RU2546243C1 - Recombinant vaccine for prevention of human papillomavirus infection and method of its preparation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant vaccine for prevention of human papillomavirus infection and method of its preparation is proposed. The vaccine is made based on VLP of main capsid protein LI HPV of types 16, 18, 56, obtained by culturing the recombinant cells of yeast Hansenula polymorpha. The recombinant cells were obtained by introducing into the genome of yeast cell of one copy of the DNA sequence encoding the capsid protein L1 HPV of type 16 or 18 or 56 under the control of a promoter of AIBN and one copy of the DNA sequence encoding the capsid protein L1 HPV of type 16 or 18, or 56, under control of the MOX promoter. The vaccine comprises an effective amount of VLP HPV16-L1, and HPV18-L1, and/or HPV56-L1, in equal proportions, the adjuvant and a physiologically acceptable diluent.

EFFECT: method enables to obtain highly-immunogenic, non-toxic vaccine without side effects.

4 cl, 6 dwg, 9 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 and 18, and / or 56, obtained by culturing recombinant cells Hansenula polymorpha yeast, 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. Depending on the type of infectious virus, which is classified into more than 70 types on the basis of DNA sequence homology, papillomavirus infection manifests itself in the form of benign warts, non-cancerous warts or papillomas on the mucous membranes of the genital organs or respiratory tract. 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. In a study conducted on the territory of the Russian Federation, it was shown that in women with a high degree of squamous intraepithelial lesion and cervical cancer, along with HPV type 16, HPV type 56 is found, and in cases of multi-infection this type of virus prevails (VII All-Russian Scientific and Practical Conference with International participation “Molecular Diagnostics - 2010”, Sat. Proceedings, ed., Academician of the Russian Academy of Medical Sciences V.I. Pokrovsky, vol. 3, pp. 373-409). According to the WHO, annually about 500 thousand women worldwide become ill with cervical cancer, more than half of these cases are fatal. Clinical manifestations of cervical cancer are not detected for a long time, while early diagnosis of human papillomavirus infection is rather 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 CV-1 mammalian cells infected with a recombinant vaccinia virus vector [Zhou J et al. Virology v. 185, p. 251-257, 1991]. Known expression of papillomavirus protein L1, capable of aggregating into virus-like particles (VLP) and having immunogenicity, in a eukaryotic system based on cultured insect cells, baculovirus system [Kimbauer R. et al. Proc. Natl. Acad. Sci USA, v. 89, p. 12180-12184, 1992]. 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, Petrov R.M. et al. [WO 2006/065166 A1, 2006] obtained capsid proteins L1 of viral serotypes 16, 18, 31 HPV, causing the most aggressive forms of diseases in humans, from recombinant yeast strains. These strains were obtained by transforming S. cerevisiae yeast cells with plasmids with genes encoding the L1 proteins of these serotypes. DNA fragments encoding these proteins were isolated from clinical material. Created yeast producers during cultivation were able to express L1 proteins in the form of virus-like particles. L1 proteins were used for the preparation of prophylactic and therapeutic vaccines.

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.

The patents RU 2445357, 2012 and RU 2445358, 2012 describe recombinant producer strains of capsid protein L1 HPV type 16 - Pichia angusta BKM Y-2988D and type 18 - Pichia angusta Y-2989D. The strains were obtained by integration into the genome of an expression plasmid containing a DNA fragment with a recombinant gene encoding HPV type L1 16 or 18, respectively, under the control of the DAK (dihydroxyacetone kinase) promoter H. polymorpha, and a selectable marker (H. polymorpha LEU2 gene). The described recombinant producer strains, having high productivity, nevertheless do not provide an antigen that is immunogenic optimal for use in the vaccine. To increase the productivity of the producer strain, the recombinant protein expression cassette is introduced into the genome of the recipient strain in a large number of copies. However, in some copies of the expression cassette during the preparation and cultivation of the strain, mutations may occur that alter the properties of the target protein. At the same time, control over the occurrence of mutations in the case of a high copy number of a gene is practically impossible. Moreover, if a certain level of synthesis is exceeded, the folding process of the target protein may be disrupted, which leads to a decrease in its yield, a deterioration in biological activity, and complicates the purification process.

A vaccine is known for creating an immune response against human papillomavirus, including virus-like particles of HPV 16, HPV 18 and at least one of the types HPV31, HPV45, HPV52 (RU 2420313, 2010). VLPs were obtained by expression in Trichoplusia ni cells infected with a recombinant baculovirus encoding the corresponding HPV LI 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.

As for the capsid protein LI HPV56, it is known from the prior art to prepare capsomeres of the indicated protein (US 6,165,471, 12/26/2000) recombinantly using a baculovirus expression system. It is also known to include the HPV56 LI protein in a multivalent vaccine directed against human papillomavirus infection (US 7,709,010, 05/04/2010). HPV VLP LIs were expressed in recombinant S. cerevisiae yeast cells. The isolated and purified VLPs were adsorbed on an aluminum adjuvant and an ISCOM-type adjuvant.

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 vaccine dose (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 vaccine (0.5 ml) contains recombinant antigens: type 16-20 μg, type 18-20 μg, MPL (3-0-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 patent RU 2206608, 2003, C12N 7/00, which describes a method for producing virus-like particles of HPV or L2 HPV protein L1 of various types, including HPV 16 and 18. 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 HPV L1 or L2 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 types 16 and 18 and / or 56 as antigen L1 and / or 56, as well as a method for its preparation. The problem was solved by creating recombinant N. Polymorpha yeast cells that stably and efficiently produce the main capsid protein of human papillomavirus type 16, 18, 56, respectively, which has the necessary biological properties (proper conformation, immunogenicity, purity) and low cost. The resulting yeast recombinant cells should provide the ability 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, and have a sufficiently high yield of the target protein, ensuring the economic feasibility of using the producer.

According to the invention, in order to obtain recombinant yeast cells producing the main capsid protein LI HPV16, two expression cassettes containing one copy of the desired gene are sequentially integrated into the genome of recipient H. polymorpha cells. The first cassette contains a DNA fragment with a recombinant gene encoding HPV16-L1, under the control of the promoter of the MOX gene (methanol oxidase) and a selective marker (H. polymorpha TRP3 gene). The second expression cassette contains a similar HPV16-L1 gene, but under the control of the promoter of the DAK gene (dihydroxyacetone kinase) of H. polymorpha yeast and a selectable marker (S. cerevisiae LEU2 gene). The transformation produces recombinant cells containing one copy of the HPV16-L1 gene under the control of the DAK promoter and one copy of the HPV16-L1 gene under the control of the MOX promoter. Moreover, artificial genes are integrated directly into the genome of recombinant cells, i.e. are part of the chromosomes of the yeast H. polymorpha. This ensures high mitotic stability of the cells, making it possible to optimize the culture conditions 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 allows you to control the absence of mutations of this gene during its introduction into the genome and during cultivation. 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 also 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 cultivation. 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.

The cells of the H. polymorpha DLT2 strain derived from DL1 (ATCC26012) or N. polymorpha CBS4732 (ATCC34438) isolated from Brazilian soil are used as transformation yeast recipient cells during transformation. These strains are of independent origin.

As a result of the selection of clones capable of synthesizing the HPV16-L1 protein, transformants that best meet the required properties were isolated. The resulting recombinant cells make it possible to isolate the HPV16-L1 protein as VLP with the correct conformation, possessing the necessary antigenic and immunogenic properties, with a rather high yield. VLP formation was confirmed using gel filtration chromatography.

Recombinant yeast cells producing VLP HPV18-L1 and recombinant yeast cells producing VLP HPV56-L1 were obtained in a similar manner.

The resulting recombinant yeast cells producing the main capsid protein of different types of HPV were cultured separately under conditions suitable for expression of the main capsid protein of HPV. 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 an VLP, L1 type 16 and 18, or L1 type 16 and 18, and 56, or L1 type 16 and 56 in an effective amount, an adjuvant, a physiologically acceptable diluent, and may contain merthiolate as a preservative. The purified VLPs 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 (improve biological properties: antigenicity, immunogenicity, proper assembly of VLP), reduce costs, and also to create a vaccine directed against types of HPV 16, 18, 56 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 pAM547 containing the recombinant gene DAK-HPV16-L1 and a selective marker LEU2 gene S. cerevisiae.

The DNA fragment DAK-HPV16 (SEQ ID NO: 1) and the preparation of the plasmid AMIpSLI (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 AMIpSLI linearized plasmid 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 Escherichia coli strain DH5α. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the DAK-HPV16 fragment was selected. Sequencing the nucleotides of this plasmid confirmed that it contains a gene encoding HPV16-L1 under the control of the H. polymorpha DAK promoter. The resulting plasmid was designated pAM547.

Example 2. Obtaining plasmid vector pKAM539A containing the recombinant gene MOX-HPV16-L1 and the incomplete TRP3 gene of H. polymorpha as a selective marker.

The MOX-HPV16 DNA fragment (SEQ ID NO: 2) and the preparation of the plasmid pTZ-MOX (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 Xhol. The hydrolysis product of the MOX-HPV16 fragment and the hydrolysis product of the pTZ-MOX plasmid 4.3 kb in length 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 Escherichia coli strain DH5α. Plasmid DNA was isolated from several grown transformants. Based on restriction analysis, a plasmid containing the MOX-HPV16 fragment was selected. The determination of the nucleotide sequence of this plasmid confirmed that it contains a gene encoding HPV16-L1 under the control of the MOX promoter H. polymorpha. The resulting plasmid was designated pKAM539A.

Example 3. Obtaining recombinant N. polymorpha cells containing the HPV16-L1 expression cassette with the MOX promoter.

The strain N. polymorpha DLT2 (Agaphonov et al., 1994, Yeast, v.10, pp. 509-513; a collection of strains of the laboratory of molecular genetics of RKNPK of the Ministry of Health of the Russian Federation) was transformed with plasmid pKAM539A hydrolyzed with restriction enzymes Es1136P and Xhol. 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 HPV16-L1 were selected. One of these transformants was designated DLT2 / pKAM539 (2).

Example 4. Obtaining recombinant H. polymorpha cells containing two HPV16-L1 expression cassettes, one with the MOX promoter and the other with the DAK promoter.

DLT2 / pKAM539 (2) cells were transformed with plasmid pAM547 digested with BsrGI restriction enzyme. 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, and the presence of the intact DAK-HPV16 gene was confirmed (see example 5) and the intact gene MOX-HPV16 (see example 6).

Example 5. Determination of the presence of an intact DAK-HPV16 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 dakFwl oligonucleotides (SEQ ID NO: 3) and AMI20 (SEQ ID NO: 4) as primers. PCR was performed using Pfu polymerase (Fermentas). In the presence of the DAK-HPV16 gene in the genome, a 1714 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 MOX-HPV16 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 oligonucleotides MOX20 (SEQ ID NO: 5) and MOX3′TRP3 (SEQ ID NO: 6) as primers. PCR was performed using Pfu polymerase (Fermentas). When the MOX-HPV16 gene was present in the genome, a 1711 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 recombinant H. polymorpha cells containing two HPV16-L1 expression cassettes, one with the MOX promoter and the other with the DAK promoter.

Fermentation of recombinant N. polymorpha yeast cells 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 HPV16-L1 protein from H. polymorpha cells.

After fermentation of the recombinant cells, the HPV16-L1 protein (SEQ ID NO: 11) 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 380 g of wet cells per liter of suspension in extraction buffer: PBS (pH 7.2) with 1.7 mM EDTA, 2 mM PMSF, 0.01% Tween-80. Next, the cells were destroyed in a Dyno-MS mill of the KDL type using glass balls with a diameter of 0.5-0.7 mm. To remove the bulk of the non-target proteins and the selection of antigen, the obtained extract was saturated with ammonium sulfate to 45% of saturation and the precipitated proteins were separated by centrifugation at 12000g for 30 minutes. The precipitate was diluted in a minimum volume of PBS buffer (pH 7.2) + 0.01% Tween-80 and dialyzed into the same buffer for 24 hours at room temperature to precipitate a portion of the remaining impurity proteins, which were then separated by centrifugation at 12000g for 30 minutes. The obtained supernatant was dialyzed into the binding buffer: PBS (pH 7.0) containing 0.33 M NaCl and 0.01% Tween-80, followed by adsorption onto the Heparin Sepharose CL-6B sorbent (GE Healthcare, USA). After washing the sorbent with the same binding buffer, the HPV16-L1 protein was eluted with a linear gradient from 0.33 to 1.5 M NaCl. The pooled antigen containing fractions (by immunoblot and SDS-PAGE) were concentrated by tangential flow ultrafiltration on a 500 KDa membrane and separated by zonal ultracentrifugation (Beckman Coulter, USA) in a glycerol / sucrose gradient. The fractions containing the target protein were combined and then purified by gel filtration on a Toyopearl HW-65 sorbent (ToyoSoda Corp., Japan). The purity of the thus obtained recombinant HPV-L1 protein was determined by SDS-PAGE electrophoresis. If the protein purity was insufficient (less than 95%), additional purification was performed using cation exchange chromatography on a Toyopearl SP-650M sorbent. The yield of the target protein was at least 50 mg per 1 liter of culture fluid.

The preparation of recombinant N. polymorpha yeast cells producing HPV18-L1 VLPs (SEQ ID NO: 12) was carried out analogously to Examples 1-4 using DAK-HPV18 DNA fragments (SEQ ID NO: 7) and MOX-HPV18 (SEQ ID NO: 8). The presence of the intact DAK-HPV18 gene in the obtained cells was confirmed according to Example 5 by the formation of a 1739 bp PCR product. The presence of the intact MOX-HPV18 gene in the obtained cells was confirmed according to Example 6 by the formation of a 1717 bp PCR product.

The preparation of recombinant H. polymorpha cells producing VLP HPV56-L1 (SEQ ID NO: 13) was carried out analogously to Examples 1-4 using DNA fragments DAK-HPV56 (SEQ ID NO: 9) and MOX-HPV56 (SEQ ID NO: 10 ) The presence of an intact DAK-HPV56 gene in the obtained cells was confirmed according to Example 5 by the formation of a PCR product of 1819 bp in length. The presence of the intact MOX-HPV56 gene in the obtained cells was confirmed according to Example 6 by the formation of a 1797 bp PCR product.

Cultivation of recombinant cells producing VLP HPV18-L1 and VLP HPV56-L1, respectively, was carried out analogously to example 7. Isolation and purification of VLP HPV18 and VLP HPV56 was carried out analogously to example 8. The yield of the target proteins is at least 20%.

The production of recombinant H. polymorpha cells producing VLP HPV16-L1, VLP HPV18-L1, VLP HPV56-L1, respectively, using N. polymorpha CBS4732 yeast cells as recipient cells, as well as their cultivation, isolation and purification of the produced recombinant proteins similar to the above examples.

Example 9. Analysis of recombinant HPV16-L1, HPV18-L1, HPV56-L1

1. The purity of recombinant HPV-L1 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 software. The purity of the selected recombinant proteins HPV-L1 is at least 95% (figa, 2A, 3A).

2. Immunospecificity of recombinant HPV16-L1, HPV18-L1, HPV56-L1 is determined by immunoblotting. Primary monoclonal antibodies to the HPV-L1 protein of serotype 16, 18, 56 (Camvir-1, AbCam, UK), respectively, stain the immunoblot with subsequent visualization using specific antibodies to mouse immunoglobulins conjugated to horseradish peroxidase according to the method of improved ECL chemiluminescence ( Amersham, UK). Recombinant antigens HPV16-L1, HPV18-L1, HPV56-L1 are presented as a protein monomer band of 55-60 kDa in size (Fig. 1B, 2B, 3B).

3. The formation of VLP recombinant proteins HPV16-L1, HPV18-L1, HPV56-L1 was confirmed by gel filtration chromatography on a TosoHaas TSK G5000PW polymer column with a diameter of 7.5 mm and a length of 60 cm, connected to a PrePW column with a diameter of 7.5 mm and a length of 7.5 cm. The pore size of the sorbent is 1000 Å, which allows you to separate the correctly collected VLP from the monomers of the protein L1. Separation was carried out in a buffer of 10 mM KH ₂ PO ₄ (pH 7.2), 0.03% Tween-20 at a flow rate of 0.6 ml / min. The process was monitored by absorbance at 280 nm. The elution time of protein L1 corresponds to the elution time of virus-like particles (Figures 4, 5, 6).

4. The immunogenicity of recombinant proteins was determined in a test on Balb / c mice weighing 12-14 g after a single vaccination with a protein sorbed on aluminum hydroxide. The value of ED / 50 (dose of antigen that causes seroconversion in 50% of mice), recombinant HPV16-L1, HPV18-L1, HPV56-L1 is not more than 100 ng, which is a good indicator of the immunogenic properties of the recombinant protein.

Cultural and morphological features of recombinant H. polymorpha cells: round 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: recombinant cells are not zoopathogenic or phytopathogenic.

Method, conditions and composition of media for the propagation of recombinant cells: 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 method for determining the activity: in the clarified homogenized cells by immunoblotting or enzyme immunoassay. The activity of recombinant cells producing HPV16-L1 and HPV18-L1 VLPs is at least 50 mg / L of culture fluid and at least 15 mg / L for HPL56-L1 VLP producer.

Isolated and purified VLPs HPV16-L1, HPV18-L1, HPV56-L1 are used to create vaccines based on them for the prevention of human papillomavirus infection.

The resulting vaccine compositions are presented in the following options:

1) The vaccine contains VLP HPV16-L1, HPV18-L1, an adjuvant and a physiologically acceptable diluent;

2) The vaccine contains VLP HPV16-L1, HPV56-L1, an adjuvant and a physiologically acceptable diluent;

3) The vaccine contains VLP HPV16-L1, HPV18-L1, HPV56-L1 adjuvant and a physiologically acceptable diluent;

4) The vaccine of options 1, 2, 3 additionally contains a preservative - thiolate.

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,

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 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 HPL56-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells, 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 and 0.13 M NaCl.

3. 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 HPV56-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

0.75 mg aluminum hydroxide gel,

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

4. 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 HPV56-L1 VLP obtained by culturing recombinant H. polymorpha yeast cells,

60 μg merthiolate, 0.75 mg aluminum hydroxide gel,

up to 0.5 ml of phosphate-buffered saline (PBS), represented by 50 mM Na-phosphate buffer, pH 6.8 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. The absence of a preservative 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 (4)

1. A recombinant vaccine for the prevention of human papillomavirus infection containing an effective amount of an HPV antigen, an adjuvant and a physiologically acceptable diluent, characterized in that, as an HPV antigen, the recombinant vaccine contains an equal proportion of HPV16-L1 VLP obtained by culturing a Hansenula polymorpha yeast cell, containing an integrated DNA fragment encoding HPV16-L1 under the control of the DAK gene promoter (SEQ ID NO: 1), and a DNA fragment encoding HPV16-L1 under the control of the MOX promoter (SEQ ID NO: 2), and VLP HPV18- L1 obtained by pu cultivating a Hansenula polymorpha yeast cell containing the integrated DNA fragment encoding HPV18-L1 in the genome under the control of the DAK gene promoter (SEQ ID NO: 7) and the DNA fragment encoding HPV18-L1 under the control of the MOX promoter (SEQ ID NO: 8), and / or VLP of HPV56-L1 obtained by culturing a Hansenula polymorpha yeast cell containing a DNA fragment encoding HPV56-L1 integrated in the genome under the control of a DAK gene promoter (SEQ ID NO: 9) and a DNA fragment encoding HPV56 -L1, under the control of the MOX promoter (SEQ ID NO: 10). 2. Recombinant vaccine for the prevention of human papillomavirus infection according to claim 1, characterized in that it additionally contains merthiolate. 3. A method of producing a vaccine for the prevention of human papillomavirus infection, comprising transforming a yeast cell with a DNA molecule encoding HPV L1, separately culturing the obtained recombinant cells producing different types of HPV-L1 VLPs under suitable conditions, isolating and purifying HPV-L1 VLPs, mixing effective the amount of different types of VLP HPV-L1 with an adjuvant and a physiologically acceptable diluent and obtaining the target product, characterized in that the Hansenula polymorpha yeast cell is transformed sequentially by expression a cassette containing a DNA fragment encoding HPV16-L1 under the control of the MOX gene promoter (SEQ ID NO: 2) and an incomplete H. polymorpha TRP3 gene, and an expression cassette containing a DNA fragment encoding HPV16-L1 under the control of a DAK gene promoter ( SEQ ID NO: 1) and a selective marker marker, S. cerevisiae LEU2 gene, isolate and purify VLP HPV16-L1, the Hansenula polymorpha yeast cell is transformed sequentially with an expression cassette containing a DNA fragment encoding HPV18-L1, under the control of the MOX gene promoter (SEQ ID NO : 8) and an incomplete TRP3 gene of H. polymorpha, and an expression cassette containing a DNA fragment, coding HPV18-L1, under the control of the DAK gene promoter (SEQ ID NO: 7) and a selective marker, S. cerevisiae LEU2 gene, were isolated and purified by HPV18-L1 VLP, the Hansenula polymorpha yeast cell was transformed sequentially with an expression cassette containing a DNA fragment encoding HPV56- L1, under the control of the promoter of the MOX gene (SEQ ID NO: 10) and the incomplete H. Polymorpha TRP3 gene, and an expression cassette containing a DNA fragment encoding HPV56-L1, under the control of the DAK promoter (SEQ ID NO: 9) and a selective marker gene LEU2 S. cerevisiae, isolate and purify VLP HPV56-L1, while isolated and purified VLP HPV16-L1 and VLP HPV18-L1, and / or VLP HPV56-L1 mixing t in equal shares. 4. The method according to p. 3, characterized in that it additionally add merthiolate.

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