RU2768044C1 - Expression vector based on adeno-associated virus with protective properties against intoxication caused by botulinum neurotoxin type a - Google Patents

Abstract

FIELD: biotechnology, immunology and microbiology.

SUBSTANCE: inventions group relates to biotechnology, immunology and microbiology. An expression vector based on an adeno-associated virus with an embedded genetic construct containing the nucleotide sequence SEQ ID NO:2 encoding a recombinant antibody SEQ ID NO:1 was created, and the use of this vector for the prevention of intoxication caused by botulinum neurotoxin type A was proposed.

EFFECT: inventions group provides the creation of a recombinant viral vector that has a lower inflammatory potential and a longer time interval of protective activity.

6 cl, 9 dwg, 8 ex

Description

Technical field

The group of inventions relates to biotechnology, immunology and microbiology. An expression vector based on an adeno-associated virus has been created that has protective properties against intoxication caused by botulinum toxin type A, as well as the use of this vector for the prevention of intoxication caused by botulinum neurotoxin type A.

prior art.

Botulism is a severe, potentially fatal toxic infection caused by the neurotoxins of the bacteria Clostridium botulinum (Gill, D. Bacterial toxins - a table of lethal amounts. Microbiol. Rev. 1982, 46(1), 86-94. PMID: 6806598; Goonetilleke, A Harris, J. Clostridial neurotoxins J. Neurol Neurosurg & Psychiatry 2004, 75, 35-39 http://dx.doi.org/10.1136/jnnp.2004.046102 PMID: 15316043). The estimated lethal dose for humans is about 10 nanograms per kilogram of body weight by inhalation and 1 microgram by oral ingestion (Arnon, S; Schechter, R; Inglesby, T; Henderson, DA; Bartlett, JG; Ascher, MS; Eitzen, E. ; Fine, AD; Hauer, J.; Layton, M.; et al. Botulinum toxin as a biological weapon - medical and public health management. JAMA 2001, 285(8), 1059-1070. http://dx.doi .org/10.1001/jama.285.8.1059 PMID: 11209178 Emmeluth, D. Botulism 2nd; New York, NY, USA; Infobase Publishing; 2010). The most common natural forms of botulism are food, wound, and infant (Goonetilleke, A; Harris, J; Clostridial neurotoxins. J. Neurol. Neurosurg. & Psychiatry 2004, 75, 35-39. http://dx.doi.org/ 10.1136/jnnp.2004.046102. PMID: 15316043). Foodborne botulism can be contracted by ingesting contaminated food, and wound form by contact with C. botulinum spores in an open wound. The infantile form occurs in children when spores of the toxin enter the gastrointestinal tract. The inhaled form of botulism does not occur naturally and can only occur in the event of a bioterrorist attack (Goonetilleke, A; Harris, J; Clostridial neurotoxins. J. Neurol. Neurosurg. & Psychiatry 2004, 75, 35-39. http://dx .doi.org/10.1136/jnnp.2004.046102.PMID: 15316043), Spickler, AR Botulism. Iowa State University, USA; 2018 Jan. Available online: http://www.cfsph.iastate.edu/DiseaseInfo/factsheets.php [accessed February 19, 2019]). The toxin consists of 3 non-covalently bound protein components: neurotoxin, hemagglutinin and protectin, providing high toxicity and resistance to aggressive environmental conditions. The toxin disrupts the process of neuronal activation, affecting the delivery of acetylcholine to the presynaptic membrane by cleaving the SNARE protein complex, which makes it impossible for the release of the neurotransmitter from synaptic vesicles, as a result of which the transmission of nerve signals stops, which leads to paralysis. There are 8 types of botulinum toxin (A-H), of which type A is the most common. About 300 cases of botulism are registered annually in Russia, 15% of which are fatal. In addition, botulinum neurotoxin is considered one of the most dangerous (US Centers for Disease Control and Prevention (CDC) - category A) of the existing bioterrorism threats. The currently available therapy for botulism has a number of disadvantages and is limited to the use of maintenance therapy, in particular, prolonged mechanical ventilation, as well as the introduction of an antitoxin obtained from hyperimmune horse serum, the use of which can cause allergic reactions, delayed-type hypersensitivity reactions and anaphylactic shock in response for non-human antibodies. For the treatment of infant botulism, the FDA has approved Baby BIG, a purified human anti-botulinum immunoglobulin (https://www.fda.gov/drugs/bioterrorism-and-drug-preparedness/products-approved-other-bioterrorism-emergencies). However, this drug has a number of limitations (https://www.drugs.com/mtm/botulism-immune-globulin.html). In addition, large fragments of IgG have a limited ability to penetrate into peripheral tissues and interact with epitopes. In most cases, the treatment of botulism is symptomatic, in which mechanical ventilation is a life-saving option (Nantel, AJ Clostridium botulinum - international program on chemical safety.Centre de Toxicologie du Québec, August, 1999. World Health Organization: Bacteria; 1999. Available online : https://www.who.int/csr/delibepidemics/clostridiumbotulism.pdf [Accessed February 8, 2019]; Patel, K.; Cai, S.; Singh, B. Current strategies for designing antidotes against botulinum neurotoxins. Expert Opin Drug Discov 2014, 9(3), 319-333 http://dx.doi.org/10.1517/17460441.2014.884066 PMID: 24520991).

Also, there are agents for the prevention of botulism, but their effectiveness and safety have not been proven (Rusnak JM, Smith LA Botulinum neurotoxin vaccines: Past history and recent developments. Hum. Vaccines. 2009;5:794-805. doi: 10.4161/hv.9420 .). However, there are risk groups such as healthcare workers, researchers, and military personnel for whom botulism vaccination is indicated (109. Smith, L.A. Botulism and vaccines for its prevention. Vaccine 2009, 27 (Suppl. S4), D33-D39).

In this regard, an urgent task is to obtain an effective and safe means of preventing intoxication caused by botulinum toxin. Currently, new approaches to vaccination against botulism are focused mainly on the creation of recombinant plasmid and viral vectors, as well as recombinant botulinum toxin subunits.

A solution is known where recombinant DNA was used as a trivalent vaccine, inducing a humoral and cellular immune response in mice against botulinum toxins A, B and E after two multiple immunizations (Scott, VL; Villarreal, DO; Hutnick, NA; Walters, JN; Ragwan , E.; Bdeir, K.; Yan, J.; Sardesai, NY; Finnefrock, AC; Casimiro, DR; et al. DNA vaccines targeting heavy chain c-terminal fragments of clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge Hum Vaccin Immunother 2015, 11, 1961-1971).

There are works on the use of a recombinant adenoviral vector for inducing a specific immune response and capable of protecting against a lethal dose of botulinum neurotoxin (Zeng, J.; Du, J.; Zhao, Y.; Palanisamy, N.; Wang, S. Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells Stem Cells 2007, 25, 1055-1061).

Research is also underway on a candidate subunit vaccine against botulinum toxin type A (Yu, CH; Song, DH; Choi, JY; Joe, HE; Jeong, WH; Hur, GH; Shin, YK; Jeong, ST A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication. Hum. Vaccin Immunother. 2018, 14, 329-336.) and a polyvalent subunit vaccine (Webb, RP; Smith, TJ; Smith, LA; Wright, PM; Guernieri, RL; Brown, JL; Skerry, JC Recombinant botulinum neurotoxin hc subunit (bont hc) and catalytically inactive clostridium botulinum holoproteins (cibont hps) as vaccine candidates for the prevention of botulism. Toxins (Basel) 2017, 9, 269).

Monoclonal and single domain antibodies can also be considered as a means of preventing botulism, however, there are difficulties associated with their limited period of circulation in the body. A work is known in which a preparation of a recombinant replication-defective human adenovirus was obtained, carrying the gene for a single-domain antibody specific for botulinum toxin type A (Mukherjee J, Dmitriev I, Debatis M, Tremblay JM, Beamer G, Kashentseva EA, Curiel DT, Shoemaker CB. Prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a VHH-based antitoxin protein PLoS One 2014 Aug 29;9(8):e106422 doi: 10.1371/journal.pone.0106422 PMID: 25170904; PMCID: PMC4149568). It has been demonstrated that the administration of this drug to mice is able to provide the expression of a single-domain antibody and induce protection against a lethal dose of botulinum toxin type A. This solution, as the closest to the claimed one, was chosen by the authors of the patent for the prototype. However, the disadvantage of the prototype is the high immunogenicity and inflammatory potential of the adenovirus vector. It is known that recombinant adenovirus is able to induce a high level of immune response to the protein expressed in its composition, as well as an immune response to adenovirus capsid proteins. All of the above will create obstacles for re-introduction of the drug into the body, after leveling the specific activity of the drug after the initial injection. In addition, the single-domain antibody expressed in the vector lacks Fc-mediated functions such as Fc-dependent cytotoxicity, Fc-dependent phagocytosis, and activation of the complement system. It should also be noted that the prototype is capable of providing full protection of mice against 10LD50 botulinum toxin for a period of about 2-2.5 months, after which only partial protection of animals is observed.

Thus, there is a need in the prior art to create a new recombinant viral vector for the delivery and expression of a single-domain antibody gene specific to botulinum toxin for the prevention of intoxication caused by botulinum toxin type A.

Disclosure of the essence of the invention

The technical objective of the claimed group of inventions is to expand the arsenal of recombinant viral vectors for the prevention of intoxication caused by botulinum neurotoxin type A.

The technical result consists in creating an adeno-associated viral vector expressing a recombinant antibody, which is a single-domain antibody fused with the Fc fragment of human IgG1, specific for botulinum neurotoxin type A (rAAV-B7-Fc), and which can be used to prevent intoxication caused by botulinum type A neurotoxin. In addition, the technical result is that a recombinant viral vector has been developed, which has a lower inflammatory potential and a longer time interval of protective activity.

This technical result is achieved by creating a recombinant adeno-associated viral vector carrying a genetic construct containing the nucleotide sequence of SEQ ID NO: 2, expressing a recombinant antibody having the amino acid sequence of SEQ ID NO: 1.

Also, the technical result is achieved by the fact that the recombinant antibody expressed in the vector specifically binds to botulinum neurotoxin type A and has a protective activity against a lethal dose of botulinum neurotoxin type A.

Also, the technical result is achieved by the fact that the introduction of the created preparation of the recombinant adeno-associated viral vector (rAAV-B7-Fc) in an effective amount ensures prolonged expression of the recombinant antibody in the body.

In addition, the introduction of the created preparation of the recombinant adeno-associated viral vector rAAV-B7-Fc in an effective amount provides long-term protection (at least 120 days) against a lethal dose of botulinum toxin type A.

A method for preventing intoxication caused by botulinum neurotoxin type A has also been developed, which consists in introducing into the body of mammals in an effective amount the obtained recombinant adeno-associated viral vector rAAV-B7-Fc.

Brief description of the drawings

In FIG. 1 is a schematic representation of the plasmid pAAV-DJ-B7-Fc,

where

5.778 bp - number, nucleotide pairs (plasmid molecular weight),

Signal peptide - nucleotide sequence of the human immunoglobulin heavy chain signal peptide,

B7 - nucleotide sequence of single-domain antibody B7,

Hinge - nucleotide sequence of the hinge region,

Fc - nucleotide sequence of the Fc fragment of human IgG1.

In FIG. 2 is a schematic representation of the amino acid sequence of a recombinant B7-Fc antibody,

where

1 - sequence of a single-domain antibody;

2 - hinged region;

3 - Fc fragment of human IgG1.

In FIG. 3 shows restriction digestion data for plasmids pAAV-DJ-B7-Fc, pAAV-DJ Vector and pHelper Vector,

where

1 - plasmid pAAV-DJ-B7-Fc, hydrolyzed at the restriction sites SacI and XbaI;

2 - plasmid pAAV-DJ Vector, digested at NcoI restriction sites;

3 - plasmid pHelper Vector, hydrolyzed at EcoRI restriction sites;

4 - plasmid pHelper Vector, hydrolyzed at HindIII restriction sites;

5 - molecular weight marker GeneRuler 1 kb DNA Ladder (ThermoFisherScientific)

6 - non-digested plasmid pAAV-DJ-B7-Fc;

7 - non-digested plasmid pAAV-DJ Vector;

3 - non-digested pHelper Vector plasmid.

In FIG. 4 shows the affinity purification chromatogram of the rAAV-B7-Fc preparation.

Y-axis - absorption at a wavelength of 280 nm, mAU

The abscissa axis is the amount of liquid passed through the chromatograph system, ml

In FIG. Figure 5 shows the results of evaluating the authenticity of the purified rAAV-B7-Fc preparation by immunoblot analysis using polyclonal antibodies to adeno-associated virus capsid proteins,

where

1,2,3 - fractions of the rAAV-B7-Fc preparation after chromatographic purification;

VP1, VP2, VP3 - surface structural proteins of adeno-associated virus.

In FIG. Figure 6 shows the results of the evaluation of the expression of the recombinant B7-Fc antibody in the composition of the purified rAAV-B7-Fc preparation by immunoblotting using monoclonal antibodies to the human IgG Fc fragment,

where

1 - molecular weight marker Precision Plus Dual Color (Bio-Rad);

2,3,4 - culture fluid samples of HEK293 cells transduced with rAAV-B7-Fc at various dosages.

In FIG. Figure 7 shows data on the study of the protective activity of various doses of the rAAV-B7-Fc preparation against 10LD50 botulinum toxin type A.

The y-axis is the survival rate of animals, %

Abscissa - groups of animals with different dosages of the drug.

- мыши, получившие 10ЛД50 ботулотоксина типа А, на 3 сутки после введения препарата или плацебо (фосфатно-солевой буфер);

- mice that received 10LD50 of botulinum toxin type A, on the 3rd day after the administration of the drug or placebo (phosphate-buffered saline);

- мыши, получившие 10ЛД50 ботулотоксина типа А, на 7 сутки после введения препарата или плацебо (фосфатно-солевой буфер);

- mice that received 10LD50 botulinum toxin type A, on the 7th day after the administration of the drug or placebo (phosphate-buffered saline);

- мыши, получившие 10ЛД50 ботулотоксина типа А, на 10 сутки после введения препарата или плацебо (фосфатно-солевой буфер);

- mice that received 10LD50 of botulinum toxin type A, on the 10th day after the administration of the drug or placebo (phosphate-buffered saline);

- мыши, получившие 10ЛД50 ботулотоксина типа А, на 14 сутки после введения препарата или плацебо (фосфатно-солевой буфер);

- mice that received 10LD50 of botulinum toxin type A, on the 14th day after the administration of the drug or placebo (phosphate-buffered saline);

- мыши, получившие 10ЛД50 ботулотоксина типа А, на 21 сутки после введения препарата или плацебо (фосфатно-солевой буфер).

- mice that received 10LD50 of botulinum toxin type A, on the 21st day after administration of the drug or placebo (phosphate-buffered saline).

In FIG. 8. The results of a study of the pharmacokinetic properties of the recombinant B7-Fc antibody, when the rAAV-B7-Fc preparation was administered to mice at a dose of 10 ¹¹ GC per animal once, are presented.

The y-axis is the concentration of the recombinant B7-Fc antibody in the blood serum of mice that received the rAAV-B7-Fc preparation, ng/ml.

The abscissa axis is the time points for taking serum samples, days.

In FIG. 9 shows the results of studying the possibility of using the rAAV-B7-Fc preparation for long-term prevention of intoxication caused by botulinum toxin type A.

The y-axis is the survival rate of animals, %.

The abscissa axis is the time points for the introduction of 10LD50 botulinum toxin type A.

- мыши, получившие препарат rAAV-B7-Fc в дозе 10¹¹ гк на животное однократно.

- mice that received the rAAV-B7-Fc preparation at a dose of 10 ¹¹ GC per animal once.

- мыши, получившие фосфатно-солевой буфер.

mice treated with phosphate-buffered saline.

Implementation of the invention

To implement the present invention, at the first stage, a plasmid system is obtained necessary for the assembly of a recombinant adeno-associated viral vector rAAV-B7-Fc. For this, a three-plasmid AVV-DJ Packaging System (Cell Biolabs, Inc) was used, containing the plasmids pAAV-DJ-EGFP carrying the green fluorescent protein genes as a transgene, pAAV-DJ-Vector carrying the rep and cap genes, and pAAV- DJ-Helper carrying the genes of the E2a, E4, VA regions of the adenovirus. The required recombinant plasmids were obtained in preparative amounts by transformation of E. coli strain Dh5α cells. Plasmids were isolated using the commercial PureLink™ HiPure Plasmid Maxiprep Kit for DNA extraction. To test the obtained plasmids, restriction hydrolysis was performed using specific restriction endonucleases. All the fragments obtained as a result of restriction hydrolysis completely correspond in terms of molecular weight to the theoretically calculated ones. The pAAV-DJ-EGFP plasmid was used to obtain a plasmid carrying the gene for a single-domain antibody fused with a human IgG1 Fc fragment and specific for botulinum neurotoxin type A. The nucleotide sequence of the single-domain antibody gene fused with the Fc-fragment of human IgG1 and specific for botulinum neurotoxin type A (SEQ ID NO: 2) was synthesized at Evrogen CJSC and cloned into the pAAV-DJ-EGFP plasmid, replacing the green fluorescent protein gene at the restriction sites EcoRI and XbaI, which makes it possible to obtain rAAV expressing this antibody in the described system. Thus, a pAAV-DJ-B7-Fc plasmid was obtained containing inverted terminal repeats of the recombinant human adeno-associated virus serotype 2, with an integrated expression cassette containing the recombinant B7-Fc antibody gene.

It is known to those skilled in the art that any other plasmid system can be used to generate a recombinant adeno-associated viral vector. Thus, any plasmid system can be used to obtain a recombinant adeno-associated viral vector containing the nucleotide sequence encoding the recombinant antibody (SEQ ID NO: 1).

It is also known to those skilled in the art that for the purposes of the present invention, the sequence of a recombinant antibody (SEQ ID NO: 1) specific for botulinum neurotoxin type A may contain amino acid substitutions that do not affect the secondary and tertiary structure of the recombinant antibody and do not affect its ability to bind and neutralize botulinum neurotoxin type A. Moreover, since only antigen-binding loops (CDR domains) of single-domain antibodies are involved in antigen binding, as is known to specialists in this field, any immunoglobulin or its analogue containing the same amino acid sequences of CDR domains falls within the scope of the present invention. Moreover, sequences of CDR domains may contain substitutions/deletions/insertions of amino acids, which, when paired with amino acid sequences, provide at least 70% homology and do not qualitatively affect the ability to bind to the antigen. Thus, any plasmid system can be used to obtain a recombinant adeno-associated viral vector containing a nucleotide sequence encoding sequences of CDR domains whose homology is at least 70% with the proposed sequences.

In addition, it is known to those skilled in the art that, for purposes of the present invention, the nucleotide sequence encoding a recombinant antibody may differ based on the principle of degeneracy of the genetic code. Thus, any plasmid systems can be used to obtain a recombinant adeno-associated viral vector containing nucleotide sequences encoding the amino acid sequence of the recombinant antibody SEQ ID NO: 1.

After transformation of competent E. coli bacteria and their lysis, supercoiled DNA isolation was performed on a PlasmidSelect Xtra Starter Kit according to the manufacturer's instructions. After chromatographic purification, plasmid DNA was reprecipitated with sodium acetate to get rid of EDTA and other buffer components capable of inhibiting the transfection process.

Further, to obtain an adeno-associated viral vector expressing a recombinant antibody specific to botulinum toxin type A, the obtained plasmids were transfected into the HEK293 cell line, in which the production and assembly of the recombinant adeno-associated virus carrying the gene of the recombinant antibody specific to botulinum toxin type A took place. Purification of the recombinant adeno-associated virus , carrying the gene of a recombinant antibody specific for botulinum toxin type A, was carried out using tangential filtration and affinity chromatography.

Those skilled in the art will recognize that other eukaryotic expression systems may be used to produce and assemble a recombinant adeno-associated viral vector for the purposes of the present invention. If necessary, the preparation method further includes isolation and purification by any method known in the art.

Further, the authors studied the quantitative and qualitative characteristics of the resulting rAAV-B7-Fc preparation, and evaluated the production of recombinant antibodies in the composition of the obtained rAAV-B7-Fc preparation.

When studying the resulting rAAV-B7-Fc preparation in vivo, the pharmacokinetics of the recombinant antibody in the blood serum of mice that received the rAAV-B7-Fc preparation was evaluated. The optimal dose of the rAAV-B7-Fc preparation was selected, which provides long-term protection against 10LD50 botulinum toxin type A. The pharmacokinetics of the recombinant B7-Fc antibody in the body of mice that received the rAAV-B7-Fc preparation was studied. The duration of the protective activity of rAAV-B7-Fc against 10LD50 botulinum toxin type A was evaluated. It was demonstrated that the protective activity of the drug starts from day 3 and is at least 120 days after administration.

In addition, a method has been developed for the prevention of intoxication caused by botulinum toxin type A, which consists in intramuscular administration of an effective amount of a preparation of a recombinant adeno-associated virus rAAV-B7-Fc carrying the gene of a recombinant antibody specific to botulinum toxin type A.

The implementation of the invention is confirmed by the following examples.

Example 1 Preparation of Plasmid Constructs

The AAV-DJ Packaging System (Cell Biolabs, Inc) consisting of pAAV-DJ Vector, pHelper Vector, and pAAV-GFP Control Vector plasmids was used to obtain the genetic constructs required for assembly and production of the recombinant adeno-associated viral vector. The nucleotide sequence encoding the single-domain antibody fused to the Fc fragment was synthesized at Evrogen CJSC and cloned into the pAAV-EGFP Control Vector plasmid, replacing the EGFP gene at the EcoRI and XbaI restriction sites, obtaining the pAAV-DJ-B7-Fc construct (Fig. . one). A schematic representation of the recombinant B7-Fc antibody is shown in Fig.2.

The resulting plasmid was grown in E. coli. To do this, the bacteria were transformed with the target plasmids by the heat shock method. Competent cells were obtained according to standard protocols. During transformation, 5-10 ng of the target plasmid DNA was added to the competent cells of the Dh5α strain and incubated on ice for 30 minutes. After incubation, the tube with cells was placed in a water bath at 42°C for 45 seconds. Then they were incubated on ice for 10 minutes, 1 ml of SOB medium was added and incubated at 37°C for 1 hour on an incubator shaker. Subsequently, using selective antibiotics, they were placed in Petri dishes on a solid medium and incubated at 37°C overnight. After 16 hours of incubation, the selected colonies were inoculated into flasks with 100 ml of LB medium and incubated in a shaker-incubator at 210 rpm for 16-18 hours at 37°C.

Target plasmid DNA derived from E. coli DH5α strains was isolated using the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen, Thermo Fisher Scientific). Isolation was performed according to the manufacturer's protocol. The resulting plasmid DNAs are subsequently identified by restriction at the appropriate restriction endonucleases (FIG. 3). Restriction hydrolysis was performed by mixing deionized water and a buffer mixture in a test tube according to the manufacturer's recommendations. Added specific restriction endonucleases and target plasmid DNA, mixed using a vortex. Incubated for 60 minutes in an incubator at the recommended temperature as described by the manufacturer for the appropriate restriction endonuclease.

The final result was evaluated using horizontal agarose gel electrophoresis (0.8%). The results were visualized using the Gel Doc EZ (Bio-Rad) gel documentation system.

Thus, in this example, a system of plasmid vectors was obtained, which is necessary for the production of a recombinant adeno-associated virus carrying the recombinant antibody gene (rAAV-B7-Fc).

Example 2 Production of rAAV in HEK293 cell line culture.

Plasmids were transfected into HEK293 cell line culture. For this, a suspension culture of HEK293 cells was cultivated in BalanCD HEK293 medium (Fujifilm Irvine Scientifics) prepared according to the manufacturer's instructions. For cultivation, 125, 500, and 1000 ml Erlenmeyer flasks with a vented cap (Corning) were used using a Multitron shaker-incubator (Infors HT) with a stirring speed of 110 rpm at an amplitude of 50 mm. Under cultivation conditions, 37°C, 80% humidity, and 5% CO _{2 were} used. Passages were carried out when the cell concentration reached 2-3*10 ⁶ cells/ml.

Transfection was carried out in 10 culture flasks per 1000 ml with a cell suspension volume of 200 ml and a cell concentration of 10 ⁶ cells/ml. Transfection was performed using PEI linear 25 kDa transfection agent (Polyscience). The final concentration of DNA used for transfection was 1 μg/ml culture medium. Opti-MEM™ I Reduced Serum Medium (Gibco™, Thermo Fisher Scientific) was used as transfection medium at a volume of 10% of the final medium volume. The transfecting agent was used at a concentration of 1 mg/ml. The volume of PEI to DNA was taken in a ratio of 3:1. The resulting mixture was incubated at room temperature for 7 minutes to form a transfection complex. After, the mixture was introduced into the cell culture. After transfection, the cells were cultured for 5 days at a stirring speed of 110 rpm at an amplitude of 50 mm. Under cultivation conditions, 37°C, 80% humidity, and 5% CO _{2 were} used. Thus, 2000 ml of the transfected culture suspension was obtained.

The quantitative yield of recombinant viral particles was assessed by the number of genomic copies in cells on day 5 after transfection using the QuickTiter ™ AAV Quantitation Kit (Cell Biolabs) according to the manufacturer's instructions. The quantitative yield of recombinant rAAV-B7-Fc was 5*10 ⁸ genomic copies per ml of culture liquid and the total yield per 2 l of culture suspension was 10 ¹² genomic copies.

Thus, as a result of the work done, a cell suspension was obtained containing recombinant adeno-associated viral particles rAAV-B7-Fc containing the gene of a single-domain antibody specific to botulinum toxin type A, fused with the Fc fragment of human IgG1.

Example 3 Purification of rAAV-B7-Fc preparation from cell suspension.

After 5 days of cultivation, the cell suspension was lysed by adding 1% Tween-20, Tris-HCl up to 20 mM, sodium chloride up to 50 mM, magnesium chloride up to 2 mM, benzonuclease (Merck) 20U per ml of culture liquid into the culture liquid. The lysis was carried out at room temperature for 4 hours with gentle agitation. After lysis, the culture liquid was clarified by centrifugation at 8000 rpm for 20 minutes and then filtered through a Sartopore 2 0.45/0.2 μm capsule filter (Sartorius Stedim Biotech).

Purification of rAAV-B7-Fc was performed using the AVB Sepharose sorbent (Cytiva Life Sciences) according to the manufacturer's instructions. The purification chromatogram of the rAAV-B7-Fc preparation is shown in FIG. 4. The resulting preparation was concentrated using 50 kDa Amicon Ultra-15 centrifuge concentrators (Merck) and sterilized with a 0.22 µm membrane filter (MF-Millipore). The concentration of viral particles (preparation) was assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) at a wavelength of 260 nm. The preparation was frozen and stored at -70°C. As a result of chromatographic purification, 4 ml of the rAAV-B7-Fc preparation was obtained with a concentration of 1 u/ml and a ratio of 260/280 = 1.2. To determine the titer of the obtained preparation, the AAVpro® Titration Kit (for Real Time PCR) Ver.2 was used according to the manufacturer's instructions. The number of genomic copies in the purified preparation was 1.64*10 ¹² /ml.

Thus, in this example, a purified preparation of rAAV-B7-Fc was obtained with a total concentration of genomic copies of 6.56*10 ¹² gc/ml.

Example 4 Evaluation of the authenticity of the rAAV-B7-Fc preparation.

To assess the authenticity of the resulting rAAV-B7-Fc preparation, an immunoblotting method was used using antibodies specific to the AAV capsid proteins. For this, the purified preparation was run on a 4-20% polyacrylamide gel (4-20% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 μl #4561096, Bio-Rad) using 4x Laemmli Sample Buffer (#161 -0747, BioRad) supplemented with 2-Mercaptoethanol (Sigma-Aldrich) for analysis under reducing conditions. Immunoblotting was performed according to the standard method, using an Amersham Protran Premium 0.45 μm NC (GE Healthcare Life science) membrane for protein transfer using the Trans-Blot Turbo Transfer System. Next, the membrane was incubated in 5% skimmed milk (Sigma-Aldrich) in PBS-T for blocking. Then, primary antibodies Adeno-Associated Virus 2 / AAV2 (VP1 + VP2 + VP3) Rabbit Polyclonal Antibody (OriGene) were added at a ratio of 1:1000 and incubated for 60 minutes. Afterward, washing was carried out three times in PBS-T. Then, a conjugate of anti-rabbit antibodies labeled with horseradish peroxidase was added in a ratio of 1:2500 and incubated for 60 minutes. After washing five times in 0.1% PBS-T and visualizing using ECL Substrate (Bio-Rad) and Amersham Imager 600 (GE Healthcare). The results of the analysis are shown in Fig. five.

From the obtained results, it follows that the resulting preparation is indeed an adeno-associated virus, since it contains all structural proteins, namely VP1, VP2, VP3.

Thus, in this example, the rAAV-B7-Fc preparation was authenticated.

Example 5. Evaluation of the transducing ability and expression of the recombinant antibody in the preparation of rAAV-B7-Fc.

To assess the transducing ability and expression of the transgene in the rAAV-B7-Fc preparation, a HEK293 cell culture was transduced with the purified preparation. To do this, cells were seeded on a 96-well plate in DMEM medium (4 mM glutamine, 10% FBS, sodium bicarbonate 3.8 g/l) in a volume of 100 μl with a concentration of 0.5*10 ⁶ cells/ml. After 4 hours, 10 μl of the rAAV-B7-Fc preparation was added after purification. After 48 hours, the culture liquid was taken for further analysis. Culture fluid was evaluated by protein electrophoresis, on a 4-20% polyacrylamide gel (4-20% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl #4561096, Bio-Rad) using 4x Laemmli Sample Buffer (# 161-0747, BioRad) supplemented with 2-Mercaptoethanol (Sigma-Aldrich) for analysis under reducing conditions. Immunoblotting was performed according to the standard method, using an Amersham Protran Premium 0.45 μm NC (GE Healthcare Life science) membrane for protein transfer using the Trans-Blot Turbo Transfer System. Next, the membrane was incubated in 5% skimmed milk (Sigma-Aldrich) in PBS-T for blocking. Then Anti-Human IgG (Fc specific) Peroxidase antibody produced in goat (Sigma-Aldrich) was added at a ratio of 1:2500 and incubated for 60 minutes. After washing five times in 0.1% PBS-T and visualizing using ECL Substrate (Bio-Rad) and Amersham Imager 600 (GE Healthcare). The results of the analysis are shown in Fig. 6.

From the obtained results, it follows that the cells transduced with the rAAV-B7-Fc preparation express the recombinant B7-Fc antibody specific for botulinum toxin type A.

Thus, in this example, the expression of a recombinant antibody specific for botulinum toxin type A was confirmed in the preparation of rAAV-B7-Fc.

Example 6. Selection of the optimal dose of the rAAV-B7-Fc preparation for the induction of protection against a lethal dose of botulinum toxin.

The purpose of this experiment was to evaluate the ability of the developed rAAV-B7-Fc preparation to protect against a lethal dose of botulinum neurotoxin type A.

The study of the protective activity of the rAAV-B7-Fc preparation was carried out on a lethal model of BALB/c 10LD50 mice intoxication with botulinum neurotoxin type A (botulinum toxin). The experiment used 40 mice 18-20 gr. Animals were divided into 4 groups, which were administered:

1) Intramuscularly, into the inner surface of the thigh, the preparation rAAV-B7-Fc at a dose of 10 ⁹ gc/animal (total 10 animals),

2) Intramuscularly, into the inner surface of the thigh, the drug rAAV-B7-Fc at a dose of 10 ¹⁰ gc / animal (total 10 animals),

3) Intramuscularly, into the inner surface of the thigh, the preparation rAAV-B7-Fc at a dose of 10 ¹¹ gc/animal (total 10 animals),

4) Intramuscularly, into the inner surface of the thigh, phosphate-buffered saline, negative control (total 10 animals).

Then, on the 3rd, 7th, 10th, 14th and 21st days after administration of the drug, mice were injected with 10LD50 of botulinum toxin type A. For each time point, 2 mice from the group were used. As a result of the experiment, it was demonstrated that the administration of the rAAV-B7-Fc preparation at a dose of 10 ⁹ gc/animal provides protection against 10LD50 botulinum toxin type A, starting from the 21st day, the administration of the rAAV-B7-Fc preparation at a dose of 10 ¹⁰ gc/animal the animal provided partial protection on the 10th day after the injection and complete on the 14th day after the injection, and finally, the administration of the rAAV-B7-Fc preparation at a dose of 10 ¹¹ gc/animal provided complete protection of mice from the 3rd day after the administration . The results of the experiment are shown in Fig. 7.

It follows from the results of this experiment that all doses of the rAAV-B7-Fc preparation have protective activity against 10LD50 botulinum toxin type A, however, a dose of 10 ¹¹ gc/animal provides protection starting from day 3 after administration. Therefore, based on the fact that a mouse weighs about 20 g, in terms of a person, the working doses of the drug will be from 5*10 ¹⁰ gk/kg of body weight to 5*10 ¹² gk/kg of body weight. At the same time, the optimal dose for the induction of complete protection from the 3rd to after administration is 5 * 10 ¹² gc / kg of weight.

Thus, in this example, the optimal dose of the rAAV-B7-Fc preparation (10 ¹¹ gc/animal) was selected, providing protection against 10LD50 botulinum toxin type A. The protective dose of the rAAV-B7-Fc preparation was determined, which ranges from 5 * 10 ¹⁰ gk / kg up to 5 * 10 ¹² gk / kg of weight.

Example 7 Study of the pharmacokinetics of recombinant B7-Fc antibody in mice treated with rAAV-B7-Fc.

To study the duration of circulation of the recombinant B7-Fc antibody, 10 BALB/c mice were injected with rAAV-B7-Fc at a dose of 10 ¹¹ gc/animal. Next, serum was taken from mice and the amount of recombinant B7-Fc antibody in it was analyzed by indirect ELISA using a kit for the quantitative determination of human IgG in blood serum "Vector-BEST", the preparation of recombinant B7-Fc antibody purified by affinity chromatography was used as standards, and in 933NA antibodies (GE healthcare, USA) were used as a conjugate. The following points were selected for analysis: before the injection, on the 21st, 45th, 60th, 90th, 120th, and 150th days after the injection. Serum samples were taken from at least two animals for each point. The results of the analysis are shown in Fig. 8. It was demonstrated that the concentration of recombinant B7-Fc antibody in serum on the 21st day after administration is about 3 ng/ml. The peak concentration of the antibody is observed on the 60th day after administration and is 15 ng/ml. After 90 days, there is a significant drop in the concentration of the B7-Fc antibody, and between the 120th and 150th days, after the administration of the rAAV-B7-Fc preparation, the antibodies are completely eliminated from the body.

Thus, this example demonstrates that the recombinant B7-Fc antibody is able to circulate in the body of mammals for at least 120 days from the moment of administration of the rAAV-B7-Fc preparation.

Example 8. Method for preventing intoxication caused by botulinum toxin type A.

In this example, the possible induction of long-term protection against botulinum toxin type A intoxication and the use of rAAV-B7-Fc as a prophylactic agent for botulinum toxin type A intoxication was studied.

In this experiment, BALB/c mice were used, which were divided into 2 groups, 30 animals each. The experimental group was injected with rAAV-B7-Fc (10 ¹¹ gc/animal) intramuscularly into the back of the thigh. Then, 10LD50 of botulinum toxin type A was administered to 3 mice from the experimental and control groups on days 0, 3, 21, 45, 60, 75, 90, 105, 120, 150 after the administration of the rAAV-B7-Fc preparation. The results of the experiment are shown in Fig. 9. As a result, it was demonstrated that animals treated with rAAV-B7-Fc are protected from a lethal dose of botulinum toxin type A from 3 to at least 120 days after administration of the drug. At the same time, the death of control animals was observed at all time points.

Thus, in this experiment, the possibility of using the rAAV-B7-Fc preparation for the prevention of intoxication caused by botulinum toxin type A was demonstrated. At the same time, the protective effect with the introduction of 5 * 10 ¹² gc / kg of body weight provides protection from 3 to 120 days after the administration of the drug .

To provide protection against 10LD50 botulinum toxin type A, this drug can be administered to a person at a concentration of 5 * 10 ¹⁰ to 5 * 10 ¹² GC per kg of body weight.

Industrial Applicability

All the above examples confirm the effectiveness of the created vector based on human adeno-associated virus expressing recombinant antibody and having protective properties against botulinum neurotoxin type A and its industrial applicability.

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SEQUENCE LIST

<110> N.F. Gamaleya Research Center for Epidemiology and Microbiology, Ministry of Health of Russia

<120> Adeno-associated expression vector

a virus that has protective properties against intoxication,

caused by botulinum neurotoxin type A.

<160> 4

<170> BISSAP1.3.6

<210> 1

<211> 356

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of the recombinant antibody

B7-Fc, which is a B7 single-domain antibody,

modified with an Fc fragment

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Ser Ser Ser Ser Tyr

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Asn Trp Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Thr Leu Gly Gly Tyr Tyr Tyr Ile Tyr Glu Tyr Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Ser Thr Met Val Arg

115 120 125

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

130 135 140

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

145 150 155 160

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

165 170 175

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

180 185 190

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

195 200 205

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

210 215 220

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

225 230 235 240

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

245 250 255

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

260 265 270

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

275 280 285

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

290 295 300

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

305 310 315 320

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

325 330 335

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

340 345 350

Ser Pro Gly Lys

355 360 365

<210> 2

<211> 1068

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of recombinant antibody B7,

which is a single-domain antibody B7, modified

Fc fragment

<400> 2

GAGGTGCAGC TGGTGGAGTC TGGGGGAGGA GCGGTGCAGG CTGGGGGCTC TTTGAGACTT 60

TCCTGTGCAG CCTCTGGACG CTCCTCCAGT AGCTATGGCA TGGGCTGGTT CCGCCAGGCT 120

CCAGGGAAGG AGCGTGAGTT TGTAGCAGCT ATTAACTGGA GTGGTGGTAG CACACACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACTGAGGAC ACGGCCGTTT ATTACTGTGC AGCAACCCTC 300

GGGGGTTACT ACTATATATA TGAGTATGAC TACTGGGGCC AGGGGACCCA GGTCACTGTC 360

TCCTCATCGA GCACCATGGT TAGATCTGAC AAAACTCACA CATGCCCACC GTGCCCAGCA 420

CCTGAACTCC TGGGGGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC 480

ATGATCTCCC GGACCCCCTGA GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT 540

GAGGTCAAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG 600

CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAG 660

GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC 720

ATCGAGAAAA CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG 780

CCCCCATCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC 840

TTCTATCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC 900

AAGACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCTACAG CAAGCTCACC 960

GTGGACAAAGA GCAGGTGGCA GCAGGGGAAC GTTCTTCTCAT GCTCCGTGAT GCACGAGGCT 1020

CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAA 1068

<---

Claims (6)

1. An expression vector based on a recombinant adeno-associated virus with an integrated genetic construct containing the nucleotide sequence of SEQ ID NO: 2 encoding the recombinant antibody of SEQ ID NO: 1. 2. The expression vector according to claim 1, characterized in that the recombinant antibody expressed in its composition, which is a single-domain antibody fused with the Fc fragment of human immunoglobulin G1. 3. The expression vector according to claim 2, characterized in that the recombinant antibody expressed in its composition specifically binds type A botulinum toxin. 4. An expression vector according to claim 1, characterized in that its administration to mammals in an effective amount ensures prolonged expression in the body of a recombinant antibody specific for botulinum toxin type A. 5. The expression vector according to claim 4, characterized in that its administration to mammals in an effective amount provides long-term protection against a lethal dose of botulinum toxin type A. 6. The use of the expression vector according to paragraphs. 1-5 for the prevention of intoxication caused by botulinum neurotoxin type A.

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