TW201834681A - Klebsiella pneumoniae capsule polysaccharide vaccines - Google Patents

Abstract

The invention provides various immunogens comprising a repeat unit of saccharide of Klebsiella pneumoniae CPS, which has a formula selected from the group consisting 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 pneumoniae and preventing infection of Klebsiella pneumoniae by using an immunogen of the invention.

Description

 Klebsiella pneumoniae capsular polysaccharide conjugate vaccine  

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

Klebsiella pneumoniae is an important pathogen causing 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 is increased to 30-40. %. Survivors of metastatic meningitis usually have severe neurological sequelae, and metastatic endophthalmitis usually affects the eye causing blindness. In addition to causing bacterial liver abscess (PLA), Klebsiella pneumoniae has also been shown to cause invasive infections, leading to abscesses in other areas (eg kidney, spleen, brain, prostate), necrotizing fasciitis And severe bacteremia pneumonia.

The study reported that the most common type of capsule for the strain causing PLA is K1 and the second is K2. In addition to these studies of bacterial liver abscess (PLA), recent studies have also indicated that K1 and K2 capsule types of Klebsiella pneumoniae can cause other necrotizing fasciitis and community-acquired bacteremia. Invasive infection of pneumonia ( Lin YT et al., BMC infectious diseases 2010, 10: 307; and Fang CT et al., 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 is also a significant problem for antibiotic resistance to broad-spectrum β-endoamines and carbapenems. Our recent studies indicate that capsular types K64 (38%), K62 (13%), K24 (8%), KN2 (7%) and K28 (6%) account for Klebs anti-carbapene antibiotics. 72% of the strains of Klebsiella pneumoniae (Carbapenem-Resistant Klebsiella pneumoniae ; CRKP), in the study can be observed that the capsule type of CRKP is concentrated.

Vaccines designed for bacterial capsules, such as vaccines for S. pneumoniae, are generally effective against infections caused by such capsular pathogens. A Klebsiella pneumoniae K1 capsular polysaccharide (CPS) vaccine was disclosed in 1985. In 1988, a invention revealed a 24-valent capsular polysaccharide (capsular polysaccharide (CPS)) vaccine against Klebsiella pneumoniae. Although Klebsiella pneumoniae K1 capsular polysaccharide (CPS) and 24-valent capsular polysaccharide (CPS) vaccines have been published many years ago, there is still no Klebsiella pneumoniae vaccine available. Previous studies have shown that polysaccharide vaccines can only induce independent immunity of T cells lacking immune memory and lack of high affinity antibodies. Thus, a Klebsiella pneumoniae capsular polysaccharide (CPS)-protein conjugate vaccine can be more effective against infections caused by the bacterium.

Past studies have indicated that K1 and K2 capsular polysaccharides (CPS) are giant molecules composed of several sugar repeating units ( YANG FL et al., The Journal of biological chemistry 2011, 286(24): 21041-21051 ). Depolymerization of capsular polysaccharide (CPS) will increase the efficiency of coupling to proteins. Some chemical agents, such as trifluoroacetic acid, ammonium hydroxide and acetic acid, which degrade capsular polysaccharide (CPS), result in capsular polysaccharide (CPS) modification (acetylation or acetone), which is important for immune response. Lost. WO20125676A1 provides an isolated phage which can infect K1 Klebsiella pneumoniae strain and has a capsular depolymerase which specifically decomposes K1 capsular polysaccharide (CPS). This enzyme will be used to develop a vaccine that is effective against Klebsiella pneumoniae infection.

Unless otherwise stated, technical terms are used according to conventional usage.

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

Abbreviations used herein: 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 joined together for stimulating or eliciting a specific immune response in an animal. In some embodiments, the immune response refers to a protective one that directly renders the animal more effective against infection by the organism to which the immunogenic conjugate is directed. 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 used to operatively link a molecular bridge of two different molecules, wherein one end of the linker is operatively linked to the first molecule and the other end is operatively linked to The 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 that elicits a prophylactic or therapeutic immune response in a subject. Typically, vaccines cause antigen-specific immune responses to pathogen antigens.

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

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

The term "antibody" as used herein encompasses both polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, altered antibodies, F(ab')2 fragments, F(ab) molecules, Fv fragments, single-stranded fragments distributed on phage. Variant (scFv), single domain antibody, chimeric antibody, humanized antibody and functional fragments thereof showing the immunological binding properties of the parent antibody molecule.

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

The present invention utilizes a capsular polysaccharide (CPS) conjugate vaccine produced by decomposing capsular polysaccharide (CPS) of Klebsiella pneumoniae conjugated to a carrier, respectively. Surprisingly, the capsular polysaccharide (CPS) conjugate vaccine of the present invention can also simultaneously induce bactericidal activity of capsular polysaccharide (CPS) antibodies. Therefore, the capsular polysaccharide (CPS) conjugate vaccine of the present invention can be used for the prevention of invasive infection caused by Klebsiella Klebsiella.

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

In one aspect, the invention provides an immunogen comprising a repeating unit of a trisaccharide of Klebsiella pneumoniae K1 capsular polysaccharide (CPS) having the following formula (I): {→4)-[2,3-( S )-pyruvate]-bD-GlcA p -(1→4)-aL- O -Ac-Fuc p -(1→3)-bD-Glc p -(1→} _m (I)

Where m is 1 to 4.

In a specific embodiment, m is 2.

The present inventors have found that the K1-ORF34 polypeptide can degrade Klebsiella Klebsiella K1 capsular polysaccharide (CPS). After degradation, the hexasaccharide of Klebsiella pneumoniae K1 capsular polysaccharide (CPS) can be obtained. The hexasaccharide degraded from K1 capsular polysaccharide (CPS) can be used as an immunogen against Klebsiella pneumoniae infection. The K1-ORF34 polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

(SEQ ID NO: 1)

In another aspect, the present invention provides an immunogen comprising a repeating unit of a tetrasaccharide of Klebsiella pneumoniae K2 capsular polysaccharide (CPS) having the following formula (II): Where n is 1 to 4.

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

The present inventors have found that the K2-ORF16 polypeptide can degrade Klebsiella Klebsiella K2 capsular polysaccharide (CPS). After degradation, a tetrasaccharide of Klebsiella pneumoniae K2 capsular polysaccharide (CPS) is obtained. The tetrasaccharide produced by the degradation of K2 capsular polysaccharide (CPS) can be used as an immunogen against Klebsiella pneumoniae infection. Accordingly, the present invention provides an isolated polypeptide or variant thereof having a degrading activity specific to a capsule of Klebsiella pneumoniae capsular type K2 strain selected from: (a a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and (B) a polynucleotide encoding at least in (i) the polypeptide coding sequence of SEQ ID NO: 2, or (ii) complementary to (i) A polynucleotide that hybridizes under stringent conditions of full length sequence. In a specific embodiment, the K2-ORF16 polypeptide has an amino acid sequence as set forth in SEQ ID NO:2.

(SEQ ID NO: 2)

A variant of the polypeptide is artificial, which comprises a 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 minor effect, ie the substitution or insertion of a retentive amino acid sequence does not significantly affect the folding and/or activity of the protein, which is typically from 1 to about 30 amino acids. Deletion, small amine or carboxy terminal extension, such as residues of amino terminal methionine, small linker peptides of up to about 20 to 25 residues, or by altering net charge or other functions, such as histidine tags, Polyhistidine tag region, epitope or binding domain.

In another aspect, the invention provides an immunogen comprising a repeating unit of a hexasaccharide of Klebsiella pneumoniae K64 capsular polysaccharide (CPS) having the following formula (III):

Where n is 1 to 4. In a specific embodiment, n is one.

In another aspect, the invention provides an immunogen comprising a repeating unit of a pentasaccharide of Klebsiella pneumoniae K62 capsular polysaccharide (CPS) having the following formula (IV):

Where n is 1 to 4. In a specific embodiment, n is 2.

In another aspect, the invention provides an immunogen comprising a repeating unit of a pentasaccharide of Klebsiella Klebsiella K24 capsular polysaccharide (CPS) having the following formula (V):

Where n is 1 to 4. In a specific embodiment, n is one.

In another aspect, the invention provides an immunogen comprising a repeating unit of a hexasaccharide of Klebsiella pneumoniae K28 capsular polysaccharide (CPS) having the following formula (VI):

Where n is 1 to 4 and n is 1.

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

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

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

The structural saccharide units and degraded capsular polysaccharide (CPS) products of K1, K2, K62, K64, K24 and K28 are as follows.

In a specific embodiment, the immunogen is conjugated to the protein carrier.

In a specific embodiment, the vaccine of the invention is a multivalent 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 an immunogen I-vector and an immunogen II-carrier mixture, wherein the immunogen I and formula (I) The immunogen II line having the formula (II) is bonded to a vector, respectively.

In a specific embodiment, the vaccine is a multivalent vaccine. In some embodiments, the vaccine is a K64 and K62 capsular polysaccharide (CPS) conjugated bivalent vaccine comprising an immunogen of Formula III and Formula IV. In another specific embodiment, the vaccine is a K64 and K62 capsular polysaccharide (CPS) conjugated bivalent vaccine comprising a mixture of an immunogen III-vector and an immunogen IV-vector, wherein the immunization with formula (III) The original III and the immunogen IV having the formula (IV) were respectively conjugated to the carrier.

The immunogen of the invention can be joined to a carrier to form a conjugate. In a specific embodiment, the conjugate is a vaccine. In another specific embodiment, the immunogenic ratio of any one of the conjugate vaccines is between 1:1.4 and 1:10.2. In a specific embodiment, the vaccine comprises from 10% by weight to 90% by weight of the immunogenic I-protein carrier or immunogen III-protein carrier and from 10% by weight to 90% by weight. Percentage of the immunogen II-protein carrier or immunogen IV-protein carrier. The vaccine preferably comprises an equal amount 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, viroid-like particles (VLPs), or combinations thereof. In a specific embodiment, the vector is a protein carrier, including but not limited to a bacterial toxoid, a toxin, an exotoxin, and a non-toxic derivative thereof, such as keyhole limpet hemocyanin (KLH), hepatitis B virus core protein, thyroid gland 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 (non-toxic diphtheria toxin mutant), cholera toxin, staphylococcus aureus exotoxin or toxoid, heat-labile Escherichia coli Enterotoxin, Pseudomonas aeruginosa exotoxin A and bacterial outer membrane proteins (eg, Neisseria meningitidis serotype B outer membrane protein complex (OMPC) and outer membrane class 3 porin (rPorB)).

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

(SEQ ID NO: 3)

Polysaccharides contain hydroxyl groups, occasionally containing carboxyl groups and amine groups, while proteins contain amine groups and carboxyl groups. A number of methods for joining proteins to polysaccharides are known in the art. If the protein carrier has two or more of the same saccharides, the saccharide can be joined to the same molecule of the protein carrier. Alternatively, the saccharide may be coupled to a different molecule of the protein carrier (each protein carrier molecule has only one saccharide bound thereto).

Considerations such as specific antigens, adjuvants (if any), age, sex, weight, type and condition of a particular animal or patient, and route of administration, etc., based on standard techniques well known to those of ordinary skill in the art, in the pharmaceutical and veterinary arts, The amount of the vaccine of the present invention to be administered to a human or animal and the method of administration can be determined.

The conjugate vaccine of the present invention can be administered to a subject in a manner consistent with conventional methods for treatment or prevention, for example, for treating or preventing a method of infection from Klebsiella pneumoniae . The present invention discloses the administration of an effective amount of a conjugate vaccine and/or other bioactive agent that is up to prophylaxis or treatment to a condition in need of such treatment, under conditions sufficient to prevent, inhibit, and/or ameliorate the infection of Klebsiella Klebsiella. The subject is a period of time. In some embodiments, upon vaccination of a conjugate vaccine of the invention, the subject successfully elicits an immune response in a subject against an antigenic immunogen of Klebsiella pneumoniae . In some embodiments, the subject to be treated is selected from a subject who has been infected with Klebsiella pneumoniae or has a risk of developing Klebsiella pneumoniae infection, for example, has been exposed or is likely to be exposed to Klebsiella pneumoniae A subject in the environment of Klebsiella pneumoniae . In an important aspect, the conjugate vaccine provided by the present invention can be used to prevent infection by Klebsiella Klebsiella.

The conjugate vaccine of the present invention can be prepared into a solution, a suspension, a tablet, a pill, a capsule, a sustained release preparation, a powder, and the like. The antigenic and immunogenic compositions can be combined with a physiologically acceptable carrier compatible therewith. These may include water, physiological saline, glucose, glycerol, ethanol, or a combination thereof. The conjugate vaccine may further contain an auxiliary substance such as a wetting or emulsifying agent or a pH buffer to further enhance the efficacy. Administration of the conjugate vaccine can include delivery by a variety of routes, such as oral, intravenous, intramuscular, intranasal, subcutaneous, and intraperitoneal administration.

In another aspect, the invention provides an antibody prepared by administering a conjugate vaccine of the invention.

The following examples are intended to illustrate and not to limit the invention. All percentages used in the present invention are by weight unless otherwise indicated. All patents, patent applications and literature references cited herein are hereby incorporated by reference.

Figure 1 (A) to (C) show the enzyme activity of the purified K2-ORF16 protein. (A) Purified K2-ORF16 protein (indicated by the arrow) was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue for protein size (thousands of ears) The phage is labeled next to the protein standard; (B) when the phage 1611E-K2-1 (left) and its K2-ORF16 protein (right) are spotted in a cover containing the K. pneumoniae 1611E strain, respectively Clear spots with translucent halo and translucent spots were observed on the porous disk of the top agar; (C) Separated with different amounts by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) K2-ORF16 protein treated extracted K2 capsular polysaccharide (CPS) and stained with Alcian blue.

Figures 2(A) to (D) show the mass and capillary electrophoresis analysis of K1 capsular polysaccharide (CPS) decomposed with K1-ORF34 protein. (A) Mass distribution of K1 capsular polysaccharide (CPS) cleaved by K1-ORF34 and isolated by Bio-Gel P-6 Gel (BIO-RAD #1504130) colloidal column; (B) K1 cleavage 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) K1 oligosaccharide nuclear magnetic resonance spectroscopy.

Figures 3 (A) to (C) show the structural analysis of K2 capsular polysaccharide (CPS) decomposed by K2-ORF16 protein. (A) Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis of K2-ORF16 protein-decomposing K2 capsular polysaccharide (CPS); (B) Electricity with a mass-to-charge ratio (m/z) of 703 Sprinkle free-primary mass spectrometry; and (C) mass-to-charge ratio (m/z) of 1365 by electrospray free mass spectrometry-mass spectrometry.

Figure 4 shows a tandem mass spectrometric analysis of the major product structure of K62 capsular polysaccharide (CPS) decomposed by K62 capsular depolymerase.

Figures 5(A) to (C) show the safety of the K1 capsular polysaccharide (CPS) conjugate vaccine. (A) The body weight of the mice one day before and one day after each immunization of the K1 capsular polysaccharide (CPS) conjugate vaccine was recorded. (B) The rectal temperature of the mice one day before and one day after the immunization of the K1 capsular polysaccharide (CPS) conjugate vaccine was recorded. (C) The liver (serum transaminase (ALT)) and kidney (blood urea nitrogen (BUN) and creatinine) functions of mice one week after each immunization with the K1 capsular polysaccharide (CPS) conjugate vaccine were determined.

Figure 6 shows mice vaccinated with K1 or K2 or K62 capsular polysaccharide (CPS) conjugate vaccines that induce antibodies against capsular polysaccharide (CPS). Different amounts of K1 or K2 (2000 Nike to 7.8125 Ng, 2 fold dilution) or K62 capsular polysaccharide (CPS) (4000 Ng to 500 Ng, 2 fold dilution) were transferred to the membrane and inoculated Serum (1:1000 dilution) obtained in mice vaccinated with K1 or K2 or K62 capsular polysaccharide (CPS) was used for dot analysis.

Figures 7A to C show the efficacy of a K1 or K2 or K62 capsular polysaccharide (CPS) conjugate vaccine in mice. Five mice immunized with K1 or K2 capsular polysaccharide (CPS) conjugate vaccine were intraperitoneally injected with 1 × 10 ⁴ colony forming units (CFU, K. pneumoniae NTUH-K2044). A) or NTUH-A4528. Vaccination with 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, logarithm Survival rate of mice with log-rank test. Five K62 capsular polysaccharide (CPS) conjugate vaccines were challenged with 5×10 ⁶ colony forming units (CFU, colony-forming unit) of K62 Klebsiella pneumoniae (C) and treated with cyclophosphamide to induce neurite Mice with sarcopenia. The K62 capsular polysaccharide (CPS) conjugate vaccine significantly protected immunocompromised mice from infection with K62 Klebsiella pneumoniae ( P = 0.0448, log-rank test).

Figures 8A and B show 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 vaccine were intraperitoneally injected with 1×10 ⁴ colony forming units (CFU, Klebsiella pneumoniae NTUH-- K2044 (A) or NTUH-A4528 (B). Vaccination with 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, logarithm Survival rate of mice with 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.

Figures 13A and B show that mice immunized with unprocessed K1 capsular polysaccharide (CPS) or K1 capsular polysaccharide (CPS) conjugate vaccine induced anti-K1 capsular polysaccharide (CPS) (A) and serum bactericidal activity ( B) antibodies. Different amounts of K1 (2000 Nike to 7.8125 Ng, two-fold dilution) were transferred to the membrane and vaccinated with unprocessed K1 capsular polysaccharide (CPS) or K1 capsular polysaccharide (CPS) vaccinated mice The serum obtained (1:1000 dilution) was used for dot analysis.

Materials and Method

Isolation of phage

Purification of phage and titer determination of phage were performed by agar-covered method; phage infectivity was determined by 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 genomic DNA and sequencing

The phage DNA of the phage 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 the phage was precipitated and cleaved, the DNA was extracted with phenol/chloroform, precipitated with ethanol, and finally sequenced by Illumina GAII to determine the genomic sequence of the phage.

Protein expression 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 using the 1611E-ORF16 forward primer (5'-CAAACATCACGGTGACGCTAGCATGACCATTATCAAACG-3'; SEQ ID NO: 4) and the 1611E-ORF16 reverse primer (5'-CTTTTAACATTTAGCACTCGAGTGTAAAATTAATAATG-3'; SEQ ID NO :5) to carry out PCR, and then decompose with NheI and XhoI restriction enzymes, and then clone the decomposed K2- orf16 fragment into the double decomposition sites of NheI and XhpI in pET28c plastid (Novagen) (also by NheI and XhoI) Restriction enzymes are broken down). The K62 capsular depolymerase was first amplified using 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 then cloned into the blunt-ended EcoRI decomposing site of pET28c plastid (Novagen). The obtained plastid was transformed into BL21 type Escherichia coli (DE3), and the recombinant His-tag protein was expressed by induction with IPTG at 0.4 mmol (mM) at 16 ° C, and was provided according to the instructions in the reagent group (Qiagen). The method for protein purification.

Purification of Klebsiella pneumoniae capsular polysaccharide (CPS)

K1, K2 and K62 capsular polysaccharides (CPS) were purified from Klebsiella pneumoniae NTUH-K2044 △ wbbO , NTUH -A4528 △ wbbO and K62 reference △ wbbO mutant strains (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 the contamination of the O polysaccharide. The bacteria were incubated on a Luria agar plate for 12 hours at 37 °C. The 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 15,000 x g for 20 minutes. The supernatant (volume) was mixed with ice-cold acetone (4 volumes) to carry out polysaccharide precipitation. After centrifugation at 12,000 xg for 20 minutes, a raw capsular polysaccharide (CPS) precipitate was obtained. The precipitate was broken up and suspended in sterile water and lyophilized. Unprocessed capsular polysaccharide (CPS) powder was decomposed by ribonuclease (Roche) and deoxyribonuclease I (Roche) for 24 hours at 37 ° C, then with proteinase K at 10 millimolar (mM) Further decomposition 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 by dialysis on a dialysis membrane having a pore size of about 8 to 10 thousand aspirate. Partially purified capsular polysaccharide (CPS) was eluted with 0.1% sodium azide in filtered pure water, followed by further purification on a TSK HW-65F column [1.6 cm (diameter) x 90 cm (height)] . Carbohydrate-containing fragments were detected by phenol-sulfuric acid method and dialyzed against pure water (molecular weight threshold: 1 thousand auricular) and concentrated by lyophilization.

Structural Analysis of Capsular Polysaccharide (CPS) of Capsular Polysaccharides

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 Cray Chemical structure of capsular polysaccharides of N. pneumoniae NTUH-K2044 (K1) and A4528 (K2).

Mass spectrometry: 0.5 μl (μl) of sample with 0.5 μl (μl) of substrate solution (20 mg/ml 2,5-dihydroxybenzoic acid (DHB)) on matrix-assisted laser desorption ionization mass spectrometry Mixing in 50% ACN and 1% aqueous solution of nitric acid (H ₃ PO ₄ ), a mixture is obtained, and the mixture is spotted on a stainless steel plate, and after air drying, the matrix-assisted laser desorption ionization is carried out in a type 4800- The time-of-flight/time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA) was analyzed in a specular reflector mode with an acceleration voltage of 20 kV and a grid voltage of 16%. A typical spectrum is produced by 1000 laser illumination. The original 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 a mass spectrometry analysis on an Ultraflex II matrix assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometer (Bruker Daltonik GmbH, Germany). Mass spectra were obtained in a reflection mode with a mass-to-charge ratio (m/z) ranging from 500 to 4,000. Mass spectrometry/mass spectrometry was performed in elevator mode. Mass spectrometry was performed by the GRC Mass Core Facility of the Central Research Institute Genomics Research Center.

Nuclear Magnetic Resonance Spectroscopy: The present invention records the results of nuclear magnetic resonance spectroscopy of small capsular polysaccharide (CPS) fragments in one heavy water; all two-dimensional nuclear magnetic resonance spectroscopy experiments were performed using standard pulse sequences provided by Bruker. The NMR spectral analysis data was processed using the up-rotation 3.1. Nuclear Magnetic Resonance Spectroscopy Experiment and Resonance Assignment: All NMR experiments were performed on a Bruker AVANCE 600 or AVANCE 800 NMR mass spectrometer (Bruker, Karlsruhe, Germany) equipped with triple ( ¹ H, ¹³ C and ¹⁵ N) resonances. The cryoprobe includes a shielded z-gradient. Two-dimensional (2D) ¹ H NMR, TOCSY and NOESY spectra were collected. Heteronuclear nuclear magnetic resonance experiments of all 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 resonance was assigned using 3D HAHB(CO)NH and HCCH-TOCSY.

Enzymatic cleavage: K1 capsular polysaccharide (CPS) is degraded by K1 lyase by β-hydrogen elimination reaction to produce a double bond between C-4 and C-5 of glucuronic acid, and determined at 232 nm Spectrophotometric change in wavelength absorbance. Three replicate assays were performed in a 1 ml volume of colorimetric tube in a UV-VIS spectrophotometer (Ultrospec 4000 UV/Visible; Amersham Pharmacia Biotech, Piscataway, NJ, USA) at 25 °C. Recombinant K1 lyase (10 μg/ml) was added to magnesium trichloride (MgCl ₂ ) containing 25 mmol (mM), pH 7.5, Tris, 3 mmol (mM) ) and a 40 μg/ml capsular polysaccharide (CPS) reaction mixture, followed by an increase in the absorbance at 232 nm wavelength for 15 minutes. The control group did not contain K1 lyase.

The reduction end is quantified by the 3,5-dinitrosalicylic acid (DNSA) assay (for K2 hydrolase): the present invention is determined by estimating the concentration of 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 was mixed with DNSA and heated to catalyze the reaction, the nitro group at the 3' end of the DNS was reduced by the device FlexStation 3 to an amine capable of measuring the absorbance at a wavelength of 535 nm. The conditions of each hydrolase can be determined by quantifying the concentration of the reducing sugar.

Capillary Electrophoresis: The present invention is subjected to capillary electrophoresis on a Beckman P/ACE MDQ capillary electrophoresis system equipped with a UV detector set at a wavelength of 230 nm. Separation and analysis were performed on a fused silica capillary (inner diameter 77 μm, total length 65 cm from the injection point to the detector 50 cm) coated by electrokinetic chromatography at 25 °C. The operating buffer consisted of sodium phosphate buffer (50 millimolar (mM), pH 9.0). The buffer was degassed by vacuum filtration through a membrane filter having a pore diameter of 0.2 μm, and oscillated in an ultrasonic water tank. Prior to each run, the capillary was first washed with 0.1 molar NaOH for 5 minutes, then with double distilled water for 5 minutes, and then treated with the operating buffer for 5 minutes. The sample to be analyzed was automatically injected using a pressure injection mode in which the sample was pressurized for 15 seconds. Capillary electrophoresis was carried out at 20 kV (about 65 mA) using normal polarity.

Decomposition of capsular polysaccharide (CPS) and DT-CRM197 carrier protein after decomposition

Method for the preparation of glycosylamine by akochetkov amination: Ammonium carbonate (3.0 g, excess) was added 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 time the reaction mixture was lyophilized until the dry weight of the residue remained constant to afford a colorless solid glycosamine, which was used without further purification.

Method for linking a thiol linker via a glycosylamine: adding 3,3'-disulfide to a glycosylamine solution (20 mg of glycosylamine dissolved in 3.0 ml of phosphate buffered saline (pH 7.4)) Substituted bis(sulfosuccinimidyl propionate, DTSSP, 20 mg, excess), followed by addition of 1 molar concentration of NaOH solution to maintain the reaction mixture at pH 7.4 to 7.8 and stirred at room temperature for 16 hours. Dithiothreitol (DTT, 20 mg, excess) was added to the above 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 and purified eluted from EtOAc EtOAc EtOAc EtOAc.

Synthesis of CRM197-maleimide (CRM197-maleimid) : Residues after removal of commercially available CRM197 (1.0 mg) salt by alternately dissolving in water and dialyzed (Amicon Ultra-0.5, 10 thousand auricular) The material was dissolved in phosphate buffered saline (pH 6.5, 1.0 ml) and transferred to a vial. To this solution was added N-ε-maleimide hexanoyloxysulfosuccinimide ester (Sulfo-EMCS, 1.0 mg, 8.22×10 ^-6 mol), then at room temperature The reaction was stirred for 2 hours and the mixture was purified again with Amicon Ultra-0.5 (10 kA). CRM197-maleimide ( CRM197-maleimid ) was stored in phosphate buffered saline (pH 7. After matrix-assisted laser desorption ionization-time-of-flight spectroscopy analysis of molecular weight and BCA determination to calculate the total amount of protein. 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-maleimide ( CRM197-maleimid ) is 61841, the number of maleimide functional groups on CRM197-maleimide ( CRM197-maleimid ) can be determined.

Synthesis of decomposed capsular polysaccharide-CRM197 (CPS-CRM197) conjugate (1 to 4): CRM197-maleimide ( CRM197-maleimid ) was dissolved in phosphate buffered saline (pH 7.2, 1.0 mg/ In ml), different amounts of decomposed capsular polysaccharide-thiol (CPS-SH) (5.0 mg/ml in phosphate buffered saline, pH 7.2) were then added to the solution. The mixture was stirred at room temperature for 2 hours. The decomposed capsular polysaccharide-CRM197 (CPS-CRM197) conjugate was purified by using Amicon Ultra-0.5 (10 thousand octaves) to remove non-reactive decomposed capsular polysaccharide-thiol (CPS- by dialysis) SH) and sodium phosphate salt. 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) is reacted with dithiothreitol (DTT) and recovered by column chromatography on LH-20. By varying the amount of decomposed capsular polysaccharide-thiol (CPS-SH), we can ligate different ratios of oligosaccharide epitopes to the CRM197 vector.

Vaccination

The capsular polysaccharide (CPS)-conjugated vaccine was diluted to a concentration of 100 μg/ml in phosphate buffered saline, and the glycolipid adjuvant C34 was dissolved in dimethylammonium (DMSO) to a concentration of 100 μg/ ML. One hundred microliters of a vaccine mixture consisting of a capsular polysaccharide (CPS) conjugate vaccine (2 micrograms of glycans) and 2 micrograms of 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 once every two weeks for a total of five times.

Detection of antibodies against capsular polysaccharide (CPS)

Different amounts of purified capsular polysaccharide (CPS) were transferred to the membrane by immunospot assay. The antibody against capsular polysaccharide (CPS) was detected by culturing the serum (1:1000 dilution) of the mouse after vaccination.

Serum bactericidal test

The mouse serum was incubated at 56 ° C for 30 minutes, and then serially diluted twice (1/2 times to 1/256 times) with physiological saline. 25 microliters (μl) of diluted serum and 12.5 microliters (μl) of bacterial suspension (100 colony forming units (CFU) were incubated at 37 ° C for 15 minutes. After the incubation, 12.5 μl (μl) of newborn rabbit complement (Pel-Freez, USA) was added and incubated at 37 ° C for 1 hour. The reaction mixture was then spread on an LB disk. After the incubation to the next day, the number of viable bacteria was counted. Serum bactericidal valence was defined as the reciprocal of serum dilution, and the bacteriocidal-suppressed mice achieved 50% bactericidal power compared to control mice vaccinated only with adjuvant.

Protection analysis

For K1 and K2 vaccines: one week after vaccination (5 mice per group), vaccinated or control mice (adjuvant only), 1 x 10 ⁴ colony forming units (CFU) were injected intraperitoneally (IP) , colony-forming unit) NTUH-K2044 or NTUH-A4528 strain. The experiment was carried out for 30 days to observe the mortality of the mice. The survival rate of the mice was analyzed by Kaplan-Meier analysis and by log-rank test; P values <0.05 were considered to be statistically significant differences. For the K62 vaccine: vaccinated or control mice (adjuvant only) were pretreated twice with cyclophosphamide (100 mg/kg) every two days. In cyclophosphamide-treated mice, the number of white blood cells and neutrophils was significantly lower. Two days after cyclophosphamide treatment (5 mice per group), these mice were intraperitoneally inoculated with a K62 reference strain of 5 x 10 ⁶ colony forming units (CFU). A 30-day observation was performed to confirm the mortality of the mice. The survival rate of the mice was analyzed by Kaplan-Meier analysis and by log-rank test; P values <0.05 were considered to be statistically significant differences. The vaccines of the other immunogens described in the present invention were all assayed in a similar manner as described above.

Embodiment 1: Isolation of phage infected with Klebsiella pneumoniae 1611E strain (K2 capsular type)

Previous studies have revealed 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 ). In order to isolate the K2 capsular degrading enzyme, phage infected with Klebsiella pneumoniae 1611E strain (K2 type capsule) were isolated from untreated water. A clear plaque with a translucent halo was detected and the phage was named 1611E-K2-1. The sensitivity of the 1611E-K2-1 phage K2 type capsule was evaluated by performing a polymerase chain reaction assay on the capsule type of the other 7 capsular type K2 strains using the wzy primer. This phage may infect all strains of the K2 type capsule.

Embodiment 2: Identification of putative capsular depolymerase

Putative K2 capsular depolymerase

The whole genome length of phage 1611E-K2-1 was determined to be 47,797 base pairs. Annotation of the genome sequence indicates that the phage is predicted to contain 17 open reading regions (ORFs) of more than 500 base pairs. Open reading region (ORF) analysis of phage 1611E-K2-1 revealed that predicted ORF16 and tail-protrusion 63D sialidase showed 46% amino acid identity, indicating that the protein may correspond to capsular depolymerase. The K2- orf16 gene was cloned into plastids and expressed in E. coli. The purity of the purified recombinant K2-ORF16 protein is shown in Figure 1A. When spotted on a plate inoculated with K. pneumoniae 1611E, the recombinant K2-ORF16 protein produced a translucent spot similar to a plaque halo (Fig. 1B). The capsular polysaccharide (CPS) was extracted from the 1611E phage, and then the K2-ORF16 protein was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Alcian blue (Fig. 1C). The results demonstrate that K2-ORF16 protein can decompose K2 capsular polysaccharide (CPS). The sensitivity of this enzyme to the K2 type capsule was further confirmed in the other seven capsular K2 strains. The results show that the K2-ORF16 protein is a K2 capsular depolymerase.

Putative K62 capsular depolymerase

Phage infected with the capsular type K10 and K62 strains were isolated from untreated water and expressed as phage Ref-K10-1. Two putative capsular depolymerases (ORF5 and ORF6) were identified from phage genome sequencing analysis. After expression of these two proteins, ORF6 was confirmed to be a K62 capsular depolymerase, which produced a translucent spot on the plate inoculated with the K62 reference strain.

Embodiment 3: Analysis of K1-ORF34 capsular depolymerase-decomposed capsular polysaccharide (CPS)

The structure of the capsular polysaccharide (CPS) decomposed by K1-ORF34 capsular depolymerase was further analyzed. The K1 capsular polysaccharide (CPS) is reduced to an oligosaccharide after being cultured with the purified K1-ORF34 protein. Separation by colloidal permeation chromatography and analysis by mass spectrometry and capillary electrophoresis revealed that most of the oligosaccharides were hexasaccharides, while the nonasaccharides were very small, ie dimers and trimers of trisaccharide units (Fig. 2A). And 2B). From the tandem mass spectrometry analysis of oligosaccharides 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, and the modification including pyruvate and acetamidine is maintained (Fig. 2C). The structure of the decomposed oligosaccharide was measured by nuclear magnetic resonance spectroscopy (Fig. 2D). K1-ORF34 decomposed oligosaccharides were still recognized by anti-K1 antisera (data not shown). Therefore, the K1-ORF34 enzyme belongs to a lytic enzyme and can be used for the development of a K1 capsular polysaccharide (CPS) conjugate vaccine.

Embodiment 4: Analysis of capsular polysaccharide (CPS) decomposed by capsular depolymerase

Analysis of K2-ORF16 capsular depolymerase-decomposed capsular polysaccharide (CPS)

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

The capsular polysaccharide (CPS) decomposed by K62 capsule depolymerase was analyzed.

The structure of K62 capsular polysaccharide (CPS) decomposed by K62 capsular depolymerase was further analyzed. After incubation with purified K62 capsular depolymerase, K62 capsular polysaccharide (CPS) is reduced to oligosaccharides. From the results of matrix-assisted laser desorption/time-of-flight mass spectrometry and tandem mass spectrometry, it can be seen that the decomposed K62 capsular polysaccharide (CPS) product is mainly composed of two repeating units (decasaccharides), and a small amount of one repeat. Unit (pentose sugar) (Figure 4). From the quality analysis point of view, K62 capsular depolymerase belongs to a hydrolase and can be used in the development of K62 capsular polysaccharide (CPS) conjugate vaccine.

The capsular polysaccharide (CPS) decomposed by the capsular depolymerase of KN2, K24, K28 and K64 was analyzed according to a similar method as described above.

Embodiment 5: capsular polysaccharide (CPS) conjugate vaccine

The major sugar chain lengths of the K1, K2, K24, K28, K62 and K64 oligosaccharides used to bind the CRM197 carrier protein are as follows: hexose (two repeating units of K1 capsular polysaccharide (CPS)), tetrasaccharide (K2 capsule) a repeating unit of polysaccharide (CPS), dodecaose (two repeating units of K62 capsular polysaccharide (CPS)), hexose (a repeating unit of K64 capsular polysaccharide (CPS)), pentasaccharide (K24 capsule) A repeating unit of polysaccharide (CPS) and a hexasaccharide (a repeating unit of K28 capsular polysaccharide (CPS)). The ratio of epitopes was determined by matrix-assisted laser desorption free/time-of-flight mass spectrometry mass spectrometry and the amount of glycans was calculated accordingly. With K1 as the representative, we can get the immunogen ratio from 1:1.4 to 1:10.2 through the change of the number of decomposed K1-capsular polysaccharide-thiol (CPS-SH); the detailed immunogen ratio of K1 vaccine is shown in the following table. Show. Immunogen ratio was determined by matrix-assisted laser desorption free/time-of-flight mass spectrometry mass spectrometry.

Embodiment 6: Toxicology test (with K1 as a representative example)

The body weight (Fig. 5A) and rectal temperature (Fig. 5B) of the mice one day before and after the immunization of the K1 capsular polysaccharide (CPS) conjugate vaccine were recorded. The sera of the mice were collected one week after the first, second and third immunizations of the K1 capsular polysaccharide (CPS) conjugate vaccine, and then the liver (serum transaminase (ALT)) and kidney (blood urea nitrogen) were measured. The function of BUN) and creatinine) (Fig. 5C). These results showed that no side effects were observed in mice immunized with three doses of K1 capsular polysaccharide (CPS) conjugate vaccine. Toxicological determination of capsular polysaccharide (CPS) of K2, K24, K28, K62 or K64 was also carried out in a manner similar to that described above.

Embodiment 7: Antibody reaction and serum bactericidal test

Serum from mice administered K1, K2 or K62 capsular polysaccharide (CPS) conjugate vaccine by intramuscular injection was 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 their original capsular polysaccharide (CPS) (Fig. 6).

The results of the bactericidal assay showed that the sera from mice immunized with the K1 capsular polysaccharide (CPS) conjugate vaccine, even with serum 1:128 diluted, could kill more than the sera from non-immunized mice. 50% of K1 bacteria (the bactericidal power is 32 to 128). The bactericidal power of K2 bacteria in the serum of mice immunized with K2 capsular polysaccharide (CPS) conjugate vaccine was 8 to 32. The bactericidal activity against K62 bacteria was detected in 40% of the sera of mice immunized with K62 capsular polysaccharide (CPS) conjugate vaccine (bactericidal power price was 8 to 32). Therefore, the capsular polysaccharide (CPS) conjugate vaccine can successfully induce the production of a capsule-type specific antibody in mice, and the antibody has a bactericidal action.

Embodiment 8: Protection test

The present invention also uses K. pneumoniae to challenge immunized mice to assess whether the vaccine has in vivo protective effects. Mice were immunized with K1, K2 capsular polysaccharide (CPS) conjugate vaccine by intramuscular injection once a week (control group only adjuvant). One week after the third immunization, mice were intraperitoneally injected with 1 x 10 ⁴ colony forming units (CFU, K. pneumoniae NTUH-K2044 or NTUH-A4528). The results of 30 days after challenge with Klebsiella pneumoniae showed that the survival rate of mice after receiving K1 or K2 capsular polysaccharide (CPS) conjugate vaccine was significantly higher than that of mice receiving adjuvant only (Fig. 7A and B).

Then, K62 bacteria in a 5×10 ⁶ colony forming unit (CFU, colony-forming unit) were used to challenge mice treated with cyclophosphamide to induce neutropenia and immunized with K62 capsular polysaccharide (CPS) conjugate vaccine. . The K62 capsular polysaccharide (CPS) conjugate vaccine significantly protected immunocompromised mice from infection with K62 bacteria (Fig. 7C).

Embodiment 9: Efficacy of K1 and K2 capsular polysaccharide (CPS) conjugated bivalent vaccine

The general capsular line K1 and K2 capsular types of Klebsiella pneumoniae strains causing invasive infections. Therefore, the protective efficacy of the bivalent vaccine (a mixture of equal amounts of K1 and K2 capsular polysaccharide (CPS) conjugate vaccines) was further examined (Figs. 8A and 8B). The mice in the immunization adjuvant group were intraperitoneally injected with 1×10 ⁴ colony forming units (CFU, colony-forming unit) of NTUH-K2044 and NTUH-A4528 Klebsiella pneumoniae, respectively, resulting in 80% And 100% mortality; on the contrary, no death was observed after intraperitoneal injection of mice immunized with the bivalent vaccine. Thus, the mice significantly protected the mice from infection with K1 and K2 Klebsiella pneumoniae after immunization with the bivalent vaccine.

Embodiment 10: Structure of capsular polysaccharide (CPS) decomposed by K64, K24, K28 and KN2 capsule depolymerase

1. K64

The decomposed K64 capsular polysaccharide (CPS) is predominant in one repeat unit (hexasaccharide) and is secondary in two repeats (dodecose). The K64 capsular polysaccharide (CPS) cleavage enzyme is a lytic enzyme since a new double bond signal is shown in the nuclear magnetic resonance spectrum of the reaction product. The tandem mass spectrometry results (hexasaccharide) of the main K64 fragment are shown in FIG.

2. K24

According to the disclosed K24 capsular polysaccharide (CPS) structure, it consists of four hexoses and one hexuronic acid. The decomposed K24 capsular polysaccharide (CPS) mass spectrum showed that the main product was acetylated modified double repeat unit oligosaccharide [mass-to-charge ratio (m/z) 1773, (Hex) ₈ (HexA) ₂ ], minor The sugar is 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 results (pentaose) are shown in FIG.

3. K28

It can be seen from the results of matrix-assisted laser desorption free/time-of-flight mass spectrometry that the decomposed product - KN 2 capsular polysaccharide (CPS) is predominant in one repeating unit (hexose). The main K28 fragment tandem mass spectrometry results (hexasaccharide) are shown in FIG.

4. KN2

Klebsiella Klebsiella KN2 belongs to the new capsular type, and its chemical structure is still not described in the literature. From the results of matrix-assisted laser desorption free/time-of-flight mass spectrometry analysis, it was found that the decomposition product-KN2 capsular polysaccharide (CPS) consisted of four hexoses and one hexuronic acid (molecular weight 1027). Furthermore, based on the mass-to-charge ratio (m/z) of matrix-assisted laser desorption free/time-of-flight mass spectrometry, the results show that the KN2 enzyme is a hydrolase. The mass-to-charge ratio (m/z) 1027 was further analyzed by electrospray free mass spectrometry-mass spectrometry. Fragments of tandem mass spectrometry confirmed that our guess was 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 free/time-of-flight mass spectrometry analysis of capsular polysaccharide (CPS) decomposed by KN2 hydrolase are shown in FIG.

Embodiment 11: Comparison of capsular polysaccharide (CPS) vaccine and capsular polysaccharide (CPS) conjugate vaccine

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

<110> DCB-USA LLC National Taiwan University

<120> capsular polysaccharide vaccine of Klebsiella pneumoniae

<130> N40580/PC0104

<150> US 62/403,365

<151> 2016-10-03

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 651

<212> PRT

<213> K1 specific phage

<400> 1

<210> 2

<211> 668

<212> PRT

<213> Phage 1611E-K2-1

<400> 2

<210> 3

<211> 535

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide; a carrier protein

<400> 3

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide; 1611E-ORF16 forward primer

<400> 4

<210> 5

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide; 1611E-ORF16 reverse primer

<400> 5

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide; Ref-K10-1 ORF6 forward primer

<400> 6

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide; Ref-K10-1 ORF6 reverse primer

<400> 7

Claims (28)

An immunogen comprising a repeating unit of a trisaccharide of Klebsiella Klebsiella K1 capsular polysaccharide having the following formula (I): {→4)-[2,3-( S )-pyruvate]-bD-GlcA p -(1→4)-aL- O- Ac-Fuc p -(1→3)-b- D-Glc p -(1→} _m (I) wherein m is 1 to 4.  The immunogen as described in claim 1, wherein m is 2.   An immunogen comprising a repeating unit of a tetrasaccharide of Klebsiella Klebsiella K2 capsular polysaccharide having the following formula (II): Where n is 1 to 4.  An immunogen according to claim 3, which contains a repeating unit (n = 1).   An immunogen comprising a repeating unit of a hexasaccharide of Klebsiella Klebsiella K64 capsular polysaccharide having the following formula (III): Where n is 1 to 4.  An immunogen according to claim 5, wherein n is 1.   An immunogen comprising a repeating unit of a pentasaccharide of Klebsiella Klebsiella K62 capsular polysaccharide having the following formula (IV): Where n is 1 to 4.  An immunogen as described in claim 7 wherein n is 2.   An immunogen comprising a repeating unit of a pentasaccharide of Klebsiella Klebsiella K24 capsular polysaccharide having the following formula (V): Where n is 1 to 4.  The immunogen of claim 9, wherein n is 1.   An immunogen comprising a repeating unit of a hexasaccharide of Klebsiella Klebsiella K28 capsular polysaccharide having the following formula (VI): Where n is 1 to 4.  The immunogen of claim 11, wherein n is 1.    An immunogen comprising a repeating unit of a hexasaccharide having Klebsiella pneumoniae KN2 capsular polysaccharide having four hexoses and a hexuronic acid having a molecular weight of about 1027.   An isolated polypeptide or variant thereof having a degrading activity specific to a capsule of Klebsiella pneumoniae capsular K2 strain selected from: (a) a fragment comprising SEQ ID NO: a polypeptide of an amino acid sequence; and (b) a polypeptide encoded by a polynucleotide which is at least in (i) the polypeptide coding sequence of SEQ ID NO: 2, or (ii) is complementary to the full length sequence of (i) A polynucleotide that hybridizes under stringent conditions.  The polypeptide of claim 14, which has the amino acid sequence of SEQ ID NO: 2.    A vaccine comprising one or more immunogens selected from the formulae (I) to (VI) as defined in claims 1, 3, 4, 7, 9 and 11.    The vaccine of claim 16, wherein the immunogen is conjugated to a carrier.    A vaccine according to claim 16 which is a K1 and K2 capsular polysaccharide-conjugated bivalent vaccine comprising an immunogen of Formula I and Formula II as defined in claims 1 and 3.    The vaccine of claim 16 which is a conjugated vaccine comprising K1 and K2 capsular polysaccharides of a mixture of an immunogen I-vector and an immunogen II-vector, wherein the vaccine is as defined in claim 1 The immunogen I of the formula (I) and the immunogen II of the formula (II) as defined in the third paragraph of the patent application are bonded to a carrier, respectively.    A vaccine according to claim 16 which is a conjugated vaccine comprising K64 and K62 capsular polysaccharides of the immunogens of formula III and formula IV as defined in claims 5 and 7 of the patent application.    A vaccine according to claim 16 which is a K64 and K62 capsular polysaccharide (CPS)-conjugated bivalent vaccine comprising a mixture of an immunogen III-vector and an immunogen IV-vector, wherein the patent is as claimed The immunogen III of the formula (III) defined in the fifth item and the immunogen IV having the formula (IV) as defined in the seventh paragraph of the patent application are respectively joined to the carrier.    The vaccine of claim 17, wherein the carrier is a protein, a peptide, a lipid, a polymer, a dendrimer, a virion, a viroid-like particle (VLP), or a combination thereof.    The vaccine of claim 17, wherein the carrier is a protein carrier.    The vaccine according to claim 23, wherein the protein carrier is a bacterial toxin, a toxin, an exotoxin and a non-toxic derivative thereof, such as keyhole limpet hemocyanin (KLH), hepatitis B virus core protein, Thyroxin, 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 (eg lysine: glutamic acid), tetanus toxoid, tetanus toxin fragment C, diphtheria toxoid, CRM (non-toxic diphtheria toxin mutant), cholera toxin, golden yellow grape Cocci exotoxin or toxoid, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A and bacterial outer membrane protein (such as Neisseria meningitidis serotype B outer membrane protein complex (OMPC) and Membrane class 3 porins (rPorB)).    The vaccine of claim 23, wherein the protein carrier is CRM197 having the amino acid sequence of SEQ ID NO: 3.    A method for eliciting an immune response against Klebsiella pneumoniae, comprising the immunogen according to any one of claims 1 to 13 or any one of claims 16 to 25 The vaccine is administered to the subject.    An antibody obtained by the method described in claim 26 of the patent application.    A method of preventing Klebsiella pneumoniae infection, comprising administering an effective amount of the immunogen according to any one of claims 1 to 13 or claiming any one of claims 16 to 25. The vaccine is administered to the subject.