RU2628693C1 - Recombinant l1hpv16 gene, recombinant pqe-l1/16 plasmid, l1hpv16 protein and their application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant gene encoding a human papillomavirus 16 type L1 protein is provided, with codons optimised for heterologous expression in E. coli strains. An expression plasmid vector pQE-L1/16 carrying this recombinant gene is presented. A method for recombinant L1HPV16 protein production using this vector, which is used to transform E. coli strains is provided. An immunogenic composition and vaccine are provided for induction of specific cellular and humoral immunity against HPV16 containing recombinant L1HPV16 protein.

EFFECT: group of inventions allows to obtain a stable purified immunogenic L1HPV16 protein, allows to reduce the cost of its preparation process for use in effective immunogenic compositions and vaccines against type 16 human papillomavirus.

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Description

The invention relates to genetic engineering, biochemistry, biotechnology and immunology. A synthetic human papilloma virus L1 protein gene of type 16 (L1HPV16), optimized for heterologous expression in non-pathogenic laboratory strains of Escherichia coli, is obtained. Based on it, a recombinant plasmid pQE-L1 / 16 is obtained, consisting of a synthetic gene of the L1HPV16 protein, including the promoter region of the early promoter of the bacteriophage T5, the L1HPV16 protein gene and T1 transcription terminator; beta-lactamase gene and bacterial replication initiation site of ColE1 type. The invention also includes a method of refolding the protein into the correct spatial structure and purifying it by gel filtration. The invention relates to the recombinant protein L1HPV16 itself, the expression vector pQE-L1 / 16 and an immunogenic vaccine composition containing the recombinant protein L1HPV16 aimed at inducing immunity against human papillomavirus (HPV). EFFECT: invention enables to obtain stable purified immunogenic protein L1HPV16, as well as to obtain effective immunogenic vaccine compositions against HPV.

To date, it has been proven that preventive vaccination is an effective way to prevent HPV infection. Currently, there are three prophylactic vaccines on the market - divalent cervarix from GSK, tetravalent gardasil and nine valent gardasil-9 from Merck & Co. The target population of these vaccines is children and young people who are not infected with HPV. In all of these vaccines, the immunogen is the L1 virus capsid protein, capable of spontaneously forming pseudovirus particles that do not have infectious activity. Pseudovirus particles are an effective form of presentation of the viral antigen to the immune system and provide a stable humoral and cellular immune response (DraperE. Et al. A randomized, observer-blinded immunogenicity trial of Cervarix® and Gardasil® human papillomavirus vaccines in 12-15 year old girls. // PloS one - 2013. - Vol. 8. - No. 5. - P. e61825; Toft L. et al. Comparison of the immunogenicity of Cervarix® and Gardasil® human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31 , HPV-33, and HPV-45 in HIV-infected adults. // Human vaccines & immunotherapeutics - 2014. - Vol. 10. - No. 5. - P. 1147-1154; Godi A. et al. Relationship between Humoral Immune Responses against HPV16, HPV18, HPV31 and HPV45 in 12-15 Year Old Girls Receiving Cervarix® or Gardasil® Vaccine. // PloSone - 2015. - Vol. 10. - No. 10. - P. e0140926; Giuliano AR et al. Immunogenicity and safety of Gardasil among mid-adult aged men (27-45 years) - The MAM Study. // Vaccine. - 2015. - Vol . 33. - No. 42. - P. 5640-5646; Gizzo S., Noventa M., Nardelli G.B. Gardasil administration to hr-HPV-positive women and their partners. // Trends Pharmacol Sci. - 2013 .-- Vol. 34. - No. 9. - P. 479-480; Luna J. et al. Long-term follow-up observation of the safety, immunogenicity, and effectiveness of Gardasil ™ in adult women. // PloS one. - 2013 .-- Vol. 8. - No. 12. - P. e83431; Petrosky E. et al. Use of 9-valent human papillomavirus (HPV) vaccine: updated HPV vaccination recommendations of the advisory committee on immunization practices. // MMWR Morb Mortal Wkly Rep. - 2015. - Vol. 64. - No. 11. - P. 300-304).

Cervarix contains L1 HPV proteins of the two most oncogenic types - 16 and 18, which cause the development of about 70% of cases of cervical cancer (cervical cancer). It is proved that cervarix significantly reduces the risk of HPV infection of types 16 and 18 and prevents the development of cervical cancer. Cervarix is indicated for women aged 9 to 26 years (Monie A. et al. Cervarix ™: a vaccine for the prevention of HPV 16, 18-associated cervical cancer. // Biologies: targets & therapy. - 2008. - Vol. 2 . - No. 1. - P. 107-113; WO 2010012780 A1).

Gardasil in its composition contains L1 proteins of four oncogenic types of HPV - 6, 11, 16 and 18. It is shown that this vaccine prevents not only the development of cervical cancer, but also cancer of the vulva and vagina, as well as papillomatosis and the formation of anogenital warts. In this regard, Gardasil is recommended for women and men aged 9 to 26 years (McLemore MR Gardasil®: introducing the new human papillomavirus vaccine. // Clinical journal of oncology nursing - 2006. - Vol. 10. - No. 5. - P. 559).

Gardasil-9 contains LI proteins of nine types of HPV. Gardasil-9 prevents the development of additional (compared to Gardasil) 20% of HPV-mediated types of anogenital cancer and benign neoplasms. Gardasil-9 is indicated for both men and women aged 9 to 26 years (Printz C. FDA approves Gardasil 9 for more types of HPV. // Cancer. - 2015. - Vol. 121. - No. 8. - P. 1156-1157).

In addition to the L1 protein itself, vaccines contain adjuvants - amorphous aluminum hydroxyphosphate sulfate, which is a stimulator of the humoral immune response (used in the vaccines gardasil and gardasil-9), as well as aluminum oxide hydrate with monophosphoryl lipid A1 from Salmonella enterica, which is an effective stimulator of cellular immune response (used in the Cervarix vaccine) (Mata-HaroV. etal. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. // Science. - 2007. - Vol. 316. - No. 5831. - P. 1628-1632; Casella CR, Mitchell T.C. Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. // Cellular and mo lecular life sciences. - 2008. - Vol. 65. - No. 20. - P. 3231-3240; Lindblad EB Aluminum compounds for use in vaccines. // Immunology and cell biology - 2004. - Vol. 82. - No. 5. - P. 497-505; Marrack P., McKee AS, Munks MW Towards an understanding of the adjuvant action of aluminum // Nature Reviews Immunology. - 2009. - Vol. 9. - No. 4. - P. 287-293). In addition, the composition of these vaccines includes suspension stabilizers polysorbate-80 and L-histidine, which maintain the properties of the suspension and increase the stability of the complex between amorphous aluminum and antigen (patent US 20050158334 A1).

The vaccines described above can significantly reduce the prevalence of HPV infection and reduce the risk of HPV-related diseases. At the same time, these vaccines have a significant drawback: their cost is quite high due to the use of expensive baculovirus (cervarix) and yeast (gardasil and gardasil-9) expression systems to obtain L1 protein and assemble pseudovirus particles. Hence the high relevance of developing new solutions to reduce the cost of the process of obtaining the L1 protein immunogen and the pseudoviral particles formed by it for use in immunogenic compositions.

The technical result is expressed in obtaining separate highly purified and stable protein components, which are characterized by a high level of expression and stability in bacterial expression systems, on the basis of which vaccines can be obtained.

An immunogenic L1 human papillomavirus protein type 16 (L1HPV16) was obtained in the expression system of non-pathogenic laboratory strains of Escherichia coli, followed by refolding of L1HPV16 and its purification by gel filtration.

A synthetic DNA sequence encoding the L1HPV16 protein was obtained. To ensure a high level of production of the recombinant protein L1HPV16 in the heterologous system, codons were optimized. The planned coding DNA sequence was different from the native DNA sequence of human papilloma virus type 16 nucleotide composition. This technique allowed us to obtain highly efficient production of the recombinant protein L1HPV16 in the insoluble fraction in amounts of about 20% of the total protein of the cell.

The claimed group of inventions allows to obtain the optimal synthetic sequence for heterologous expression of DNA encoding the recombinant protein L1HPV16, providing a high level of production of protein L1HPV16 in non-pathogenic laboratory expression strains of E. coli; to carry out a simple and effective purification of the L1HPV16 protein, which is able to assemble into pseudovirus particles, and in this form, efficiently and specifically induce a humoral and cellular immune response to type 16 human papillomavirus; create immunogenic compositions based on a recombinant protein using alumina (Al) hydrate gel and monophosphoryl lipid A1 from Salmonella enterica (MPLA) as adjuvants.

This result is achieved due to the synthesis of the recombinant protein L1HPV16 in the cells of laboratory E. coli strains carrying the recombinant plasmid pQE-L1 / 16, as well as by creating a complex preparation L1HPV16 (5 or 0.5 μg) with Al adjuvants (200 μg) and MPLA (1 μg).

The essence of the invention lies in the fact that the plasmid pQE-L1 / 16 contains the following structural elements essential for its functioning:

a) the artificial gene L1HPV16, consisting of:

- the promoter region of the early promoter of the T5 bacteriophage (10-54 bp), which provides efficient transcription of mRNA;

- a recombinant gene encoding the L1 protein of human papillomavirus type 16 (117-1524 bp);

- untranslated region of the termination of transcription of the bacterial operon, which ensures the efficient termination of transcription of the L1HPV16 gene (2150-2236 bp);

c) the bacterial bla gene, which is under the control of the AmpR promoter (3477-4442 bp), encoding the beta-lactamase protein, a selective marker for the transformation of E. coli strain;

d) a bacterial site of replication initiation of ColE1 type, which ensures replication of the plasmid in E. coli strain: L1HPV16 (2718-3306 bp).

Thus, a recombinant plasmid pQE-L1 / 16 (4542 bp) was created that encodes a recombinant protein L1HPV16, which has the ability to spontaneously form pseudovirus particles. The plasmid contains the following structural elements: an artificial gene of the full-sized protein L1 of human papillomavirus type 16, including the promoter region of the early promoter of the bacteriophage T5 (45 bp), the gene of the chimeric protein L1HPV16 (1524 bp) and the T1 transcription terminator (87 bp .n.); the beta-lactamase gene (966 bp) and the bacterial replication initiation site of ColE1 type (589 bp).

By formulating the preparation of the recombinant protein L1HPV16 in the form of pseudovirus particles in combination with adjuvants (gel of aluminum oxide hydrate and monophosphoryl lipid A1 from S. enterica), the technical result is achieved by creating a recombinant protein L1HPV16, as well as by obtaining an immunological vaccine composition in which the recombinant L1HPV16 protein is contained in a complex with MPLA and Al particles.

The immunogenicity of the complex vaccine L1HPV16-Al-MPLA in mice was investigated. The mice were immunized subcutaneously with the vaccine composition three times at two-week intervals, and after a week the humoral and cellular immune responses were compared with those of mice immunized with equal amounts of L1HPV16 protein. A pronounced specific humoral and cellular immune response to a composition containing L1HPV16 with the addition of Al and MPLA was detected.

The technical result achieved by the implementation of the invention is to obtain a high level of production of the L1HPV16 protein, which, after refolding and purification, forms stable pseudovirus particles with the ability to induce a humoral and cellular immune response. In addition, the technical result is an effective immunogenic vaccine composition that effectively and specifically induces a humoral and cellular immune response to type 16 human papillomavirus.

The invention is illustrated by the following examples below. Genetic engineering and microbiological manipulations, amplification and DNA sequencing were carried out according to standard methods (Maniatis T., Fritsch E., Sambrook J. Molecular cloning, M .: Mir, 1984; DNA cloning. Methods. Edited by D. Glover, Per . English, M .: Mir, 1988; Saiki RK, Gelfand DH, Stoffel S. etal. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988, Vol. 239, No. 4839, pp. 487 -491; Sanger F., Nicklen S., Coulson AR DNA sequencing with chain-terminating inhibitors (DNA polymerase / nucleotide sequences / bacteriophage 4X174). Proc. Nat. Acad. Sci. USA, 1977, Vol. 74, No. 12 , pp. 5463-5467).

Example 1

Obtaining plasmid pQE-L1 / 16

The synthetic DNA sequence encoding the L1HPV16 protein gene was designed so that the nucleotide composition of the codons was optimized for heterologous expression in a non-pathogenic laboratory strain of E. coli, while the amino acid composition of the synthesized recombinant protein was not changed (sequence No. 1). To clone a DNA fragment, the gene sequence was flanked by restriction endonucleases BamHI and KpnI. To initiate and stop the broadcast, start and termination codons were provided.

The DNA fragment encoding the L1HPV16 protein was synthesized by the solid-phase amidophosphite method (ZAO Evrogen, RF) on an AppliedBiosystemsABI 3900 synthesizer, followed by cloning of the duplex DNA into a pAL-TA vector. The primary structure of the resulting construct was confirmed by sequencing.

To obtain a construct encoding the L1HPV16 gene sequence, the plasmids pALTA-L1HPV16 and pQE6 were hydrolyzed with restriction endonucleases BamHI and KpnI at 37 ° C in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 100 mM sodium chloride and 0.1 mg / ml BSA, for 1 hour. Restriction products were separated on a 1.2% agarose gel. A fragment of plasmid pALTA-L1HPV16 isolated from an agarose gel (1521 bp) was ligated with a fragment of plasmid pQE6 (3028 bp) isolated from an agarose gel using bacteriophage T4 DNA ligase. Using electroporation, the obtained ligase mixture was used to transform competent E. coli M15 cells [pREP4]. After transformation, clones were selected on LB agar medium containing antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by alkaline lysis and analyzed by restriction mapping. Clones of plasmid DNA pQE-L1 / 16 (4542 bp, sequence No. 2) containing the DNA sequence encoding the full-length protein L1HPV16 were selected. The primary structure of the resulting construct was confirmed by sequencing. The plasmid pQE-L1 / 16 is shown in FIG. one.

Example 2

Obtaining, refolding and purification of the recombinant protein L1HPV16

The E. coli cell culture was besieged, weighed, washed with 10-fold volume of washing buffer A: 20 mM Tris-HCl, pH 7.5, 50 mM sodium chloride, 0.5 mM EDTA, 1 mM PMSF. After washing, E. coli cells were again resuspended in a 10-fold volume of the same buffer and lysozyme was added at the rate of 25 μg / ml cell suspension. Incubated for 30 min at room temperature, stirring occasionally. The suspension was voiced for 5 min using a BandelinSonopuls ultrasonic disintegrator (20% amplitude, 5 sec pulse, 3 sec pause). Centrifuged for 30 min at 10,000 g and a temperature of 5 ° C. The target protein is in inclusion bodies (TB) in the sedimentary fraction. The supernatant was decanted, the sediment fraction was weighed and washed again with 10 times the volume of washing buffer A. The washed TB was dissolved in 10 times the volume of washing buffer A + 5-20 mM DTT + 1-5% sodium lauryl sarcosinate. The content of the target protein in the washed TV was about 80%. After solubilization of the target protein from TB, the solution was clarified by centrifugation at 10,000 g. To reduce the detergent content, the solution was dialyzed against washing buffer A + 10 mM DTT + 0.06% sodium lauryl sarcosinate, 0.02% Tween-20. Further protein purification was carried out by gel filtration on a ToyopearlHW-65 sorbent, equilibrated with washing buffer A + 0-10 mM DTT + 0-1% sodium lauryl sarcosinate, 0-1% Tween-20. The quantitative characteristics of the cleaning steps are shown in the table:

Example 3

Verification of the assembly of the L1HPV16 protein, the formation of pentamers and pseudovirus particles.

Copper grids (200 mesh) were coated with collodion (4% solution of nitrocellulose in amyl acetate) and sprayed with charcoal. Next, the nets were ionized in a glow discharge to hydrophilize the surface. Samples of L1HPV16 protein, dissolved in phosphate-buffered saline at a concentration of 50 μg / ml, were applied to the surface of the nets in a volume of 15 μl and contrasted with a 2% aqueous solution of uranyl acetate or a 1.5% aqueous solution of phosphofungstate. The analysis was performed on a JEM-1400 TEM transmission electron microscope (JEOL, Japan) at an accelerating voltage of 80 kV. Electronic images were obtained using an OlympusQuemesa digital camera (Olympus-SIS, Germany) and iTEM software (Olympus Soft Imaging Solutions GmbH, Germany). Using electron microscopy, pseudovirus particles with a diameter of 40-50 nm were detected in a L1HPV16 protein sample (Fig. 2).

Example 4

Verification of the interaction of the L1HPV16 protein with mono- and polyclonal specific antibodies

The interaction of L1HPV16 with specific polyclonal antibodies was checked by enzyme-linked immunosorbent assay. Enzyme immunoassay plates were loaded with 50 μg of a solution of purified refolded L1HPV16 protein in PBS with a concentration of 1 μg / ml and incubated for 2 hours at 25 ° C, after which they were washed three times with PBS with the addition of 0.02% Tween-20. The free binding sites were blocked with a 5% solution of fetal bovine serum in PBS (2 h at 25 ° C), followed by washing with PBS with the addition of 0.02% Tween-20. The primary antibodies to L1HPV16 were serum rabbit antibodies immunized with the Gardasil vaccine. Binding to primary antibodies was carried out for 2 hours at 25 ° C, followed by three times washing with PBS, 0.02% Tween-20. Binding to goat's secondary antibodies to rabbit immunoglobulins conjugated to horseradish peroxidase (Imtek, Russia) was carried out for 2 h at 25 ° C, followed by three times washing with PBS, 0.02% Tween-20. Secondary antibodies were detected using a chromogenic substrate of 3.3 ', 5.5'-tetramethibenzidine for 10 minutes at a temperature of 25 ° C. The reaction was stopped with 0.16 M aqueous sulfuric acid. The luminescence intensity of the chromogenic substrate was measured at a wavelength of 450 nm using a Multiskan ™ FC MicroplatePhotometer (ThermoFisher, USA). Background signal correction was carried out at a wavelength of 620 nm. An enzyme-linked immunosorbent assay showed that purified refolded L1HPV16 is efficiently recognized by the polyclonal antibodies obtained against the components of the Gardasil vaccine (Fig. 3A).

The ability of commercially available CamVir-1 monoclonal antibodies (clone 289-16981, Meridian LifeScience, USA) to efficiently recognize the L1HPV16 protein obtained and purified by the methods proposed in this application was demonstrated using the Western blot method (Fig. 3 B).

Example 5

Testing the immunogenicity of the vaccine composition based on the protein preparation L1HPV16 in mice.

The following preparations were administered subcutaneously to female balb / c mice three times at a two-week interval:

1) 5 μg of purified recombinant protein L1HPV16;

2) 0.5 μg of purified recombinant protein L1HPV16;

3) 5 μg purified recombinant protein L1HPV16 with the addition of 200 μg Al and 1 μg MPLA;

4) 0.5 μg of purified recombinant protein L1HPV16 with the addition of 200 μg of Al and 1 μg of MPLA.

All injectable preparations were dissolved in 100 μl of sterile phosphate-buffered saline. One week after the last immunization, blood was taken from the animals and spleen resection was performed with splenocytes isolation to determine the cellular immune response (the amount of interferon-gamma synthesized by mouse splenocytes after stimulation with the antigen was estimated). An immunogenic composition containing 0.5 μg or 0.5 μg L1HPV16 + 200 μg Al + 1 μg MPLA is optimal both for the induction of a cellular immune response (from 300 to 1630 pg / ml interferon-gamma depending on the animal), and to induce the formation of specific antibodies (determination by enzyme immunoassay: titer of about 1/60000).

Thus, the results indicate that the resulting vaccine composition leads to a specific induction of a humoral and cellular immune response.

Claims (9)

1. Recombinant gene with sequence No. 1 encoding a human papilloma virus L1 protein of type 16, with codons optimized for heterologous expression in E. coli strains. 2. Expression plasmid vector pQE-L1 / 16 with sequence No. 2, a physical map shown in figure 1, carrying the recombinant gene according to claim 1. 3. A method of obtaining a recombinant protein L1HPV16, including: - obtaining protein L1HPV16 in the form of inclusion bodies in expression strains of E. coli transformed with the expression plasmid vector according to claim 2; - washing inclusion bodies, solubilization of L1HPV16 and its refolding; - protein purification by gel filtration. 4. An immunogenic composition for the induction of specific cellular and humoral immunity against HPV16, containing the recombinant protein L1HPV16 with a molecular weight of 56.47 kDa, pI 8.64, obtained by the method according to claim 3, an alumina hydrate gel and monophosphoryl lipid A from S. enterica. 5. A vaccine that causes specific cellular and humoral immunity against HPV16, containing the recombinant protein LlHPV16 with a molecular weight of 56.47 kDa, pI 8.64, obtained by the method according to claim 3, in an amount of 0.5-5 μg, aluminum oxide hydrate gel 200 μg and monophosphoryl lipid A from S. enterica, 1 μg. 6. A method of inducing specific cellular and humoral immunity against HPV16, comprising vaccinating balb / c mice three times, subcutaneously, with an interval of 2 weeks with the vaccine according to claim 5.

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