RU2501568C1 - Recombinant vaccine for prevention of viral hepatitis e in animals - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to genetic engineering. What is presented is a recombinant vaccine for the prevention of viral hepatitis E in animals. The vaccine contains a capside protein (rtHEV-ORF2) prepared by culture of the recombinant Hansenula polymorpha yeast strain KBT-11/pHEV-001 containing a DNA sequence integrated into a yeast cell genome and coding a fragment of an amino acid sequence in position 86 to 607 of the capside protein of hepatitis E virus genotype 3 (rtHEV-ORF2) under the control of a MOX gene promoter. The vaccine contains an effective amount of the protein rtHEV-ORF2, an oil adjuvant and a physiologically acceptable solvent.

EFFECT: prepared vaccine is high-immunogenic, non-toxic, and possesses no side effects.

2 cl, 5 ex

Description

The invention relates to the field of biotechnology, namely genetic engineering, and for the creation of a recombinant vaccine for the prevention of viral hepatitis E in animals, containing as its active component the capsid protein of the hepatitis E virus (HEV) obtained by culturing the strain of yeast Hansenula polymorpha KBT- 11 / pHEV-001.

Hepatitis E is a viral infection belonging to the conditional group of fecal-oral hepatitis, characterized by liver damage, acute cyclic course, often with high fever, arthralgia, as well as severe manifestation in pregnant women. The reservoir and source of infection is a person, patient, or carrier. Recently, a zoonotic reservoir has been considered as a source of infection for most people, since hepatitis E virus with genotype 3 has also been found in wild and domestic pigs. A few studies conducted in the Russian Federation also demonstrated the presence of antibodies to the hepatitis E virus in people who do not travel to endemic areas, which indicates the contact of the population with the virus. One of the possible explanations for the high detection rate of antibodies in such individuals is infection from animals, since it is known that in vivo the virus can infect such wild and domestic animals as wild boars, deer, pigs, sheep, chickens, etc.

Hepatitis E virus belongs to the Hepeviridae family of the genus Hepevirus. Virus particles are spherical formations with a diameter of 27-34 nm, built from identical structural elements, and deprived of the outer shell. Viral particles react with serum antibodies from infected individuals from geographically different regions, suggesting the widespread prevalence of HEV. Serologically and molecularly, genetic HEV is different from other types of hepatitis viruses. The sequence of the HEV genome was first determined in 1991 (Tam A.W. et.al., 1991 Virology, 185, 120-131). The hepatitis E virus genome is represented by single-stranded RNA of positive polarity. Analysis of the genome sequence showed that its size is 7.2 kb, and it contains 3 open reading frames (ORFs), each of which encodes the synthesis of a specific protein or group of proteins. ORF1 encodes the non-structural proteins of the virus responsible for virus replication, ORF2 encodes the structural capsid protein of the virus (ORF2 protein), ORF3 encodes an auxiliary protein, the function of which has not yet been determined. Using mapping, it was found that linear antigenic epitopes are localized in the polypeptides encoded by ORF2 and ORF3 (Khydyakov Y.E. et al., Virology 1993, 194, 89-96). The recombinant protein ORF2 is capable of self-assembly into virus-like particles (VLP), similar in their antigenic properties to the capsid protein of an infectious virus.

Phylogenetic analysis, based on the full length of the genome sequence of some HEV isolates, indicates the existence of 4 different genotypes, each of which dominates in a particular geographical area. Genotype 1 includes strains from Asia and Africa, genotype 2 - a Mexican strain and several variants from Africa, genotypes 3 and 4 include human and pig HEV strains from industrial countries and Asia. Genotypes 1 and 2 were found only in humans, while genotypes 3 and 4 in humans, as well as pigs and other animals. Genotype 3 is ubiquitous; genotype 4 is more common in China and Japan. The discovery of viruses similar to HEV in pigs (pork HEV) and chickens (avian HEV) suggests that the virus belongs to zoonosis. Despite the existence of different genotypes, one HEV serotype has been identified. All virus subtypes have major cross-reactive epitopes that allow the development of a capsid protein-based recombinant vaccine that is effective against many strains of HEV.

Currently, the development of recombinant vaccines, along with monitoring and identification of HEV carriers, is a promising area in the fight against infection caused by HEV. The difficulties hindering the development of effective vaccines against hepatitis E virus are primarily associated with the development of optimal expression systems for the production of recombinant proteins with immunogenic properties of natural proteins.

Examples of use for the production of recombinant HEV proteins of the bacterial expression system based on E. coli are presented in the scientific and patent literature. Thus, it is known to obtain a recombinant chimeric protein trpE-C2 containing 439 C-terminal amino acids of the HEV genoform ORF2 protein of genotype 1 fused to the N-terminal region of tryptophan synthetase in a bacterial expression system in E. coli (Kamili S., Virus Research 2011, 161 (1): 93-100). The protein expressed in E. coli has high immunoreactivity and contains cross-reactive epitopes recognized by antibodies to HEV. As a result of vaccination of monkeys with trpE-C2 protein adsorbed on aluminum hydroxide, the induction of a humoral immune response was detected.

The preparation of an immunoreactive pE2 polypeptide originating from the carboxyterminal region of a protein encoded by the ORF2 genome of the Chinese strain D11092 is described. This polypeptide, due to the presence of a conformational antigenic determinant, is capable of forming active homodimers from monomers (US 7,204,989, 04.17.2007). The pE2 polypeptide was expressed in E. coli cells using the pGEX vector. When cloning in this vector, the sequence encoding pE2 was linked in frame with the glutathione 3-transferase (GST) gene. The result was a hybrid gene encoding a polypeptide with GST at the N-terminal end and pE2 at the C-terminal end. Such a composite protein obtained by expression of a hybrid gene in E. coli cells was soluble and could be isolated from the cell lysate by binding to glutathione agarose. The pE2 polypeptide was separated from GST using a specific protease. The pE2 polypeptide expressed in bacterial systems is considered as a potential candidate for HEV vaccines. The proposed vaccine for immunization against HEV contains an immunologically effective amount of pE2, a pharmacologically acceptable carrier and an adjuvant. The vaccine can be administered orally or parenterally.

Despite the fact that the bacterial expression system is used to obtain recombinant proteins quite widely, there are drawbacks to its use associated with the solubility of proteins produced in E. coli, as well as with the difficulties encountered during serological immunoassays. In addition, recombinant fragments of the HEV capsid protein expressed in bacterial cells have a significantly shortened amino acid sequence, which can negatively affect their antigenic properties. In this regard, it is more preferable to use a eukaryotic expression system to obtain recombinant HEV proteins, in particular baculovirus or yeast.

It is known to use a 549 amino acid residue of the ORJF2 protein of the Mexican and Burmese strains as the HEV antigen. This fragment was designated as "antigen 62K" (US 6,291,641, 09/18/2001). The antigen was produced in a baculovirus expression system. For this, a recombinant baculovirus was obtained, which transfected insect cells with SF9. Transfected cells were cultured either in suspension or in a monolayer. Recombinant antigen 62K was obtained mainly in soluble form. The isolated antigen showed immunoreactivity with the serum of individuals infected with HEV, and was able to form VLP according to electron microscopic data.

US Pat. No. 6,458,562 (10/01/2002), considered as the closest analogue, describes the preparation of various truncated variants of ORF2 protein from the Pakistan HEV line (SAR-55) in the eukaryotic expression system. The obtained proteins at the N-terminus begin with the 112th amino acid, and at the C-terminus end in the amino acid region 578-607 or 660 of the wild-type ORF2 protein (as in US Pat. No. 6,787,145, September 7, 2004) and are capable of forming VLP. Viral RNA was isolated from a biological sample collected from primates infected with SAR-55. To obtain the ORF2 protein, a baculovirus vector was created by cloning the nucleotide sequence encoding the ORF2 protein into pBlueBac and transfected with it SF9 cells. After culturing the transfected cells under conditions suitable for expression of the recombinant ORF2 protein, the protein was isolated and purified using anion exchange chromatography and gel filtration. Using the ELISA method, it was shown that the ORF2 protein expressed in the baculovirus system binds to antibodies produced in response to infection with various strains of HEV and does not bind to antibodies produced in response to hepatitis A, B, C, D. viruses. ORF2 protein used as an antigen in immunoassays, as well as an active antigen in a vaccine. The effective dose of the recombinant ORF2 protein is preferably 10-50 μg. The hepatitis E virus vaccine also contains a pharmaceutically acceptable carrier, adjuvant, and may contain a stabilizer (polyethylene glycol, sucrose, etc.). The vaccine is intended for both human immunization against HEV and for use in veterinary medicine.

Currently, two candidate HEV vaccines have reached clinical trials. One of them, designated rHEV (GlaxoSmithKline Biological), contains as its active principle a recombinant HEV antigen with a molecular weight of 56 kDa obtained in a baculovirus expression system (Kamili S., Virus Research 2011, 161 (1): 93-100). The antigen used in the HEV vaccine has an amino acid sequence corresponding to amino acid residues 112-607 of the wild-type ORF2 protein. The vaccine study involved 2,000 anti-HEV-negative volunteers from the Nepal Army who were divided into an experimental (20 μg rHEV) and control group (20 μg placebo). Vaccination was carried out according to the scheme of 0, 1 and 6 months. Those receiving all 3 doses of the vaccine had a clinically pronounced acute hepatitis score compared with those receiving placebo. Despite the demonstrated effectiveness of the vaccine, many issues related to the use of the vaccine remain unclear. Thus, the safety and efficacy of using the vaccine in women, the elderly, pregnant women and patients with chronic liver diseases have not been studied, and the efficacy of this vaccine containing the antigen of genotype 1 origin against infections caused by HEV strains of other genotypes has not been investigated.

Another candidate vaccine (HEV239, China) contains a more shortened HEV antigen (amino acid residues 376-606 of the ORF2 protein) of genotype 1 obtained by expression in E. coli (Kamili S., Virus Research 2011, 161 (1): 93-100). The vaccine provided protection in primates against hepatitis E caused by genotypes 1 and 4. Clinical studies of a bacterial recombinant vaccine conducted in South China, an area endemic for genotype 1 and 4, showed the safety, immunogenicity and effectiveness of the vaccine in the general population. In addition, it was found that a vaccine based on genotype 1 provides cross-immunity against genotype 4 in humans. However, clinical trials cannot be considered complete, since many questions remained unclear. So, the duration of the protective effect of the vaccine, the safety of vaccination in pregnant women and people with chronic liver diseases have not been studied. The use of these vaccines to protect animals is not described.

Significant disadvantages of these vaccines include the fact that the vaccines protect against a clinically pronounced form of hepatitis, but not against HEV infection. Such vaccines cannot have a significant effect on the epidemiology of HEV infection in endemic areas.

Thus, due to the lack of specific therapy and licensed vaccines against hepatitis E virus in both humans and animals, it remains relevant to develop an effective recombinant vaccine containing the active HEV antigen, which has the immunogenic properties of a natural protein.

The objective of the invention is to develop a highly immunogenic, non-toxic, non-side-effect vaccine for the prevention of viral hepatitis E in animals based on the hepatitis E virus capsid protein of the ubiquitous genotype 3 found in humans and animals. The problem is solved by creating an effective recombinant yeast producer of the specified protein. The effectiveness of a recombinant yeast producer refers to the possibility of obtaining the target product with the necessary biological properties and low cost.

According to the invention, to obtain a recombinant truncated shortened capsid protein rtHEV-ORF2 (recombinant truncated HEV-ORF2), a recombinant yeast strain producing this protein is created, for which an expression plasmid containing a DNA fragment with a recombinant gene encoding rtHEV-ORF2 under the control of the promoter Hansenula polymorpha methanol oxidase and a selectable marker (Saccharomyces cerevisiae LEU2 gene) are integrated into the genome of the recipient strain H. polymorpha DL1-L. As a result of the selection of clones capable of synthesizing the rtHEV-ORF2 protein, the transformant with the highest productivity was identified, designated KBT-11 / pHEV-001. The artificial gene is integrated into the genome, i.e. is part of one of the H. polymorpha chromosomes. This ensures high mitotic stability of the strain, making it possible to optimize the conditions for its cultivation without taking into account the risk of accumulation of cells that have lost the ability to synthesize a foreign protein.

The resulting recombinant strain of H. polymorpha allows you to achieve higher levels of synthesis of the recombinant protein rtHEV-ORF2 with simpler methods for producing high density cultures.

The strain was deposited under the number KBT-11 / pHEV-001, 12.24.2011 in the collection of microorganisms of NPK Kombiotekh CJSC. The strain is new and is neither described in the patent nor in the scientific and technical literature.

The resulting recombinant producer strain was cultured under conditions suitable for expression of the rtHEV-ORF2 protein. After isolation and purification, the recombinant antigen was used as part of a vaccine to prevent viral hepatitis E in animals. The recombinant vaccine contains rtHEV-ORF2 protein in an effective amount, an oil adjuvant, and may contain thiolate as a preservative. By effective amount is meant an amount of the active principle of the present invention that is sufficient to protect animals from HEV infection. The exact amount will depend on the specific circumstances and can be estimated by a person skilled in the art using known techniques.

The invention can be illustrated by the following examples.

In the given examples, all genetic engineering operations were performed according to standard procedures and instructions of companies producing enzymes and sets for manipulating DNA in vitro. The transformation of Escherichia coli and Hansenula polymorpha cells was carried out according to the previously described methods (Inoue H. et.al., Gene, 1990, 96: 23-28. And Bogdanova AI et.al., Yeast. 1995, 11 (4): 343- 53, respectively). The synthesis of DNA fragments, as well as the determination of the nucleotide sequence were carried out by CJSC Evrogen, Moscow. A synthetic MOX-rtHEV-ORF2 double-stranded DNA fragment containing the MOX promoter H. polymorpha and an open reading frame encoding the rtHEV-ORF2 polypeptide corresponding to the 523 amino acid fragment from position 86 to 607 of the amino acid sequence of the HEV genotype 3 capsid protein (SEQ ID NO) was used : one).

Example 1. Obtaining a pHEV1 expression vector containing the MOX gene promoter and the sequence encoding the rtHEV-ORF2 polypeptide

To obtain the expression vector pHEV1, the pAM270 plasmid was used, which contains the gene encoding β-lactamase, and bacterial origin of replication to maintain the plasmid in E. coli cells, as well as S. cerevisiae LEU2 gene as a yeast selective marker and a sequence providing autonomous plasmid replication in Hansenula polymorpha cells. The synthetic MOX-rtHEV-ORF2 fragment (SEQ ID NO: 2) and plasmid pAM270 were digested with restriction enzymes BsrGI and BglII. Hydrolysis DNA fragments were purified by agarose gel electrophoresis. A reaction mixture was prepared to conduct a ligase reaction with a total volume of 10 μl containing 20 ng of isolated fragments, a single ligase buffer and 1 unit of T4 ligase DNA. The mixture was incubated at room temperature for 2 hours, after which it was used to transform E. coli strain DH5alpha. Transformants were selected on LB medium (1% Tryptone, 0.5% yeast extract, 1% NaCl) containing ampicillin (100 mg / L). Plasmid DNA preparations were obtained from several transformants. A plasmid containing the insert of the MOX-rtHEV-ORF2 fragment was selected based on restriction analysis. To confirm the absence of unwanted mutations in the cloned insert, its nucleotide sequence was determined. As a result, the pHEV1 plasmid was obtained containing the gene encoding rtHEV-ORF2 under the control of the MOX promoter and the S. cerevisiae LEU2 gene as a selective marker.

Example 2. Obtaining a producer strain of H. polymorpha expressing rtHEV-ORF2

The strain N. polymorpha DL1-L, which is an auxotroph of leucine, was grown for 15-18 hours in YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) at 37 ° C. The resulting culture was diluted 100 times with fresh YPD medium and incubated for 4 hours under the same conditions. Cells from 200 μl of the obtained culture were pelleted by centrifugation, washed with sterile distilled water and suspended in 30 μl of buffer T (10 mM Tris-HCl, pH 7.4; 100 mM CH ₃ COOLi; 0.5 mM EDTA). To the suspension, 1 μg of pHEV1 plasmid, 5 μg of carrier DNA (fragmented and denatured herring DNA) and 60 μl of a 70% solution of polyethylene glycol 4000 were added. All components were mixed and incubated for 30 min at 30 ° C and 18 min at 45 ° C. The mixture was diluted in 1 ml of YPD medium and incubated at 37 ° C for 1 hour. Cells were plated on Petri dishes with YNB-D medium (0.67% Yeast Nitrogen Base [Difco], 2% glucose and 2% agar) and incubated for several days until transformant colonies appeared. The cells of the grown transformants were scattered with a draining stroke on fresh YNB-D plates to obtain individual colonies. Among the grown subclones, the fastest growing subclones were selected and were again redistributed with an exhausting stroke into YNB-D plates. The procedure was repeated several times until the colonies growing during sieving became homogeneous in size.

The subclones of transformants thus obtained were tested for their ability to express rtHEV-ORF2 protein. One of the transformants with the highest productivity was designated KBT-11 / pHEV-001.

Cultural and morphological features of the strain: round-shaped cells, small in size, form large round colonies with a pronounced convex middle on an agarized YPD medium.

Storage - at -70 ° C in the form of a suspension of cells in a sterile 15 or 50% glycerol solution.

Genetic features: the strain is not zoopathogenic or phytopathogenic.

Method, conditions and composition of media for the propagation of the strain: incubation by pumping at 30 ° C in a nutrient medium composition: 2% peptone, 1% yeast extract, 2% glucose.

The method for determining the activity: in the clarified homogenized cells by immunoblotting or enzyme immunoassay. The strain activity is not less than 100 mg / l of culture fluid.

Example 3. Cultivation of the recombinant strain KBT-11 / pHEV-001

The fermentation of the strain producing yeast H. polymorpha was carried out in three stages at a temperature of 30 ° C. At the first stage, in the regime of periodic fermentation, biomass was increased in a culture medium containing 4% yeast extract, 2% bacto-peptone and 4% glycerol. After consumption of all glycerin by yeast (determined by a sharp increase in the concentration of dissolved oxygen), they switched to periodic fermentation with fed glycerol (fed-batch). When the raw biomass reached a density of 120 g / l (after about 28-32 hours of fermentation), they went to the induction stage by adding the inductor in portions of 0.5 volume% of the volume of the culture medium every 2 hours. The induction stage lasted from 2 to 5 days, depending on the level of antigen accumulation in yeast cells, determined by immunoblotting.

Example 4. Isolation and purification of the recombinant protein rtHEV-ORF2

After fermentation, cells from the culture fluid were precipitated by centrifugation at 4000 g for 15 minutes at + 6 ° C. The precipitated biomass was washed by resuspension and re-centrifugation in extraction buffer (PBS, pH 7.2, with the addition of 1.7 mM EDTA, 2 mM PMSF, 0.5% [v / v] Tween-20). After washing, the biomass was resuspended in an extraction buffer at a concentration of 380 g of wet cells per liter of suspension and cells were destroyed in a Dyno-Mill KDL mill using glass balls with a diameter of 0.5-0.7 mm. Undamaged cells and debris were besieged by centrifugation at 12000 g, 30 minutes at + 6 ° C.

To the obtained clarified homogenate were added polyethylene glycol (PEG-8000) up to 6% [w / v] and NaCl up to 400 mM and incubated for 18 hours with constant stirring at + 4 ° C. The precipitated proteins, including rtHEV-ORF2, were separated by centrifugation at 12000 g for 30 minutes. The precipitate was dissolved in 5 volumes of PBS buffer and the pH was adjusted to 8.0 with sodium hydroxide solution. Insoluble proteins and aggregates precipitated for 30 minutes at 12,000 g. Using tangential ultrafiltration on a 300 KDa membrane, low molecular weight proteins were removed from the supernatant and simultaneously transferred to ion exchange chromatography buffer (50 mM Tris-HCl, pH 8.0, 10 mM NaCl), followed by applying to a chromatographic column with a strong anion-exchange coarse porous sorbent MacroCap-Q (GE, USA). Unbound proteins were washed with ion exchange chromatography buffer containing 50 mM NaCl. The rtHEV-ORF2 protein was eluted with a linear gradient of 50 to 500 mM NaCl in 10 column volumes at a linear speed of 100 cm / h.

The pooled fractions containing the rtHEV-ORF2 target protein (by immunoblot and SDS-PAGE) were concentrated by ultrafiltration on a 300 KDa membrane and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a sucrose gradient from 10 to 50% w / w in PBS buffer (pH 7.2). Fractions containing the target protein were purified from aggregates and transferred to a buffer of 10 mM NaH ₂ PO ₄ , pH 7.2, 0.1% Tween-20 using gel filtration on a Toyopearl HW-65F sorbent (ToyoSoda Corp., Japan). The purity of the thus obtained recombinant protein was determined by electrophoresis. The yield of the target protein was at least 50 mg of rtHEV-ORF2 protein per kilogram of crude biomass.

Example 5. Analysis of the recombinant rtHEV-ORF2 antigen

The purity of the recombinant rtHEV-ORF2 was determined using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) by staining with Coomassie Brilliant Blue R-250. The purity of the isolated recombinant antigen was at least 95%.

The immunospecificity of recombinant rtHEV-ORF2, as well as the level of expression during fermentation, was determined by immunoblotting. Primary monoclonal antibodies to the HEV capsid protein of genotype 3 (ab1011241, AbCam, UK) were stained with an immunoblot followed by visualization with specific antibodies to mouse immunoglobulins conjugated to horseradish peroxidase according to the improved ECL chemiluminescence technique (Amersham, UK). The recombinant rtHEV-ORF2 antigen on the immunoblot is presented as a protein band of 50-60 kDa in size.

The degree of aggregation and the approximate size of virus-like particles were determined by size exclusion chromatography on a TSK G5000PW analytical column (ToyoSoda Corp., Japan) in 10 mM NaH ₂ PO ₄ buffer, pH 7.2, 0.03% Tween-20. The first sharp peak elutes at ~ 0.4 column volumes and is a protein aggregate. The subsequent gentle peak, eluting at ~ 0.55 column volumes, consists of virus-like particles of rtHEV-ORF2. Peaks corresponding to low molecular weight proteins, including recombinant antigen monomers, elute in the range of 0.7–0.9 column volumes.

The molecular weight of the rtHEV-ORF2 protein, determined by polyacrylamide gel electrophoresis in the presence of SDS, is 56 kDa.

The isolated rtHEV-ORF2 antigen was used to create a vaccine based on it to prevent viral hepatitis E in animals. As an adjuvant, the vaccine contains a mixture of mineral oils (paraffin oil, liquid paraffin) and a surfactant (mannitol oleate) or Montanide ISA-70 (or ISA 888, or 71 RVG, or 71 VG) oil adjuvant manufactured by SEPPIC (France). To prepare the vaccine, an antigen solution in phosphate-buffered saline (PBS) 50 mM, 0.13 M NaCl, pH 6.8 is emulsified with an oil adjuvant at a component ratio of 30:70 (v / v), respectively. Emulsification is carried out in two stages: 1) obtaining a pre-emulsion in the reactor at a speed of 250 rpm; 2) emulsification on the apparatus "Silverson-450 L" (Sweden) or using a colloidal mill. The vaccine is obtained in the form of an emulsion "water-oil-water" with the most homogeneous particles. The following are examples of compositions of the vaccine obtained on the basis of the selected antigen.

a) The vaccine for the prevention of viral hepatitis E contains (1 ml):

0.3 ml of phosphate-buffered saline (PBS), represented by 50 mM Na-phosphate buffer, pH 6.8 and 0.13 M NaCl, containing 20 μg of rtHEV-ORF2 protein obtained by cultivation of the H. polymorpha yeast strain KBT-11 / pHEV-001,

0.7 ml Montanide ISA-70 Oil Adjuvant;

b) The vaccine for the prevention of viral hepatitis E contains (1 ml):

0.3 ml of PBS (50 mM Na-phosphate buffer, pH 6.8 and 0.13 M NaCl) containing 30 μg of rtHEV-ORF2 protein obtained by cultivation of the H. polymorpha strain KBT-11 / pHEV-001,

0.7 ml Montanide ISA-888 Oil Adjuvant;

c) The vaccine for the prevention of viral hepatitis E contains (1 ml):

0.3 ml of PBS (50 mM Na-phosphate buffer, pH 6.8 and 0.13 M NaCl) containing 50 μg of rtHEV-ORF2 protein obtained by culturing the H. polymorpha strain KBT-11 / pHEV-001, 70 μg of merthiolate

0.7 ml Montanide ISA-70 Oil Adjuvant.

According to the invention, a vaccine based on the isolated antigen can be obtained both with the introduction of the preservative of thiolate and without it. The absence of a preservative does not affect the quality of the vaccine.

The immunogenicity of the recombinant vaccine was determined by immunization of Balb / c mice. The immunogenicity of the vaccine in the test on mice weighing 12-14 g is 0.05-0.1 μg per ED 50 (the dose of the vaccine that causes seroconversion in 50% of mice).

The toxicity of the vaccine was determined by injecting white mice with 1.0 ml (20 μg) of the vaccine intraperitoneally and 1.0 ml (20 μg) of the vaccine subcutaneously in guinea pigs. Observation over 7 days showed that the vaccine is non-toxic.

Claims (2)

1. Recombinant vaccine for the prevention of viral hepatitis E in animals, containing an effective amount of the capsid protein of the hepatitis E virus, an adjuvant and a physiologically acceptable diluent, characterized in that the vaccine contains rtHEV-ORF2 protein with the amino acid sequence of SEQ ID NO: 1 obtained by culturing a recombinant strain of the yeast Hansenula polymorpha KBT-11 / pHEV-001, and as an adjuvant an oil adjuvant, in the following ratio of components, vol.%:
rtHEV-ORF2 protein thirty oil adjuvant 70 2. The recombinant vaccine according to claim 1, characterized in that it further comprises a thiolate.

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