RU2681174C1 - Method of obtaining a recombinant vaccine for prevention of humanpapilloma virus infection, recombinant vaccine - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, particularly to genetic engineering. Proposed recombinant vaccine for the prevention of human papillomavirus infection and how to obtain it. Vaccine is based on the VLP of the main capsid protein L1 of HPV types 16, 18, 6, 11, obtained by cultivation of recombinant yeast strains Hansenula polymorpha. Recombinant strains producing VLP HPV11-L1 and VLP HPV6-L1, were obtained by introducing into the genome of a yeast cell one copy of a DNA sequence encoding an HPV type 11 or 6 L1 capsid protein under the control of the AIBN promoter, and one copy of the DNA sequence that encodes the capsid protein L1 HPV type 11 or 6 under the control of MOX promoter. Vaccine contains an effective amount VLP HPV11-L1, HPV6-L1, HPV16-L1 and HPV18-L1, adjuvant and physiologically acceptable diluent.EFFECT: proposed method allows to obtain a highly immunogenic, non-toxic, non-adverse vaccine.6 cl, 4 dwg, 2 tbl, 3 ex

Description

The invention relates to the field of biotechnology, namely genetic engineering, and relates to a recombinant vaccine for the prevention of human papillomavirus infection containing recombinant human papillomavirus L1 capsid protein type 16, 18, 6, 11 as an active ingredient obtained by culturing recombinant yeast strains Hansenula polymorpha, as well as a method for producing a vaccine.

The human papilloma virus (HPV - Human Papilloma Virus), affecting epithelial cells, causes various diseases in humans. Based on differences in DNA sequence, more than 200 types of papilloma virus are isolated. According to the WHO, more than 630 million people are carriers of human papillomavirus infection, of which 190 million have clinical manifestations. Human papillomavirus type 16 and 18 causes epithelial dysplasia of the genital mucosa, it is these types of papilloma virus that are associated with most pre-invasive and invasive carcinomas of the anogenital sphere (cervical, vaginal, vulvar and anal cancers), of which cervical cancer is one of the most common and dangerous types of tumors in women. About 500 thousand women worldwide fall ill with cervical cancer every year, more than half of these cases are fatal. The human papilloma virus types 6 and 11 are not classified as oncogenic: papillomavirus infection in this case manifests itself in the form of benign warts, non-cancerous warts or papillomas on the mucous membranes of the genital organs or respiratory tract. 30 million new cases of genital warts associated with human papillomavirus infection are reported annually in the world. The clinical manifestations of the infection are not detected for a long time, at the same time, the early diagnosis of human papillomavirus infection is quite complicated. That is why, in the fight against the spread of such a serious disease, preference is given to the prevention of human papillomavirus infection, in particular, by inducing an immune response in humans with vaccination.

The papilloma virus belongs to the family of papoviruses (Papoviridae), has a diameter of 40-50 nm. The virus capsid is formed from 72 protein capsomeres consisting of the main protein L1 and minor protein L2. The genome of the virus is presented in the form of circular double-stranded DNA, which contains 8 early genes (E1-E7) and two late genes encoding the capsid proteins L1 and L2. The main L1 protein (55-60 kDa) is able to assemble itself into virus-like particles (VLP, virus-like particles), similar in structure to virions, but not containing viral DNA. Protein L1 has a high immunogenicity and induces the formation of antibodies that neutralize the infectious virus.

Currently, recombinant HPV L1 proteins are promising immunogenic components for the development of prophylactic vaccines against dangerous types of papillomavirus. Such recombinant vaccines are safe because they do not contain a potentially oncogenic viral genome. The recombinant L1 protein expressed in a eukaryotic cell is capable of self-assembly into a capsid-like structure, mainly reflecting VLP, morphologically and antigenically equivalent to authentic virions. Immunization with similar VLPs promotes the production of antibodies that neutralize the virus, resulting in protection against HPV infection.

The difficulties encountered in the development of effective human papillomavirus vaccines are primarily associated with the development of optimal expression systems for the production of recombinant HPV proteins with immunogenic properties of natural HPV L1 proteins capable of forming conformationally correct VLPs.

It is known to produce HPV type 16 L1 and L2 proteins by culturing mammalian CV-1 cells infected with a recombinant vaccinia virus vector [Zhou J et al., Virology v. 185, p. 251-257, 1991]. The expression of papillomavirus protein L1 is known, including types 6 and 11, which can spontaneously assemble into virus-like particles and possess immunogenicity in a eukaryotic system based on cultured insect cells (baculovirus system) [Kirnbauer R. et al., Proc.Natl.Acad. Sci USA, v. 89, p. 12180-12184, 1992], US 8, 062, 642, US 9, 050, 287, US 9, 745, 351, US 6,165,471. The scientific and patent literature presents examples of the use of the Saccharomyces cerevisiae yeast expression system for the production of recombinant HPV proteins, which are preferred due to the internationally recognized yeast safety, the ability to produce large amounts of protein in the native conformation. So, in patent RU 2206608, 2003, C 12 N 7/00 describes the creation of a yeast producer of L1 HPV or L2 HPV protein of various types, including HPV 6 and 11, using the example of Saccharomyces cerevisiae. Cloning of the HPV L1 and L2 genes was carried out using genomic DNA extracted from Caski cells (ATCS No. CRL 1550). An expression plasmid yeast vector containing the DNA sequence encoding the corresponding polypeptide was constructed under the control of the GAL10 promoter and transformed S. cerevisiae cells with it. The ability of recombinant S. cerevisiae cells to express L1 or L2 HPV proteins was analyzed by immunoblotting.

The use of methylotrophic yeast Pichia pastoris is known to produce HPV16-L1 [Bazan S.B. et al., Arch. Virol. 2009, 154 (10), 1609-17]. A gene encoding HPV16-L1 was introduced into the P. pastoris genome under the control of a methanol-regulated promoter. This gene had an optimized codon composition. Purification of the L1 protein was performed using heparin-sepharose chromatography followed by the assembly of virus-like particles. The formation of biologically active VLPs was confirmed by immunoelectron microscopy and hemagglutination.

A vaccine is known for creating an immune response against human papillomavirus, including virus-like particles of HPV16, HPV18 and at least one of the types HPV31, HPV45, HPV52 (RU 2420313, 2010). VLPs were obtained by expression in Trichoplusia ni cells infected with recombinant baculovirus encoding the corresponding HPV L1 gene. After purification, each type of VLP was independently adsorbed onto aluminum hydroxide to give adsorbed concentrated monovalents, which were then mixed in the desired ratio. The dosage of the vaccine components varies depending on the condition, gender, age, weight of the individual, route of administration, and may be 1-100 μg of each VLP.

US Pat. No. 7,709,010, 05/04/2010 describes the creation of a multivalent vaccine against human papillomavirus infection, including VLP L1 of various types of HPV, including types 6, 11. VLPs were obtained by expressing the capsid protein L1 in recombinant S. cerevisiae yeast cells. The isolated and purified VLPs were adsorbed on an aluminum adjuvant and an ISCOM-type adjuvant. The advantage of the expression system based on the yeast S. cerevisiae is the relatively low cost and easy adaptation to large-scale growth in fermenters.

Known recombinant vaccine for the prevention of human papillomavirus infection containing as antigen L1 VLP HPV types 16 and 18, and / or 56 (RU 2546213, 02/13/2015). HPV VLPs were obtained by culturing recombinant H. Polymorpha yeast cells.

Most vaccines known in the art for the prevention of human papillomavirus infection have not yet passed the necessary clinical trials. There are currently two approved vaccines: the Gardasil quadrivalent prophylactic vaccine manufactured by Merck Sharp & Dohme B.V. and Cervarix Bivalent Vaccine manufactured by Glaxo SmithKline Biologicals S.A.

Gardasil (LS-002293, 11.24.06) is a recombinant quadrivalent vaccine consisting of virus-like particles of HPV 6, 11, 16, 18 types. VLPs were obtained by expressing L1 viral capsid proteins in Saccharomyces cerevisiae yeast cells, purified and adsorbed on an aluminum adjuvant (amorphous aluminum hydroxyphosphate sulfate). Each dose of vaccine (0.5 ml) contains recombinant antigens: type 6 - 20 μg, type 11 - 40 μg, type 16 - 40 μg, type 18 - 20 μg, as well as 225 μg of aluminum adjuvant and buffer solution. The recommended course of vaccination consists of three doses (0 -2 - 6).

Cervarix (LSR - 006423/08, 08/11/08) is a recombinant bivalent vaccine consisting of virus-like particles of types 16 and 18 HPV. VLPs were obtained using recombinant baculoviruses in a cell culture of Trichoplusia ni. Each dose of the vaccine (0.5 ml) contains recombinant antigens: type 16-20 μg, type 18 - 20 μg, MPL (3-O-desacyl-4-monophosphoryl lipid A) - 50 μg, aluminum hydroxide - 0.5 mg .

Despite the fact that these vaccines are already in use, there are still many unexplored issues related to their use. So, the long-term results of vaccination have not been studied, there is no data on the correlation of the resulting antibodies with a protective effect. It is also noted that a large number of specific antibodies are produced during vaccination, but not all antibodies are able to neutralize the papilloma virus. The disadvantages of vaccines include reactogenicity, side effects, restrictions on use, and high cost.

The closest analogue is the patent RU 2206608, 2003, C 12 N 7/00, which describes a method for producing virus-like particles of L1 HPV or L2 HPV protein of different types. The method includes constructing an expression plasmid yeast vector containing a DNA sequence encoding the corresponding polypeptide under the control of the GAL10 promoter and transforming Saccharomyces cerevisiae cells, culturing the transformed cell, collecting VLP from the transformed cell and purifying the VLP by chromatography. The ability of recombinant S. cerevisiae cells to express L1 or L2 HPV proteins was analyzed by immunoblotting. To create a vaccine against human papillomavirus infection, purified VLPs were mixed with a pharmaceutically acceptable carrier, stabilizer or adjuvant. The vaccine contains 0.1-100 μg, preferably 1-20 μg of active antigen. The disadvantages of using an expression system based on Saccharomyces cerevisiae include the instability of recombinant cells associated with the relatively high frequency of loss of an autonomous plasmid during cell division. When the strain is cultured under nonselective conditions for the plasmid, this leads to the accumulation of cells that do not contain plasmids and are unable to synthesize the target protein. This significantly complicates the cultivation of the recombinant strain and increases the cost of the target product. To induce the expression of recombinant proteins, a rather expensive reagent, galactose, is used, which increases the cost of the target product.

The objective of the invention is to provide a highly immunogenic, non-toxic, non-side-effect recombinant vaccine for the prevention of human papillomavirus infection containing HPV type 16, 18, 6, 11 L1 VLP antigen, as well as a method for its preparation. The problem was solved by creating recombinant strains of H. Polymorpha yeast that stably and efficiently produce the main capsid protein of human papillomavirus type 11, 6, respectively, possessing the necessary biological properties (proper conformation, immunogenicity, purity) and low cost. VLP L1 types 16 and 18 were obtained according to patent RU 2546213. The yeast producer should be able to control the absence of mutations in the expressed foreign gene, have a controlled expression level of the recombinant gene, to ensure high efficiency of folding of the synthesized polypeptide with the formation of the natural conformation of the resulting product, have a sufficiently high yield target protein, ensuring the economic feasibility of using the producer.

To obtain a recombinant producer strain of the main HPV11 L1 capsid protein, two expression cassettes containing one copy of the desired gene are sequentially integrated into the genome of the recipient strain of H. polymorpha DLT2. The first cassette contains a DNA fragment with a recombinant gene encoding HPV11-L1, under the control of the promoter of the MOX gene (methanol oxidase) and a selectable marker (H. polymorpha TRP3 gene). The second expression cassette contains a similar HPV11-L1 gene, but under the control of the DAK gene promoter (the gene encodes dihydroxyacetone kinase) of the H. polymorpha yeast and a selective marker (S. cerevisiae LEU2 gene). Transformation yields a recombinant strain containing one copy of the HPV11-L1 gene under the control of the MOX promoter and one copy of the HPV11-L1 gene under the control of the DAK promoter. Moreover, the artificial genes are integrated directly into the genome of the producer strain, i.e. are part of the chromosomes of the yeast H. polymorpha. 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. In addition, the presence of only one copy of the recombinant gene in each expression cassette with a unique promoter and terminal region makes it possible to control the absence of mutations of this gene during its introduction into the genome and during cultivation of the strain. The absence of mutations is controlled by analyzing the nucleotide sequence of the products of the polymerase chain reaction using primers unique to specific sequences in the coding region of each expression cassette.

An advantage of the proposed invention is the possibility of obtaining a stably high level of synthesis of the target protein without compromising the efficiency of its folding. The use of expression cassettes with two different promoters (MOX and DAK), the maximum activity of which is achieved under different cultivation conditions, provides a more uniform synthesis of the product during the cultivation of the strain. At the same time, the level of protein synthesis is not exceeded, which can lead to a possible violation of the laying efficiency and, as a result, a decrease in the strain productivity.

As a result of the selection of clones capable of synthesizing the HPV11-L1 protein, a transformant that best meets the required properties was isolated. The resulting recombinant strain allows one to achieve higher levels of recombinant protein synthesis with simpler methods for producing high density cultures, as well as to isolate the HPV11-L1 protein as VLP with the correct conformation, possessing the necessary antigenic and immunogenic properties, with a sufficiently high yield. VLP formation was confirmed using transmission electron microscopy.

The strain was deposited November 21, 2017 in the collection of NPK Kombioteh CJSC under the number KBT-17 / rPV-112. The strain is new and is neither described in the patent nor in the scientific and technical literature.

Similarly, a recombinant strain of H. polymorpha yeast producing HPV6-L1 VLP was obtained. The strain was deposited November 21, 2017 in the collection of NPK Kombioteh CJSC under the number KBT-17 / rPV-062. The strain is new and is neither described in the patent nor in the scientific and technical literature.

The resulting recombinant yeast strains producing a major HPV type 6.11 capsid protein, respectively, were cultured separately under conditions suitable for expression of the main HPV capsid protein. After isolation and purification, recombinant antigens were used as part of the vaccine for the prevention of human papillomavirus infection. The recombinant vaccine contains, in the form of VLP, L1 protein type 16 and 18, 6, 11 in an effective amount, an adjuvant, a physiologically acceptable diluent, and may contain thiolate as a preservative, as well as L-histidine and Tween-80 as a stabilizer. VLP L1 type 16 and 18 were obtained according to RU 2546213. Isolated and purified VLP L1 type 16 and 18 had no mutations, had a purity of at least 95%, had antigenic activity and immune specificity. The purified 4 types of VLP L1 were adsorbed on an adjuvant (aluminum hydroxide) and mixed in the desired ratio, or the purified VLPs were initially mixed in the desired ratio and then adsorbed on the adjuvant. By effective amount is meant an amount of the active principle of the present invention that is sufficient to protect against HPV 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 technical result of the proposed invention is to increase the immunogenicity of the vaccine (to improve the biological properties: antigenicity, immunogenicity, proper assembly of VLP), reduce costs, and also to create a vaccine directed against HPV types 16,18,6,11 common in the Russian Federation.

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. Transformation of Escherichia coli and Hansenula polymorpha cells was carried out according to the previously described methods (Inoue et al, 1990, Gene, 96: 23-28. And Bogdanova et al., 1995, Yeast 11: 343-353, respectively). The synthesis of DNA fragments, as well as the determination of the nucleotide sequence, were carried out by ZAO Evrogen, Moscow. Synthetic DNA fragments were used, the nucleotide sequences of which are shown in the List of sequences.

Example 1. Obtaining plasmid vector p11HPV3 containing the recombinant gene DAK-HPV11-L1 and a selective marker gene LEU2 S. cerevisiae.

The DNA fragment DAK-HPV11 (SEQ ID NO: 1) and the preparation of the plasmid AMIpSL1 (Agaphonov et al., 1999, Yeast 15: 541-551) containing the S. cerevisiae LEU2 gene as a yeast selective marker was digested with restriction enzymes BsrGI and HindIII . The resulting preparations of the recombinant gene DNA fragment and the linearized plasmid AMIpSL1 were introduced into the reaction mixture for ligation at a concentration of 30 ng / μl. After incubation with T4 DNA ligase for 2 hours, 2 μl of the reaction mixture was used to transform E. coli strain DH5α. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the DAK-HPV11 fragment was selected. The determination of the nucleotide sequence of this plasmid confirmed that it contains a gene encoding HPV11-L1 under the control of the H. polymorpha DAK promoter. The resulting plasmid was designated p11HPV3.

Example 2. Obtaining plasmid vector p11HPV2 containing the recombinant gene MOX-HPV11-L1 and the incomplete gene TRP3 H. polymorpha as a selective marker.

The MOX-HPV11 DNA fragment (SEQ ID NO: 2) and the preparation of the pTZ-MOX plasmid (Agaphonov et al., 1995, Yeast, 11: 1241-1247) containing the incomplete H. polymorpha TRP3 gene as a yeast selective marker were hydrolyzed restriction enzymes BglII and XhoI. The product of hydrolysis of the MOX-HPV11 fragment and the product of hydrolysis of the plasmid pTZ-MOX 4.3 kbp were isolated after electrophoretic separation in agarose gel. The obtained DNA preparations were introduced into the reaction mixture for ligation at a concentration of 30 ng / μl. After incubation with T4 DNA ligase for 2 hours, 2 μl of the reaction mixture was used to transform E. coli strain DH5α. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the MOX-HPV11 fragment was selected. The determination of the nucleotide sequence of this plasmid confirmed that it contains a gene encoding HPV11-L1 under the control of the MOX promoter H. polymorpha. The resulting plasmid was designated p11HPV2.

Example 3. Obtaining a strain of H. polymorpha containing the expression cassette HPV11-L1 with the MOX promoter.

Strain H. polymorpha DLT2 [Agaphonov et al., 1994, Yeast v10, pp. 509-513; the collection of strains of the laboratory of molecular genetics of RKNPK of the Ministry of Health of the Russian Federation] was transformed with plasmid p11HPV2 hydrolyzed by restriction enzymes Ecl136II and XhoI. Transformants were selected on a medium containing 6.7 g / l of a mixture of salts and vitamins "Yeast Nitrogen Base" (Difco), 20 g / l of D-glucose, 20 g / l of agar and 60 mg / l of leucine. Among the obtained transformants, clones capable of producing HPV11-L1 were selected. One such transformant was designated DLT2 / p11HPV2.

Example 4. Obtaining a strain of H. polymorpha containing two expression cassettes HPV11-L1, one of which is with the MOX promoter, and the other with the DAK promoter.

The DLT2 / p11HPV2 strain (Example 3) was transformed with the plasmid p11HPV3 hydrolyzed by the restriction enzyme BsrGI. Transformants were selected on a medium containing 6.7 g / l of a mixture of salts and vitamins "Yeast Nitrogen Base" (Difco), 20 g / l of D-glucose and 20 g / l of agar. One of the obtained transformants was designated KBT17 / pPV-112. This transformant was confirmed to have the intact MOX-HPV11 gene (see Example 5) and the intact DAK-HPV11 gene (see Example 6).

Example 5. Determination of the presence of an intact gene MOX-HPV11 in the genome of H. polymorpha transformants.

Genomic DNA preparations were obtained from the analyzed clones and used as a template for polymerase chain reaction (PCR) with oligonucleotides MOX20 (SEQ ID NO: 3) and MOX3’TRP3 (SEQ ID NO: 4) as primers. PCR was performed using Pfu polymerase (Fermentas). In the presence of the MOX-HPV11 gene in the genome, a 1696 bp PCR product was formed. To confirm the intactness of this gene, the nucleotide sequence of the obtained PCR product was determined.

Example 6. Determination of the presence of an intact DAK-HPV11 gene in the genome of H. polymorpha transformants.

Genomic DNA preparations were obtained from the analyzed clones and used as a template for polymerase chain reaction (PCR) with dakFw1 oligonucleotides (SEQ ID NO: 5) and AMI20 (SEQ ID NO: 6) as primers. PCR was performed using Pfu polymerase (Fermentas). In the presence of the DAK-HPV11 gene in the genome, a 1718 bp PCR product was formed. To confirm the intactness of this gene, the nucleotide sequence of the obtained PCR product was determined.

Example 7. Cultivation of a strain of H. polymorpha KBT17 / pPV-112, containing two expression cassettes HPV11-L1, one of which is with the MOX promoter, and the other with the DAK promoter.

The fermentation of the strain producing yeast H. polymorpha was carried out in two stages. At the first stage, in the fed-batch mode at a temperature of 30 ° C, biomass was increased in a culture medium containing 4% yeast extract, 2% bacto-peptone and 4% glycerol. When the density of the raw biomass reached 150 g / l (after about 28-32 hours of fermentation), the inductor was added to a concentration of 0.5-0.8% and maintained at this level for 48-72 hours.

Example 8. Isolation and purification of recombinant HPV11-L1 protein from H. polymorpha cells.

After fermentation of the producer strain, the HPV11-L1 protein (amino acid sequence — SEQ ID NO: 7) was isolated according to the published procedure [Kim H.J. et al., Protein Expression and Purification, v. 70, p. 68-74, 2010] with minor changes. Cells from the culture fluid were besieged by centrifugation at 4000 g for 15 minutes at 4 ° C. The precipitated biomass was resuspended to a concentration of 300 g of wet cells per liter of suspension in extraction buffer: PBS (pH 7.2) with 1.7mM EDTA, 2mM PMSF, 0.01% Tween-80. Next, the cells were destroyed in a Dyno-Mill mill of the KDL type using glass balls with a diameter of 0.5-0.7 mm. To remove the bulk of non-target proteins, the resulting cell lysate was saturated with ammonium sulfate to 35% of saturation and the precipitated proteins were separated by centrifugation at 12000g for 30 minutes. The pellet was resuspended in 9 volumes of PBS buffer (pH 7.2) + 0.01% Tween-80 to achieve 5% saturation with ammonium sulfate and incubated for 24 hours at room temperature to completely dissolve the target protein and precipitate a portion of the impurity proteins, which were then separated by centrifugation at 12000g within 30 minutes. The resulting supernatant was dialyzed into the binding buffer: PBS (pH 6.2) containing 0.2 M NaCl and 0.01% Tween-80, followed by adsorption onto the sorbent SP Sepharose FF (GE Healthcare, USA). After washing the sorbent with the same binding buffer, the HPV11-L1 protein was eluted with a linear gradient from 0.2 M to 1.5 M NaCl. The pooled antigen-containing fractions (by immunoblot and SDS-PAGE) were concentrated by tangential flow ultrafiltration on a Pellicon Biomax 300 KDa membrane (Millipore, USA) and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a glycerol / sucrose gradient. Fractions containing the target protein were combined and then purified from glycerol and sucrose by gel filtration on a Toyopearl HW-65F sorbent (ToyoSoda Corp., Japan). The purity of the HPV11-L1 recombinant protein thus obtained was determined by SDS-PAGE electrophoresis. If the protein purity was insufficient (less than 95%), additional purification was performed using anion exchange chromatography on a Toyopearl DEAE-650M sorbent (ToyoSoda Corp., Japan).

As a result of these operations, it is possible to isolate about 15-20% of the L1 protein present in the cell lysate (table 1). The yield of purified HPV11-L1 was at least 50 mg / kg wet yeast biomass.

Table 1. Typical balance of isolation and purification of HPV11-L1. The concentration of total protein in the first stages was determined by the Biuret method. In the remaining stages, according to the Lowry method. The content and purity of HPV11-L1 was determined by immunoblotting and SDS-PAGE electrophoresis.

HPV11-L1
(mg / ml) HPV11-L1
(mg) Total protein
(g) Purity
% Exit
% Cell lysate 0.21 930 245.1 0.4 one hundred 5-35% SA extract 0.11 640 22.4 2.9 69 SP Sepharose FF 0.56 350 0.84 41.7 38 Toyopearl HW-65F 0.84 171 - > 95 eighteen

Example 9. The preparation of a recombinant strain of H. polymorpha yeast KBT-17 / pV-062 producing HPV6-L1 VLP (SEQ ID NO: 10) was carried out analogously to Examples 1-4 using DAK-HPV6 DNA fragments (SEQ ID NO: 8 ) and MOX-HPV6 (SEQ ID NO: 9). The presence of an intact DAK-HPV6 gene in the obtained cells was confirmed according to Example 6 by the formation of a 1715 bp PCR product. The presence of the intact MOX-HPV6 gene in the obtained cells was confirmed according to Example 5 by the formation of a PCR product of 1693 bp in length. To confirm the intactness of this gene, the nucleotide sequence of the resulting product was determined.

The cultivation of H. polymorpha strain KBT-17 / pV-062 producing HPV6-L1 VLP was carried out analogously to example 7. Isolation and purification of HPV6 VLP was carried out analogously to example 8 (table 2). The yield of purified HPV6-L1 was at least 50 mg / kg wet yeast biomass.

Table 2. Balance of isolation and purification of HPV6-L1. The concentration of total protein in the first stages was determined by the Biuret method. In the remaining stages, according to the Lowry method. The content and purity of HPV6-L1 was determined by immunoblotting and SDS-PAGE electrophoresis.

HPV6-L1
(mg / ml) HPV6-L1
(mg) Total protein
(mg) Purity
% Exit
% Cell lysate 0.23 880 295000 0.3 one hundred 5-45% SA extract 0.14 660 26450 2.5 75 SP Sepharose FF 0.47 380 1050 36 43 Toyopearl HW-65 0.75 152 - 95 17

Example 10. Analysis of recombinant antigens HPV11-L1, HPV6-L1

1) The purity of the recombinant target protein is determined by polyacrylamide gel electrophoresis under reducing conditions (SDS-PAGE) by staining with Coomassie Brilliant Blue R-250 and analyzing the intensity of the bands of scanned gels using the NIH Image computer program. The purity of the isolated recombinant HPV-L1 proteins is at least 95% (FIG. 1A HPV11-L1, FIG. 2A HPV6-L1).

2) Immunospecificity of recombinant antigens is determined by immunoblotting. Primary antibodies to the HPV6-L1 protein (cat # CABT-B8785, Creative Diagnostics, USA) or the HPV11-L1 protein (cat # CABT-B8787, Creative Diagnostics, USA) stain the immunoblot with subsequent visualization using specific antibodies to mouse immunoglobulins conjugated with horseradish peroxidase, according to the method of improved chemiluminescence ECL (Amersham, UK). The recombinant HPV-L1 antigen is presented as a protein monomer band in the region of 55 kDa (Fig. 1B HPV11-L1, Fig. 2B HPV6-L1).

3) The formation of VLP recombinant antigens HPV11-L1 and HPV6-L1 was confirmed by transmission electron microscopy (Fig. 3, 4). The presented samples were studied by the negative contrast method (an aqueous solution of phosphoric tungsten acid was used as a contrast) with distilled water washing, in a transmission electron microscope JEOL 100B (Japan) at an accelerating voltage of 80 kV, shooting was carried out at instrumental magnifications from 15000 to 59000. The average size of virus-like particles amounted to ~ 50nm.

4) Immunogenicity of recombinant HPV-L1 was determined in a test on Balb / c mice weighing 12-14 g after a single vaccination with an antigen sorbed on aluminum hydroxide. The ED / 50 value (ED / 50 is the dose of antigen that causes seroconversion in 50% of mice) is less than 100 ng, which is a good indicator of the immunogenic properties of the recombinant protein.

Cultural and morphological features of the strains: 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 30-50% glycerol solution.

Genetic features: Strains are not zoopathogenic or phytopathogenic.

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

The conditions and composition of the medium for fermentation: pumping at 30 ° C and pH 5.0-5.5 in a nutrient medium containing up to 4% glycerol.

The strain activity was determined in the clarified cell homogenizate by immunoblotting or enzyme immunoassay. The activity of the strains is not less than 15 mg / l of culture fluid.

Isolated and purified VLPs HPV16-L1, HPV18-L1, HPV6-L1, HPV11-L1 are used to create vaccines based on them for the prevention of human papillomavirus infection. VLP of 4 types of papilloma virus is mixed in the presence of an adjuvant and a physiologically acceptable diluent. The vaccine may additionally contain a preservative - merthiolate, as well as stabilizers L-histidine and tween-80.

Specific examples of vaccine compositions for the prevention of papillomavirus infection:

1. The vaccine for the prevention of human papillomavirus infection contains:

20 μg of HPL16-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV18-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV6-L1 VLP obtained by culturing strain KBT-17 / pV-062,

20 μg VLP HPV11-L1 obtained by culturing strain KBT-17 / pV-112

0.5 mg aluminum hydroxide gel,

up to 0.5 ml of phosphate-buffered saline (PBS), represented by 50 mM Na-phosphate buffer, pH 6.8-7.2 and 0.13 M NaCl.

2. The vaccine for the prevention of human papillomavirus infection contains:

20 μg of HPL16-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV18-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV6-L1 VLP obtained by culturing strain KBT-17 / pV-062,

20 μg VLP HPV11-L1 obtained by culturing strain KBT-17 / pV-112

0.5 mg aluminum hydroxide gel,

60 mcg of thiol,

up to 0.5 ml of phosphate-buffered saline (PBS), represented by 50 mM Na-phosphate buffer, pH 6.8-7.2 and 0.13 M NaCl.

3. The vaccine for the prevention of human papillomavirus infection contains:

40 μg of HPV16-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV18-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

20 μg of HPV6-L1 VLP obtained by culturing strain KBT-17 / pV-062,

40 μg VLP HPV11-L1 obtained by culturing strain KBT-17 / pV-112

0.5 mg aluminum hydroxide gel,

780 mcg L-histidine,

50 mcg Tween-80,

up to 0.5 ml of phosphate-buffered saline (PBS), represented by 50 mM Na-phosphate buffer, pH 6.8-7.2 and 0.13 M NaCl.

According to the invention, vaccines for the prevention of human papillomavirus infection can be obtained both with the introduction of a preservative - thiolate and without it. This also applies to the stabilizers L-histidine and tween-80. The lack of preservative and stabilizers in the vaccine does not affect its quality.

The immunogenicity of recombinant vaccines, as determined in the test on Balb / c mice weighing 12-14 g per ED / 50 (the dose of antigen that causes seroconversion in 50% of mice), is not more than 100 ng.

For comparison, the immunogenicity of the Gardasil quadrivalent vaccine (a commercially available vaccine) was determined. The data obtained showed that the relative potency (the ratio of the dose expressed in ED / 50, analogue to ED / 50 of the test vaccine) of the vaccine according to the invention in comparison with the analogue is not less than 1.0.

Vaccine toxicity was determined by injecting white mice with 0.5 ml (60 μg) of the vaccine intraperitoneally and 0.5 ml (60 μg) of the vaccine subcutaneously in guinea pigs. Observation over 7 days showed that the vaccine is not toxic.

Claims (7)

1. A method of obtaining a vaccine for the prevention of human papillomavirus infection, comprising transforming a yeast cell with a DNA molecule encoding HPV L1, preparing recombinant strains producing different types of HPV-L1 VLPs, separately culturing the obtained producer strains under suitable conditions, isolating and purifying HPV-VLPs L1, mixing an effective amount of HPV-L1 VLP of various types with an adjuvant and a physiologically acceptable diluent, and obtaining the target product, characterized in that I transform the Hansenula polymorpha yeast cell t sequentially with an expression cassette containing a DNA fragment encoding HPV11-L1 under the control of a promoter of the MOX gene (SEQ ID NO: 2) and an incomplete H. polymorpha TRP3 gene, and an expression cassette containing a DNA fragment encoding a HPV11-L1 under control of a promoter DAK gene (SEQ ID NO: 1) and a selective marker, S. cerevisiae LEU2 gene, produce a recombinant strain KBT17 / pPV-112 producing HPV11-L1 VLP, the Hansenula polymorpha yeast cell is transformed sequentially with an expression cassette containing a DNA fragment encoding HPV6-L1 under the control of the promoter of the MOX gene (SEQ ID NO: 9) and partial n H.Polymorpha TRP3, and an expression cassette containing a DNA fragment encoding HPV6-L1, under the control of the promoter of the DAK gene (SEQ ID NO: 8) and a selective marker gene S.U. cerevisiae LEU2, receive the recombinant strain KBT-17 / pV-062 producing VLP HPV6-L1, isolated and purified VLP HPV11-L1 and VLP HPV6-L1 are mixed with VLP HPV16-L1 and VLP HPV18-L1. 2. The method according to p. 1, characterized in that it further add merthiolate. 3. The method according to p. 1, characterized in that they additionally add L-histidine and tween - 80. 4. A recombinant vaccine for the prevention of human papillomavirus infection containing an effective amount of HPV antigen, an adjuvant and a physiologically acceptable diluent, characterized in that, as an HPV antigen, the recombinant vaccine contains a mixture of HPL11-L1 VLP obtained by culturing a recombinant strain of Hansenula polymorpha yeast KBT17 / pPV-112 containing the integrated DNA fragment encoding HPV11-L1 under the control of the DAK gene promoter (SEQ ID NO: 1) and the DNA fragment encoding HPV11-L1 under the control of the MOX promoter (SEQ ID NO: 2 ), VLP of HPV6-L1 obtained by culturing a recombinant strain of the Hansenula polymorpha yeast KBT-17 / pPV-062 containing a DNA fragment encoding HPV6-L1 integrated into the genome under the control of the DAK gene promoter (SEQ ID NO: 8), and a fragment DNA encoding HPV6-L1 under the control of the MOX promoter (SEQ ID NO: 9), VLP HPV16-L1 and VLP HPV18-L1. 5. Recombinant vaccine for the prevention of papillomavirus infection according to claim 4, characterized in that it additionally contains merthiolate. 6. Recombinant vaccine for the prevention of human papillomavirus infection according to claim 4, characterized in that it additionally contains L-histidine and Tween - 80.

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