SE447789B - PROCEDURE FOR THE PREPARATION OF SUMMARY-MENINGO-ENCEPHALITIS VIRUS VACCINES AND BY THE PROCEDURE VACCINES PREPARED - Google Patents

Description

g447 789 type according to the invention in that a) the culture of the virus enterprises in suspensions of avian embryonic cells, in particular chicken embryonic cells, in a manner known per se, b) that before the purification of the suspension enterprises an inactivation of the virus by treatment down f -propiolactone or formalin and by the addition of protanine sulphate constituents are precipitated as sediments faster than FSME virus, and which are separated by centrifugation and c) after ultracentrifugation check using sucrose as density gradient the fractions are collected. which at 260 nm show an increased extinction and which contain parts with a sedimentation constant in the range of 200 S.

In a preferred embodiment, the suspension containing the virus before the continuous density gradient ultracentrifugation is separated more rapidly as ISHB virus sedimenting constituents by the addition of protamine sulphate.

Conveniently, the virus suspension is concentrated before the continuous density gradient ultracentrifugation by ultrafiltration.

As the density gradient, a sucrose solution of 0- $ 0% is preferably used. when using such a sucrose solution, at 260 nl a clearly increased extinction has the fraction corresponding to the density of an approximately 40 percent sucrose solution. According to a preferred embodiment, the virus peak fractions obtained after the density gradient ultracentrifugation are diluted with a human albumin-containing buffer, thereby achieving an increased stability of the viral antigen.

The continuous density gradient ultracentrifugation is a recently developed purification and concentration method of smallest parts due to its density and its sedimentation properties. This technique consists in removing particulate particles from a suspension in a continuous ultracentrifugation process, by penetrating the sealing gradients due to the effect of centrifugal force, while the proportions not settling under these conditions remain in the suspension (effluent). The particles penetrated into the sealing gradients travel during the centrifugation process towards the place corresponding to its own density, whereby a concentration of these particles is achieved here. This method was used for the first time for purification of the influenza virus, which has a sedimentation constant of about 800 S. The method is described in detail in the "Journal of Virology", December 1967, pages 1207 - 1216, article "Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation "by CB Reimer, RS Baker, RM van Frank, TE Newlin, GB Cline and NG Anderson.

Since the FSME virus has a much lower sedimentation constant, namely a value of only 200 S, a relatively low throughput speed is achieved according to the invention.

The procedure is performed in detail as follows: Chicken embryonic cells are suspended in a cell culture medium (TCM 199) (Tissue Culture Medium 199), infected with the virus 447 789 and kept under aerobic conditions at a temperature of between 25 and 38 ° C for one to five days in suspension. .

Thereafter, cells and cell constituents are separated by centrifugation; the resulting virus suspension is inactivated by formalin or ββ-propiolactone and then concentrated by ultrafiltration. In addition to the FSME virus, the suspension contains various impurities, which partly settle faster and slower than the FSME virus. The faster sedimenting proportions are separated by precipitation with protamine sulphate before the actual continuous density gradient ultracentrifugation process is carried out.

The process is carried out in detail in such a way that half of the buffer solution in the rotor of the ultracentrifuge filled with buffer solution, suitably with phosphate buffer solution at 3,000 rpm is displaced by a 50% sucrose solution, whereby a density gradient of 0-50% sucrose is formed. Thereafter, the virus containing the suspension is allowed to flow through the rotor at a rotor speed of 000 revolutions per minute at a flow rate of suitably 3 liters / hour. Under the selected conditions, Iden penetrates most of the virus particles into the density gradient. As soon as all the material has passed the apparatus, it is centrifuged for another hour, while at the same time a phosphate buffer solution is burned to pass. The rotor is then stopped carefully and the rotor contents are divided into individual fractions. Thereafter, the extinction of each fraction is determined at 260 nm and at the same time the sucrose content of the fraction is determined. The localization of the virus in the density gradient is made possible by its extinction at 260 nm. The virus binds in the area around 40% sucrose and here causes a clear increase in the extinction at 260 nm.

The attached diagram shows this context, from which the extinction of the individual fractions appears in relation 447 789 to the density gradient. The density gradient of the sucrose solution is between 0 - 50%.

The dashed curve represents the distribution of the extinction at 260 nm in the individual fractions. Within the range around 40% sucrose, fractions 10, ll and 12 have an increased, fraction ll, the maximum extinction. In this fraction there are almost exclusively 200 S virus particles without noticeably faster or slower than the virus sedimenting contaminants. The peak corresponds to a sucrose concentration of 39%. The additional peak at fraction number 25, which corresponds to a sucrose concentration of less than 10%, indicates contaminants which are discarded. The diagram also shows the relative protective participation, Eastställ in direct * mouse protection experiments) of the individual factions through different high pillars. The fraction ll thus has the highest relative protective effect.

The collected virus-containing fractions are now diluted to advantage with a human albumin-containing buffer to improve the stability of the virus antigen and then further processed into vaccines in the usual manner.

The recovered vaccines have a higher purity and can be much better tolerated by humans than the preparations prepared according to known methods. This is shown in the following table, in which in the first column - expressed as a percentage - the recatniums are divided into vaccines according to known methods and in the second column according to the present method.

In the case of fresh inoculations with vaccines prepared according to the invention, no undesirable reactions whatsoever, either locally or systematically, have been observed.

G1 447 789 Preparations prepared according to known methods- According to inventive Local pain up to 72 z 36 z General reactions Headache, fatigue up to 64% 25% Fever (total) up to 68% 19% 37.3 to 3s ° c up to see i 14 f: 38 to 39 ° c up to 27 in 4 æ more than 39 ° c up to 9 in 1 i The vaccines prepared according to the proposed procedure are characterized by a low protein content, apart from added human albumin. At the same antigenic effect, this protein content is many times, at least 10 times less than with preparations used hitherto. Also the specific protein content = protein content (human albumin not included) in micrograms per immunization dose] is clearly lower in vaccines prepared according to the invention, as can be seen from the following comparisons.

According to the invention, specific batches of immunization dose obtained in relation to known preparations are in the range between 250 and 500 pg of protein per immunization dose.

Claims (5)

10 15 20 25 447 789 P a t e n t k r a v 1. A method of producing an early summer meningoencephalitis virus (FSME virus) vaccine by culturing the virus in tissue cultures, separating the cells and cell constituents by centrifuging, concentrating and purifying the virus by continuous density gradient ultra centrifugation and preparation of the substrate into a vaccine, characterized in that a) the culture of the virus is carried out in suspensions of avian embryonic cells, in particular chicken embryonic cells, in a manner known per se, b) that an inactive pre-purification of the suspension is carried out - ringing of the virus by treatment with β-propiolactone or formalin and by the addition of protamine sulphate, precipitating constituents which precipitate faster than FSME virus, and which are separated by centrifugation and c) the fractions which at 260 nm show an increased extinction and which contain parts with a sediment ion constant in the range of 200 S. Process according to Claim 1, characterized in that the virus suspension is concentrated by ultrafiltration before the continuous density gradient ultracentrifugation. 3. A method according to claim 1 or 2, characterized in that the virus peak fraction obtained after the continuous density gradient ultracentrifugation is diluted with a human albumin-containing buffer solution. 447 789 Vaccine prepared according to claims 1-3, characterized in that the vaccine contains a maximum of 20 .mu.g / ml of protein in addition to human albumin. Vaccine according to claim 4, characterized in that the specific protein content - apart from human albumin - ie. the protein content of fug per immunization dose does not exceed 20.