DE1617545C2 - Use of informational ribonucleic acid as a vaccine - Google Patents

Description

The invention relates to the field of immunization of macroorganisms (humans and animals), hereinafter also referred to as the "vaccinee".

At present, either so-called dead vaccines, killed virulent pathogens, or live vaccines, non-killed avirulent pathogens are used. Next to it are Toxides (detoxified Bacteria), toxins and subunits of viruses in use as vaccines. These conventional vaccines place high demands on the care of the manufacturer. In the case of dead vaccines, inactivation be carefully monitored, as insufficient inactivation of the natural antigen leads to undesirable effects Side effects lead to the vaccinee, on the other hand through the inactivation of the natural antigen up can be altered or damaged to some extent. Insufficient attenuation of the Viruses in vaccines made from viruses that are capable of replication could have unmistakable effects. In the The manufacture of vaccines therefore requires complex, time-consuming and costly controls. Nevertheless, intolerance reactions have been described.

All previous immunization methods have in common that the vaccinee ultimately has an antigen is applied so that in vivc, d. H. Antigen processing first takes place in the organism of the vaccinee must in order to produce the immunizing antibodies. The invention is based on the knowledge that the Disadvantages of the previous immunization methods can be attributed to this antigen application. Consequently it is the object of the invention to provide an antigen-free vaccine for the immunization of macroorganisms to deliver.

This object is achieved according to the invention by using informational ribonucleic acid, which by stimulating immunologically competent cells of the species to be immunized (human or animal) or cell-free systems obtained therefrom with antigen particles with subsequent isolation known separation methods and sterilization has been obtained, when optionally mixed with adjuvants Vaccine for the production of antigen-specific antibodies in the species concerned.

In a fundamental departure from all previous immunization methods, the invention thus provides now for active immunization not the antigen itself, but an informative ribonucleic acid (RNS), d. H. one that contains the entire blueprint for the desired antibodies.

It is already known that immunologically competent cells, for example peritoneal exudate

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b ^r > cells, informational active ribonucleic acid (RNA) can isolate, if these cells were previously stimulated in vitro with an antigen. Cell-free systems made from peritoneal exudate cells are also able to synthesize an informational RNA after an antigen stimulus. The informational RNS has already been checked for its informational content. It was found that it is able to induce cultures of lymphatic cells as well as cell-free systems from spleen and peritoneal exudate cells to synthesize antibodies (Z. med. Mikrobiol. Immunol. 152, 1-19; 20-32; 33-44; 112-133; 262-272 (1966), ZbI. Bakter. LOrig. 199, 315-328 (1966)).

Surprisingly, it has now been found that such informationally active RNA can be found in macroorganisms (Humans and animals) can inject and that these macroorganisms following the injection Form antibodies and develop a cell-bound, i.e. resilient, immunity.

The use of such an informational RNA as a vaccine has been compared to the usual Vaccines Significant Benefits:

1. The informational RNA is free from contamination. Side reactions in the vaccinee are therefore not to fear.

2. Post-vaccination immunity is achieved quickly because the macroorganism is directly affected by the A blueprint for the antibody synthesis is conveyed and an antigen processing thereby in the macroorganism becomes unnecessary.

3. Immunizations can be achieved that were previously not possible due to a lack of suitable vaccines because the informational RNA can be synthesized in vitro.

To produce such an informational RNA in vitro, the procedure is that one immunologically competent cells or cell-free systems from immunologically competent cells with a corresponding Antigen is stimulated, the informational ribonucleic acid is isolated from it and then sterilized. The process products can optionally be mixed with an adjuvant.

Under "immunologically competent cells" are to be understood those cells that are able to a primary antigenic stimulus to respond with the formation of informational RNA. The competent Cells are found in the lymph, in lymph nodes, in the spleen, in exudates - for example in the peritoneal exudate - and especially in the peripheral leukocytes (blood). The cells can be more human or of animal origin, depending on the vaccine obtained according to the invention in humans or To be applied to animals. For the production of a vaccine for a certain species is therefore of immunologically competent cells of the species concerned assumed. Human informational For example, RNA can be obtained from peripheral leukocytes, which are produced as a by-product in the Plasma recovery incurred.

Antigens and toxins are used to stimulate the immanologically competent cell material Bacteria, viruses or their subunits as well as snake venom, etc.

As bacterial starting materials are z. B. Tetanus and Diphtcrietoxin called, also the metabolism or Decay products of Bercletella pertussis, Myco-

bacterium tuberculosis as well as salmonella, shigella, Brucella and streptococci: viruses and their subunits, e.g. B. Influenza viruses, influenza hemaglutinin, Polioviruses or their protein envelope, measles, rabies, foot-and-mouth disease, smallpox, swine fever, distemper, Hepatitis contagiosacanis and encephalitis viruses.

A cellular one is used to obtain the informational RNA and to produce the vaccine System, one can generally proceed as follows:

A cell culture of immunologically competent cells (e.g. human leukocytes) is stimulated with an optimal dose of the antigen to be determined beforehand (usually one antigen particle per 100 cells) and lasted for 5-60 minutes, preferably about 20 minutes, at about 37 ° C incubated. The cell culture is then rapidly ^{cooled to at least 0 °} C. and washed. The informational RNA is isolated from the washed cells by known methods, for example by means of phenol, so that it is free of proteins and nucleases. For sterilization, the aqueous RNA suspension is sterile-filtered or ^{heated to about 100 °} C. for about 5 minutes, with it being dialyzed beforehand if necessary to remove traces of phenol. The product obtained is the vaccine; it can be mixed with an adjuvant such as aluminum hydroxide or aluminum phosphate.

If one assumes a cell-free system, one proceeds advantageously as follows.

Washed, immunologically competent cells (e.g. human or animal leukocytes from the peripheral blood) are dissolved in a buffer solution with a molarity of 0.01-0.15, preferably about 0.1 with a pH of 7.6-8.6, preferably added about 7.8. A Tris / maleic acid or Tris / HCl buffer is best. The cells are destroyed by freezing and thawing several times and the cell homogenate is ^{centrifuged for about 30 minutes at 30,000 g and 0} ° C. or below ⁰ ° C. The supernatant obtained after centrifugation is the cell-free system which contains all the soluble components of the cells. It is then stimulated with an optimal dose of an appropriate antigen and ^{incubated at about 37 °} C. for 2-60 minutes, preferably for about 10 minutes. Further processing takes place as above.

The inventive use of informational ribonucleic acid offers numerous advantages for the Manufacture of vaccines. The process step of inactivation, which is necessary in the customary production of vaccines, is omitted. Side reactions (allergic reactions) and vaccination damage are not to be feared, as the vaccines are absolutely antigen-free. the Starting materials for the process can easily be obtained in larger quantities. The cost of the Procedural are minor.

The tolerance of the informational ribonucleic acid of the influenza virus was tested on mice. The mice received 0.25 ml of an aqueous suspension of the informational ribonucleic acid intraperitioneally administered. The mice showed no reaction. The vaccine used according to the invention therefore does not work toxic.

To determine the effectiveness, a protection test was carried out on mice and that The product used in the invention is an egg-bred flu virus according to the prior art contrasted. The preparation of the two preparations to be compared (both without adjuvant) was used assumed the same amount in each case. As can be seen from the table below, that is according to the invention used vaccine more effectively than one made according to the prior art.

preparation

protection against

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(Load capacity)

Informative RNA (Antigen Free) Influenza Virus Bred from Egg

10'- ⁶ LD ₅₀ 10 '° LD ₅₀

The table shows the result of a stress test. A larger number of comparing the two - "> the preparations immunized mice increasing doses of the active influenza virus has been exposed Here, the value rose to LD ₅ O (that is the dose lethal to 50% of the animals) mice at those obtained with. the preparation according to the prior art were immunized to the

10 times the value for non-immunized mice, but for the informational RNA used in ^accordance with the invention to 10 times this value.

example 1

The leukocytes are made from 100 ecm of human blood isolated. They are washed with physiological saline solution and then in a 0.1 molar

in Tris / maleic acid buffer of pH 7.8, which is also 0.01 molar of KCl, 0.005 molar of magnesium acetate and 0.006 molar of 2-mercaptoethanol is added. The cell suspension is frozen 10 times and thawed. The cell homogenate is at 30,000 g and

i "> 0 ⁰ C for 30 minutes. The sediment is discarded, the cell-free system is mixed with ΙΟ ^-3 units of the receptor for the phage κ (kappa) from Serratia marcescens and ^{incubated in a water bath at 37 0} C for 10 minutes. After that, the RNA will go to Kirby

■ "> isolated.

The determination of the antibody synthesis brought about by the vaccine obtained in the cellular (A) and cell-free (B) system is carried out as follows:

A) Examination of the informational RNS in one

⁴ 'cellular system:

^{A lymph cell culture with 10 7} cells / culture is set up from human lymph nodes. 0.3 ml of an aqueous solution of the above vaccines with an optical density of 0.004 are added to this lymph cell culture

><> given, measured at 260 πψ. After two days, the supernatant from the cell culture is separated off and tested for its antibody content in the receptor neutralization test. 10 ¹⁰ antibody molecules were detected per ml of tissue culture fluid.

> ^r > B) Testing of the RNA in a cell-free system from human leukocytes:

A cell-free system is produced as described above. The deoxyribonucleic acid present in this system is broken down with deoxyribonuclease.

^bf i As a result of this treatment, the cell-free system is no longer able to synthesize informational RNA or antibodies after the addition of antigen. It is therefore used as a test system for the informational content of an RNA preparation that is used as a vaccine

^b ^ should be suitable. 0.1 ecm of such a cell-free system is added 0.1 ecm of an RNA preparation with an optical density of 0.004 (at 260 μm). The batch is at 37 ° C for 30 minutes

incubated; then the amount of antibodies synthesized after the addition of the RNA is measured in the receptor neutralization test. The antibody titer was 10 ^q antibody per ml tissue culture fluid.

Example 2

According to Example 1, a cell-free system was created from human leukocytes with equine Milford hemagglutinin stimulated and made a vaccine from it as described. With this vaccine were cell-free Systems made from human leukocytes for antibody synthesis. There could be 5 x 10 "antibody molecules / ml be detected.

Example 3

According to Example 1, a culture of peripheral leukocytes from rhesus monkeys was stimulated with equine Milford hemagglutinin and a vaccine (informational RNA) was produced therefrom. This vaccine was injected in an amount of 0.5 ml (intramuscular dilution 1/20, optical density 0.002) to two rhesus monkeys. The two immunized monkeys had an antibody titre of 6.0 · 10 ⁹ and 12.0 · 10 ¹ * per ml of serum. The two control animals did not have any

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Antibodies raised against equine Milford hemagglutinin.

Example 4

According to Example 1, a culture of peritoneal cells from NMRI mice was stimulated with equine Milford hemagglutinin and a vaccine (informational RNA) was produced therefrom. NMRI mice were injected intraperitoneally with this vaccine. Such animals were found to be immune to stress infection with the above types of virus. The serum contained 6.7 x 10 ⁹ antibodies per ml, the controls produced no antibodies.

Example 5

According to Example 1, a cell-free system of spleen cells from rhesus monkeys with hemagglutin in the Influenza viruses of types A, Ai, A2, Swine, B Lee and PR 8 stimulates and a vaccine is made from it. Rhesus monkeys were injected with this vaccine. With Vaccine (informational RNA) treated animals then formed antibodies as in Example 3 described.

Claims (1)

Claim: Use of informational ribonucleic acid obtained by stimulating immunological competent cells of the Spe- -> to be immunized zies (human or animal) or cell-free systems with antigen particles obtained therefrom, subsequent Isolation with known separation methods and sterilization has been obtained, as if necessary with Adjuvants added to vaccines for the formation of antigen-specific antibodies in the affected area Species.