DK143201B - PROCEDURE FOR THE PRODUCTION OF VIRUS STEMS THAT STIMULATES THE NATURAL DEPARTMENTS OF A HOST ORGANISM WITHOUT THEREFORE AT THE SAME TIME ACTING ANTIGENT - Google Patents

Description

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The present invention relates to a method of producing viral strains which stimulate the natural defense mechanisms of a host organism without thereby simultaneously acting antigenically.

5 By "antigenic effect" is meant herein and hereinafter the ability to trigger an immunological cross-reaction with starting virus.

It is known that the natural primary defense mechanisms against viral infections are mainly triggered by the production of interferon. However, it cannot be ruled out that other, unknown factors also play a role.

Interferons are proteins that, in vertebrates, elicit a nonspecific and non-immunological response to viral infections. For example, when viruses enter the organism of a warm-blooded animal, it is first and foremost cells of the so-called "reticuloendothelial system" that, over a few hours, produce large amounts of interferon and produce a high interferon level in the circuit.

This "circulating interferon" is rapidly distributed in the body and further prevents the spread of the viral infection, e.g. a secondary infection.

It is already known that interferon formation in an organism can be stimulated with both active and inactive viruses, ie. viruses that can no longer multiply, cf. G. Bodo: "The Natural Science" 58 (1971) 425-429, J.L. Le Clero and J. Cogniaux: "Le Clerc, Acta Virol" 9 (1965) 18-24, S. Hermodsson: "Acta path, microbiol. Scand." 62 (1964), 224-238 and M. Harris: "Science" 170 (1970), 1068-1070.

The disadvantage of the therapeutic use of these methods consists in the simultaneous induction of virus-specific antibodies in the host organism. Thereby, repeated use of such viruses is hampered or hindered to stimulate nonspecific mitigation mechanisms 35 due to the risk of allergy and anaphylactic shock at certain time intervals.

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There are a number of additional, completely different microorganisms and substances that can induce interferon formation in the organism. Examples of this include bacteria, endotoxins, phytohemaglutinins, natural and synthetic ribonucleic acids, e.g. poly-inosine-polycytidylic acid (poly I: C) as well as certain synthetic polymers with anion character such as e.g. polyvinyl sulfate, polyacrylic and polymethacrylic acid, and pyran copolymers, cf. Y.K.S. Murthy and H.P. Other: "Angew. Chem. Internat." 10 9th Edition (1970) 480-488. These substances all have the weighty disadvantage of being too toxic, for example. are not physiologically degradable (synthetic polymers) or have other strong side effects (poly I: C) so that they are clinically unusable, cf. Y.K.S. Murthy and H.P.

15 Other: "Angew. Chem. Internat.", 9th edition (1970), 480--488, "Nature" 223 (1969), 715-718.

From the disclosure of Danish Patent No. 119,724, a method for preparing an antigen-free vaccine is known in which immunologically competent cells or preferably cell-free systems of immunologically competent cells with an antigen are grown for 2 to 60 minutes at ca. 37 ° C, isolates the formed informational ribonuclein acid in known manner, e.g. with phenol, and sterilizing and optionally adding a carrier. In this known method, it is intended in the shortest possible time to induce immunocompetent cells to form antibodies to shorten the relatively long time it takes for a body to produce antibodies themselves.

It has now been found that the detectable specific immune response of the host organism to a viral strain can disappear with complete retention of a nonspecific defense mechanism when a corresponding virus strain is attenuated by reviewing a sufficient number of tissue cultures on cell cultures in which that virus can be propagated. , in a manner known per se, e.g. by the method described in German Publication No. 2,033,946.

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The consequence is that a virus so attenuated upon administration does not elicit the clinical symptoms characteristic of the virus in question in a host organism, or respond with an immune response by formation of specific, detectable antibodies. The nonspecific stimulation of a defense mechanism, e.g. in the form of interferon induction, however, is retained. Furthermore, such attenuated viruses are well compatible and do not exhibit the 10 toxicity symptoms described for the above substances.

The invention thus relates to a method characterized in that a virus strain selected from infectious pustular vulvovaginitis IPV virus strain, Aujeszky / Pseudorabies virus strain S / T and influenza virus A 1 strain FM 1 undergoes per se known methods. tissue culture passages until, by known test methods known per se, no virus-specific immune response to the starting virus is detected, but only a nonspecific interferon induction.

Surprisingly, it has been found that the virus strains attenuated by sufficiently further passage, according to the invention, no longer exhibit the frequently observed immunological cross-reaction with the starting virus, i.e. does not exhibit "antigenic effect" as defined above, but only the desired nonspecific interferon induction. Thereby, the clinical symptoms of the use of insufficiently attenuated virus strains are avoided. Thus, the process of the invention constitutes enrichment of the pharmaceutical.

The virus strains attenuated by the method of the invention can be used for prophylaxis and treatment of viral infections in both human and veterinary medicine.

The viral solutions prepared by the process of the invention can be formulated according to conventional methods and administered as e.g. solution, syrup, emulsion, suspension, spray, ointment, paste, cream, lotion, aerosol and tablets.

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The compositions are prepared in a manner known per se, e.g. by diluting the virus attenuated by the process of the invention with suitable solvents and / or inert, non-toxic, solid, semi-solid or liquid carriers, optionally using emulsifying and / or dispersing, spraying or propellants and using suitable stabilizers.

Examples of such solvents, carriers, emulsifiers or dispersants are mentioned here: water, non-toxic, organic solvents or diluents such as paraffins (eg petroleum fractions), plant oils (e.g. peanut / sesame oil) ), alcohols (e.g., ethyl alcohol or glycerol) and glycols (e.g., propylene glycol or polyethylene glycol), solid carriers such as e.g. natural stone flour (e.g., high-dispersion silicic acid, silicates), sugar (e.g., cane, milk and grape sugar), emulsifiers such as nonionic and anionic emulsifiers (e.g., polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, alkyl sulfonates and arylsulfonates), dispersants (e.g., lignin, sulfite waste liquor, methyl cellulose, starch and polyvinylpyrrolidone) and lubricants (e.g., magnesium stearate, talc, stearic acid and sodium lauryl sulfate).

Stabilizers include: amino acids, sugars, proteins, polysaccharides and polyalkylene glycols. These stabilizers can be used in both aqueous solution and lyophilized state.

The attenuated viruses or the drugs produced by the method according to the invention can be used in conventional manner, e.g. orally, intramuscularly, subcutaneously or locally, especially on all human and animal mucous membranes.

When used in human medicine, 0.1 to 5 ml, preferably 0.5 to 2 ml, is administered by tramuscular and

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5 poison in spray form 0.5-5 ml, preferably 1-2 ml, of a solution containing 300-800, preferably 400-500 CCA units per ml.

When using the virus strains 2 in question in the veterinary field, the dosage range depends on the individuals to be vaccinated as well as the administration of two.

The process according to the invention is illustrated in more detail in the following examples. It is noted that the vaccine used contains a total of 10 ^ - 10® KiD ^ ** ^ units per ml, preferably 10®-107 KiD,. g units per ml.

For intramuscular administration to cattle, it has been found appropriate to use 0.5-10 ml, preferably 1-5 ml vaccine of the above concentration. When administered in spray form, 1-15 ml, preferably 2-6 ml, is used.

For intramuscular administration to pigs, it has been found appropriate to use 0.5-10 ml, preferably 1.5-5 ml, of a vaccine of the above concentration. When administered in spray form, pigs use 1.5-10 ml, preferably 2-5 ml.

Example 1

The description described in German Publication No. 2,033,946, attenuated via 200 tissue culture passages

Muu j undergoes another 150 tissue culture passages in the manner described therein.

No clinical symptoms occur when applied to the respiratory and genital mucosa of the animals. It is not possible by any of the commonly used methods, after the usual period of 4 weeks after inoculation with the starting chicken cell agglutination unit, etc.)

KiD = culturally infectious dose 35 xxx) -j.pv _ infectious pustular vulvaquinitis = Exanthema coitale vesiculosum bovis = bladder rash.

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6 virus to detect reactive antibodies in serum. However, there may be detected interferon formation, which has caused the animals to be protected before the virulent virus infection.

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Example 2 In 4 herds - 1 insemination station and 3 breeding use - the animals suffer partly from cough and partly from nonspecific genital infections, apparently not from viral origin. Following vaccination with the interferon-inducing virus strain described in Example 1, the affected animals are completely healed within a few weeks. Humoral t immune response to exit virus is absent.

Example 3

The AUJESZKY / Pseudora biliary virus strain S / T attenuated at 300 passages is inoculated partly intranasally and intramuscularly to pig populations suffering from nonspecific enzootic pneumonia in the respiratory tract. After 3 weeks, the disease symp- 20 tumors - as opposed to control animals - have disappeared and the specific immune response to the starting virus is absent.

Example 4 2- Influenza virus A 1 - strain FM 1 is adapted to primary monkey kidney cultures and undergoes 350 tissue culture passages.

With thus attenuated virus solutions, 20 subjects aged 30-40 years are vaccinated whose serum antibody titers against A 1 - FM 1 are <1:32, each with X 1

XX) Interferon can be detected by conventional methods such as the plaque reduction method, reduction of the viral hemag-glutin titer or by quantitative hemadsorption method.

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143201 500 CCA units per ml. The administration is done in 10 people subcutaneously and in 10 people intranasally.

4 days after vaccination, the experimental treatment of the vaccinated subjects as well as 10 control subjects of the same age group with serum antibody titers <1:32 is performed by intranasal injection of 500 CCA units per day. ml of a pathogenic A 1 - FM 1 virus solution.

In the drawing, FIG. 1 the size of the serum antibody titer (ordinates) in the 20 different subjects prior to vaccination.

FIG. 2 shows the same titers 14 days after the active virus treatment.

The vaccinated individuals are protected against the infection, while 9 of the 10 control subjects exhibit influenza symptoms.

FIG. 3 shows the serum antibody titers of the 10 control subjects before the active treatment.

FIG. 4 shows the serum antibody titer 14 days after the active virus treatment. Only with control person no.

20 6 no flu symptoms are found.

In the serum of the vaccinated subjects, no increase in the virus-specific antibody titer was observed (Fig. 2). The antibody titer against A 1 - FM 1 in the convalescence serum is between 1:32 and 1: 128 (Fig. 4).