 ^50 (mouse ) Influenza vaccine from viruses cultivated from eggs (A2/Asia) 101 ' 00 I350(moU8e) The following Examples illustrate the invention but they are not intended to limit it thereto: Example 1 : Leukocytes were isolated from 100 ml of human blood. They were washed with physiological NaCl solution and then taken up in a 0. 1 molar Tris/maleio acid buffer having a pH-value of 7.8 and bound additionally to 0.01 mol of KC1, 0.005 mol of magnesium acetate and 0.006 mol of 2-mercapto-ethanol. The cell suspension was ten times freezed and thawed.

The homogenous cell mass was then centrifuged for 30 minutes at 30000 g and at 0° C. The sediment was rejected, the oell- free system was combined with 10"' units of the reoeptor of thephage (kappa) from Serratia maroescens and incubated for 10 minutes on a water bath at 37° C. The RNA was then isolated according to irby.

The determination of the antibody synthesis provoked by the vaccine in a cellular (A) and a cell-free (B) system was made as follows: A) Test of the informatory RNA in a oellular system: A lymph cell culture having 10 cells/culture was produced from human lymph nodes. To this lymph cell culture, there was added 0.3 ml of an aqueous solution of the above vaccine having an optical density of 0.0004, measured at 260 mu . After 2 days, the supernatant of the cell culture was separated and tested in the receptor-neutralisation test for its antibody content. 10^ antibody molecules per ml of tissue culture liquid were found.

B) Test of the RNA in a cell-free system of human leukocytes: A cell-free system was prepared as described above. The desoxyribonucleic acid present in this system was degraded with desoxyribonuclease. By this treatment, the cell-free system was no longer able to synthesize informationally active RNA or antibodies after the addition of antigens. Therefore, it was suitable as test system for the informational content of an RNA preparation, intended to be used as a vaccine. To 0.1 ml of suoh a cell-free system, there was added 0.1 ml of a RNA preparation having an optical density of 0.004 (at 260 nju). The batch was incubated for 30 minutes at 37° Cj then, the quantity of antibodies synthesized after the addition of the antibody titer was 10 antibodies per ml of tissue culture liquid.

Example 2: A cell-free system from human leukocytes was stimulated in the manner described in Example 1 with Equine-Milford hemagglutinine and a vaccine was prepared from the resulting homogenous mass. This vaccine caused cell-free systems from human leukocytes to synthesize antibodies. 5 x 10 antibody molecules/ml could be proved.

Example 3: A "culture of peripheral leukocytes of Rhesus monkeys was stimulated as described in Example 1 with Equine-Milford hemagglutinine and from the resulting homogenous mass there was prepared a vaccine (informationally active RNA). This vaccine was injected in a dose of 0.5 ml (dilution intramuscularly 1/20, optical density 0.002) to Rhesus monkeys. The two immunized monkeys showed an antibody titer of 6.0 x 10 9 q and 12.0 x 10 , respectively,per ml of serum. The control animals had not formed antibodies against Equine-Milford hemagglutinine.

Example t A culture of peritoneal cells of NMRI-mice was stimulated as described in Example 1 with Equine-Milford hemagglutinine and from the homogenous mass there was prepared a vaccine (informationally active RA). This vaccine was injected peritoneally to NMRI-mice. Upon ohallenge infection with the above types of viruses, the animals proved to be immune. The q serum contained 6.7 x 10 antibodies per ml, the oontrol animals had not formed antibodies.

Example 5: A cell-free system of spleen cells of Rhesus monkeys was influenza viruses of the types A, A_j , A2, Swine, B Lee and PR 8, and a vaccine was prepared from the resulting homogenous mass. This vaccine was injected to Rhesus monkeys. The animals treated with the vaocine (informationally active R A) formed antibodies as already described in Example 3.

Claims (11)

P.Af 30168/2 WHAT IS CLAIMED IS:

1. A vaccine which is antigen-free and comprises an aqueous suspension of informationa.lly active ribonucleic acid (as hereinbefore defined),

2. A vaccine as claimed in Claim 1, which also contains an adjuvant.

3· A vaccine as claimed in Claim 2, wharein the adjuvant is aluminium hydroxide or aluminium phosphate,

. A vaccine as claimed in any one of Claims 1-3» whclh also contains a pharmaceutically suitable carrier,

5. A vaccine as claioed in Claim 1 and described in any one of the Examples herein.

6. A process «Hor the preparation of a vaccine as claimed in Claim 1 wherein an aqueous suspension of informationally active ribonucleic acid is obtained by stimulating with an antigen corresponding to the required inf¾praationally active RNA: - (a) a system of immunologically competent cells (as hereinBefore defined )or (b) a cell-free system obtained from immunologically competent cells, and isolation an aqueous suspension of the informationally active RNA.

7. A process as claimed in Claim 6, wherein an optimal dose of the antigen is used. P/A 30168/2

8. A process as claimed in Claim 6 or Claim 7, wherein the Immunologically competent cells are any of those specifically described herein*

9. A process as claimed in any one of Claims 6 to 8, wherein the antigen is any one of those specifically described herein.

10. A process as claimed in Claim 6, conducted substantially as described herein.

11. A process as claimed in Claim 6, conducted substantially ad described in any one of the Examples herein. Attorney for Applicant