Pneumonia vaccine

Abstract

The invention relates to the field of health care, and more particularly to the prevention of infections that cause pneumonia and other illnesses using an immunogenic protein containing fragments of the proteins FliC, PspA, OmpA and PE, connected by rigid linkers, wherein: - the FliC protein fragment contains SEQ ID NOs: 2 and 3 connected to one another by a flexible linker; - the PspA protein fragment contains SEQ ID NO: 4; the OmpA protein fragment contains SEQ ID NOs: 5-8 connected to one another by flexible linkers; - the PE fragment contains SEQ ID NO: 9. Also proposed are a host cell and a method for producing the protein. The immunogenic protein is suitable for use as a broad-spectrum polyvalent vaccine and exhibits a marked protective effect against Streptococcus pneumoniae, Haemophilus influenzae and Klebsiella pneumoniae, as well as providing for production efficiency and safety.

Description

[0001] Pneumonia vaccine

[0002] Field of technology to which the invention relates

[0003] The invention relates to a hybrid protein immunogen against bacterial pneumonia, intended for use in pharmaceuticals, medicine, and other technical fields. In addition to the protein, a host cell, a method for producing the protein, and other technical solutions are also proposed.

[0004] Information on the prior art

[0005] Pneumonia is a group of infectious diseases caused by bacteria, viruses, or fungi that affect the alveoli and interstitial tissue of the lung. Pneumonia affects approximately 450 million people annually, with mortality estimated at 1.4 million cases in 2010 and 3 million cases in 2016.

[0006] Pneumonia is especially dangerous for the elderly and children, representing the leading infectious cause of death in children worldwide. According to the WHO, in 2019, pneumonia accounted for 14% of all deaths among children under 5 (740,180 children) and 22% of deaths among children aged 1-5.

[0007] The main causative agents of bacterial pneumonia are the gram-positive coccus Streptococcus pneumoniae (up to 70% of the total number of pneumonia cases), the gram-negative coccobacillus Haemophilus influenzae (10-15% of the total number of pneumonia cases), and the gram-negative bacillus Klebsiella pneumoniae (a common causative agent of hospital pneumonia and pneumonia in patients with alcoholism and diabetes).

[0008] Bacterial pneumonia can be treated with antibiotics, but many circulating strains have become multidrug-resistant, so public health efforts must focus on prevention, primarily through vaccination.

[0009] Currently registered vaccines against pneumococcal infections include polysaccharide vaccines, such as Pneumovax 23 (Merck Sharp & Dohme (MSD), USA), and conjugate vaccines (polysaccharide-protein), such as Prevenar 13 (Pfizer Ireland Pharmaceuticals, Ireland) or Synflorix (GlaxoSmithKline Biologicals, France). These vaccines are polyvalent, containing 23, 13, and 10 polysaccharides of Streptococcus pneumoniae from different serotypes, respectively.

[0010] Currently, only conjugate vaccines are used against Haemophilus influenzae infections, for example, Pentaxim and Act-HIB (Sanofi Pasteur, France), Hiberix and Infanrix Hexa (GlaxoSmithKline Biologicals, France).

[0011] There are no approved vaccines against infections caused by Klebsiella pneumoniae for human use. One of the problems associated with the use of both polysaccharide and conjugate vaccines is that they offer no protection against infections caused by other serotypes and non-typeable strains. Therefore, developers focus on combining antigens from different serotypes as much as possible, which complicates the vaccine composition and production process. For example, over 100 serotypes have been described for S. pneumoniae, of which no more than 23 are included in vaccines. Furthermore, a process known as serotype substitution is observed, in which serotypes for which there are no vaccines begin to occupy the niche of serotypes for which vaccines exist. For example, for H. influenzae, six capsular serotypes and non-typeable strains lacking a capsule are known. After the widespread use of vaccination against H.Influenzae type b has seen a decline in the number of infectious diseases it causes. However, the number of illnesses associated with non-typeable strains is increasing.

[0012] Another common problem with the use of polysaccharide vaccines is reduced or absent immunogenicity when administered to children, especially under 2 years of age. Polysaccharide vaccines typically induce a response of short-lived B cells that differentiate into antibody-secreting plasma cells; however, many polysaccharide vaccines do not generate new memory B cells but deplete the pool of existing ones, leading to reduced efficacy of subsequent vaccination (Pollard, A., Perrett, K. & Beverley, P. Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines. Nat Rev Immunol 9, 213–220 (2009)).

[0013] It is important to note that young children, especially those in their first year of life, are most vulnerable to bacterial infections, particularly pneumococcal infections, and from a public health perspective, it is especially important to have vaccines that are effective specifically for this group.

[0014] Conjugate vaccines offer some limited advantages in this regard; for example, they can be used in children. The carrier protein in these vaccines is taken up by polysaccharide-specific B cells and presented to T cells, which in turn stimulate the production of plasma cells and memory B cells.

[0015] Despite all the advantages of conjugate vaccines, their prophylactic effect is limited to the serotypes whose antigens are included in the vaccine. This is a serious drawback, especially for patients in high-risk groups. Healthcare professionals primarily rely on additional vaccines against other serotypes, for example, administering a polysaccharide vaccine (such as Pneumovax 23) after a conjugate vaccine (such as Prevenar 13). Clearly, such regimens are undesirable from a patient perspective and increase the burden on the healthcare system, particularly in the context of the growing anti-vaccination movement and increasing antibiotic resistance among modern strains.

[0016] It is also important to have an expanded arsenal of such vaccines to have a choice when vaccinating certain subgroups of patients who have contraindications or sensitivity to a particular vaccine.

[0017] Polyvalent polysaccharide and conjugate vaccines are essentially a mixture of multiple active components, which complicates production and quality control. This same drawback will also apply to mixed peptide vaccines, which use separate molecules for individual antigens.

[0018] Therefore, the development of vaccines targeting multiple types of pneumonia pathogens is promising. Recombinant proteins are one of the modern platforms for vaccine creation. This technology allows for the use of the entire pathogen protein, its fragments, or a chimeric protein (a fusion of several proteins from the same or different pathogens). Selecting the most immunogenic and conserved epitopes potentially expands the vaccine's spectrum of activity and makes it serotype-independent. One method for developing such vaccines is the construction of a chimeric protein containing immunogenic regions of the pathogen's surface proteins. Despite the promise of this technology, specialists face the challenging task of designing a chimeric construct, as it will not always possess the properties of the fragments it is composed of. In other words, the immunogenicity of individual fragments does not mean that they will be immunogenic when fused into a single protein.

[0019] A hybrid protein is known from RU2510281, which includes immunogenic fragments of the proteins PspA (aa 160-262 gb|EHD89266.1 | pneumococcal surface protein A [Streptococcus pneumoniae]), Sprl895 (ao 94-161 ref|ZP 01836139.1| LysM domain protein [Streptococcus pneumoniae]), PsaA (ao 238-309 gb|AAF70667.1| PsaA [Streptococcus pneumoniae]), as well as flagellin components as an adjuvant (aa 1-169 gb|AAB33952.1| flagellin {alternatively spliced} [Salmonella typhimurium], aa 311-405 gb|AAB33952.1| flagellin (alternatively spliced) [Salmonella typhimurium]) linked by flexible bridges. The immunogenicity and efficacy of the protein in terms of survival of mice infected with Streptococcus pneumoniae were demonstrated.

[0020] At the same time, there is a need for improved immunogenic recombinant proteins suitable for use as vaccines and targeting a wider range of pathogens that cause pneumonia. Specifically, there is a need for vaccines effective against Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae. Given the lack of registered vaccines against Klebsiella pneumoniae, there is also a specific need for such candidate vaccines. Finally, given the predominance of polysaccharide and conjugate vaccines, there is a need to expand the arsenal of available pneumonia vaccines, particularly those based on recombinant proteins.

[0021] The protein disclosed in this description allows us to solve the above problems.

[0022] The protein disclosed in this application differs from the prior art known from RU2510281 in that it utilizes a different and single S. pneumoniae antigen (a fragment of only one protein), also utilizes K. pneumoniae and H. influenzae antigens, and utilizes a different adjuvant (different fragments of the FliC protein). These differences lead to a number of unexpected advantages, described below.

[0023] The essence of the invention

[0024] Brief description of the invention

[0025] In a first aspect, the invention relates to an immunogenic protein comprising fragments of the FliC, PspA, OmpA and PE proteins connected by rigid linkers, wherein:

[0026] - the FliC protein fragment comprises, from the N- to the C-terminus, SEQ ID NO:2 and 3, connected to each other by flexible linkers;

[0027] - the PspA protein fragment includes SEQ ID NO:4;

[0028] - the OmpA protein fragment includes SEQ ID NO: 5, 6, 7 and 8, connected to each other by flexible linkers;

[0029] - fragment PE includes SEQ ID NO:9.

[0030] In some particular embodiments, rigid linkers are independently selected from, but not limited to, (EAAAK) П , where n = 1-5; [A(EAAAK) n A]k, where k = 1 or 2, n = 1-5; A(EAAAK)4ALEA(EAAAK)4A; R к (HR) п , where k = 0 or 1, n = 2-34, X is any amino acid; and flexible linkers are independently selected, but not limited to, from (GGGGS)n, where n = 1-9, (GGGGS)nAS, where n = 1-9, AGGGS(GGGGS) n , where n = 1-8, AGGGS(GGGGS) n AS, where n = 1-8, G n , where n = 5-20, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, GGSSG, (GS) n , where n = 3-8,

[0031] GGGGSLVPRGSGGGGS, GGSGGHMGSGG, GG(SGG) n , where n = 1-3, GGSGGGGG, (GGGSE)3GGG, AAGAATAA, GSGGGTGGGSG, (GS)4GGSG, GSGGGTGGGSG, GS(GGS)4, GSGGSGSGGSGGSG.

[0032] The order of fragments of said protein fragments from N- to C-terminus can be selected from the following: FliC-PspA-OmpA-PE, FliC-OmpA-PE-PspA, FliC-PE-PspA-OmpA, FliC-OmpA-PspA-PE, FliC-PspA-PE-OmpA, FliC-PE-OmpA-PspA, PspA-OmpA-PE-FliC, OmpA-PE-PspA-FliC, PE- PspA-OmpA-FliC, OmpA-PspA-PE-FliC, PspA-PE-OmpA-FliC, PE-OmpA-PspA-FliC, PspA-FliC-OmpA-PE, OmpA-FliC-PE-PspA, PE-FliC-PspA-OmpA, OmpA-FliC-PspA-PE, PspA-FliC-PE-OmpA, PE-FliC-OmpA-PspA, PspA-OmpA-FliC-PE, OmpA-PE-FliC-PspA, PE-PspA-FliC-OmpA, OmpA-PspA-FliC-PE, PspA-PE-FliC-OmpA, PE-OmpA-FliC-PspA; preferably FliC-PspA-OmpA-PE.

[0033] In a preferred embodiment, said immunogenic protein comprises or consists of the sequence SEQ ID NO: 1.

[0034] In a second aspect, the invention relates to a host cell that contains a polynucleotide or vector encoding said immunogenic protein. The host cell is capable, in particular, of producing the immunogenic protein.

[0035] In a third aspect, the invention relates to a method for producing said immunogenic protein, comprising culturing said host cell under conditions conducive to the production of said protein and isolating the protein. If necessary, the method further comprises purifying the protein and / or producing a preparation (or pharmaceutical substance) of the protein.

[0036] In other aspects, the following objects are proposed.

[0037] A polynucleotide encoding said immunogenic protein and a vector encoding said immunogenic protein. Preferably, the vector is an expression vector. Preferably, the vector is a plasmid.

[0038] A composition comprising the said immunogenic protein and, if necessary, one or more excipients. The composition is, without limitation, a pharmaceutical, in particular a pharmaceutical immunogenic (vaccine) composition. Most preferably, the pharmaceutical composition is intended for vaccinating against pneumonia, in particular bacterial pneumonia, and / or infections caused by Streptococcus pneumoniae, Haemophilus influenzae, and / or Klebsiella pneumoniae.

[0039] A kit comprising said protein or said composition, a means for administering the protein or composition to a subject, instructions for use, and, optionally, auxiliary substances and / or devices. Preferably, the kit is a pharmaceutical kit, in particular a vaccination kit; most preferably, the kit is intended for vaccinating against pneumonia, in particular against bacterial pneumonia, and / or against infections caused by Streptococcus pneumoniae, Haemophilus influenzae, and / or Klebsiella pneumoniae. A method of vaccinating, comprising administering said protein or composition to a subject in need thereof, or using said kit. In particular, the method is intended for vaccinating against pneumonia, in particular against bacterial pneumonia, and / or against infections caused by Streptococcus pneumoniae, Haemophilus influenzae, and / or Klebsiella pneumoniae.

[0040] The invention provides for the use of the said protein, composition, and kit for vaccinating against pneumonia, in particular bacterial pneumonia, and / or infections caused by Streptococcus pneumoniae, Haemophilus influenzae, and / or Klebsiella pneumoniae. The protein and composition can be used as a pharmaceutical substance for the production of pharmaceutical compositions (medicines, preparations).

[0041] Finally, the protein of the invention can be used for diagnostic purposes and in laboratory testing. In particular, the protein can be used as an antigen in immunoassays, such as in an immunoassay for detecting the presence of antibodies to Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae in a subject's sample, comprising incubating the sample with the protein of the invention and detecting binding of the protein to the antibodies. In some embodiments, in such applications, the protein is conjugated with a detectable label.

[0042] As a result of meticulous research, the inventors were able to identify specific immunogenic fragments of proteins from the main pathogens causing pneumonia—Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae—that elicit an effective protective immune response. As demonstrated in the experimental portion of the application, the protein does provide protective activity against each pathogen. Thus, for the first time, a single vaccine against Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae has been developed. Furthermore, the present invention solves a long-standing problem and provides the first vaccine with demonstrated efficacy against Klebsiella pneumoniae.

[0043] The protein's prophylactic effect is no worse than that of vaccines routinely used in healthcare (such as Prevenar 13), and even slightly superior. This means, firstly, that this is the first recombinant polyvalent subunit vaccine designed for the effective prevention of pneumonia and other conditions and diseases caused by the above-mentioned pathogens. Secondly, due to its protective effect, this recombinant polyvalent subunit vaccine could replace polysaccharide (conjugate) vaccines used in healthcare, offering a number of additional advantages for manufacturers, patients, and healthcare in general:

[0044] - overcomes the disadvantage inherent in polysaccharide vaccines, which is associated with the fact that their protective effect is limited exclusively to those serotypes of bacteria whose antigens are included in the composition of the drug;

[0045] - the possibility of pediatric use of vaccines is expanding, especially in children under 2 years of age;

[0046] - the spectrum of protective action of vaccines has been expanded due to the selection of conservative epitopes that are common among a wide range of strains, regardless of their serological affiliation;

[0047] - the production of protein does not require the cultivation of pathogenic strains, which means greater safety for production personnel and for patients, and increased quality control requirements in vaccine production are avoided or alleviated;

[0048] - the technology for producing recombinant polyvalent subunit vaccines is simpler than the production of polysaccharide vaccines, provides greater pharmaceutical purity and the use of various strains of known producers with high productivity, which allows for a reduction in the costs of purification and other production stages and, ultimately, provides a more affordable vaccine for the healthcare system;

[0049] - the technology for producing a recombinant subunit vaccine, which combines multiple antigenic determinants in a single molecule, is further simplified compared to a mixture of individual molecules that must be separately obtained, isolated, and purified, which leads to more complex production and higher costs to achieve the same purity as a drug with a single active component.

[0050] It is important to note that the unexpectedly successful combination of antigens against three pathogens in a single molecule offers advantages related to reducing the burden on patients and the healthcare system, namely, avoiding multiple different vaccinations, i.e. reducing the number of visits to the doctor, which increases compliance (adherence to the treatment regimen by patients), especially in children, reduces the risk of side effects and complications, and reduces government costs while expanding the coverage of preventive programs.

[0051] In addition to the above, the inventors unexpectedly discovered that the efficacy of the protein against Streptococcus pneumoniae is comparable to that of the protein known from RU2510281, which contains several antigens of this pathogen. This made it possible to improve the protein design by simultaneously incorporating antigens from additional pathogens.

[0052] The protein according to the invention allows vaccination using a lower dose, which makes the vaccine cheaper and more accessible to patients and the healthcare system.

[0053] Detailed description of the invention

[0054] Terminology

[0055] All terms used have the meanings accepted in the given field, unless a more specific or different interpretation is defined in this document or does not follow from the context.

[0056] The terms "chimeric", "hybrid", "recombinant" are used interchangeably in relation to the protein of the invention and generally mean a protein that is created by combining parts (proteins or fragments thereof) taken from different sources.

[0057] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to natural or synthetic polymers formed from nucleotide residues. Polynucleotides include DNA, RNA, and modified forms.

[0058] "Antigens" and "antigenic determinants" refer to a molecule or molecular structure recognized by an antigen receptor, such as antibodies or their fragments, T-cell receptors, and other binding structures. Antigens contain one or more epitopes—characteristic structural features of the antigen (antigenic determinants). An antigen can be an immunogen, meaning it can induce humoral or cellular immunity.

[0059] A protein "fragment" is defined as a sequence derived from a single protein (FliC, PspA, OspA, PE). Fragments of FliC and OspA, in turn, contain a number of fragments (also called "subfragments") derived from the corresponding protein. All fragments are separated by linkers.

[0060] When using terms in the singular, it is implied, where the context permits, that the plural is also possible, and vice versa.

[0061] The terms "including," "containing," "having," and the like mean that, in addition to the specified elements, other unspecified (nonessential) elements may be present. In each case, these terms also encompass the meaning of "consisting of," i.e., in one embodiment, they imply that the described object is characterized by the presence of only the specified elements.

[0062] The numerical values ​​mentioned imply both the exact value indicated and an approximate value corresponding to it (the terms “about”, “approximately”, “approximately” are also used for this), which is determined by a deviation upward and / or downward by 10, 5, 2 or 1%, or, in the case of parameters that can be measured with a certain accuracy, within the limits of measurement error characteristic of traditional measuring instruments.

[0063] The default intervals provided imply that both the boundary values ​​of the intervals and all values ​​contained in the interval are directly disclosed.

[0064] The bioengineering methods and techniques of the present invention are generally performed in accordance with conventional techniques well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

[0065] References to amino acid residues used in this application are designated by a conventional one-letter or three-letter code (see, e.g., Lehninger, Biochemistry, 2 nd edition, Worth Publishers, New York, 1975, p. 72).

[0066] Pneumonia is defined as any form of acute respiratory infection affecting the alveoli and / or distal bronchial tree of the lungs and includes community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), which includes ventilation pneumonia associated with mechanical ventilation (MV), as well as aspiration pneumonia and pneumonia associated with community healthcare facilities.

[0067] By “prevention”, “prophylaxis”, “warning” are meant in a broad sense the prevention of a disease (in particular, pneumonia), the elimination or mitigation of one or more symptoms of a disease, the elimination of risk factors for the disease and / or the reduction of the risk of complications of infection and / or disease.

[0068] The terms "subject" and "patient" as used herein refer to any mammal, including animals such as livestock, and humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human. "Pharmaceutical compositions" broadly encompass any formulations, including both manufactured formulations (e.g., pharmaceutical substances) and ready-to-use products (medicines). "Pharmaceutical" or "pharmaceutically acceptable" refers to chemical compounds and compositions that are suitable for administration to a mammal, particularly humans, in a manner that reduces or avoids adverse, allergic, or other undesirable reactions.

[0069] Antigenic determinants

[0070] PspA

[0071] PspA is a surface protein that is almost universally present among strains of all serotypes of S. pneumoniae. Its N-terminal fragment (amino acids 1–288) is an α-helix located on the bacterial surface, and its C-terminal fragment (amino acids 289–578) is attached to phosphocholine residues of the bacterial cell wall (Khan N, Jan AT. Towards Identifying Protective B-Cell Epitopes: The PspA Story. Front Microbiol. 2017 May 2;8:742). Based on the amino acid sequences of the α-helical domain, the entire protein diversity is divided into three families and six clades exhibiting cross-reactivity (Aceil J, Avci FY. Pneumococcal Surface Proteins as Virulence Factors, Immunogens, and Conserved Vaccine Targets. Front Cell Infect Microbiol. 2022 May 12; 12:832254). Antibodies to PspA are ubiquitous in people who have recovered from pneumococcal infection. Immunization of mice with recombinant PspA also provides protection against bacterial carriage in the nasopharynx and against invasive infections.

[0072] PspA inhibits the activation of C3 convertases of the classical and alternative pathways of the complement cascade. PspA interacts with factor B, influencing the formation of C3 convertase and prevents the binding of C-reactive protein to phosphocholines of the bacterial cell wall, thereby inhibiting the activation of the classical pathway of the complement cascade (Mukerji R, Mirza S, Roche AM, Widener RW, Croney CM, Rhee DK, Weiser JN, Szalai AJ, Briles DE. Pneumococcal surface protein A inhibits complement deposition on the pneumococcal surface by competing with the binding of C-reactive protein to cell-surface phosphocholine. J Immunol. 2012 Dec 1;189(11):5327–35). Blocking PspA with antibodies accelerates complement-mediated pneumococcal clearance (Andre GO, Converse TR, Politano WR, Ferraz LF, Ribeiro ML, Leite LC, Darrieux M. Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity. Front Microbiol. 2017 Feb 20;8:224).Also, anti-PspA antibodies enhance phagocytosis of pneumococci (Ren B, Li J, Genschmer K, Hollingshead SK, Briles DE. The absence of PspA or the presence of antibody to PspA facilitates the and complement-dependent phagocytosis of pneumococci in vitro. Clin Vaccine Immunol. 2012 Oct;19(10):1574-82).

[0073] In addition to disrupting the complement cascade, PspA binds to the multifunctional protein lactoferrin, which is widely present in the secretory fluids of the mucous membranes. Lactoferrin is one of the components of the body's immune system, participates in the nonspecific humoral immunity system, regulates the functions of immunocompetent cells, and is an acute-phase protein of inflammation (Senkovich O, Cook WJ, Mirza S, Hollingshead SK, Protasevich II, Briles DE, Chattopadhyay D. Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein A provides insight into microbial defense mechanism. J Mol Biol. 2007 Jul 20;370(4):701-13). The antibacterial properties of lactoferrin are due to its ability to bind iron, which is necessary for bacterial growth. Lactoferrin binds lipopolysaccharides of bacterial walls and initiates peroxidation, leading to a change in membrane permeability and subsequent lysis of bacterial cells.At the same time, lactoferrin has a stimulating effect on phagocytosis and influences complement activity (Ling JM, Schryvers AB. Perspectives on interactions between lactoferrin and bacteria. Biochem Cell Biol. 2006 Jun;84(3):275-81).

[0074] The N-terminal fragment of PspA (amino acids 193–288) effectively protects bacterial cells from the bactericidal action of lactoferrin (Shaper M, Hollingshead SK, Benjamin WH Jr, Briles DE. PspA protects Streptococcus pneumoniae from killing by apolactoferrin, and antibody to PspA enhances killing of pneumococci by apolactoferrin. Infect Immun. 2004 Sep;72(9):5031–40). The negatively charged surface of PspA specifically interacts with a highly cationic motif in lactoferrin. Binding of PspA blocks the surface activity of lactoferrin, preventing its penetration into the bacterial membrane.

[0075] In strains of serotypes 3 and 19F, PspA also functions as an adhesion protein, binding to glyceraldehyde-3-phosphate dehydrogenase on the surface of dying host cells. It has also been shown to bind lactate dehydrogenase, enhancing bacterial virulence by altering metabolic activity.

[0076] The present invention uses a fragment of PspA corresponding to positions 164-288 (according to PDB III 2PMS).

[0077] OtrA

[0078] OmpA is a transmembrane protein of the outer membrane of K. pneumoniae. It has a traditional P-barrel structure with four long loops located outside the cell (Renault M, Saurel O, Czaplicki J, Demange P, Gervais V, Lohr F, Rat V, Piotto M, Milon A. Solution state NMR structure and dynamics of KpOmpA, a 210-residue transmembrane domain possessing a high potential for immunological applications. J Mol Biol. 2009 Jan 9;385(1): 117–30). This protein has a number of functions that provide virulent and pathogenic properties of the bacterium and is expressed at very high levels: the number of its molecules in a cell can reach one hundred thousand.

[0079] Through its outer loops 1 and 2, OmpA binds to cerebral vascular endothelial cells and epithelial cells, thus acting as an adhesin. The same outer loops of OmpA can bind to the complement system protein C4bp, which activates factor 1, which cleaves the C3b and C4b proteins, completely deactivating the complement cascade. Bacteria secrete antiapoptotic factors and suppress the expression of proinflammatory cytokines, including interleukin 8 (Smith SG, Mahon V, Lambert MA, Fagan RP. A molecular Swiss army knife: OmpA structure, function, and expression. FEMS Microbiol Lett. 2007 Aug; 273(l): ll l). When infected with a strain mutated in its gene, the body develops a strong inflammatory response with higher levels of proinflammatory mediators and also eliminates such a pathogen more quickly (March C, Moranta D, Regueiro V, Llobet E, Tomas A, Garmendia J, Bengoechea JA.Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells. J Biol Chem. 2011 Mar 25;286(12):9956-67).

[0080] Incubation with purified OmpA in vitro activates Langerhans cells, antigen-presenting cells of the epidermis and mucosa, by upregulating MHC class II, CD86, and CCR7 expression and by enhancing cell migration across an artificial basement membrane in response to MIP-3P (simulating passage through the basement membrane en route to the lymph nodes). The Langerhans cell response is part of the host response to bacterial invasion (Godefroy S, Corvaia N, Schmitt D, Aubry JP, Bonnefoy JY, Jeannin P, Staquet MJ. Outer membrane protein A (OmpA) activates human epidermal Langerhans cells. Eur J Cell Biol. 2003 Apr; 82(4): 193–200).

[0081] In the present invention, fragments of OmpA corresponding to positions 27-52, 73-92, 120-139, 164-184 (according to PDB III 2K0L) are used.

[0082] RE

[0083] Membrane protein E (PE) is a surface protein of H. influenzae. It consists of six P-sheets, eight loops, and one C-terminal α-helix. In the monomer, antiparallel P-sheets form a P-sheet. Among 186 analyzed strains, including clinical NTHi (non-typeable H. influenzae strains), encapsulated H. influenzae, and strains from collections, amino acid sequence similarity was 96.9%–100%. Protein E-deficient mutants attach significantly less strongly to epithelial cells than wild-type strains, whereas recombinant PE and E. coli strains expressing it successfully attach to cells (Singh B, Brant M, Kilian M, Hallstrom B, Riesbeck K. Protein E of Haemophilus influenzae is a ubiquitous highly conserved adhesin. J Infect Dis. 2010 Feb l;201(3):414-9).

[0084] Amino acids 84–108 of the PE protein constitute the central epithelial cell-binding domain. Immunization with even this PE fragment accelerates NTHi clearance in the lungs. This amino acid region is responsible for binding vitronectin of the extracellular matrix, while amino acids 41–68 can bind laminin of the basement membrane, which is exposed upon damage to the epithelial layer. This same region of PE binds plasminogen, which, nevertheless, can be converted into functionally active plasmin even in the bound state (Barthel D, Singh B, Riesbeck K, Zipfel PF. Haemophilus influenzae uses the surface protein E to acquire human plasminogen and to evade innate immunity. J Immunol. 2012 Jan 1;188(1):379–85).

[0085] Vitronectin is used by bacterial cells as a messenger molecule for cell entry (Duell BL, Su YC, Riesbeck K. Host-pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen. FEBS Lett. 2016 Nov;590(21):3840-3853. doi: 10.1002 / 1873-3468.12351). The ability to bind vitronectin protects bacteria from attacking factors of the immune system. This is associated with the ability of vitronectin to inhibit the formation of the membrane attack complex C5b-C9 of complement (Singh B, Al-Jubair T, Morgelin M, Thunnissen MM, Riesbeck K. The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors. Infect Immun. 2013 Mar; 81 (3): 801-14). Mutants lacking the PE protein are more sensitive to the action of immune components of blood serum. The conservation of this region of PE is extremely high. The similarity of regions from amino acids 84 to 108 was complete among the 186 strains studied.In 98.4% of NTHi strains, the protein was expressed regardless of the growth phase.

[0086] Vitronectin and plasminogen bind to PE competitively. These two associated plasma proteins allow bacteria to control the complement system at the level of C3b degradation via plasminogen and the terminal stages of the complement cascade via vitronectin.

[0087] The present invention utilizes the PE fragment corresponding to positions 24-160 (according to PDB III 6GUS). Adjuvant

[0088] Adjuvants are an important component of vaccines, acting as immunostimulants and antigen delivery systems. Immunostimulants act as danger signaling molecules by binding to pattern-recognition receptors, including Toll-like receptors (TLRs), leading to the maturation and activation of antigen-presenting cells (APCs), stimulating the production of antigen and costimulatory signals, which, in turn, enhance adaptive immune responses. On the other hand, adjuvants can also be antigen carrier materials that facilitate antigen presentation by prolonging its bioavailability, as well as by delivering antigens to lymph nodes or APCs (Zhao T, Cai Y, Jiang Y, He X, Wei Y, Yu Y, Tian X. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther. 2023 Jul 19;8(1):283).

[0089] TLR1, TLR2, TLR4, TLR5, and TLR6 receptors are located on the surface of cell membranes. Their main functions are to recognize components of microbial membranes, namely proteins, lipids, and lipoproteins. Activation of these TLRs on the surface of antigen-producing cells (APCs) by the corresponding ligands leads to the induction of proinflammatory cytokines IL-1p, TNF-a, and IL-6 and the formation of an immune response along the Th1 or Th2 pathway.

[0090] A TLR5 agonist is the protein flagellin, whose monomers are non-covalently linked to form a helical flagellar filament used by bacteria for locomotion. TLR5 recognizes monomeric forms of flagellin from organisms of the β- and γ-proteobacteria classes. Binding induces MyB88-dependent signaling and activates the proinflammatory transcription factor NF-kB in epithelial cells, monocytes, and dendritic cells, leading to the production of TNF-a and activation of the immune response against flagellated bacteria (Yoon SI, Kurnasov O, Natarajan V, Hong M, Gudkov AV, Osterman AL, Wilson IA. Structural basis of TLR5-flagellin recognition and signaling. Science. 2012 Feb 17;335(6070):859–64). In addition, flagellin can be recognized by the receptors NLRC4 and NAIP5, activating the formation of the inflammasome (Cui B, Liu X, Fang Y, Zhou P, Zhang Y, Wang Y. Flagellin as a vaccine adjuvant. Expert Rev Vaccines. 2018 Apr;17(4):335-349).

[0091] Functionally, S. enterica flagellin FliC is divided into several fragments. Its highly conserved N- and C-terminal fragments, also designated D0 and D1, are required for flagellum export and self-assembly, while the hypervariable central region (domains D2 and D3) is highly variable in size and amino acid sequence and is deletion-tolerant (Malapaka RR, Adebayo LO, Tripp BC. A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility. J Mol Biol. 2007 Jan 26;365(4): 1102–16). Deletion of the hypervariable fragment reduces flagellin’s intrinsic immunogenicity but has no effect on its TLR5-dependent immunostimulatory activity (Biedma et al., 2019). The critical sites for flagellin binding to TLR5 are fragments 79-117 and 408-439.

[0092] The widespread expression of TLR5 on epithelial cells makes flagellin a good adjuvant for mucosal surfaces (Biedma ME, Cayet D, Tabareau J, Rossi AH, Ivicak-Kocjan K, Moreno G, Errea A, Soulard D, Parisi G, Jerala R, Berguer P, Rumbo M, Sirard JC. Recombinant flagellins with deletions in domains DI, D2, and D3: Characterization as novel immunoadjuvants. Vaccine. 2019 Jan 21;37(4):652–663). Nasal administration of flagellin enhances T-cell responses, as well as systemic and secretory antibody responses to co-administered antigens. Importantly, the presence of antibodies to flagellin itself did not affect its efficacy as an adjuvant (Van Maele L, Fougeron D, Janot L, Didierlaurent A, Cayet D, Tabareau J, Rumbo M, Corvo-Chamaillard S, Boulenouar S, Jeffs S, Vande Walle L, Lamkanfi M, Lemoine Y, Erard F, Hot D, Hussell T, Ryffel B, Benecke AG, Sirard JC. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal Immunol. 2014 May;7(3):489-500).TLR5 was found to be present on the surface of bipotent macrophage and osteoclast precursors, which circulate in small numbers in the blood. Upon intranasal administration of flagellin, these precursors are recruited to the lungs and rapidly differentiate into macrophages. Recruitment likely occurs through the chemokine CCL2, secreted by epithelial cells, for which CCR2 receptors are also present on the precursors. This pathway of macrophage precursors may act alongside monocyte recruitment and enhance the body's defense against bacterial infection in the mucosa (Lei X, Palomero J, de Rink I, de Wit T, van Baalen M, Xiao Y, Borst J. Flagellin / TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages. Front Immunol. 2021 Mar 19; 12:621665).

[0093] In the present invention, FliC fragments corresponding to positions 1-176, 210-327 (according to GenBank ID ABJ98818.1) are used.

[0094] Linkers

[0095] Protein fragments are separated by rigid and flexible amino acid linkers. Fragments belonging to the same protein (see FliC, OmpA), i.e., subfragments, are connected by flexible linkers (non-limiting examples are given below), while the remaining linkers are connected by rigid linkers. The inventors discovered that the combination of flexible and rigid linkers within the structure of the chimeric protein with the proposed fragments simultaneously enables a potentially native-like folding and structural separation for fragment recognition by the immune system.

[0096] Flexible linkers are defined by specialists as sequences that provide some level of mobility or interaction for the fragments being joined. For this reason, they are primarily composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The inclusion of polar amino acids can maintain linker stability in aqueous solutions by forming hydrogen bonds with water molecules.

[0097] By rigid linkers are meant those that provide distance between fragments to maintain their independent functioning. The rigid structure of such linkers is due to the presence of a helical conformation, for example, an α-helix, or the content of several proline residues. As a rule, rigid linkers are those that are characterized by a helicity (e.g., α-helicity) of at least about 80% (Li, Gang et al. (2016). Construction of a linker library with widely controllable flexibility for fusion protein design. Applied microbiology and biotechnology. 100). In one embodiment, a helicity below 80% allows the linker to be used as a flexible one, however, it is preferable to use linkers with a helicity below about 60, 50, 40, 30, 20, 15, 10, 5%, and most preferably without helicity (i.e., about 0%).Modeling of the linker structure and its classification as rigid or flexible can be carried out by specialists, for example, using the Discovery Studio 2.5 program (Accelrys, USA).

[0098] Non-exhaustive examples of flexible linkers include: (GGGGS) n , where n = 1-9, (GGGGS)nAS, where n = 1-9, AGGGS(GGGGS) n , where n = 1-8, AGGGS(GGGGS) n AS, where n = 1-8, G n , where n = 5-20 (preferably 6 or 8), KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, GGSSG, (GS) n , where n = 3-8,

[0099] GGGGSLVPRGSGGGGS, GGSGGHMGSGG, GG(SGG) n , where n = 1-3, GGSGGGGG, (GGGSE)3GGG, AAGAATAA, GSGGGTGGGSG, (GS)4GGSG, GSGGGTGGGSG, GS(GGS)4, GSGGSGSGGSGGSG.

[0100] Most preferably, the flexible linkers are selected from (GGGGS) n , where n is preferably 1-5, even more preferably 2, 3, 4 or 5.

[0101] Non-exhaustive examples of hard linkers include: (EAAAK)П , where n = 1-5; [A(EAAAK) n A]k, where k = 1 or 2, n = 1-5; A(EAAAK)4ALEA(EAAAK)4A; R к (HR) п , where k = 0 or 1, n = 2-34, X is any amino acid, preferably Ala, Lys or Glu, in particular, but not limited to, Pk(XP) n can be selected from P(AP)2-34, (AP)r-34, P(LP)2-34, (LP)r-34, P(EP)r-34, (EP)r-34, in particular PAPAP.

[0102] Most preferably, the rigid linkers are selected from (EAAAK) П , where n is preferably 1-5, even more preferably 2, 3, 4 or 5.

[0103] The above index n may take any integer value within the range specified for a particular linker, for example if n = 1-9 then any of 1, 2, 3, 4, 5, 6, 7, 8, 9 is implied.

[0104] It should be noted that flexible units may be present in rigid linkers, provided that the overall rigidity of the structure is maintained, for example, at least 80% of the helicity of the linker is preserved. In particular, the (GGGGS) unit may be present in combination with four (EAAAK) units, for example (EAAAK)4GGGGS, (EAAAK)3GGGGSEAAAK, (EAAAK)2GGGGS(EAAAK)2, EAAAKGGGGS(EAAAK)3. Similarly, rigid units may be included in flexible linkers if the overall flexibility of the structure is ensured. Using the above units as an example, the following flexible linkers are permissible, without limitation: for example (GGGGS)4EAAAK,

[0105] (GGGGS)3EAAAKGGGGS, (GGGGS)2EAAAK(GGGGS)2, GGGGSEAAAK(GGGGS)3.

[0106] The total length of the linker, flexible or rigid, between any of the fragments (or subfragments), may preferably be from 5 to 50 amino acid residues, such as from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6. Most preferably, the length of the linker is 10-15 amino acid residues.

[0107] Effects of spontaneous schizophrenia (Chen X, Zaro JL, Shen WC. Fusion). [ PMC free article ] [ PubMed ] [ Cross Ref ] Arai R, Ueda H, Nagamune T. Design of the linkers which effectively separate domains of a bifunctional protein. [ PubMed ] Jiang S, Galimidi RP, Keeffe JR, Bjorkman PJ, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014; 2013 Feb;22(2):153-67, Linkers in Biomacromolecules Ed. Расположение фрагментов

[0108] The arrangement of the fragments FliC, PspA, OmpA and PE in the hybrid protein is not critical; in particular, the order of the fragments of the said protein fragments from N- to C-terminus can be selected from the following:

[0109] FliC-PspA-OmpA-PE, FliC-OmpA-PE-PspA, FliC-PE-PspA-OmpA, FliC-OmpA-PspA-PE, FliC-PspA-PE-OmpA, FliC-PE-OmpA-PspA, PspA-OmpA-PE-FliC, OmpA-PE-PspA-FliC, PE- PspA-OmpA-FliC, OmpA-PspA-PE-FliC, PspA-PE-OmpA-FliC, PE-OmpA-PspA-FliC, PspA- FliC-OmpA-PE, OmpA-FliC-PE-PspA, PE-FliC-PspA-OmpA, OmpA-FliC-PspA-PE, PspA- FliC-PE-OmpA, PE-FliC-OmpA-PspA, PspA-OmpA-FliC-PE, OmpA-PE-FliC-PspA, PE-PspA-FliC-OmpA, OmpA-PspA-FliC-PE, PspA-PE-FliC-OmpA, PE-OmpA-FliC-PspA.

[0110] Of course, fragments belonging to one protein ("subfragments") are grouped together, i.e. FliC subfragments and OmpA subfragments are grouped together, respectively. In particular, it is advisable to arrange the subfragments of the FliC protein in order from the N- to the C-terminus as follows: SEQ ID NO: 2, SEQ ID NO: 3. The arrangement of the OmpA subfragments is not critical and can be selected from the following combinations, from the N- to the C-terminus (SEQ ID NO): 5-6-7-8, 5-6-8-7, 5-7-8-6, 5-7-6-8, 5-8-6-7, 5-8-7-6,

[0111] 6-5-7-8, 6-5-8-7, 7-5-8-6, 7-5-6-8, 8-5-6-7, 8-5-7-6, 6-7-5-8, 6-8-5-7, 7-8-5-6, 7-6-5-8, 8-6-5-7, 8-

[0112] 7-5-6, 6-7-8-5, 6-8-7-5, 7-8-6-5, 7-6-8-5, 8-6-7-5, 8-7-6-5; preferably arranged in the order 5-6-7-8.

[0113] Preferably, the N-terminus of the protein of the invention comprises a methionine amino acid residue, either directly adjacent to the first amino acid residue of the first fragment, or adjacent to it via one or more amino acid residues. In a preferred embodiment of the protein according to SEQ ID NO: 1, the methionine residue is directly adjacent to the FliC fragment.

[0114] Protein execution options

[0115] The protein of the invention may be an isolated protein, i.e., a single molecule, or be part of another molecule, such as part of a protein or conjugate.

[0116] In one embodiment of the invention, a protein may be conjugated to another molecule, for example, to increase solubility, to prevent or reduce proteolysis, to enable detection, etc. The groups, molecules, fragments mentioned herein may be attached to the C- or N-terminus of the protein of the invention or to an amino acid residue within the sequence of the protein of the invention. Molecules that can be conjugated to the protein of the invention include, but are not limited to, biotin, polyethylene glycol, nucleic acids, polysaccharides, and proteins of other organisms. Conjugation may be direct or indirect (e.g., via a linker). The use of biotin, which binds tightly to avidin and streptavidin, may be useful for the isolation and / or purification of the protein; biotin may be attached to the N-terminus or the side chain of a lysine or glutamic acid residue, as is known in the art.

[0117] A protein, either at its N- or C-terminus or within its sequence, may contain additional functional sequences, such as peptide tags, which are designed to alter solubility (e.g., increase it), purify or immobilize the protein (particularly affinity tags), or signal peptides, such as those used to transport the peptide to a specific location within or outside the producer cell, or for protein visualization / tracking. Peptide tags are typically short sequences, but in some cases they can represent entire proteins, such as enzymes. Without limitation, the protein may contain the following tags: His-tag (6-8 histidine residues), Myc tag (EQKLISEEDL), V5 tag (GKPIPNPLLGLDST), GST (glutathione-8-transferase), Arg tag (5-6 arginine residues), FLAG-tag (DYKDDDDK or DYKDHD-G-DYKDHD-I-DYKDDDDK), HA (YPYDVPDYA), streptavidin-binding peptide, Strep-tag, Strep-tag II (WSHPQFEK), Twin-Strep Tag, etc.Additional information regarding peptide tags and the nuances of their use in hybrid proteins is well known to specialists (Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26: 54–61; Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, NJ) 1129: 147–168; Xi Han, Wenbo Ning, Xiaoqiang Ma, Xiaonan Wang, Kang Zhou. Improving protein solubility and activity by introducing small peptide tags designed with machine learning models. Metabolic Engineering Communications, Volume 11, 2020).

[0118] After purification, peptide tags are removed using known methods, such as proteases. Self-cleaving tags can be added to the protein sequence; in particular, when combined with a suitable affinity tag, they enable purification, cleavage, and separation of the desired protein in a single step.

[0119] In addition, it is possible to construct the protein according to the invention with the addition of fragments that improve expression, solubility and secretion or localization in the periplasmic space of the cell, for example, those obtained from ecotin, maltose-binding protein, Z-domain of protein A of Staphylococcus aureus, albumin-binding domain of protein G of streptococci, cellulose-binding domain of proteins of Cellulomonas fimi, disulfide bond oxidoreductase DsbA, barnase - an inactive version of extracellular RNase of Bacillus amyloliquefaciens - and others.

[0120] In general, the protein of the invention may contain 1 or more additional amino acid residues at the N- or C-terminus, functional or non-functional.

[0121] The above-considered functionalization of the protein according to the invention will be determined primarily by the specific purposes for which the protein is planned to be used, the methods of production and purification, etc.

[0122] Preferably, the protein of the invention does not contain any amino acid residues other than the above-mentioned FliC, PspA, OmpA, PE fragments and linkers, optionally with an N-terminal methionine, at least after protein production. This embodiment is optimal for use as a vaccine. For diagnostic and research applications, a specialist may, if necessary, incorporate one or more of the above-mentioned or other known modifications into the protein.

[0123] Method of obtaining

[0124] The protein of the invention can be obtained by standard microbiological methods for the production of recombinant proteins known to those skilled in the art (Rosano GL, Ceccarelli EA. Recombinant protein expression in microbial systems. Front Microbiol. 2014 Jul 8; 5: 341; Wingfield PT. Overview of the purification of recombinant proteins. Curr Protoc Protein Sci. 2015 Apr 1; ​​80: 6.1.1-6.1.35; Saraswat, Mayank, Musante, Luca, Ravida, Alessandra, Shortt, Brian, Byrne, Barry, Holthofer, Harry, Preparative Purification of Recombinant Proteins: Current Status and Future Trends, BioMed Research International, 2013, 312709).

[0125] The protein has a mass of less than 100 kDa (in particular, the protein according to SEQ ID N0: 1 has a mass of about 78 kDa), which makes it easy to obtain in various expression systems, especially in bacterial ones.

[0126] In general, the method for producing a protein according to the invention may include the steps of culturing microorganisms (host cells) containing a polynucleotide or vector encoding said protein under conditions conducive to the production of said protein; isolating the protein, and, if necessary, purifying it.

[0127] In particular, the method may include the steps of:

[0128] - cultivation of the specified microorganism until a certain amount of protein is produced;

[0129] - separation of microorganism cells from the culture medium; - destruction of cells;

[0130] - separation of protein from cellular material;

[0131] - protein purification;

[0132] - obtaining a dosage form.

[0133] Cell disruption can be accomplished by grinding in a homogenizer (e.g., ultrasonic or milled), or by treatment with a lysing or chemical agent, or a combination of both. Protein separation, particularly in the form of inclusion bodies, is accomplished using an extraction solution. Protein refolding then occurs.

[0134] Purification is carried out using chromatography. Anion exchange and hydrophobic interaction chromatography are preferably used. Most preferably, anion exchange, hydrophobic interaction, and then anion exchange chromatography are used sequentially. Dialysis is performed if necessary.

[0135] For anion exchange chromatography, a sorbent containing quaternary ammonium (Q), diethylaminoethyl (DEAE), or diethylaminopropyl (ANX) functional groups can be selected, such as Q Sepharose, DEAE Sepharose, or ANX Sepharose. For hydrophobic chromatography, a sorbent containing hydrophobic groups such as phenyl, octyl, or butyl can be selected, such as Butyl Sepharose, Sepharose High Performance, Sepharose Fast Flow, or SOURCE 15.

[0136] The protein of the invention may be prepared as a concentrated or diluted (ready-to-use) aqueous solution containing the protein of the invention at the required concentration and, optionally, a buffer, such as a carbonate-bicarbonate buffer or PBS (phosphate buffer). The solution may contain other additional components deemed useful by those skilled in the art; for example, the solution may be a premix for use in an immunoassay.

[0137] Alternatively, the protein of the invention can be obtained as a lyophilized preparation. Lyophilization methods and the excipients used are well known to those skilled in the art (C. Challener, “For Lyophilization, Excipients Really Do Matte,” BioPharm International 30 (1) 2017).

[0138] Polynucleotide, vector, cell

[0139] The invention also includes a polynucleotide encoding the protein of the invention. The polynucleotide may be isolated (a nucleic acid molecule) or part of a larger nucleic acid molecule or vector. The polynucleotide may be used to introduce a sequence encoding the protein of the invention into a cell, clone it, transcribe it within the cell, and express (produce) the protein. The polynucleotide may be DNA or RNA, preferably DNA.

[0140] The nucleotide sequence of the nucleic acid corresponds, taking into account the genetic code, to the sequence of the protein according to the invention. Due to the degeneracy of the genetic code, multiple sequence variants of the polynucleotide encoding the same protein may exist, all of which are within the scope of the invention. The nucleotide sequence encoding the protein according to the invention can be optimized to ensure stable and high-level expression using methods known in the art.

[0141] In the present invention, a nucleic acid molecule also refers to a hybrid gene comprising, in functional linkage, at least one promoter, a coding sequence, and a terminator, wherein the promoter and terminator are functional in the selected host organism. The hybrid gene may additionally contain elements that regulate transcription, translation, and protein maturation, such as an additional promoter, a sequence encoding a signal or transit peptide, and transcriptional activators (enhancers).

[0142] The choice of regulatory elements depends on the host cell in which they are to function, and those skilled in the art are able to select the necessary regulatory elements without further experimentation (in particular, those described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press).

[0143] Promoters that may comprise the hybrid gene according to the invention are either constitutive or inducible. For example, a universally effective promoter used for expression in mammalian cells is the CMV (cytomegalovirus) promoter.

[0144] In a preferred embodiment, the polynucleotide of the invention comprises a start codon and restriction sites suitable for cloning into the selected vector.

[0145] In another embodiment, the invention relates to a vector comprising the above-mentioned nucleic acid and which thus encodes the protein of the invention. The purpose of vectors is known to those skilled in the art and the invention is not limited to a particular purpose. The vector may be a cloning vector, a transcription vector and / or an expression vector. Preferably, the vector is an expression vector, i.e. it contains at least a promoter and a ribosome binding site in addition to the said nucleic acid; accordingly, the vector is intended for the expression of the fusion protein of the invention. The vector may be a plasmid, a viral vector (retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, etc.), a YAC (yeast artificial chromosome), a BAC (bacterial artificial chromosome), a HAC (human artificial chromosome), bacteriophages and other types of vectors.The vector may be circular or linear, single-stranded or double-stranded. Preferably, the vector is a plasmid. Examples of ready-to-use plasmids are known to those skilled in the art (e.g., the database http: / / genome-www.stanford.edu / vectordb / vector_pages / plasmid_pages / Plasmid.html). Examples of plasmids that allow relatively easy introduction of expression cassettes are well known in the art and can be purchased (WO 1990002189, US20140065699, US20010016351).

[0146] The vector may be capable of integrating into the cell genome. Since a vector is essentially a nucleic acid, whether or not enclosed in an envelope (as in viruses), all aspects disclosed above with respect to the nucleic acid of the invention also apply to the vector, in particular with respect to the coding and regulatory elements. The vector may include genes for transformation markers indicating transformation of the host cell by the vector, and these are known to those skilled in the art (see, for example, WO1991002071, WO1995006128, WO1996038567 and WO1997004103).

[0147] Examples of vectors from which the vector of the invention can be obtained include, but are not limited to, pBR322, pET24a, pET22a, pUC19, pGEX-3X, AAV (adeno-associated virus), baculovirus, etc. Many vectors are commercially available (see, for example, https: / / www.genscript.com / expression-vector-selection-guide.html).

[0148] A cell, which may also be referred to as a host cell, comprises the vector or nucleic acid disclosed herein and is therefore capable of carrying the genetic information for the fusion protein of the invention and expressing it. The cell may be used for both production purposes—for obtaining (producing) the protein of the invention—and for maintaining the genetic information in a culture collection. In other words, the cell's intended use is not limited.

[0149] Here, cells are understood to mean cell strains or lines, or a combination of them.

[0150] Thus, the protein according to the invention can be produced in cells, produced in the required quantities and isolated from the cells and / or culture medium. The cell is not limited in terms of the type of organism from which it is obtained and may be a prokaryotic or eukaryotic cell, such as yeast such as S. cerevisiae, bacteria such as E. coli, such as Escherichia coli strain DH5a, genotype F- (p801acZAM15 A(lacZYA-argF)U169 recAl endAl hsdR17(rK- mK+) phoA supE44 X-thi-1 gyrA96 relAl, Escherichia coli strain BL21 (DE3), genotype F- ompT hsdSB (rB- mB-) gal dem (DE3), Rosetta™ strains such as Rosetta™(DE3), Rosetta™(DE3)pLacI, Rosetta™ 2, Rosetta™ 2(DE3), Rosetta™ 2(DE3)pLysS, Rosetta™ 2(DE3) Singles™, Rosetta™ 2(DE3)pLysS Singles™, Rosetta™ 2(DE3)pLacI; insect cells, animal cells, such as mammalian cells, in particular human or hamster (CHO).In some embodiments, the cell is suitable for producing a lysate for cell-free production of the protein of the invention.

[0151] Compositions, sets

[0152] A pharmaceutical composition refers to a mixture containing an active component—a protein according to the present invention—in an effective amount, along with other components, in particular one or more physiologically / pharmaceutically acceptable excipients. The pharmaceutical composition should have a composition and form suitable for the desired route of administration of the active component to the body and promote the manifestation of the biological effect.

[0153] A pharmaceutically acceptable excipient is a solid, soft or liquid filler, diluent, stabilizer, preservative, antioxidant, solvent, surfactant, emulsifier, buffer, colorant, acidity regulator or other excipient of any type.

[0154] Liquid dosage forms may, for example, be a solution or suspension of the protein of the invention in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like. In one embodiment, aerosols or sprays are used for mucosal administration, for example, for intranasal or intrapulmonary administration. In one embodiment, solutions or suspensions of the protein of the invention in a sterile aqueous solution, in aqueous propylene glycol, or in a pharmaceutically acceptable oil are used for parenteral administration. Aqueous solutions should have an appropriate buffer capacity, where appropriate, for example, using PBS buffer; if necessary, the liquid diluent can be made isotonic, for example, using an appropriate saline solution (e.g., sodium chloride) or glucose.Aqueous solutions are particularly suitable for intranasal, intratracheal, intrapulmonary, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. All sterile aqueous media used can be readily prepared by standard methods known to those skilled in the art. Practical methods for preparing such dosage forms are known or will be obvious to those skilled in the art, for example, from Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 16. th Edition, 1980.

[0155] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of solid carriers include lactose, magnesia, sucrose, cyclodextrin, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and lower alkyl ethers of cellulose. Examples of liquid carriers include syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water. Additionally, the carrier or diluent can include any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0156] The pharmaceutical compositions of the invention may be in the form of a lyophilisate suitable for the preparation of a solution by adding sterile water or another suitable solvent immediately before administration of the composition.

[0157] The composition may contain one or more additional adjuvants (in addition to FliC fragments), such as aluminum hydroxide, aluminum phosphate, calcium phosphate, etc.

[0158] The kit is a conveniently combined set of components that necessarily include the protein of the invention (for example, in the form of a composition), and, if necessary, one or more of the following components: additional substances necessary for creating a dosage form ready for administration, for example, water or another solvent, for example, a buffer solution; instructions for use, means for administration, for example, a syringe, dropper, pipette, dispenser, device for creating an aerosol or spray; a disinfectant, etc. The kit is packaged in suitable packaging, and the components are contained in appropriate containers, for example, vials, blisters, ready-to-use devices (syringes, sprayers, etc.).

[0159] Vaccination, dosage, route of administration

[0160] The protein may be administered parenterally or orally. In particular, the route of administration may be selected, without limitation, from intramuscular, subcutaneous, intranasal, intrapulmonary, intratracheal, buccal, vaginal, rectal, intravenous, and other routes. Accordingly, the vaccine may be administered, without limitation, by injection, drops, or aerosol (inhalation). There is no age limit for vaccination with the protein according to the invention. It is most preferable to vaccinate children under 2 years of age and elderly individuals aged 70 years and older. As a rule, children should be vaccinated as early as possible; for example, according to the national vaccination schedule, the first vaccine administration should be administered at 2 months of age, followed by subsequent doses at 4 and 6 months, followed by a booster dose at 12-15 months.The vaccination schedule may be adjusted by the physician depending on the circumstances, including previous illnesses, immune status, previous vaccinations, etc.

[0161] For adults, the frequency of administration may be, for example, 2 or 3 times. The interval between the first and second administrations may be 1-2 months, and between the second and third administrations, 2-6 months. Subsequently, a booster dose may be optionally administered 6-20 months later, for example, 10-15 months after the last vaccine administration.

[0162] The vaccination dose may, without limitation, be 10-50 μg protein / dose. In particular, the dose may be, in μg protein / dose: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0163] The purpose of vaccination is determined by the pathogens against which the vaccine is directed. This means that the purpose of vaccination with the protein of the invention is not limited to the prevention of pneumonia, but also any other diseases and conditions caused by or otherwise associated with infection with Streptococcus pneumoniae, Haemophilus influenzae and / or Klebsiella pneumoniae, including, but not limited to, otitis, meningitis, sinusitis, rhinitis, conjunctivitis, osteomyelitis, peritonitis, pericarditis, brain abscess, subcutaneous phlegmon, endocarditis, pleurisy, sepsis, arthritis, bronchial asthma, bronchitis (including chronic).

[0164] The method of vaccinating with the protein of the invention may include subsequent or prior vaccination with another vaccine, such as a polysaccharide, conjugate or peptide vaccine.

[0165] The vaccination method and composition of the invention may be for the prevention of an infection and the disease or condition it causes, or for therapy, such as for the treatment or alleviation of a chronic disease. The invention contemplates both eliciting an immune response against an infection and enhancing the immune response. The method of prevention or therapy comprises administering an effective amount of the protein or composition of the invention. Sequence listing

[0166] Table 1. Sequences

[0167] List of figures, drawings and other materials

[0168] Fig. 1 : Map of plasmid pET24a_PneumChim. Legend: op - plasmid replication origin; KanR - kanamycin resistance gene; flori - bacteriophage fl replication origin; bom - mobility base (for plasmid transfer during conjugation); lacl - lactose repressor; lacl promoter - lacl gene promoter; T7 promoter - promoter for bacteriophage T7 RNA polymerase; lac operator - lacl repressor binding site; PneumChim - target protein; 6*His - polyhistidine tag (out of frame); T7 terminator - transcription terminator for bacteriophage T7 RNA polymerase; Ndel, Xhol - restriction sites.

[0169] Fig. 2: Percentage of surviving mice vaccinated with the protein of the invention (“vaccine”) and control (“control”) after infection with S. pneumoniae.

[0170] Fig. 3: Percentage of surviving mice vaccinated with the protein of the invention (“vaccine”) and control (“control”) after infection with K. pneumoniae.

[0171] Fig. 4: Percentage of surviving mice vaccinated with the protein of the invention (“vaccine”) and control (“control”) after infection with H. influenzae.

[0172] Fig. 5: Percentage of mice surviving after infection with S. pneumoniae after being vaccinated with the protein of the invention (“vaccine”), the Prevenar 13 vaccine (“prevenar”) and the control (“control”). Information confirming the possibility of carrying out the invention Example 1. Obtaining the protein Plasmid

[0173] Plasmid DNA containing the target insert was constructed de novo at GenScript (USA). A plasmid containing the sequence encoding the PneumChim vaccine protein (plasmid pET24a_PneumChim) was constructed using pET24a. The insertion was carried out at sites recognized by the Ndel and XhoI restriction enzymes. The plasmid map is shown in Fig. 1. Plasmid characteristics are summarized in Table 2. The complete nucleotide sequence of the plasmid is presented as SEQ ID NO: 10.

[0174] Table 2. Characteristics of the pET24a_PneumChim plasmid

[0175] Cell transformation

[0176] Preparation of competent cells was carried out according to the standard procedure. Escherichia coli cells of strains DH5a or BL21 (DE3), stored at -70 °C, were picked up with an inoculated loop and transferred to a conical flask with 25 ml of LB-M medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride). The flasks were incubated at 37 °C and 180 rpm for 16 hours in an ES-20 / 60 shaker-incubator (BioSan, Latvia). Then 1 ml of the overnight culture was transferred to a flask with 50 ml of fresh LB-M medium and incubated at 37 °C and 180 rpm in a shaker-incubator until the optical density at a wavelength of 600 nm (OD600) reached 0.6-0.8 (about 2.5 h). The cells were cooled in an ice bath for 15 min and centrifuged for 5 min at 5000 g and 4°C. The supernatant was discarded, and the cells were carefully resuspended in 50 ml of cold sterile 10% (v / v) glycerol solution in an ice bath. The cells were centrifuged under the same conditions, washed again with glycerol solution, and then centrifuged again under the same conditions.The pellet was resuspended in 500 µl of sterile 10% glycerol solution. Twenty-five µl of the cell suspension was poured into 1.5 ml tubes and frozen at -70°C.

[0177] E. coli cells were transformed with plasmid DNA using electroporation (Lessard, 2013). Electrocompetent E. coli cells were thawed in an ice bath. 100 ng of plasmid DNA was added and mixed gently. The cells were transferred to a chilled electroporation cuvette with a 1 mm interplate spacing, capped, and inserted into a Gene Pulser Xcell electroporator (Bio-Rad, USA). Transformation was performed under the following conditions: 1.8 kV, 25 μF, 200 Ω. The cells were then immediately transferred to 1 ml of SOC medium, mixed, and incubated at 37 °C for 1 h in a Termit solid-state thermostat (DNA-Technology, Russia). Next, 50-200 µl of cells were transferred to Petri dishes with solid LB-M medium (10 g / l peptone, 5 g / l yeast extract, 10 g / l sodium chloride, 20 g / l agar) with the addition of kanamycin (50 µg / ml, LB-M-Km). The dishes were incubated at 37 °C for 16 h in a BINDER BD 115 thermostat (BINDER, Germany). The grown colonies were transferred to new dishes with LB-M medium with the appropriate antibiotic.

[0178] Plasmid DNA was isolated from successfully transformed E. coli DH5a cells. The colony was transferred to a flask containing 20 ml of LB-M-Km liquid medium and incubated at 37 °C and 180 rpm for 16 h in a shaker-incubator. Two ml of the overnight culture were transferred to a 2 ml tube and centrifuged at 5000 g for 5 min in an Eppendorf 5418R minicentrifuge (Eppendorf, Germany). The supernatant was discarded, and another 2 ml of the overnight culture was placed in the same tube and centrifuged under the same conditions. Plasmid DNA was isolated from the resulting pellet using the Plasmid Miniprep kit (Eurogen, Russia) according to the manufacturer's instructions. DNA was eluted in a volume of 50 µl. The presence of DNA was controlled using electrophoresis in 1% agarose gel stained with ethidium bromide.

[0179] Protein expression

[0180] Protein expression was tested in successfully transformed E. coli BL21 (DE3) cells according to the standard protocol (Kielkopf et al., 2021). The colony was transferred to a flask with 20 ml of liquid LB-M-Km medium and incubated at 37 °C and 180 rpm for 16 h in a shaking incubator. Next, 1 ml of the overnight culture was transferred to a flask with 50 ml of fresh LB-M medium with the appropriate antibiotic and incubated at 37 °C and 180 rpm in a shaking incubator until an OD600 of 0.6-0.8 was reached (approximately 2.5 h). A 1 ml sample of the culture was removed from the flask, transferred to a 1.5 ml tube, and centrifuged at 5000*g for 5 min in a minicentrifuge. The supernatant was removed, the pellet was frozen at -20°C, and then used as a negative control (without induction). A solution of 10, 50, or 100 µl of IPTG (Isopropyl-PDl-thiogalactopyranoside) (0.5 mol / l) was added to the flask. Thus, the final IPTG concentration in the culture was 0.1, 0.5, or 1 mmol / l. The culture was then incubated in a shaker-incubator under the same conditions.After 4 and 16 hours of induction (IPTG addition), 1 ml of culture was collected and cells were pelleted under the same conditions. The resulting pellets were resuspended in 100 μl of urea solution (8 mol / L urea, 50 mmol / L Tris-HCl, pH 8.0).

[0181] The presence of protein expression was monitored using polyacrylamide gel electrophoresis (PAAG) under denaturing conditions in a non-uniform (stepwise) buffer system (Laemmli, 1970).

[0182] Samples of cell suspension, inclusion body solution, or protein were mixed with sample loading buffer (0.25 mol / L Tris, 8% SDS, 40% glycerol, 20% β-mercaptoethanol, 0.4% bromophenol blue, 0.4 mol / L DTT) in a 3:1 ratio and then boiled for 5 min. The resulting samples were loaded into the gel in volumes ranging from 1 to 20 μL.

[0183] Tris-glycine buffer (25 mmol / L Tris, 192 mmol / L glycine, 0.1% SDS) was used for electrophoresis. The following separating gel composition was used: 1.5 mol / L Tris-HCl buffer + 0.4% SDS pH 8.8 - 3.75 ml; 10% ammonium persulfate - 50 μl; TEMED - 10 μl; 29.2% acrylamide + 0.8% bisacrylamide solution - until the desired acrylamide content in the solution was achieved; water - up to 15 ml. The following stacking gel composition was used: 0.5 mol / L Tris-HCl buffer + 0.4% SDS pH 6.8 - 1.25 ml; 29.2% acrylamide + 0.8% bisacrylamide solution - 0.65 ml; water - 3.05 ml, 10% ammonium persulfate - 25 μl; TEMED - 5 μl.

[0184] Electrophoresis was carried out for 60-80 min at a constant voltage of 150 V. The gels were washed with water for 10 minutes, then stained with Coomassie R-250 (0.1% Coomassie R-250, 10% acetic acid, 50% methanol) for 5-15 min and washed with 10% acetic acid until the background became bleached and clear protein bands appeared.

[0185] In E. coli BL21 (DE3) pET24a_PneumChim cells, IPTG induction resulted in PneumChim protein expression, with the induction being visually identical with the addition of different IPTG concentrations. After 16 hours of induction, protein expression remained at the same level.

[0186] Next, an overnight culture of the strain confirmed to have expression was prepared in 50 ml of LB-M-Km liquid medium and incubated at 37 °C and 180 rpm for 16 h in a shaking incubator. Next, 5 ml of the overnight culture was transferred to a flask with 250 ml of fresh LB-M medium with the appropriate antibiotic and incubated at 37 °C and 180 rpm in a shaking incubator until an OD600 of 0.6-0.8 was reached. 50 μl of IPTG solution (0.5 mol / L) were added to the flask. Thus, the final IPTG concentration in the culture was 0.1 mmol / L. The culture was then incubated in a shaker-incubator under the same conditions for 16 hours. Cells were pelleted by centrifugation at 5000*g for 5 min in an Eppendorf 581 OR centrifuge (Eppendorf, Germany). The supernatant was removed, and the cell pellet was frozen and stored at -20°C.

[0187] After biomass growth, protein expression was rechecked and inclusion bodies were washed. The soluble fraction of non-target proteins migrates into the supernatant, thus enriching the inclusion bodies for the target protein.

[0188] The biomass was resuspended in a buffer containing 50 mmol / L Tris-HCl + 5 mmol / L EDTA + 1 mmol / L PMSF, pH 8.0, at a rate of 20 ml of buffer per 1 g of biomass. The biomass was disrupted by ultrasound for 2 min using an UP400St ultrasonic homogenizer (Hielscher, Germany). The solution was centrifuged for 10 min at 20,000*g and 4 °C in an Eppendorf 5810R centrifuge. The pellet was dissolved in a buffer containing 50 mmol / L Tris-HCl + 5 mmol / L EDTA + 1 mmol / L PMSF + 0.5 mol / L sodium chloride + 2% Tween-20, pH 8.0, then sonication and centrifugation were repeated under the same conditions. The precipitate was dissolved in the same buffer and sonication and centrifugation were repeated under the same conditions.

[0189] The final precipitate was dissolved in a buffer containing 8 mol / L urea + 50 mmol / L Tris-HCl, pH 8.0, at a rate of 20 ml of solution per 1 g of inclusion bodies. Fractions were analyzed by electrophoresis under denaturing conditions and by HPLC.

[0190] Refolding and cleaning

[0191] The protein solution in urea was diluted 10-fold with 50 mmol / L Tris-HCl + 0.1% Triton X-100, pH 8.0, and stirred for 2 h on an MSH-300 magnetic stirrer (BioSan, Latvia). The solution was centrifuged for 10 min at 20,000 g and 4 °C. The supernatant was filtered through a capsule filter (polyethersulfone, pore diameter 0.45 μm; Technofilter, Russia).

[0192] For chromatographic purification, the AKTA Purifier 100 (GE Heathcare, USA) and NGC Quest™ 100 (BioRad, USA) systems with conductometric and single-wavelength optical detectors were used. Absorption detection was performed at a wavelength of 280 nm.

[0193] The following solutions were used for ion exchange chromatography:

[0194] - buffer A: 50 mmol / l Tris-HCl + 0.1% Triton X-100, pH 8.0;

[0195] - buffer B: similar to buffer A + 1 mol / l sodium chloride.

[0196] A HiTrap Q HP 5 ml chromatography column (Cytiva, Sweden) was washed with 1 CV (column volume) of buffer B and then equilibrated with 5 CV of buffer A. 1000 ml of the refolded protein solution was applied, and the column was then washed with 2 CV of buffer A. The protein was eluted with 15% buffer B, then with a gradient of 15-45% buffer B, 5 ml / min, 10 min. Fractions were analyzed by electrophoresis under denaturing conditions and by HPLC, the concentration was measured by the BCA method and spectrophotometrically.

[0197] Next, purification was performed using hydrophobic interaction chromatography. Dry sodium chloride was added to the eluted protein to a final concentration of 2.5 mol / L. The following solutions were used for hydrophobic interaction chromatography:

[0198] - buffer A: 2.5 mol / l sodium chloride + 50 mmol / l Tris-HCl, pH 8.0;

[0199] - buffer B: 50 mmol / l Tris-HCl, pH 7.6.

[0200] A HiPrep Butyl FF 16 / 10 chromatography column (CV 20 ml) (Cytiva, Sweden) was washed with 1 CV of buffer B and then equilibrated with 5 CV of buffer A. 20 ml of the protein solution was applied, and the column was then washed with 2 CV of buffer A. The protein was eluted with a gradient of 0-100% buffer B, 8 ml / min, 25 min. Fractions were analyzed by electrophoresis under denaturing conditions and by HPLC, the concentration was measured by the BCA method and spectrophotometrically.

[0201] Next, dialysis and final purification were performed using ion-exchange chromatography. One hundred milliliters of the solution was transferred to a dialysis bag and dialyzed against a 10 mmol / L sodium acetate solution, pH 6.3, at a ratio of 1:50 with constant stirring using a magnetic stirrer. After 8 hours, the dialysis buffer was replaced with fresh one.

[0202] The dialyzed protein was purified by ion exchange chromatography using the following solutions:

[0203] - buffer A: 10 mmol / l sodium acetate, pH 6.3;

[0204] - buffer B: similar to buffer A + 1 mol / l sodium chloride.

[0205] A HiPrep DEAE FF 16 / 10 chromatography column (CV 20 ml) (Cytiva, Sweden) was washed with 1 CV of buffer B and then equilibrated with 5 CV of buffer A. The dialyzed protein solution was applied, and the column was then washed with 2 CV of buffer A. The protein was eluted with a gradient of 0-50% buffer B, 5 ml / min, 10 min. Fractions were analyzed by electrophoresis under denaturing conditions and by HPLC; concentration was measured by the BCA method and spectrophotometrically.

[0206] During purification, the protein underwent three chromatographic purification steps using liquid chromatography based on different principles. In the first step, Q Sepharose, a strong anion-exchange sorbent, was used. The protein was eluted from the column at a conductivity of 22 mS / cm. In the second step, Butyl Sepharose, a hydrophobic sorbent, was used. The protein was eluted from the column at a conductivity of 70 mS / cm. In the final step, DEAE Sepharose, a weak anion-exchange sorbent, was used. The protein was eluted from the column at a conductivity of 21 mS / cm.

[0207] After three chromatography steps, purified recombinant PneumChim protein was obtained at a concentration of 1.3 mg / ml. Thus, from 10 g of E. coli BL21 (DE3) pET24a_PneumChim strain biomass, a total of 25 mg of PneumChim protein was obtained.

[0208] The protein retention time by HPLC analysis was 15.2±0.1 min. Protein purity, assessed by HPLC, was 88.85%. A qualitative LAL (Limulus amebocyte lysate pyrogenicity test) showed that the protein endotoxin content was less than 200 EU / mg (EU – endotoxin units). The protein has a half-life in E. coli of over 10 h, which is due to the presence of N-terminal methionine. Additional protein characteristics are presented in Table 3.

[0209] Table 3. Physicochemical properties of protein

[0210] * - If all cysteines form Cys-Cys pairs

[0211] ** - If all cysteines are in the reduced state Cys-H

[0212] To confirm the authenticity of the protein, two Western blots were performed using antibodies with different specificities.

[0213] Western blotting was performed according to the standard method (Mahmood, 2012). Protein samples (0.1–1 μg per lane) were loaded onto the gel and electrophoresed in PAAT under denaturing conditions. The protein was transferred to a polyvinylidene fluoride (PVDF) membrane (0.2 μm pore size) by electrotransfer. The membrane was soaked in transfer buffer (10% methanol, 12.5 mmol / L Tris, 96 mmol / L glycine, 0.05% sodium dodecyl sulfate) for 15 min, then dipped in methanol, and a transfer cassette consisting of filter paper, membrane, gel, and a second layer of filter paper was assembled. Transfer was performed in an electrophoresis chamber in transfer buffer with buffer cooling using a cold pack at 100 V for 90 min. After transfer, the membrane was incubated in loading buffer (3% sodium caseinate, 0.1% Tween-20, PBS) for 15 min with shaking on an MR-1 rocker shaker (BioSan, Latvia). The membrane was then incubated in fresh loading buffer with the addition of primary antibodies for 1 hour.The membrane was washed with blocking buffer three times for 5 minutes each. The membrane was then incubated in blocking buffer with labeled secondary antibodies for 1 hour. The membrane was washed with blocking buffer three times for 5 minutes each. Before detection, the membrane was rinsed with water, placed on a support, and a solution of 3,3',5,5'-tetramethylbenzidine (TMB) was added until a blue color appeared.

[0214] To detect PneumChim, which contains a flagellin fragment, primary antibodies were used: Rabbit polyclonal to Flagellin IgG (ab93713, Abeam, USA), 1:10000. Goat-anti-Rabbit IgG polyclonal, HRP-conjugated (12-348, Millipore, USA) at a dilution of 1:5000.

[0215] A band corresponding to the PneumChim protein is clearly visible on the membrane.

[0216] Protein concentration was determined colorimetrically using bicinchoninic acid using Pierce™ BCA Protein Assay Kits (Thermo, USA) according to the manufacturer's protocol. A fresh working solution was prepared by mixing solution A and solution B in a 50:1 ratio. 2 ml of the working solution were added to 100 µl of protein samples and standards, mixed, and incubated at 37°C for 30 min.

[0217] The optical density of the samples was then measured at a wavelength of 562 nm. A protein-free sample was used as a reference. Protein concentration was calculated using a calibration curve constructed using bovine serum albumin.

[0218] The bacterial endotoxin content in the solution was determined using a qualitative gel-clot test (Sitnikov, 2007). A control endotoxin standard with a concentration of 0.25 EU / ml was prepared by preparing a series of serial dilutions of a standard stock endotoxin solution (Pyrotell, USA) with a dilution step of no more than 1:10. All dilutions were performed with pyrogen-free sterile water in pyrogen-free tubes (Pyrotell, USA). Only a fresh dilution of the standard was used for testing.

[0219] The maximum permissible dilution of the protein sample was calculated using the formula:

[0220] СхХ МДР= — л where

[0221] MDR - maximum permissible dilution;

[0222] C - protein concentration, mg / ml; X - LAL reagent sensitivity, EU / ml

[0223] X – maximum value of endotoxin content, EU / mg.

[0224] The endotoxin content limits of 50 EU / mg, 100 EU / mg, 200 EU / mg, and 500 EU / mg were tested. The MDR was calculated and a convenient value close to the MDR was selected. A series of serial protein dilutions was prepared, each diluted by no more than 10 times, with the final dilution being 2-fold. All dilutions were performed using pyrogen-free sterile water in pyrogen-free tubes. Only freshly diluted samples were used for testing.

[0225] Test tubes were prepared. One hundred microliters of the test sample (the final dilution, two replicates) or pyrogen-free water (negative control) were added to the bottom of a pyrogen-free tube. To prepare the inhibition control, 50 microliters of the 0.25 EU / ml endotoxin control standard and 50 microliters of the test sample (the penultimate dilution) were added to the pyrogen-free tube. Next, 100 microliters of LAL reagent solution with a sensitivity of 0.125 EU / ml (Pyrotell, USA) were added to the samples. The tubes were capped and incubated for 60±3 min at 37±1°C in a solid-state thermostat.

[0226] The results were assessed by the presence or absence of a dense thrombus at the bottom of the test tube when the test tube was inverted.

[0227] If there was no thrombus in the negative control tube, a thrombus was present in the positive control tube, and there was no thrombus in both tubes with the protein sample, the endotoxin content in the protein was assumed to be less than the selected value of X EU / mg.

[0228] The resulting protein demonstrated stability, ensuring the shelf life of the finished vaccine for more than 2 years and suitability for inclusion in medicinal products.

[0229] Example 2. Application of protein

[0230] The study was performed on inbred BALB / c mice (females, 8 weeks old, weighing 18-20 g). All animals were kept in illuminated conditions with controlled temperature. Mice were provided with standard commercial chow and water ad libitum.

[0231] The animals were divided into six groups of 10 animals each. Three experimental groups were administered the PneumChim vaccine protein subcutaneously at a dose of 10 μg in 0.1 ml of PBS buffer. Three control groups were administered a placebo subcutaneously in the form of 0.1 ml of PBS buffer. Injections were performed twice, three weeks apart.

[0232] Seven weeks after the first immunization, mice were injected with pathogenic organisms intranasally in the amount of 10 7CFU. For each pathogen (S. pneumoniae, K. pneumoniae, I. influenzae), one vaccinated group and one placebo-treated group were infected. The statistical significance of differences was assessed using the log-rank test (also known as the Mantel-Cox test).

[0233] The results are shown in the table below (K - control, V - vaccine) and summarized in Fig. 2-Fig. 4.

[0234] Table 4

[0235] Vaccination provided a significant protective effect against each pathogen. All survival differences were statistically significant (p < 0.05).

[0236] The studied dose of protein did not have a toxic effect on mice.

[0237] An additional experiment compared the efficacy of the PneumChim protein and the Prevenar 13 vaccine (Pfizer Ireland Pharmaceuticals). The experimental conditions were identical to those in the previous example. The results are summarized in Fig. 5. The protective effect of the protein according to the invention was comparable to and even slightly superior to that of the Prevenar vaccine.

[0238] The above description of specific embodiments of the invention and examples of its implementation are intended merely to illustrate the invention and should not be construed as limiting the scope of the patent holder's rights, which are defined solely by the claims. Various modifications of the present invention, in addition to those described above, will be apparent to those skilled in the art, given their prior knowledge.

Claims

Invention formula 1. An immunogenic protein containing fragments of FliC, PspA, OmpA and PE proteins connected by rigid linkers, wherein: - the FliC protein fragment comprises, from the N- to the C-terminus, SEQ ID NO:2 and 3, connected to each other by a flexible linker; - the PspA protein fragment includes SEQ ID NO:4; - the fragment of the protein OshpA includes SEQ ID NO: 5, 6, 7 and 8, connected to each other by flexible linkers; - fragment PE includes SEQ ID NO:

9.

2. The protein according to I. 1, wherein the rigid linkers are independently selected from (EAAAK) П , where n = 1-5; [A(EAAAK) n A]k, where k = 1 or 2, n = 1-5; A(EAAAK)4ALEA(EAAAK)4A; R к (HR) п , where k = 0 or 1, n = 2-34, X is any amino acid; and flexible linkers are independently selected from (GGGGS)n, where n = 1-9, (GGGGS)nAS, where n = 1-9, AGGGS(GGGGS) n , where n = 1-8, AGGGS(GGGGS) n AS, where n = 1-8, G n, where n = 5-20, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, GGSSG, (GS) n , where n = 3-8, GGGGSLVPRGSGGGGS, GGSGGHMGSGG, GG(SGG) n , where n = 1-3, GGSGGGGG, (GGGSE)3GGG, AAGAATAA, GSGGGTGGGSG, (GS)4GGSG, GSGGGTGGGSG, GS(GGS)4, GSGGSGSGGSGGSG.

3. Protein according to I. 1, containing the sequence SEQ ID NO:

1.

4. A host cell for producing a protein according to any of paragraphs 1-3, which contains a polynucleotide or vector encoding said protein.

5. A method for producing a protein according to any of paragraphs 1-3, comprising culturing a host cell according to paragraph 4 under conditions conducive to the production of said protein, and isolating the protein.

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