RU2445117C2 - Method for preparing combined bivalent, tissue-culture, inactivated, concentrated, purified vaccine for preventing hemorrhagic fever and renal syndrome - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to a method for preparing a combined bivalent, tissue-culture, inactivated, concentrated, purified vaccine for preventing hemorrhagic fever and renal syndrome (HFRS). Substance of the invention involves reproduction of strains "ПУУ-ТКД/VERO" and "ДОВ-ЕАТ/VERO" of PUUMALA and DOBRABA viruses in a multilayer passaged culture of VERO-line grivet monkey's kidneys involving microfiltration of a virus-containing culture fluid to remove cell debris, virus inactivation by formalin, concentration by ultrafiltration and chromatography purification of the inactivated concentrated from ballast proteins, aluminium hydroxide sorption.

EFFECT: provided creation of the vaccine to be applied in European Russia and other European countries.

8 cl, 3 ex

Description

The invention relates to the field of biotechnology, in particular to a vaccine for the prevention of viral infections, namely, hemorrhagic fever infection with renal syndrome (HFRS).

4 viruses are the causative agents of HFRS, of which 2 PUMALA and GOOD viruses cause a serious illness only in the European part of Russia and other European countries, while the other two HANTAAN and CEUL viruses exist and infect humans, usually only in Asian countries, mainly in China. Currently, commercial HFRS vaccines are produced only in China, however, none of these vaccines can be used in the European regions of Russia and other European countries, because they are made on the basis of the HANTAAN and CEUL viruses, which do not have a protective effect against of viruses "PUMALA" and "GOOD" [Yong-K. Chu, et. al., A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus, Journal of virology, Oct. 1995, p. 6417-6423; E.A. Tkachenko and S.G. Drozdov. Hantaviruses and hantavirus fevers. Epidemiology and vaccine prevention 2002, 6, p.14-18].

It is known that the difficulties associated with the development of vaccines against the viruses "PUMUMALA" and "GOOD" are due to the peculiarities of the reproduction of strains of these viruses in sensitive cell systems. They are characterized by a rather long breeding cycle, a low level of production and the absence of a cytopathogenic effect.

The aim of the invention is to obtain for the first time in the world a bivalent, cultured, inactivated, concentrated, purified vaccine against viruses "PUMUMALA" and "GOOD".

The vaccine production process includes reproduction of the virus in cell culture, collection of the virus-containing substrate, inactivation of the virus, concentration and purification of the inactivated virus, and sorption of the purified virus onto aluminum hydroxide.

To obtain a virus-containing substrate use the line of transplantable kidney cells of the green monkey VERO, certified and recommended by WHO and GISK them. L.A. Tarasevich as a possible substrate for the production of inactivated vaccines. The accumulation of the producer culture is achieved by serial passages of cell culture recovered from the "bank" of seed cells. A cell culture for virus infection is grown in 1 L roller bottles in a Needle MEM medium with a double set of vitamins and amino acids and 5% serum from cow fruits. For infection of cells using strains PUU-TKD / VERO and DOB-EAT / VERO viruses "PUMUMALA" and "GOOD", deposited in the State collection of viruses (Research Institute of Virology named after D.I. Ivanovsky RAMS) dated November 24, 2009 under No. 1026 and No. 1027, respectively. Infection is carried out 3-4 days after sieving the cells that formed a dense monolayer, without signs of degeneration and contamination by extraneous microflora. Before infection, cells are washed twice with Hanks' working solution, after which a pool of virus is added to the bottles, previously diluted with Igla 2MEM medium to a concentration that ensures a multiplicity of infection of 0.1-0.5 IU / cell with an inoculum volume of 15 ml per 1 liter roller bottle. In order to adsorb the virus onto cells, infected bottles are placed in a roller unit and incubated for 1 hour at a temperature of (37 ± I) ° С. After this time, 100 ml of cell growth-promoting medium Needle MEM with a double content of amino acids and vitamins (without adding blood serum of cow fruits) is introduced into each bottle and again placed in a roller unit in a thermostatic room with a temperature of (37 ± I) ° С. The collection of virus-containing material is carried out on the 6th, 7th, 8th, 9th, 10th day after infection and up to 5 collections of virus-containing liquid are obtained from one monolayer of cells. Only the collection of virus-containing culture fluids in which the infectious titer of the virus is at least 5.5 lg FOU / ml is subject to mixing. The virus contained in the combined culture fluid is subjected to formalin inactivation. The optimal mode of inactivation of the infectious activity of viruses includes exposure to a virus-containing substrate at a temperature of 6 ± 2 ° C in the presence of a 0.025% formalin solution for 30 days.

The inactivated pooled collection (antigen-containing culture fluid) is concentrated by ultrafiltration in a tangential flow using a concentration system with a cut-off limit of 100 kDa and a filter area of 0.1 to 0.5 m ² . The ultrafiltration process is carried out until the initial volume of antigen-containing culture fluid is reduced by 70-100 times.

At the first stage of purification of the inactivated virus concentrate, the ballast components are removed by clarification filtration using a Poly Pro XL (CUNO) filter with pore sizes and surface areas of 0.6 μm and 0.14 m ^2, respectively. The second step is gel filtration using Sepharose 6 FF and an ACT chromatograph purifier (GE Healthcare). To optimize the processes carried out at each of these stages, the parameters of the interaction of the virus with different porous sorbents are preliminarily determined. A gel filtration scheme for purifying the virus is selected, resulting in highly active preparations that satisfy the purity requirements of the final product.

After sterilizing filtration through a Millipore filter with a pore size of 0.22 μm, the chromatographically purified fraction is diluted with a buffer solution in accordance with its antigenic activity and used to obtain monovalent vaccines against the viruses PUMUMALA and GOOD. To prepare a bivalent vaccine against these viruses, the antigenic activity of each vaccine is determined and their working doses (RD) are calculated. In accordance with a pre-established relationship between the amount of specific antigen contained in the vaccine and its immunogenic activity, the minimum immunizing dose (MFA), i.e., the maximum dilution of the initial preparation, which ensures the production of virus-neutralizing antibodies in 50% of animals, corresponds to 8 antigenic units (AE). Dilution of the original drug containing 4 MID or 32 AE is taken as 1 RD of a monovalent vaccine. After reducing the equal volumes of monovalent preparations in dilutions containing 1 RD for each of the monovaccines, and adding the aluminum hydroxide adsorbent (final concentration 1.5 ± 0.5 mg / ml), the ready-made combined (bivalent) vaccine for the PUMALA viruses is obtained and GOOD.

According to the WHO requirements, vaccines made on the basis of transplantable cell cultures strictly limit the residual DNA content: it should not exceed 10 pg per 1 dose of the vaccine to exclude the potential for its oncogenic and transforming effect. There are restrictions on the content of heterologous proteins such as cattle serum proteins in the final product.

Compliance with national and international requirements for medical immunobiological preparations administered to people is achieved by the fact that the claimed method involves infection of HFRS viruses with cell pathogens with subsequent reproduction and release of the virus into the culture medium, its inactivation, concentration, purification and sorption on adjuvant. At the stage of reproduction of the virus, culture medium without the addition of cattle serum is used. Specific safety (absence of a live virus), bacteriological sterility of the vaccine are achieved using a hard mode of inactivation of the virus with formalin and filtration through a filter bag with a final pore size of 0.22 μm. The absence of tumorigenicity is ensured due to a sharp decrease in the content of cellular DNA at the stage of gel filtration (less than 10 pg in one vaccine dose). At almost all stages of vaccine manufacture, from virus strains to the finished vaccine form, a number of well-known and new physicochemical and molecular biological tests, as well as animal and cell culture controls, are provided. This system reliably ensures the safety of the new vaccine, the high level and stability of its immunological activity.

The purpose of the invention is the creation of technology for the manufacture of vaccine preparations that can be used for specific prophylaxis of HFRS. A known analogue [Y.Choi et. al. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. Vaccine 21 (2003) 1867-1873], where VERO E6 cells were cultured in spinner flasks on a Cytodex 3 microcarrier. After collection of the virus-containing liquid, centrifugation was performed to remove cell detritus. Concentration was carried out on ultrafiltration cassettes (Millipore) with a molecular weight of passable particles of 300,000 Daltons and subsequent ultracentrifugation at 80,000 g. The pellet was resuspended in Tris-HCl buffer and sucrose gradient ultracentrifugation was performed. The disadvantage of this method of obtaining and purification of viral concentrate is that centrifugation is used twice. The use of high-speed centrifugation cannot be considered technological in the scale of industrial production of vaccine preparations, since requires a lot of time and additional conditions that ensure the safety of personnel with especially dangerous viruses, which include HFRS agents. Another analogue of the present invention can be considered a patent [Huang Yongcheng et. al. Production of vaccine for epidemic hemorrhagic fever using prinmary kidney cills of golden hamster. CN 1109782], where the primary culture of Golden Hamster kidney cells served as a substrate for the production of virus-containing culture fluid. The collected virus-containing liquid was filtered and inactivated with formaldehyde. The concentration of the viral antigen was carried out by ultracentrifugation in a sucrose gradient followed by gel filtration to remove sucrose. Purification was carried out using ion exchange chromatography on a Cellufine Sulfate gel. The disadvantages of this method of obtaining a viral concentrate can be considered the use of a primary culture of Golden Hamster kidney cells. The use of primary cell cultures is associated with the risk of microbial and viral contamination of the target product. It can be noted that the use of primary cell culture in the production of viral vaccines is associated with the use of a large number of animals, and therefore, with the cost of the technological process. The use of transplantable cell cultures as a substrate for the reproduction of viruses can reduce the risk of microbial and viral contamination of the target product and significantly reduce the cost of the final product. Transferred cell cultures provide more standard virus reproduction results compared to primary cell cultures. The use of sucrose gradient ultracentrifugation requires an additional sucrose purification step. Sucrose gradient ultracentrifugation of virus concentrates allows efficient separation of viral particles from ballast proteins, but requires a large number of pieces of equipment to operate on an industrial scale. In the work [Agafonov A.P. and others. A method of obtaining concentrates of viruses that cause hemorrhagic fever and having immunogenic and protective activity. RU 2029561] the authors cultivate the Marburg virus on a culture of human fibroblast cells with the addition of human serum albumin. The resulting virus-containing liquid is filtered on filters MFA-C-0.6-1.5. After filtration, it is concentrated on an ultrafiltration system 20 times. Purification is carried out using diafiltration with a ten-fold volume of Hanks solution. A significant difference between this example of obtaining viral concentrates and the claimed invention is the fact that in the process of reproduction of HFRS virus viruses in VERO cells, serum of the fetal cattle is not used. After diafiltration of the viral concentrate, the authors determine that the concentration of total protein in the preparation is 2.5 ± 0.5 mg / ml, whereas in the claimed method, after chromatographic purification of the virus-containing material, the total protein content in it is 0.2-0.3 μg / ml The use of chromatographic purification in the proposed method allows to reduce the concentration of total protein by about ten times. An analogue of the present invention can be considered the article [Immunization effect of purified bivalent vaccine to haemorrhagic fever with renal syndrome manufactured from primarycultured hamster kidney cells. Chin Med J. 2005 118 (9), 766-768], where viral strains isolated in China are cultured in a primary hamster kidney cell culture. The resulting virus-containing liquid is inactivated with formaldehyde and purification is carried out on a column with Sepharose 4 FF. A significant difference between the method described above and the claimed method are: the use of a primary cell culture as a substrate for the reproduction of viruses, as well as the absence of a stage of concentration of the virus-containing liquid. The claimed method uses a transplantable cell culture, which reduces the risk of microbial and viral contamination of the virus-containing fluid. In addition, the use of the preliminary stage of concentration of the virus-containing liquid allows the use of a virus-containing liquid with medium and low values of the titer of the virus, which in industrial production can increase the efficiency of the process. Storage of virus concentrates requires smaller areas of refrigeration and freezers, which is another positive aspect of the application of the stage of concentration of virus-containing liquid. It should be noted that the chromatographic purification of viral concentrates, the concentration index of which can be 100-200 times, requires correspondingly less time than chromatographic purification of the initial volume of virus-containing liquid.

A comparison of the claimed method with known analogues shows that distinctive features from prototypes can achieve the goal. Distinctive features are: the reproduction of hantaviruses on a transplanted VERO cell culture without the addition of cattle fetal serum, a five-fold collection of virus-containing fluid from one monolayer of cells, clarification of the virus-containing fluid from cell detritus on filters with pores from 0.6 μm to 0.22 μm, concentration on ultrafiltration cassettes 100-200 times, chromatographic purification on columns with Sepharose 4 FF or Sepharose 6 FF.

Thus, the invention consists in a method for producing inactivated purified viral concentrates for the preparation of vaccine preparations on an industrial scale.

Example 1

Virus-containing culture fluid is obtained by reproducing the PUUM-TKD / VERO strain of the PUMALA virus in a monolayer transplanted green monkey kidney cell culture (VERO). A monolayer of cells is obtained in 1 liter roller bottles in a MEM Eagle medium with a double set of vitamins and amino acids and 5% of the blood serum of cow fruits. After infection of monolayer cultures, a medium containing no serum components is used to support viral reproduction. Virus-containing fluid collection begins on day 6. From one monolayer of cells receive from 3 to 5 collections of virus-containing fluid. The resulting virus-containing liquid with an average titer of 5.5 lg CFU / ml is clarified using ultrafiltration cassettes (Millipore) with a molecular weight of passable particles of 100,000 Daltons and an area of 0.2 m ² , inactivated with formalin at a concentration of 1: 4000 at 6 ° C for 30 days and concentrate 70-200 times. The inactivated concentrate is purified on a 2.6 / 70 column with Sepharose 6 Fast Flow at a rate of 7 ml / min. The concentration of total protein in the purified concentrate is 200 ± 50 μg / ml. The purified inactivated concentrate is filtered through a 0.22 μm filter and used to prepare a monovalent vaccine adsorbed onto aluminum hydroxide.

Example 2

Virus-containing culture fluid is obtained by reproducing the ADV-EAT / VERO strain of the ADVANCES virus in a monolayer transplanted green monkey kidney cell culture (VERO). A monolayer of cells is obtained in 1 liter roller bottles in a MEM Eagle medium with a double set of vitamins and amino acids and 5% of the blood serum of cow fruits. After infection of monolayer cultures, a medium containing no serum components is used to support viral reproduction. Virus-containing fluid collection begins on day 6. From one monolayer of cells receive from 3 to 5 collections of virus-containing fluid. The resulting virus-containing liquid with an average titer of 5.5 lg CFU / ml is clarified using ultrafiltration cassettes (Millipore) with a molecular weight of passable particles of 100,000 Daltons and an area of 0.2 m ² , inactivated with formalin at a concentration of 1: 4000 at 6 ° C for 30 days and concentrate 70-200 times. The inactivated concentrate is purified on a 2.6 / 70 column with Sepharose 6 Fast Flow at a rate of 7 ml / min. The concentration of total protein in the purified concentrate is 200 ± 50 μg / ml. The purified inactivated concentrate is filtered through a 0.22 μm filter and used to prepare a monovalent vaccine adsorbed onto aluminum hydroxide.

Example 3

After reducing (before adsorption on aluminum hydroxide) equal volumes of monovalent preparations in dilutions containing 1 RD for each of the monovaccines, and adding an adsorbent-aluminum hydroxide (final concentration 1.5 ± 0.5 mg / ml), a ready-made combined (bivalent ) the vaccine against viruses "PUMUMALA" and "GOOD". The specific activity (immunogenicity) of the bivalent vaccine is tested in mice. The concentrate adsorbed on the surface of the gel of aluminum hydroxide is administered to Balb / c mice of 10-12 weeks of age, weighing 18-20 g in a volume of 0.5 ml subcutaneously three times with a 2-week interval. As a control, Balb / c mice were used, which were injected with aluminum hydroxide without viral antigen at the same time. 2 weeks after the last immunization, blood is taken from the orbital vein in animals and serum is prepared from it. In the obtained blood serum titers of virus-specific antibodies are determined. As a result of the study of sera, it was shown that in Balb / c mice, on day 28, after the first immunization, neutralizing antibodies to the PUMALA and GOOD viruses were detected. The results obtained in experiments on immunization of mice make it possible to assert that the inactivated and purified concentrate of the viruses PUMUMALA and ADVANCE induces the production of humoral immunity in mice, that is, it has high immunogenicity.

Claims (8)

1. A method of obtaining a combined bivalent, cultural, inactivated, concentrated, purified vaccines for the prevention of hemorrhagic fever with renal syndrome by reproducing the PUUM-TKD / VERO and ADD-EAT / VERO strains of the PUMALA and WELF viruses adapted for reproduction in a monolayer transplantable green monkey kidney cell culture, VERO line, including microfiltration of virus-containing culture fluid to remove cell detritus, inactivation of the virus with formalin, concentration of ultrafiltration minutes and purifying the inactivated concentrate ballast proteins by chromatography on aluminum hydroxide sorption. 2. The method according to claim 1, characterized in that the reproduction of viruses on transplantable cell culture of VERO is achieved without the addition of serum of the fetal cattle. 3. The method according to claim 1, characterized in that to obtain the viral mass, a five-fold collection of virus-containing liquid is carried out from one monolayer of cells. 4. The method according to claim 1, characterized in that specific safety is achieved by inactivation of live virus with a 0.025% formalin solution at a temperature of (6 ± 2) ° C. 5. The method according to claim 1, characterized in that microfiltration is carried out on filters with a pore size of from 0.6 to 0.22 μm. 6. The method according to claim 1, characterized in that the inactivated antigen-containing culture fluid is subjected to concentration by ultrafiltration in a tangential flow using a concentration system with a cutoff limit of 100 kDa and a filtration area of 0.1 to 0.5 m ² . 7. The method according to claim 1, characterized in that the chromatographic purification is carried out on Sepharose 4 Fast Flow or Sepharose 6 Fast Flow. 8. The method according to claim 1, characterized in that bacteriological sterility is achieved by filtration through a filter bag with a final pore size of 0.22 μm.