GB2038179A

GB2038179A - Meningo-encephalitis virus vaccines

Info

Publication number: GB2038179A
Application number: GB7942857A
Authority: GB
Original assignee: Immuno AG; Immuno AG fuer Chemisch Medizinische Produkte
Current assignee: Oesterreichisches Institut fuer Haemoderivate
Priority date: 1978-12-22
Filing date: 1979-12-12
Publication date: 1980-07-23
Also published as: SE7910054L; FR2444466B1; SE447789B; ATA922078A; BE880732A; GB2038179B; YU311779A; CS223975B2; DE2950004C2; IT7928341A0; CH644271A5; AT358167B; YU42204B; FR2444466A1; SU1003738A3; SU1318149A3; DE2950004A1; IT1126676B

Abstract

Meningo-encephalitis virus (TBE-virus) vaccines are produced by culturing the virus in tissued cultures or in suspensions of avian embryo cells, separating the cells and cell debris by centrifugation, inactivating and concentrating the virus, purifying the suspension containing the virus by subjecting it to a continuous flow density gradient ultracentrifugation in which the gradient content is divided into fractions and the fractions exhibiting an increased absorbance at 260 nm and containing particles with a sedimentation constant in the range of 200 S are collected, and processing the purified suspension into vaccines.

Description

Method of producing early-summer meningo encephalitis virus (tick-borne meningo-encephalitis virus, TBE-virus) vaccines, and vaccines produced according to this method The invention relates to a method of producing early-summer meningo-encephalitis virus (tick borne meningo-encephalitis - TBE-virus) vaccine by culturing the virus in tissue cultures or in suspen sions of avian embryo cells, in particular in in suspensions of avian embryo cells, in particular in suspensions of chick embryo cells, separation of the cells and of cell debris by centrifugation, inactiva tion, concentration and purification of the virus, and processing the purified suspension to administrable preparations.

With a known method of such kind for preparing TBE-virus vaccine, as discribed in British patent No.

1,453,035, after centrifugation of the suspension, a virus-containing suspension in obtained in which undesired contaminations are still present, which may lead to side reactions when applying the vac cine. Therefore, it is necessary to subject the virus containing suspension to a purification method, e.g.

to a hydroxyl-apatite chromatography. The known method works with considerable losses. Yet, they have not succeeded in reaching the desired degree of purity.

The invention aims at avoiding the disadvantages and difficulities described and has its object to obtain early-summer meningo-encephalitis virus vaccine with an improved degree of purity and an improved yield with which, when being applied, the occurrence of side reactions is strongly reduced.

This object is achieved according to the invention with a method of the initially defined kind in that the virus-containing suspension is subjected to a con tinuous flow density gradient ultracentrifugation for purification, the gradient content is divided into frac tions, and the fractions that show an increased absorbance at 260 nm and contain particles having a sedimentation constant in the region of 200 S are collected.

According to a preferred embodiment, compo nents that sediment more rapidly than TBE-virus are separated from the virus-containing suspension prior to the continuous flow density gradient ultracentrifugation bythe addition of protamine sul fate.

Advantageously, the virus-containing suspension is inactivated prior to the continuous flow density gradient ultracentrifugation, preferably by formalin orp-propiolactone. Suitably, the suspension can be concentrated by ultrafiltration.

-As the density gradient, preferably a sucrose solu tion of 0-50 % is used. When using such a sucrose solution, the fraction exhibiting a clearly increased absorbance at 260 nm corresponds to the density of an approximately 40 % sucrose solution. But other density gradients can be used as well, such as a potassium tartrate solution, a cesium chloride solu tion or a glycerine solution. The fractions corres ponding to the density of the virus contain the virus particles with a sedimentation constant in the region of 200 S, thus exhibiting an increased absorbance at 260 nm.

According to a preferred embodiment the virus peak fractions that are obtained after the density gradient ultracentrifugation are diluted with a human albumin containing buffer, thus effecting an increased stability of the virus antigen.

The continuous flow density gradient ultracentrifugation is a recently developed purification and concentration technique of minute particles on grounds of their density and their sedimentation characteristics. It consists in that in a continuous flow ultracentrifugation process particulate portions are removed from a suspension by penetrating into the density gradient on account of the centrifugal force acting upon them, while the portions that do not sediment under these conditions remain in the suspension (effluent). The particles having entered into the density gradient during the centrifugation process migrate to that spot which corresponds to their own density, a concentration of these particles thus being caused there. The method was first applied for the purification of the influenza virus, which has a sedimentation constant of approximately 800 S.It is described in detail in "Journal of Virology", Dec. 1967, pages 1207 to 1216, Article on "Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation" by C. B. Reimer, R. S. Baker, R. M. van Frank, T. E. Newlin, G. B. Cline and N. G. Anderson.

Since the TBE-virus has a much lower sedimentation constant, i.e. a value of 200 S only, the flow rate is kept relatively low according to the invention.

The method is carried out in detail as follows: Chick embryo cells are suspended in a cell culture medium (TCM 199-Tissue Culture Medium 199), infected with the virus and kept in suspension under aerobic conditions at a temperature of between 25 and 38"C, between one and five days. Then cells and cell debris are separated by centrifugation; the virus suspension obtained is inactivated by means of formalin orp-propiolactone and then concentrated by ultrafiltration. The method steps of the inactivation and concentration can be carried out also.in the opposite order. The suspension, in addition to the TBE-virus, contains various impurities which sediment more rapidly for one part and more slowly for the other part, than the TBE-virus.Advantageously, the portions that sediment more rapidly are separated by precipitation with protamine sulfate, before the continuous flow density gradient ultracentrifugation method proper will be carried out.

The method is carried out in detail for instance in that in the rotor, which is filled with a buffer solution, suitably with a phosphate buffer solution, of the ultracentrifuge, half of the buffer solution is displaced at 3,000 revolutions per minute by a 50% sucrose solution, a density gradient of 0-50 % sucrose thus forming. Then the virus-containing suspension is allowed to flow through the rotor at a rotor speed of 35,000 revolutions per minute at a flow rate of suit ably I/h. Underthechosen conditions, the main part of the virus particles penetrate into the density gradient. As soon as the total material has passed through the apparatus, centrifugation is continued for one further hour, while at the same time a phos phate buffer solution is allowed to run through.Then the rotor is carefully stopped and the rotor content is divided into individual fractions. Then the absor bance is determined for each fraction at 260 nm, and the content of sucrose in the fraction is found out simultaneously. The localisation of the virus in the density gradient is made possible by its absorbance at 260 nm. The virus bands in the region of 40 % sucrose, thus causing there a distinct increase of the absorbance at 260 nm.

In the diagram annexed, this relationship is illustrated, the absorbance of the individual fractions in relation to the density gradient being visible. The density gradient of the sucrose solution ranges from Oto50%.

The dot-and-dash curve represents the distribution of the absorbance at 260 nm in the individual fractions. In the region of 40 % sucrose, fractions 10, 11 and 12 have an increased absorbance, fraction 11 having the maximum absorbance. In this fraction there are almost exclusively 200 S virus particles without noticeable impurities sedimenting more quickly or slowly than the virus. The peak corresponds to a sucrose concentration of 39 %. Afurther peak at fraction number 25, which corresponds to a sucrose concentration of less than 10 %, shows impurities, which are rejected. In the diagram also the relative protective potency (determined in the direct mouse protection test) of the individual fractions is illustrated by differently high columns.Thus, fraction 11 has the highest relative protection potency.

The virus-containing fraction collected are now advantageously diluted with a human albumin containing buffer in orderto improve the stability of the virus antigen, and is then processed into vaccine in a conventional manner.

The vaccines gained have a higher purity and are substantially more compatible for man than the preparations produced according to the conventional methods. This is reflected in the following table, in the first column of which - evaluated in per cents reactions with vaccines produced according to conventional methods, and in the second column of which reactions with vaccines produced according to the method of the invention, are indicated. With restorative vaccinations with vaccines produced according to the invention, no undesired reactions, neither local nor systemic, could be observed.

Preparations produced conventionally according to invention Local pain up to 72 % 36% General reactions headache, tiredness up to 64 % 25% Fiever (total) up to 68 % 19% 37.3 to 38 C up to 36 % 14% 38 to 39"C up to 27 % 4% more than 39 C upto 9% 1% The vaccines produced according to the method of the invention are characterised by a low protein content, apart from the human albumin added. This pro-.

tein content is lower by a multiple, at least the tenfold, than with preparations used so far, with the same antigenic potency. Also the specific protein content[= protein content (human albumin not included) in microgrammes per immunisation dose] is distinctly lower with the vaccines of the invention, as can be derived from the following com,3arisons: Charges according Specific protein content to invention in pg per immunisation dose 1 12 2 5 3 6 4 7 5 15 Compared to this, these values with conventional preparations like the range of between 250 to 500.

Claims

1. A method of producing early-summer meningo-encephalitis virus (TBE-virus) vaccines which method comprises culturing the virus in tissued cultures or in suspensions of avian embryo cells, separating the cells and cell debris by centrifugation, inactivating and concentrating the virus, purifying the suspension containing the virus by subjecting it to a continuous flow density gradient ultracentrifugation in which the gradient content is divided into fractions and the fractions exhibiting an increased absorbance at 260 nm and containing particles with sedimentation constant in the range of 200 S are collected, and processing the purified sus pension into vaccines.

2. A method according to claim 1, wherein the suspension containing the virus is subjected to a continuous flow density gradient ultracentrifugation is one from which components sedimenting more rapidly than TBE-virus have already been separated by the addition of protamine sulfate.

3. Amethod according to claim 1, wherein the virus in the suspension containing the virus is inacti voted prior to the suspension being subjected to the continuous flow density gradient ultracentrifugation.

4 A method according to claim 3, wherein the virus is inactivated with formalin orp-propiolactone.

5. A method according to any one of Claims 1 to 4, wherein prior to being subjected to the continuous flow density gradient ultracentrifugation, the virus in the said suspension is inactivated and the suspen sion is concentrated by ultrafiltration.

6. A method according to any one of claims 1 to 5, wherein a sucrose solution of Q-50% is used as the density gradient.

7. A method according to any of Claims 1 to 6, whereintheviruspeakfractionsobtained afterthe continuous flow density gradient ultracentrifugation are diluted with a buffer solution which contains human albumin.

8. A method according to any of the preceding claims, wherein the avian embryo cells are chick embryo cells.

9. A method according to Claim 1 substantially as hereinbefore described.

10. A method according to Claim 1 substantially as hereinbefore described, with reference to the accompanying drawing.

11. A vaccine when produced by a method as claimed in any one of the preceding claims.

12. A vaccine according to Claim 11, containing at most 20 yg/ml of protein, exclusive of human albumin.

13. A vaccine according to Claim 12, wherein the specific protein content, exclusive of human albumin, amounts to at most 20,at per immunisation dose.

14. A vaccine according to Claim 11 substantially as hereinbefore described.