DE3019554A1 - Attenuated live vaccine prodn. - using virus produced by trans-infection, i.e. by infection of a tissue culture with viral nucleic acid - Google Patents

Abstract

In the prodn. of attenuated viruses for live vaccines, the virus present in the vaccine is obtd. by transfection, i.e. by infection of tissue cultures with previously isolated viral nucleic acid with the addn. of a substance which increases the infectiousness of the viral nucleic acid. The viral nucleic acid is pref. that isolated from members of the Picorna virus group Coxsackie A(1-24) and B(1-6), Echo(1-34), Polio 1,2,3 and Rhino(1-80). The pref. infectiousness-increasing additive is DEAE-dextran (molecular weight 500,000) in a concn. of 250 meg/ml. The rapid process gives predictable attenuation.

Description

Process for the production of pathogenicity by means of transfection

weakened viruses for live vaccines in the fight against virus-related Infectious diseases in humans comes from the use of so-called live vaccines, i. H. Vaccines that are weakened in their pathogenicity, but fully reproducible in the person being vaccinated (-attenuated) pathogens contain very special meaning.

The live vaccines against polio, measles, rubella, Called mumps and yellow fever.

So far, the recovery of attenuated vaccine viruses has only been accomplished lengthy passage of the wild virus in the animal and for the multiplication of the virus non-optimal tissue culture systems possible, with those leading to attenuation Steps were unpredictable and found in arduous trials through trial and error had to be.

Surprisingly, it has been shown that attenuated viruses, as they are needed for the production of live vaccines, specifically in comparison can gain a short time by extracting the nucleic acid of the virus and in suitable cell cultures with the addition of the infectivity of the nucleic acid increasing substances used to trigger virus production (transfection) As an example, in Table 1 are corresponding findings for Coxsackievirus of the type A7 shown.

The concentration of infectious virus, expressed in that for 50% the submitted cells infectious virus dose (TCID50), was for a wild virus Coxsackie A7 and an attenuated Coxsackie obtained therefrom by transfection A7 determined.

Graduated dilutions of these virus suspensions were made to groups administered to newborn suckling mice within 24 hours of birth (per animal 0.1 ml virus suspension subcutaneously dorsally, 12 animals per virus dilution). For one Observation time of 14 days was the rate of deaths as a result of infection Animals and determined the virus dilution according to the method of Reed and Muench, which- just sufficed to kill 50% of the animals.

Thereafter, the dose corresponding to this dilution of infectious Virus (TCID50) calculated. It was found that about 508 of the animals were killed 105 times more transfected virus was required than in the case of the Wild virus. This means an approximately 105-fold weakening of the pathogenic effect of the Ooxsackievirus as a result of the transfection.

Because of the well-known parallelism of the pathogenicity of Coxsackie viruses in suckling mice and humans it can be concluded from this that the attenuated virus in humans as a vaccine virus with full immunogenicity and no pathogenic effect would be applicable.

Example: 12.5 ml of Coxsackievirus A7 (TCIDS0 / 0.2 ml) were after addition of 18 SDS (sodium dodecyl sulfate) with 12.5 ml of phenol saturated with water 600C shaken vigorously for 5 minutes. The phases were centrifuged in a Minifuge II in the 3350 rotor (20 ', 40C,' 4000 rpm).

The aqueous layer was gently washed with a ribonuclease-free Pasteur pipette peeled off and treated again as described. After that, that became Phenol is removed by shaking 7 times with 40 ml of cold, buffer-saturated ether. The ether was then ribonuclease-free by bubbling nitrogen through it Pasteur pipette eliminated.

Appropriate cell cultures (HSp-2) were made with PBS (0.15 M NaCl, 0.01 M Phosphate pH 7.0) or nHank's saline solution washed very thoroughly to remove any serum residues completely removed. Thereafter, the cultures were 30 'at room temperature with 250 μg / ml DEAE-dextran (5 ml on 680 cm "cell lawn) was incubated Pour off the 5 ml of the ribonucleic acid suspended in 250 ug / ml DEAE-dextran Cells added (3 ml nucleic acid per 680 cm2 cell lawn) and 60 'at room temperature incubated.

Then 300 ml of tßedius Eagle with Earle's salts, 2% calf serum, 100 IcE. Penicillin and 100 mcg streptomycin / ml added and incubated at 370C until a complete cytopathic effect occurs 4 - 5 days after infection.

The virus obtained from the transfection with ribonucleic acid was detached from the cell detritus by repeated freezing and thawing and this through Centrifugation separated. This was followed by a TCID50 determination and a bacterial one Sterility control.

Table 1 Compared the pathogenicity of wild virus Coxsachie A / and the attenuated virus obtained therefrom by transfection in a sucking test.

TCID50 Number of infectious HEp-2 cells virus units (TCID50) just enough Coxsachie A7 (wild) 106.16 104.98 to kill 50% of the animals Coxsachie A7 (trans- 107.39 1010.1 ferred)

Claims (1)

Claims Claim 1: A method for producing attenuated viruses for live vaccines, characterized in that s3afi is present in the vaccine Virus by transfection (infection of tissue cultures with previously isolated virus nucleic acid obtained with the addition of the infectivity of the viral nucleic acid increasing substance) will. Claim 2: The method according to claim 1, characterized in that the viral nucleic acid of members of the picornavirus group Coxsackie A (1 - 24) and B (1 - 6), Echo (1 - 34), Polio 1, 2, 3 and rhino (1 - 80) are obtained. Claim 3: The method according to claim 1, characterized in that as an additive which increases the infectivity of the nucleic acid when inoculating the Cell cultures DEAE-Dextran (MW 500,000) used in a concentration of 250 uq / nl will.