RU2808965C1 - Recombinant protein for immunization against feline coronavirus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the recombinant production of immunogenic protein of feline coronavirus, and can be used in veterinary medicine to obtain a vaccine against feline coronavirus. The following is proposed: a new vaccination strategy based on the use of a prophylactic medicinal product based on the conservative recombinant nucleocapsid protein of feline coronavirus (N-FCoV) with SEQ ID NO.: 2 and a specific adjuvant, which is a mixture of squalane, α-tocopherol (vitamin E) and polysorbate-80, which effectively induces a T-cell response against feline coronavirus.

EFFECT: obtaining recombinant protein for immunization against feline coronavirus.

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Description

Field of invention

The group of inventions relates to biotechnology and immunology, namely to immunogenic genetic constructs aimed at stimulating an immune response against the feline coronavirus FCoV.

Description of the Prior Art

Feline coronavirus (FCoV) is a complex RNA virus belonging to the Coronaviridae family. The structure of the virus consists of a nucleocapsid containing the viral genome and an outer shell. These two elements stabilize and protect the RNA genome [Masters P.S., Perlman S. Coronaviridae. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA, pp.825-858, 2013]. The FCoV genome encodes four major structural proteins: surface spike protein (S), nucleocapsid protein (N), envelope protein (E), and membrane protein (M); and seven non-structural proteins: two replicase proteins (1a and 1b) and five accessory proteins (3a, 3b, 3c, 7a and 7b) [de Barros B.D.C.V., Castro C.M.O.D., Pereira D., et al. First complete genome sequence of a feline Alphacoronavirus 1 strain from Brazil. Microbiol Resour Announc. 2019; 8:e01535-18; Tekes G., Thiel H.J. Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis. Adv Virus Res. 2016; 96:193-218].

Based on differences in the structure of the gene encoding the surface protein S (spike), feline coronavirus is divided into two pathotypes: the less lethal causative agent of coronavirus enteritis (FECV - Feline enteritis coronavirus), which is widespread among wild and domestic animals, and the disease caused by it occurs relatively easy and can become chronic; and the feline infectious peritonitis virus (FIPV Feline infectious peritonitis virus), which is less common, but can cause a systemic lethal disease [Shchelkanov M.Yu., Popova A.Yu., Dedkov V.G. and others. History of the study and modern classification of coronaviruses (Nidovirales: Coronaviridae). Infection and immunity.2020; 10(2): 221-246].

Protein S is a large glycoprotein (180-220 kDa) that forms peplomeres that protrude from the surface of virions and are responsible for receptor binding, induction of intercellular fusion, fusion of the viral envelope with target cell membranes and induction of neutralizing antibodies [Jaimes J.A., Whittaker G.R. Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function. Virology. 2018; 517: 108-121]. Depending on the amino acid sequence of this protein and neutralizing antibodies, the virus is divided into serotypes I and II [Kipar A., Meli M.L. Feline infectious peritonitis: still an enigma? Veterinary pathology. 2014; 51(2): 505-526; Lewis C.S., Porter E., Matthews D., et al. Genotyping coronaviruses associated with feline infectious peritonitis. Journal of General Virology. 2015; 96:1358-1368]. Serotype I is the original and predominant type with S protein derived from FCoV. Strains from this serotype are more widespread and have a higher epidemiological significance [Benetka V., Kubber-Heiss A., Kolodziejek J., et al. Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis. Vet Microbiol. 2004; 99(1): 31-42; Pedersen N.C. A review of feline infectious peritonitis virus infection: 1963-2008. Journal of Feline Medicine & Surgery. 2009; 11(4): 225-258]. Serotype II viruses are less common, and unlike serotype I viruses, their genome is formed by recombination between at least two different viruses, as confirmed by in vitro and in vivo experiments [Herrewegh A.A.P.M., Smeenk I., Horzinek M.C., et al. Feline Coronavirus Type II Strains 79-1683 and 79-1146 Originate from a Double Recombination between Feline Coronavirus Type I and Canine Coronavirus. Journal of Virology. 1998; 72(5): 4508-4514]. Serotype I is distributed primarily in Europe and North America, while serotype II is more common in Asian and African countries. There is also data on co-infections with FCoV I and II [An D. J., Jeoung H. Y., Jeong W., et al. Prevalence of Korean cats with natural feline coronavirus infections. Virol J. 2011; 8: 455]. Both FCoV serotypes can occur in two antigenically and morphologically indistinguishable pathotypes: FECV and FIPV [Shiba N., Maeda K., Kato N., et al. Differentiation of feline coronavirus type I and II infections by virus neutralization test. Veterinary Microbiology. 2007; 124(3-4): 348-352]. The S protein is the main regulator of the FCoV infectious process in the host cell and determines the virulence of FCoV: mutations of this protein (together with mutations of ORF non-structural proteins) determine the transition of FECV to FIPV [Jaimes J.A., Whittaker G.R. Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function. Virology. 2018; 517: 108-121].

FCoV disease affects populations of domestic and wild cats around the world. Seropositivity to FCoV is 20-60% among domestic cats and up to 90% in households with several cats or shelters [Gilmutdinov R.Ya., Malev A.V. Coronavirus infections of wild felines (Felidae). Current issues of zoology, ecology and nature conservation. 2021; 3: 36-41; Paltseva E.D., Pleshakova V.I. Coronaviruses in the domestic cat population. Bulletin of Omsk State Agrarian University. 2022; 1(45): 94-101]. The routes of transmission of FCoV can be either vertical (transplacental route) or horizontal (fecal-oral route). Seasonality is not recorded. The source of the infectious agent is sick, recovered animals and virus carriers, in which the virus is excreted in feces and saliva. Kittens most often become infected through colostrum or feces. The disease affects young and older animals more often [Paltseva E.D., Pleshakova V.I. Coronaviruses in the domestic cat population. Bulletin of Omsk State Agrarian University. 2022; 1(45): 94-101].

Feline coronavirus enteritis is a disease caused by the FECV pathotype. It has low pathogenicity and does not threaten the life of the animal. The clinical sign of the disease is diarrhea, but the infection can also be asymptomatic [Pedersen N.C. A review of feline infectious peritonitis virus infection: 1963-2008. Journal of Feline Medicine & Surgery. 2009; 11(4): 225-258].

However, in 8-10% of cases, low pathogenic FECV mutates into FIPV, causing feline infectious peritonitis, which has a mortality rate of up to 90% [Pedersen N.C. A review of feline infectious peritonitis virus infection: 1963-2008. Journal of Feline Medicine & Surgery. 2009; 11(4): 225-258].

Feline infectious peritonitis (FIP), along with Dengue and Ebola hemorrhagic fevers, equine infectious anemia, RSV respiratory tract infection and several other human and animal diseases, is a disease characterized by antibody-dependent enhancement (ADE) [ Mironov A.A., Supotnitsky M.V., Lebedinskaya E.V. The phenomenon of antibody-dependent increase in infection in vaccinated and recovered patients. Biological products. 2013; 3:1225].

ADE occurs when antibodies produced during an immune response recognize and bind to a pathogen but fail to prevent infection. Instead, antibodies act as a Trojan horse, allowing the pathogen to enter cells (monocytes and macrophages). As part of these cells, the virus quickly spreads to the mesenteric lymph nodes and throughout the body. A high degree of viremia leads to the deposition of immune complexes and macrophages containing viral particles around small venules, causing immune-mediated vasculitis and focal granulomatous damage to all organs [Cloutier M., Nandi M., flisan A.U., et al. ADE and hyperinflanimation in SARS-CoV2 infection-comparison with dengue hemorrhagic fever and feline infectious peritonitis. Cytokine. 2020; 136:155256]. This leads to complications of FIP such as renal failure, accompanied by neurological symptoms of damage to the brain and spinal cord, parenchymal jaundice and toxic liver dystrophy, interstitial pneumonia, atelectasis and emphysema, general intoxication of the body, persistent fever and death [Kulikov E.V., Vatnikov Yu.A., Sakhno N.V. et al. Pathological characteristics of viral peritonitis in cats. Russian Journal of Agricultural and Socio-Economic Sciences. 2017; 64(4): 270-280].

There is practically no modern research devoted to the development of an ideal and effective vaccine against feline coronavirus, due to the long-term study of classical approaches to the development of vaccines, which either did not have a protective effect or, on the contrary, caused an increase in infection [Pedersen N.C. Animal virus infections that defy vaccination: equine infectious anemia, caprine arthritis-encephalitis, maedi-visna, and feline infectious peritonitis. Adv. Vet. Sci. Sotr. Med. 1989; 3:413-428]. Vaccination with closely related heterologous live strains of CoV did not provide protection at all [Barlough J.E., Johnson-Lussenburg S.M., Stoddart S.A., et al. Experimental inoculation of cats with human coronavirus 229E and subsequent challenge with feline infectious peritonitis virus. Can. J. et al. Med. 1985; 49: 303-307]. Similar to inactivated and recombinant subunit vaccines against FCoV [Napeta B.J., Rottier P.J.M., de Groot R.J. Coronaviruses. Caister Academic Press; UK: Norfolk. Caister Academic Press: 2007. Feline coronaviruses: a tale of two-faced types], immunization with FECV, low-virulent FIPV, or sublethal amounts of virulent FIPV caused only partial protection [Pedersen N.C, Black J.W. Attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus. Am. J. Vet. Res. 1983; 44: 229-234; Pedersen N.C., Floyd K. Experimental studies with three new strains of feline infectious peritonitis virus: FIPV-UCD2, FIPV-UCD3, and FIPV-UCD4. Compend. Contin. Educ. Pract. Vet. 1985; 7:1001-1011], which often led to ADE and the so-called early death syndrome. The most promising results were obtained with recombinant FIPV mutants lacking the ORF3abc or ORF7ab regions, which provided 100% and 80% protection after lethal homologous challenge [Haijema B.J., Volders H., Rottier P.J. Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis. J. Virol. 2004; 78: 3863-3871], respectively. However, no follow-up studies using these vaccine candidates have been published.

The only registered vaccine for the preventive immunization of healthy cats against viral peritonitis, Vanguard® Feline FIP Intranasal produced by Zoetis (formerly Pfizer Animal Health, USA), also known as Primucel FIP® and FELOCELL FIP, is not recommended for use by many veterinary organizations, such as " American Association of Feline Practitioners" [Scherk M.A., Ford R.B., Gaskell R.M., et al. 2013 AAFP feline vaccination advisory panel report. Journal of feline medicine and surgery. 2013; 15(9): 785-808], and may cause an ADE effect. Vanguard® Feline FIP Intranasal contains an attenuated strain of feline peritonitis virus, DF2-FIPV. Since it is known that stimulating a systemic IgG response only aggravates the course of FIP without providing adequate protection [Cloutier M., Nandi M., Ihsan A.U., et al. ADE and hyperinflammation in SARS-CoV2 infection-comparison with dengue hemorrhagic fever and feline infectious peritonitis. Cytokine. 2020; 136: 155256], an alternative local vaccination strategy was adopted. The Vanguard® Feline FIP Intranasal vaccine is administered intranasally to stimulate a response with IgA immunoglobulins, which prevent viral invasion in the oropharynx and do not intensify the pathological process [Tizard I.R. Vaccination against coronaviruses in domestic animals. Vaccine. 2020; 38(33):5123-5130]. According to the instructions for use, the drug can only be used to vaccinate animals over 16 weeks of age. In highly endemic situations where kittens become infected with FCoV early in life or in utero, this vaccine regimen is ineffective. In addition, intranasal vaccination actually increases both local IgA antibody titers and IgG titers to the FCoV S protein in the blood serum. According to a study by Fred W. Scott, 48 of 49 cats vaccinated intranasally with the Primucel FIP® vaccine had an increase in serum IgG titer [Scott F.W. Evaluation of risks and benefits associated with vaccination against coronavirus infections in cats. Advances in Veterinary Medicine. 1999; 41: 347]. It can also lead to ADE in vaccinated animals and is a limiting factor in the use of intranasal vaccines.

Today, FIP is a highly lethal and widespread disease of cats for which no effective vaccines have been developed and there is no treatment approved by regulatory authorities in leading countries. This leads to the illegal distribution on the “black market” of experimental samples of drugs that have not undergone full-scale preclinical and clinical studies [https://abc7.com/cats-covid-coronavirus-cure/6253361/, accessed 07/22/2023].

Various evidence suggests that robust T cell-mediated immunity is required to confer long-lasting immunity against FCoV. Animals that survived acute infection had robust T-cell responses during later stages of infection. In addition, surviving individuals were resistant to subsequent infection with the pathogenic FCoV virus [Mustaffa-Kamal F., Liu F.L., Pedersen N.C., Sparger E.E. Characterization of antiviral T cell responses during primary and secondary challenge of laboratory cats with feline infectious peritonitis virus (FIPV). BMC Vet Res. 2019; 15 (1): 165]. In FIP, inhibition of T-cell-mediated processes occurs, which does not occur in FCoV seropositive cats that do not have FIP [Malbon A. J., Russo G., Burgener C, et al. The Effect of Natural Feline Coronavirus Infection on the Host Immune Response: A Whole-Transcriptome Analysis of the Mesenteric Lymph Nodes in Cats with and without Feline Infectious Peritonitis. Pathogens. 2020; 9(7): 524]. And in a cohort of laboratory cats resistant to the development of FIP, both during primary infection and during secondary reinfection with the virus, similar virus-specific T-cell immunological correlates of protection were observed, possibly providing long-term protection against the virus [Mustaffa-Kamal F., Liu N ., Pedersen N.C. et al. Characterization of antiviral T cell responses during primary and secondary challenge of laboratory cats with feline infectious peritonitis virus (FIPV). BMC Vet Res. 2019; 15: 165].

Thus, works before 2003 mention the study of the N-protein of feline coronavirus as an antigen in viral vectors (pox virus - Vennema N., de Groot R.J., Harbor D.A. et al. Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens. Virology. 1991; 181: 327-335; raccoon pox virus - Wasmoen T. L., Kadakia N. P., Unfer R. C. et al. Protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus. Adv. Exp. Med. Biol. 1995; 280: 221-228; plasmid vector - Glansbeek H. L., Haagmans B. L., te Lintelo E. G. et al. Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus. J. Gen. Virol. 2002; 83: 1-10; or a cell lysate containing a protein expressed by a baculovirus - Hohdatsu T., Yamato N., Ohkawa T. et al. Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP. Vet Microbiol. 2003 Dec 2; 97(1): 31-44; patent application US 20120107390 A1). The vector vaccine studied by the group of Vennema et al., like the DNA vaccine by Glansbeek et al. did not have a protective effect. Vector vaccine Wasmoen et al. was effective against low-dose infection with the FIPV virus, however, it is difficult to assess the contribution of the vaccine specifically to this effect, since after immunization the authors first carried out infection with the FECV coronavirus, which does not cause severe consequences, and only after that with the FIPV coronavirus, which causes peritonitis. The most promising data were obtained by the group T. Hohdatsu et al. After vaccination with SF-9 (Spodoptera frugiperda) insect cell lysate containing baculovirus-expressed FIPV N protein together with an adjuvant (Felidovac PCR inactivated feline vaccine containing aluminum hydroxide adjuvant), cats achieved 62.5% protection against lethal FIPV infection. Quite low (62.5%), but still the effectiveness of the cell lysate can be explained by the presence in its composition of an adjuvant based on aluminum hydroxide, which effectively stimulates the humoral immune response, but does not effectively stimulate the antigen-specific T-cell response, in addition, in the lysate in parallel, the Felidovac PCR vaccine was administered, which also stimulated the humoral immune response.

Thus, the technical problem to be solved by the present invention is to create a means for immunization against coronavirus in cats.

The essence of the invention

One of the technical problems that the present invention aims to solve is the creation of an immunogen against feline coronavirus (FCoV).

A solution to the problem may be the development of a new effective vaccination strategy based on the use of a prophylactic drug based on the conservative recombinant nucleocapsid protein of feline coronavirus (N-FCoV) and a specific adjuvant, which is a mixture of squalane, α-tocopherol (vitamin E) and polysorbate-80, which effectively induces a T-cell response.

The technical result is an immune response against feline coronavirus.

The present group of inventions provides a recombinant feline coronavirus nucleocapsid protein, hereinafter also referred to as N-FCoV, containing the amino acid sequence SEQ ID NO:2, which can be used to immunize cats against FCoV. The recombinant protein of the present invention may additionally contain 6-10 histidine residues, called histidine tag (His-tag).

Also provided is a recombinant nucleic acid encoding a recombinant protein of the present invention. Said nucleic acid in one embodiment of the invention contains the nucleotide sequence SEQ ID NO:l with a length of 1128 bp.

Said nucleic acid may be incorporated into any of the pET family of plasmid expression vectors or the like. These plasmid expression vectors are used to transform Escherichia coli cell strains to express the recombinant N-FCoV protein.

Also, the group of inventions proposes a recombinant plasmid expression vector pET-28a(+)-N-FCoV, intended for producing a recombinant Escherichia coli strain - a producer of the recombinant N-FCoV protein. The said recombinant plasmid expression vector with a size of 6406 bp, according to the map presented in Fig. 1, contains:

KanR is a kanamycin resistance gene that provides resistance of E. coli transformants to the selective antibiotic kanamycin (coordinates 5032-5847);

ori (ori, origin of replication) replication initiation site (coordinates 4322 4910);

lad - The transcriptional repressor lad binds to the lac operator to inhibit transcription in E. coli (coordinates 1810 2892). This inhibition can be reversed by adding lactose or isopropyl-r-O-thiogalactopyranoside (IPTG);

lad promoter - promoter for binding RNA polymerase transcribing the lad gene (coordinates 1732-1809);

T7 promoter promoter of RNA polymerase of bacteriophage T7 (coordinates 1405 1423);

lac operator operator for binding LacI (coordinates 1380-1404);

RBS (ribosome binding site) is an effective ribosome binding site from gene 10 of bacteriophage T7 (coordinates 1344-1349);

T7 terminator - transcription terminator of RNA polymerase of bacteriophage T7 (coordinates 26-73);

NcoI/XhoI fragment containing the artificial gene SEQ ID NO: 1, used to obtain the N-FCoV protein, encoding an artificial immunogen protein (coordinates 164-1336). The nucleotide sequence of the plasmid expression vector pET-28a(+)-N-FCoV includes the sequence SEQ ID NO: 3.

The group of inventions also offers a strain of Escherichia coli - a producer of the recombinant N-FCoV protein, obtained by transformation of competent cells. E'. coli strain BL21[DE3] with the plasmid expression vector of the present invention.

The group of inventions also proposes a composition for immunization against feline coronavirus containing recombinant N-FCoV protein, squalane, α-tocopherol and polysorbate 80 in a weight ratio of 1: 750: 250: 50.

The group of inventions also proposes a method of using the said composition for the prevention of coronavirus infection in cats, which consists in administering the composition to cats at the rate of 25 mcg of protein per cat twice with an interval of 21 days.

The invention is illustrated by the following graphics:

In FIG. Figure 1 shows a map of the plasmid expression vector pET-28a(+)-N-FCoV.

In FIG. Figure 2 shows an electropherogram of E. coli cell lysates containing pET-28a-N-FCoV plasmid DNA after induction of expression and without induction. 1 - Bio-Rad Precision Plus Protein Standards marker; 2 - negative control without induction of expression; 3 total cell lysate after induction of expression with 1 mM IPTG. The arrow indicates the target protein.

In FIG. Figure 3 shows an electropherogram of soluble and insoluble fractions of E. coli cell lysates containing pET-28a-N-FCoV plasmid DNA after induction of expression and subsequent separation into fractions by centrifugation. 4 marker Bio-Rad Precision Plus Protein Standards; 5 total cell lysate without centrifugation; 6

- soluble fraction (supernatant after centrifugation); 7 - insoluble fraction (precipitate after centrifugation). The arrow indicates the target protein.

In FIG. Figure 4 presents the result of assessing the Th1 cell (T helper 1) immune response to stimulation of peripheral blood mononuclear cells (PBMCs) isolated from immunized cats with the recombinant N-FCoV protein. Control - medium, N-FCoV+T-adjuvant - N-FCoV with T-cell adjuvant, N-FCoV+B-adjuvant - N-FCoV with B-cell adjuvant.

Detailed description of the invention

The present group of inventions provides a recombinant nucleocapsid protein of the feline coronavirus N-FCoV containing the amino acid sequence SEQ ID NO: 2, which can be used to immunize cats against FCoV.

Said N-FCo V protein can be used as an antigen to produce a vaccine or immunogenic composition intended to produce immunity against feline coronavirus.

The recombinant N-FCoV protein included in the vaccine is conservative and common to both serotypes and pathotypes of feline coronavirus, that is, the creation of a vaccine based on it can provide the most complete protection of animals, regardless of their geographic residence, as well as in both mono- and and coinfections with serotypes I and II. The recombinant protein of the present invention can be produced by any method known in the art for producing polypeptides and proteins, including, but not limited to, recombinant methods such as synthesis in cells transfected or transduced with expression vectors encoding the polypeptide and others, as well as methods of chemical synthesis. The recombinant protein of the present invention induces an immune response, both humoral and cellular, against feline coronavirus, thus achieving the technical result of the invention.

In one embodiment, the recombinant protein of the present invention may further comprise 6 to 10 histidine residues, referred to as a His-tag. The histidine tag allows the additional use of affinity chromatography to purify the recombinant protein of the present invention [see, for example, Spriestersbach A, Kubicek J, Schafer F, et al. Purification of His-Tagged Proteins. Methods Enzymol. 2015;559:1-15. doi:10.1016/bs.mie.2014.11.003].

Also provided is a recombinant nucleic acid encoding said recombinant protein. The recombinant N-FCoV protein of the present invention can be encoded by nucleic acids with different nucleotide sequences due to the degeneracy of the genetic code in accordance with the standard genetic code table [see, for example, Griffiths A.J.F. et al., An Introduction to Genetic Analysis. New York: W.H. Freeman; 2000]. In one embodiment, the invention provides an artificial nucleic acid for producing recombinant N-FCoV protein. Said nucleic acid encodes the recombinant N-FCoV protein and contains the nucleotide sequence SEQ ID NO: 1, 1128 bp long. Said nucleic acid can be included in expression vectors known in the art for producing recombinant protein - PCoV, such as plasmid expression vectors, viral expression vectors, etc.

Said nucleic acid may be incorporated into any of the pET family of plasmid expression vectors or the like. These plasmid expression vectors are used to transform Escherichia coli cell strains to express the recombinant N-FCoV protein.

Also, the group of inventions proposes a recombinant plasmid expression vector pET-28a(+)-N-FCoV, intended for producing a recombinant Escherichia coli strain - a producer of recombinant N-FCoV protein. The said recombinant plasmid expression vector with a size of 6406 bp, according to the map presented in Fig. 1, contains:

KanR kanamycin resistance gene, which provides resistance of E. coli transformants to the selective antibiotic kanamycin (coordinates 5032-5847);

ori (ori, origin of replication) - replication initiation site (coordinates 4322-4910);

The lad transcriptional repressor lad binds to the lac operator to inhibit transcription in E. coli (coordinates 1810–2892). This inhibition can be reversed by adding lactose or isopropyl-r-O-thiogalactopyranoside (IPTG);

lad promoter promoter for binding RNA polymerase transcribing the lad gene (coordinates 1732-1809);

T7 promoter - promoter of RNA polymerase of bacteriophage T7 (coordinates 1405-1423);

lac operator - operator for binding LacI (coordinates 1380-1404);

RBS (ribosome binding site) effective ribosome binding site from gene 10 of bacteriophage T7 (coordinates 1344 1349);

T7 terminator - transcription terminator of RNA polymerase of bacteriophage T7 (coordinates 26-73);

NcoI/XhoI is a fragment containing an artificial gene SEQ ID NO: 1, encoding the recombinant N-FCoV protein (coordinates 164-1336).

The nucleotide sequence of the plasmid expression vector pET-28a(+)-N-FCoV includes the sequence SEQ ID NO:3. Said plasmid expression vector of the present invention produces transformed E. coli cell lines that synthesize the N-FCoV protein containing the amino acid sequence of SEQ ID NO:2. Thus, the recombinant N-FCoV protein of the present invention contains the amino acid sequence SEQ ID NO:2 with a length of 376 aa. and can be produced using a plasmid expression vector containing the nucleic acid of the present invention.

The group of inventions also offers a strain of Escherichia coli - a producer of the recombinant N-FCoV protein, obtained by transformation of competent cells. E'. coli strain BL21[DE3] with a plasmid expression vector according to the present invention. The resulting producer strain demonstrates stable growth in suspension culture and expression of the recombinant protein of the present invention in soluble form.

The recombinant N-FCoV protein of the present invention can be used to obtain a vaccine against FCoV in the form of a composition containing a solvent, as well as an adjuvant and other auxiliary substances, for example, stabilizers or preservatives, buffer solutions, or in the form of a lyophilisate, for example, using lyoprotector or without it.

The group of inventions also proposes a composition for immunization against feline coronavirus containing recombinant N-FCoV protein, squalane, α-tocopherol and polysorbate 80 in a weight ratio of 1: 750: 250: 50.

Squalane, α-tocopherol and polysorbate 80 are an oil-in-water emulsion adjuvant. Similar adjuvants containing the fully metabolized lipid squalane, which forms an emulsion, and the immunostimulant α-tocopherol (vitamin E) effectively induce the development of a T-cell immune response to a co-administered antigen [Del Giudice G, Rappuoli R., Didierlaurent A.M. Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Seminars in Immunology. 2018; 39: 14-21]. The adjuvant properties of this emulsion are due to its ability to recruit and activate antigen-presenting cells. With the introduction of such an adjuvant, an increase in antigen-specific synthesis of Th1 (IFN-γ) and Th2 cytokines (IL-5 and IL-13) occurs, which ensures activation of the T-cell immune response to the administered antigen [Andreev Yu.Yu., Toptygina A. P. Adjuvants and immunomodulators in vaccines. Immunology. 2021; 42(6): 720-729; Morel S. et al. Adjuvant System AS03 containing a-tocopherol modulates innate immune response and leads to improved adaptive immunity. Vaccine. 2011; 29(13): 2461-2473].

The group of inventions also proposes a method of using the said composition for the prevention of coronavirus infection in cats, which consists in administering the composition to cats at the rate of 25 mcg of protein per cat twice with an interval of 21 days.

The invention will now be illustrated by non-limiting examples.

Example 1. Preparation of a plasmid expression vector containing a protein gene

All full-length amino acid sequences of the nucleocapsid protein of the feline coronavirus FCoV were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/guide/al1/, access date 07/18/2023). The sequences were aligned using Geneious Prime 2023.1 and a consensus protein sequence was determined (SEQ ID NO:2). A consensus sequence is an artificial protein sequence containing at each position an amino acid residue that is most often found in several homologous sequences. It is the result of multiple sequence alignment in which homologous sequences are compared to each other. Accordingly, the consensus sequence of the recombinant N-FCoV protein of the present invention has the properties of the sequences used in the alignment.

The resulting consensus amino acid sequence was used to design an artificial nucleotide sequence taking into account codon preference and codon pair preference for E. coli. The resulting nucleotide sequence at the 5' and 3' ends was flanked by sequences for recognition by the endonucleases Ncol (CCATGG) and XhoI (CTCGAG), necessary for integration into plasmid DNA. Synthesis of a nucleic acid containing the sequence of the present invention and insertion at the indicated restriction sites into the plasmid expression vector pET-28a(+) was carried out according to standard methods.

The resulting plasmid expression vector was sequenced using primers T7 promotor (TAATACGACTCACTATAGGG), T7 Terminator (GCTAGTTATTGCTCAGCGG) to confirm the nucleotide sequence; the sequence did not contain insertions, deletions or substitutions.

The nucleotide sequence of the resulting DNA corresponded to the sequence of SEQ ID NO: 1.

Example 2. Creation of a strain producing recombinant N-FCoV protein

To obtain a strain producing the recombinant N-FCoV protein, competent cells of E. coli strain BL21[DE3] were transformed by electroporation according to standard methods. Vector pET28a, shown in SEQ ID NO:3, contains a promoter for T7 RNA polymerase, and the strain used contains in the genome the T7 RNA polymerase gene under the control of the lacUV5 promoter, which allows inducing the expression of the recombinant protein using IPTG or lactose.

In this way, an E. coli producer strain incorporating the nucleic acid of the present invention was obtained.

The resulting producer strain from the working bank was sown on Petri dishes with selective LB agar medium. After incubating the Petri dishes for 16 hours at plus 37°C, a single colony was subcultured into liquid LB24 medium containing 30 μg/ml kanamycin sulfate, 1% glucose and 1% glycerol. Cultivated in a shaker incubator at plus 37°C, 250 rpm for 16 hours. The next day, the overnight culture was diluted with LB24 medium containing 30 μg/ml kanamycin sulfate and 0.2% glucose to an OD _{of 600 nm} ≈0.1. After which they were cultured for 1.5 3 hours at plus 30°C, 220 rpm in a thermal shaker until an OD _{of 600 nm} ≈0.6-0.8 was achieved. When the cells reached the beginning of the logarithmic growth phase, the temperature in the incubator shaker was reduced to 25°C and IPTG was added to a final concentration of 1 mM. After adding the inducer, the bacteria were cultivated for 16-18 hours at plus 25°C, 220 rpm. After completion of the cultivation process, the expression level was analyzed using vertical disk electrophoresis in polyacrylamide gel (PAGE) under denaturing conditions.

The electropherogram of E. coli cells containing pET-28a-N-FCoV plasmid DNA after induction of expression and without induction (Fig. 2) shows that the target protein is present in the total cell lysate sample after induction of expression with 1 mM IPTG. This indicates that the resulting producer strain expresses a recombinant plasmid expression vector and synthesizes and also secretes recombinant N-FCoV protein into the culture fluid. Expression of DNA with an optimized nucleotide sequence provided a high level of the target protein - about 20% of total cellular proteins. Cell lysate of the producer strain without IPTG induction was used as a control; the target protein band is absent in the electropherogram, which indicates the absence of uncontrolled expression of the target sequence.

Example 3. Preparation of recombinant N-FCoV protein

E. coli biomass containing the synthesized N-FCoV protein was lysed on ice in 20 mM Tris-HCl buffer, pH 8.5, 0.5 M NaCl at the rate of 10 ml of buffer per 1 g of wet cell biomass using an I10- ultrasonic homogenizer 840 under the following conditions: exposure time 4 minutes, power 100%, frequency 43.5 kHz, pulse - 10 seconds, pause - 5 seconds. The total lysate was collected into a clean tube for subsequent analysis using vertical disk PAGE under denaturing conditions. The lysed biomass was pelleted by centrifugation for 15 min at 10,000 g at plus 4°C. The supernatant was filtered through a 0.22 μm filter and used for further purification. A sample of the supernatant was taken into a clean tube for subsequent analysis of the soluble fraction using vertical disk electrophoresis in PAGE under denaturing conditions. The sediment in the tubes was resuspended in a lysis buffer solution and centrifuged for 30 min at 10,000 g at plus 4°C. The supernatant was poured off, and the resulting precipitate was resuspended in a lysis buffer solution; a sample of the suspension was taken into a clean test tube for subsequent analysis of the insoluble fraction by vertical disk electrophoresis in PAGE under denaturing conditions.

The electrophoregram of E. coli cell lysate fractions containing pET-28a-N-FCoV plasmid DNA, after induction of expression and subsequent separation into fractions by centrifugation (Fig. 3), shows that the target protein is present in the sample of the total cell lysate before centrifugation and in the soluble fraction (supernatant after centrifugation) and is absent in the insoluble fraction (precipitate after centrifugation). Thus, it was shown that when expressed in E. coli cells, the N-FCoV protein is in the soluble fraction, which indirectly indicates its correct three-dimensional structure.

To carry out chromatographic purification, the supernatant was applied to a column containing 20 ml of Ni Sepharose High Performance sorbent (GE Healthcare, USA), equilibrated with ten volumes of buffer solution A (20 mM Tris HCl, pH 8.5, 0.5 M NaCl), using AKTA Explorer chromatograph. After applying the sample to the column, it was washed with buffer solution A at a rate of 3 ml/min until the absorbance values at a wavelength of 280 nm reached a plateau. After this, 10% buffer solution B (20 mM Tris - HCl, pH 8.5, 0.15 M NaCl, 0.5 M imidazole) was applied to the column in order to wash away weakly bound impurities, while the flow rate was 3 ml /min. After the peak was released, the column was washed with 100% buffer solution B in order to wash away the target product. Fractions with absorbance greater than 50 mAU (optical density units) were analyzed by vertical disk electrophoresis in a polyacrylamide gel under denaturing conditions.

The target protein is released at an imidazole concentration of 0.5 M (100% buffer solution B). Fractions containing the target protein were combined.

Concentrated fractions with a volume of 30 ml were applied to a column containing a sorbent for size exclusion chromatography Sephacryl S-200HR (GE Healthcare, USA), equilibrated with buffer solution B (20 mM Tris-HCl, pH 8.5, 100 mM NaCl). The flow rate was 0.75 ml/min, the elution volume was 1000 ml.

Fractions with an absorbance greater than 50 mAU were analyzed by vertical disk electrophoresis in a polyacrylamide gel under denaturing conditions.

The N-FCoV protein solution obtained after the second stage was dialyzed against buffer (NaH ₂ PO ₄ 0.010 M, KH ₂ PO ₄ 0.0018 M, NaCl 0.137 M, KCl 0.0027 M, pH 7.4).

The protein yield was 50 mg/l with a purity of more than 95%.

Example 4. Determination of the protective properties of the N-FCoV protein

The study of the protective (protective) properties of the N-FCoV protein was carried out on laboratory animals. The study was conducted on non-linear, non-SPF (Specific Pathogen Free) cats of both sexes. The cats were 14-18 weeks old at the time of immunization, weighing 800-1200 g. The animals were kept in separate groups in vivarium conditions in compliance with basic zoohygienic requirements. Before immunization, cats were tested for the absence of FCoV, FPV, FHV, FCV, FIV, FELV viruses by ELISA and PCR using commercial kits.

Laboratory animals were divided into 6 groups:

group 10M 5 females immunized with protein with water-oil adjuvant; group 20M - 5 males immunized with protein with water-oil adjuvant; group 30A - 5 females immunized with protein with aluminum hydroxide adjuvant;

group 40A 5 males immunized with protein adjuvanted with aluminum hydroxide;

group 5Kk and - 3 non-immunized females; group 3 non-immunized males.

Animals from groups 10M, 20M, 30A, 40A were immunized twice with an interval of 21 days with a composition containing 25 μg of protein and 18.75 mg of squalane, 6.25 mg of α-tocopherol and 1.25 mg of polysorbate 80 in 1 ml for groups 10M and 20M, or a composition containing in 1 ml 25 μg of protein and an adjuvant based on aluminum hydroxide (Alhydrogel® 2%, Brenntag Biosector A/S) in a ratio of 1: 2 (adjuvant: antigen) for groups 30A and 40A. The control group was injected with saline solution. 21 days after the last immunization, the animals were oronasally injected with the FCoV virus at a dose of 1000 (3.0 lg) TCD50 per animal (a dose that infects 50% of the cell culture with a cytopathic effect in 50% of the monolayer cells). 1 day before the introduction of the virus, intravital blood sampling was performed in laboratory animals to determine the titer of specific antibodies and analyze the induction of a specific T-cell immune response.

The animals were observed for 6 months. The day of death and the rate of manifestation of clinical signs of the onset of the disease (infectious peritonitis) were determined. The following parameters were analyzed and assessed every day:

- depression (inactivity for 3 days in a row, 1 point);

- anorexia (not eating food for 3 days in a row, 1 point);

- neurological disorders (1 point).

The following parameters were analyzed and assessed every week:

- fever (40.1°C, 1 point);

- jaundice (yellow plasma, 1 point);

- lymphopenia (lymphocyte count less than 0.5*10 ⁹ /l, 1 point);

- weight loss (loss of 2.5% of body weight, 1 point).

The results of calculating the coefficient of manifestation of clinical signs of the onset of the disease (infectious peritonitis) and the day of death after infection are presented in Table 1.

During the analysis of the data obtained, it was found that in cats immunized with the protein of the present invention together with a water-oil adjuvant that stimulates the T-cell response, there was no mortality in all groups, the protective (protective) effectiveness was 100%, the average disease manifestation rate was only 0.60±0.55 and 0.80±0.50. In the group immunized with the protein of the present invention together with the adjuvant aluminum hydroxide, which stimulates the humoral B-cell immune response, the mortality and protective efficacy were 50%, the average disease incidence rate was 3.60 ± 4.10 and 4.40 ± 4.04 . All animals in the control groups died.

Example 5. Determination of antibody titer

The titer of antibodies produced after administration of the protein according to the invention was determined using the following procedure.

After two immunizations, on day 41, blood was collected from all animals. The blood was centrifuged at 4000 rpm and the serum was collected for ELISA.

Previously, 200 μl of a 5 μg/ml solution of the recombinant N-FCoV protein of the present invention (hereinafter referred to as AG) in carbonate buffer, pH 9.6, was added to each well of a microtiter plate. The plates were incubated overnight at 4°C. Next, the AG solution was removed from the wells and ₂₀₀ μl of blocking buffer (10 mM _H2PO4 , 150 mM NaCl, 10% casein, 5% sucrose) was added to each well. Incubated for 30 min at 37°C. The contents of the wells were carefully removed. 100 μl of dilution solution (RR) (1× TBS, 0.1% casein) was added to all wells of the plate except the first row; 125 μl of serum solution diluted 100 times RR was added to the first row and titrated in increments of x5. The plates were incubated for 1 hour at 37°C in a thermostat. After removing the contents, the plates were washed 3 times with wash buffer (WB) (1xTBS, 0.05% Tween 20). Conjugate of goat anti-cat IgG antibodies with horseradish peroxidase (Sigma, USA) was diluted PP 1:20000. Add 100 μl of conjugate per well. The plates with the conjugate were incubated for 30 minutes at 37°C. After removing the conjugate, the plates were washed 4 times with PB. 100 μl of tetramethylbenzidine (TMB) solution was added to each well and left for 15 minutes, after which the reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ to each well. Optical density (OD) readings were performed on an Epoch spectrophotometer plate reader (Biotek) at a wavelength of 450 nm. The results are shown in tables 25.

The titer is the maximum serum dilution at which a positive result is still detected. An optical density value that is more than 2 times higher than the OD value for serum from non-immunized animals at the same dilution is considered positive.

According to the results obtained, presented in tables 2-5, it can be seen that the average titer of AT to AG one month after the start of immunization in the OM groups was 1:27500, in the OA groups - 1:52500. The antibody titer in the groups immunized with the recombinant protein with an adjuvant that stimulates a humoral response is significantly higher than in the groups with an adjuvant that stimulates a T-cell response.

Example 6 Determination of T-cell immune response

After two immunizations, blood was collected from all animals on day 41. Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate™-15 density gradient centrifugation tubes (RUO) (STEMCELL Technologies, USA).

Selected PBMCs were inoculated at 5-10 ⁶ cells/ml in a volume of 200 μl of RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 U/ml penicillin and streptomycin. , into the wells of a 96-well culture plate and incubated in a thermostat at 37°C in an atmosphere of 5% CO ₂ for 7 days. To stimulate antigen-specific synthesis of interferon gamma (IFNγ), the N-FCoV protein of the present invention was added, and cells in the culture medium were used as a negative control.

The concentration of IFNγ in the conditioned culture liquid after stimulation of cells with N-FCoV protein was determined using a commercial ELISA test system Feline IFN gamma ELISA Kit (Abeam, USA). The sensitivity of the method is 0.24 ng/ml, the measurement range is 0.24 - 60 ng/ml.

The measurement results are presented in Fig. 4. N-FCoV protein together with a T-cell adjuvant significantly (p<0.01) causes a stronger T-cell response compared to the B-cell adjuvant.

Thus, using the claimed group of inventions, it is possible to achieve an immune response against feline coronavirus, including, in a particular case, a cellular immune response, including, in a particular case, a protective immune response.

--->

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cggacaaaaaaccggaagaactgtctgttaccctggttgaagcgtacaccgacgttttcgacgacaccca

ggttgaaatgatcgacgaagttaccaactaatgactcgagcaccaccaccaccaccactgagatccggct

gctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttg

gggcctctaaacgggtcttgaggggttttttgctgaaagggaggaactatatccggat</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (10)

1. Recombinant protein for immunization against feline coronavirus, containing the amino acid sequence SEQ ID NO:2. 2. Protein according to claim 1, additionally containing a histidine tag of 6-10 histidine residues. 3. Nucleic acid encoding a protein according to any one of paragraphs. 1 or 2. 4. Nucleic acid according to claim 3, containing the sequence SEQ ID NO:1. 5. An expression vector for transformation of Escherichia coli cell strains, containing a nucleic acid according to any one of paragraphs. 3 or 4. 6. The vector according to claim 5, which is a plasmid expression vector. 7. The vector of claim 6, which includes SEQ ID NO:3. 8. Escherichia coli strain - a recombinant protein producer according to claim 1, obtained by transforming competent cells of E. coli strain BL21[DE3] with a plasmid expression vector according to claim 6. 9. Composition for immunization against feline coronavirus, containing the recombinant protein according to claim 1, squalane, α-tocopherol and polysorbate 80 in a weight ratio of 1: 750: 250: 50. 10. A method of using the composition according to claim 9 for the prevention of coronavirus infection in cats, which consists in administering the composition to cats at the rate of 25 mcg of protein per cat twice with an interval of 21 days.