A vaccine for the prevention of Klebsiella pneumoniae infection and its application

Abstract

This invention relates to a fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention of Klebsiella pneumoniae infection. The invention provides vaccines expressing the PstS antigen alone, as well as vaccines expressing a fusion protein of YidR-PstS or PstS-YidR, further enhanced with Fc domains, STABILON, and other elements. The invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and uses. The vaccine of this invention can attenuate tissue lesions caused by Klebsiella pneumoniae infection, possesses good immunogenicity, and provides effective prevention and immune protection, demonstrating high efficiency in preventing Klebsiella pneumoniae infection and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to a vaccine for preventing Klebsiella pneumoniae infection and its application. Background Technology

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae (KP) is a Gram-negative bacillus widely distributed in nature and also present in the respiratory and intestinal tracts of healthy humans and animals. It can cause infection when the body's immunity is weakened or when prolonged use of antibiotics leads to dysbiosis. Types of infection mainly include pneumonia, meningitis, liver abscess, endophthalmitis, urinary tract infections, wound infections, and sepsis. In recent years, Klebsiella pneumoniae has become the second most prevalent opportunistic pathogen after Escherichia coli, and is a significant potential pathogen causing both community-acquired and nosocomial infections.

[0003] Klebsiella pneumoniae can be classified into 82 K serotypes based on capsular polysaccharide (K antigen). Among them, K1, K2, and K5 serotypes are closely related to serious infectious diseases in humans and animals. K1 serotypes typically cause Friedlanderella pneumonia, particularly as a complication of purulent liver abscess. K2 and K5 serotypes typically cause community-acquired pneumonia. Rhinosclerosing disease is associated with serotype K3. Clinically isolated Klebsiella pneumoniae subspecies from atrophic rhinitis mostly originate from serotype K4 and very rarely from serotype K5. K54 and K57 serotypes are associated with invasive liver abscess syndrome. Compared to other serotypes, K1, K2, K54, and K57 are highly virulent strains and are commonly found in pneumonia cases.

[0004] Furthermore, Klebsiella pneumoniae can be serotyped based on lipopolysaccharide (LPS) antigens. Currently, nine LPS O antigen serotypes have been identified in Klebsiella pneumoniae, with O1 and O2 being the most common. The O1 antigen is typically associated with K1 and K2 capsule types, making it the most frequently detected O antigen type in highly virulent Klebsiella pneumoniae (hvKP) strains. Studies have found that the O2 serotype is prevalent in most drug-resistant Klebsiella pneumoniae strains, with the O1 serotype being more prevalent in highly invasive strains, and O1-type Klebsiella pneumoniae exhibiting stronger immune evasion capabilities.

[0005] There is an urgent need for the development of a vaccine against Klebsiella pneumoniae. An effective and safe vaccine is of great significance in reducing the incidence of infection caused by this bacterium, reducing medical costs for families and health systems, and alleviating the global burden of disease.

[0006] Due to the lack of commercially available vaccines, current clinical treatment for Klebsiella pneumoniae infections still primarily relies on antibiotics. However, antibiotic treatment has become a major driving force behind the emergence and spread of highly resistant and virulent strains. To overcome this predicament, researchers are actively developing new vaccines. For example, existing patent CN118063571A (publication date 2024-05-24) discloses a method for producing a high-yield Klebsiella pneumoniae O2 serotype nanopolysaccharide conjugate vaccine; patent CN111448205A (publication date 2019-06-06) discloses a synthetic sugar of general formula (I) related to serotypes O1, O2, O2ac, and O8 O-polysaccharides and carbapenem-resistant Klebsiella pneumoniae ST258 O-polysaccharides, and their conjugates, for the prevention and control of Klebsiella pneumoniae serotypes O1, O2a, O2ac, O2aeh, O2afg, and O8. These polysaccharide vaccines aim to develop vaccines that target specific serotypes of Klebsiella pneumoniae. However, there are already more than 80 K antigen serotypes and 9 O antigen serotypes for pathogenic Klebsiella pneumoniae, and more serotypes may emerge in the future. The development and iteration of polysaccharide vaccines are difficult, there are numerous limitations in the synthesis of polysaccharides from multiple serotypes, and the degree of cross-immunity protection is not high, making it difficult to meet the current market demand for Klebsiella pneumoniae vaccines.

[0007] Phosphate-binding protein PstS is part of the phosphate-specific transport (Pst) system, located in the bacterial periplasmic space, and has a high affinity for phosphate. Previous studies have demonstrated that PstS can serve as a biosensor for phosphate contamination. For example, patent CN114965403A (publication date 2022-08-30) discloses a method for constructing a phosphate biosensor using the PstS protein from *Pseudomonas aeruginosa*. Furthermore, in some pathogenic bacteria, PstS expression is associated with the production of virulence factors and participates in biofilm formation and host cell adhesion; however, no studies have reported that PstS can be used as a protective antigen for *Klebsiella pneumoniae* in vaccine development.

[0008] In conclusion, developing a safe, effective, and easily manufactured vaccine that provides broad-spectrum protection against Klebsiella pneumoniae is of significant scientific and practical value. Such a vaccine could not only prevent various human infections but also potentially be used for animal disease prevention, aiming to reduce the incidence of Klebsiella pneumoniae infections and bringing significant benefits to public health and the economy. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention proposes a novel fusion protein design scheme, aiming to develop a highly efficient and broad-spectrum vaccine for the prevention of Klebsiella pneumoniae infection, capable of simultaneously combating multiple serotypes of Klebsiella pneumoniae. The purpose of this invention is to provide a novel fusion protein for the prevention of Klebsiella pneumoniae infection, an immunogenic composition, a recombinant vaccine, and its molecular architecture design and applications. Starting from the highly conserved target sites PstS and YidR of Klebsiella pneumoniae, this invention designs a fusion protein by combining immunoepitaphtoids. Through creative screening, the stable structural domains of PstS and YidR proteins were ultimately selected to construct the fusion protein. Further optimization was then performed on the connection sequence of PstS and YidR, and the addition of Fc domains, STABILON elements, etc. This invention reveals that the novel fusion protein molecule can attenuate tissue lesions caused by Klebsiella pneumoniae infection, exhibits good immunogenicity, and provides effective prevention and immune protection, efficiently preventing Klebsiella pneumoniae infection. This invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and applications.

[0010] One aspect of the present invention provides an antigenic protein for preventing Klebsiella pneumoniae infection, characterized in that it comprises a PstS antigenic protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] Another aspect of the present invention provides an antigen protein for preventing Klebsiella pneumoniae infection, characterized in that it comprises a YidR antigen protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] Another aspect of the present invention provides a fusion protein, characterized in that it comprises any one of the following three proteins:

[0013] (1) Fusion protein A: contains PstS antigen protein or fragments thereof;

[0014] (2) Fusion protein B: From the N-terminus to the C-terminus, it contains: YidR antigen protein or fragment thereof, PstS antigen protein or fragment thereof;

[0015] (3) Fusion protein C: From the N-terminus to the C-terminus, it contains: PstS antigen protein or fragment thereof, YidR antigen protein or fragment thereof;

[0016] Furthermore, the amino acid sequence of the PstS antigen protein is shown in SEQ ID NO.1, and the amino acid sequence of the YidR antigen protein is shown in SEQ ID NO.2.

[0017] Furthermore, the PstS and / or YidR antigen proteins or fragments thereof are derived from bacteria.

[0018] Furthermore, the bacteria are Gram-negative.

[0019] Furthermore, the Gram-negative bacteria are Enterobacteriaceae.

[0020] Furthermore, the Enterobacteriaceae bacteria mentioned are Klebsiella pneumoniae.

[0021] Furthermore, the N-terminus of the fusion protein also includes a signal peptide and / or the Fc domain of human immunoglobulin heavy chain constant region γ1 protein (IGHG1).

[0022] Furthermore, the C-terminus of the fusion protein also contains the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

[0023] Furthermore, the signal peptide is derived from human azurocidin protein, the amino acid sequence of which is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of IGHG1 is shown in SEQ ID NO: 4, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0024] Furthermore, the different elements of the fusion protein may optionally be linked by a GGS linker sequence, a spacer sequence, and / or a GS linker sequence.

[0025] Further, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 8.

[0026] Furthermore, the fusion protein comprises any one of the following three proteins:

[0027] (1) Fusion protein A: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen protein or fragment thereof, GS linker, STABILON;

[0028] (2) Fusion protein B: From the N-terminus to the C-terminus, it contains the following components in sequence: signal peptide, Fc domain, GGS linker, YidR antigen protein or fragment thereof, spacer sequence, PstS antigen protein or fragment thereof, GS linker, and STABILON.

[0029] (3) Fusion protein C: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen protein or fragment thereof, spacer sequence, YidR antigen protein or fragment thereof, GS linker, STABILON.

[0030] Further, the amino acid sequence of the PstS antigen protein is shown in SEQ ID NO.1, the amino acid sequence of the YidR antigen protein is shown in SEQ ID NO.2, the amino acid sequence of the signal peptide is shown in SEQ ID NO:3, the amino acid sequence of the Fc domain is shown in SEQ ID NO:4; the amino acid sequence of the STABILON is shown in SEQ ID NO.5, the amino acid sequence of the spacer sequence is shown in SEQ ID NO:6, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO:7, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO:8.

[0031] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises nucleic acid encoding an antigen protein or a fusion protein as described in any one of the present invention.

[0032] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0033] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0034] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0035] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0036] Furthermore, the prokaryotic vector comprises an Escherichia coli vector.

[0037] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0038] Furthermore, the eukaryotic vector includes yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0039] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0040] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0041] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0042] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0043] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0044] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0045] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0046] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more antigen proteins according to any one of the present invention, and / or one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention.

[0047] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0048] Furthermore, the immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Klebsiella pneumoniae.

[0049] Furthermore, the other medications used to prevent diseases caused by Klebsiella pneumoniae include antibiotics.

[0050] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more antigen proteins according to any one of the present invention, and / or one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention.

[0051] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0052] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0053] Another aspect of the present invention provides the use of one or more antigen proteins described in any one of the present inventions, and / or one or more fusion proteins described in any one of the present inventions, and / or one or more recombinant nucleic acid molecules described in the present inventions, and / or one or more recombinant gene expression cassettes described in the present inventions, and / or one or more recombinant vectors described in the present inventions, and / or one or more recombinant host cells described in the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present inventions, and / or one or more recombinant vaccines described in the present inventions in the preparation of a medicament for the prevention of disease caused by Klebsiella pneumoniae.

[0054] Another aspect of the present invention provides a method for preventing disease caused by Klebsiella pneumoniae, characterized in that it includes administering to a subject one or more antigen proteins described in any one of the present inventions, and / or one or more fusion proteins described in any one of the present inventions, and / or one or more recombinant nucleic acid molecules described in the present inventions, and / or one or more recombinant gene expression cassettes described in the present inventions, and / or one or more recombinant vectors described in the present inventions, and / or one or more recombinant host cells described in the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present inventions, and / or one or more recombinant vaccines described in the present inventions.

[0055] Furthermore, the diseases caused by Klebsiella pneumoniae include pulmonary infections and extrapulmonary infections; preferably, the extrapulmonary infections include urinary tract infections, sepsis, meningitis, liver abscess, wound infections, surgical infections, skin infections, abdominal infections, respiratory tract infections, endophthalmitis, and cystitis.

[0056] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0057] 1. This invention discovers that novel fusion molecules possess excellent immunogenicity and can provide immune protection, making them suitable for the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of this invention can provide effective immune protection against Klebsiella pneumoniae infection.

[0058] 2. This invention can serve as a universal antigenic framework for nucleic acid vaccines or subunit vaccines, exhibiting strong immunogenicity. Compared to traditional vaccines, it is not limited by serotype and can induce broad-spectrum immune protection.

[0059] 3. This invention utilizes reverse vaccinology technology to screen candidate targets with broad-spectrum protective effects, preferentially selecting the PstS antigen for vaccine design. The PstS antigen is then truncated, and comparative experiments verify that this antigen, used alone or as part of a fusion protein, can induce immune protection against Klebsiella pneumoniae in mice. For the YidR antigen, specific amino acid mutations at specific sites in the known YidR sequence enhance its antigenicity and other properties.

[0060] 4. Example 5 of the present invention shows that the fusion molecular vaccines A, B, and C based on the present invention can effectively promote the high abundance expression and secretion of bacterial proteins in eukaryotic cells, and the expressed protein structures are correct and stable, which is conducive to the presentation of immune epitopes, thereby further improving the immune protection effect and providing a new technology for the design of immunotherapeutic drugs for Klebsiella pneumoniae multivalent vaccines.

[0061] 5. The mouse nasal droplet infection model experiment in this embodiment of the invention showed that, in lung tissue, the bacterial load in mice vaccinated with the vaccine of this invention was significantly lower than that in the PBS control group. Statistical analysis showed that this reduction was statistically significant (P<0.001). In particular, with vaccine C, the bacterial load in the lung tissue of mice immunized with vaccine C was reduced by approximately 180-fold compared to the PBS group. This result demonstrates that the vaccine based on this invention can effectively reduce Klebsiella pneumoniae colonization in the lungs and provide effective local immune protection.

[0062] 6. Further analysis using tissue sections and HE staining further confirmed the protective effect of the vaccine of this invention. Specifically, the alveolar structure of normal mice remained intact, with no abnormalities in the epithelial septa, and no obvious lesions or inflammatory infiltration. In the PBS group, the alveolar septa thickened, the alveolar morphology and structure were largely disrupted, and inflammatory cell infiltration was observed. Mice vaccinated with the vaccine of this invention showed even lower alveolar septal thickness and more intact alveolar structure. In particular, the vaccine C group showed better alveolar structural integrity and even lower alveolar septal thickness. This demonstrates that the vaccine based on this invention can significantly reduce tissue lesions caused by Klebsiella pneumoniae infection.

[0063] 7. Based on the combined results of bacterial load and histopathological examination, the vaccine of this invention exhibits a dual protective effect: on the one hand, it significantly reduces the number of bacteria in lung tissue, and on the other hand, it effectively alleviates tissue lesions caused by infection. This comprehensive protective effect lays a solid foundation for the potential clinical application of the vaccine of this invention. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of the molecular structure of vaccine A, vaccine B, and vaccine C used in the comparative experiment in this embodiment of the invention.

[0065] Figure 2This is a schematic diagram of a circular plasmid containing the target gene of the vaccine of this invention.

[0066] Figures 3A-3C This invention provides quality control peak diagrams and purity test results of the nucleic acid vaccines used in comparative experiments. Figure 3A This is a quality control peak diagram and purity test results for nucleic acid vaccine A. Figure 3B This is a quality control peak diagram and purity test results for nucleic acid vaccine B. Figure 3C This is a peak diagram of the quality control of nucleic acid vaccine C and the results of purity testing.

[0067] Figures 4A-4B This is a comparative experiment using HEK293 cells transfected with a nucleic acid vaccine to detect in vitro expression via Western blot (WB). Figure 4A The results of Western blot analysis of the in vitro expression of nucleic acid vaccine A transfected into HEK293 cells. Figure 4B The results of in vitro expression detection of nucleic acid vaccines B and C transfected into HEK293 cells were obtained by Western blot analysis.

[0068] Figure 5 This is a schematic diagram of the immunization, challenge, and sampling process for a mouse nasal drop infection experiment.

[0069] Figures 6A-6B The results show the bacterial load in the lung tissue of mice after immunization and challenge; among which... Figure 6A These are comparative photographs of the bacterial load (number of colonies on agar plates) in the lung tissues of mice in the vaccine-immunized group, PBS group, and negative control group after immunization and challenge. Figure 6B This is a comparison of the bacterial load (number of colonies on agar plates) in the lung tissues of mice in the vaccine-immunized group, PBS group, and negative control group after immunization and challenge.

[0070] Figure 7 The results of staining of pathological tissue sections of lung tissue from mice in each experimental group after immunization and challenge. Detailed Implementation

[0071] Terms and Definitions

[0072] The term "Klebsiella pneumoniae" refers to Klebsiella pneumoniae KP, also known as Friedlanderella pneumoniae, is a Gram-negative, rod-shaped, facultative anaerobic bacterium belonging to the Enterobacteriaceae family.

[0073] The term "Klebsiella pneumoniae infection" refers to various diseases caused by Klebsiella pneumoniae, including lung infections, urinary tract infections, sepsis, meningitis, liver abscess, wound infections, surgical infections, skin infections, abdominal infections, respiratory tract infections, endophthalmitis, cystitis, etc.

[0074] The term "PstS" refers to the phosphate-specific transport system substrate-binding protein (PstS), an important component of the phosphate transport system of Klebsiella pneumoniae. Preferably, the amino acid sequence of PstS is shown in SEQ ID NO: 1.

[0075] The term "YidR" refers to a conserved protein in Klebsiella pneumoniae containing the DUF3748 domain. Its function is not fully elucidated, but it may be involved in bacterial stress responses and biofilm formation. Preferably, the amino acid sequence of YidR is shown in SEQ ID NO: 2.

[0076] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immune responses can be measured by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0077] The terms "administration" or "vaccination" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous route, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injection), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes.

[0078] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0079] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be used to transform into a suitable host cell.

[0080] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide that is used to express, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences.

[0081] The term "mRNA" refers to messenger RNA, also known as "messenger RNA," which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0082] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) located upstream of the coding sequence.

[0083] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule.

[0084] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells.

[0085] The terms "individual," "patient," or "subject" include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0086] The terms "transformation," "transfection," and "transduction" have the meanings commonly understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0087] The term "drug combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, as well as to provide a method for its effective absorption in a subject.

[0088] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0089] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0090] This invention discloses a novel method for preparing a Klebsiella pneumoniae vaccine and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0091] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0092] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0093] Example 1: PstS antigen screening

[0094] Because Klebsiella pneumoniae antigens are serotype-dependent, this invention employs a reverse vaccinology strategy to screen for potential Klebsiella pneumoniae antigens. The main objective of the screening is to identify antigens that are highly expressed in the transcriptome and are highly conserved across different Klebsiella pneumoniae subtypes.

[0095] The specific screening steps are as follows:

[0096] (1) Collect transcriptome data of different serotypes of Klebsiella pneumoniae.

[0097] (2) Bioinformatics analysis of transcriptome data to identify genes with high abundance expression.

[0098] (3) The conservation of these highly expressed genes across different serotypes was analyzed using comparative genomics methods.

[0099] Through the aforementioned systematic screening, this invention ultimately identified several candidate antigens, including PstS and YidR. Subsequently, based on immunoepitaxy prediction analysis, PstS and YidR were evaluated and determined to be the optimal candidate antigens.

[0100] Example 2: Design and Construction of Recombinant Nucleic Acid Vaccine Containing PstS and YidR Antigens

[0101] The screening results in Example 1 allow for the design of three types of vaccines: single expression of PstS antigen, single expression of YidR antigen, and fusion expression of PstS and YidR. Since both PstS and YidR are outer membrane proteins of Klebsiella pneumoniae and require secretory expression, different combinations can affect protein conformation and secretory expression efficiency. Therefore, there are two possible combinations for PstS and YidR fusion expression: YidR-PstS and PstS-YidR.

[0102] This invention verifies the effectiveness of the PstS antigen and the fusion expression of the PstS and YidR antigens, and truncates the PstS antigen. Furthermore, to improve immunogenicity, specific amino acid mutations were performed on the known YidR sequence. Figure 1 As shown, three vaccines were designed: (1) vaccine A, which expresses the PstS antigen alone; (2) vaccine B, which expresses the YidR-PstS fusion protein; and (3) vaccine C, which expresses the PstS-YidR fusion protein. The three vaccines were compared with each other to verify the immunoprotective effect of the PstS antigen, whether the fusion protein could be successfully expressed, and whether there was an additive effect in their protective effects.

[0103] To prepare the above three recombinant nucleic acid vaccines, an exemplary schematic diagram of the vaccine molecular structure involved in the embodiments is shown below. Figure 1 As shown, from the N-terminus to the C-terminus, the following elements are sequentially included: signal peptide, Fc domain, target antigen region, and STABILON. These elements can be directly linked or linked through different linkers, such as GGS linker, spacer sequence, or GS linker. Figure 1 The illustrated vaccines A, B, and C differ in their target antigen regions. The target antigens are PstS antigen, YidR antigen, and PstS antigen (linked by a spacer sequence), respectively. In order to prepare vaccines capable of producing... Figure 1 The recombinant nucleic acid vaccine for the protein shown first constructs a gene expression cassette for expressing the antigen sequence described in this invention. The expression cassette, from the 5' end to the 3' end, sequentially includes: a 5' UTR, a CDS region, a 3' UTR, and a PolyA region, wherein the CDS region contains the fusion molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector suitable for in vitro RNA transcription, such as... Figure 2 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine was obtained.

[0104] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0105] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0106] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGS linker sequence shown in SEQ ID NO: 7, and the PstS antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4, the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0107] Step b: Construct a nucleic acid vaccine architecture carrier.

[0108] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0109] Step c: Prepare recombinant plasmids.

[0110] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention. Figure 2 (As shown).

[0111] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention

[0112] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-YidR antigen-PstS antigen-STABILON”. The human IGHG Fc domain and YidR antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The YidR antigen and PstS antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The PstS antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the YidR antigen is shown in SEQ ID NO: 2. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0113] Step b: Construct a nucleic acid vaccine architecture carrier.

[0114] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0115] Step c: Prepare recombinant plasmids.

[0116] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine B preparation vector based on the present invention. Figure 2 (As shown).

[0117] (3) Preparation vector of recombinant nucleic acid vaccine C based on the present invention

[0118] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-YidR antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The PstS antigen and YidR antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The YidR antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the YidR antigen is shown in SEQ ID NO: 2. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0119] Step b: Construct a nucleic acid vaccine architecture carrier.

[0120] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0121] Step c: Prepare recombinant plasmids.

[0122] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine C preparation vector based on the present invention. Figure 2 (As shown).

[0123] Table 1. Protein amino acid sequences involved in this invention.

[0124] amino acid sequence and sequence number PstS antigen AASLTGAGATFPAPVYAKWADTYQKETGNKVNYQGIGSSGGVKQIIANTVDFGASDAPLADDKLTQEGLFQFPTVIGGVVLAVNLPGVKSGELVLDGKTLGDIYLGKIKKWDDEAIAKLNPGLKLPSQNIAVVRRADGSGTSFVFTSYLSKVNEEWKSKIGA GSTVNWPTGLGGGKGNDGIAAFVQRLPGSIGYVEYAYAKQNNLAYTKLVSADGKPVSPTEDNFANAAKGVDWSKSFAQDLTNQKGENAWPITSTFILVHKATNKPEQTAEVLKFFDWAYKNGGKEANALDYATLPESVVEQVRAAWKTNVKDSSGKALY (SEQ IDNO: 1) YidR antigen MKQVTFAPRHHQLTNINTWTPDSQWLVFDVRPSGASFTGETIERVNVNSGTVETIYRATQGAHVGVVTVHPTQERYVFIHGPERPDAQWQYDFHHRRGVVAFQ GAVENLDAMDITPPYTPGALRGGSHVHVYSPNGQFVSFTYNDHVLHERDPALDLRNVGVAAPYGPVTPQGQHPREYGGSHWCVLVSRTAPAPAPGSDEINRAY EEGWVGNHALAFIGDTLAENGDKVPELFIVDLPQDEAGWKQPGGAPLAGAAAAMPAPPAGVAQRRLTFAHHRRYPGLVNVPRHWVRANPQATAIAFLMRDDAG VVQLWLISPQGGEPRQLTHHASGIQSAFNWHPSGEWLGFALEDRIACCHAGTGDITFLTDTHAHAPSADAIVFSPDGKQIAWMEEVDGYRQLWVTQTGR (SEQ ID NO: 2) signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 3) Fc domain of human IGHG EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4) STABILON KDGKKDKKEEDKK (SEQ ID NO: 5) Interval sequence GGSGGGGSGG (SEQ ID NO: 6) GGS linker GGSGGSGGSG (SEQ ID NO: 7) GS linker GSGSGSG (SEQ ID NO: 8)

[0125] Example 3: Preparation of the recombinant nucleic acid vaccine of the present invention

[0126] (1) Preparation of capped mRNA vaccines

[0127] Step a: Linearize the vector plasmid used in Example 2 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0128] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0129] (2) Preparation of uncapped mRNA vaccines

[0130] Step a: Linearize the vector plasmid used in Example 2 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0131] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0132] (3) DNA vaccine preparation

[0133] Step a: Amplify the vector plasmid used in Example 2 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0134] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0135] Example 4: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0136] Vaccine A (recombinant nucleic acid vaccine A based on the present invention), vaccine B (recombinant nucleic acid vaccine B based on the present invention), and vaccine C (recombinant nucleic acid vaccine C based on the present invention) were prepared using the capped mRNA vaccine preparation method described in Example 3. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiments was greater than or equal to 85%. The quality control peak diagram of the recombinant nucleic acids based on the present invention is shown below. Figure 3A , Figure 3B , Figure 3C As shown. Specifically, it is described as follows: (1) The recombinant nucleic acid vaccine A based on the present invention has a purity of 85.2%, and the quality control peak diagram and purity test results are as follows. Figure 3A (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 85.8%, and the quality control peak diagram and purity test results are as follows: Figure 3B (3) The recombinant nucleic acid vaccine C based on the present invention has a purity of 85.8%, and the quality control peak diagram and purity test results are as follows: Figure 3C The purity levels described above all meet the quality requirements for cell transfection experiments and vaccine production.

[0137] Example 5: In vitro expression effect of the recombinant nucleic acid of the present invention

[0138] The vaccine from Example 4 was transfected into HEK293T cells using cell transfection reagents. After 48 hours of in vitro culture, the protein was collected and subjected to Western blot analysis. The molecular weights of the proteins of vaccine A, vaccine B, and vaccine C were calculated, as shown in Table 2.

[0139] Table 2. Protein molecular weight of vaccines A, B, C, and D

[0140] vaccine Protein molecular weight (kDa) A 65.7 B 111.3 C 111.3

[0141] Figure 4A and Figure 4B The results of in vitro expression of WB (Western blot) in HEK293 cells transfected with vaccines A, B, and C are presented respectively. The expressed antigens are all humoral immune antigens, which should theoretically be significantly expressed in the supernatant.

[0142] As shown in Figure 4, the target protein signal was detected in the supernatant of vaccines A, B, and C, and the molecular weights were all as expected. This demonstrates that the fusion protein based on the present invention can not only be successfully translated and correctly folded in eukaryotic cells, but also has a stable structure and can be secreted extracellularly.

[0143] according to Figure 4A The results show that the PstS antigen can be expressed and secreted at extremely high levels when expressed alone, therefore vaccine A can be used as a humoral immunity vaccine. According to... Figure 4B The results showed that both vaccine B (YidR-PstS) and vaccine C (PstS-YidR) could be detected in the supernatant. Among them, the lysed protein signal of vaccine C was lower, indicating that the secretion efficiency of vaccine C was better than that of vaccine B.

[0144] The comparative analysis above shows that all three vaccines can be successfully expressed and secreted, and theoretically, all can present the correct antigenic epitopes and activate humoral immunity. Considering that vaccines B and C use the same antigen and do not require repeated verification, vaccines A and C, which has better secretion efficiency, were chosen for subsequent animal experiments to verify their immunoprotective effects. Simultaneously, the immunoprotective effects of monoantigen and multiantigen fusion proteins will be compared to determine if there are differences.

[0145] Example 6: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse nasal droplet infection model.

[0146] To verify whether the nucleic acid vaccine based on the present invention has an immune protective effect, this embodiment conducted an immunization and challenge comparison experiment on mice immunized with vaccine A (vaccine A immunization group), mice immunized with vaccine C (vaccine C immunization group), and unimmunized mice (PBS group). One mouse was not immunized or challenged and served as a negative control group.

[0147] Sixteen 6-8 week old BALB / c mice, weighing 18-25g, were selected for the experiment and housed in individual cages with constant temperature and humidity. The mice were acclimatized to the environment for 7 days prior to the experiment. The temperature in the housing was 20-26℃, and the humidity was 40-70%. A day-night cycle was implemented, with light from 8:00 AM to 8:00 PM and darkness from 8:00 PM to 8:00 AM the following day. Sufficient feed was continuously provided, with unlimited access to sterile water via a continuous water bottle. After acclimatization, the mice were randomly assigned to groups, and each mouse was ear-tagged. Details are shown in Table 3. Dosages in this table and below refer to the amount of active ingredient.

[0148] Table 3. Grouping and Immunization Procedure for Mouse Immunization Experiment in Example 6

[0149]

[0150] Mice in each group were immunized twice according to the immunization protocol in Table 3. On Day 35, mice were administered 100 μl of a mixture of 50 mg / ml penicillin and streptomycin antibiotics by gavage once daily for 3 days. On Day 35, mice were administered 50 μl (1×10⁻⁶) of a mixture of 50 mg / ml penicillin and streptomycin antibiotics by gavage. 7 Mice were infected with CFU (Chronic Klebsiella pneumoniae) via intranasal instillation. Mice were sacrificed on Day 38 for lung tissue collection. Bacterial load and histopathological sections were analyzed to comprehensively evaluate the vaccine's immunoprotective effect. The immunization, challenge, and sampling schedule is as follows: Figure 5 As shown.

[0151] 10 mg of lung tissue was aseptically harvested from each mouse and homogenized on ice with sterile PBS buffer until no significant precipitation occurred. Blood was diluted 1:1000 with sterile PBS buffer, and 100 μl of the diluted solution was evenly spread onto solid culture medium and incubated for 24 h. Bacterial clones were counted and photographed afterward to observe the protective effect of the vaccine on mice. Results are as follows: Figure 6A and Figure 6B As shown.

[0152] Figure 6A These are comparative photos of the bacterial load (number of colonies on agar plates) in the lung tissue of mice after immune challenge. The results show that the bacterial load in the negative control group was 0, confirming the validity of the comparative experiment. Figure 6B This image shows a comparison of the bacterial load (number of colonies on agar plates) in the lung tissues of mice in the vaccine-immunized group, PBS group, and negative control group after immunization and challenge. The results show that, compared to the PBS group, the Klebsiella pneumoniae load in the vaccine-immunized groups (immunized with vaccine A and vaccine C) was significantly reduced, with vaccine C showing the lowest load, almost completely eliminated. Compared to the PBS group, the bacterial load in the lung tissue of mice immunized with vaccine A decreased by approximately 18-fold, and the bacterial load in the lung tissue of mice immunized with vaccine C decreased by approximately 180-fold.

[0153] Lung tissue was collected from each mouse, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Pathological changes in the lung tissue were observed under a microscope. The results are as follows: Figure 7 As shown. Figure 7Comparison of lung tissue sections (HE staining) from mice after immunization with the virus showed that the alveolar structure of normal mice was intact, the epithelial septa were normal, and there were no obvious lesions or inflammatory infiltrations. In the PBS group, the alveolar septa were thickened, the alveolar morphology and structure were basically destroyed, and inflammatory cell infiltration was present. In the vaccine A immunization group, the alveolar septa were partially thickened, and some inflammatory cell infiltration was observed. Compared with the vaccine A immunization group, the alveolar structure of the vaccine C immunization group was better intact, and the alveolar septa were thinner. Analysis of these results indicates that mice immunized with both vaccines A and C of this invention showed resistance to Klebsiella pneumoniae infection, proving that the vaccines of this invention can induce immune protection against Klebsiella pneumoniae in mice and prevent Klebsiella pneumoniae infection. Among these, vaccine C showed better protective effects than vaccine A.

[0154] In summary, the PstS antigen, PstS antigen, and YidR antigen fusion protein provided by this invention can all induce effective protective immunity in mouse model animals, demonstrating good preventive effects against Klebsiella pneumoniae infection. Furthermore, compared to the PstS antigen alone, the fusion protein exhibits better immunoprotective efficacy, demonstrating an antigen potency additive effect. Therefore, this invention not only provides a novel antigen design option but also offers a multi-antigen fusion expression strategy, effectively improving vaccine protective potency, filling a gap in current Klebsiella pneumoniae vaccine development, and possessing extremely high commercial value and broad application prospects.

[0155] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein, characterized in that, Selected from either of the following two proteins: Fusion protein B: From N-terminus to C-terminus, the sequence is optionally linked by spacer sequences: signal peptide, Fc domain, YidR antigen protein, PstS antigen protein, STABILON; Fusion protein C: From N-terminus to C-terminus, the sequence is optionally linked by spacer sequences: signal peptide, Fc domain, PstS antigen protein, YidR antigen protein, STABILON; The amino acid sequence of the PstS antigen protein is shown in SEQ ID NO.1, the amino acid sequence of the YidR antigen protein is shown in SEQ ID NO.2, the amino acid sequence of the Fc domain is shown in SEQ ID NO.4, and the amino acid sequence of the STABILON is shown in SEQ ID NO.

5.

2. The fusion protein according to claim 1, characterized in that, The signal peptide is derived from human azurocidin protein, and its amino acid sequence is shown in SEQ ID NO:

3.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the spacer sequence is shown in any one of SEQ ID NO: 6, 7, or 8.

4. A recombinant nucleic acid molecule, characterized in that, The nucleic acid encoding the fusion protein of any one of claims 1-3.

5. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule of claim 4.

6. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 4 or the recombinant gene expression cassette of claim 5.

7. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 4, or the recombinant gene expression cassette of claim 5, or the recombinant vector of claim 6.

8. An immunogenic composition or pharmaceutical composition, characterized in that, It comprises one or more fusion proteins according to any one of claims 1-3, and / or one or more recombinant nucleic acid molecules according to claim 4, and / or one or more recombinant gene expression cassettes according to claim 5, and / or one or more recombinant vectors according to claim 6, and / or one or more recombinant host cells according to claim 7.

9. The immunogenic composition or pharmaceutical composition according to claim 8, characterized in that, The immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

10. The immunogenic composition or pharmaceutical composition according to claim 9, characterized in that, The immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Klebsiella pneumoniae.

11. A recombinant vaccine, characterized in that, The composition comprises one or more fusion proteins according to any one of claims 1-3, and / or one or more recombinant nucleic acid molecules according to claim 4, and / or one or more recombinant gene expression cassettes according to claim 5, and / or one or more recombinant vectors according to claim 6, and / or one or more recombinant host cells according to claim 7, and / or one or more immunogenic compositions or pharmaceutical compositions according to any one of claims 8-10.

12. The recombinant vaccine according to claim 11, characterized in that, The recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

13. Use of one or more fusion proteins of any one of claims 1-3, and / or one or more recombinant nucleic acid molecules of claim 4, and / or one or more recombinant gene expression cassettes of claim 5, and / or one or more recombinant vectors of claim 6, and / or one or more recombinant host cells of claim 7, and / or one or more immunogenic compositions or pharmaceutical compositions of any one of claims 8-10 in the preparation of a medicament for the prevention of diseases caused by Klebsiella pneumoniae.

14. Use of fusion protein A in the preparation of a medicament for the prevention of disease caused by Klebsiella pneumoniae, wherein the fusion protein A comprises, optionally, the following components linked by spacer sequences from N-terminus to C-terminus: a signal peptide, an Fc domain, a PstS antigen protein, and STABILON; wherein, The amino acid sequence of the PstS antigen protein is shown in SEQ ID NO.1, the amino acid sequence of the Fc domain is shown in SEQ ID NO: 4, and the amino acid sequence of the STABILON is shown in SEQ ID NO.

5.

15. The use according to claim 13 or 14, characterized in that, The diseases caused by Klebsiella pneumoniae are selected from pulmonary infections or extrapulmonary infections.

16. The use according to claim 15, characterized in that, The extrapulmonary infections are selected from urinary tract infections, sepsis, meningitis, wound infections, surgical infections, skin infections, abdominal infections, respiratory infections, and endophthalmitis.

17. The use according to claim 16, characterized in that, The urinary tract infection is selected from cystitis; the abdominal infection is selected from liver abscess.

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