RU2502745C2 - Anti-flu nanoantibody, recombinant viral vectors and pharmaceutical compositions for prophylaxis and therapy of a-type flu - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes a nanoantibody (a single-domain antibody) specifically bound to hemagglutinin of A-type flu virus H5N2 and suppressing infection of this virus, which is characterised with aminoacid sequence. Besides, a viral, an adenoviral, an adeno-associated and a lentiviral vector for expression of a nanoantibody and a composition for suppression of progress of infection of A-type flu virus H5N2 containing a nanoantibody and a viral vector are described.

EFFECT: invention can be further used in therapy of infection of A-type flu virus H5N2.

6 cl, 12 dwg, 18 ex, 1 tbl

Description

The technical field of the present invention

The present invention relates to virology and involves the use of antibodies that are nanoantibodies for the prevention and treatment of influenza, as well as the use of the genes of these antibodies in combination with viral vectors (vectors based on recombinant adenoviruses, adeno-associated viruses, lentiviruses) and their expression in the body of mammals for the prevention and treatment of influenza.

BACKGROUND OF THE INVENTION

Influenza viruses can cause epidemic emergencies. These viruses are widespread in nature and can infect humans, many types of mammals (horses, pigs, seals, etc.), as well as all types of birds. Respiratory diseases caused by influenza viruses in some cases can occur with complications and result in death.

Influenza viruses are characterized by high antigenic variability. The surface virion glycoproteins, hemagglutinin and neuraminidase, are most susceptible to changes. Two ways to change them are known. The first is antigenic drift. Under the pressure of population immunity, mutations that allow you to get out of control of the immune system give the virus a serious advantage and become fixed. As a result, new antigenic variants of hemagglutinin and neuraminidase are constantly replacing each other. As a result, epidemics arise, as protection against previous strains of viruses, including the same subtype, is insufficient to neutralize the new strain. Unfortunately, the use of modern subunit and inactivated vaccines does not solve this problem, because provides good protection only from the strain from which they were derived. Therefore, currently, to protect the population from new epidemic strains, it is constantly necessary to create new vaccines.

The second way to change influenza viruses is antigenic shift, i.e. a change in the antigenic formula of the virus as a result of the replacement of the gene (s) and corresponding protein (s). The mechanism of antigenic shift is based on reassortment or recombination of genes, which can occur during joint infection with two or more viruses [Kochergin-Nikitsky K.S. // Analysis of gene interaction when crossing a low pathogenic avian influenza virus subtype H5 and a highly productive strain of human influenza virus. M., GU Research Institute of Virology. D.I. Ivanovsky RAMS, 2007]. Shift changes, as a rule, affect the antigenic structure of hemagglutinin, less often neuraminidase. Thus, at irregular time intervals, pandemic variants of influenza viruses with new antigenic and biological properties that cause severe illness and death of people appear [Lvov D.K. // Sov. Med. Rev. E. Virol. Rev. 1987. V. 2. P.15-37; Webster R.G., Bean W.J., German O.T., et al. // Microbiol. Rev. 1992. V. 56. P.152-179]. For example, the pandemic of the "Spanish woman" - the H1N1 flu virus in 1918-1920. led to the death of about 50 million people around the world. In June 2009, the World Health Organization (WHO) assigned the sixth degree of pandemic threat to the new H1N1 virus, cases of which were detected in early April 2009. This virus is a reassortant containing the genes of avian, human and swine viruses [Shinde V., Bridges C. B., Uyeki T. M. // The new England journal of medicine. 2009. V. 360; 25. P.2616-2625]. With the emergence of a new pandemic strain, the creation of a vaccine takes too much time, which does not allow you to quickly stop the spread of a dangerous virus. Thus, despite the large-scale preventive measures being taken, including vaccination, in many countries of the world, including Russia, seasonal outbreaks of influenza are recorded annually, covering all segments of the population from children to the elderly and senile. To solve this problem, new approaches to the prevention and treatment of influenza are needed.

In addition to flu vaccines, there is now a wide selection of medicines. However, it should be noted that prophylaxis with such drugs does not have sufficient effectiveness, in addition, many drugs have a wide range of contraindications and can cause adverse reactions.

Currently, several groups of drugs are used for the treatment and prevention of influenza, among which M2-channel inhibitors and neuraminidase inhibitors occupy a special place. Of the drugs of the first group in our country, Remantadine was used for many years, which had a pronounced therapeutic and preventive effect in case of influenza caused by type A. However, the widespread use of M2-channel inhibitors led to the emergence of resistant strains of influenza viruses resistant to Remantadine. Of the drugs of the second group, zanamivir and oseltamivir are registered in Russia. Zanamivir is not suitable for widespread use in clinical practice, as can be used only in the form of inhalation, which is unacceptable for preschool children and elderly patients. In addition, a number of adverse reactions are possible, including bronchospasm and laryngeal edema. Another drug, oseltamivir (Tamiflu) has established itself as a highly effective and safe drug, but one of the most fundamental drawbacks of this drug is the need for its early use (most effective when taken in the first 36 hours of the disease).

The problem of timely access to medical care and early treatment of respiratory viral infections is one of the most acute practical health problems. According to the Research Institute of Influenza, Russian Academy of Medical Sciences, the majority of patients seek medical help only on the 2nd-3rd day, when it becomes obvious that the patient is not ill with a simple cold and that treatment cannot be limited to antipyretic drugs, but patients with severe forms of influenza and viral pneumonia enter the clinic of the Institute of Influenza RAMS only on the 5th day. In this regard, neuraminidase inhibitors cannot be widely used in real conditions of an emerging epidemic.

Thus, we can conclude that today there are no effective and safe drugs for the treatment or prevention of influenza.

The solution to this problem can be the creation of drugs based on antibodies obtained against type A influenza virus. The action of these drugs is based on the introduction of antibodies to the protective antigen of the pathogen into the body, which can protect the host from infection. This approach has been applied since the first half of the 20th century. Until the late 1960s in the USSR, hyperimmune horse serum was relatively widely used for the prevention and treatment of influenza. However, often as an adverse reaction, this drug caused an allergy, and in this regard, he had to be abandoned. But the very idea of using antiviral antibodies in the blood of people is still being implemented as blood products. These drugs are called immunoglobulins. The most effective and common drug in Russia is “specific influenza immunoglobulin” from the blood of donors specially and repeatedly vaccinated against influenza. In addition to anti-influenza antibodies specifically stimulated by vaccination, donor immunoglobulin contains antibodies to many common infectious agents, including respiratory viruses. In recent years, there have been reports of the formation of antibodies to the introduced human protein. The drug is not without other side effects: fever, allergization: from the appearance of a rash to the development of a serious condition and anaphylactic shock.

Immunoglobulin preparations obtained by genetic engineering methods lack some of these drawbacks. Thus, the effectiveness of monoclonal single chain antibodies (scFv) obtained against anthrax protein toxin [Mabry R., Infect Immun. 2005: 73: 8362-8368.]. However, extremely high doses of protein are used, which is a significant limitation. Humanized single chain nanoantibodies are not rejected by the human body and do not cause an allergic reaction. However, this type of antibody has several disadvantages: the high cost of their production and the limitations of genetic engineering manipulations.

Currently, a technology has been developed to obtain, using the methods of genetic engineering, another type of antibody - recombinant nanoantibodies of a given specificity. It is based on the production of nucleotide sequences of the genes of noncanonical antibodies of animals of the Camelidae family. These antibodies are a dimer of only one shortened (the first constant region of CH1 is absent) immunoglobulin heavy chain. For the specific recognition and binding of antigen proper, in this case, only one variable domain of this antibody is necessary and sufficient.

The full equivalent of the term "nanoantibodies" for the purposes of the present invention is the widely used designation "nanobody", introduced by ABLYNX (NANOBODYTM), as well as "single-domain mini-antibody" and "single-domain nanoantibody".

Obtained by this technology nanoantibodies have the following properties [Tillib S.V. Camel Mini Antibodies is an effective tool for research, diagnosis and therapy. Molecular Biology, 2011; 45 (1): 77-85]:

1) High solubility and stability (in a wide range of temperatures and acidity of the medium).

2) Improved cell permeability due to small size (~ 2 × 4 nm, 13-15 kDa).

3) The ability to form paratypes that are unusual for classical antibodies, allowing them to bind to recesses and active centers of proteins.

4) The presence of a proven method for the production and selection of nanoantibodies.

5) The possibility of economical operating time in large quantities. Nanoantibodies can be produced in the periplasm of E. coli bacteria.

6) The possibility of genetic engineering modifications for polyvalent nanoantibodies.

7) Low immunogenicity.

Another positive feature of these antibodies is the possibility of their "humanization" without a noticeable loss of their specific activity, by making a small number of point substitutions of amino acids [Vincke C., Loris R., Saerens D., et al. // J. Biol. Chem. 2009.V. 284. No. 5. P.3273-3284]. This opens up the potential for widespread use of nanoantibodies as passive immunization agents to prevent the development of various dangerous infectious diseases [Wesolowski J., Alzogaray V., Reyelt J. et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009; 198, 157-174.].

Thus, it was shown (WO 2007/052242; Prendergast Patrick; Composition and method for the treatment of viral infection using camelid heavy chain antibodies) that antibodies obtained against the influenza virus and selected for their specificity for neuraminidase can protect birds from the development of diseases, caused by influenza viruses of various strains. This technical solution as the closest to the claimed by the composition of the active substance of the pharmaceutical composition and the method of introducing it into the body is selected by the authors of the present for the prototype.

The disadvantages of the prototype are:

1) Antibodies to neuraminidase of the influenza virus have a weak neutralizing activity. Effective virus neutralization with such a drug can be provided only if it is circulated for a long time in the body. This leads to the need to introduce large doses of drugs into the body to achieve a positive effect, as well as to the need for repeated administration of the drug.

2) To prevent the occurrence of various immune reactions to a foreign protein (antibody), introduced in large quantities due to its weak virus-neutralizing activity, an administration scheme is used, according to which the drug is administered twice, and the second time together with a drug that inhibits TNF-α. Moreover, repeated administration is often difficult for patients.

Thus, in the prior art there is an urgent need to develop a pharmaceutical composition that serves for the prevention and treatment of influenza.

The present invention was the creation of:

1) a nanoantibody that can effectively bind a certain epitope of the hemagglutinin of the influenza virus and, thereby, block the development of influenza infection,

2) a vector expressing a nanoantibody that can effectively bind a certain epitope of hemagglutinin of the influenza virus and, thereby, block the development of influenza infection,

3) a composition consisting of a nanoantibody and an adenoviral vector expressing a nanoantibody that can efficiently bind a certain hemagglutinin epitope of the influenza virus and, thereby, block the development of influenza infection.

The problem is solved by the claimed invention due to the fact that they create an anti-flu nanoantibody that specifically binds to a certain type A influenza virus hemagglutinin epitope, suppresses type A influenza virus infection. A viral vector expressing an anti-flu nanoantibody is created that can effectively bind a specific hemagglutinin epitope of influenza virus and, thereby, block the development of influenza infection. Moreover, the viral vector may be an adenoviral vector expressing an anti-flu nanoantibody, which is able to efficiently bind a certain epitope of the hemagglutinin of the influenza virus and, thereby, block the development of influenza infection. The viral vector may also be an adeno-associated vector expressing an anti-flu nanoantibody that is able to efficiently bind a specific hemagglutinin epitope of influenza virus and thereby block the development of influenza infection. The viral vector may be a lentiviral vector expressing an anti-flu nanoantibody that is able to efficiently bind a specific hemagglutinin epitope of the influenza virus and thereby block the development of influenza infection. A composition is proposed consisting of an effective amount of an anti-flu nanoantibody and a viral vector expressing an anti-flu nanoantibody capable of efficiently binding the hemagglutinin of the influenza virus and thereby block the development of influenza infection.

Disclosure of the present invention

The above objective of the present invention is solved in that the development of the drug is carried out using specially prepared nanoantibodies that bind a certain epitope of the surface protein of the hemagglutinin of the influenza virus in such a way that the infectious activity of influenza A virus is violated (blocked and neutralized).

The following were selected as methods of application for the active substance (antibody): use of nanoantibodies in the form of a suspension in a pharmaceutically acceptable solvent; the use of recombinant viral vectors as a carrier for the active substance: based on adenoviruses, adeno-associated viruses and lentiviruses expressing nanoantibody genes capable of binding and / or neutralizing influenza virus; the use of compositions containing both a viral vector with the nanoantibody gene and nanoantibody (in the form of a protein).

The choice of implementation routes in order to obtain a pharmaceutical composition with the claimed properties is due to the following factors.

The proposed technical solution provides for the use of a special nanoantibody as an active ingredient in the pharmaceutical composition, which is able to efficiently bind to a certain hemagglutinin epitope of the influenza virus in such a way that the infectious activity of the virus is violated (blocked, neutralized). Most currently used neutralizing antibodies (classical antibodies consisting of 4 immunoglobulin chains) have an affinity for this influenza hemagglutinin protein. Antibodies against hemagglutinin are able to prevent the penetration of the virus into the cell and, thus, prevent the development of infection. In contrast, the effect of antibodies against neuraminidase is carried out at the level of exit of mature particles of the virus from the cell and therefore only can reduce the period of the disease and its intensity. Due to the fact that it is antibodies to hemagglutinin that can have a pronounced neutralizing activity and exert their effect against the influenza virus immediately after it enters the body, that is, they are able to prevent the development of infection, nanoantibodies obtained with influenza virus hemagglutinin and possessing virus-neutralizing activity were used as an active substance for creating a pharmaceutical composition for the prevention and treatment of influenza.

The proposed solution provides a method of using nanoantibodies, including the use of viral vectors as a carrier for the active substance. Viral vectors are recombinant viruses whose genome includes the target gene (nanoantibody gene) with a set of regulatory elements. The following recombinant viruses are most often used as viral vectors: adenoviruses [Shmarov M.M., Tutykhina I.L., Logunov D.Yu. et al. Induction of a protective immune response in mice vaccinated with CELO recombinant avian adenovirus expressing rabies virus glycoprotein G. Journal of Microbiology, Epidemiology and Immunology, 2006; 4: 69-71; Tutykhina I.L., Shulpin M.I., Chvala I.A. et al. Construction of recombinant CELO adenoviruses expressing the avian influenza A virus hemagglutinin gene and studying the possibility of their use as vaccines for protection against avian influenza A virus H5N1 and H7N1. Molecular Genetics, Microbiology and Virology 2011 .; Tutykhina I.L., Shmarov M.M., Logunov D.Yu. et al. Design and prospects for the use in medicine of recombinant adenoviral nanostructures. Russian nanotechnology. 2009; 4 (11-12): 82-92; Liu M.A. Immunologic basis of vaccine vectors. Immunity. 2010; 33 (4): 504-15; Lasaro M.O., ErtI H.C. New insights on adenovirus as vaccine vectors. Mol ther. 2009: 17 (8): 1333-9], retroviruses [Liu M.A. Immunologic basis of vaccine vectors. Immunity. 2010; 33 (4): 504-15; Pincha M., Sundarasetty B.S., Stripecke R. .. Lentiviral vectors for immunization: an inflammatory field. Expert Rev Vaccines. 2010; 9 (3): 309-321; Negri D.R., Michelini Z., Cara A. Toward integrase defective lentiviral vectors for genetic immunization. Curr HIV Res. 2010; 8 (4): 274-2810]. Adeno-associated viruses [Sun J.Y., Anand-Jawa V., Chatterjee S., Wong K.K. Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther. 2003: 10 (11): 964-76]. Among the existing delivery systems of antigens, viral vectors occupy a special place, since they have the following properties:

- have a natural mechanism of interaction with the cell and penetration into the cell;

- transport foreign genetic material into the cell nucleus;

- able to provide long-term expression of antigen;

- the viral envelope performs a protective function for the genetic material encoding the antigen.

When introduced into the body, viral vectors penetrate into various types of cells, where they express the genes integrated into the DNA. In this regard, viral vectors are a widely used tool for delivering genes into human and animal cells [R. Harrop, et al., Advanced Drug Delivery Reviews, V.58, 1. 8, 2006, P.931-947].

Along with the above, viral vectors are highly efficient in expressing the target gene in various types of cells, and are safe for humans and animals.

Thus, the use of viral vectors as a carrier for the active substance of the claimed composition allows delivery of target protein genes to the body, which, in turn, provides an extension of the circulation period of the target gene product in the body.

As a carrier, viral vectors obtained on the basis of recombinant human adenovirus of the 5th serotype are used. Adenoviral vectors are known to be used most often in practice. Currently, vaccines against various bacterial (tularemia, tuberculosis, brucellosis, etc.) and viral (human immunodeficiency virus, human papillomavirus, rabies virus, Ebola, etc.) human pathogens [Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, Zganiacz A, Hitt M, Xing Z. Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J Immunol. 2004 Nov 15; 173 (10): 6357-65 .; Richardson JS, Yao MK, Tran KN, Croyle MA, Strong JE, Feldmann H, Kobinger GP. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine. Plos one. 2009; 4 (4): e5308. Epub 2009 Apr 23 .; Li WH, Zhang Y, Wang SH, Liu L, Yang F. Recombinant replication-defective adenovirus based rabies vaccine. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2003 Dec; 25 (6): 650-4 .; Lees CY, Briggs DJ, Wu X, Davis RD, Moore SM, Gordon C, Xiang Z, ErtI HC, Tang DC, Fu ZF. Induction of protective immunity by topic application of a recombinant adenovirus expressing rabies virus glycoprotein. Vet Microbiol. 2002 Apr 2; 85 (4): 295-303]. Thus, adenoviral vectors containing the nucleotide sequence of human influenza virus hemagglutinin were obtained for further use as a vaccine for humans and animals against a pathogenic strain of influenza virus (Vaccine. 2005, V 23, p. 1029-1036). The H5N1 avian influenza virus hemagglutinin gene was included in RPAN DNA based on human adenovirus of the 5th serotype also with the aim of using the resulting product as a vaccine for humans, mammals and birds (Vaccine 2007, J. of Virology, 2006, V. 80, No. 4 , p. 1959-1964). Adenoviral vectors were also used for gene therapy of tumors when the tumor suppressor gene p53 was cloned into their DNA (US 7,033,750). In addition, two drugs, which are adenoviral vectors based on human adenovirus of the 5th serotype, are approved for use in oncology.

The use of viral vectors as carriers gives the pharmaceutical composition the following advantages:

- Using viral vectors, the problem of instability of antibody preparations (introduced into the body in the form of proteins) can be solved.

Viral vectors are stable, can be lyophilized, can be stored at 4 ° C for a long time without loss of activity.

Technologies for producing viral vectors on an industrial scale are economically viable and allow you to get drugs with a high titer. The process of obtaining a new type of viral vector takes several weeks, which can allow you to quickly respond to changing epidemiological conditions in the shortest possible time [R. Harrop, et al., Advanced Drug Delivery Reviews, V.58, 1. 8, 2006, P.931 -947].

- The use of viral vectors allows to ensure the development of a protective level of antibodies in 96 hours after administration. Moreover, the protective level of antibodies is maintained in the body for at least 2 weeks [Kasuya K., Mol Ther. 2005 Feb; 11 (2): 237-44; Sofer-Podesta C., Infect Immun. 2009 Apr; 77 (4): 1561-8; Chiuchiolo M.J., J Infect Dis. 2006 Nov 1; 194 (9): 1249-57.] For adenoviral vectors and several months for adeno-associated and lentiviral vectors [Viral vectors for gene transfer: a review of their use in the treatment of human diseases. WaltherW, Stein U. Drugs. 2000 Aug; 60 (2): 249-71].

As a carrier, viral vectors derived from the recombinant adeno-associated virus of the 2nd serotype (AAV-2) are used. This is known to be a non-pathogenic human virus, which is often used in practice to create gene therapy drugs [AAV-mediated gene therapy for liver diseases: the prime candidate for clinical application? van der Laan LJ, Wang Y, Tilanus HW, Janssen HL, Pan Q. Expert Opin Biol Ther. 2011 Mar; 11 (3): 315-27; Adeno-associated virus (AAV) based gene therapy for eye diseases. Wang S, Liu P, Song L, Lu L, Zhang W, Wu Y. Cell Tissue Bank. 2011 May; 12 (2): 105-10; Gene therapy for ischemic heart disease. Hinkel R, Trenkwalder T, Kupatt C. Expert Opin Biol Ther. 2011 Jun; 11 (6): 723-37].

Currently, vaccines against various human pathogens are being developed based on adeno-associated viral vectors. Thus, adeno-associated viral vectors containing the nucleotide sequence of the type A H1N1 influenza virus hemagglutinin were obtained for further use as a vaccine for humans and animals against the pathogenic strain of the influenza virus [Vaccine protection against lethal homologous and heterologous challenge using recombinant AAV vectors expressing codon-optimized genes from pandemic swine origin influenza virus (SOIV). Sipo I, Knauf M, Fechner H, Poller W, Planz O, Kurth R, Norley S. Vaccine. 2011 Feb 11; 29 (8): 1690-9]. Adeno-associated viral vectors were also used for gene therapy of hemophilia (expressed factor IX), tumors (expressed diphtheria toxin and pro-apoptotic factor PUMA) and other diseases [Novel cytotoxic vectors based on adeno-associated virus. Kohlschutter J, Michelfelder S, Trepel M. Toxins (Basel). 2010 Dec; 2 (12): 2754-68; AAV provides an alternative for gene therapy of the peripheral sensory nervous system. BeutIerAS. Mol ther. 2010 Apr; 18 (4): 670-3; Muscle-directed gene therapy for hemophilia B with more efficient and less immunogenic AAV vectors. Wang L, Louboutin JP, Bell P, Greig JA, Li Y, Wu D, Wilson JM. J Thromb Haemost. 2011 Oct; 9 (10): 2009-19]. Moreover, it was shown that adeno-associated viral vectors carrying HIV-1 nanoantibody genes can prevent HIV-1 infection in animals [Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro . Abdel-Motal UM, Sarkis PT, Man T, Pudney J, Anderson DJ, Zhu Q, Marasco WA.PLoS One. 2011; 6 (10): e26473].

As the carrier, viral vectors derived from the recombinant human immunodeficiency virus (HIV-1) are used. It is known that this vector is often used in practice to create gene therapeutic drugs [Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Dropulic B. Hum Gene Ther. 2011 Jun; 22 (6): 649-57]. Thus, lentiviral vectors were used for gene therapy of diseases of the nervous system (expressed various genes), tumors (expressed shRNA STAT-3) and other diseases [STAT3 knockdown reduces pancreatic cancer cell invasiveness and matrix metalloproteinase-7 expression in nude mice. Li H, Huang C, Huang K, Wu W, Jiang T, Cao J, Feng Z, Qiu Z. PLoS One. 2011; 6 (10): e25941; Manipulation of gene expression in the central nervous system with lentiviral vectors. Sun B, Gan L. Methods Mol Biol. 2011: 670: 155-68; Gene therapy for ocular diseases. Liu MM, Tuo J, Chan CC. Br J Ophthalmol. 2011 May; 95 (5): 604-12].

Moreover, lentiviral vectors carrying the genes of chimeric antibodies to the TRAIL receptor have been shown to cause tumor size reduction [2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice. Li M, Wu Y, Qiu Y, Yao Z, Liu S, Liu Y, Shi J, Zheng D. Mol Ther. 2011 Sep 20].

The use of drugs, which are a viral vector that carries the nanoantibody gene in the DNA, which can neutralize the influenza virus of the type of the current strain, allows you to provide protection after 48-96 hours against the currently circulating influenza virus strain. Moreover, the use of a pharmaceutical composition consisting of one type of viral vector carrying the nanoantibody gene and protein preparations of the corresponding single-domain nanoantibody allows protecting the body during the period immediately after drug administration, which lasts at least 3-4 weeks.

Thus, it is advisable to use a complex preparation having two components: nanoantibodies against influenza virus type A and a recombinant viral vector expressing the gene of nanoantibodies against influenza virus type A.

Such a drug will have the following advantageous characteristics:

1) The basis of the drug are antibodies - the most effective natural antiviral substances.

2) A short course of administration (1 time), which reduces the risk of side effects and intolerance.

3) Effectiveness of action at various stages of the development of the infectious process.

4) The possibility of using as a supplement to late vaccination of people at risk in the first 2 weeks after vaccination (for the period of antibody production).

5) The possibility of use for persons with immunodeficiency who may give an insufficient immune response to vaccination.

6) The possibility of use for the elderly, for whom the effectiveness of vaccination is reduced and reaches 50-70%, as an addition to vaccination.

7) The possibility of use for unvaccinated persons who are in contact with sick relatives and neighbors.

8) The possibility of use for those who, for whatever reason, were not vaccinated on time.

9) The cost of the course of use of such a drug is comparable to the cost of one dose of vaccine.

The present invention combines the advantages of the various approaches mentioned and relates to single-domain nanoantibodies that bind / neutralize the influenza virus, as well as to a method for using such antibodies as by using recombinant viral vectors containing an expression cassette containing a nucleotide sequence as an active carrier (antibody) encodes a nanoantibody that binds to the influenza virus, and by creating a pharmaceutical composition consisting of nanoantibody p otiv influenza virus and rekmbinantnogo viral vector expressing this nanoantibody.

In accordance with one preferred embodiment of the present invention, the influenza virus is a type A influenza virus characterized by the antigenic formula H1N (1-9).

In accordance with another preferred embodiment of the present invention, the influenza virus is a type A influenza virus characterized by the antigenic formula H3N (1-9) or H5N (1-9).

Furthermore, the present invention relates to a pharmaceutical composition for the prevention or treatment of influenza, which is a suspension of the recombinant viral vectors disclosed above in a pharmaceutically acceptable solvent or excipient. Such pharmaceutically acceptable solvents and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, Ed., MackPubIishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds. , Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

The use of such a pharmaceutical composition makes it possible to achieve transient expression of nanoantibodies in a patient, which, due to their unique properties described in detail above, are capable of providing sufficiently reliable immune defense against the influenza virus. One of ordinary skill in the art will understand that the use of such a pharmaceutical composition is effective mainly for the prevention of infection.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of influenza, which is a suspension of the recombinant viral vectors and nanoantibodies disclosed above that bind to an influenza virus of the same type and antigenic formula as the nanoantibodies encoded by expression cassettes contained in said recombinant viral viruses vectors in a pharmaceutically acceptable solvent or excipient.

The use of such a pharmaceutical composition provides a sufficiently reliable immune defense against the influenza virus, not only as a preventive measure, but also after the patient is infected with the influenza virus. Of course, the earlier the claimed pharmaceutical composition is applied at an earlier stage of infection, the more effective this treatment will be.

In addition, the present invention relates to a method for the prevention or treatment of influenza, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of one of the pharmaceutical compositions described above.

According to one preferred embodiment of the present invention, said pharmaceutical composition is administered intranasally.

In accordance with more specific embodiments of the present invention, intranasal administration is in the form of drops or in the form of a spray (aerosol).

The authors of the present invention proceed from the fact that, as is known to the average person skilled in the art, the primary, initially obtained sequences of nanoantibodies can serve as source modules for more complex multimodule preparations. It is possible to combine in one multivalent derivative two, three or more monovalent primary nanoantibodies. These nanoantibodies combined into one construct can bind to the same epitope of the target antigen, as well as to its different epitopes or even to different target antigens. It is also possible to combine in one design nanoantibodies and other molecules or drugs to obtain multifunctional drugs [Conrath KE, Lauwereys M, Wyns L, Muyldermans S. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem. 2001 Mar 9; 276 (10): 7346-50; Zhang J, Tanha J, Hirama T, Khieu NH. To R, Tong-Sevinc H, Stone E, Brisson JR, MacKenzie CR. Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J Mol Biol. 2004 Jan 2; 335 (1): 49-56; Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H. Efficient cancer therapy with a nanobody-based conjugate. Cancer Res. 2004 Apr 15; 64 (8): 2853-7; Baral TN, Magez S, Stijlemans B, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, De Baetselier P. Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor. Nat Med. 2006 May; 12 (5): 580-4; Coppieters K, Dreier T, Silence K, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum. 2006 Jun; 54 (6): 1856-66]; multimerization by introducing additional amino acid sequences of interacting protein domains, such as leucine zippers [Harbury P. B., Zhang T., Kirn P. S., et al. A switch between two-, three- and four-stranded coiled coils in GCN4 leucine zipper mutants. Science, 1993, 262: 1401-1407; Shirashi T., Suzuyama k., Okamoto H. et al. Increased cytotoxicity of soluble Fas ligand by fusing isoleucine zipper motif. Biochem. Biophys. Res. Communic. 2004, 322: 197-202; Chenchik A., Gudkov A., Komarov A., Natarajan V. Reagents and methods for producing bioactive secreted peptides. 2010. US Patent Application 20100305002], sequences of small proteins forming stable complexes [Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Pluckthun A. Design of multivalent complexes using the barnase * barstar module. Nat Biotechnol. 2003, 21 (12): 1486-92].

To modulate the properties of a single-domain nanoantibody preparation, for example, to increase the in vivo lifetime (in the patient’s blood), additional amino acid sequences, protein sequences (such as, for example, serum albumin), or another nanoantibody that specifically binds to major and long-lived protein in human blood (eg, albumin or immunoglobulin) [Gibbs WW. Nanobodies. Sci am. 2005 Aug; 293 (2): 78-83; Harmsen MM, Van Solt CB, Fijten HP, Van Setten MC. Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins. Vaccine 2005 Sep 30; 23 (41): 4926-34; Coppieters K, Dreier T, Silence K, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum. 2006 Jun; 54 (6): 1856-66.].

It will be apparent to one of ordinary skill in the art that such modifications and other antibody variants that underlie the present invention fall within the scope of the present invention as they are structural and functional variants of nanoantibodies. Thus, the authors of the present invention understand the term "single-domain nanoantibodies" as primary, initially obtained, "minimal" amino acid sequences of nanoantibodies, and their modifications resulting from the mentioned adaptation or "formatting", and their variants.

Brief Description of the Figures

Figure 1 shows the nucleotide and amino acid sequence of a selected single-domain anti-flu nanoantibody capable of specifically binding and blocking influenza A. SEQ ID NO: 1 is the nucleotide sequence encoding an anti-flu nanoantibody. The corresponding amino acid sequence of the anti-flu nanoantibody (SEQ ID NO: 2) was derived from it. In the indicated amino acid sequence, hypervariable regions of CDR1, CDR2, and CDR3 are underlined respectively (from left to right, from the N- to the C-terminus), which are the main determining binding specificity for regions of similar antigen-recognizing molecules.

Figure 2 illustrates the results of an enzyme-linked immunosorbent assay for recognition of a selected single-domain nanoantibody ("anti-flu") of an influenza virus preparation immobilized in the wells of an immunological plate. Cells with immobilized chicken ovalbumin protein were used as controls.

Figure 3 presents a graph of the survival of mice after intranasal administration of a single-domain anti-flu nanoantibody and subsequent infection with A / Mallard duck / PA / 10218/84 (H5N2) avian influenza virus versus time after infection.

The solid line with a black square marker is 20 μl of an intranasal solution of a single-domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a white square marker is 20 μl of an intranasal phosphate buffer solution.

Figure 4 presents a graph of the dependence of the body weight of mice after intranasal administration of a single-domain anti-flu nanoantibody and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection.

The solid line with a black square marker is 20 μl of an intranasal solution of a single domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a cross marker is 20 μl of an intranasal solution of phosphate buffer.

Figure 5 presents a graph of the survival of mice after infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) and subsequent intranasal administration of a solution of single-domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse.

The solid line with a black square marker is 20 μl of an intranasal solution of a single-domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a white square marker is 20 μl of an intranasal phosphate buffer solution.

Figure 6 shows a graph of the change in body weight of mice after infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) and subsequent intranasal administration of a solution of single-domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse.

The solid line with a black square marker is 20 μl of an intranasal solution of a single domain anti-flu nanoantibody in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a cross marker is 20 μl of an intranasal solution of phosphate buffer.

Figure 7 presents a graph of the survival of mice after intranasal administration of the recombinant adenoviral vector Ad5-anti-flu and subsequent infection with the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection.

The solid line with a black square marker is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad5-anti-flu at a dose of 10 ⁷ PFU / mouse, the solid line with a marker in the form of a cross is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad-null, not containing the insertion of the target gene in the expression cassette at a dose of 10 ⁷ PFU / mouse, a solid line with a marker in the form of a white square - 20 μl intranasally 1 × PBS.

On Fig presents graphs of the dependence of the change in body weight of mice after intranasal administration of the recombinant adenoviral vector Ad5-anti-flu and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) from time to time after infection.

The solid line with a black square marker is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad5-anti-flu at a dose of 10 ⁷ PFU / mouse, the solid line with a marker in the form of a cross is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad-null, not containing the insertion of the target gene in the expression cassette at a dose of 10 ⁷ PFU / mouse, a solid line with a marker in the form of a white square - 20 μl intranasally 1 × PBS.

Figure 9 presents graphs of the dependence of the survival of mice after intranasal administration of the recombinant adeno-associated vector AAV-anti-flu and their subsequent infection with the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection.

The solid line is 20 μl of the intranasal preparation of the recombinant adeno-associated vector AAV-anti-flu at a dose of 10 ¹¹ copies of the genome / mouse, the dash-dot line is 20 μl of the intranasal preparation of the recombinant adeno-associated vector AAV-null that does not contain the target gene insert in the expression cassette , at a dose of 10 ¹¹ Copies of the genome / mouse, dash-dot line with two points - 20 μl intranasally 1 × PBS.

Figure 10 presents graphs of the dependence of the change in body weight of mice after intranasal administration of the recombinant adeno-associated vector AAV-anti-flu and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection.

The solid line is 20 μl of the intranasal preparation of the recombinant adeno-associated vector AAV-anti-flu at a dose of 10 ¹¹ copies of the genome / mouse, the dash-dot line is 20 μl of the intranasal preparation of the recombinant adeno-associated vector AAV-null, which does not contain the target gene insert in the expression cassette, at a dose of 10 ¹¹ copies of the genome / mouse, dash-dot line with two points - 20 μl intranasally 1 × PBS.

11 shows graphs of the dependence of the survival of mice after intranasal administration of the recombinant lentiviral vector LV-anti-flu and their subsequent infection with the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection.

The solid line with a black square marker is 20 μl of the intranasal preparation of the recombinant lentiviral vector LV-anti-flu at a dose of 10 ⁶ p24 pg / ml / mouse, the solid line with the cross marker is 20 μl of the intranasal preparation of the recombinant lentiviral vector LV-null containing no insertion of the target gene in the expression cassette at a dose of 10 ⁶ p24pg / ml / mouse, a solid line with a white square marker is 20 μl intranasally 1 × PBS.

On Fig presents graphs of the dependence of the change in body weight of mice after intranasal administration of the recombinant lentiviral vector LV-anti-flu and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) from time after infection.

The solid line with a black square marker is 20 μl of the intranasal preparation of the recombinant lentiviral vector LV-anti-flu at a dose of 10 ⁶ p24 pg / ml / mouse, the solid line with the cross marker is 20 μl of the intranasal preparation of the recombinant lentiviral vector LV-null containing no insertion of the target gene in the expression cassette at a dose of 10 ⁶ p24pg / ml / mouse, a solid line with a white square marker - 20 μl intranasally 1 × PBS.

Examples of the implementation of the present invention

Example 1

Obtaining a library of variable domains of single-chain antibodies

Immunization

Bactrian camel Camelus bactrianus was subsequently immunized 4 times by subcutaneous administration of antigenic material mixed with an equal volume of incomplete Freund's adjuvant. The antigen used was the preparation of the inactivated avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) from the Museum of FSBI NIIEM named after N.F. Gamalei ”of the Ministry of Health and Social Development of Russia and the preparation of the bird flu virus H5N2 (strain A / 17 / Utka / Potsdam / 86/92) obtained from the FSUE NPO Mikrogen of the Ministry of Health and Social Development of Russia, Irkutsk). The second immunization was carried out 3 weeks after the first, then two more immunizations were carried out with an interval of two weeks. Blood sampling (150 ml) was performed 6 days after the last injection. To prevent coagulation of the taken blood, 50 ml of standard saline solution (PBS) containing heparin (100 units / ml) and EDTA (3 mM) was added.

Blood was diluted 2 times with standard saline (PBS) containing 1 mm EDTA. 35 ml of diluted blood solution was layered on a step of a special medium (Histopaque-1077, Sigma) with a density of 1.077 g / ml and a volume of 15 ml and centrifuged for 20 min at 800 xg. Mononuclear cells (lymphocytes and monocytes) were selected from the plasma / Histopaque interphase, and then washed with a PBS solution containing 1 mM EDTA.

Total RNA from B lymphocytes was isolated using TRIzol reagent (Invitrogen). Then, on a column of oligo (dT) cellulose from total RNA, poly (A) -containing RNA was purified. RNA concentration was determined using a biophotometer (Eppendorf) and the quality of the isolated RNA was checked by electrophoresis in 1.5% formaldehyde agarose gel.

The reverse transcription reaction was carried out according to the standard protocol [Sambrook et al., 1989] using reverse transcriptase H ^- M-MuLV and oligo primer (dT) 15 as a seed.

Reverse transcription products were used as a template in a two-step polymerase chain reaction, and the resulting amplification products were cloned at the Ncol (Pstl) and NotI sites into a phagemid vector as previously described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006]. The selection procedure was also carried out similarly to those in the indicated works. It was based on the phage display method, in which the bacteriophage M13CO7 (New England Biolabs, USA) was used as an assistant phage.

Example 2

Selection of nanoantibodies that specifically recognize influenza A virus

The selection of nanoantibodies was carried out by phage display using the preparation of inactivated avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) immobilized at the bottom of the wells of a 96-well ELISA plate. Used polystyrene immunological plates with high sorption MICROLON 600 (Greiner Bio-One). For blocking, 1% BSA (Sigma-Aldrich, USA) and / or 1% skim milk (Bio-Rad, USA) in PBS were used. The selection and subsequent amplification of selected phage particles (containing the single-domain nanoantibody gene inside, and the expressed nanoantibody as a part of surface phage protein pIII) was repeated, as a rule, three times in succession. All manipulations were performed as described in publications Tillib S.V., Ivanova T.I., Vasiliev L.A. 2010. Fingerprint analysis of the selection of "nanoantibodies" by the phage display method using two variants of assistant phages. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman C., Atarhouch T., Muyldermans S. et al. Nature 1993; 363: 446-448 .; Nguyen V.K., Desmyter A., Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens D., Kinne J., Bosmans E., Wernery U., Muyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer U., Zolghadr K., Tillib S., et al. Nature Methods 2006; 3: 887-889].

The clone sequences of the selected nanoantibodies were grouped according to the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of amplified nanoantibody sequences in parallel with three frequently digested restriction endonucleases (Hinfl, Mspl, Rsal). Then, selected clones representing each of the groups differing in fingerprint were used for the analytical production of nanoantibodies, the activity of which was checked by enzyme immunoassay for effective binding of the inactivated avian influenza virus drug A / Mallard duck / PA / 10218/84 (H5N2) immobilized in the cell of an immunological tablet )

As a result, a sequence encoding an anti-flu nanoantibody was selected (FIG. 1).

Nanoantibody Products

The cDNA sequences of the selected single-domain anti-flu nanoantibody were cloned into an expression plasmid vector — modified pHEN6 vector [Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S. Beta-lactamase inhibitors derived from single- domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother. 2001; 45: 2807-12], which allows the addition of a single-domain (His) nanoantibody of the 6-epitope to the C-terminus (immediately after the HA-epitope encoded in the pHEN6 vector). Due to the presence of the signal peptide sequence (pelB) at the N-terminus of the expressed sequence, the produced recombinant protein (nanoantibody) accumulates in the periplasm of bacteria, which allows it to be efficiently isolated by the osmotic shock method without destroying the bacterial cells themselves. The production of nanoantibodies was carried out in E. coli (strain BL21). Expression was induced by adding 1 mM indolyl-beta-D-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37 ° C or overnight at 29 ° C. Nanoantibodies were isolated from the periplasmic extract using affinity chromatography on Ni-NTA agarose using the QIAExpressionist purification system (Q1AGEN, USA).

Demonstration of the binding of a single domain nanoantibody with influenza virus

The ability of a single domain nanoantibody anti-flu to bind influenza virus was tested using an enzyme-linked immunosorbent assay with immobilized recombinant inactivated avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) according to the standard protocol. As a control, wells with immobilized chicken ovalbumin protein (non-specific protein) were used. The detection of a single domain nanoantibody associated with the influenza virus was carried out using anti-HA mouse antibodies, secondary antibodies to mouse immunoglobulins conjugated to horseradish peroxidase, and the chromogenic substrate ABTS (2,2'-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid , Sigma).

Figure 2 presents the results of the analysis, from which it follows that the anti-flu nanoantibody specifically binds to the influenza virus preparation, but does not bind to ovalbumin or BSA, used as a blocking agent.

The selected sequence of a single domain anti-flu nanoantibody was then cloned into the genome of a recombinant adenoviral vector.

Example 3

Creation of the plasmid construct pShuttle-CMV-anti-flu with portions of the genome of human adenovirus 5th serotype to obtain a recombinant adenoviral vector carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to the influenza virus

To obtain the pShuttle-CMV-anti-flu plasmid construct, the pShuttle-CMV plasmid construct with portions of the 5th serotype human adenovirus genome for the production of recombinant adenoviral vectors included in the AdEasy Adenoviral vector system (Stratagene) system is used as a vector Cat. No. 240009). The pShuttle-CMV plasmid construct is hydrolyzed at the EcoRV restriction endonuclease site, and then the nucleotide sequence of a single domain nanoantibody is inserted to obtain, respectively, the pShuttle-CMV anti-flu construct. The nucleotide sequence of a single domain anti-flu nanoantibody for insertion into pShuttle-CMV is obtained by hydrolysis of the pAL-anti-flu plasmid construct at EcoRV restriction endonuclease sites. The presence of the single domain anti-flu nanoantibody gene in the pShuttle-CMV-anti-flu plasmid construct is confirmed by restriction analysis using endonucleases EcoRI, NotI and EcoRV and PCR.

Example 4

Obtaining a recombinant adenoviral vector Ad5-anti-flu carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to the influenza virus

The preparation of the recombinant adenoviral vector Ac15-anti-flu is carried out according to the method of "AdEasy Adenoviral vector system" ("Stratagene" Cat No. 240009). The presence of the single domain anti-flu nanoantibody gene in the corresponding recombinant adenoviral vector Ad5 anti-flu is confirmed by PCR.

Next, the titer of the preparation of the recombinant adenoviral vector Ad5-anti-flu is determined by plaque formation on a 293 cell culture (human embryonic kidney cells).

Example 5

Neutralization of influenza virus with a single-domain anti-flu nanoantibody in an in vitro cell culture

The virus neutralization reaction was staged using an MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium is replaced with serum-free. For the reaction, the bird flu virus A / Mallard duck / PA / 10218/84 (H5N2) is used at a dose of 100 TCD50 per well. Dilutions of the virus and the anti-flu antibody tested are performed in serum-free DMEM. 50 μl of a virus suspension is added to the wells of a 96-well plate, then equal volumes of two-fold dilutions of serum are added to each well, starting at 1:10. The plate is incubated at 37 ° C for 1 hour. After incubation, the mixture of virus and serum is added to each well of the cell plate to adsorb the virus on the cells. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the tablets and replaced with MEM medium containing 0.2% BSA and trypsin at a concentration of 1 μg / ml and replaced with fetal bovine serum. The results of the virus-neutralization reaction are taken into account after 72 hours after the onset of CPD in cell culture according to the method of Reed and Mench. The studied anti-flu antibody neutralizes the influenza virus in the concentration range from 0.009 μg / ml to 2.5 μg / ml.

Example 6

Neutralization of influenza virus by a single-domain anti-flu nanoantibody expressed by the recombinant adenoviral vector Ad5-anti-flu in an in vitro cell culture

The expression of a single domain anti-flu nanoantibody is performed in A549 cell lines. For this, A549 cells are seeded into the wells of a 48 well plate (Costar, USA) in the amount of 104 cells per well, after which serum-free medium 293AGT (Invitrogen, USA) is added to the cells and incubated at 37 ° C and 5% CO ₂ for 20-24 hours. Then the cells are transduced with the preparation of the recombinant adenoviral vector Ad5-anti-flu at a dose of 5 PFU / cell. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours, after which medium is taken from the wells and replaced with a new portion of medium 293AGT. Transduced cells are incubated for 72 hours, then the culture medium is selected. Thus, preparations of a culture medium containing Ad5-anti-flu recombinant adenoviral vector anti-flu nanoantibodies are obtained.

The virus neutralization reaction was performed using the MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium is replaced with serum-free. For the reaction, the bird flu virus A / Mallard duck / PA / 10218/84 (H5N2) is used at a dose of 100 TCD50 per well. Dilutions of the virus and culture medium containing the anti-flu antibody tested are produced in serum-free DMEM. 50 μl of the virus suspension is added to the wells of a 96-well plate, then equal volumes of two-fold dilution of the culture medium are added to each well, starting at 1:10. The plate is incubated at 37 ° C for 1 hour. After incubation, a mixture of the virus and culture medium is added to each well of the cell plate to adsorb the virus on the cells. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the tablets and replaced with MEM medium containing 0.2% BSA and trypsin at a concentration of 1 μg / ml and replaced with fetal bovine serum. The results of the virus-neutralization reaction are taken into account after 72 hours after the onset of CPD in cell culture according to the method of Reed and Mench. Culture medium preparations containing the Ad5-anti-flu recombinant adenoviral vector anti-flu nanoantibody anti-flu neutralize the influenza virus at a 1: 100 dilution.

Example 7

The use of single-domain nanoantibodies anti-flu for the prevention of diseases caused by influenza virus

In order to prevent diseases caused by type A influenza virus, Balb / c mice, females weighing 18 g are divided into 2 groups of 50 mice in each group:

- the first group of mice injected with 20 μl of a solution of anti-flu nanoantibodies in phosphate buffer dose of 2.5 μg / ml,

- the second group of mice injected with 20 μl of intranasal phosphate buffer.

1 hour after the administration of the above preparations, the mice were infected with 25 LD50 of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) adapted for their virulent properties for the mouse model. Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival of mice and the change in their weight are taken into account.

The protection of mice against the A / Mallard duck / PA / 10218/84 (H5N2) virus is 100% in the group to which the single-domain anti-flu nanoantibody preparation was administered (see FIG. 3). Also, a decrease in body weight was not observed in these mice. Curves of weight loss in mice of each of the two above groups after infection with influenza virus are presented in figure 4.

Thus, intranasal administration of a single-domain anti-flu nanoantibody completely protects mice from infection with influenza virus at a dose of 25 LD50.

Example 8

The use of single-domain anti-flu nanoantibodies for the treatment of diseases caused by influenza virus

For the treatment of diseases caused by type A influenza virus, 100 Balb / c mice, 18 g females, are infected with 25 LD50 adapted for their virulent properties for the mouse model of bird flu virus A / Mallard duck / PA / 10218/84 (H5N2) . Infection is performed intranasally under mild ether anesthesia.

4 hours after the introduction of the above preparations, the mice are divided into 2 groups of 50 mice in each group:

- the first group of mice injected with 20 μl of a solution of nanoantibodies anti-flu in phosphate buffer dose of 2.5 μg / ml

- the second group of mice injected with 20 μl of intranasal phosphate buffer. Within 14 days after infection, the survival of mice and the change in their weight are taken into account.

The protection of the mice against the A / Mallard duck / PA / 10218/84 (H5N2) virus is 100% in the group to which the single-domain anti-flu nanoantibody preparation was administered (see FIG. 5). Also, a decrease in body weight was not observed in these mice. The curves of decrease in body weight in mice of each of the two above groups are presented in Fig.6.

Thus, intranasal administration of a single-domain anti-flu nanoantibody completely protects mice from infection with influenza virus at a dose of 25 LD50.

Example 9

The use of the recombinant adenoviral vector Ad5-anti-flu for the prevention of diseases caused by influenza virus

In order to prevent diseases caused by influenza virus, Balb / c mice, females weighing 18 g are divided into 3 groups of 50 mice in each group:

- the first group of mice injected with 20 μl of an intranasally colloidal solution of the recombinant adenoviral vector Ad5-anti-flu in phosphate buffer at a dose of 10 ⁷ PFU / mouse,

- the second group of mice injected with 20 μl of an intranasally colloidal solution of a recombinant adenoviral vector Ad-null that does not contain the insertion of the target gene in the expression cassette in phosphate buffer at a dose of 10 ⁷ PFU / mouse,

- the third group of mice injected with 20 μl of intranasal phosphate buffer.

72 hours after the administration of the above preparations, the mice were infected with 25 LD50 of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) adapted for their virulent properties for the mouse model. Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival of mice and the change in their weight are taken into account.

The protection of mice against virus A / Mallard duck / PA / 10218/84 (H5N2) is 100% in the group to which the preparation of the recombinant adenoviral vector Ad5-anti-flu was administered (see Fig. 7). Also, a decrease in body weight was not observed in these mice. Curves of weight loss in mice of each of the three above groups after infection with the influenza virus are presented in Fig. 8.

Thus, the intranasal administration of the recombinant adenoviral vector Ad5-anti-flu fully protects mice from infection with influenza virus at a dose of 25 LD50.

Example 10

The use of a pharmacological composition consisting of a single-domain anti-flu nanoantibody and a recombinant adenoviral vector Ad5-anti-flu for the prevention and treatment of diseases caused by influenza virus

In order to prevent and treat diseases caused by the influenza virus, Balb / c mice, females weighing 18 g are divided into 18 groups of 20 mice in each group:

Group 1, 2 and 3 - each animal is injected with 20 μl of an intranasal preparation containing 3 μg of anti-flu nanoantibodies and 10 ⁷ PFU of the recombinant adenoviral vector Ad5-anti-flu,

4, 5 and 6 group, each animal is injected with 20 μl of an intranasal preparation containing 3 μg of BSA and 10 ⁷ PFU of recombinant adenoviral vector Ad5-anti-flu,

7, 8 and 9 group - each animal is injected with 20 μl of an intranasal preparation containing 3 μg of anti-flu nanoantibodies and 10 ⁷ PFU of the recombinant adenoviral vector Ad5-null,

10, 11 and 12 group - each animal is injected with 20 μl of an intranasal preparation containing 3 μg of BSA and 10 ⁷ PFU of the recombinant adenoviral vector AdS-null,

13, 14 and 15 group - each animal is injected with 20 μl of intranasal phosphate buffer,

Group 16, 17 and 18 — intact mice.

In groups 1, 4, 7, 10, 13, 16, 1 hour after the administration of the above preparations, the mice were infected with 25 LD50, adapted in their virulent properties for the mouse model, the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of mice and the change in their body weight are taken into account.

In groups 2, 4, 8, 11, 14, 17, 72 hours after the administration of the above preparations, mice were infected with 25 LD50, adapted in their virulent properties for the mouse model, the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of mice and the change in their body weight are taken into account.

In groups 3, 6, 9, 12, 15, 18, 14 days after the administration of the above preparations, mice were infected with 25 LD50, adapted for their virulent properties for the mouse model, the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of mice and the change in their body weight are taken into account.

The protection of mice against the virus A / Mallard duck / PA / 10218/84 (H5N2) is 100% in groups No. 1-3, 5-8 (see table 1). Also, in these mice there is no decrease in body weight (see table 1).

Table 1. The survival and weight change of mice after intranasal administration of a pharmacological composition consisting of a single domain anti-flu nanoantibody and recombinant adenoviral vector Ad5-anti-flu, and their subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2 )

Table 1 Group number The maximum decrease in body weight in relation to the initial body weight, on average per group,% The number of surviving mice,% one 4,5 one hundred 2 4,5 one hundred 3 5 one hundred four 22 0 5 6 90 6 5 one hundred 7 4,5 one hundred 8 4,5 thirty 9 40 0 10 40 0 eleven 40 0 12 40 0 13 40 0 fourteen 40 0 fifteen 40 0 16 40 0 17 45 0 eighteen 40 0

Thus, the intranasal administration of a pharmacological composition consisting of nanoantibodies and recombinant adenoviral vectors Ad5-anti-flu fully protects against type A influenza virus infection in all selected infection modes, in contrast to using only anti-flu antibodies and only Ad5-anti- vector flu ..

Example 11

Creation of the plasmid construct pAAV-IRES-anti-flu with regions of the genome of the adeno-associated virus to obtain an adeno-associated vector carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to the influenza virus.

To obtain the pAAV-IRES-anti-flu plasmid construct, the pAAV-IRES-hrGFP plasmid construct with portions of the adeno-associated vector genome for producing adeno-associated vectors, included in the AAV Helper-Free System (Agilent), is used as a vector. Technologes, cat # 240071).

The plasmid construct pAAV-IRES-hrGFP is hydrolyzed at the sites for restriction endonucleases EcoRI, Sail, and then the nucleotide sequence of a single domain nanoantibody is inserted to obtain the construct pAAV-IRES-anti-flu. The nucleotide sequence of a single-domain anti-flu nanoantibody for insertion into pAAV-IRES-hrGFP is obtained by hydrolysis of the pAL-anti-flu plasmid construct at EcoRV restriction endonuclease sites. The presence of the single domain anti-flu nanoantibody gene in the corresponding plasmid construct pAAV-IRES-anti-flu is confirmed by restriction analysis using endonuclease EcoRI, Sail and PCR.

Example 12

Obtaining an adeno-associated AAV-anti-flu vector carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to the influenza virus.

Obtaining the adeno-associated vector AAV-anti-flu is carried out according to the method of "AAV Helper-Free System" (Agilent Technologes, cat # 240071). The presence of the single-domain anti-flu nanoantibody gene in the corresponding adeno-associated AAV-anti-flu vector is confirmed by PCR.

Next, the titer of the preparation of the adeno-associated vector AAV-anti-flu is determined by ELISA using a Progen kit (cat # PRAAV1).

Example 13

Neutralization of influenza virus by a single-domain anti-flu nanoantibody expressed by the adeno-associated AAV-anti-flu vector in an in vitro cell culture.

The expression of a single domain anti-flu nanoantibody is performed in A549 cell lines. For this, A549 cells are seeded into the wells of a 48 well plate (Costar, USA) in the amount of 104 cells per well, after which serum-free medium 293AGT (Invitrogen, USA) is added to the cells and incubated at 37 ° C and 5% CO ₂ for 20-24 hours. Then the cells are transduced with the preparation of the adeno-associated vector AAV-anti-flu in a dose of 1x10 ⁴ copies of the genome / cell. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours, after which medium is taken from the wells and replaced with a new portion of medium 293AGT. Transduced cells are incubated for 72 hours, then the culture medium is selected. Thus, a culture medium preparation is prepared containing the anti-flu nanoantibody expressed by the adeno-associated vector AAV-anti-flu.

The virus neutralization reaction was staged using an MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium is replaced with serum-free. For the reaction, the bird flu virus A / Mallard duck / PA / 10218/84 (H5N2) is used at a dose of 100 TCD50 per well. Dilutions of the virus and culture medium containing the anti-flu antibody tested are produced in serum-free DMEM.

50 μl of a virus suspension is added to the wells of a 96-well plate, then equal volumes of two-fold dilutions of serum are added to each well, starting at 1:10. The plate is incubated at 37 ° C for 1 hour. After incubation, a mixture of the virus and culture medium is added to each well of the cell plate to adsorb the virus on the cells. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the tablets and replaced with MEM medium containing 0.2% BSA and trypsin at a concentration of 1 μg / ml and replaced with fetal bovine serum. The results of the virus-neutralization reaction are taken into account after 72 hours after the onset of CPD in cell culture according to the method of Reed and Mench. A culture medium preparation containing the anti-flu nanoantibody expressed by the adeno-associated vector AAV-anti-flu neutralizes the influenza virus at a 1: 100 dilution.

Example 14

The use of the adeno-associated vector AAV-anti-flu for the prevention and treatment of diseases caused by influenza virus

In order to prevent diseases caused by influenza virus, Balb / c mice, females weighing 18 g are divided into 3 groups of 50 mice in each group:

- the first group of mice injected with 20 μl of an intranasally colloidal solution of adeno-associated vectors AAV-anti-flu in a phosphate buffer dose of 10 ¹¹ copies of the genome / mouse,

- the second group of mice injected with 20 μl of an intranasally colloidal solution of adeno-associated AAV-null vectors that does not contain the insertion of the target gene in the expression cassette in phosphate buffer at a dose of 10 ¹¹ copies of the genome / mouse,

- the third group of mice injected with 20 μl of intranasal phosphate buffer.

72 hours after the administration of the above preparations, the mice were infected with 25 LD50 of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) adapted for their virulent properties for the mouse model. Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival of mice and the change in their weight are taken into account.

The protection of mice against the A / Mallard duck / PA / 10218/84 (H5N2) virus is 100% in the group to which the preparation of the recombinant adeno-associated AAV-anti-flu vector was administered (see Fig. 9). Also, in these mice there is no decrease in body weight. Curves of weight loss in mice of each of the three above groups after infection with influenza virus are presented in figure 10.

Thus, the intranasal administration of the preparation of the recombinant adeno-associated vector AAV-anti-flu completely protects against infection with the influenza virus.

Example 15

Obtaining a plasmid construct pLV-anti-flu with portions of the lentivirus genome to obtain a lentiviral vector LV-anti-flu carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to type A influenza virus

To obtain the plasmid construct pLV-anti-flu as a vector, the plasmid construct pLV ("Cellecta") is used to produce lentiviral vectors ("Cellecta" Cat. No. CVPRC-PS). The plasmid construct pLV is hydrolyzed at the site for the restriction endonuclease EcoRV, and then the nucleotide sequence of a single domain nanoantibody is inserted to obtain the construct pLV-anti-flu. The nucleotide sequence of a single-domain anti-flu nanoantibody for insertion into pLV is obtained by hydrolysis of the plasmid construct pAL-anti-flu at EcoRV restriction endonuclease sites. The presence of the single domain anti-flu nanoantibody gene in the plasmid construct pLV-anti-flu is confirmed by restriction analysis using endonuclease EcoRI, NotI and EcoRV and PCR.

Example 16

Obtaining a lentiviral vector of LV-anti-flu carrying an expression cassette containing a nucleotide sequence that encodes an anti-flu nanoantibody that binds to type A influenza virus

Obtaining a lentiviral vector LV-anti-flu is carried out according to the method of "LENTI-SMART" ("Invivogen" Cat. No. Itsint-5). The presence of the single domain anti-flu nanoantibody gene in the LV-anti-flu lentiviral vector is confirmed by PCR.

Next, titers of the preparation of the lentiviral vector LV-anti-flu are determined by ELISA using the Lenti-X ^TM p24 Rapid Titer Kit (Clontech, cat. # 632200).

Example 17

Neutralization of influenza virus with a single-domain anti-flu nanoantibody expressed by the LV-anti-flu lentiviral vector in vitro cell culture

The expression of a single domain anti-flu nanoantibody is performed in A549 cell lines. For this, A549 cells are seeded into the wells of a 48 well plate (Costar, USA) in the amount of 104 cells per well, after which serum-free medium 293AGT (Invitrogen, USA) is added to the cells and incubated at 37 ° C and 5% CO ₂ for 20-24 hours. Then the cells are transduced with preparations of the lentiviral vector LV-anti-flu at a dose of 5 p24 pg / ml. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours, after which medium is taken from the wells and replaced with a new portion of medium 293AGT. Transduced cells are incubated for 72 hours, then the culture medium is selected. Thus, preparations of a culture medium containing LV-anti-flu recombinant lentiviral vector anti-flu nanoantibody are obtained.

The virus neutralization reaction was staged using an MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium is replaced with serum-free. For the reaction, the bird flu virus A / Mallard duck / PA / 10218/84 (H5N2) is used at a dose of 100 TCD50 per well. Dilutions of the virus and culture medium containing the anti-flu antibody tested are produced in serum-free DMEM. 50 μl of a virus suspension is added to the wells of a 96-well plate, then equal volumes of two-fold dilution of the ultural medium are added to each well, starting at 1:10. The plate is incubated at 37 ° C for 1 hour. After incubation, a mixture of the virus and culture medium is added to each well of the cell plate to adsorb the virus on the cells. Cells are incubated at 37 ° C and 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the tablets and replaced with MEM medium containing 0.2% BSA and trypsin at a concentration of 1 μg / ml and replaced with fetal bovine serum. The results of the virus-neutralization reaction are taken into account after 72 hours after the onset of CPD in cell culture according to the method of Reed and Mench. The preparation of the culture medium containing the anti-flu nanoantibody expressed by the recombinant lentiviral vector LV-anti-flu ensures neutralization of the influenza virus at a dilution of 1: 100.

Example 18

The use of the lentiviral vector LV-anti-flu for the prevention of diseases caused by influenza virus

In order to prevent diseases caused by the influenza virus of Balb / c mice, females weighing 18 g are divided into 3 groups of 50 mice in each group:

- the first group (Group 1) of mice injected with 20 μl of an intranasally colloidal solution of the recombinant lentiviral vector LV-anti-flu in phosphate buffer at a dose of 10 ⁶ p24pg / ml / mouse,

- the second group (Group 2) of mice is injected with 20 μl of an intranasally colloidal solution of recombinant lentiviral vectors LV-nuII that does not contain the target gene insert in the expression cassette in a phosphate buffer at a dose of 10 ⁶ p24 pg / ml / mouse,

- the third group (Group 3) of mice injected with 20 μl of intranasal phosphate buffer.

14 days after the administration of the above preparations, the mice were infected with 25 LD50 of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) adapted for their virulent properties for the mouse model. Infection is performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival of mice and the change in their weight are taken into account.

The protection of mice against virus A / Mallard duck / PA / 10218/84 (H5N2) is 100% in the group to which the preparations of the recombinant lentiviral vector LV-anti-flu were administered (see Fig. 11). Also, in these mice there is no decrease in body weight. The curves of weight loss in mice of each of the three above groups after infection with the influenza virus are presented in Fig.12.

Thus, the intranasal administration of the recombinant lentiviral vector LV-anti-flu fully protects against infection with the influenza virus.

Thus, the above examples show that the tasks are completed, and this proves the possibility of using nanoantibodies that specifically bind to type A influenza hemagglutinin, viral vectors expressing nanoantibodies that specifically bind to type A influenza hemagglutinin, and a pharmaceutical composition consisting of the above nanoantibodies and an adenoviral vector for the treatment and prevention of influenza A.

Claims (6)

1. Nanoantibody having the amino acid sequence of SEQ ID NO: 2, specifically binding to H5N2 type A influenza virus hemagglutinin and inhibiting H5N2 type A influenza virus infection. 2. The viral vector for expression of the nanoantibody according to claim 1, containing the nanoantibody gene according to claim 1. 3. The viral vector according to claim 2, characterized in that it is an adenoviral vector expressing the nanoantibody according to claim 1, which is able to efficiently bind a certain epitope of influenza hemagglutinin and thereby block the development of influenza infection. 4. The viral vector according to claim 2, characterized in that it is an adeno-associated vector expressing a nanoantibody according to claim 1, which is able to efficiently bind a specific epitope of hemagglutinin of the influenza virus and, thereby, block the development of influenza infection. 5. The viral vector according to claim 2, characterized in that it is a lentiviral vector expressing the nanoantibody according to claim 1, which is able to efficiently bind a certain hemagglutinin epitope of the influenza virus and, thereby, block the development of influenza infection. 6. A composition for suppressing the development of H5N2 influenza A virus infection, comprising an effective amount of a nanoantibody according to claim 1 and a viral vector according to claim 2.

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