TWI824571B - Klebsiella pneumoniae capsule polysaccharide vaccines - Google Patents

Abstract

The invention provides various immunogens comprising a repeat unit of saccharide of Klebsiella pneumoniaeCPS, which has a formula selected from the group consisitng of Formulae (I) to (VI) as described herein. Also provided are vaccines comprising one or more immunogens selected from Formula (I) to (VI) and methods of eliciting an immune response against a Klebsiella pneumoniaeand preventing infection of Klebsiella pneumoniaeby using an immunogen of the invention.

Description

Klebsiella pneumoniae capsular polysaccharide vaccine

The present invention relates to the field of vaccines. In particular, the present invention relates to a vaccine against Klebsiella pneumoniae infection comprising a conjugate of oligosaccharide units of the capsular polysaccharide of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important pathogen that causes various diseases in the human body. Recently, community-acquired bacterial liver abscess (PLA) caused by Klebsiella pneumoniae has become a global disease. In bacterial liver abscess (PLA) with Klebsiella pneumoniae, the mortality rate is 10%, while in bacterial liver abscess (PLA) with metastatic meningitis, the mortality rate increases to 30-40 %. Survivors of metastatic meningitis often have severe neurological sequelae, while metastatic endophthalmitis often affects the eye leading to blindness. In addition to causing bacterial liver abscesses (PLAs), Klebsiella pneumoniae has also been shown to cause invasive infections, leading to abscesses in other sites (e.g., kidney, spleen, brain, prostate), necrotizing fasciitis and severe bacteremic pneumonia.

Research reports indicate that the most common capsule type of strains causing PLA is K1, followed by K2. In addition to these studies on bacterial liver abscess (PLA), recent studies also indicate that the K1 and K2 capsular types of Klebsiella pneumoniae can also cause other diseases such as necrotizing fasciitis and community-acquired bacteremia. Invasive infections of pneumonia (Lin YT et al., BMC infectious diseases 2010, 10:307; and Fang CT etal., Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2007, 45(3):284- 293 ).

In addition, Klebsiella pneumoniae causes approximately 10% of nosocomial infections and increasing resistance to antibiotics such as broad-spectrum β-lactams and carbapenems is a significant problem. Our recent study indicates that capsule types K64 (38%), K62 (13%), K24 (8%), KN2 (7%) and K28 (6%) account for 10% of carbapenem-resistant Klebsiella In 72% of Carbapenem-Resistant Klebsiella pneumoniae (CRKP) strains, a concentration of CRKP capsule types was observed in the study.

Vaccines designed to target bacterial capsules, such as those for Streptococcus pneumoniae, are often effective against infections caused by these encapsulated pathogens. In 1985, a Klebsiella pneumoniae K1 capsular polysaccharide (CPS) vaccine was disclosed. In 1988, an invention disclosed a Klebsiella pneumoniae 24-valent capsular polysaccharide (capsular polysaccharide (CPS)) vaccine. Although Klebsiella pneumoniae K1 capsular polysaccharide (CPS) and 24-valent capsular polysaccharide (CPS) vaccines were disclosed many years ago, until now, there is still no available Klebsiella pneumoniae vaccine. Previous studies have shown that polysaccharide vaccines can only induce independent immunity of T cells lacking immune memory and lack the production of high-affinity antibodies. Therefore, a Klebsiella pneumoniae capsular polysaccharide (CPS)-protein conjugate vaccine may be more effective against infections caused by this bacterium.

Past studies have pointed out that K1 and K2 capsular polysaccharides (CPS) are giant molecules composed of several sugar repeating units ( Yang FL etal., The Journal of biological chemistry 2011, 286(24):21041-21051 ). Depolymerization of capsular polysaccharide (CPS) will increase the efficiency of conjugation to proteins. Some chemical reagents, such as trifluoroacetic acid, ammonium hydroxide, and acetic acid, although they degrade capsular polysaccharide (CPS), lead to the modification (acetylation or acetonation) of capsular polysaccharide (CPS) that is important for immune responses. loss. WO20125676A1 provides an isolated phage that can infect K1 Klebsiella pneumoniae strains and has a capsular depolymerase that specifically breaks down K1 capsular polysaccharide (CPS). This enzyme will be used to develop a vaccine that is effective against Klebsiella pneumoniae infections.

Unless otherwise stated, technical terms are used in this article in accordance with conventional usage.

The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Likewise, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

Abbreviations used in this article: GlcAp: glucuronic acid; Fucp: fucose; Glcp: glucose; Manp: mannose; Rhap: rhamnose; Galp: galactose; GlcUAp: GlcAp: glucuronic acid.

The term "conjugate" as used herein refers to a composition consisting of two heterologous molecules linked together for the purpose of stimulating or eliciting a specific immune response in an animal. In some embodiments, the immune response is protective, which directly renders the animal more effective against infection by the organism targeted by the immunogenic conjugate. A specific embodiment of the immunogenic conjugate refers to a vaccine, such as a conjugate vaccine.

The term "linker" as used herein refers to a compound or moiety of a molecular bridge used to operably link two different molecules, wherein one end of the linker is operably linked to the first molecule and the other end is operably linked to Second molecule. Two different molecules can be attached to the linker in a stepwise manner.

The term "vaccine" as used herein refers to a pharmaceutical composition capable of eliciting a prophylactic or therapeutic immune response in a subject. Typically, vaccines elicit an antigen-specific immune response to pathogen antigens.

The term "oligosaccharide" as used herein refers to compounds containing two or more monosaccharide units. Oligosaccharides are considered to have reducing and non-reducing ends.

The term "protein carrier" as used herein refers to a protein, peptide or fragment thereof coupled or conjugated to an oligosaccharide, which enhances the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater extent than the oligosaccharide alone.

The term "antibody" as used herein encompasses both polyclonal and monoclonal antibody preparations and also includes hybrid antibodies, altered antibodies, F(ab')2 fragments, F(ab) molecules, Fv fragments, and single-chain fragments distributed on phage Variants (scFv), single domain antibodies, chimeric antibodies, humanized antibodies and functional fragments thereof that display the immunobinding properties of the parent antibody molecule.

The term "immunogen" as used herein refers to a protein or portion thereof capable of inducing an immune response in a mammal, such as a mammal that is infected or is at risk of infection with a pathogen. Administration of the immunogen induces protective immunity and/or active immunity against the pathogen of interest.

The present invention is a capsular polysaccharide (CPS) conjugated vaccine produced by utilizing decomposed capsular polysaccharide (CPS) of Klebsiella pneumoniae separately conjugated with a carrier. Surprisingly, the capsular polysaccharide (CPS) conjugate vaccine of the present invention can also induce capsular polysaccharide (CPS) antibodies with bactericidal activity at the same time. Therefore, the capsular polysaccharide (CPS) conjugate vaccine of the present invention can be used to prevent invasive infections caused by Klebsiella pneumoniae.

The present invention provides various immunogens comprising repeating units selected from the Klebsiella pneumoniae capsular polysaccharide (CPS) of formulas (I) to (VI) as described herein.

In one aspect, the invention provides an immunogen comprising repeating units of the trisaccharide of Klebsiella pneumoniae K1 capsular polysaccharide (CPS) having the following formula (I):

In a specific embodiment, m is 2.

The present invention finds that K1-ORF34 polypeptide can degrade Klebsiella pneumoniae K1 capsular polysaccharide (CPS). After degradation, the hexasaccharide of Klebsiella pneumoniae K1 capsular polysaccharide (CPS) can be obtained. Hexasaccharides degraded from K1 capsular polysaccharide (CPS) can be used as immunogens against Klebsiella pneumoniae infections. The K1-ORF34 polypeptide has the amino acid sequence shown in SEQ ID NO:1. MALIRLVAPERVFSDLASMVAYPNFQVQDKITLLGSAGGDFTFTTTASVVDNGTVFAVPGGYLLRKFVGPAYSSWFSNWTGIVTFMSAPNRHLVVDTVLQATSVLNIKSNSTLEFTDTGRILPDAAVARQVLNITGSAPSVFVPLAADAAAGSKVITVAAGALSAVKGTYLYLRSNKLCDGGPNTYGVKISQIRKVVGVSTSGGVTSIRLDK ALHYNYYLSDAAEVGIPTMVENVTLVSPYINEFGYDDLNRFFTSGISANFAADLHIQDGVIIGNKRPGASDIEGRSAIKFNNCVDSTVKGTCFYNIGWYGVEVLGCSEDTEVHDIHAMDVRHAISLNWQSTADGDKWGEPIEFLGVNCEAYSTTQAGFDTHDIGKRVKFVRCVSYDSADDGFQARTNGVEYLNCRAYRAAMDGFASNTGVAFPIY RECLAYDNVRSGFNCSYGGGYVYDCEAHGSQNGVRINGGRVKGGRYTRNSSSHIFVTKDVAETAQTSLEIDGVSMRYDGTGRAVYFHGTVGIDPTLVSMSNNDMTGHGLFWALLSGYTVQPTPPRMSRNLLDDTTGIRGVATLVAGEATVNARVRGNFGSVANSFKWVSEVKLTRLTFPSSAGALTVTSVAQNQDVPTPNPDLNSFVIRSSNAADVSQVA WEVYL (SEQ ID NO:1)

In another aspect, the invention provides an immunogen comprising repeating units of the tetrasaccharide of Klebsiella pneumoniae K2 capsular polysaccharide (CPS), having the following formula (II): where n ranges from 1 to 4.

In a specific embodiment, the immunogen contains one repeating unit (ie, n is 1).

The present invention finds that K2-ORF16 polypeptide can degrade Klebsiella pneumoniae K2 capsular polysaccharide (CPS). After degradation, the tetrasaccharide of Klebsiella pneumoniae K2 capsular polysaccharide (CPS) can be obtained. The tetrasaccharide produced after the degradation of K2 capsular polysaccharide (CPS) can be used as an immunogen against Klebsiella pneumoniae infection. Therefore, the present invention provides an isolated polypeptide or a variant thereof, which has a degrading activity specific to the capsule of Klebsiella pneumoniae capsular type K2 strain, the polypeptide or a variant thereof being selected from the following The group consisting of (a) and (b): (a) a polypeptide comprising the amino acid sequence SEQ ID NO: 2; and (b) a polypeptide encoded by a polynucleotide, wherein The polynucleotide is a polynucleotide that hybridizes to (i) the coding sequence of the polypeptide SEQ ID NO: 2, or (ii) the full-length complementary sequence of (i), at least under high stringency conditions. In a specific embodiment, the K2-ORF16 polypeptide has the amino acid sequence shown in SEQ ID NO: 2. MTIIKRADLGRPLTWDELDDNFQQVDDLTAAASAAVLSASASATAAAGSATNSLNSANSAASSADDAAASATVAINALMNSTFEPADFDFTSGGTLDSTDRNKAVYNPADNNWYSWSGILPKIVTAATDPTADSNWKPRTDQLLRQNLASSVIPGTSLVTHSDGIHLDDYIEIFNRRTKFIMPEDFPGTDTEQLQSALSYAKSNRVNVVLQAGKTYYVTGSQGLEVD LGYYSFESPNGIAYIDFTGCTATYCLWVHSSRPYPDGSENHCTSMRGIKFKSSVKGIGQRLLLTGNNNDSSNGTYNGDCKIENCMFSTADIVLGASNSTWRYKFINCGFMMESTGGTYAMHFPAGISDSGESVTFQNCKIFDMKGCPILVECASFAIGMPGTSVLNTPIKITGSGAMVIMDSAANIENPGASAWYRYGEVTGTGARLILNGCTLVCNNPSLQTKPLFYV GANAFIDVTLVKTPGNDYLFQNGDEGLRTFVEGDGYVTASHCIGDILSGVGNIPLHKSLNPTLNPGFETGDLSSWSFNNQGSASQTCVVGTAYKKTGTYGARMTSFGSLSCFLTQKVKVTQHGYYSTTCQINTITAGTGTTAGSLTITFYNRDGNALQAGASSNFTNTPSGWQSVGRFIQGRVPQAAEYCEVSFRCREGAVIDVDNFIINFT (SEQ ID NO:2)

Variants of the polypeptide are man-made and comprise the substitution, deletion and/or insertion of one or more amino acids of the polypeptide of SEQ ID NO:2. Preferably, the amino acid change has a small impact, that is, the substitution or insertion of a retaining amino acid sequence does not significantly affect the folding and/or activity of the protein, which is usually a small change of 1 to about 30 amino acids. Deletions, small amine or carboxyl-terminal extensions, such as residues of the amine-terminal methionine, small linker peptides of up to about 20 to 25 residues, or by changing net charge or other functionality, such as histidine tags, Polyhistidine tag region, epitope or binding domain.

In another aspect, the present invention provides an immunogen comprising the repeating unit of the hexasaccharide of Klebsiella pneumoniae K64 capsular polysaccharide (CPS), having the following formula (III): where n ranges from 1 to 4. In a specific embodiment, n is 1.

In another aspect, the invention provides an immunogen comprising repeating units of the pentasaccharide of Klebsiella pneumoniae K62 capsular polysaccharide (CPS), having the following formula (IV): where n ranges from 1 to 4. In a specific embodiment, n is 2.

In another aspect, the invention provides an immunogen comprising repeating units of the pentasaccharide of Klebsiella pneumoniae K24 capsular polysaccharide (CPS), having the following formula (V): where n ranges from 1 to 4. In a specific embodiment, n is 1.

In another aspect, the invention provides an immunogen comprising repeating units of the hexasaccharide of Klebsiella pneumoniae K28 capsular polysaccharide (CPS), having the following formula (VI): Where n ranges from 1 to 4, and n is 1.

In another aspect, the invention provides an immunogen comprising a repeating unit of the hexasaccharide of Klebsiella pneumoniae KN2 capsular polysaccharide (CPS) having four hexasaccharides and one hexuronic acid , its molecular weight is approximately 1027.

WO20125676A1 describes enzymes for the degradation of capsular polysaccharides (CPS) of K24, K28, K62, K64 and KN2.

In another aspect, the invention provides a vaccine comprising one or more immunogens selected from formulas (I) to (VI).

The structural sugar units and degraded capsular polysaccharide (CPS) products of K1, K2, K62, K64, K24 and K28 are as follows. sugar unit structure Degradation products of capsular polysaccharide (CPS) K1 trisaccharide Six sugars (two units) K2 Tetrasaccharide Tetrasaccharide (one unit) K62 Five sugars Ten sugars (two units) K64 Six sugars Six sugars (one unit) K24 Five sugars Five sugars (one unit) K28 Six sugars Six sugars (one unit)

In a specific embodiment, the immunogen and protein carrier are conjugated to each other.

In a specific embodiment, the vaccine of the invention is a polyvalent vaccine. In some embodiments, the vaccine is a K1 and K2 capsular polysaccharide (CPS) conjugated bivalent vaccine comprising the immunogens of Formula I and Formula II. In another embodiment, the vaccine is a K1 and K2 capsular polysaccharide (CPS) conjugated bivalent vaccine comprising a mixture of Immunogen I-vector and Immunogen II-vector, wherein Immunogen I and The immunogens II of formula (II) are respectively conjugated to a carrier.

In a specific embodiment, the vaccine is a polyvalent vaccine. In some embodiments, the vaccine is a K64 and K62 capsular polysaccharide (CPS) conjugated bivalent vaccine comprising immunogens of Formula III and Formula IV. In another specific embodiment, the vaccine is a K64 and K62 capsular polysaccharide (CPS) conjugated bivalent vaccine, including a mixture of an immunogen III-vector and an immunogen IV-vector, wherein the immune response of formula (III) Protein III and immunogen IV of formula (IV) are separately conjugated to the carrier.

The immunogen of the present invention can be conjugated to a carrier to form a conjugate. In a specific embodiment, the conjugate is a vaccine. In another specific embodiment, the ratio of either immunogen in the conjugate vaccine is from 1:1.4 to 1:10.2. In a specific embodiment, the vaccine includes 10% (weight percent) to 90% (weight percent) immunogen I-protein carrier or immunogen III-protein carrier and 10% (weight percent) to 90% (weight percent) %) of the Immunogen II-protein carrier or the Immunogen IV-protein carrier. The vaccine preferably contains equal amounts of an immunogen I-protein carrier or an immunogen III-protein carrier and an immunogen II-protein carrier or an immunogen IV-protein carrier.

Suitable vectors are well known in the art and may include, for example, proteins, peptides, lipids, polymers, dendrimers, virions, virus-like particles (VLPs), or combinations thereof. In a specific embodiment, the carrier system protein carrier includes but is not limited to bacterial toxoids, toxins, exotoxins and non-toxic derivatives thereof, such as keyhole limpet hemocyanin (KLH), hepatitis B virus core protein, thyroid Globulin, albumin (BSA), human serum albumin (HSA) and ovalbumin), pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), tuberculin purified protein derivative (PPD) ; Transferrin-binding protein, polyamino acid, tetanus toxoid, tetanus toxin fragment C, diphtheria toxoid, CRM (nontoxic diphtheria toxin mutant), cholera toxin, Staphylococcus aureus exotoxin or toxoid, E. coli heat-labile enterotoxin, Pseudomonas aeruginosa exotoxin A and bacterial outer membrane proteins such as Neisseria meningitidis serotype B outer membrane protein complex (OMPC) and outer membrane porin class 3 (rPorB).

In a specific embodiment, the vector is a protein carrier. In another specific embodiment, the protein carrier system is CRM197. The amino acid sequence of CRM197 is shown in SEQ ID NO:3. GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD KTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPI AGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS (SEQ ID NO:3)

Polysaccharides contain hydroxyl groups and occasionally carboxyl and amine groups, whereas proteins contain amine and carboxyl groups. Many methods of conjugating proteins to polysaccharides are known in the art. If the protein carrier has two or more identical sugars, the sugars can be conjugated to the same molecules of the protein carrier. Alternatively, the saccharides may be individually coupled to different molecules of the protein carrier (each protein carrier molecule having only one saccharide bound to it).

In accordance with standard techniques well known to those of ordinary skill in the art in the pharmaceutical and veterinary fields, taking into account such factors as the specific antigen, adjuvants (if any), age, sex, weight, species and condition of the particular animal or patient, and route of administration, The amount of the vaccine of the invention to be administered to a human or animal and the method of administration can be determined.

The conjugation vaccine of the present invention can be administered to a subject in a manner consistent with conventional methodologies for treatment or prevention, for example, methods for treatment or prevention of infection from Klebsiella pneumoniae . The present invention discloses that under conditions sufficient to prevent, inhibit and/or ameliorate Klebsiella pneumoniae infection, a conjugate vaccine and/or other biologically active agents at an effective dose for prevention or treatment are administered to patients in need of such treatment. Subject for a period of time. In some embodiments, after the subject is vaccinated with the conjugate vaccine of the present invention, the immune response against the Klebsiella pneumoniae antigenic immunogen in the subject is successfully induced. In some embodiments, the subject being treated is selected from a subject who has been infected with Klebsiella pneumoniae or is at risk of developing a Klebsiella pneumoniae infection, e.g., has been exposed or is at risk of being exposed to Klebsiella pneumoniae Klebsiella pneumoniae environment. In an important aspect, the conjugation vaccine provided by the invention can be used to prevent infection by Klebsiella pneumoniae.

The conjugated vaccine of the present invention can be prepared into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, powders, etc. Antigens and immunogenic compositions may be mixed with physiologically acceptable carriers that are compatible therewith. These can include water, physiological saline, glucose, glycerol, ethanol, or combinations thereof. The conjugated vaccine may further contain auxiliary substances, such as wetting or emulsifying agents or pH buffering agents, to further increase efficacy. Administration of the conjugate vaccine may include delivery by various routes, such as oral, intravenous, intramuscular, intranasal, subcutaneous, and intraperitoneal administration.

In another aspect, the invention provides antibodies produced by administering a conjugation vaccine of the invention.

The following embodiments are only used to illustrate the present invention but not to limit the present invention. Unless otherwise stated, all percentages used herein are by weight. All patents, patent applications and literature references cited herein are incorporated by reference.

Materials and methods

Isolate phage

Phage purification and phage titer determination were performed using agar overlay method; phage infectivity was determined using spot test ( Lin TL, Hsieh PF, Huang YT, et al. Isolation of a bacteriophage and its depolymerase specific for K1 capsule of Klebsiella pneumoniae: implication in typing and treatment. The Journal of infectious diseases 2014; 210:1734-44 ).

Phage genome DNA and sequencing

The genomic DNA of the bacteriophage was extracted with QIAGEN's lambda reagent set, and the method was modified from the following literature ( Lin TL, Hsieh PF, Huang YT, et al. Isolation of a bacteriophage and its depolymerase specific for K1 capsule of Klebsiella pneumoniae: implication in typing and treatment. The Journal of infectious diseases 2014; 210:1734-44 ). After precipitating and lysing the phage, the DNA was extracted with phenol/chloroform and precipitated with ethanol. Finally, the genome sequence of the phage was determined by Illumina GAII sequencing.

Protein representation and purification

Recent studies have revealed the expression and purification of recombinant His-tagged K1-ORF34 protein ( Lin TL , Hsieh PF , Huang YT , et al . Isolation of a bacteriophage and its defolymerase specific for K1 capsule of Klebsiella pneumoniae : implicationin typing and treatment .Journal of infectious diseases 2014; 210 : 1734-44 ) . The K2- orf16 fragment was amplified by first using the 1611E-ORF16 forward primer (5'-CAAACATCACGGTGACGCTAGCATGACATTATCAAACG-3'; SEQ ID NO: 4) and the 1611E-ORF16 reverse primer (5'-CTTTTAACATTTAGCACTCGAGTGTAAAATTAATAATG-3'; SEQ ID NO :5) to perform PCR, and then digest it with NheI and XhoI restriction enzymes, and then clone the digested K2- orf16 fragment into the NheI and Restriction enzymes break it down). To amplify K62 capsular depolymerase, the Ref-K10-1 ORF6 forward primer (5'-ATGAATAAGATGTTTACCCAG-3'; SEQ ID NO: 6) and the Ref-K10-1 ORF6 reverse primer (5'- AATTGGGCGAAGGCGTTCAAAC-3'; SEQ ID NO: 7) was subjected to PCR and cloned into the blunted EcoRI cleavage site of the pET28c plasmid (Novagen). The obtained plasmid was transformed into BL21 Escherichia coli (DE3), induced with 0.4 millimol concentration (mM) IPTG at 16°C overnight to express the recombinant His-tagged protein, and the recombinant His-tagged protein was expressed according to the instructions provided in the reagent set (Qiagen). method for protein purification.

Klebsiella pneumoniae capsular polysaccharide ( CPS ) purification

Capsular polysaccharides (CPS) of K1, K2 and K62 were purified from Klebsiella pneumoniae NTUH-K2044Δ wbbO , NTUH-A4528Δ wbbO and K62 reference Δ wbbO mutant strains respectively (Hsieh PF, Lin TL, Yang FL , et al. Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess. PloS one 2012;7:e33155) to eliminate O-polysaccharide contamination. Bacteria were cultured on Luria agar plates at 37°C for 12 hours. Bacteria were collected and placed in sterile water (weight/volume = 1/10) and heated at 100°C for 10 minutes. After cooling, the bacterial solution was centrifuged at 15,000xg for 20 minutes. Mix the supernatant (volume) with ice-cold acetone (4 volumes) for polysaccharide precipitation. After centrifugation at 12,000xg for 20 minutes, an unprocessed capsular polysaccharide (CPS) pellet was obtained. The pellet was broken up and suspended in sterile water and freeze-dried. Raw capsular polysaccharide (CPS) powder was decomposed by ribonuclease (Roche) and deoxyribonuclease I (Roche) at 37 °C for 24 h and then mixed with proteinase K at 10 millimolar concentration (mM). Further decomposition occurs in hydroxymethylaminomethane-HCl (pH 7.4) for 6 to 8 hours. After denaturation at 100°C for 10 minutes, the supernatant was dialyzed and purified using a dialysis membrane with a pore size of approximately 8 to 10 kilodaltons and freeze-dried. Partially purified capsular polysaccharide (CPS) was eluted with 0.1% sodium azide in filtered pure water and further purified on a TSK HW-65F column [1.6 cm (diameter) × 90 cm (height)] . The carbohydrate-containing fragments were detected by the phenol-sulfuric acid method and dialyzed against pure water (molecular weight threshold: 1 kilodalton) and concentrated by freeze-drying.

Capsular polysaccharide (capsular polysaccharide) CPS ) Structural analysis

Previous studies ( Yang FL et al., The Journal of biological chemistry 2011, 286(24):21041-21051; Corsaro MM et al., Carbohydrate research 2005, 340(13):2212-2217 ) have revealed that Cray Chemical structures of capsular polysaccharides of C. burnetii NTUH-K2044 (K1) and A4528 (K2).

Mass spectrometry: For matrix-assisted laser desorption ionization mass spectrometry, 0.5 microliters (μl) of sample was first mixed with 0.5 microliters (μl) of matrix solution (20 mg/ml 2,5-dihydroxybenzoic acid (DHB) ) mixed in 50% ACN and 1% nitric acid aqueous solution (H ₃ PO ₄ ) to obtain a mixed solution. The mixed solution was placed on a stainless steel plate. After air drying, it was subjected to 4800 matrix-assisted laser desorption ionization - Analysis was performed on a time-of-flight/time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA) in specular reflector mode with an acceleration voltage of 20 kV and a grid voltage of 16%. A typical spectrum is generated from 1000 laser shots. Raw spectra were processed by baseline subtraction and noise removal using Data-Explorer software (Applied Biosystems) ( Tsai CF et al., Neuromethods 2011, 57:181-196 ). Measurements were performed on an Ultraflex II matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometer (Bruker Daltonik GmbH, Germany) in primary mass spectrometry. Mass spectra were acquired in reflection mode with mass-to-charge ratios (m/z) ranging from 500 to 4,000. Mass spectrometry/mass spectrometry analyzes were performed in elevator mode. Mass spectrometry analysis was performed by the GRC Mass Core Facility of the Genome Research Center of Academia Sinica.

Nuclear magnetic resonance spectroscopic analysis: The present invention records the nuclear magnetic resonance spectroscopic analysis results of small capsular polysaccharide (CPS) fragments in heavy water; all two-dimensional nuclear magnetic resonance spectroscopic analysis experiments are performed using the standard pulse sequence provided by Bruker. NMR spectroscopic analysis data were processed using spin-up 3.1. NMR spectroscopy experiments and resonance assignments: All NMR experiments were performed on a Bruker AVANCE 600 or AVANCE 800 NMR mass spectrometer (Bruker, Karlsruhe, Germany) equipped with triple ( ^1H , ^13C and ^15N ) resonances Cryoprobe, including a shielded z-gradient. Two-dimensional (2D) ¹ H NMR, TOCSY and NOESY spectra were collected. All heteronuclear NMR experiments of recombinant proteins were performed as required. Sequence-specific assignment of backbone atoms was achieved by independent connectivity analysis of CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, and C(CO)NH. ¹ H resonances were assigned using 3D HAHB(CO)NH and HCCH-TOCSY.

Enzymatic cleavage: K1 capsular polysaccharide (CPS) is degraded by K1 lyase through β-hydrogen elimination reaction to generate the double bond between C-4 and C-5 of glucuronic acid and measured at 232 nm Spectrophotometric changes in wavelength absorbance values. Determinations were performed in triplicate in a UV-VIS spectrophotometer (Ultrospec 4000 UV/Visible; Amersham Pharmacia Biotech, Piscataway, NJ, USA) at 25 °C in colorimetric tubes with a volume of 1 ml. Recombinant K1 lyase (10 μg/ml) was added to a solution containing 25 mM Tris, pH 7.5, and 3 mM MgCl ₂ ) and 40 μg/ml capsular polysaccharide (CPS) in the reaction mixture, and then measured the increase in absorbance at 232 nm wavelength for 15 min. The control group did not contain K1 lyase.

Quantification of the reducing end using the 3,5 -dinitrosalicylic acid ( DNSA ) assay (for K2 hydrolase): This method is determined by estimating the concentration of the 3,5-dinitrosalicylic acid reducing sugar. Hydrolase activity (DNAS) (Sigma-Aldrich) ( Danner M et al., European journal of biochemistry / FEBS 1993, 215(3):653-661; Miller GL et al., Analytical Chemistry 1959, 31(3): 426-428 ). When the sample is mixed with DNSA and heated to catalyze the reaction, the nitro group at the 3' end of the DNS is reduced by the FlexStation 3 device to an amine whose absorbance can be measured at 535 nm. The conditions for each hydrolase can be determined by quantifying the concentration of reducing sugars.

Capillary electrophoresis: Capillary electrophoresis is performed on a Beckman P/ACE MDQ capillary electrophoresis system equipped with a UV detector set at a wavelength of 230 nanometers. Separation and analysis were performed at 25°C in electrodynamic chromatography-coated fused silica capillaries (inner diameter 77 μm, total length 65 cm, 50 cm from injection point to detector). The working buffer consisted of sodium phosphate buffer (50 millimolar (mM), pH 9.0). The buffer solution was vacuum filtered and degassed through a membrane filter with a pore diameter of 0.2 μm, and oscillated in an ultrasonic water tank. Before each execution, the capillary was cleaned with 0.1 molar NaOH for 5 minutes, then with double-distilled water for 5 minutes, and then treated with operating buffer for 5 minutes. The sample to be analyzed is automatically injected using pressure injection mode, where the sample is pressurized for 15 seconds. Perform capillary electrophoresis at 20 kV (approximately 65 mA) using normal polarity.

Decomposed capsular polysaccharide ( CPS )and DT-CRM197 conjugation of carrier proteins

Method for preparing glycosylamines by kochetkov amination: Add ammonium carbonate (3.0 g, excess) to a reducing sugar aqueous solution (20 mg of reducing sugar dissolved in 3.0 ml of double distilled water). The resulting suspension was sealed and stirred at room temperature for 7 days, after which the reaction mixture was freeze-dried until the dry weight of the residue remained constant to obtain the glycosylamine as a colorless solid and used without further purification.

To connect thiol linkers via glycosylamine: Add 3,3'-disulfide to the glycosylamine solution (20 mg of glycosylamine dissolved in 3.0 ml of phosphate buffered saline (pH 7.4)). Substitute bis(sulfosuccinimidylpropionate, DTSSP, 20 mg, excess), then add 1 molar NaOH solution to maintain the reaction mixture at pH 7.4 to 7.8, and stir at room temperature for 16 hours. Dithiothreitol (DTT, 20 mg, excess) was added to the aforementioned solution, and the mixture was stirred at 40°C for 1-2 hours. Finally, the solvent was removed under reduced pressure, and the residue was purified by Sephadex LH-20 column chromatography and eluted with double distilled water to obtain the desired product: decomposed capsular polysaccharide-thiol (CPS-SH).

Synthesis of CRM197 -maleimid : After removal of salts from commercially available CRM197 (1.0 mg) by alternate dissolution in water and dialysis (Amicon Ultra-0.5, 10 kDa ) , the residual Dissolve in phosphate buffered saline (pH 6.5, 1.0 ml) and transfer to sample vial. Add N-ε-maleimid (maleimid) hexanoyloxysulfosuccinimide ester (Sulfo-EMCS, 1.0 mg, 8.22×10 ^-6 mol) to this solution, and then incubate at room temperature. The reaction was stirred for 2 hours and the mixture was purified using Amicon Ultra-0.5 (10 kilodaltons). After using matrix-assisted laser desorption ionization-time of flight spectroscopy to check the molecular weight and BCA determination to calculate the total amount of protein, CRM197-maleimid was stored in phosphate-buffered saline (pH 7. 2, 1.0 mg/ml), used in the next step. Based on data from matrix-assisted laser desorption ionization-time of flight spectroscopy, the number of functional groups of maleimide can be calculated. For example, when the molecular weight of CRM197 -maleimid is 61841, the number of maleimine functional groups on CRM197-maleimid can be determined.

Synthesis of decomposed capsular polysaccharide -CRM197 ( CPS-CRM197 ) conjugates ( 1 to 4 ): Dissolve CRM197-maleimid in phosphate buffered saline (pH 7.2, 1.0 mg/ ml), then varying amounts of decomposed capsular polysaccharide-thiol (CPS-SH) (5.0 mg/ml in phosphate buffered saline, pH 7.2) were added to the solution. The mixture was stirred at room temperature for 2 hours. The cleaved capsular polysaccharide-CRM197 (CPS-CRM197) conjugate was purified by using Amicon Ultra-0.5 (10 kDa) to remove non-reactive cleaved capsular polysaccharide-thiol (CPS-CRM197) by dialysis. SH) and sodium phosphate. The carbohydrate incorporation rate of the obtained decomposed capsular polysaccharide-CRM197 (CPS-CRM197) conjugate was determined by matrix-assisted laser desorption ionization-time of flight spectroscopy. The non-reactive decomposed capsular polysaccharide-thiol (CPS-SH) reacts with dithiothreitol (DTT) and can be recovered through LH-20 column chromatography purification. By varying the amount of cleaved capsular polysaccharide-thiol (CPS-SH), we can conjugate different ratios of oligosaccharide epitopes to the CRM197 vector.

Vaccination

The capsular polysaccharide (CPS)-conjugated vaccine was diluted in phosphate-buffered saline to a concentration of 100 μg/ml, and the glycolipid adjuvant C34 was dissolved in dimethylsulfoxide (DMSO) to a concentration of 100 μg/ml. ml. 100 μl of a vaccine mixture consisting of capsular polysaccharide (CPS) conjugated vaccine (2 μg glycan) and 2 μg adjuvant was administered intramuscularly (IM) to mice. For K1 and K2 vaccines: Five-week-old female BALB/c mice received the vaccine three times, one week apart. For the K62 vaccine: Five-week-old female BALB/c mice were vaccinated every two weeks for five times.

Against capsular polysaccharide ( CPS ) detection of antibodies

Different amounts of purified capsular polysaccharide (CPS) were transferred to the membrane by immunospot assay. Antibodies against capsular polysaccharide (CPS) were detected by culturing sera from vaccinated mice (1:1000 dilution).

Serum bactericidal test

After incubating the mouse serum at 56°C for 30 minutes, make two-fold serial dilutions (1/2 times to 1/256 times) with physiological saline. Incubate 25 μl of diluted serum and 12.5 μl of bacterial suspension (100 colony-forming units (CFU)) at 37°C for 15 minutes. After incubation, 12.5 microliters (μl) of newborn rabbit's complement (Pel-Freez, USA) was added and incubated at 37°C for 1 hour. The reaction mixture was then spread on an LB pan. After culturing for the next day, the number of surviving bacteria was counted. Serum bactericidal potency was defined as the reciprocal of serum dilution, and bacterial-complement-serum from vaccinated mice achieved 50% bactericidal potency compared to control mice vaccinated with adjuvant only.

Protection power analysis

For K1 and K2 vaccines: One week after vaccination (5 mice per group), including vaccinated or control mice (adjuvant only), inoculate 1 × 10 ^colony forming units (CFU) intraperitoneally (IP) , colony-forming unit) NTUH-K2044 or NTUH-A4528 strain. The experiment was carried out for 30 days to observe the mortality of mice. The survival rates of mice were analyzed by Kaplan-Meier analysis and log-rank test; P values <0.05 were considered to have statistically significant differences. For K62 vaccine: mice in the vaccinated group or control group (adjuvant only) were pretreated with cyclophosphamide (100 mg/kg) twice every two days. The numbers of white blood cells and neutrophils were significantly lower in cyclophosphamide-treated mice. Two days after cyclophosphamide treatment (5 mice per group), these mice were inoculated intraperitoneally with 5 × ¹⁰ colony-forming units (CFU) of the K62 reference strain. Observations were conducted for 30 days to confirm the mortality of mice. The survival rates of mice were analyzed by Kaplan-Meier analysis and log-rank test; P values <0.05 were considered to have statistically significant differences. Vaccines with other immunogens described in the present invention are all tested using similar methods as described above. Embodiment 1 : Isolation of phages infecting Klebsiella pneumoniae strain 1611E ( K2 capsular type)

Previous studies have revealed the K1 capsular depolymerase (K1-ORF34) from the K1-specific phage NTUH-K2044-K1-1 ( Lin TL et al., The Journal of infectious diseases 2014, 210(11):1734 -1744 ). To isolate the K2 capsule-degrading enzyme, phages infecting Klebsiella pneumoniae strain 1611E (K2-type capsule) were isolated from untreated water. Clear plaques with a translucent halo were detected, and the phage was named 1611E-K2-1. The susceptibility of the K2 type capsule of 1611E-K2-1 bacteriophage was evaluated by polymerase chain reaction assay on the capsule types of seven additional capsular type K2 strains using the wzy primer. This phage may infect all K2-capsulated strains. Embodiment 2 : Identification of putative capsular depolymerases

Presumptive identification K2 capsular depolymerase

The full genome length of phage 1611E-K2-1 was determined to be 47,797 base pairs. Annotation of the genome sequence shows that this phage is predicted to contain 17 open reading regions (ORFs) of over 500 base pairs. Open reading region (ORF) analysis of phage 1611E-K2-1 showed that the predicted ORF16 showed 46% amino acid identity with tail protrusion 63D sialidase, indicating that this protein may correspond to a capsular depolymerase. The K2- orf16 gene was cloned into plasmids and expressed in E. coli. The purity of the purified recombinant K2-ORF16 protein is shown in Figure 1A. When spotted on plates inoculated with Klebsiella pneumoniae 1611E, the recombinant K2-ORF16 protein produced translucent spots that resembled a halo of plaques (Fig. 1B). Capsular polysaccharide (CPS) was extracted from 1611E phage, and the K2-ORF16 protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Alcian blue (Fig. 1C). The results demonstrated that the K2-ORF16 protein can break down K2 capsular polysaccharide (CPS). The sensitivity of this enzyme to K2-type capsules was further confirmed in seven additional capsular-type K2 strains. The results showed that the K2-ORF16 protein is a K2 capsular depolymerase.

Presumptive identification K62 capsular depolymerase

Phages infecting the capsular K10 and K62 strains were isolated from untreated water and designated phage Ref-K10-1. Two putative capsular depolymerases (ORF5 and ORF6) were identified from phage genome sequencing analysis. Following the presentation of these two proteins, ORF6 was confirmed to be the K62 capsular depolymerase that produced translucent spots on plates inoculated with the K62 reference strain. Embodiment 3 : Analysis of Capsular Polysaccharide ( CPS ) Decomposed by K1-ORF34 Capsular Depolymerase

The structure of capsular polysaccharide (CPS) decomposed by K1-ORF34 capsular depolymerase was further analyzed. K1 capsular polysaccharide (CPS) is reduced to oligosaccharides after incubation with purified K1-ORF34 protein. Separation by colloidal permeation chromatography and analysis by mass spectrometry and capillary electrophoresis showed that most of the oligosaccharides are hexasaccharides, while a very small amount of nonasaccharides are dimers and trimers of trisaccharide units (Figure 2A and 2B). From the tandem mass spectrometry analysis of an oligosaccharide with a molecular weight of 1145, it can be seen that glucuronic acid is reduced to a double-bond derivative, which is absorbed at a wavelength of 232 nm, maintaining modifications including pyruvation and acetylation (Fig. 2C). The structure of the decomposed oligosaccharides was determined by NMR spectroscopy (Fig. 2D). Oligosaccharides cleaved by K1-ORF34 could still be recognized by anti-K1 antiserum (data not shown). Therefore, the K1-ORF34 enzyme is a lytic enzyme and can be used in the development of K1 capsular polysaccharide (CPS) conjugated vaccines. Embodiment 4 : Analysis of Capsular Polysaccharide ( CPS ) Decomposed by Capsular Depolymerase

analyze K2-ORF16 Capsular polysaccharide broken down by capsular depolymerase ( CPS )

The structure of K2 capsular polysaccharide (CPS) cleaved by K2-ORF16 capsular depolymerase was further analyzed. After incubation with purified K2-ORF16 protein, K2 capsular polysaccharide (CPS) is reduced to oligosaccharides. The spectral results showed that two oligosaccharide compositions with different molecular weights (mass-to-charge ratio (m/z) of 703 and 1365) were released after enzyme degradation (Figure 3A), and were analyzed by electrospray dissociation mass spectrometry-mass spectrometry. The analysis was examined further (Figures 3B and 3C). According to the previously revealed structure of K2 capsular polysaccharide (CPS), the main product of the capsular polysaccharide (CPS) decomposed by K2-ORF16 is a tetrasaccharide (one repeating unit), and the secondary product is an octasaccharide (two repeating units). Therefore, K2-ORF16 enzyme is a hydrolase and can be used to develop K2 capsular polysaccharide (CPS) conjugate vaccine.

analyze K62 Capsular polysaccharide broken down by capsular depolymerase ( CPS ).

The structure of K62 capsular polysaccharide (CPS) decomposed by K62 capsular depolymerase was further analyzed. K62 capsular polysaccharide (CPS) is reduced to oligosaccharides after incubation with purified K62 capsular depolymerase. From the analysis results of matrix-assisted laser desorption free/time-of-flight mass spectrometry and tandem mass spectrometry, it can be seen that the decomposed K62 capsular polysaccharide (CPS) product is mainly two repeating units (deca-saccharide), and a small amount of one repeating unit unit (pentasaccharide) (Fig. 4). From the quality analysis point of view, K62 capsular depolymerase is a hydrolase and can be used in the development of K62 capsular polysaccharide (CPS) conjugated vaccine.

Capsular polysaccharide (CPS) broken down by the capsular depolymerases of KN2, K24, K28 and K64 was analyzed according to a similar method as described above. Embodiment 5 : Capsular polysaccharide ( CPS ) conjugation vaccine

The main sugar chain lengths of the K1, K2, K24, K28, K62 and K64 oligosaccharides used to conjugate the CRM197 carrier protein are as follows: hexasaccharide (two repeating units of K1 capsular polysaccharide (CPS)), tetrasaccharide (K2 capsular polysaccharide) polysaccharide (CPS)), dodecose (two repeating units of K62 capsular polysaccharide (CPS)), hexasaccharide (one repeating unit of K64 capsular polysaccharide (CPS)), pentasaccharide (K24 capsular polysaccharide) polysaccharide (CPS)) and hexasaccharide (K28 a repeating unit of capsular polysaccharide (CPS)). The ratio of epitopes is determined by matrix-assisted laser desorption/time-of-flight mass spectrometry and the amount of glycans is calculated accordingly. Taking K1 as a representative, by changing the amount of decomposed K1-capsular polysaccharide-thiol (CPS-SH), we can get the immunogen ratio from 1:1.4 to 1:10.2; the detailed immunogen ratio of K1 vaccine is as shown in the table below Show. Immunogen ratios were determined by matrix-assisted laser desorption/time-of-flight mass spectrometry. Embodiment 6 : Toxicology test (taking K1 as a representative example)

The body weight (Fig. 5A) and rectal temperature (Fig. 5B) of mice were recorded one day before and one day after each immunization with the K1 capsular polysaccharide (CPS) conjugate vaccine. Sera from mice were collected one week after the first, second, and third immunizations with K1 capsular polysaccharide (CPS) conjugate vaccine, and then liver (serum transaminase (ALT)) and kidney (blood urea nitrogen ( BUN) and creatinine) functions (Fig. 5C). These results showed that no side effects were observed in mice immunized with three doses of the K1 capsular polysaccharide (CPS) conjugate vaccine. Toxicological determination of capsular polysaccharide (CPS) of K2, K24, K28, K62 or K64 was also performed according to a method similar to that described above. Embodiment 7 : Antibody response and serum bactericidal test

Sera from mice administered K1, K2, or K62 capsular polysaccharide (CPS) conjugate vaccines by intramuscular injection were collected for antibody detection and bactericidal assays. The results of the immunospot assay showed that antibodies induced by the capsular polysaccharide (CPS) conjugate vaccine could interact with its original capsular polysaccharide (CPS) (Figure 6).

Results from the bactericidal assay showed that sera from mice immunized with the K1 capsular polysaccharide (CPS) conjugate vaccine, even at a 1:128 dilution, killed > 50% K1 bacteria (bactericidal power price 32 to 128). The bactericidal potency of K2 bacteria in sera from mice immunized with the K2 capsular polysaccharide (CPS) conjugate vaccine ranged from 8 to 32. Bactericidal activity against K62 bacteria (bactericidal potency valence 8 to 32) was detected in 40% of the sera of mice immunized with the K62 capsular polysaccharide (CPS) conjugated vaccine. Therefore, the capsular polysaccharide (CPS) conjugate vaccine can successfully induce the production of capsular type-specific antibodies in mice, and the antibodies have bactericidal effects. Embodiment 8 : Protection test

The present invention also uses K. pneumoniae to challenge immunized mice to evaluate whether the vaccine has a protective effect in vivo. Mice were immunized with K1 and K2 capsular polysaccharide (CPS) conjugate vaccines by intramuscular injection once a week (the control group was only given adjuvant). One week after the third immunization, mice were infected intraperitoneally with 1×10 ^colony -forming unit (CFU) of Klebsiella pneumoniae NTUH-K2044 or NTUH-A4528. Results 30 days after challenge with Klebsiella pneumoniae showed that the survival rate of mice receiving K1 or K2 capsular polysaccharide (CPS) conjugate vaccine was significantly higher than that of mice receiving adjuvant alone (Figure 7A and B).

Mice treated with cyclophosphamide to induce neutropenia and immunized with the K62 capsular polysaccharide (CPS) conjugate vaccine were then challenged with 5 × ¹⁰ colony-forming units (CFU) of K62 bacteria. . The K62 capsular polysaccharide (CPS) conjugate vaccine significantly protected immune-compromised mice from infection with K62 bacteria (Fig. 7C). Embodiment 9 : Efficacy of K1 and K2 capsular polysaccharide ( CPS ) conjugated bivalent vaccines

The common capsular types of Klebsiella pneumoniae strains that cause invasive infections are the K1 and K2 capsular types. Therefore, the protective efficacy of the bivalent vaccine, a mixture of equal amounts of K1 and K2 capsular polysaccharide (CPS) conjugated vaccines, was further examined (Figures 8A and 8B). After the mice in the immunization adjuvant group were infected by intraperitoneal injection with 1×10 ⁴ colony-forming unit (CFU, colony-forming unit) of Klebsiella pneumoniae of NTUH-K2044 and NTUH-A4528, 80% of the and 100% mortality; in contrast, no deaths were observed in mice immunized with the bivalent vaccine following intraperitoneal infection. Therefore, mice immunized with the bivalent vaccine were significantly protected from infection by both K1 and K2 Klebsiella pneumoniae. Embodiment 10 : Structure of capsular polysaccharide ( CPS ) decomposed by K64 , K24 , K28 and KN2 capsular depolymerases

1.K64

The decomposed K64 capsular polysaccharide (CPS) is major in one repeating unit (hexasaccharide) and minor in two repeating units (dodecaccharide). K64 capsular polysaccharide (CPS) cleavage enzyme is a lytic enzyme due to the new double bond signal shown in the NMR spectrum of the reaction product. The results of tandem mass spectrometry analysis of the main K64 fragment (hexasaccharide) are shown in Figure 9.

2.K24

According to the published structure of K24 capsular polysaccharide (CPS), it is composed of four hexose sugars and one hexuronic acid. The mass spectrum of the decomposed K24 capsular polysaccharide (CPS) showed that the main product was an acetylation-modified double repeating unit oligosaccharide [mass-to-charge ratio (m/z) 1773, (Hex) ₈ (HexA) ₂ ], and the minor single product was The sugars are acetylated (mass-to-charge ratio (m/z) 907 and 865), which shows that the K24 enzyme acts as a hydrolase. The main K24 fragment tandem mass spectrometry analysis results (pentasaccharide) are shown in Figure 10.

3. K28

From the results of matrix-assisted laser desorption/time-of-flight mass spectrometry analysis, it can be seen that the decomposition product-KN 2 capsular polysaccharide (CPS) is dominant in one repeating unit (hexasaccharide). The main K28 fragment tandem mass spectrometry results (hexasaccharide) are shown in Figure 11.

4.KN2

Klebsiella pneumoniae KN2 belongs to a new capsule type, and there is still no literature describing its chemical structure. From the results of matrix-assisted laser desorption/time-of-flight mass spectrometry analysis, it can be seen that the decomposition product-KN2 capsular polysaccharide (CPS) is composed of four hexose sugars and one hexuronic acid (molecular weight 1027). In addition, based on the mass-to-charge ratio (m/z) results of matrix-assisted laser desorption/time-of-flight mass spectrometry analysis, the results showed that the KN2 enzyme is a hydrolase. The mass-to-charge ratio (m/z) 1027 was further analyzed by electrospray dissociation mass spectrometry-mass spectrometry. Tandem mass spectrometry analysis of the fragments confirmed our suspicions to be correct. In the near future, gas chromatography–mass spectrometry (GC-MS) analysis of KN2 capsular polysaccharide (CPS) components and their linkers will be used to elucidate the detailed chemistry of KN2 capsular polysaccharide (CPS). structure. The results of matrix-assisted laser desorption/time-of-flight mass spectrometry analysis of capsular polysaccharide (CPS) decomposed by KN2 hydrolase are shown in Figure 12. Embodiment 11 : Comparison of capsular polysaccharide ( CPS ) vaccine and capsular polysaccharide ( CPS ) conjugated vaccine

Comparison of induction of anticapsular polysaccharide (CPS) antibodies and serum bactericidal in mice immunized with K1 unprocessed capsular polysaccharide (CPS) (2 mg) or K1 capsular polysaccharide (CPS) conjugated vaccine (2 mg) activity (Fig. 13A and B). The amount of anti-K1 antibodies induced in mice that received the K1 capsular polysaccharide (CPS) conjugate vaccine was higher than that in mice that received K1 unprocessed capsular polysaccharide (CPS). Therefore, the serum bactericidal activity of mice receiving the K1 capsular polysaccharide (CPS) conjugate vaccine was also significantly higher.

without

Figure 1 (A) to (C) shows the enzyme activity of purified K2-ORF16 protein. (A) Purified K2-ORF16 protein (indicated by arrow) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. Its protein size (kilodalars) Endocytosis) is marked next to the protein standard; (B) When phage 1611E-K2-1 (left) and its K2-ORF16 protein (right) are respectively spotted on an overlay containing K. pneumoniae ( K. pneumoniae ) 1611E strain Clear spots with translucent halos and translucent spots can be observed when placed on a multi-well plate of top agar; (C) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation of different amounts of Extracted K2 capsular polysaccharide (CPS) treated with K2-ORF16 protein and stained with Alcian blue.

Figure 2 (A) to (D) shows mass and capillary electrophoresis analysis of K1 capsular polysaccharide (CPS) proteolytically digested by K1-ORF34. (A) Mass distribution of K1 capsular polysaccharide (CPS) cleaved by K1-ORF34 and separated by Bio-Gel P-6 Gel (BIO-RAD #1504130) colloidal column; (B) K1 cleaved by K1-ORF34 Capillary electrophoresis of capsular polysaccharide (CPS); (C) Tandem mass spectrometry analysis of K1 capsular polysaccharide (CPS) fragment (molecular weight: 1,145); (D) H NMR spectrum of K1 oligosaccharide.

Figures 3(A) to (C) show structural analysis of K2 capsular polysaccharide (CPS) cleaved by K2-ORF16 proteolytic process. (A) Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis of K2 capsular polysaccharide (CPS) broken down by K2-ORF16 proteins; (B) Electron with a mass-to-charge ratio (m/z) of 703 Sprinkle ionization-primary mass spectrometry analysis; and (C) Electrospray ionization mass spectrometry-mass spectrometry analysis with a mass-to-charge ratio (m/z) of 1365.

Figure 4 shows tandem mass spectrometry analysis of the structure of the main product of K62 capsular polysaccharide (CPS) degraded by K62 capsular depolymerase.

Figures 5(A) to (C) show the safety profile of the K1 capsular polysaccharide (CPS) conjugate vaccine. (A) The body weight of mice was recorded one day before and one day after each immunization with K1 capsular polysaccharide (CPS) conjugate vaccine. (B) Rectal temperatures were recorded in mice one day before and one day after each immunization with K1 capsular polysaccharide (CPS) conjugate vaccine. (C) Hepatic (serum transaminases (ALT)) and renal (blood urea nitrogen (BUN) and creatinine) functions were measured in mice one week after each immunization with the K1 capsular polysaccharide (CPS) conjugate vaccine.

Figure 6 shows the induction of anti-capsular polysaccharide (CPS) antibodies in mice vaccinated with K1, K2, or K62 capsular polysaccharide (CPS) conjugate vaccine. Different amounts of K1 or K2 (2000 nanograms to 7.8125 nanograms, 2-fold dilution) or K62 capsular polysaccharide (CPS) (4000 nanograms to 500 nanograms, 2-fold dilution) were transferred to the membrane and separated from the inoculation Serum (1:1000 dilution) obtained from mice vaccinated with K1, K2, or K62 capsular polysaccharide (CPS) was used for blot analysis.

Figure 7 A to C shows the efficacy of K1 or K2 or K62 capsular polysaccharide (CPS) conjugate vaccines in mice. Five mice immunized with K1 or K2 capsular polysaccharide (CPS) conjugate vaccine were each intraperitoneally inoculated with 1×10 ⁴ colony-forming unit (CFU) of Klebsiella pneumoniae NTUH-K2044 ( A) or NTUH-A4528. Vaccination with the K1 or K2 capsular polysaccharide (CPS) conjugate vaccine significantly increased infection with K. pneumoniae NTUH-K2044 ( P = 0.0144, log-rank test) or NTUH-A4528 ( P = 0.0023, log Survival rate of mice using log-rank test. Five animals were vaccinated with K62 capsular polysaccharide (CPS) conjugated vaccine and treated with cyclophosphamide to induce neutropenia, challenged with 5 × 10 ⁶ colony-forming units (CFU) of K62 Klebsiella pneumoniae (C). Cytopenic mice. The K62 capsular polysaccharide (CPS) conjugate vaccine significantly protected immunocompromised mice from K62 K. pneumoniae infection ( P = 0.0448, log-rank test).

Figure 8A and B shows the efficacy of K1 and K2 capsular polysaccharide (CPS) conjugated bivalent vaccines in mice. Five mice immunized with K1 and K2 capsular polysaccharide (CPS) conjugated bivalent vaccines were each intraperitoneally injected with 1×10 ⁴ colony-forming unit (CFU) of Klebsiella pneumoniae NTUH- K2044(A) or NTUH-A4528(B). Vaccination with the K1 or K2 capsular polysaccharide (CPS) conjugate vaccine significantly increased infection with K. pneumoniae NTUH-K2044 ( P = 0.0143, log-rank test) or NTUH-A4528 ( P = 0.0023, log Survival rate of mice using log-rank test.

Figures 9 to 12 show the spectra of K64 capsular polysaccharide (CPS), K24 capsular polysaccharide (CPS), K28 capsular polysaccharide (CPS) and KN2 capsular polysaccharide (CPS), respectively.

Figure 13 A and B shows the induction of anti-K1 capsular polysaccharide (CPS) (A) and serum bactericidal activity in mice vaccinated with unprocessed K1 capsular polysaccharide (CPS) or K1 capsular polysaccharide (CPS) conjugate vaccine. (B) Antibodies. Different amounts of K1 (2000 nanograms to 7.8125 nanograms, two-fold dilution) were transferred to the membrane and vaccinated mice were conjugated with unprocessed K1 capsular polysaccharide (CPS) or K1 capsular polysaccharide (CPS). The serum obtained (1:1000 dilution) was used for ink blot analysis.

          Sequence Listing <![CDATA[<110> National Taiwan University]]> Academia Sinica <![CDATA[<120> Klebsiella pneumoniae capsular polysaccharide vaccine]]> <![CDATA[<130> 106134175]]> <![CDATA[<150> US 62/403,365]]> <![CDATA[<151> 2016-10-03]]> <![CDATA[<150> TW 106134175]]> <! [CDATA[<151> 2017-10-03]]> <![CDATA[<160> 7 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1] ]> <![CDATA[<211> 651]]> <![CDATA[<212> PRT]]> <![CDATA[<213> K1 specific phage]]> <![CDATA[<400> 1 ]]> Met Ala Leu Ile Arg Leu Val Ala Pro Glu Arg Val Phe Ser Asp Leu 1 5 10 15 Ala Ser Met Val Ala Tyr Pro Asn Phe Gln Val Gln Asp Lys Ile Thr 20 25 30 Leu Leu Gly Ser Ala Gly Gly Asp Phe Thr Phe Thr Thr Thr Ala Ser 35 40 45 Val Val Asp Asn Gly Thr Val Phe Ala Val Pro Gly Gly Tyr Leu Leu 50 55 60 Arg Lys Phe Val Gly Pro Ala Tyr Ser Ser Trp Phe Ser Asn Trp Thr 65 70 75 80 Gly Ile Val Thr Phe Met Ser Ala Pro Asn Arg His Leu Val Val Asp 85 90 95 Thr Val Leu Gln Ala Thr Ser Val Leu Asn Ile Lys Ser Asn Ser Thr 100 105 110 Leu Glu Phe Thr Asp Thr Gly Arg Ile Leu Pro Asp Ala Ala Val Ala 115 120 125 Arg Gln Val Leu Asn Ile Thr Gly Ser Ala Pro Ser Val Phe Val Pro 130 135 140 Leu Ala Ala Asp Ala Ala Ala Gly Ser Lys Val Ile Thr Val Ala Ala 145 150 155 160 Gly Ala Leu Ser Ala Val Lys Gly Thr Tyr Leu Tyr Leu Arg Ser Asn 165 170 175 Lys Leu Cys Asp Gly Gly Pro Asn Thr Tyr Gly Val Lys Ile Ser Gln 180 185 190 Ile Arg Lys Val Val Gly Val Ser Thr Ser Gly Gly Val Thr Ser Ile 195 200 205 Arg Leu Asp Lys Ala Leu His Tyr Asn Tyr Tyr Leu Ser Asp Ala Ala 210 215 220 Glu Val Gly Ile Pro Thr Met Val Glu Asn Val Thr Leu Val Ser Pro 225 230 235 240 Tyr Ile Asn Glu Phe Gly Tyr Asp Asp Leu Asn Arg Phe Phe Thr Ser 245 250 255 Gly Ile Ser Ala Asn Phe Ala Ala Asp Leu His Ile Gln Asp Gly Val 260 265 270 Ile Ile Gly Asn Lys Arg Pro Gly Ala Ser Asp Ile Glu Gly Arg Ser 275 280 285 Ala Ile Lys Phe Asn Asn Cys Val Asp Ser Thr Val Lys Gly Thr Cys 290 295 300 Phe Tyr Asn Ile Gly Trp Tyr Gly Val Glu Val Leu Gly Cys Ser Glu 305 310 315 320 Asp Thr Glu Val His Asp Ile His Ala Met Asp Val Arg His Ala Ile 325 330 335 Ser Leu Asn Trp Gln Ser Thr Ala Asp Gly Asp Lys Trp Gly Glu Pro 340 345 350 Ile Glu Phe Leu Gly Val Asn Cys Glu Ala Tyr Ser Thr Thr Gln Ala 355 360 365 Gly Phe Asp Thr His Asp Ile Gly Lys Arg Val Lys Phe Val Arg Cys 370 375 380 Val Ser Tyr Asp Ser Ala Asp Asp Gly Phe Gln Ala Arg Thr Asn Gly 385 390 395 400 Val Glu Tyr Leu Asn Cys Arg Ala Tyr Arg Ala Ala Met Asp Gly Phe 405 410 415 Ala Ser Asn Thr Gly Val Ala Phe Pro Ile Tyr Arg Glu Cys Leu Ala 420 425 430 Tyr Asp Asn Val Arg Ser Gly Phe Asn Cys Ser Tyr Gly Gly Gly Gly Tyr 435 440 445 Val Tyr Asp Cys Glu Ala His Gly Ser Gln Asn Gly Val Arg Ile Asn 450 455 460 Gly Gly Arg Val Lys Gly Gly Arg Tyr Thr Arg Asn Ser Ser Ser His 465 470 475 480 Ile Phe Val Thr Lys Asp Val Ala Glu Thr Ala Gln Thr Ser Leu Glu 485 490 495 Ile Asp Gly Val Ser Met Arg Tyr Asp Gly Thr Gly Arg Ala Val Tyr 500 505 510 Phe His Gly Thr Val Gly Ile Asp Pro Thr Leu Val Ser Met Ser Asn 515 520 525 Asn Asp Met Thr Gly His Gly Leu Phe Trp Ala Leu Leu Ser Gly Tyr 530 535 540 Thr Val Gln Pro Thr Pro Pro Arg Met Ser Arg Asn Leu Leu Asp Asp 545 550 555 560 Thr Gly Ile Arg Gly Val Ala Thr Leu Val Ala Gly Glu Ala Thr Val 565 570 575 Asn Ala Arg Val Arg Gly Asn Phe Gly Ser Val Ala Asn Ser Phe Lys 580 585 590 Trp Val Ser Glu Val Lys Leu Thr Arg Leu Thr Phe Pro Ser Ser Ala 595 600 605 Gly Ala Leu Thr Val Thr Ser Val Ala Gln Asn Gln Asp Val Pro Thr 610 615 620 Pro Asn Pro Asp Leu Asn Ser Phe Val Ile Arg Ser Ser Asn Ala Ala 625 630 635 640 Asp Val Ser Gln Val Ala Trp Glu Val Tyr Leu 645 650 <![CDATA[<210> 2]]> <![ CDATA[<211> 668]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Phage 1611E-K2-1]]> <![CDATA[<400> 2]]> Met Thr Ile Ile Lys Arg Ala Asp Leu Gly Arg Pro Leu Thr Trp Asp 1 5 10 15 Glu Leu Asp Asp Asn Phe Gln Gln Val Asp Asp Leu Thr Ala Ala Ala 20 25 30 Ser Ala Ala Val Leu Ser Ala Ser Ala Ser Ala Thr Ala Ala Ala Gly 35 40 45 Ser Ala Thr Asn Ser Leu Asn Ser Ala Asn Ser Ala Ala Ser Ser Ala 50 55 60 Asp Asp Ala Ala Ala Ser Ala Thr Val Ala Ile Asn Ala Leu Met Asn 65 70 75 80 Ser Thr Phe Glu Pro Ala Asp Phe Asp Phe Thr Ser Gly Gly Thr Leu 85 90 95 Asp Ser Thr Asp Arg Asn Lys Ala Val Tyr Asn Pro Ala Asp Asn Asn 100 105 110 Trp Tyr Ser Trp Ser Gly Ile Leu Pro Lys Ile Val Thr Ala Ala Thr 115 120 125 Asp Pro Thr Ala Asp Ser Asn Trp Lys Pro Arg Thr Asp Gln Leu Leu 130 135 140 Arg Gln Asn Leu Ala Ser Ser Val Ile Pro Gly Thr Ser Leu Val Thr 145 150 155 160 His Ser Asp Gly Ile His Leu Asp Asp Tyr Ile Glu Ile Phe Asn Arg 165 170 175 Arg Thr Lys Phe Ile Met Pro Glu Asp Phe Pro Gly Thr Asp Thr Glu 180 185 190 Gln Leu Gln Ser Ala Leu Ser Tyr Ala Lys Ser Asn Arg Val Asn Val 195 200 205 Val Leu Gln Ala Gly Lys Thr Tyr Tyr Val Thr Gly Ser Gln Gly Leu 210 215 220 Glu Val Asp Leu Gly Tyr Tyr Ser Phe Glu Ser Pro Asn Gly Ile Ala 225 230 235 240 Tyr Ile Asp Phe Thr Gly Cys Thr Ala Thr Tyr Cys Leu Trp Val His 245 250 255 Ser Ser Arg Pro Tyr Pro Asp Gly Ser Glu Asn His Cys Thr Ser Met 260 265 270 Arg Gly Ile Lys Phe Lys Ser Ser Val Lys Gly Ile Gly Gln Arg Leu 275 280 285 Leu Leu Thr Gly Asn Asn Asn Asp Ser Ser Asn Gly Thr Tyr Asn Gly 290 295 300 Asp Cys Lys Ile Glu Asn Cys Met Phe Ser Thr Ala Asp Ile Val Leu 305 310 315 320 Gly Ala Ser Asn Ser Thr Trp Arg Tyr Lys Phe Ile Asn Cys Gly Phe 325 330 335 Met Met Glu Ser Thr Gly Gly Thr Tyr Ala Met His Phe Pro Ala Gly 340 345 350 Ile Ser Asp Ser Gly Glu Ser Val Thr Phe Gln Asn Cys Lys Ile Phe 355 360 365 Asp Met Lys Gly Cys Pro Ile Leu Val Glu Cys Ala Ser Phe Ala Ile 370 375 380 Gly Met Pro Gly Thr Ser Val Leu Asn Thr Pro Ile Lys Ile Thr Gly 385 390 395 400 Ser Gly Ala Met Val Ile Met Asp Ser Ala Ala Asn Ile Glu Asn Pro 405 410 415 Gly Ala Ser Ala Trp Tyr Arg Tyr Gly Glu Val Thr Gly Thr Gly Ala 420 425 430 Arg Leu Ile Leu Asn Gly Cys Thr Leu Val Cys Asn Asn Pro Ser Leu 435 440 445 Gln Thr Lys Pro Leu Phe Tyr Val Gly Ala Asn Ala Phe Ile Asp Val 450 455 460 Thr Leu Val Lys Thr Pro Gly Asn Asp Tyr Leu Phe Gln Asn Gly Asp 465 470 475 480 Glu Gly Leu Arg Thr Phe Val Glu Gly Asp Gly Tyr Val Thr Ala Ser 485 490 495 His Cys Ile Gly Asp Ile Leu Ser Gly Val Gly Asn Ile Pro Leu His 500 505 510 Lys Ser Leu Asn Pro Thr Leu Asn Pro Gly Phe Glu Thr Gly Asp Leu 515 520 525 Ser Ser Trp Ser Phe Asn Asn Gln Gly Ser Ala Ser Gln Thr Cys Val 530 535 540 Val Gly Thr Ala Tyr Lys Lys Thr Gly Thr Tyr Gly Ala Arg Met Thr 545 550 555 560 Ser Phe Gly Ser Leu Ser Cys Phe Leu Thr Gln Lys Val Lys Val Thr 565 570 575 Gln His Gly Tyr Tyr Ser Thr Thr Cys Gln Ile Asn Thr Ile Thr Ala 580 585 590 Gly Thr Gly Thr Thr Ala Gly Ser Leu Thr Ile Thr Phe Tyr Asn Arg 595 600 605 Asp Gly Asn Ala Leu Gln Ala Gly Ala Ser Ser Asn Phe Thr Asn Thr 610 615 620 Pro Ser Gly Trp Gln Ser Val Gly Arg Phe Ile Gln Gly Arg Val Pro 625 630 635 640 Gln Ala Ala Glu Tyr Cys Glu Val Ser Phe Arg Cys Arg Glu Gly Ala 645 650 655 Val Ile Asp Val Asp Asn Phe Ile Ile Asn Phe Thr 660 665 <![CDATA[<210> 3]]> <![CDATA[<211> 535]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence]] > <![CDATA[<220>]]> <![CDATA[<223> Synthetic polypeptide; a carrier protein]]> <![CDATA[<400> 3]]> Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 180 185 190 Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 305 310 315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr 435 440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525 Leu Phe Phe Glu Ile Lys Ser 530 535 <![CDATA[<210> 4]]> <![CDATA [<211> 39]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Synthetic polynucleotide; 1611E-ORF16 forward primer]]> <![CDATA[<400> 4]]> caaacatcac ggtgacgcta gcatgaccat tatcaaacg 39 <![CDATA[<210> 5]]> <![CDATA[ <211> 38]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > Synthetic polynucleotide; 1611E-ORF16 reverse primer]]> <![CDATA[<400> 5]]> cttttaacat ttagcactcg agtgtaaaat taataatg 38 <![CDATA[<210> 6]]> <![CDATA[< 211> 21]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic polynucleotide; Ref-K10-1 ORF6 forward primer]]> <![CDATA[<400> 6]]> atgaataaga tgtttaccca g 21 <![CDATA[<210> 7]]> <![CDATA[ <211> 22]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > Synthetic polynucleotide; Ref-K10-1 ORF6 reverse primer]]> <![CDATA[<400> 7]]> aattgggcga aggcgttcaa ac 22
      

Claims (15)

A Klebsiella pneumoniae capsular polysaccharide vaccine, which contains: an immunogen containing the following formula (II) , formula (III) , formula (IV) , formula (V) , formula (VI) or combinations thereof of sugar repeating units, wherein n is 1 to 4, and the immunogen is in the broken down capsular polysaccharide-thiol form. The vaccine according to claim 1, wherein the immunogen contains the sugar repeating unit of formula (II), and n is 1. The vaccine according to claim 1, wherein the immunogen contains the sugar repeating unit of formula (III), and n is 1. The vaccine according to claim 1, wherein the immunogen contains the sugar repeating unit of formula (IV), and n is 2. The vaccine according to claim 1, wherein the immunogen contains the sugar repeating unit of formula (V), and n is 1. The vaccine according to claim 1, wherein the immunogen contains the sugar repeating unit of formula (VI), and n is 1. A Klebsiella pneumoniae capsular polysaccharide vaccine, which is a capsular polysaccharide conjugated bivalent vaccine containing the immunogens of formula (III) and formula (IV) as described in claim 1. A Klebsiella pneumoniae capsular polysaccharide vaccine, which is a capsular polysaccharide formed from a mixture of the immunogen of formula (III), the immunogen of formula (IV) and a carrier as described in claim 1 Conjugated bivalent vaccine, wherein the immunogen of formula (III) and the immunogen of formula (IV) are respectively conjugated to the carrier. The vaccine according to claim 8, wherein the carrier protein, peptide, lipid, polymer, dendrimer, virion, virus-like particle (VLP) or a combination thereof. The vaccine according to claim 9, wherein the carrier is a protein carrier. The vaccine according to claim 10, wherein the protein carrier system is bacterial toxoid, toxin, exotoxin and non-toxic derivatives thereof or combinations thereof. The vaccine according to claim 10, wherein the protein carrier system is selected from the group consisting of keyhole hemocyanin (KLH), hepatitis B virus core protein, thyroglobulin, albumin, human serum albumin (HSA), egg Albumin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), tuberculin purified protein derivative (PPD), transferrin-binding protein, polyamino acids, tetanus toxoid, tetanus toxin fragment C, Diphtheria toxoid, CRM (nontoxic diphtheria toxin mutant), cholera toxin, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat-labile enterotoxin, Pseudomonas aeruginosa exotoxin A, bacterial outer membrane proteins, meninges Neisseria serotype B outer membrane protein complex (OMPC) and outer membrane porin class 3 (rPorB). The vaccine according to claim 10, wherein the protein carrier system has CRM197 of the amino acid sequence shown in SEQ ID NO: 3. Use of a vaccine as described in claim 1, 7 or 8 for preparing a pharmaceutical composition that triggers an immune response against Klebsiella pneumoniae. The use of a vaccine as described in claim 1, 7 or 8 for preparing a pharmaceutical composition for preventing Klebsiella pneumoniae infection.