RU2791256C2 - Immunogenic compositions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to an immunogenic composition containing (i) a framework polymer comprising a polymer and one or more antigenic polysaccharides conjugated with a polymer; and (ii) one or more polypeptide antigens in a non-covalent complex with a polymer and/or antigenic polysaccharide; in which the polymer is a capsule polysaccharide Klebsiella spp. K19; and in which each of one or more antigenic polysaccharides is an O-antigenic polysaccharide (OPS) of K. pneumoniae and/or P. aeruginosa; and in which each of one or more polypeptide antigens is independently selected from: flagellin P. aeruginosa, PcrV P. aeruginosa, MrkA K. pneumoniae, their antigenic fragments and their combinations; and moreover, the immunogenic composition is characterized by the fact that after administration to the subject it causes an immune response against Klebsiella and/or Pseudomonas, also refers to the immunogenic composition containing (a) a framework polymer comprising (i) a polymer containing the capsule polysaccharide Klebsiella spp. K19; and (ii) one or more antigenic polysaccharides containing the O-antigenic polysaccharide (OPS) Klebsiella and/or Pseudomonas conjugated with the capsule polysaccharide Klebsiella spp. K19; and (b) one or more polypeptide antigens in a non-covalent complex with a polymer due to at least one pair of affine molecules, including (i) the first affine molecule associated with the polymer; and (ii) a second affine molecule complementary to the first molecule and associated with one or more polypeptide antigens, where each of one or more polypeptide antigens is independently selected from: flagellin P. aeruginosa, PcrV P. aeruginosa, MrkA K. pneumoniae, their antigenic fragments or their combinations; and moreover, the immunogenic composition is characterized by the fact that after administration to the subject it causes an immune response against Klebsiella and/or Pseudomonas, also refers to vaccine compositions, also refers to a pharmaceutical composition containing one or more immunogenic compositions or vaccine compositions and a pharmaceutically acceptable carrier, where the pharmaceutical composition differs in that when administered to the patient it causes the immune response against Klebsiella and/or Pseudomonas also refers to the method for immunizing a patient against an infection caused by Klebsiella, and to the method for immunization of the patient against infection caused by P. aeruginosa.

EFFECT: group of inventions provides the development of technologies for the prevention and/or treatment of nosocomial infections and ensures the production of vaccines with sufficient immunogenicity to create immunity and prevent colonization.

71 cl, 98 dwg, 36 tbl, 16 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No. 62/524,315, filed June 23, 2017, and U.S. Provisional Application No. 62/633,807, filed February 22, 2018, the contents of which are incorporated herein by links in full.

BACKGROUND TO THE INVENTION

Nosocomial infection, also called nosocomial infection, is the main problem that patients face today. Surveys conducted in the United States in 183 hospitals (Magill et al., 2014) showed that of 11,282 patients, 452 had one or more healthcare-associated infections (4.0%; 95% CI, 3.7 -4.4). Of the 504 such infections, the most common were pneumonia (21.8%), surgical site infections (21.8%), and gastrointestinal infections (17.1%). Clostridium difficile was the most commonly reported pathogen (causing 12.1% of healthcare-associated infections). Medical device-associated infections (i.e., central catheter-associated bloodstream infection, catheter-associated urinary tract infection, and ventilator-associated pneumonia) that have traditionally been targeted by healthcare-associated infection prevention programs accounted for 25, 6% of such infections. The inventors estimate that there were 648,000 patients in emergency hospitals in the United States of America (USA) in 2011 with 721,800 healthcare-associated infections.

With the surge in multidrug-resistant bacteria (MLR), it has become difficult to treat nosocomial infections. Clinicians have to resort to more toxic and less effective antibiotics. However, the Centers for Disease Control and Prevention (CDC) estimates that ~23,000 deaths occur each year from infections caused by MDR bacteria.

Thus, there remains a need for effective technologies for the prevention and/or treatment of such infections.

SUMMARY OF THE INVENTION

The present invention addresses the problem of the lack of appropriate technologies for the prevention and/or treatment of nosocomial infections. Among other things, the present invention addresses the problems of obtaining vaccines with sufficient immunogenicity to confer immunity and prevent colonization.

In particular, the present disclosure relates to compositions and methods for the prevention and/or treatment of nosocomial infections in patients.

In particular, the present invention relates to immunogenic compositions containing, for example, (i) a backbone polymer comprising a polymer and one or more antigenic polysaccharides conjugated to the polymer; and (ii) one or more polypeptide antigens in a non-covalent complex with a polymer or antigenic polysaccharide.

In some embodiments, the implementation of the immunogenic composition may contain one or more antigenic polysaccharides conjugated to the polymer using a linker. In some embodiments, the immunogenic composition may comprise one or more antigenic polysaccharides conjugated to a polymer without a linker. In some embodiments, one or more antigenic polysaccharides are derived from Gram-negative bacteria and/or Gram-positive bacteria. In some embodiments, the one or more antigenic polysaccharides are derived from Klebsiella, Pseudomonas and/or E. coli antigenic polysaccharides.

In some embodiments, one or more of the potential for opsonization or immunogenicity of one or more antigenic polysaccharides is increased relative to a previously determined level, as measured by ELISA and/or in a functional antibody assay. In some embodiments, one or more of the potential for opsonization or immunogenicity of one or more antigenic polysaccharides is increased relative to a control composition, as measured by ELISA and/or in a functional antibody assay. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the vaccine composition and not contain the backbone polymer present in the vaccine composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the vaccine composition and not contain the backbone polymer present in the vaccine composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the vaccine composition and/or an antigenic polysaccharide present in the vaccine composition and not contain a polymer present in the vaccine composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the pharmaceutical composition and not contain the backbone polymer present in the pharmaceutical composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the pharmaceutical composition and not contain the backbone polymer present in the pharmaceutical composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the pharmaceutical composition and/or the antigenic polysaccharide present in the pharmaceutical composition and not contain the polymer present in the pharmaceutical composition.

In some embodiments, one or more of the potential for opsonization or immunogenicity of one or more antigenic polysaccharides is increased by at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a previously determined level, as measured by ELISA and/or functional analysis of antibodies. In some embodiments, the previously determined level is the pre-immune level. In some embodiments, the epitope valency of one or more antigenic polysaccharides is at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold greater than that of the native polysaccharide.

In some embodiments, one or more polypeptide antigens may be carrier proteins for one or more antigenic polysaccharides. In some embodiments, upon administration to a patient, the immunogenic composition causes the production of antibodies against one or more pathogens in the patient at a higher level than the composition containing the antigenic polysaccharide and not containing the backbone polymer, as measured by ELISA. In some embodiments, upon administration to a patient, the immunogenic composition causes the production of antibodies against one or more pathogens in the patient at a higher level than the composition containing the polypeptide antigen and not containing the backbone polymer, as measured by ELISA. In some embodiments, one or more pathogens are selected from Klebsiella, Pseudomonas , and E. coli . In some embodiments, upon administration to a patient, the immunogenic composition elicits an immune response against one or more of Klebsiella, Pseudomonas , and E. coli .

In some embodiments, the polypeptide antigen is, or contains, a bacterial polypeptide, a fungal polypeptide, and/or a viral polypeptide, or combinations thereof. In some embodiments, the polypeptide antigen is, or contains, a polypeptide, or an immunogenic fragment thereof, from Klebsiella, Pseudomonas, and/or E. coli . In some embodiments, the polypeptide antigen is, or contains, a polypeptide derived from a Klebsiella, Pseudomonas and/or E. coli polypeptide.

In some embodiments, the polymer is, or contains, a polysaccharide, a polypeptide, or a synthetic dendrimer. In some embodiments, the implementation of the polymer has a size of from about 50 kDa to about 2000 kDa. In some embodiments, the implementation of the polymer is, or contains, capsular polysaccharide from gram-negative or gram-positive bacteria. In some embodiments, the capsular polysaccharide is, or contains, a capsular polysaccharideklebsiella, exopolysaccharidePseudomonasand/or capsular polysaccharideEscherichia. In some embodiments, the polymer is, or contains, a capsular polysaccharideKlebsiella spp. K19. In some embodiments, the implementation of the polymer is, or contains, linear poly-L-lysine or L-lysine dendrimer. In some embodiments, the polymer is, or contains, a synthetic monosaccharide or oligosaccharide dendrimer.

In some embodiments, one or more polypeptide antigens are in a non-covalent complex with a polymer and/or one or more antigenic polysaccharides by forming at least one pair of complementary affinity molecules, comprising (i) a first affinity molecule associated with the polymer or one or more antigenic polysaccharides; and (ii) a complementary affinity molecule associated with one or more polypeptide antigens. In some embodiments, a pair of complementary affinity molecules is selected from the group consisting of: biotin/biotin-binding protein, antibody/antigen, enzyme/substrate, receptor/ligand, metal/metal-binding protein, carbohydrate/carbohydrate-binding protein, lipid/ a lipid-binding protein; and a His-tag/His-tag-binding molecule. In some embodiments, the first affinity molecule is biotin or a derivative thereof. In some embodiments, the complementary affinity molecule is a biotin binding protein or a biotin binding domain thereof. In some embodiments, the biotin binding protein is rizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, neutravidin, captavidin™, or a combination thereof.

In some embodiments, the first affinity molecule is cross-linked or covalently linked to a polymer or one or more antigenic polysaccharides. In some embodiments, the first affinity molecule is cross-linked or covalently linked to the backbone polymer polymer. In some embodiments, the first affinity molecule is cross-linked or covalently linked to one or more antigenic polysaccharides. In some embodiments, the first affinity molecule is cross-linked or covalently linked to the backbone polymer polymer and to one or more antigenic polysaccharides. In some embodiments, the implementation of the polymer is, or contains, poly-L-lysine. In some embodiments, the one or more antigenic polysaccharides are, or contain, a polysaccharide from one or more of Klebsiella, Pseudomonas , and E. coli.

In some embodiments, the one or more antigenic polysaccharides are, or contain, K. pneumoniae type O1, O2, O3, O5 OPS, or a combination thereof. In some embodiments, the one or more antigenic polysaccharides are or comprise P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11, O12 OPS, or a combination thereof. In some embodiments, at least one of the antigenic polysaccharides is, or contains, a P. aeruginosa PsL exopolysaccharide. In some embodiments, PsL is, or contains, at least one epitope that binds a monoclonal antibody containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identity with the sequence of SEQ ID NO: 4, and/or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identity with SEQ ID NO: 5.

In some embodiments, at least one polypeptide antigen is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. with the sequence of SEQ ID NO: 1 ( K. pneumoniae type I fimbrial protein), or an immunogenic fragment thereof. In some embodiments, at least one polypeptide antigen is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. with the sequence of SEQ ID NO: 2 (conservative fimbrial protein type III MrkA K. pneumoniae ), and/or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 3, or an immunogenic fragment thereof. In some embodiments, at least one polypeptide antigen is, or comprises, P. aeruginosa FliC subtype A flagellin, P. aeruginosa FliC subtype B flagellin, or an immunogenic fragment thereof. In some embodiments, at least one polypeptide antigen is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. with the sequence of SEQ ID NO: 6 ( P. aeruginosa FliC subtype A1 flagellin), an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 9 ( P. aeruginosa FliC subtype A2 flagellin), an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity with the sequence of SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin), or an immunogenic fragment thereof. In some embodiments, at least one polypeptide antigen is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment.

In some embodiments, the immunogenic composition of the invention is, or contains, (a) a backbone polymer comprising (i) a polymer containing a capsular polysaccharideKlebsiella spp. K19; and (ii) one or more antigenic polysaccharides comprising a polysaccharideKlebsiella orPseudomonas, conjugated with capsular polysaccharideKlebsiella spp. K19; and (b) one or more polypeptide antigens in a non-covalent complex with a polymer by forming at least one pair of complementary affinity molecules, including (i) a first affinity molecule associated with the polymer; and (ii) a complementary affinity molecule associated with one or more polypeptide antigens. In some embodiments, one or more antigenic polysaccharides are, or contain, OPS type O1, O2, O3, O5K. pneumoniae, or a combination of them. In some embodiments, one or more antigenic polysaccharides are, or contain, OPS type O1, O2, O3, O4, O5, O6, O10, O11P. aeruginosa, or a combination of them. In some embodiments, the first affinity molecule is biotin or a derivative thereof, and the complementary affinity molecule is, or contains, a biotin binding protein or a biotin binding domain thereof. In some embodiments, the biotin binding protein is, or contains, rizavidin or its biotin binding domain. In some embodiments, one or more polypeptide antigens are, or contain, the D2 domain of FliC subtype B flagellinP. aeruginosa, lacking a TLR5-binding motif, PcrVP. aeruginosa, MrkAK. pneumoniae, their antigenic fragments, or their combination.

In some embodiments, at least one pair of complementary affinity molecules comprises a fusion protein containing (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 (FlaB flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or its immunogenic fragment. In some embodiments, the fusion protein is, or contains, a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 24 (Rhavi-FlaB-Domain2-MrkA-His).

In some embodiments, at least one pair of complementary affinity molecules comprises a fusion protein containing (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 ( P. aeruginosa FliC subtype B flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof , and (iii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 ( PcrV P. aeruginosa ), or its immunogenic fragment. In some embodiments, the fusion protein is, or contains, a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 26 (Rhavi-FlaB-Domain2-PcrV-His).

In particular, the present invention relates to vaccine compositions. In some embodiments, the implementation of the vaccine composition may contain one or more immunogenic compositions. In some embodiments, the implementation of the vaccine composition may contain a pharmaceutically acceptable carrier.

In some embodiments, the implementation of the vaccine composition may contain one or more of (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition may contain one or more of (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition may contain one or more of (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition may contain one or more of (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition may contain one or more of: (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (j) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition may contain one or more of (a) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (j) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; and (l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer. In some embodiments, the Rhavi-FlaBD2-MrkA fusion protein is, or contains, a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 24 (Rhavi-FlaB-Domain2-MrkA-His). In some embodiments, the Rhavi-FlaBD2-PcrV fusion protein is, or contains, a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 26 (Rhavi-FlaB-Domain2-PcrV-His).

In some embodiments, the OPS of each immunogenic composition is present in a weight ratio of about 1:1. In some embodiments, the backbone polymer of each immunogenic composition is present in a weight ratio of about 1:1. In some embodiments, the combined OPS and CPS of each immunogenic composition are present in a weight ratio of about 1:1. In some embodiments, the OPS of each immunogenic composition is present in an amount of about 1 μg. In some embodiments, the framework polymer of each immunogenic composition is present in an amount of about 1 μg. In some embodiments, the total OPS and CPS of each immunogenic composition are present in an amount of about 1 μg. In some embodiments, the OPS of each immunogenic composition is present in an amount of about 5 μg. In some embodiments, the backbone polymer of each immunogenic composition is present in an amount of about 5 μg. In some embodiments, the total OPS and CPS of each immunogenic composition are present in an amount of about 5 μg. In some embodiments, the backbone polymer is, or contains, a BP-1 backbone. In some embodiments, the backbone polymer is, or contains, a BP-1.2 backbone. In some embodiments, the backbone polymer is, or contains, a BP-2a backbone. In some embodiments, the backbone polymer is, or contains, a BP-2b backbone. In some embodiments, the backbone polymer is, or contains, a BP-3 backbone.

In some embodiments, the one or more polypeptide antigens are, or contain, one or more P. aeruginosa proteins, or variants thereof, and wherein the complementary affinity molecule is, or contains, a biotin-binding protein or a biotin-binding domain thereof. In some embodiments, at least one of the polypeptide antigens is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 7 (flagellin FlaB P. aeruginosa ), or its immunogenic fragment. In some embodiments, at least one of the polypeptide antigens is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 6 ( P. aeruginosa flagellin FlaA1), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 9 (flagellin FlaA2), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 (FlaB flagellin D2 domain lacking a TLR5-binding motif), or an immunogenic fragment thereof, or a polypeptide containing an amino acid a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 11 (FlaA1 flagellin D2 domain lacking TLR5 binding motif), or an immunogenic fragment thereof, or a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the SEQ ID sequence NO: 12 (FlaA2 flagellin D2 domain lacking TLR5-binding motif), or its immunogenic fragment.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 10 (FlaB flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 (FlaA1 flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12 (FlaA2 flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising a biotin binding protein or a biotin binding domain thereof and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV secretory system type III (TTSS) P. aeruginosa ), or its immunogenic fragment.

In some embodiments, at least one pair of complementary affinity molecules is, or includes, a fusion protein containing (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin), or an immunogenic fragment thereof, and ( iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof.

In some embodiments, at least one pair of complementary affinity molecules is, or includes, a fusion protein containing (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 ( P. aeruginosa FliC subtype B flagellin D2 domain lacking TLR5 binding motif) , or an immunogenic fragment thereof, and (iii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the SEQ sequence ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof.

In some embodiments, at least one pair of complementary affinity molecules is, or includes, a fusion protein containing (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin), or an immunogenic fragment thereof, and ( iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO : 3 (MrkA K. pneumoniae ), or its immunogenic fragment.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising (i) a biotin binding protein or a biotin binding domain thereof, (ii) a polypeptide comprising an amino acid sequence having at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 ( P. aeruginosa FliC subtype B flagellin D2 domain lacking TLR5 binding motif) , or an immunogenic fragment thereof, and (iii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof.

In some embodiments, at least one complementary affinity molecule pair is, or includes, a fusion protein comprising (i) a biotin binding protein or a biotin binding domain thereof, (ii) a polypeptide comprising an amino acid sequence having at least 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof, and (iii) a polypeptide , containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3 ( MrkA K. pneumoniae ), or its immunogenic fragment.

In some embodiments, upon administration to a patient, the immunogenic composition causes the production of antibodies that recognize native MrkA against K. pneumoniae expressing MrkA. In some embodiments, upon administration to a patient, the vaccine composition causes the production of antibodies that recognize native MrkA against K. pneumoniae expressing MrkA.

In some embodiments, the implementation of the pharmaceutical composition contains one or more immunogenic compositions or vaccine compositions described in the present invention. In some embodiments, the implementation of the pharmaceutical composition contains a pharmaceutically acceptable carrier. In some embodiments, the implementation of the pharmaceutical composition contains one or more adjuvants. In some embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, phosphated aluminum hydroxide, TLR agonists such as TLR2 agonist saponin (QS21) or porins, TLR4 agonists such as, for example, monophosphoryl lipid A (MPL), and TLR5 agonists such as flagellins, and so on.

In some embodiments, the pharmaceutical composition is formulated for intramuscular, intraperitoneal, intradermal, and/or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for injection. In some embodiments, upon administration to a patient, the pharmaceutical composition induces a Th1 and/or Th17 cellular response. In some embodiments, upon administration to a patient, the pharmaceutical composition elicits an opsonic/bactericidal response against one or more of Klebsiella , Pseudomonas , and E. coli . In some embodiments, upon administration to a patient, the pharmaceutical composition results in a reduction in the rate of transmission of infection and/or colonization of mucosal surfaces by one or more of Klebsiella, Pseudomonas , and E. coli. In some embodiments, upon administration to a patient, the pharmaceutical composition results in a reduction in the rate of transmission of infection and/or colonization of the GI tract by one or more of Klebsiella , Pseudomonas , and E. coli .

Some embodiments relate to methods for immunizing a patient against an infection.Klebsiella, comprising administering to a patient an effective amount of an immunogenic composition of the invention. Some embodiments relate to methods for immunizing a patient against an infection.Klebsiella, comprising administering to a patient an effective amount of a vaccine composition of the invention. Some embodiments relate to methods for immunizing a patient against an infection.Klebsiellacomprising administering to a patient an effective amount of a pharmaceutical composition of the invention.

Some embodiments relate to methods for immunizing a patient against P. aeruginosa infection, comprising administering to the patient an effective amount of an immunogenic composition of the invention. Some embodiments relate to methods for immunizing a patient against P. aeruginosa infection, comprising administering to the patient an effective amount of a vaccine composition of the invention. Some embodiments relate to methods for immunizing a patient against P. aeruginosa infection, comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

Some embodiments relate to methods for immunizing a patient against an E. coli infection, comprising administering to the patient an effective amount of an immunogenic composition of the invention. Some embodiments relate to methods for immunizing a patient against an E. coli infection comprising administering to the patient an effective amount of a vaccine composition of the invention. Some embodiments relate to methods for immunizing a patient against an E. coli infection, comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

In some embodiments, upon administration to a patient, the immunogenic composition elicits an immune response against Gram-negative and/or Gram-positive bacteria. In some embodiments, upon administration to a patient, the vaccine composition elicits an immune response against Gram-negative and/or Gram-positive bacteria. In some embodiments, upon administration to a patient, the pharmaceutical composition induces an immune response against Gram-negative and/or Gram-positive bacteria. In some embodiments, the Gram-negative bacteria are selected from Klebsiella, Pseudomonas, E. coli , and combinations thereof. In some embodiments, the implementation of the immune response is the production of antibodies or B-cell response. In some embodiments, the immune response is a CD4+ T cell response, including a Th1, Th2, or Th17 response, or a CD8+ T cell response, or a CD4+/CD8+ T cell response. In some embodiments, the implementation of the immune response is the production of antibodies or B-cell response; and T cell response.

Some embodiments relate to antibody compositions comprising antibodies raised in a mammal immunized with an immunogenic composition of the invention. Some embodiments relate to antibody compositions comprising antibodies raised in a mammal immunized with a vaccine composition of the invention. Some embodiments relate to antibody compositions comprising antibodies raised in a mammal immunized with a pharmaceutical composition of the invention. In some embodiments, the antibody composition comprises at least one antibody selected from the group consisting of monoclonal antibodies and anti-idiotypic antibodies. In some embodiments, the antibody composition contains an isolated gamma globulin fraction.

In some embodiments, the invention relates to methods for purifying an O-antigenic polysaccharide from one or more Klebsiella cellular components, wherein the cellular components include a protein and/or nucleic acid, comprising the steps of: contacting one or more cellular components with an oxidizing agent, to obtain a mixture , while the pH of the mixture is about 3-5; separating the O-antigenic polysaccharide from cellular components; and extracting the OPS, wherein the OPS is substantially free of other cellular components and contains a free aldehyde at its reducing end. In some embodiments, the oxidizing agent is sodium nitrite, the acid is acetic acid, and the reducing end of the OPS is a free aldehyde containing 2,5-anhydromannose residue.

In some embodiments, the invention relates to methods for purifying an O-antigenic polysaccharide from one or more cellular components of gram-negative bacteria, wherein the cellular components include a protein and/or nucleic acid, including: contacting one or more cellular components with an acidic reagent, obtaining a mixture , while the pH of the mixture is about 3-5, heating the mixture to a temperature of from about 80°C to about 100°C; separating the O-antigenic polysaccharide from cellular components; and extracting the OPS, wherein the OPS is substantially free of other cellular components and contains a ketone at its reducing end.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one non-biotinylated O-antigenic polysaccharide, wherein the O-antigenic polysaccharide contains a primary amino group, and at least one biotinylated polysaccharide, comprising: chemical binding of the primary amino group O- an antigenic polysaccharide by mixing an O-antigenic polysaccharide with a partially oxidized polysaccharide containing aldehyde groups and biotin containing a primary amine to form a mixture; and reductively aminating the mixture with a reducing reagent to form a BP-1 polymer backbone, wherein the polysaccharide is biotinylated and the chemically bound O-antigenic polysaccharide is not biotinylated. Some embodiments relate to a BP-1 polymer backbone made by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one non-biotinylated O-antigenic polysaccharide, wherein the O-antigenic polysaccharide contains a primary amino group, and at least one biotinylated polysaccharide, comprising: chemical binding of the primary amino group O- antigenic polysaccharide by mixing an O-antigenic polysaccharide with a polysaccharide, biotinylated and oxidized, obtained first by treating the carboxylate groups of the polysaccharide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as biotin hydrazide, followed by treatment of the polysaccharide with an oxidizing agent; and reductively aminating the mixture with a reducing reagent to form a BP-1.2 polymer backbone, wherein the polysaccharide is biotinylated and the chemically bound O-antigenic polysaccharide is not biotinylated. In some embodiments, the primary group on the O-antigenic polysaccharide is the α-amino group of L-alanine of the outer core of the OPS from P. aeruginosa , or an amino group that has been introduced into the reducing end of the O-antigenic polysaccharide by chemically linking a short spacer molecule containing the primary group at each end, by reductive amination with a reducing agent. In some embodiments, the oxidizing reagent for oxidizing the polysaccharide is sodium periodate, the diamine-containing short spacer is adipic acid dihydrazide, and the reducing reagent for reductive amination is sodium cyanoborohydride. Some embodiments relate to a BP-1.2 polymer backbone made by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one non-biotinylated O-antigenic polysaccharide, wherein the O-antigenic polysaccharide contains a primary amino group, and at least one biotinylated polysaccharide, comprising: chemical binding of the primary amino group O- antigenic polysaccharide by mixing an O-antigenic polysaccharide with a biotinylated and CDAP-activated polysaccharide obtained first by treating the carboxylate groups of the polysaccharide with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and biotin with hydrazide, followed by activation of the biotinylated CDAP polysaccharide to form a BP-1.3 polymer backbone, the polysaccharide being biotinylated and the chemically bound O-antigenic polysaccharide being non-biotinylated. In some embodiments, the primary group on the O antigenic polysaccharide is the P. aeruginosa OPS outer core L-alanine α-amino group or an amino group that has been introduced into the reducing end of the O antigenic polysaccharide by chemically linking a short spacer molecule containing the primary group on each end, by reductive amination with a reducing reagent. In some embodiments, the diamine-containing short spacer is adipic acid dihydrazide and the reductive amination reducing agent is sodium cyanoborohydride. Some embodiments relate to a BP-1.3 polymer backbone made by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one biotinylated O-antigenic polysaccharide and at least one biotinylated polysaccharide, including: a short spacer containing a primary amine at each end with an O-antigenic polysaccharide by reductive amination to form an O-antigenic polysaccharide spacer molecule, or using non-derivatized OPS containing the α-amino group of L-alanine, mixing the O-antigenic polysaccharide, containing a primary amine, with a partially oxidized polysaccharide, to obtain a mixture; reductive amination of the mixture to form an OPS backbone; and derivatizing the OPS backbone with CDAP and biotin containing a primary amine, resulting in a backbone polymer wherein the polysaccharide and chemically bound O-antigenic polysaccharide are biotinylated. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one biotinylated O-antigenic polysaccharide and at least one non-biotinylated polysaccharide, comprising: biotinylating an O-antigenic polysaccharide containing one or more core KDO and/or heptose fragments , using CDAP and biotin containing a primary amine, to obtain a mixture of biotinylated OPS; partial oxidation of the mixture of biotinylated OPS with sodium periodate to introduce aldehydes into core KDO and/or heptose moieties; reductive amination of the biotinylated mixture with adipic acid dihydrazide; mixing the biotinylated and aminated OPS with the partially oxidized polysaccharide to form a mixture; and reductive amination of the mixture; resulting in a backbone polymer wherein the polysaccharide is not biotinylated and the chemically bound O-antigenic polysaccharide is biotinylated. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one biotinylated O-antigenic polysaccharide and a PLL polymer, including: reductive amination of an O-antigenic polysaccharide containing terminal aldehydes or alpha-KDO, ε-NH ₂ groups of the PLL polymer , to obtain an OPS-PLL polymer; derivatization of OPS with CDAP to obtain a mixture of derivatives; carrying out the reaction of a mixture of derivatives with biotin containing a primary amine; and treating, or not treating, the mixture with an acylating agent to block unreacted ε-NH ₂ groups of the PLL polymer; resulting in a backbone polymer wherein the N-acetylated-PLL blocked backbone, or unblocked PLL, is not biotinylated and the chemically bound O-antigenic polysaccharide is biotinylated. In some embodiments, the acylating agent is acetic anhydride or acetyl chloride. In some embodiments, the reducing agent is sodium cyanoborohydride. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one non-biotinylated O-antigenic polysaccharide, wherein the O-antigenic polysaccharide contains an azido group at its reducing end, and at least one biotinylated polysaccharide containing alkyne groups, comprising : Catalyzed linking of an O-antigenic polysaccharide containing an azido group at its reducing end with a biotinylated alkyne-containing polysaccharide to form an OPS backbone polymer, wherein the polysaccharide is biotinylated and the click chemistry linked O- the antigenic polysaccharide is not biotinylated. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one non-biotinylated O-antigenic polysaccharide, wherein the O-antigenic polysaccharide contains an aldehyde or ketone group at its reducing end, and at least one biotinylated polysaccharide containing primary amino group obtained by a click chemistry reaction in the presence of a catalyst, wherein the biotinylated polysaccharide contains alkyne groups with a low molecular weight compound containing an azido group at one end and a free amino group at the other end, including: chemical binding of an O-antigenic polysaccharide containing aldehyde or ketone groups by mixing an O-antigenic polysaccharide with a biotinylated polysaccharide containing primary amino groups, and reductively aminating the mixture with a reducing reagent, resulting in a backbone polymer, wherein the polysaccharide is biotinylated and the chi the micably linked O-antigenic polysaccharide is not biotinylated. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the invention relates to methods for producing a backbone polymer comprising at least one biotinylated O-antigenic polysaccharide, wherein the biotinylated O-antigenic polysaccharide contains an azido group at its reducing end, and at least one non-biotinylated alkyne-containing polysaccharide, comprising : linking by a click chemistry reaction in the presence of a catalyst a biotinylated O-antigenic polysaccharide containing an azido group at its reducing end to a non-biotinylated polysaccharide containing alkynes, resulting in a backbone polymer, the polysaccharide being non-biotinylated and bound by a click chemistry reaction The O-antigenic polysaccharide is biotinylated. In some embodiments, the catalyst for the click chemistry coupling of the O-antigenic polysaccharide and the polysaccharide via an alkyne azide cycloaddition is copper sulfate. In some embodiments, the alkyne group for derivatization of the polysaccharide is selected from the group consisting of 1-amino-3-butyne, 1-amino-4-pentine, DBCO-amine, and alkyne-PEG4-amine. In some embodiments, the azide group for derivatization of the polysaccharide is selected from the group consisting of azido-PEG3-amine, azidopropylamine, and azidobutylamine. Some embodiments relate to a backbone polymer obtained by the methods disclosed in the present invention.

In some embodiments, the fusion protein is, or contains, a biotin binding domain and a polypeptide that is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% identity with any sequence from SEQ ID NO: 16-26. In some embodiments, the fusion protein is, or contains, a biotin binding domain and a polypeptide that is, or contains, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% identity with any of SEQ ID NOs: 1-3 or 6-26.

In some embodiments, a method for making a MAPS variant comprises complexing at least one backbone polymer described herein with at least one fusion protein described herein. In some embodiments, the MAPS variant is MAPS BP-1, MAPS BP-1.2, MAPS BP-1.3, MAPS BP-2a, MAPS BP-2b, or MAPS BP-3.

In some embodiments, a method for assessing the immunogenicity of an immunogenic composition of the present invention comprises assessing, measuring, and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokine levels, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis , infections of burn wounds, gastrointestinal tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

In some embodiments, a method for evaluating the efficacy of an immunogenic composition of the present invention comprises evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokines, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis, infections of burn wounds, GI tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

In some embodiments, a method for assessing the immunogenicity of a vaccine composition described in the present invention includes assessing, measuring and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokines, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis, infections of burn wounds, GI tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

In some embodiments, a method for evaluating the efficacy of a vaccine composition of the present invention includes evaluating, measuring, and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokines, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis, infections of burn wounds, GI tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

In some embodiments, a method for assessing the immunogenicity of a pharmaceutical composition described in the present invention includes assessing, measuring and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokines, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis, infections of burn wounds, GI tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

In some embodiments, a method for evaluating the effectiveness of a pharmaceutical composition described in the present invention includes assessing, measuring and/or comparing an immune response using one or more in vitro bioassays, including analysis of B-cell and T-cell responses, for example, antibody levels by ELISA, functional antibody levels by FC, OFU, and other functional tests such as determination of agglutination, motility, cytotoxicity, Th1/Th17 cells and cytokines, as well as in vivo assays in animal models of nosocomial disease (pneumonia, sepsis, infections of burn wounds, GI tract and surgical field), such as analysis of bacterial clearance, passive and active protection after infection with nosocomial pathogens that are the target of the immunogenic composition. In some embodiments, the implementation of the immune response is compared with the response to the control composition. In some embodiments, the control composition may contain the antigenic polysaccharide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain the antigenic polypeptide present in the immunogenic composition and not contain the backbone polymer present in the immunogenic composition. In some embodiments, the control composition may contain an antigenic polypeptide present in the immunogenic composition and/or an antigenic polysaccharide present in the immunogenic composition and not contain a polymer present in the immunogenic composition.

List of common abbreviations

ADH: adipic acid dihydrazide; PMR: resistance to antimicrobial agents; UE: conventional unit; BB: frame; BP: framework polymer; CDAP: 1-cyano-4-dimethylaminopyridinium tetrafluoroborate; CDC: Centers for Disease Control and Prevention; CDM: environment with a defined chemical composition; CF: cystic fibrosis; CP: general part; CPS: capsular polysaccharide; CFU: colony forming units; COPS: core O-polysaccharide; HT: cholera toxin; DNA: deoxyribonucleic acid; DTT: dithiothreitol; EC: Escherichia coli ; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA: enzyme immunoassay; ELISpot: cellular enzyme immunoassay; ETEC: enterotoxic E. coli ; PC: flow cytometry; PF: flow phase; GI: gastrointestinal; GOB: gram-negative bacteria; GOP: gram-negative rods; H-NMR: proton nuclear magnetic resonance; HA: hemagglutination; UN: killed by heat; HL-60 cells: human promyelocytic leukemia cells; HPLC: high performance liquid chromatography; IATS: International Antigen Serotyping System; ICU: intensive care unit; IOC: ion exchange chromatography; IFN: interferon; IL: interleukin; i / m: intramuscularly; i/n: intranasally; i/b: intraperitoneally; in / in: intravenously; IV IG: intravenous immunoglobulin; Ig: immunoglobulin; KDO: keto acid group; KP: Klebsiella pneumoniae ; KO: Klebsiella oxytoca ; LAL: horseshoe crab amebocyte lysate: LPS: lipopolysaccharide; mAb: monoclonal antibody; MALLS: analysis of multi-angle laser light scattering; MAPS: Multiple Antigen Presentation System; MDR: multiple drug resistance; MHC: major histocompatibility complex; MM: molecular weight; NAPLL: N-acetyl-poly-L-lysine; NaBH ₃ CN: sodium cyanoborohydride; NaIO ₄ : sodium metaperiodate; NHS: N-hydroxysuccinimide; SNP: normal human serum; NMR: nuclear magnetic resonance; OD: optical density; OMP: outer membrane protein; OFA: analysis of opsonophagocytic activity; OFU: opsonophagocytic killing; OPS: O-polysaccharide; PA: Pseudomonas aeruginosa ; PCR: polymerase chain reaction; PBS: phosphate-buffered saline; PEG: polyethylene glycol; PMN: polymorphonuclear leukocytes; PLL: poly-L-lysine; p/o: orally; PPG: polypropylene glycol; PRO-CN: control carrier protein; CT: pertussis toxin; RCB: research cell bank; KP: refractive index; RIA: radioimmunoassay; RNA: ribonucleic acid; rpm: revolutions per minute; PBT: serum bactericidal test; s / c: subcutaneously; SDS: sodium dodecyl sulfate; SDS-PAGE: polyacrylamide gel electrophoresis with sodium dodecyl sulfate: SEC: size exclusion chromatography; TFF: flow filtration along the flow; TLR: toll-like receptor; TNF: tumor necrosis factor; TTSS: secretory system of the third type, UV: ultrafiltration; USA: United States of America; WFI: water for injection.

BRIEF DESCRIPTION OF THE DRAWINGS

The ideas of the present invention described in the present description will be better understood by studying the following description of various illustrative embodiments in conjunction with the accompanying drawings. It should be understood that the drawings described below are for illustrative purposes only and should in no way limit the scope of the present invention.

Figure 1A schematically shows the structure of the inner core (top) and outer core (bottom) of lipopolysaccharide fromP. aeruginosa(PA), an arrow labeled "cleavage" indicates the acetic acid-mediated cleavage site between "keto acid groups" (KDO) and the lipid A molecule. international typing standards (IATS) for PA core types. Figure 1C shows the structure of the lipopolysaccharide (LPS) core polysaccharide portionK. pneumoniaewith a deaminating cleavage site between core glucosamine and galacturonic acid residues upon treatment of LPS with sodium nitrite and acetic acid (nitrous acid), resulting in 2,5-anhydromannose with an aldehyde group at C-1 at the reducing end of the common portion (CP) of OPS. Figure 1D shows the chemical structure of the repeating units of some of the OPSK. pneumoniae (KP) with 2,5-anhydromannose at the reducing end of CP OPS. Figure 1E shows the chemical structure of the capsular polysaccharide repeating unit (CPS)K. oxytoca(KO) K19 which is identical to CPSK. pneumoniae K19et al., 2013), thus both are referred to interchangeably in the present invention as KP K19 CPS. Figure 1F shows the H-Nuclear Magnetic Resonance (H-NMR) spectrum at 950 MHz of purified KP K19 CPS.

Figure 2A is a schematic of an exemplary chemical process for preparing KP/PA OPS labeled with adipic acid dihydrazide (ADH) and coupled by reductive amination to oxidized CPS, with selective biotinylation on CPS (BP-1). Figure 2B is a schematic representation of an exemplary chemical process for the preparation of KP/PA CPS/OPS BP-1.2 where biotin residues are bonded to the carboxylate groups of the CPS backbone polymer during reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (EDC-NHS), and the ADH-labeled OPS is linked to the biotinylated backbone polymer CPS by reductive amination with oxidized periodate and biotinylated CPS. Figure 2C is a schematic representation of an exemplary chemical process for the preparation of KP/PA CPS/OPS BP-1.3 where biotin residues are bonded to the CPS carboxylate groups of the backbone polymer during reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (EDC-NHS), followed by binding of the ADH-labeled OPS to the biotinylated and CDAP-activated CPS backbone polymer.

Figure 3 is a schematic representation of an exemplary chemical method for producing ADH-labeled OPS bound to oxidized CPS followed by the addition of biotin residues on CPS and OPS (BP-2a).

Figure 4 is a schematic representation of an illustrative chemical method for producing OPS associated with oxidized CPS, in which biotin residues are selectively associated with OPS (BP-2b).

Figure 5A is a schematic of an exemplary OPS scaffold polymer chemistry with poly-L-lysine (PLL) polymer in which biotin residues are selectively introduced into OPS and unreacted free ε-amino groups of PLL are blocked to produce a biotinylated OPS and N-acetylated scaffold polymer. PLL (NAPLL) (BP-3). Figure 5B shows three schemes for the preparation of exemplary backbone polymers. Scheme 1 and Scheme 2 show two variants of the chemical method for obtaining CPS/OPS scaffold 1 (BP-1) using click chemistry; Scheme 3 shows the chemistry using click chemistry to prepare the CPS/OPS scaffold 2b (BP-2b).

Figure 6A shows the results of an exemplary size exclusion chromatography (SEC), high performance liquid chromatography (HPLC) (Waters BioAlliance with a Phenomenex BioSep SEC-4000 column and a flow rate of 1 ml/min in PBS pH 7.4 containing sodium azide (0.02% elution profiles of oxidized CPS KP K19, OPS-ADH derivatized KPO1 (curve 1), KPO5 (curve 5), PAO6 (curve 6), PAO10 (curve 10). Phenomenex BioSep SEC-4000 and a flow rate of 1 ml/min in PBS, pH 7.4 containing sodium azide (0.02%), elution profiles (left): OPS_ADH; oxidized KP K19 CPS_OX (indicated on the figure as K19_OX); and K19 CPS_OX:OPS_ADH framework polymer and (right): K19 CPS_OX:OPS_ADH framework polymer after molecular sizing by either SEC or ultrafiltration (UV) using a membrane with a nominal molecular weight cut-off (NMWCO) of 100 kDa. Figure 6C shows the results of an ELISA antigenicity study with PA COPS or PA COPS: K19 BP-1 constructs using hyperimmune rabbit serum after immunization. heat killed (HH)Pseudomonas aeruginosa (after the fourth immunization). COPS enterotoxicE. coli (ETEC) was used as a negative control in the ELISA. Strong reactivity of PA COPS K19 BP-1 with OPS-specific immune seraPseudomonas aeruginosa, indicates that the OPS epitopes in the backbone polymer are conserved.

Figure 7A is a schematic of an exemplary MAPS BP-1 complex with a rizavidin fusion protein and OPS BP-1 or BP-1.2; Figure 7B is a schematic representation of an exemplary MAPS BP-2a complex with a fusion protein of rizavidin and OPS BP-2a; Figure 7C is a schematic representation of an exemplary MAPS BP-2b complex with a fusion protein of rizavidin and OPS BP-2b; Figure 7D is a schematic representation of the MAPS BP-3 complex with rizavidin fusion protein and OPS BP-3; Figure 7E is a schematic representation of the MAPS complex with rizavidin fusion protein and native OPS.

Figure 8A is an exemplary flow diagram of a subsequent purification process for OPS from KP and OPS from PA. Figure 8B shows the results of an illustrative SEC-HPLC (Waters BioAlliance with a Phenomenex BioSep SEC-4000 column used at a flow rate of 1 ml/min in PBS, pH 7.4 containing sodium azide (0.02%); profiles of purified KP OPS and K19 CPS superimposed on refractive index (RI) curves, from left to right: K19 CPS, O1, O3, O2 and O5 Figure 8C shows the results of an illustrative SEC-HPLC (Waters BioAlliance with a Phenomenex BioSep SEC-4000 column used at a flow rate of 1 ml /min in PBS pH 7.4 containing sodium azide (0.02%) Purified PA OPS profiles superimposed on CP curves, from left to right: O2, O6, O3, O4, O5, O1, O10 and O11.

Figure 9A is an exemplary flow diagram of a subsequent KO CPS K19 purification process. Figure 9B shows the gene construct for an exopolysaccharide overproducing PsL Pseudomonas strain. Figure 9C is a flow diagram of the subsequent purification process for PsL PA exopolysaccharide.

Figure 10, top panel, shows an exemplary SEC elution profile on a Superdex-200 column of BP-2a MAPS complexes of the Rhavi-FlaBD2-MrkA fusion protein with K19:KPO2-ADH BP-2a. Figure 10, bottom panel, shows the results of screening by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining for total protein of fractions eluted from the SEC column, samples were processed without heating in sodium dodecyl sulfate (SDS) buffer for samples containing 10 mM dithiothreitol (DTT) prior to SDS-PAGE so that the MAPS complexes can remain intact.

Figures 11A-11C show examples of SDS-PAGE analysis and total protein staining of exemplary variants of MAPS complexes with BP-1 made using different fusion proteins. Figure 11A shows the results of exemplary SDS-PAGE variants of MAPS complexes with BP-1 formed from the fusion protein of rizavidin (PRO-CN) alone with PA O10 OPS BP-1 (lanes 4 and 5), PA O1 OPS BP-1 (lanes 6 and 7) and PA O2 OPS BP-1 (lanes 8 and 9). Figure 11B shows the results of exemplary SDS-PAGE variants of MAPS complexes with BP-1 formed from rhavidin-FlaB-PcrV (Rhavi-FlaB-PcrV) protein alone with PA O10 OPS BP-1 (lanes 4 and 5), PA O1 OPS BP-1 (lanes 6 and 7) and PA O2 OPS BP-1 (lanes 8 and 9). Figure 11C shows the results of exemplary SDS-PAGE variants of MAPS complexes with BP-1 formed from the protein rizavidin-FlaB-D2-MrkA (Rhavi-FlaBD2-MrkA) with PA O10 OPS BP-1 (lanes 4 and 5), PA O1 OPS (tracks 6 and 7) and PA O2 OPS BP-1 (tracks 8 and 9). In Figures 11A-11C, rizavidin fusion protein alone was used as a control (lanes 1 and 2). Each of the samples was treated with heat (by boiling) (lanes 1, 4, 6 and 8) and without heat (lanes 2, 5, 7 and 9). MAPS complex variants with BP-1 did not break down without heating, and the protein in MAPS complex variants with BP-1 remained at the very top of the gel. Upon boiling, variants of the MAPS complexes with BP-1 were destroyed, the protein was released, and the protein dimer also dissociated. Protein molecular weight markers were also included in the experiment (lane 3) and the molecular weight of the standards is indicated to the left of the gel in kDa.

Figure 12A is a schematic representation of rhizavidin-antigen fusion proteins that are derived from Pseudomonas (PcrV; FlaB; FlaA2; FlaA1; FlaB-PcrV; FlaBD2; FlaA2-D2) or Klebsiella (MrkA), or fusion proteins derived from both Klebsiella , and from Pseudomonas (FlaB-D2-MrkA; PcrV-MrkA). Figure 12B shows the biological activity of the toll-like receptor (TLR) 5 against native Pseudomonas flagellins and Rhavi fusion proteins, alone or as a variant of the MAPS complex with pneumococcal polysaccharides (PnPS) 6B, in this assay, HEK293-NFkB:Luc cells responded to flagellin, but not a single medium . These results indicate that the rhizavidin and flagellin constructs (FlaB, FlaA1, FlaA2), alone or in combination with MAPS and PnPS 6B, stimulated TLR5 activity, hence the protein was correctly folded.

Figures 13A-13B compare the ability of MAPS complexes made with different rhizavidin-antigen fusion proteins and pneumococcal capsular polysaccharides (PnCPS) to elicit an anti-CPS 19A antibody response (Figure 13A) or an anti-CPS 6B antibody (Figure 13B ) in rabbits, as measured by ELISA. Samples obtained before immunization (P0), two weeks after the first immunization (P1) and two weeks after the second immunization (P2) were evaluated. The measured IgG response was expressed in arbitrary units (AU) based on the standard curve, counting AU per ml as 12500. The geometric mean is indicated as a horizontal black line with error bars for the 95% confidence interval. Figure 13A, upper panel: immunoglobulin (IgG) production response to PnCPS 19A with PRO-CN; rizavidin alone (Rhavi); rizavidin-FlaB-D2 (FlaBD2); rizavidin-FlaA2-D2 (FlaA2D2); rizavidin-FlaB-PcrV (FlaB-PcrV); rizavidin-FlaB-D2-MrkA (FlaBD2-MrkA); rizavidin-PcrV-MrkA (PcrV-MrkA). Figure 13A, bottom panel: PRO-CN; rizavidin-PcrV (PcrV); rizavidin-FlaB (FlaB); rizavidin-FlaA1 (FlaA1); rizavidin-FlaA2 (FlaA2); rizavidin-MrkA (MrkA). Figure 13B, top panel: IgG production response to PnCPS 6B with PRO-CN; rizavidin alone (Rhavi); rizavidin-FlaB-D2 (FlaBD2); rizavidin-FlaA2-D2 (FlaA2D2); rizavidin-FlaB-PcrV (FlaB-PcrV); rizavidin-FlaB-D2-MrkA (FlaBD2-MrkA); rizavidin-PcrV-MrkA (PcrV-MrkA). Figure 13B, bottom panel: PRO-CN; rizavidin-PcrV (PcrV); rizavidin-FlaB (FlaB); rizavidin-FlaA1 (FlaA1); rizavidin-FlaA2 (FlaA2); rizavidin-MrkA (MrkA).

Figure 14 shows the immune response in rabbits immunized with rizavidin-antigen fusion proteins (100 μg protein in Freund's adjuvant). IgG against immunization antigen in rabbits as measured by ELISA in serum samples obtained before immunization (P0) and two weeks after the second immunization (P2). The measured IgG response was expressed in arbitrary units (AU) based on the standard curve, counting AU per ml as 12500. The geometric mean is indicated as a horizontal black line. Titers are in AU for Rhavi-FlaB-D2 (FlaB-D2) proteins; Rhavi-FlaA2-D2 (FlaA2-D2); Rhavi-FlaB-PcrV (FlaB-PcrV); Rhavi-FlaB-D2-MrkA (FlaB-D2-MrkA) and Rhavi-PcrV-MrkA (PcrV-MrkA).

Figure 15 compares immunogenicity in rabbits of native OPS divalent PA O6 and O10 preparations in MAPS complexes with rizavidin PRO-CN fusion protein or rizavidin (Rhz). IgG titers against O6 or O10 OPS are presented as ELISA units before (P0), after one (P1) or two injections (P2).

Figure 16 compares the immunogenicity in rabbits of 2-valent KP O1 and KP O5 OPS BP-1 preparations; monovalent KP O1 BP-2a, as well as KP O1 BP-2b, all in variants of MAPS complexes with the PRO-CN rhizavidin fusion protein and with aluminum phosphate. IgG titers against KP O1 or KP O5 OPS are presented as ELISA units before (P0), after one (P1) or two injections (P2).

Figure 17 compares the immunogenicity in rabbits of preparations: 2-valent PA O6, O10 OPS BP-1 in complex with PRO-CN; PA O6, O10 OPS BP-1 in complex with PRO-CN (without alum); PA O6, O10-BP-1 (no protein, e.g. no PRO-CN); PA O6, O10 OPS BP-1 (no MAPS; e.g., no biotinylation, but backbone polymer and PRO-CN components included); and monovalent PA-O6 OPS BP-1 in MAPS BP-1 complexes with PRO-CN rhizavidin aluminum phosphate fusion protein, unless otherwise indicated. Anti-PA O6 and anti-PA O10 OPS IgG titers are presented as ELISA units before (P0), after one (P1) or two injections (P2).

Figure 18A compares opsonophagocytic killing (OPK) results obtained using human promyelocytic leukemia cells (HL-60), Klebsiella cells strain 12-0581 (O1:K62), rabbit immune sera (from AFV651 and AFV667 rabbits) to: KP O1 OPS BP-1 PRO-CN MAPS before and after the second immunization (left); KP O1 OPS BP-2a/rhizavidin PRO-CN MAPS before and after the second immunization (right). Increased killing was observed for anti-BP-2a sera when comparing immune sera with pre-immune sera from the same rabbit. Figure 18B compares the results of OFU obtained with HL-60 cells, Pseudomonas strain PA IATSO6 cells, rabbit immune serum to bivalent PA O6, O10-OPS BP-1 PRO-CN MAPS and monovalent PA IATSO6-OPS BP-1 PRO- CN MAPS. Separate tested rabbit sera resulted in OFU of PA IATSO6 cells, rabbit AFV643 serum resulted in a prozone effect, indicating a high titer of anti-OPS antibodies. Figure 18C shows that four separate rabbit immune sera (P2) prepared using a 2-valent PA O6 vaccine, PA O10 OPS BP-1-PRO-CN-MAPS, induced strong RFU of PA O10 bacteria on HL-60 cells.

On Figure 19A comparison of whole cell binding results obtained by flow cytometry (FC)Klebsiellastrain B5055 (O1:K2) with separate rabbit sera to PRO-CN variants MAPS KPO1, O5 OPS BP-1 (left) and KPO1 OPS BP-2a (right). These QC results for binding to whole bacteria showed a similar trend with ELISA titers towards purified KPO1 OPS, ie, low titer, low QC binding. A small number of double positive events indicated poor antibody binding or limited OPS exposure on the cell surface.Klebsiellastrain B5055. On Figure 19B (top panel) scatter plot shown PTs for a separate rabbit serum (AFV667) to PRO-CN BP-2a MAPS complex with KPO1 OPS BP-2a comparing weak binding to (B5055) and (K. pneumoniae,strain Caroli, O1:K2) and strong binding to non-encapsulated cells of strainsKlebsiella (CVD 3001) O1. Figure 19B (lower panel) shows the total binding events determined by the QC method with the same three strains.Klebsiella O1 serum from a rabbit vaccinated with 2-valent KP O1, O5 OPS BP-1 PRO-CN MAPS. These binding data indicate that the amount of CPS affecting OPS exposure may be dependent on growth conditions.

Figure 20 compares the results of PAK (O6:FlaA1) whole bacteria Pseudomonas (O6:FlaA1) binding to (left) rabbit serum to 2-valent PAO6-PAO10 to native OPS-PRO-CN (pooled serum from 3 rabbits, after the second immunization) obtained by PC. ); (middle) rabbit serum to 2-valent PAO6-PAO10 OPS BP-1-PRO-CN MAPS (pooled sera from 3 rabbits, after second immunization); (right) rabbit serum to monovalent PAO6 - IATSO6 - OPS BP-1-PRO-CN MAPS (pooled sera from 3 rabbits, after second immunization); stronger binding by LC results was observed for PAO6 OPS BP-1 MAPS compared to the native form of OPS MAPS.

Figure 21A shows the QC data for rabbit anti-FlaB binding to PA O1 of the FlaB-expressing strain O5: the right panel shows the scatterplot of the data. with a high percentage of labeled PA O1 population in sera after the third immunization (P3 immunization; pooled sera from 10 rabbits) in rabbits vaccinated with rizavidin-FlaB-D2 (no TLR5 binding region) when compared to pre-immunization sera (P0) (left panel ). Figure 21B, top panel, shows the LC results for a single P3 rabbit serum against rizavidin-FlaB-D2; central panel showing results for a single P3 rabbit serum against rizavidin-FlaB-PcrV; bottom panel showing results for a single P3 rabbit serum against rizavidin-FlaB-D2-MrkA.

Figure 22 shows the results of PFU with HL-60 cells of target strain Pseudomonas strain PAO1 (O5: expressing FlaB) with pooled rabbit anti-Rhavi-FlaB-His immune sera compared with positive control anti-PA-FlaB anti-PAO1 FlaB antibody and polyclonal human intravenous immunoglobulin (IV IG) and negative control (no antibody).

Figure 23 shows blocking of adhesion of KP O5 6997 (origin South Africa) to A549 cells (lung carcinoma) at different dilutions of anti-FlaBD2-MrkA rabbit serum. Serum against FlaBD2-MrkA (after the third immunization, P3) inhibited the binding of Klebsiella strain 6997 to A549 cells, in contrast to pre-immune serum. This indicates that the MrkA protein component in the FlaBD2-MrkA SB was correctly folded and caused the production of functional antibodies.

Figure 24A shows anti-PcrV antibody protection of A549 cells against cytotoxic intoxication by Pseudomonas strain PAK (O6:FlaA1). Monolayers of A549 cells were infected with Pseudomonas strain PAK (O6:FlaA1) for 4 hours in the presence of 10% serum from rabbits (3 pooled sera) vaccinated with the Rhavi-FlaB-PcrV fusion protein. Anti-Rhavi-FlaB-PcrV (P3, after third immunization) serum protected cells from Pseudomonas strain PAK intoxication compared to pre-immune serum, as evidenced by fewer rounded and detached cells. In Figure 24B, a monolayer of A549 cells was infected with Pseudomonas strain PAK for 4 hours in the presence of sera from NCB006 Rhavi-FlaB-PcrV (3 pooled sera) at various dilutions. Cells were washed with PBS and incubated for 1 hour with neutral red. Cells were lysed and dye uptake in each well was recorded. Anti-Rhavi-FlaB-PcrV (P3) serum protected cells from cytotoxic intoxication with Pseudomonas strain PAK, compared to pre-immune serum, up to a dilution of 1:40.

Figure 25 shows the results of a cytotoxicity assay using A549 cells and anti-FlaB-PcrV rabbit serum (after the third immunization; P3) with different strains of Pseudomonas : in the case of highly toxic PA IATS O1 (closed diamonds), there was no serum protection against cytotoxicity; in the case of cytotoxic PA M2 O5 (closed circles), there was no serum protection; in the case of cytotoxic PA 17002 O10 (closed triangles), serum protection was clearly observed; in the case of non-cytotoxic PA IATS O6 (closed triangles), there was no toxicity.

Figure 26 is a Kaplan-Meier plot of the survival of burnt mice followed by infection with 28 colony forming units (CFU) of FlaB-expressing Pseudomonas strain M-2 (O5) injected into a burn wound. In this model, CD-1 mice were passively injected with pre-immune rabbit serum or anti-FlaB rabbit serum and either mimicked phosphate buffered saline (PBS) infection or infected with Pseudomonas strain M-2 in PBS. Mice treated with anti-FlaB immune sera were protected (5/5, or 100% survival) for 10 days or more from wound infection with Pseudomonas strain M-2, while all mice from other groups except falsely infected mice died from infection of the wound.

Figure 27 shows clearance and organ loading in a mouse model of KP infection. Mice (CD1; groups of 3) were passively injected intraperitoneally (ip) with rabbit anti-KPO1 BP-2a OPS-PRO-CN MAPS serum and then infected intravenously (iv) with Klebsiella strain KP B5055 O1:K2 (9× 10 ⁴ CFU). The number of viable KP in the spleen was measured 14 hours after infection. There was a clear decrease in the number of viable KP in the spleens of mice treated with immune serum (P2) compared to the spleens of mice that received pre-immune serum (P0). These data suggest that the anti-OPS O1 antibody raised against the backbone polymer in the MAPS variant vaccine was able to clear microorganisms from mouse tissues and thus had functional protective activity in vivo .

Figure 28 is a Kaplan-Meier plot of survival when mice are passively protected with administered rabbit anti-KPO1 BP-1 OPS-PRO-CN MAPS serum prior to infection with Klebsiella strain B5055 (O1:K2) grown in salicylate to reduce capsule expression. CD1 mice (5/group) were injected ip with 0.1 ml pooled rabbit immune (P2) or pre-immune (P0) serum, followed by ip bacterial challenge with 2×10 ⁴ cfu of Klebsiella strain B5055 (O1:K2). Shown are Kaplan-Meier plots of survival for mice treated with serum P0 or P2 against KPO1 BP-1 OPS-PRO-CN MAPS. Serum P2 protected animals in the model, with mice treated with serum against KPO1 BP-1 OPS-PRO-CN MAPS surviving in greater numbers and for a longer time than mice treated with serum P0.

Figure 29 shows ELISA results of end-point anti-KP OPS IgG titers in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 (5 μg PS from each BP type in the vaccine, treatment group 5) compared to 4-valent KP vaccine (5 μg PS from each type of BP in the vaccine, experimental group 4).

Figure 30 shows ELISA results of end-point anti-PA OPS IgG titers in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 (5 μg PS from each BP type in the vaccine, treatment group 5) compared to 8-valent PA vaccine (5 μg PS from each type of BP in the vaccine, experimental group 4).

Figure 31 shows ELISA results of endpoint titers of anti-PA OPS IgG in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 (e.g., 60 μg total PS in vaccine; 5 μg PS from each BP type in vaccine), compared with 12-valent KP/PA vaccine 1 at (1 μg PS from each type of BP in the vaccine, experimental group 6).

Figure 32 shows individual IgG titers against each PS in 12-valent KP/PA vaccine 1 at a dose of 5 μg (eg, 60 μg total PS in the vaccine; 5 μg PS from each type of BP in the vaccine).

Figure 33 shows individual IgG titers against each PS in 1 μg 12-valent KP/PA vaccine 1 (eg, 12 μg total PS in vaccine; 1 μg PS from each BP type in vaccine).

Figure 34 shows the increase in the induction of individual anti-OPS IgG in the case of a 12-valent KP/PA vaccine at a dose of 5 μg.

Figure 35 compares the increase in the induction of individual anti-OPS IgG in the case of a 12-valent KP/PA vaccine at a dose of 5 μg PS per dose of BP and 1 μg PS per dose of BP.

Figure 36 shows ELISA results of end-point anti-PA and KP OPS IgG titers in pooled sera from rabbits immunized with 60 μg 12-valent KP/PA vaccine 2, 5 μg PS from each BP type per vaccine dose (experimental group 1), versus 12 µg 12 µg KP/PA vaccine 2, 1 µg PS from each BP type in the vaccine (treatment group 2).

Figure 37 shows individual IgG titers against each PS in 12-valent KP/PA vaccine 2 at a dose of 5 μg.

Figure 38 shows the increase in the induction of individual anti-OPS IgG in the case of 12-valent KP/PA vaccine 2 at a dose of 5 μg.

Figure 39 shows the results of an ELISA assay of rabbit anti-FlaBD2 IgG induced by 12-valent KP/PA vaccine 1, 12-valent KP/PA vaccine 2, 8-valent PA vaccine and 4-valent KP vaccine.

Figure 40 shows the results of an ELISA assay of rabbit anti-MrkA IgG induced by administration of 12-valent KP/PA vaccine 1, 12-valent KP/PA vaccine 2, 8-valent PA vaccine and 4-valent KP vaccine.

Figure 41 shows the results of an ELISA assay of rabbit anti-PcrV IgG induced by 12-valent KP/PA vaccine 1.

Figure 42 shows the increase in induction of individual anti-carrier protein IgGs induced by administration of 12-valent KP/PA vaccine 1, 12-valent KP/PA vaccine 2, 8-valent PA vaccine and 4-valent KP vaccine.

Figure 43 shows the increase in the induction of a response to FlaB caused by the administration of various vaccine preparations in three studies of immunogenicity in rabbits: NCB007, NCB008 and NCB012.

Figure 44 shows the increase in induction of a response to MrkA caused by the administration of various vaccine preparations in three rabbit immunogenicity studies: NCB007, NCB008 and NCB012.

Figure 45 shows the increase in PcrV response induction induced by the administration of various vaccine preparations in three rabbit immunogenicity studies: NCB007, NCB008 and NCB012.

Figure 46 shows the absence of TLR5 agonist biological activity in carrier proteins containing only FlaB domain 2.

Figure 47 shows the results of analysis of PFU with human neutrophils KP O2 K-strain 7380.

Figure 48A shows the results of an uptake and RFU assay with the J774 macrophage cell line in the presence of gentamicin. Figure 48B shows the results of the uptake and PFU analysis with the J774 macrophage cell line in the absence of gentamicin.

Figure 49 shows the results of the gentamicin protection assay for PA and KP opsonized with pooled sera from rabbits immunized with 12-valent vaccine 1. Immune serum (P2) mediates bacterial uptake by J774 macrophage-like cells, in contrast to pre-immune serum (P0).

Figure 50 shows the results of microscopic analysis of bacterial uptake by macrophage-like J774 cells using GFP-labeled PA. PA are actively taken up after exposure to immune serum.

Figure 51 shows that pooled sera from rabbits treated with a 5 μg dose of 12-valent KP/PA vaccine 1 (total 60 μg PS) stimulated bacterial uptake by phagocytic cells.

Figure 52 shows the cell survival, in percent, in the HL60 PFU with KP O2 strain 7380 assay.

Figure 53 presents the results of analyzes demonstrating that antibodies to PcrV protect cells from cytotoxicity. When mixing the PA strain (without FlaB) with 12-valent immune sera (pool 10, high vaccine dose 1), reduced cytotoxicity to cells was observed compared to using pre-immune sera from the same rabbits.

Figure 54 shows that cells treated with pooled sera from rabbits immunized at 5 μg (e.g. total 60 μg PS in vaccine; 5 μg PS from each BP type in vaccine) of 12-valent KP/PA vaccine 1 show reduced motility strain PA O5 FlaB through anti-FlaB antibody compared to cells treated with pre-immune serum.

Figure 55 shows that pooled sera from rabbits immunized with a 4-valent KP vaccine (5 μg dose, experimental group 4) without PA OPS resulted in increased aggregation of FlaB-expressing PA, indicating that the antibody generated against a FlaB-containing carrier protein is functional.

Figure 56 shows pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1, pooled sera from rabbits immunized with 8-valent PA vaccine (MrkA is the only KP antigen in the 8-valent PA vaccine), pooled sera from rabbits , immunized with a 4-valent KP vaccine, block the adhesion of KP O5 strain 6997 to A549 cells. The data suggest that the cause is an anti-MrkA antibody induced by the carrier protein FlaBD2-MrkA.

The Figure 57 shows the results of the analysis of blood clearance for 3 mice infected with KP O1 strain B5055, 1 hour and 4 hours after infection.

Figure 58 shows organ loading in the liver and spleen of mice after infection with KP O1 strain B5055.

The Figure 59 shows the results of the blood clearance analysis for 3 mice infected with KP O5 strain 6997, 1 hour and 4 hours after infection.

Figure 60 shows organ loading in the spleen of mice after infection with KP O5 (strain 6997).

Figure 61 shows the results of a passive organ loading defense assay in the spleen of mice showing the clearance of KP O2 (strain KPN-12) from the circulatory system of mice treated with sera from rabbits immunized with 12-valent KP/PA vaccine 1.

Figure 62 shows various illustrative optimized protocols and results of passive protection experiments in CD-1 mice with preimmune and immune sera against Klebsiella O2 K2 from the NCB012 study.

Figure 63 shows the induction of IL-17a in stimulated spleen cells from mice immunized with the MAPS KP O1 vaccine followed by bacterial infection with KP O1:K2 strain B5055.

Figure 64 shows the experimental protocol and results of a PA O6 colonization study of the GI tract of rats immunized with two doses of MAPS PA O6 vaccine followed by bacterial challenge.

Figure 65 is a schematic representation of the structure of KP OPS Gal-I, Gal-II and Gal-III epitopes.

Figure 66 shows binding to cells expressing KP D-Gal-III and KP D-Gal-I epitopes as determined by flow cytometry in sera from animals immunized with 4-valent KP PRO-CN MAPS vaccine.

Figure 67 shows the results of flow cytometry analysis demonstrating that antibodies in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 bind to two of the three ST258 strains from the bacteremic isolates and also bind to three of the four serotype strains not present in the vaccine with mutant MrkA alleles.

Figure 68 shows individual anti-OPS IgG titers after immunization with BP-1 (group 1) and BP-1.2 (group 2) KP/PA 4-valent MAPS vaccine.

Figure 69 shows the IgG response to carrier protein observed when pooled sera against MAPS BP-1 (Group 1) and BP-1.2 MAPS (Group 2) are compared.

Figure 70 shows individual sera antibody responses to the FlaBD2 carrier protein for BP-1 and BP-1.2 (KP/PA, 4-valent), respectively.

Figure 71 shows individual sera antibody responses to the MrkA carrier protein for BP-1 and BP-1.2 (KP/PA, 4-valent), respectively.

Definitions

In this application, unless otherwise indicated by the context, (i) the term in the singular can be considered to mean "at least one"; (ii) "or" can be understood as "and/or"; (iii) the terms "comprising" and "comprising" can be understood as covering the specified components or steps, presented either alone or together with one or more additional components or steps; (iv) the terms "about" and "approximately" are to be considered as allowing for a standard deviation, as understood by those skilled in the art; and (v) when specifying ranges, endpoints are included.

Approximately: The term "about", as used in the present invention in relation to the value, refers to the value, which is similar, in the context of the specified value. Typically, those skilled in the art familiar with the context will understand the relative degree of difference encompassed by the term "about" in this context. For example, in some embodiments, the term "about" may cover a range of values that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the indicated value.

Introduction: As used herein, the term "administration" generally means administering a composition to a patient, or system, to effect delivery of the agent that is, or included in, the composition. Those skilled in the art are familiar with a variety of methods that can, under appropriate circumstances, be used to administer to a patient, eg, a human. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, and so on. In some specific embodiments, administration may be bronchial (eg, by bronchial instillation), buccal, dermal (which may be, or include, for example, one or more of topical administration to the skin, intradermal, subcutaneous, transdermal, and so on), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, specific organ (eg, liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (eg, by intratracheal instillation), vaginal, intravitreal and so on. In some embodiments, the implementation of the introduction may include the introduction of a single dose. In some embodiments, the implementation of the introduction may include the introduction of a fixed number of doses. In some embodiments, administration may include discontinuous (eg, multiple doses separated by time) and/or intermittent (eg, separate doses separated by equal time intervals) dosing. In some embodiments, administration may include continuous dosing (eg, perfusion) for at least a selected period of time.

Means: In the present invention, the term "agent" can generally be used to refer to a compound or entity of any chemical class, including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or a combination or complex thereof. Under certain circumstances, as will be understood from the context by those skilled in the art, the term may be used to refer to an entity that is, or contains, a cell or microorganism, or a fraction, extract, or component thereof. Alternatively, or additionally, as will be clear from the context, the term may be used to refer to a natural product, in that it occurs and/or can be obtained in nature. In some cases, again, as will be clear from the context, the term can be used to refer to one or more objects that are artificial in that they are designed, constructed and/or created by human hand, and/or do not occur in nature. In some embodiments, the agent may be used in isolated or pure form; in some embodiments, the agent may be used in its raw form. In some embodiments, potential agents may be provided as kits or libraries, for example, which may be screened to identify or characterize the active agents within them. In some instances, the term "agent" may refer to a compound or entity that is, or contains, a polymer; in some cases, the term may refer to a compound or entity containing one or more polymeric fragments. In some embodiments, the term "agent" may refer to a non-polymer compound or entity and/or substantially free of any polymer and/or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity devoid of or substantially free of any polymer moiety.

Amino acid: In the broadest sense used in the present invention, the term "amino acid" refers to any compound and/or substance that can be included in the polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, the amino acid has the general structure H₂N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is a La-amino acid. The term "standard amino acid" refers to any of the twenty standard L-amino acids commonly found in natural peptides. The term "non-standard amino acid" refers to any amino acid that differs from the standard amino acids, whether obtained by synthesis or from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, may have a structural modification from the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., an amino group, a carboxyl group, one or more protons, and/or a hydroxyl group) relative to the overall structure. In some embodiments, such a modification may, for example, alter the circulatory half-life of a polypeptide containing a modified amino acid as compared to a polypeptide containing an otherwise identical, unmodified amino acid. In some embodiments, the implementation of such a modification does not significantly change the corresponding activity of the polypeptide containing the modified amino acid, in comparison with the polypeptide containing an otherwise identical, unmodified amino acid. As will be clear from the context, in some embodiments, the implementation of the term "amino acid" can be used to refer to a free amino acid; in some embodiments, it can be used to refer to an amino acid residue of a polypeptide.

Antibody: As used herein, the term "antibody" refers to a polypeptide containing canonical immunoglobulin sequence elements sufficient to confer the specific binding property of a particular target antigen. As is known in the art, intact antibodies naturally produced are about 150 kDa tetramer molecules composed of two identical heavy chain polypeptides (each about 50 kDa) and two identical light chain polypeptides (each about 25 kDa), which are connected to each other in a structure commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the ends of a Y-shaped structure), followed by three constant domains: CH1, CH2 and a carboxy-terminal CH3 (located at the base of the "Y" stem). A short region, known as the "crossover", connects the variable and constant regions of the heavy chain. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bridges in this hinge region connect two heavy chain polypeptides to each other in an intact antibody. Each light chain consists of two domains - an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, they are separated from each other by another "junction". Intact antibody tetramers consist of two heavy chain-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds connect the hinge regions of the heavy chains to each other so that the dimers are linked to each other to form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure called an "immunoglobulin chain fold" that is formed from two beta-pleated sheets (eg, 3-, 4-, or 5-stranded sheets) packed opposite each other in a tight, anti-parallel beta-barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2 and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3 and FR4). When natural antibodies are folded, the FR regions form beta-sheet structures that create a structural framework for the domains, the CDR loop regions, both from the light and heavy chains, converge in three-dimensional space, forming one hypervariable antigen-binding site located at the “Y” end structures. The Fc region of natural antibodies binds to elements of the complement system as well as to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and/or other binding characteristics of Fc regions to Fc receptors can be modulated by glycosylation or other modification. In some embodiments, antibodies made and/or used in accordance with the present invention contain glycosylated Fc domains, including Fc domains modified or engineered using such glycosylation. For the purposes of the present invention, in specific embodiments, any polypeptide or complex of polypeptides comprising sufficient sequences of immunoglobulin domains that are found in naturally occurring antibodies may be referred to and/or used as an "antibody", whether or not the polypeptide is naturally produced (e.g. , produced by an organism in response to an antigen), or produced by recombinant methods, chemical synthesis, or using another artificial system or methodology. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, and so on, as is known in the art. In addition, as used herein, the term "antibody", in appropriate embodiments, may mean (unless otherwise indicated or evident from the context) any of the constructs or formats known in the art or developed for using the structural and functional features of an antibody in an alternative presentation. For example, in some embodiments, an antibody used according to the present invention has a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (eg Zybodies^® and so on); antibody fragments such as Fab fragments, Fab' fragments, F(ab')₂-fragments, Fd'-fragments, Fd-fragments, as well as selected CDR areas or their sets; single chain Fv; polypeptide-Fc fusion products; single domain antibodies (eg single domain shark antibodies such as IgNAR or fragments thereof); camel antibodies; masked antibodies (for example, Probodies^®); immunopharmaceuticals based on a small-sized modular protein (SmallModularImmunoPharmaceuticals ("SMIP™"); single-stranded or tandem diabodies (TandAb^®); VHH; anticalin^®; nanobody^® minitel; BiTE^®; proteins containing ankyrin repeats, or darpins^®; avimers^®; DART; TCR-like antibodies;, adnectins^®; affiliates^®; transtel^®; affiliate^®; TrimerX^®; microproteins; finomers^®, centirins^® and calbitors^®. In some embodiments, the antibody may lack the covalent modification (eg, addition of a glycan) that it would have if produced naturally. In some embodiments, the antibody may have a covalent modification (eg, attachment of a glycan, payload (eg, detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant group (eg, polyethylene glycol, etc.).

Antigen: Used in the present invention, the term "antigen" means (i) an agent that causes an immune response; and/or (ii) an agent that binds to a T cell receptor (eg, when presented with an MHC molecule) or an antibody. In some embodiments, the antigen elicits a humoral response (eg, including the production of antigen-specific antibodies); in some embodiments, the antigen elicits a cellular response (eg, involving T cells whose receptors specifically interact with the antigen). In some embodiments, the antigen elicits a humoral response and a cellular response. In some embodiments, an antigen binds to an antibody, and may or may not elicit a specific physiological response in the body. Typically, an antigen may be, or comprise, any chemical molecule such as, for example, a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, polymer (in some embodiments, other than a biological polymer (e.g., other than a nucleotide or amino acid polymer) and so on. In some embodiments, the antigen is, or contains, a polypeptide. In some embodiments, the implementation of the antigen is, or contains, a polysaccharide. Those skilled in the art will appreciate that, in general, the antigen may be provided in isolated or pure form, or alternatively may be provided in raw form (e.g., together with other materials, e.g., in an extract such as a cell extract or other relatively untreated antigen-containing source). In some embodiments, the antigens used in the present invention are provided in raw form. In some embodiments, the antigen is a recombinant antigen.

Connected with: Two objects are "associated" with each other, as the term is used in the present invention, if the presence, level and/or form of one is correlated with the corresponding characteristics of the other. In some embodiments, two or more objects are physically "connected" if they interact, directly or indirectly, such that they are and/or remain in physical proximity to each other. In some embodiments, two or more entities that are physically linked are covalently linked; in some embodiments, two or more entities that are physically bonded to each other are not covalently bonded to each other, but are bonded non-covalently, for example, through interaction affinity, hydrogen bonds, van der Waals forces, hydrophobic interactions, magnetism, and as well as their combinations.

Framework polymer: As used herein, the term "framework polymer" means a polymer to which one or more polysaccharides (eg, antigenic polysaccharides) are conjugated, or may be conjugated. In some embodiments, the backbone polymer is a polymer and one or more polysaccharides (eg, antigenic polysaccharides) conjugated to the polymer.

Binding: It should be understood that as used in the present invention, the term "binding", as a rule, means non-covalent association between, or among, two or more objects. "Direct linking" includes physical contact between objects or fragments; indirect bonding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts - including cases where interacting entities or fragments are studied in isolation or in the context of more complex systems (e.g., in covalent or other association with a carrier and/or in biological system or cell).

Carrier protein: Used in the present invention, the term "carrier protein" means a protein or peptide that causes or enhances the immune response associated with it, or complexed with it, hapten. In some embodiments, the immune response elicited or enhanced by the carrier protein is a response to a hapten. In some embodiments, no significant response develops to the carrier protein itself; in some embodiments, the response to the carrier protein can be detected. In some embodiments, the carrier protein is complexed with one or more other molecules.

Click chemistry: As used herein, the term "click chemistry" refers to a fast and high throughput reaction such as that described by K. B. Sharpless in 2001. In some embodiments, the click chemistry reaction produces by-products that can be removed without the use of chromatography. In some embodiments, the click chemistry reaction produces only by-products that can be removed without the use of chromatography. In some embodiments, the click chemistry reaction is stereospecific. In some embodiments, the click chemistry reaction can be carried out using one or more easily removable solvents. In some embodiments, the click chemistry reaction can be carried out using one or more weak solvents. In some embodiments, the click chemistry reaction uses a copper-catalyzed azide-alkyne cycloaddition reaction to join one or more molecular entities. In some embodiments, the implementation of one or more molecular entities is not limited to molecular sizes. In some embodiments, the click chemistry reaction may be bioorthogonal; for example, neither the reactants nor the functional groups of their products interact with the functionalized biomolecules, and/or the reaction proceeds readily under mild, non-toxic conditions, eg, at room temperature and preferably in water. In some embodiments, the click chemistry reaction can be a copper-catalyzed Huisgen cycloaddition, an azide-alkyne [3+2] bipolar cycloaddition, a Staudinger ligation, or an azide-phosphine ligation. In some embodiments, click chemistry can be used to link two polymers (eg, two nucleic acids, or a nucleic acid and a protein or peptide, or two glycopolymers, etc.) to each other.

Colonization (eg, mucosal or GI tract): Used in the present invention, the term "colonization" means the ability of a microorganism to create a niche in the anatomical zone, for example, the mucous membrane, the site of injury, organ, and so on.

Combination Therapy: Used in the present invention, the term "combination therapy" refers to situations where the patient is simultaneously exposed to two or more therapeutic regimens (for example, two or more drugs). In some embodiments, two or more modes may be applied simultaneously; in some embodiments, such regimens may be administered sequentially (eg, all "doses" of the first regimen are administered prior to the administration of any of the doses of the second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administering" a combination therapy may include administering one or more agents or treatments to a patient receiving another agent(s) or treatment(s) in combination. To be clear, combination therapy does not require that the individual agents be co-administered in the same composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active fragments thereof, may be co-administered in a combined composition, or even in a combined compound (eg, as part of a single chemical complex or covalently bonded entity).

Composition: Those skilled in the art will appreciate that the term "composition" as used herein can be used to refer to a discrete physical object that contains one or more specific components. As a rule, unless otherwise indicated, the composition may be in any form - for example, gas, gel, liquid, solid, and so on.

Derivative: Used in the present invention, the term "derivative" means a structural analogue of the reference substance. That is, a "derivative" is a substance that has a significant structural similarity to the reference substance, such as a common core or consensus structure, but also differs in some discrete features. In some embodiments, a derivative is a substance that can be obtained from a reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be obtained by carrying out a synthetic process substantially similar (eg, having many common steps) to that which results in a reference substance.

Domain: Used in the present invention, the term "domain" means a section or part of the object. In some embodiments, a "domain" is associated with a particular structural and/or functional feature of an entity such that when the domain is physically separated from the rest of the original entity, it retains the particular structural and/or functional feature to a significant extent or all of it. Alternatively, or additionally, a domain may be, or include, a portion of an entity that, when separated from that (original) entity and associated with another entity (the recipient), substantially retains and/or transfers to the recipient entity one or more structural and/or functional features that characterize it in the original object. In some embodiments, a domain is a section or part of a molecule (eg, a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, the domain is a section of a polypeptide; in some such embodiments, the domain is characterized by a specific structural element (e.g., a specific amino acid sequence or sequence motif, α-helical structure, β-sheet structure, coiled structure, random helical structure, etc.), and/or a specific functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, and so on).

Dosage form or standard dosage form: Those skilled in the art will appreciate that the term "dosage form" may be used to refer to a physically discrete unit of an active agent (eg, therapeutic or diagnostic agent) for administration to a patient. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is the amount of a unit dose (or whole portion thereof) suitable for administration in accordance with the administration regimen that has been selected to achieve the desired or beneficial result when administered to the relevant population (i.e., in accordance with the therapeutic dosing regimen ). Those skilled in the art will appreciate that the total amount of therapeutic composition or agent administered to a particular patient is determined by one or more practitioners and may be administered in a variety of dosage forms.

Dosing regimen: Those skilled in the art will appreciate that the term "dosing regimen" may be used to refer to a set of unit doses (typically more than one) individually administered to a patient, typically at some time interval. In some embodiments, the implementation of a particular drug has a recommended dosing regimen, which may include the introduction of one or more doses. In some embodiments, the implementation of the dosing regimen includes the introduction of multiple doses, with each of them administered after a certain period of time. In some embodiments, the implementation of the time intervals between the introduction of individual doses are the same; in some embodiments, the implementation of the dosing regimen includes the introduction of multiple doses, and there are at least two different time intervals between the introduction of individual doses. In some embodiments, all doses in a dosing regimen are the same with respect to the amount administered. In some embodiments, different doses in a dosing regimen are different with respect to the amount administered. In some embodiments, the dosing regimen comprises administering a first dose in a first dose amount followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, the dosing regimen comprises administering a first dose in a first dose amount, followed by administering one or more additional doses in a second dose amount, the same as the first dose amount. In some embodiments, the implementation of the dosage regimen leads to the achievement of the desired or beneficial result when administered to the appropriate population (ie, is a therapeutic dosage regimen).

Epitope: As used herein, the term "epitope" encompasses any fragment that is specifically recognized by an immunoglobulin binding component (eg, antibody or receptor). In some embodiments, an epitope consists of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen assumes the appropriate three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically located in space in close proximity to each other when the antigen assumes such a conformation. In some embodiments, at least some of these chemical atoms or groups physically move away from each other when the antigen assumes an alternative conformation (eg, linearizes).

Exopolysaccharide: As used in the present invention, the term "exopolysaccharide" refers to natural high molecular weight polymers composed of sugar residues and secreted by microorganisms into their environment. Exopolysaccharides may be referred to interchangeably as extracellular polysaccharides (EPS). IPNs provide the functional and structural integrity of biofilms and are considered to be a fundamental component that determines the physicochemical properties of a biofilm. VPS make up from 50% to 90% of the total organic matter of biofilms.

Fragment: A "fragment" of a material or object described in the present invention has a structure that includes a discrete part of the whole, but lacks one or more fragments found in the whole object. In some embodiments, the implementation of the fragment consists of such a discrete part. In some embodiments, the implementation of the fragment includes a discrete part of the whole, while the discrete part has one or more functional characteristics in common with the whole object. In some embodiments, the implementation of the fragment consists of such a discrete part. In some embodiments, the implementation of the fragment consists of, or contains a characteristic structural element or group found in the whole object. In some embodiments, the polymer fragment contains, or consists of, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 , 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more, monomeric units (e.g., residues) that are present in the entire polymer. In some embodiments, the polymer fragment contains, or consists of, at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, monomeric units (e.g., residues) that are present as a whole polymer. In some embodiments, the implementation of the whole material or object may be called the "original" whole.

Homology: Used in the present invention, the term "homology" refers to a common relationship between polymer molecules, for example, between nucleic acid molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% identity. , 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% similarity. , 75%, 80%, 85%, 90%, 95% or 99%.

Identity: Used in the present invention, the term "identity" refers to a common relationship between polymeric molecules, for example, between nucleic acid molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymer molecules are considered "substantially identical" to each other if their sequences share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% identity. , 70%, 75%, 80%, 85%, 90%, 95% or 99%. Percent identity between two nucleotide or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (for example, deletions can be made in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes) . In specific embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides are then compared at the corresponding positions. If a position in the first sequence is occupied by the same residue (eg, nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences depends on the number of identical positions in both sequences, considering the number of deletions and the length of each deletion that must be made to optimally align the two sequences. Comparing sequences and determining percent identity between two sequences can be performed using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the Myers-Miller algorithm (CABIOS, 1989, 4: 11-17), which is included in the ALIGN program (version 2.0). In some exemplary embodiments, nucleotide sequence comparisons performed using the ALIGN program use a PAM120 residue substitution weight table, a deletion extension penalty of 12, and a deletion insertion penalty of 4. Alternatively, percent identity between two nucleotide sequences can be determined using the GAP program in GCG software package using the NWSgapDNA.CMP matrix.

"Improvement", "increase", "inhibition" or"decrease" : Used in the present invention, the terms "improvement", "increase", "inhibition", "decrease", or their grammatical equivalents, indicate a change in values relative to the original level or other benchmark. In some embodiments, the relevant benchmark may be, or include, a score in a particular system (e.g., in one individual) under otherwise comparable conditions except for the presence (e.g., before and/or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, the implementation of the corresponding benchmark may be, or include, the indicator in a comparable system, which is known or expected to respond in a certain way in the presence of the corresponding means or influence.

Selected: The term used in the present invention means a substance and/or object that was (1) separated from at least some of the components with which it was associated when it was originally obtained (either in nature and/or under experimental conditions), and /or (2) designed, produced, obtained and/or made by human hand. The isolated substances and/or objects can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were originally associated. In some embodiments, committed funds are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% %, about 99% or more about 99% pure. In the present invention, a substance is referred to as "pure" if it is substantially free of other components. In some embodiments, as understood by those skilled in the art, the substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffer, diluent , water and so on); in such embodiments, the percentage isolation or purity of a substance is calculated without taking into account such carriers or excipients. As one example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" if a) by virtue of its origin, or source of origin, it is not associated with some or all of the components that accompany him in his natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that naturally produces it; c) it is expressed or otherwise associated with components from a cell or other expression system not belonging to the species that naturally produces it. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than that which naturally produces it is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide to which one or more purification methods have been applied can be considered an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is naturally associated; and/or b) with which it was associated when it was originally produced.

Linker: Used in the present invention, the term "linker" means a part of a multi-component means, which connects different components to each other. For example, those skilled in the art will appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes an amino acid sequence between such domains that links them together. In some embodiments, the implementation of the polypeptide containing the element of the linker, in general, has the structure of the General formula S1-L-S2, where S1 and S2 may be the same or different, and represent two domains linked to each other by a linker. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 in length. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the implementation of the linker is characterized in that it does not take on a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can be appropriately used in constructing polypeptides (e.g., fusion polypeptides) are known in the art (Holligeret al., 1993; Poljak, 1994).

Pharmaceutical composition: Used in the present invention, the term "pharmaceutical composition" means a composition in which the active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a dosage unit suitable for administration in a therapeutic regimen that has a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including forms adapted for: oral administration, e.g., liquid dosage forms (aqueous or non-aqueous solutions or suspensions), tablets, e.g. and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, in the form of, for example, a sterile solution or suspension, or a sustained release formulation; topical application, for example, in the form of a cream, ointment, controlled release patch or spray applied to the skin, lungs or mouth; intravaginal or intrarectal administration, for example, in the form of a pessary, cream or foam; sublingual; eye; transdermal administration; nasal, pulmonary administration and administration to other mucous surfaces.

Pharmaceutically acceptable: In the present invention, the term "pharmaceutically acceptable" as used in relation to the carrier, diluent or excipient that is used to formulate the composition disclosed in the present invention means that the carrier, diluent or excipient must be compatible with the other ingredients of the composition and not harm the recipient.

Polysaccharide: As used herein, the term "polysaccharide" means a polymeric carbohydrate molecule composed of long chains of monosaccharide units linked together by glycosidic bonds, which, upon hydrolysis, breaks down into its constituent monosaccharides or oligosaccharides. Polysaccharides have a structure ranging from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, structural polysaccharides such as cellulose and chitin, and microbial polysaccharides and antigenic polysaccharides found in microorganisms, including but not limited to OPS, CPS, COPS, and LPS.

Prevention or Prevention: In the present invention, when used in connection with the occurrence of a disease, disorder and/or condition, the terms "prevention" or "prevention" means reducing the risk of developing a disease, disorder and/or condition, and/or delaying the onset of one or more signs or symptoms of a disease, disorders or conditions. Prevention can be considered complete if the onset of the disease, disorder, or condition has been delayed by a predetermined period of time.

Protein: As used in the present invention, the term "protein" means a polypeptide (ie, a chain of at least two amino acids linked together by peptide bonds). Proteins may include moieties other than amino acids (eg, may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those skilled in the art will appreciate that a "protein" may be a complete polypeptide chain that is produced by a cell (with or without a signal sequence) or may be a characteristic portion thereof. Those skilled in the art will appreciate that a protein may sometimes contain more than one polypeptide chain, such as linked by one or more disulfide bonds or otherwise linked. The polypeptides may contain L-amino acids, D-amino acids, or both, and may have any of a variety of amino acid modifications or analogues known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and so on. In some embodiments, proteins may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

Polypeptide: As used herein, the term "polypeptide" is generally the art-recognized designation for a polymer of at least three amino acids. Those skilled in the art will appreciate that the term "polypeptide" should be generic enough to include not only polypeptides having the entire sequence recited in the present invention, but also polypeptides that are functional fragments (i.e., fragments that retain at least at least one activity) of such complete polypeptides. In addition, those skilled in the art will appreciate that protein sequences generally can tolerate some degree of change without disrupting activity. Thus, any polypeptide that retains activity and has, in general, at least about 30-40% sequence identity, often more than about 50%, 60%, 70%, or 80%, and also typically includes at least at least one area with a much higher degree of identity, often more than 90% or even 95%, 96%, 97%, 98% or 99% in one or more highly conservative areas, usually including at least 3-4 and often up to 20 amino acids or more, with another polypeptide of the same class, is encompassed by the appropriate term "polypeptide" as used herein. The polypeptides may contain L-amino acids, D-amino acids, or both, and may have any of a variety of amino acid modifications or analogues known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and so on. In some embodiments, proteins may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

Prevention: Used in the present invention, the term "prevention" means delaying the onset of development and/or reducing the frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder, or condition is observed in the population predisposed to a disease, disorder, or condition. Prevention can be considered complete if the onset of the disease, disorder, or condition has been delayed by a predetermined period of time.

Protein: As used in the present invention, the term "protein" means a polypeptide (ie, a chain of at least two amino acids linked together by peptide bonds). Proteins may include moieties other than amino acids (eg, may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those skilled in the art will appreciate that a "protein" may be a complete polypeptide chain that is produced by a cell (with or without a signal sequence) or may be a characteristic portion thereof. Those skilled in the art will appreciate that a protein may sometimes contain more than one polypeptide chain, such as linked by one or more disulfide bonds or otherwise linked. The polypeptides may contain L-amino acids, D-amino acids, or both, and may have any of a variety of amino acid modifications or analogues known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and so on. In some embodiments, proteins may contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

Recombinant: As used herein, the term "recombinant" means polypeptides that have been designed, engineered, produced, expressed, created, manufactured and/or isolated by recombinant methods, for example, polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant combinatorial library of human polypeptides; polypeptides derived from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or has been otherwise altered to express a gene or genes or gene components that encode and/or direct the expression of a polypeptide or one or more than its components, parts, elements or domains; and/or polypeptides obtained, expressed, created or isolated by any other methods, including splicing or ligation of selected elements of nucleotide sequences with each other, chemical synthesis of selected elements of the sequence and/or otherwise obtaining a nucleic acid that encodes and/or directs the expression of a polypeptide or one or more of its components, parts, elements or domains. In some embodiments, one or more of such selected sequence elements occur naturally. In some embodiments, one or more of such selected sequence elements are constructedin silico. In some embodiments, one or more of such selected sequence elements are obtained by mutagenesis (e.g.,in vivo orin vitro) a known sequence element, for example, from a natural or synthetic source, such as, for example, present in the germline of the original organism of interest (eg, human, mouse, etc.).

Reference: Used in the present invention, the term "reference" describes the standard, or control, which is compared. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested and/or determined at substantially the same time that the object of interest is tested or determined. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is defined or characterized under conditions or circumstances comparable to those under which the evaluation is performed. Those skilled in the art will recognize when there is sufficient similarity to justify reliance on, and/or comparison with, a particular candidate reference or control.

Answer: As used herein, the term "response" to treatment can mean any beneficial change in a patient's condition that is caused by, or correlates with, treatment. Such change may include stabilizing the condition (eg, preventing deterioration that would have occurred in the absence of treatment), reducing the symptoms of the condition and/or improving the prospects for treatment of the condition, and so on. It can mean the patient's response or the tumor's response. Tumor or patient response can be measured according to a variety of criteria, including clinical criteria and objective criteria. Response evaluation methods include, but are not limited to, clinical examination, positron emission tomography, chest x-ray, CT scan, MRI, ultrasound, endoscopic, laparoscopic, determination of the presence and level of tumor markers in a patient sample, cytology and/or histological analysis. Many of these methods are aimed at determining the size of the tumor or otherwise determine the total tumor burden. Methods and guidelines for assessing response to treatment are described in Therasse et. al. (2000) "New guidelines to evaluate the response to treatment in solid tumors", European Organization for Research and Treatment of Cancer, US National Cancer Institute, National Cancer Institute of Canada. The exact response criteria may be chosen in any appropriate manner, provided that when comparing groups of tumors and/or patients, compared groups are evaluated based on the same or comparable criteria to determine the degree of response. One skilled in the art will be able to select appropriate criteria.

Risk: As will be clear from the context, "risk" of developing a disease, disorder, and/or condition means the likelihood that a particular individual will develop a disease, disorder, and/or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample, or group of reference samples, is associated with a known risk of a disease, disorder, condition, and/or event. In some embodiments, reference samples, or a group of reference samples, are obtained from individuals comparable to a particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

Serotype: Used in the present invention, the term "serotype", also called serovar, refers to a certain variation among species of bacteria or viruses, or among the immune cells of different individuals. These micro-organisms, viruses or cells are collectively classified based on their cell surface antigens, which makes it possible to classify the micro-organisms epidemiologically at the subspecies level. A group of serovars with common antigens may be called a serogroup or sometimes a serocomplex.

Short oligosaccharide: For the purposes of the present invention, an oligosaccharide is generally considered "short" if it has less than 4, or less than 3, residues in any linear chain.

Patient: Used in the present invention, the term "patient" means an organism, usually a mammal (eg, human, in some embodiments, including a human before birth). In some embodiments, the implementation of the patient suffers from a corresponding disease, disorder or condition. In some embodiments, the patient is predisposed to a disease, disorder, or condition. In some embodiments, the patient exhibits one or more symptoms or signs of a disease, disorder, or condition. In some embodiments, the patient does not exhibit any symptoms or signs of the disease, disorder, or condition. In some embodiments, the patient is a patient with one or more features indicative of a predisposition to or risk of developing a disease, disorder, or condition. In some embodiments, the patient is a patient. In some embodiments, the patient is an individual who has been diagnosed and/or is being treated and/or has been treated.

Predisposition: An individual who is "predisposed" to a disease, disorder, or condition is at risk of developing the disease, disorder, or condition. In some embodiments, the individual who is predisposed to the disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, the individual who is predisposed to the disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, the individual who is predisposed to the disease, disorder, or condition is an individual who has been exposed to conditions associated with the development of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and/or condition is a risk based on an estimate of the population (eg, family members of individuals suffering from the disease, disorder, or condition).

Reducing symptoms: In accordance with the present invention, there is a "reduction in symptoms" when one or more symptoms of a particular disease, disorder or condition are reduced in magnitude (eg, intensity, severity, etc.) and/or frequency. To be clear, delaying the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

Therapeutically effective amount: Used in the present invention, the term "therapeutically effective amount" means an amount that produces the desired effect for which it was introduced. In some embodiments, the term means an amount that is sufficient when administered to a population suffering from or predisposed to a disease, disorder and/or condition, in accordance with a therapeutic dosage regimen for the treatment of the disease, disorder and/or condition. In some embodiments, a therapeutically effective amount is an amount that results in a reduction in the frequency and/or severity of, and/or a delay in the onset of, one or more symptoms of a disease, disorder, and/or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not necessarily mean that a particular individual has been successfully treated. Rather, a therapeutically effective amount may be that amount which provides a particular desired pharmacological response in a significant number of patients when administered to patients in need of such treatment. In some embodiments, a reference to a therapeutically effective amount may be a reference to an amount measured in one or more specific tissues (e.g., tissue affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). and so on). Those skilled in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapeutic agent may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount of an agent may be formulated and/or administered in multiple doses, eg, as part of a dosage regimen. In some embodiments, a therapeutically effective amount may mean an amount of an immunogenic composition or vaccine that is sufficient to elicit an immune response. For example, "therapeutically effective amount" means a non-toxic but sufficient amount that can be used to treat, reduce or prevent infection and/or disease (eg, bacterial infection, pneumococcal infection, and so on) in any patient.

Treatment: As used herein, the term "treatment" (and also "treat" or "therapy") refers to any use of therapy that results in partial or complete reduction, alleviation, mitigation, inhibition, delay in onset, reduction in severity, and/or reduction the frequency of one or more symptoms, signs and/or causes of a particular disease, disorder and/or condition. In some embodiments, such treatment may be administered to a patient who is not showing signs of the relevant disease, disorder, and/or condition and/or to a patient who is showing only early signs of the disease, disorder, and/or condition. Alternatively, or additionally, such treatment may be administered to a patient who exhibits one or more of the recognized features of a relevant disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a patient who has been diagnosed as suffering from a relevant disease, disorder, and/or condition. In some embodiments, the treatment may be applied to a patient who is known to have one or more predisposition factors that are statistically correlated with an increased risk of developing the corresponding disease, disorder and/or condition.

Option: Used in the present invention in the context of molecules, such as nucleic acids, proteins or small molecules, the term "variant" means a molecule that has significant structural identity with the reference molecule, but differs structurally from this reference molecule, for example, the presence or absence, or level one or more chemical groups compared to a reference molecule. In some embodiments, the variant also differs functionally from its reference molecule. As a rule, the decision about whether there is reason to consider a particular molecule as a "variant" of the reference molecule is based on the degree of its structural identity with the reference molecule. As understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a single molecule that shares one or more of these characteristic structural elements, but differs in at least one aspect from the reference molecule. As a few examples, a polypeptide may have a characteristic element of the sequence, consisting of a plurality of amino acids having certain positions relative to each other in linear or three-dimensional space and/or contributing to the formation of a particular structural motif and/or biological function; a nucleic acid may have a characteristic element of the sequence, consisting of a plurality of nucleotide residues having certain positions relative to each other in linear or three-dimensional space. In some embodiments, the variant polypeptide or nucleic acid may differ from the reference polypeptide or nucleic acid due to one or more amino acid or nucleotide sequence differences and/or one or more chemical group differences (e.g., carbohydrates, lipids, phosphate groups) , which are covalently linked components of the polypeptide or nucleic acid (eg, which are attached to the backbone of the polypeptide or nucleic acid). In some embodiments, the variant polypeptide or nucleic acid has a common sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97% or 99%. In some embodiments, the variant polypeptide or nucleic acid differs in at least one characteristic sequence element from the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities with the reference peptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference peptide or nucleic acid. In some embodiments, the implementation of the variant polypeptide, or nucleic acid, has a reduced level of one or more types of biological activity, compared with the reference polypeptide, or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference peptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but has few sequence changes at specific positions. Typically less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% residues per option replaced, inserted or deleted in comparison with the reference. In some embodiments, the variant polypeptide or nucleic acid has about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue relative to a reference. Often, a variant polypeptide or nucleic acid has a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues that are involved in a particular biological activity). ) in comparison with the standard. In some embodiments, the variant polypeptide or nucleic acid has no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions, and, in some embodiments, no additions or deletions, compared to a reference. In some embodiments, the variant polypeptide or nucleic acid has less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the implementation of the reference polypeptide, or nucleic acid, occurs in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

Vaccination: Used in the present invention, the term "vaccination" means the introduction of a composition designed to initiate an immune response, for example, to a disease-causing agent. For the purposes of the present invention, vaccination can be carried out before, during and/or after exposure to the disease-causing agent and, in specific embodiments, before, during and/or shortly after exposure to the agent. In some embodiments, the implementation of the vaccination includes several introductions, suitably spaced apart in time, the vaccine composition.

DETAILED DESCRIPTION OF SPECIFIC IMPLEMENTATION OPTIONS

The present invention relates generally to compositions, systems and methods that include complexed proteins and polysaccharides, eg protein-polysaccharide complexed vaccines. Such complexes can be used, for example, to induce and/or enhance an immune protective response in patients at risk of nosocomial infection.

A nosocomial infection is an infection acquired from a hospital or other health care organization within at least about 48 hours of admission. To emphasize both hospital and non-hospital settings, hospital-acquired infection is sometimes referred to as healthcare-associated infection (HAI or HAI). In some embodiments, the nosocomial infection may be acquired in a hospital, nursing home, rehabilitation center, clinic, or other clinical setting. In some embodiments, the nosocomial infection is clinically transmitted to a predisposed patient in a variety of ways. In some embodiments, healthcare personnel may spread a nosocomial infection other than transmission through contaminated equipment, linens, or airborne droplets. In some embodiments, the source of the nosocomial infection may be the external environment, another infected patient, personnel who may be infected, or, in some cases, the source of the nosocomial infection may be unknown. In some cases, the organism causing nosocomial infection originates from the patient's own skin microbiota, becoming opportunistic after surgery or other procedures that compromise the skin's protective barrier. Although a patient's own skin may be the source of infection, such an infection is still considered nosocomial because it develops in a healthcare setting.

Resistance to antimicrobial agents

People are increasingly understanding and appreciating the role of vaccines in tackling the problem of antimicrobial resistance (AMR). Vaccines can reduce the prevalence of resistance by reducing the need for antimicrobial use, and can reduce its consequences by reducing the overall number of cases. By reducing the number of pathogens that may be responsible for a particular clinical syndrome, vaccines may allow the use of narrower spectrum antibiotics for empiric therapy. These effects can be enhanced by herd immunity, extending protection to unvaccinated people in the population. Since much of the selection for resistance is due to selection for uninvolved normal microflora, vaccination can reduce the pressure of selection for resistance even for pathogens not included in the vaccine. Some vaccines have had a disproportionate effect on drug-resistant clones of the target species, a beneficial effect that could have been more thoughtfully exploited in vaccine development (Lipsitch and Siber, 2016). The beneficial effects of vaccines in controlling MTCT for each of these mechanisms can be enhanced by indirect protection, or herd immunity (Fine, 1993), which occurs when vaccinated individuals do not become infected or colonized and thus do not transmit the pathogen to others. . In this case, the number of infections, resistant infections and the use of antimicrobial agents can be reduced not only for vaccinated individuals, but also for their contacts. Finally, for vaccines against microorganisms that asymptomatically colonize the nasopharynx, skin, intestines, or other areas of the body, there is a theoretical possibility that the reduction in microbial population density due to vaccination would reduce the possibility of genetic exchange of resistance elements (Levin et al., 1979; Levin and Cornejo, 2009).

Vaccines of particular interest are those that target the most important HCAI pathogens, which are often resistant to multiple antibiotics (CDC, 2013; Chang et al., 2015). The most common causative agents of HAIs include multidrug-resistant gram-negative bacteria; recent publications report isolates from around the world that have become resistant to the drugs of last resort, polymyxin B and colistin (Du et al., 2015: Liu et al., 2016). Resistance to first and second line agents is also a problem in Gram-positive organisms. Candida species is an important causative agent of mucosal and disseminated infections in immunocompromised patients, as well as after antimicrobial therapy (CDC, 2013). Gram-negative E. coli, Pseudomonas , Klebsiella and Acinetobacter species are also becoming increasingly resistant to many antibiotics (McGann et al., 2016; Collignon, 2009; Agodi et al., 2011, WHO report, 2014). To address this knowledge gap, CDC began a three-stage study in 2009 to design and conduct a multilevel survey on HAI prevalence and antimicrobial use. Prevalence studies have been carried out in other countries to understand the extent and depth of the problem associated with such an infection. CDC's efforts culminated in 2011 with a large-scale survey that estimated the prevalence of HCAI in acute care hospitals, determined the prevalence of these infections by site of infection and pathogen as shown in Table 1, and provided updated estimates of the national burden of these infections (Magill et al., 2014).

Surveys were conducted in the US in 183 hospitals (Magill et al., 2014). Of 11282 patients, 452 had 1 or more HCAIs (4.0%; 95% CI, 3.7-4.4). Of the 504 such infections, the most common types were pneumonia (21.8%), surgical field infections (21.8%), and gastrointestinal (GI) infections (17.1%). C. difficile was the most commonly reported pathogen (causing 12.1% of healthcare-associated infections). Medical device infections (i.e., central catheter-associated bloodstream infection, catheter-associated urinary tract infection, and ventilator-associated pneumonia), which have traditionally been targeted by HCAI prevention programs, accounted for 25.6% of these infections. The inventors estimate that there were 648,000 patients in emergency hospitals in the United States of America (USA) in 2011 with 721,800 healthcare-associated infections. Vaccination against K. pneumoniae , P. aeruginosa , E. coli , or a combination of these, in the hospital setting would significantly reduce the incidence of the most common HAIs, such as pneumonia, surgical site infections, and urinary tract infections, and reduce the prevalence of resistance to antibiotics, as shown in Table 1.

Table 1: Multilevel estimation of HAI prevalence: by type of infection (Magill et al., 2014)

pathogen All plots Pneumonia Surgical field infections RBI IMP Gastrointestinal infections K. pneumoniae 9.9% 11.8% 13.6% 8% 23.1%* 1.2% P. aeruginosa 7.1% 12.7%* 6.4%* 4% 10.8%* 1.2% A. baumannii 1.6% 3.6%* 1.8% 0% 0% 0% E. coli 9.3% 2.7% 12.7%* 10% 27.7%* 1.2% S. aureus 10.7% 16.4%* 15.5%* 14% 3.1% 1.2% C. difficile 12.1% 0% 0% 0% 0% 70.9%* Candida 6.3% 3.6% 2.7% 22%* 4.6% 3.5% ^* Most antibiotic resistant bacteria are in these groups

Immunogenic complexes

The present invention relates to immunogenic complexes that include backbone polymers, polysaccharides and polypeptides.

In some embodiments, the immunogenic complexes are multiple antigen presentation systems (MAPS). Some MAPS systems have previously been described in WO 2012/155007. Typically, the immunogenic complex described in the present invention is a novel variant of MAPS-type complexes that include (i) a backbone polymer comprising a polymer and one or more polysaccharides (eg, antigenic polysaccharides) conjugated to the polymer; and (ii) one or more polypeptides (eg, antigenic and/or carrier polypeptides) in a non-covalent complex with a polymer and/or antigenic polysaccharide. Some exemplary backbone polymers are shown in the immunogenic complexes shown in Figures 7A-7D. In some embodiments, the variant MAPS complex is BP-1 MAPS. In some embodiments, the variant MAPS complex is BP-1.2 MAPS. In some embodiments, the variant MAPS complex is MAPS BP-2a. In some embodiments, the variant MAPS complex is BP-2b MAPS. In some embodiments, the variant MAPS complex is BP-3 MAPS.

In some embodiments, one or more polypeptide antigens are in a non-covalent complex with a backbone polymer and/or an antigenic polysaccharide conjugated to said polymer by forming at least one affinity molecule/complementary affinity molecule pair comprising (i) a first affinity a molecule associated with a polymer and/or antigenic polysaccharide; and (ii) a complementary affinity molecule associated with one or more polypeptide antigens. In some embodiments, one or more polysaccharides are chemically bonded to a polymer. In some embodiments, one or more antigenic polysaccharides are derived from Gram-negative bacteria and/or Gram-positive bacteria. In some embodiments, the one or more microbial antigenic polysaccharides are derived from K. pneumoniae, P. aeruginosa, and E. coli antigenic polysaccharides.

In some embodiments, one or more polypeptides are covalently linked (eg, fused) to a complementary affinity molecule as described herein.

In some embodiments, one or more antigenic polysaccharides are conjugated to a backbone polymer. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 0.01 to about 10 by weight. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 0.01 to about 0.1 by weight. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 0.01 to about 1 by weight. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 0.1 to about 1 by weight. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 0.1 to about 10 by weight. In some embodiments, one or more antigenic polysaccharides are conjugated to the backbone polymer in a ratio of about 1 to about 10 by weight.

In some embodiments, one or more antigenic polysaccharides are derived from one or more pathogens. In some embodiments, one or more antigenic polysaccharides are derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24 or 25 serotypes or strains of the pathogen.

In some embodiments, the framework polymer contains one or more affinity molecules conjugated to the framework polymer. In some embodiments, the antigenic polysaccharides comprise one or more affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the backbone polymer and the antigenic polysaccharides are conjugated to one or more affinity molecules. In some embodiments, the backbone polymer is conjugated to one or more affinity molecules and the antigenic polysaccharides are not conjugated to the affinity molecule. In some embodiments, the backbone polymer is not conjugated to an affinity molecule and the antigenic polysaccharides are conjugated to one or more affinity molecules. In some embodiments, the affinity molecules include biotin or biotin derivatives.

In some embodiments, the framework polymer contains a plurality of affinity molecules conjugated to the framework polymer. In some embodiments, the antigenic polysaccharides comprise a plurality of affinity molecules conjugated to the antigenic polysaccharides. In some embodiments, the framework polymer and the antigenic polysaccharides are conjugated to a plurality of affinity molecules. In some embodiments, the backbone polymer is conjugated to a plurality of affinity molecules and the antigenic polysaccharides are not conjugated to the affinity molecule. In some embodiments, the backbone polymer is not conjugated to an affinity molecule and the antigenic polysaccharides are conjugated to a plurality of affinity molecules. In some embodiments, the affinity molecules include biotin or biotin derivatives.

In some embodiments, the implementation of the polysaccharides and/or polypeptides that can be included in the immunogenic complexes obtained by recombinant methods or methods of synthesis. In some embodiments, the polysaccharides and/or polypeptides that may be included in the immunogenic complexes are isolated and/or obtained from natural sources. Illustrative polysaccharides and/or polypeptides are described below.

Klebsiella

The vast majority of Klebsiella infections are associated with hospitalization. As opportunistic pathogens, Klebsiella spp . mainly attack immunocompromised individuals who are hospitalized and suffer from severe underlying diseases such as diabetes mellitus or chronic obstructive pulmonary disease. The causative agent of nosocomial infections Klebsiella is mainly K. pneumoniae , the most important species of the genus for medicine. It is estimated that Klebsiella spp . is the causative agent in 8% of all nosocomial bacterial infections in the US and Europe (Podschun and Ullmann, 1998). There were no significant geographic differences in incidence, with the exception of hypervirulent K1-encapsulated strains, which occur predominantly in Asia and are rare in Europe and the Western Hemisphere. In the US, Klebsiella accounts for 3-7% of all hospital-acquired bacterial infections, making it one of the eight most important infectious pathogens in hospitals (Horan et al., 1988; Schaberg et al., 1991); and data from the United Kingdom (Bergogne-Berezin, 1995) and from Germany (Ullmann, 1986) are surprisingly similar to those reported by the CDC. Thus, in some embodiments, the immunogenic complex described herein comprises one or more Klebsiella.beta . polysaccharides and/or polypeptides.

In some embodiments, the immunogenic complex described herein comprises one or more polysaccharides from LPS and/or CPS polysaccharides.Klebsiella. In some embodiments, the polysaccharides from the LPS are OPS. In some embodiments, the polysaccharides from the LPS are COPS. Capsules are produced by almost all strainsKlebsiella; they are the outermost layer of surface structures in contact with the host environment and have been shown to be highly immunogenic and non-toxic (Cryzet al., 1985). The lack of a vaccine against CPSKlebsiella pneumoniae (KP) is a large number of capsular (K) antigens (more than 80 different antigens). However, in studies of the frequency of occurrence of capsular types among bacteremic isolatesKlebsiella, only 25 serotypes were found to account for 70% of all bacteremic strains (Cryzet al., 1986a). Based on these sero-epidemiological data, a 24-valent KP CPS vaccine was formulated, which was subsequently proven to be safe and immunogenic (Edelmanet al., 1994). However, simple polysaccharides alone are not immunogenic enough to induce and maintain a long-term and effective memory response, and often must be conjugated to proteins to be effective. The development of a 24-valent polysaccharide conjugate KP vaccine is complex and problematic because combining 24 glycoconjugates in one vaccine may require balancing the immunogenicity of each component.

Owing to its endotoxic properties, LPS is considered important in the pathology of septicemia. Until recently, it was generally believed that O antigens LPSKlebsiella masked by CPS, and therefore not exposed to the bacterial surface. However, several studies have demonstrated surface exposure and antibody accessibility of O antigens in strains expressing specific capsular serotypes (Tomaset al., 1988). Unlike K antigens, only eight O types are known, with O1 being the most common O type in clinical isolates. The four KP isolates (O1, O2, O3, O5) account for the majority of clinical isolates (Hansenet al., 1999). It has been reported that administration of monoclonal antibodies (mAbs) to the O1 antigenKlebsiella produced a protective effect in a mouse model of lethal endotoxemia (Mandineet al., 1990).

In some embodiments, the immunogenic complex described herein comprises a Klebsiella pili type 3 protein or polypeptide. Unlike other pili, type 3 pili cause agglutination of tannin-treated erythrocytes. Although its name, mannose-resistant, Klebsiella -like hemagglutinin (MR/K-HA), implies that this fimbrial type is synthesized only by Klebsiella , more recent studies have demonstrated that type 3 pili are present in many genera of intestinal bacteria ( Clegg and Gerlach, 1987). In addition, type 3 pili are apparently not identical across all genera of Enterobacteriaceae, as serological studies have shown significant antigenic diversity (Old and Adegbola, 1985). Initially described as adhesion organelles of Klebsiella that inhabit plant roots, these pili were later found to be able to bind to various human cells. K. pneumoniae strains expressing type 3 pili attach to endothelial cells, airway epithelium, and uroepithelial cells (Hornick et al., 1992; Tarkkanen et al., 1997; Würker et al, 1990). In the kidney, these pili mediate bacterial adhesion to tubular basement membranes, Bowman's capsules, and renal blood vessels (Tarkkanen et al., 1990). MrkA is the main protein of the type III fimbrial complex and is involved in host cell adhesion and biofilm formation (Murphy and Clegg, 2012), a strategy used by bacterial pathogens upon infection (Burmolle et al., 2008). MrkA, but not adhesin (MrkD), has previously been shown to promote biofilm formation (Langstraat et al., 2001). The MrkA antigen has a high degree of sequence conservation across different KP isolates and is generally available as an extracellular target (Wang et al., 2016). MrkA of the two most predominant pathogenic isolates, K. pneumoniae and Klebsiella oxytoca , have 95% homology. In addition, MrkA homology in representative members of the Enterobacteriaceae family is >90% and thus these sequence data provide the potential for developing a MrkA-based strategy against K. pneumoniae and all Gram-negative bacteria.

Polysaccharides of Klebsiella pneumoniae

In some embodiments, the implementation of the immunogenic complex described in the present invention includes one or more polysaccharidesK. pneumoniae. Apart from CPS (e.g. K-antigen), OPS is another important virulence factor forK. pneumoniae. There are at least 9 subtypes of OPSK. pneumoniae, each with a different polysaccharide chemical structure. In some embodiments, the OPS polysaccharides are included in the immune complex described in the present invention. Each serotype designation used in the present invention is indicated in accordance with the Reference and Research Center for the studyEscherichia AndKlebsiellacooperating with the World Health Organization (WHO).

In some embodiments, the immunogenic complex comprises one or more polysaccharidesKlebsiella, which are polysaccharides, or derivatives thereof, from one or more serotypesK. pneumoniaeselected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8 and O12. In some embodiments, the immune complex comprises polysaccharides, or derivatives thereof, from one or more of the O1, O2, O3, and O5 serotypes.Klebsiella, or combinations thereof. In some embodiments, the immune complex comprises polysaccharides, or derivatives thereof, from each of the O1, O2, O3 and O5 serotypesKlebsiella.

In some embodiments, the immunogenic complex comprises one or more polysaccharidesKlebsiella, which are, or derived from, OPS. In some embodiments, the OPS is, or is derived from, serotype O1, O2, O3, O5Klebsiella, as well as their variants and/or combinations. In some embodiments, the implementation of the immune complex includes OPSKlebsiella, selected from, or derived from, one or more of the O1, O2, O3 and O5 serotypes, or combinations thereof. In some embodiments, the implementation of the immune complex includes OPSKlebsiella from, or derived from, each of the O1, O2, O3 and O5 serotypes.

In some embodiments, the immunogenic complex comprises one or more Klebsiella polysaccharides that are, or are derived from, CPS. In some embodiments, the implementation of the CPS is, or derived from K1, K2, K10, K16 and K19 Klebsiella . In some embodiments, the CPS is or is derived from K19 Klebsiella .

Polypeptides of Klebsiella pneumoniae

In some embodiments, the implementation of the immunogenic complex described in the present invention includes one or more polypeptidesK. pneumoniae. In some embodiments, the polypeptideK. pneumoniae is a polypeptide, or derivative, of type I fimbrial proteinK. pneumoniae, or its immunogenic fragment. In some embodiments, the polypeptideK. pneumoniae is, or contains, the polypeptide of SEQ ID NO: 1 published in Gerlachet al., 2012, J Bacteriology, or an immunogenic fragment thereof. In some embodiments, the polypeptideK. pneumoniae is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the sequence of SEQ ID NO: 1, or an immunogenic fragment thereof.

SEQ ID NO: 1 K. pneumoniae type I fimbrial protein

mkiktlamivvsalslsstaaladtttvnggtvhfkgevvnaacavdagsidqtvqlgqvrsaklatagstssavgfniqlddcdttvatkasvafagtaidssnttvlalqnsaagsatnvgvqildntgtplaldgatfsaattlndgpniipfqaryyatgaatagianadatfkvqye

In some embodiments, the K. pneumoniae polypeptide is a polypeptide, or derivative, of the K. pneumoniae conserved type III fimbrial protein MrkA, or an immunogenic fragment thereof. In some embodiments, the K. pneumoniae polypeptide is, or contains, a polypeptide of SEQ ID NO: 2, or an immunogenic fragment thereof. In some embodiments, the K. pneumoniae polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 2, or an immunogenic fragment thereof.

SEQ ID NO: 2 Conserved fimbrial protein type III MrkA K. pneumoniae

MKKVLLSAAMATAFFGMTAAHAADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYQ

In some embodiments, the K. pneumoniae polypeptide is a polypeptide, or derivative, of the K. pneumoniae MrkA fimbrial protein type III, or an immunogenic fragment thereof, e.g., a repeat sequence fragment to provide stabilizing interactions with the monomeric MrkA protein. In some embodiments, the K. pneumoniae polypeptide is, or contains, a polypeptide of SEQ ID NO: 3, or an immunogenic fragment thereof. In some embodiments, the K. pneumoniae polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3, or an immunogenic fragment thereof.

SEQ ID NO: 3 K. pneumoniae type III stabilized fimbrial protein MrkA

ADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYVSQGGGGGADTTVGK

Pseudomonas

Pseudomonas is the main causative agent of HCAI, particularly in immunocompromised patients due to neutropenia and in mechanically ventilated patients, and is the leading cause of death in patients with cystic fibrosis. Pseudomonas infection also occurs in burn wounds, eye injuries (eg, cornea), trauma, and so on. Thus, in some embodiments, the immunogenic complex described herein comprises one or more Pseudomonas polysaccharides and/or polypeptides.

In some embodiments, the immunogenic complex described herein comprises Pseudomonas O antigen. Previously, Wellcome Research focused on developing a vaccine based on this antigen by combining purified extracts containing the O antigen from 16 out of 20 serotypes into a PEV-1 vaccine. Results from a total of five phase 1-2 clinical trials were published from 1976 to 1984 (Jones et al., 1976, 1978, 1979, 1980; Langford and Hiller, 1984). Safety data from the vaccine indicate that it was well tolerated. Seroconversion was relatively constant in vaccinated groups of healthy volunteers, as well as patients with burns or cystic fibrosis (CF). Vaccination had a variable effect on burn wound colonization, from negligible or none to significant. In one study, blood cultures showed an almost 10-fold reduction in bacteria due to vaccination. In every study involving patients with burns, there was a reduction in mortality that ranged from 70-100%, although the number of deaths due to P. aeruginosa is not always accurately determined. In CF studies (Langford and Hiller, 1984) there appeared to be no clinical benefit or difference in P. aeruginosa colonization upon vaccination, and no correlation between ELISA antibody titers and clinical or bacteriological parameters. An octavalent O-antigen-exotoxin A conjugate vaccine (Aerugen) was developed by the Swiss Serum Institute, Berna Biotech and Crucell and has gone through three Phase 1-2 clinical trials with published results (Schaad, et al., 1991; Lang, et al . al., 1995; Cryz et al., 1994). Their intramuscular (IM) vaccine appeared to be well tolerated, and seroconversion for all serotypes was found to be significant in all studies. Vaccination resulted in a significant reduction in colonization in most studies, and no PA-related deaths were reported in the vaccine or placebo groups. In the Crucell phase 3 study of 476 patients with CF, the results for the vaccine did not reach the desired end point and did not replicate the results obtained in previous studies.

In some embodiments, the immunogenic complex described herein comprises a Pseudomonas flagellum, or an antigenic fragment thereof. Early efficacy studies (Holder et al., 1982; Holder and Naglich, 1986; Montie, et al., 1987) found that both type a and b flagellum could confer type-specific protection against death in murine burn models. , however, the type b flagellum seems to have been somewhat more efficient in this. Preclinical studies by IMMUNO AG (Crowe, et al., 1991) used individual vaccines consisting of a0, a0a1a2, a0a2, a0a3, a0a3a4 and b flagellum types and determined that the b antigen appears to be the best immunogen , providing a higher degree of protection in a mouse model of immune deficiency than type a preparations. The results of a phase 1 study in the same publication showed that all of these immunogens produced serum antibodies with high titers and long duration of action, as did a single type b immunogen in a subsequent phase 2 study (Döring, et al., 1995), where they also observed seroconversion of IgG, IgA and sIgA in bronchoalveolar washings. Administration of the combined type a (a0a1a2) + type b flagella vaccine to patients with CF (phase 3) resulted in the production of high titers and long acting serum IgG. However, although there was some protective effect against P. aeruginosa lung infection, the presence of non-vaccine related serotypes prevented a clear interpretation of the result (Döring and Dorner, 1997; Döring, et al. 2007). In a subsequent review, Döring and Pier (2008) suggested that a bivalent flagellar vaccine may not be optimal, and yet there appears to be no data available for a single vaccine containing the type b flagellum and all of the type a subtypes.

In some embodiments, the immunogenic complex described herein comprises Pseudomonas flagellin, or an antigenic fragment thereof. Research on the flagellar subunit, flagellin, has not advanced beyond the exploratory phase. The studies included purified flagellin (Campodonico, et al., 2010; Faezi, et al., 2013), flagellin DNA vaccine (Saha, et al., 2007), and a flagellin/polymannuronic acid conjugate (Campodonico , et al. ., 2011). While all of these are immunogenic and protective, they do not appear to reach the levels that flagellar drugs achieve. Passive immunization using anti-type b sera (Barnea, et al., 2006), anti-type a mAb (Barnea, et al., 2009), and anti-type a sera (Faezi, et al., 2011) has also been studied. All drugs resulted in reduced mortality in a mouse burn model.

In some embodiments, the implementation of the immunogenic complex described in the present invention includes PcrV Pseudomonas (type III secretory system), or antigenic fragment. A key virulence factor associated with disease severity is the type III secretory system (T3SS) of P. aeruginosa , which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the end of the T3SS injectisomal complex, is essential for T3SS function and is a well-established target in animal models for the development of immunological prevention strategies targeting P. aeruginosa . Based on previous results from PcrV studies in which active and passive immunization (Sawa, et al., 1999; Holder, et al., 2001; Shime, et al., 2001; Frank, et al., 2002) demonstrated protection in various animal models, "Humaneered™, high affinity pegylated Fab'" (KB-001) has been developed in KaloBios and has been studied as a passive therapy in preclinical (Baer, et al., 2009) and clinical trials (Francois et al., 2012 ; Milla et al., 2014). Although both murine and humaneered mAbs and their Fab' fragments have been shown to be effective in animal studies, no significant differences in P. aeruginosa colonization scores, symptoms, or spirometry were found in the KB-001 clinical trial, and a new improved version of KB-001A for the treatment of P. aeruginosa infection in patients with cystic fibrosis did not meet key efficacy outcomes in a phase 2 human clinical trial (ClinTrials.gov, 2014). With regard to use as an active vaccine, little research has been done with the PcrV protein antigen prior to the present invention.

In some embodiments, the immunogenic complex described herein comprises Pseudomonas exopolysaccharide. In some embodiments, the implementation of the immune complex described in the present invention includes PsL Pseudomonas . This exopolysaccharide is important for the adhesion of P. aeruginosa to mammalian cells and for the formation and maintenance of biofilms produced by nonmucoid and mucoid isolates of P. aeruginosa . The PsL polysaccharide holds biofilm cells together, thus, this is the important role of PsL in adhesion (Ma et al., 2006). Functional screening revealed that mAbs against a single PsL epitope exhibit excellent activity in RFU and cell adhesion assays, and provide significant protection in many animal models.

The polysaccharide PsL is an antibody-accessible, serotype-independent surface element that is common in both nonmucoid and mucoid clinical isolates (DiGiandomenico et al., 2012). In animals, anti-PsL antibodies mediate OFU in the presence of effector cells and inhibit the binding of P. aeruginosa to epithelial cells. Presumably, the anti-PsL specific mAb Cam-003 passively protected and reduced the bacterial load in mouse organs in a dose-dependent manner (DiGiandomenico et al., 2012). The mAb Cam-003 was also protective against multiple P. aeruginosa serotypes in a murine pneumonia model. Similar results were obtained in mouse models of corneal scarification or burn, in both cases Cam-003 was effective in reducing infection. MEDI 3902 is a bispecific antibody targeting the serotype-independent virulence factor TTSS PcrV and the exopolysaccharide persistence factor PsL (DiGiandomenico et al., 2014). The Phase 1 safety study of MEDI 3902 for the prevention of Pseudomonas pneumonia (ventilator-associated pneumonia) has been completed (https://clinicaltrials.gov/NCT02255760). With regard to use as an active vaccine, little research has been done with the antigenic PsL exopolysaccharide prior to the present invention.

Pseudomonas aeruginosa polysaccharides

In some embodiments, the implementation of the immunogenic complex described in the present invention includes one or more polysaccharidesP. aeruginosa. Approximately 20 O antigen serotypes are known as determined by IATS. The O-antigenic polysaccharide molecule is a non-toxic potential target for vaccine development. In some embodiments, the immunogenic complex described herein comprises one or more OPS, LPS, and/or exopolysaccharides.Pseudomonas. In some embodiments, the polysaccharides from the LPS are OPS. In some embodiments, the polysaccharides from the LPS are COPS.

In some embodiments, the immunogenic complex comprises one or more polysaccharidesPseudomonas, which are polysaccharides, or derivatives thereof, from one or more serotypesP. aeruginosaselected from O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19 and O20 (IATS). In some embodiments, the immune complex comprises one or more polysaccharidesPseudomonas, which are polysaccharides, or derivatives thereof, from one or more serotypesP. aeruginosaselected from O1, O2, O3, O4, O5, O6, O10 and O11, or a combination thereof. In some embodiments, the immune complex comprises polysaccharidesPseudomonas, which are polysaccharides, or their derivatives, from each of the serotypesP. aeruginosaselected from O1, O2, O3, O4, O5, O6, O10 and O11.

In some embodiments, the immunogenic complex comprises one or more Pseudomonas polysaccharides that are, or are derived from, OPS. In some embodiments, OPS is OPS, or a derivative thereof, from Pseudomonas type O1, O2, O3, O4, O5, O6, O7, O8, O9, O1O, O11, O12, O13, O14, O15, O16, O17, O18, O19 and O2O, or combinations thereof. In some embodiments, OPS is OPS, or a derivative thereof, from Pseudomonas type O1, O2, O3, O4, O5, O6, O1O, or O11, or combinations thereof. In some embodiments, OPS is OPS, or a derivative thereof, from each of Pseudomonas type O1, O2, O3, O4, O5, O6, O1O, or O11.

In some embodiments, the immunogenic complex comprises one or more Pseudomonas polysaccharides that are, or are derived from, a capsular or capsular-like extracellular polysaccharide. In some embodiments, the capsular or capsular-like polysaccharide is a polysaccharide, or a derivative thereof, from Pseudomonas alginate, Psl or Pel.

In some embodiments, the immunogenic complex comprises one or more Pseudomonas polysaccharides that are, or are derived from, an exopolysaccharide. In some embodiments, the exopolysaccharide is, or is derived from, PsL. In some embodiments, PsL contains at least one epitope that binds a monoclonal antibody comprising the sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5. In some embodiments, a monoclonal antibody that binds at least one PsL epitope is is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the sequence of SEQ ID NO: 4, or its PsL-binding fragment. In some embodiments, a monoclonal antibody that binds at least one PsL epitope is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identity with the sequence of SEQ ID NO: 5, or its PsL-binding fragment. In some embodiments, a monoclonal antibody that binds at least one PsL epitope is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identity with the sequence of SEQ ID NO: 4, or a PsL-binding fragment thereof, and a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence SEQ ID NO: 5, or a PsL-binding fragment thereof.

Sequence of SEQ ID NO: 4 in PsL-binding mAb Cam-003

QVRLQQSGPGLVKPSETLSLTCTVSGGSTSPYFWSWLRQPPGKGLEWIGYIHSNGGTNYNPSLKSRLTISGDTSKNQFSLNLSFVTAADTALYYCARTDYDVYGPAFDIWGQGTMVTV

Sequence of SEQ ID NO: 5 in PsL-binding mAb Cam-003

SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL

Pseudomonas aeruginosa polypeptides

In some embodiments, the implementation of the immunogenic complex described in the present invention includes one or more polypeptidesP. aeruginosa. In some embodiments, the polypeptideP. aeruginosa is a polypeptide, or derivative thereof, of flagellin subtype A1 FliCP. aeruginosa, or its immunogenic fragment. In some embodiments, the polypeptideP. aeruginosa is, or contains, a polypeptide of SEQ ID NO: 6, or an immunogenic fragment thereof. In some embodiments, the polypeptideP. aeruginosa is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the sequence of SEQ ID NO: 6, or an immunogenic fragment thereof.

SEQ ID NO: 6 P. aeruginosa subtype A1 flagellins

MALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFKADGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFSDGDTISYVSKAGKDGSGAITSAVSGVVIADTGSTGVGTAAGVTPSATAFAKTNDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLR

In some embodiments, the P. aeruginosa polypeptide is a P. aeruginosa FliC subtype B flagellin polypeptide, or derivative thereof, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, a polypeptide of SEQ ID NO: 7, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 7, or an immunogenic fragment thereof.

SEQ ID NO: 7 Flagellin subtype B FliC P. aeruginosa

MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLR

In some embodiments, the P. aeruginosa polypeptide is a P. aeruginosa FliC subtype B flagellin D2 domain polypeptide, or a derivative thereof, lacking a TLR5 binding motif, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, the polypeptide of SEQ ID NO: 10, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 10, or an immunogenic fragment thereof.

SEQ ID NO: 10 P. aeruginosa FliC subtype B flagellin D2 domain lacking TLR5 binding motif

GSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSTGTQAAK

In some embodiments , the P. aeruginosa polypeptide is a P. aeruginosa secretory system type III PcrV virulence factor (TTSS) virulence factor polypeptide, or derivative thereof, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, a polypeptide of SEQ ID NO: 8, or an immunogenic fragment thereof. In some embodiments, the P. aeruginosa polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 8, or an immunogenic fragment thereof.

SEQ ID NO: 8 PcrV Type III Secretory System (TTSS) Virulence Factor P. aeruginosa

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI

Escherichia coli

Mortality due to E. coli sepsis is 20%-40% even with optimal antimicrobial therapy and supportive care (Grandsen et al., 1990; Kreger et al., 1980; Rayner and Willcox, 1988; Bryan et al., 1983; Cross et al., 1983). Clearly, there is a need for new preventive and therapeutic approaches, including adjunctive treatment with antibodies to these and other Gram-negative bacteria. Although O-antigen-specific antibodies protect against infection by homologous bacterial strains, researchers believe that immunotherapy with such antibodies is of limited value due to the diversity of Gram-negative bacteria serotypes (Baumgartner, 1990). However, epidemiological studies of bacterial serotypes associated with extraintestinal invasive E. coli infection have found that a limited number of O-serogroups are responsible for approximately 60% of bacteremia cases (Grandsen et al., 1990; Cross et al., 1984; McCabe et al., 1978). Accordingly, there remains a need for effective technologies for the prevention and/or treatment of such infections.

Methods for purification of polysaccharides

In some embodiments, the invention relates to methods for purifying one or more of the polysaccharides described herein from one or more bacterial cellular components. In some embodiments, the methods include purifying the O antigenic polysaccharide from one or more bacterial cellular components. In some embodiments, the methods include purifying the CPS from one or more bacterial cellular components.

In some embodiments, the bacteria are gram negative. In some embodiments, the bacteria representK. pneumoniae,P. aeruginosa AndE. coli.

In some embodiments, the cellular components include a protein. In some embodiments, the cellular components comprise a nucleic acid. In some embodiments, the cellular components include lipids. In some embodiments, the cellular components include polysaccharides. In some embodiments, the cellular components are part of the lysate.

In some embodiments, a method for purifying a polysaccharide from one or more bacterial cellular components includes contacting the cellular components with an oxidizing agent and an acid to form a mixture. In some embodiments, the oxidizing agent is sodium nitrite. In some embodiments, the acid is acetic acid. In some embodiments, the pH of the mixture is from about 3 to about 5. In some embodiments, the method includes separating the O-antigenic polysaccharide from one or more cellular components in the mixture. In some embodiments, the separated OPS contains a free aldehyde at its reducing end. In some embodiments, the separated OPS contains a free aldehyde containing 2,5-anhydromannose moiety at its reducing end. In some embodiments, the method includes recovering OPS, wherein the recovered OPS is substantially free of one or more other cellular components. In some embodiments, the recovered OPS contains a free aldehyde at its reducing end. In some embodiments, the recovered OPS contains a free aldehyde containing 2,5-anhydromannose residue at its reducing end.

In some embodiments, a method for purifying a polysaccharide from one or more Gram-negative bacterial cellular components comprises contacting the cellular components with an acidic reagent to form a mixture. In some embodiments, the pH of the mixture is from about 3 to about 5. In some embodiments, the method includes heating the mixture to a temperature of from about 80°C to about 135°C. In some embodiments, the method includes separating the core and O antigenic polysaccharide (COPS) from one or more cellular components in the mixture. In some embodiments, the implementation of the separated COPS contains KDO at its recovery end. In some embodiments, the separated COPS contains a ketone in the KDO at its reducing end. In some embodiments, the method includes recovering COPS, wherein the recovered COPS is substantially free of one or more other cellular components. In some embodiments, the recovered COPS contains a ketone at its reducing end.

Pairs of affine molecules

As described herein, in some embodiments, the immune complexes of the invention comprise one or more polypeptides in a non-covalent complex with one or more polymers and/or one or more antigenic polysaccharides. In some embodiments, one or more polypeptides form a complex through affinity interaction with one or more polymers and/or one or more antigenic polysaccharides. In some embodiments, the immune complexes of the invention comprise one or more polypeptides in a non-covalent complex with one or more polymers and/or one or more antigenic polysaccharides formed by one or more affinity molecule/complementary affinity molecule pairs. In some embodiments, the immune complex comprises (i) a first affinity molecule described herein conjugated to one or more polymers and/or conjugated to one or more antigenic polysaccharides, and (ii) a fusion protein that is, or contains, a complementary affinity molecule described in the present invention, and a polypeptide. Upon binding of the first affinity molecule and the complementary affinity molecule, one or more polypeptides form a non-covalent complex with one or more polymers and/or one or more antigenic polysaccharides.

In some embodiments, the affinity molecule/complementary affinity molecule pair is selected from one or more of: biotin/biotin binding protein, antibody/antigen, enzyme/substrate, receptor/ligand, metal/metal binding protein, carbohydrate/carbohydrate binding protein , a lipid/lipid-binding protein, and a His-tag/His-tag-binding molecule.

In some embodiments, the first affinity molecule is biotin (or a derivative or fragment thereof) and the complementary affinity molecule is a biotin-binding protein, or a biotin-binding domain thereof. In some embodiments, the biotin-binding protein is rizavidin, avidin, streptavidin, bradavidin, tamavidin, lentiavidin, zebavidin, neutravidin, captavidin™, or biotin-binding fragments thereof, or a combination thereof. In some embodiments, the biotin-binding protein is rizavidin, or a biotin-binding fragment thereof. In some embodiments, the biotin-binding protein comprises a polypeptide of SEQ ID NO: 13, or a biotin-binding fragment thereof. In some embodiments, the biotin-binding protein comprises a polypeptide of SEQ ID NO: 14, or a biotin-binding fragment thereof.

Fusion proteins

In some embodiments, the implementation of the immune complex described in the present invention includes a fusion protein that is, or contains, a complementary affinity molecule described in the present invention, and one or more polypeptides described in the present invention. In some embodiments, such fusion proteins have carrier properties and/or antigenic properties. In some embodiments, the fusion protein is, or contains, a biotin-binding protein, or a biotin-binding domain thereof, and a polypeptide.

In some embodiments, the biotin-binding protein comprises a polypeptide of SEQ ID NO: 13, or a biotin-binding fragment thereof. In some embodiments, the polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 13, or its biotin-binding fragment. In some embodiments, the biotin-binding protein comprises a polypeptide of SEQ ID NO: 14, or a biotin-binding fragment thereof. In some embodiments, the polypeptide is, or contains, a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 14, or its biotin-binding fragment.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and one or more polypeptides derived from one or more of K. pneumoniae, P. aeruginosa, and E coli.

In some embodiments, the fusion protein contains one or more linkers and/or a histidine tag. In some embodiments, the implementation of the linker contains a polypeptide containing the amino acid sequence of SEQ ID NO: 15 (GGGGSSS). In some embodiments, the linker contains a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 15.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 7 ( P. aeruginosa flagellin FlaB), or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 6 ( P. aeruginosa flagellin FlaA1), or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9 ( P. aeruginosa flagellin FlaA2), or an immunogenic fragment thereof.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 ( P. aeruginosa FlaB flagellin D2 domain lacking a TLR5-binding motif), or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 ( P. aeruginosa FlaA1 flagellin D2 domain lacking the TLR5-binding motif), or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12 ( P. aeruginosa FlaA2 flagellin D2 domain lacking a TLR5-binding motif), or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV secretory system type III (TTSS) P. aeruginosa ), or its immunogenic fragment.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin), or an immunogenic fragment thereof, and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 23. In some embodiments, the fusion protein contains a complementary affinity molecule described in the present invention (for example, a biotin-binding protein described in the present invention), and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10 ( P. aeruginosa FliC subtype B flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof, and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ) , or its immunogenic fragment. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 26.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin), or an immunogenic fragment thereof, and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2 ( K. pneumoniae conserved MrkA), or an immunogenic fragment thereof.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10 ( P. aeruginosa FliC subtype B flagellin D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof, and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 3 (stabilized MrkA K. pneumoniae ), or its immunogenic fragment. In some embodiments, the fusion protein comprises the polypeptide of SEQ ID NO: 24.

In some embodiments, the fusion protein comprises a complementary affinity molecule as described herein (e.g., a biotin binding protein as described herein) and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof, and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3 ( K. pneumoniae stabilized MrkA), or an immunogenic fragment thereof.

Framework polymers

As described herein, in some embodiments, the immune complexes of the invention comprise a backbone polymer comprising a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the polymer is, or contains, a polysaccharide, a polypeptide, or a synthetic dendrimer. In some embodiments, the implementation of the polymer is, or contains, poly-L-lysine (PLL). In some embodiments, the implementation of the polymer is, or contains, linear poly-L-lysine or L-lysine dendrimer. In some embodiments, the polymer is a CPS derived from Gram-negative or Gram-positive bacteria. In some embodiments, the CPS is K. pneumoniae CPS, P. aeruginosa exopolysaccharide, and/or E. coli CPS. In some embodiments, the polymer is CPS KP K19. In some embodiments, the polymer is a synthetic monosaccharide or oligosaccharide dendrimer. In some embodiments, the implementation of the polymer has a size of from about 50 KD to about 2000 KD. In some embodiments, the implementation of the polymer has antigenic properties. In some embodiments, the implementation of the polymer is not antigenic. In some embodiments, the implementation of the polymer is, or contains, one or more antigenic polysaccharides. In some embodiments, the one or more antigenic polysaccharides may be K. pneumoniae OPS, P. aeruginosa OPS, or a combination thereof.

Framework polymer 1 (BP-1)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (eg, biotin) conjugated (eg, cross-linked or covalently linked) to the polymer. In some embodiments, the implementation of the polymer is a polysaccharide. In some embodiments, the antigenic polysaccharide is an O antigenic polysaccharide, a core O antigen, or a core polysaccharide. In some embodiments, the framework polymer includes at least one polymer (eg, a polysaccharide) that is biotinylated and at least one antigenic polysaccharide (eg, at least one O-antigenic polysaccharide) that is not biotinylated.

In some embodiments, a backbone polymer that includes at least one biotinylated polymer (e.g., a biotinylated polysaccharide) and at least one non-biotinylated O-antigenic polysaccharide is prepared by combining a non-biotinylated O-antigenic polysaccharide that contains a primary amino group (naturally or with a linker), with a partially oxidized polysaccharide containing an aldehyde group and biotin containing a primary amine, to obtain a mixture; and reductively aminating the mixture with a reducing agent to chemically bond the O-antigenic polysaccharide (i.e., antigenic polysaccharide) to the polysaccharide (i.e., polymer), thereby forming a backbone polymer that includes a biotinylated polysaccharide chemically bonded to a non-biotinylated antigenic polysaccharide ( i.e., non-biotinylated O-antigenic polysaccharide).

In some embodiments, the O-antigenic polysaccharide is chemically bonded to a partially oxidized polymer (eg, polysaccharide) using a spacer. In some embodiments, the spacer is a short spacer. In some embodiments, the short spacer contains a primary amine at each end of the spacer. In some embodiments, the spacer may be adipic acid dihydrazide. In some embodiments, the polymer (eg, polysaccharide) is biotinylated and the chemically linked O-antigenic polysaccharide is not biotinylated.

In some embodiments, the implementation of the O-antigenic polysaccharide contains a primary amino group. In some embodiments, the primary amino group is the L-alanine α-amino group of the outer core of the P. aeruginosa O-polysaccharide, or an amino group that has been introduced into the reducing end of the O-antigenic polysaccharide by chemically linking a spacer molecule containing a primary group at each end by reductive amination with a reducing reagent. In some embodiments, an O-antigenic polysaccharide containing a primary amino group is chemically bonded by mixing an O-antigenic polysaccharide containing a primary amino group with a partially oxidized polymer (e.g., a polysaccharide) containing an aldehyde group and biotin containing a primary amine to form a mixture; and reductively aminating the mixture with a reducing agent to form a backbone polymer, wherein the polymer (eg, polysaccharide) is biotinylated but the chemically bound O-antigenic polysaccharide is not biotinylated.

In some embodiments, the polymer (eg, polysaccharide) is a partially oxidized oxidizing agent. In some embodiments, the oxidizing agent is sodium periodate. In some embodiments, the reducing agent comprises sodium cyanoborohydride or sodium borohydride.

Framework polymer 1.2 (BP-1.2)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (eg, biotin) conjugated (eg, cross-linked or covalently linked) to the polymer. In some embodiments, the implementation of the polymer is a polysaccharide. In some embodiments, the antigenic polysaccharide is an O antigenic polysaccharide, a core O antigen, or a core polysaccharide. In some embodiments, the framework polymer includes at least one polymer (eg, a polysaccharide) that is biotinylated and at least one antigenic polysaccharide (eg, at least one O-antigenic polysaccharide) that is not biotinylated.

In some embodiments, a backbone polymer that includes at least one biotinylated polymer (e.g., a biotinylated polysaccharide) and at least one non-biotinylated O-antigenic polysaccharide is prepared by combining a non-biotinylated O-antigenic polysaccharide that contains a primary amino group (naturally or with a linker), with a partially oxidized polysaccharide containing one or more aldehyde groups and one or more biotin residues introduced into the orthogonal site by reaction with carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and using carboxylates of uronic acid residues polysaccharide, and reductively aminating the mixture with a reducing reagent to chemically link the O-antigenic polysaccharide (i.e., antigenic polysaccharide) with the polysaccharide (i.e., polymer), thereby forming a backbone polymer that includes a biotinylated polysaccharide chemically associated with non-bio tinylated antigenic polysaccharide (i.e., non-biotinylated O-antigenic polysaccharide). In some embodiments, the O-antigenic polysaccharide is chemically bonded to a partially oxidized polymer (eg, polysaccharide) using a spacer. In some embodiments, the spacer is a short spacer. In some embodiments, the short spacer contains a primary amine at each end of the spacer. In some embodiments, the spacer may be adipic acid dihydrazide. In some embodiments, the polymer (eg, polysaccharide) is biotinylated and the chemically linked O-antigenic polysaccharide is not biotinylated.

In some embodiments, the implementation of the O-antigenic polysaccharide contains a primary amino group. In some embodiments, the primary amino group is the L-alanine α-amino group of the outer core of the P. aeruginosa O-polysaccharide, or an amino group that has been introduced into the reducing end of the O-antigenic polysaccharide by chemically linking a spacer molecule containing a primary group at each end by reductive amination with a reducing reagent. In some embodiments, an O-antigenic polysaccharide containing a primary amino group is chemically bonded by mixing an O-antigenic polysaccharide containing a primary amino group with a partially oxidized polymer (e.g., a polysaccharide) containing an aldehyde group and biotin containing a primary amine to form a mixture; and reductively aminating the mixture with a reducing agent to form a backbone polymer, wherein the polymer (eg, polysaccharide) is biotinylated but the chemically bound O-antigenic polysaccharide is not biotinylated.

In some embodiments, the polymer (eg, polysaccharide) is a partially oxidized oxidizing agent. In some embodiments, the oxidizing agent comprises sodium periodate. In some embodiments, the reducing agent comprises sodium cyanoborohydride or sodium borohydride.

In some embodiments, an OPS containing an aldehyde or ketone is aminated at its reducing end, using a short spacer containing a primary amine at each end (e.g., adipic acid dihydrazide ADH), by reductive amination with sodium cyanoborohydride (NaBH3CN), to produce a hydrazide-derivatized OPS. In some embodiments, the KP19 CPS backbone is biotinylated with one or more biotin residues introduced into the orthogonal site by reacting carboxylates of polysaccharide uronic acid residues with carbodiimide (EDC), N-hydroxysuccinimide (NHS), and a biotin derivative containing a primary amine (such as, for example, , amine-PEG3-biotin). In some embodiments, the biotinylated CPS KP 19 polymer is then partially oxidized with periodate to give one or more aldehyde groups, mixed with derivatized OPS hydrazide, and reductively aminated with a reducing agent such as sodium cyanoborohydride (NaBH3CN) to form a BP-1.2 backbone polymer, containing one or more biotin fragments on the uronic acid residues of the polymer backbone and without biotin on the O-antigenic polysaccharide.

Framework polymer 1.3 (BP-1.3)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (eg, biotin) conjugated (eg, cross-linked or covalently linked) to the polymer. In some embodiments, the implementation of the polymer is a polysaccharide. In some embodiments, the antigenic polysaccharide is an O antigenic polysaccharide, a core O antigen, or a core polysaccharide. In some embodiments, the framework polymer includes at least one polymer (eg, a polysaccharide) that is biotinylated and at least one antigenic polysaccharide (eg, at least one O-antigenic polysaccharide) that is not biotinylated.

In some embodiments, a backbone polymer that includes at least one biotinylated polymer (e.g., a biotinylated polysaccharide) and at least one non-biotinylated O-antigenic polysaccharide is prepared by combining a non-biotinylated O-antigenic polysaccharide that contains a primary amino group (naturally or with a linker), with a CDAP-activated polysaccharide with one or more biotin residues introduced into the orthogonal site by reaction with carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and using carboxylates of polysaccharide uronic acid residues, to form a backbone polymer , which includes a biotinylated polysaccharide chemically linked to a non-biotinylated antigenic polysaccharide (i.e., a non-biotinylated O-antigenic polysaccharide). In some embodiments, the O-antigenic polysaccharide is chemically bonded to a CDAP-activated polymer (eg, polysaccharide) using a spacer. In some embodiments, the spacer is a short spacer. In some embodiments, the short spacer contains a primary amine at each end of the spacer. In some embodiments, the spacer may be adipic acid dihydrazide. In some embodiments, the polymer (eg, polysaccharide) is biotinylated and the chemically linked O-antigenic polysaccharide is not biotinylated.

In some embodiments, an OPS containing an aldehyde or ketone is aminated at its reducing end, using a short spacer containing a primary amine at each end (e.g., adipic acid dihydrazide ADH), by reductive amination with sodium cyanoborohydride (NaBH3CN), to produce a hydrazide-derivatized OPS. In some embodiments, the KP19 CPS backbone is biotinylated with one or more biotin residues introduced into the orthogonal site by reacting carboxylates of polysaccharide uronic acid residues with carbodiimide (EDC), N-hydroxysuccinimide (NHS), and a biotin derivative containing a primary amine (such as, for example, , amine-PEG3-biotin). In some embodiments, the biotinylated CPS KP 19 polymer is then activated with CDAP, mixed with derivatized OPS hydrazide, to form a BP-1.3 backbone polymer containing one or more biotin moieties on the backbone uronic acid residues, and no biotin on the O-antigenic polysaccharide.

Framework polymer 2a (BP-2a)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (e.g., biotin) conjugated (e.g., crosslinked or covalently linked) to a polymer, and includes a first affinity molecule described herein (e.g., biotin) conjugated (eg, cross-linked or covalently linked) to one or more antigenic polysaccharides. In some embodiments, the implementation of the polymer is a polysaccharide. In some embodiments, the antigenic polysaccharide is an O-antigenic polysaccharide. In some embodiments, the backbone polymer includes at least one polymer (eg, polysaccharide) that is biotinylated and at least one O-antigenic polysaccharide that is biotinylated.

In some embodiments, a backbone polymer that includes at least one biotinylated polymer (e.g., biotinylated polysaccharide) and at least one biotinylated O-antigenic polysaccharide is prepared by combining an O-antigenic polysaccharide with a partially oxidized polymer (e.g., polysaccharide) containing an aldehyde group to give a mixture; and reductively aminating the mixture with a reducing reagent for chemically bonding the O-antigenic polysaccharide to a polymer (eg, polysaccharide) to form a backbone polymer that includes the O-antigenic polysaccharide and the polymer (eg, polysaccharide); as well as derivatizing the backbone polymer with CDAP or another cyanylating reagent (e.g., CNBr) and biotin containing a primary amine to form a backbone polymer that includes a biotinylated polymer (e.g., a biotinylated polysaccharide) chemically bonded to a biotinylated O-antigen and a polymeric polysaccharide. .

In some embodiments, the O-antigenic polysaccharide is chemically bonded to the partially oxidized polysaccharide backbone using a spacer. In some embodiments, the spacer is a short spacer. In some embodiments, the short spacer contains a primary amine at each end of the spacer. In some embodiments, the spacer may be adipic acid dihydrazide.

In some embodiments, the implementation of the O-antigenic polysaccharide contains a primary amino group. In some embodiments, the primary amino group is the L-alanine α-amino group of the outer core of the P. aeruginosa O-polysaccharide, or an amino group that has been introduced into the reducing end of the O-antigenic polysaccharide by chemical bonding of a short spacer molecule containing a primary group at each end, by reductive amination with a reducing reagent.

In some embodiments, the polymer (eg, polysaccharide) is a partially oxidized oxidizing agent. In some embodiments, the oxidizing agent comprises sodium periodate. In some embodiments, the reducing agent comprises sodium cyanoborohydride or sodium borohydride.

Framework polymer 2b (BP-2b)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (eg, biotin) conjugated (eg, cross-linked or covalently linked) to an antigenic polysaccharide. In some embodiments, the implementation of the polymer is a polysaccharide. In some embodiments, the antigenic polysaccharide is an O-antigenic polysaccharide. In some embodiments, the backbone polymer includes at least one polymer (eg, polysaccharide) that is not biotinylated and at least one biotinylated O-antigenic polysaccharide.

In some embodiments, a backbone polymer that includes at least one non-biotinylated polymer (e.g., a non-biotinylated polysaccharide) and at least one biotinylated O-antigenic polysaccharide is produced by biotinylation of an O-antigenic polysaccharide containing O-polysaccharide repeats and one or more core alpha-KDO and/or heptose fragments, using CDAP and biotin containing a primary amine, to obtain a mixture of biotinylated O-antigenic polysaccharide; partially oxidizing the biotinylated OPS mixture with sodium periodate to introduce aldehydes into core KDO and/or heptose fragments and/or OPS residues; reductive amination of the biotinylated polysaccharide with adipic acid dihydrazide; mixing biotinylated and aminated OPS with a partially oxidized polymer (eg, polysaccharide) to form a mixture; and reductive amination of the mixture; to provide a backbone polymer that includes a polymer (eg, polysaccharide) that is not biotinylated and a chemically linked O-antigenic polysaccharide that is biotinylated.

In some embodiments, the O-antigenic polysaccharide is a partially oxidized oxidizing agent. In some embodiments, the oxidizing agent comprises sodium periodate. In some embodiments, the reducing agent comprises sodium cyanoborohydride or sodium borohydride.

Framework Polymer 3 (BP-3)

In some embodiments, the implementation of the immunogenic complex described in the present invention includes a backbone polymer, including a polymer conjugated to one or more antigenic polysaccharides. In some embodiments, the backbone polymer comprises a first affinity molecule described herein (eg, biotin) conjugated (eg, cross-linked or covalently linked) to an antigenic polysaccharide. In some embodiments, the polymer is a poly-L-lysine (PLL) polymer. In some embodiments, the antigenic polysaccharide is an O-antigenic polysaccharide. In some embodiments, the backbone polymer comprises at least one biotinylated O-antigenic polysaccharide and a PLL polymer.

In some embodiments, a backbone polymer that includes at least one non-biotinylated polymer (e.g., a non-biotinylated PLL polymer) and at least one biotinylated O-antigenic polysaccharide is obtained by reductive amination of an O-antigenic polysaccharide containing terminal aldehydes or KDOs, with ε-NH ₂ groups of the PLL polymer to form an OPS-PLL polymer; treating, or not treating, the mixture with an acylating agent to block unreacted ε-NH ₂ groups of the PLL polymer; derivatizing OPS with CDAP to give a mixture of derivatives; and carrying out the reaction of a mixture of derivatives with biotin containing a primary amine; to form a backbone polymer, wherein the blocked N-acetylated-PLL or non-blocked PLL polymer is not biotinylated and the chemically bound O-antigenic polysaccharide is biotinylated.

In some embodiments, the acylating agent is acetic anhydride or acetyl chloride.

In some embodiments, a click chemistry reaction is used to couple a polymer (e.g., a polysaccharide) having an alkyne functional group on its carboxylate groups to oxidized polysaccharide aldehydes on an O-antigenic polysaccharide derivatized with an azido group at its reducing end. The click-chemistry coupling of the 2 polysaccharides is carried out with a catalyst in an aqueous medium. Click chemistry coupling of the O-antigenic polysaccharide to the polysaccharide can be used in reversible ways, either with an azido group attached to the OPS and an alkyne group attached to the polysaccharide, or vice versa. Biotination can be carried out using such chemistry selectively either on the polysaccharide to form backbone polymer 1 or to the O-antigenic polysaccharide to form backbone polymer 2b.

In some embodiments, the catalyst for the alkyne azide cycloaddition is copper sulfate. In some embodiments, the alkyne group for derivatization of the polysaccharide is selected from the group consisting of 1-amino-3-butyne, 1-amino-4-pentine, DBCO-amine, and alkyne-PEG4-amine.

In some embodiments, the azide group for derivatization of the O-antigenic polysaccharide at its reducing end is selected from the group consisting of amino-PEG-azide, azido-PEG3-amine, azidopropylamine, and azidobutylamine.

The use of immunogenic complexes

In some embodiments, the immunogenic complex described herein, which includes one or more antigenic polysaccharides, is characterized in that one or more of the potential for opsonization or immunogenicity of one or more antigenic polysaccharides is increased from a previously determined level, as measured by ELISA and/ or in a functional antibody assay. In some embodiments, one or more of the potential for opsonization or immunogenicity of one or more antigenic polysaccharides is increased by at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a previously determined level, as measured by ELISA and/or functional analysis of antibodies. In some embodiments, the previously determined level is the pre-immune level. In some embodiments, the epitope valency of one or more antigenic polysaccharides is at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold that of the native polysaccharide. In some embodiments, one or more polypeptide antigens are carrier proteins for one or more antigenic polysaccharides.

In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, induces the production of antibodies against one or more pathogens in the patient at a level higher than that of a composition containing only the antigenic polysaccharide and no backbone polymer, as measured by ELISA. In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, induces the production of antibodies against one or more pathogens in the patient at a level greater than that of a composition containing only the backbone polymer and no antigenic polysaccharide, as measured by ELISA.

In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, induces an immune response against one or more pathogens in the patient at a level greater than that of a composition containing only the antigenic polysaccharide and no backbone polymer. In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, induces an immune response against one or more pathogens in the patient at a level greater than that of a composition containing a backbone polymer and no antigenic polysaccharide. In some embodiments, the implementation of the immune response is the production of antibodies or B-cell response. In some embodiments, the immune response is a CD4+ T cell response, including a Th1, Th2, or Th17 response, or a CD8+ T cell response, or a CD4+/CD8+ T cell response. In some embodiments, the implementation of the immune response is the production of antibodies or B-cell response; and T cell response.

In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, causes the production of antibodies against one or more pathogens in the patient at a higher level than a composition containing only a polypeptide antigen and not containing a backbone polymer, as measured by ELISA. . In some embodiments, one or more pathogens are selected from K. pneumoniae, P. aeruginosa, and E. coli . In some embodiments, the immunogenic complex described herein, upon administration to a patient, elicits an immune response against one or more of K. pneumoniae, P. aeruginosa, and E. coli .

In some embodiments, the implementation of the immunogenic complex described in the present invention, after administration to the patient, causes an immune response against gram-negative and/or gram-positive bacteria. In some embodiments, the Gram-negative bacteria are selected fromK. pneumoniae, P. aeruginosa, E. coli., as well as their combinations. In some embodiments, the immunogenic complex of the present invention, upon administration to a patient, causes the production of antibodies that recognize native MrkA on expressing MrkA.K. pneumoniae.

In some embodiments, the implementation of the immunogenic complex described in the present invention contains one or more polypeptide antigens. In some embodiments, one or more of the polypeptide antigens is a bacterial polypeptide, a fungal polypeptide, and/or a viral polypeptide. In some embodiments, the one or more polypeptide antigens is a polypeptide from Klebsiella, Pseudomonas and/or E. coli .

In some embodiments, at least one of the antigenic polysaccharides is a P. aeruginosa PsL exopolysaccharide. In some embodiments, PsL contains at least one epitope that binds a monoclonal antibody containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 4, and/or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO : 5 (Cam-003). In some embodiments, at least one polypeptide antigen contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO : 1 ( K. pneumoniae type I fimbrial protein). In some embodiments, at least one polypeptide antigen contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO : 2 ( K. pneumoniae type III conserved fimbrial protein MrkA), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 3 monomeric stabilized MrkA, or immunogenic fragment. In some embodiments, the at least one polypeptide antigen is P. aeruginosa FliC subtype A flagellin, P. aeruginosa FliC subtype B flagellin, or an immunogenic fragment thereof. In some embodiments, at least one polypeptide antigen contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO : 6 (subtype A1), an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 9 (subtype A2), or an immunogenic fragment thereof.

Preparation of immunogenic complexes

The present invention relates to methods for preparing the immunogenic complexes described in the present invention. In some embodiments, a method for making immunogenic complexes comprises complexing at least one biotinylated backbone polymer with at least one biotin-binding fusion protein. In some embodiments, the fusion protein contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NOs: 16- 26.

Diseases, disorders and conditions

In some embodiments, upon administration to a patient, an immunogenic complex of the present invention (e.g., a composition comprising an immunogenic complex of the present invention) causes the patient to produce antibodies against one or more pathogens at a higher level than a composition containing antigenic polysaccharide, and does not contain a backbone polymer, when measured by ELISA. In some embodiments, one or more pathogens are selected from Klebsiella, Pseudomonas , and E. coli .

In some embodiments, upon administration to a patient, an immunogenic complex of the present invention (eg, a composition comprising an immunogenic complex of the present invention) elicits an immune response against one or more pathogens in the patient. In some embodiments, upon administration to a patient, the immunogenic complex of the present invention elicits an immune response against one or more pathogens in the patient at a higher level than a composition containing an antigenic polysaccharide and no backbone polymer. In some embodiments, one or more pathogens are selected from Klebsiella, Pseudomonas , and E. coli .

In some embodiments, after administration to a patient, an immunogenic complex of the present invention (eg, a composition comprising an immunogenic complex of the present invention) is used to treat or prevent an infection. In some embodiments, the infection is a burn wound infection, GI infection, operative field infection, urinary tract infection, corneal infection, skin and/or soft tissue infection, diabetic ulcer infection, medical device (e.g., catheter, surgical implant) infection. , prosthesis). In some embodiments, after administration to a patient, the immunogenic composition described herein is used to treat or prevent pneumonia, sepsis, and/or colonization.

Immunogenic Compositions and Formulations

The invention also relates to compositions containing one or more of the immunogenic complexes described in the present invention. For example, an immunogenic composition, such as a vaccine composition, may contain one or more of the immunogenic complexes described in the present invention. In some embodiments, such compositions may contain multiple immunogenic complexes of the same type described in the present invention. Additionally or alternatively, such compositions may contain a plurality of immunogenic complexes of more than one type described in the present invention. In some embodiments, the immunogenic complexes described herein are formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition may be a vaccine. In some embodiments, the implementation of the pharmaceutical composition contains a pharmaceutically acceptable carrier.

In some embodiments, the implementation of the vaccine composition is a polyvalent or multivalent vaccine. In some embodiments, the implementation of the valency of the vaccine composition means the number of types of immunogenic complexes present in the vaccine composition. The valence of the vaccine described in the present invention is not limited in relation to the total number of antigens present in the specified pharmaceutical composition, immunogenic complex or vaccine, or in relation to the number of strains of pathogens against which the introduction of the specified pharmaceutical composition, immunogenic complex, immunogenic composition or vaccine composition, can induce a protective immune response. As a non-limiting example, a 12-valent vaccine composition may contain more than 12 antigenic components (eg, peptide and/or polysaccharide components), and can induce a protective immune response against more than 12 pathogens or pathogenic strains.

In some embodiments, the implementation of the vaccine composition contains 1-50 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 1-25 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 1-15 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 1-10 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 1-5 types of immunogenic complexes. In some embodiments, the implementation of the vaccine is a polyvalent vaccine.

In some embodiments, the implementation of the vaccine composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49 or 50 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 1 type of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 2 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 4 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 6 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 8 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 10 types of immunogenic complexes. In some embodiments, the implementation of the vaccine composition contains 12 types of immunogenic complexes. In some embodiments, the vaccine composition contains two or more types of immunogenic complexes in such amounts that the mass of OPS in the vaccine composition from each immunogenic composition is different, for example, they are present in a weight ratio other than about 1:1. In some embodiments, the vaccine composition contains two or more types of immunogenic complexes in such amounts that the weight of OPS in the vaccine composition from each immunogenic composition is approximately the same, for example, they are present in a weight ratio of approximately 1:1. In some embodiments, the mass of OPS in the vaccine from each immunogenic composition is approximately 1 μg. In some embodiments, the mass of OPS in the vaccine from each immunogenic composition is approximately 2 μg. In some embodiments, the mass of OPS in the vaccine from each immunogenic composition is approximately 3 μg. In some embodiments, the mass of OPS in the vaccine from each immunogenic composition is approximately 4 μg. In some embodiments, the mass of OPS in the vaccine from each immunogenic composition is approximately 5 μg.

In some embodiments, the vaccine composition contains two or more types of immunogenic complexes in such amounts that the total weight of OPS and CPS in the vaccine composition from each immunogenic composition is different, e.g., they are present in a weight ratio other than about 1:1. In some embodiments, the vaccine composition contains two or more types of immunogenic complexes in such amounts that the total weight of OPS and CPS in the vaccine composition from each immunogenic composition is different, e.g., they are present in a weight ratio other than about 1:1. In some embodiments, the vaccine composition contains two or more types of immunogenic complexes in such amounts that the total mass of OPS and CPS in the vaccine composition from each immunogenic composition is approximately the same, for example, they are present in a weight ratio of approximately 1:1. In some embodiments, the total mass of OPS and CPS in the vaccine from each immunogenic composition is approximately 1 μg. In some embodiments, the total mass of OPS and CPS in the vaccine from each immunogenic composition is approximately 2 μg. In some embodiments, the total mass of OPS and CPS in the vaccine from each immunogenic composition is approximately 3 μg. In some embodiments, the total mass of OPS and CPS in the vaccine from each immunogenic composition is approximately 4 μg. In some embodiments, the total mass of OPS and CPS in the vaccine from each immunogenic composition is approximately 5 μg.

In some embodiments, the implementation of the vaccine composition contains two or more types of immunogenic compositions selected from

(a) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(c) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g') an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h') an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(j) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And

(l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition contains two or more types of immunogenic compositions selected from

(a) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(a') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(c') an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e) an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g') an immunogenic composition containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(i') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(j) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(j') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated Klebsiella spp. capsular polysaccharide. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(k') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(l') an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition contains

(a) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(j) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And

(l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition contains

(a) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(b) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(c) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(d) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(e) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(f) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(g) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(h) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(i) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(j) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer;

(k) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And

(l) an immunogenic composition containing a backbone polymer comprising a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and a Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer.

In some embodiments, the implementation of the vaccine composition contains two or more types of immunogenic compositions selected from

(a) an immunogenic composition comprising a KP O1 OPS and a FlaBD2-MrkA fusion protein;

(a') an immunogenic composition comprising KP O1 OPS and a FlaBD2-PcrV fusion protein;

(b) an immunogenic composition comprising KP O2 OPS and a FlaBD2-MrkA fusion protein;

(b') an immunogenic composition comprising KP O2 OPS and a FlaBD2-PcrV fusion protein;

(c) an immunogenic composition comprising a KP O3 OPS and a FlaBD2-MrkA fusion protein;

(c') an immunogenic composition comprising KP O3 OPS and a FlaBD2-PcrV fusion protein;

(d) an immunogenic composition comprising a KP O5 OPS and a FlaBD2-MrkA fusion protein;

(d') an immunogenic composition comprising KP O5 OPS and a FlaBD2-PcrV fusion protein;

(e) an immunogenic composition comprising PA O1 OPS and a FlaBD2-MrkA fusion protein;

(e') an immunogenic composition comprising PA O1 OPS and a FlaBD2-PcrV fusion protein;

(f) an immunogenic composition comprising a PA O2 OPS and a FlaBD2-MrkA fusion protein;

(f') an immunogenic composition comprising PA O2 OPS and a FlaBD2-PcrV fusion protein;

(g) an immunogenic composition comprising PA O3 OPS and a FlaBD2-MrkA fusion protein;

(g') an immunogenic composition comprising PA O3 OPS and a FlaBD2-PcrV fusion protein;

(h) an immunogenic composition comprising a PA O4 OPS and a FlaBD2-MrkA fusion protein;

(h') an immunogenic composition comprising PA O4 OPS and a FlaBD2-PcrV fusion protein;

(i) an immunogenic composition comprising a PA O5 OPS and a FlaBD2-MrkA fusion protein;

(i') an immunogenic composition comprising PA O5 OPS and a FlaBD2-PcrV fusion protein;

(j) an immunogenic composition comprising a PA O6 OPS and a FlaBD2-MrkA fusion protein;

(j') an immunogenic composition containing PA O6 OPS and a FlaBD2-PcrV fusion protein;

(k) an immunogenic composition comprising PA O10 OPS and a FlaBD2-MrkA fusion protein;

(k') an immunogenic composition comprising PA O10 OPS and a FlaBD2-PcrV fusion protein;

(l) an immunogenic composition comprising PA O11 OPS and a FlaBD2-MrkA fusion protein; And

(l') immunogenic composition containing PA O11 OPS and FlaBD2-PcrV fusion protein.

In some embodiments, one or more types of immunogenic compositions further comprise one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the vaccine composition comprises an immunogenic composition selected from

(a) an immunogenic composition comprising a combination of K. pneumoniae type O1, O2, O3, O5 OPS and a FlaBD2-MrkA fusion protein; And

(b) an immunogenic composition containing a combination of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS and a FlaBD2-MrkA fusion protein.

In some embodiments, one or more types of immunogenic compositions further comprise one or more polymers. In some embodiments , the K. pneumoniae OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) an immunogenic composition comprising

(i) one or more of K. pneumoniae type O1, O2, O3, O5 OPS, and any combination thereof; And

(ii) FlaBD2-MrkA fusion protein; and/or

(b) an immunogenic composition containing

(i) one or more of O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa OPS, and any combination thereof; And

(ii) FlaBD2-MrkA fusion protein.

In some embodiments, one or more types of immunogenic compositions further comprise one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the vaccine composition comprises an immunogenic composition selected from

(a) an immunogenic composition comprising a combination of K. pneumoniae type O1, O2, O3, O5 OPS, a FlaBD2-PcrV fusion protein, and a FlaBD2-MrkA fusion protein; And

(b) an immunogenic composition comprising a combination of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS , a FlaBD2-PcrV fusion protein, and a FlaBD2-MrkA fusion protein.

In some embodiments, the implementation of the vaccine further includes one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to a polymer. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to a polymer. In some embodiments, the implementation of the polymer includes a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ; And

(b) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, one or more OPS are conjugated to one or more polymers. In some embodiments, one or more polypeptides are conjugated to one or more polymers. In some embodiments, the OPS and one or more polypeptides are conjugated to one or more polymers. In some embodiments, the implementation of the polymer includes a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(b) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, one or more polypeptides are conjugated to one or more polymers. In some embodiments, the OPS and one or more polypeptides are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ;

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, one or more polypeptides are conjugated to one or more polymers. In some embodiments, the OPS and one or more polypeptides are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ; And

(b) FlaBD2-MrkA fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(b) FlaBD2-MrkA fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ;

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) FlaBD2-MrkA fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ; And

(b) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(b) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ;

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ;

(b) FlaBD2-MrkA fusion protein; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some OPS embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(b) FlaBD2-MrkA fusion protein; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some OPS embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more OPS types O1, O2, O3, O5 of K. pneumoniae ;

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) FlaBD2-MrkA fusion protein; And

(d) FlaBD2-PcrV fusion protein.

In some embodiments, the vaccine composition further comprises one or more polymers. In some embodiments, the OPS is conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some OPS embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more of O1, O2, O3, O5 K. pneumoniae OPS , wherein the K. pneumoniae OPS is conjugated to one or more polymers; And

(b) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, one or more polypeptides are conjugated to one or more polymers. In some embodiments, the OPS and one or more polypeptides are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more polymers; And

(b) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, one or more polypeptides are conjugated to one or more polymers. In some embodiments, the OPS and one or more polypeptides are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is conjugated to one or more first polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more second polymers; And

(c) one or more polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the SEQ sequences ID NO: 1-3, 6-12 or 16-26.

In some embodiments, one or more polypeptides are conjugated to one or more first polymers. In some embodiments, one or more polypeptides are conjugated to one or more second polymers. In some embodiments, one or more polypeptides are conjugated to one or more first polymers and one or more second polymers. In some embodiments, one or more of the first polymers and one or more of the second polymers are the same. In some embodiments, one or more of the first polymers and one or more of the second polymers are different. In some embodiments, one or more of the first polymers comprise a capsular polysaccharide from a first pathogen. In some embodiments, the one or more second polymers comprise a capsular polysaccharide from a second pathogen. In some embodiments, the first pathogen and the second pathogen are different bacteria. In some embodiments, the first pathogen and the second pathogen are the same bacteria of the same serotype. In some embodiments, the first pathogen and the second pathogen are the same bacteria of different serotypes. In some embodiments, one or more first polymers include PLL. In some embodiments, one or more of the second polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more of O1, O2, O3, O5 K. pneumoniae OPS , wherein the K. pneumoniae OPS is conjugated to one or more polymers; And

(b) FlaBD2-MrkA fusion protein.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more polymers; And

(b) FlaBD2-MrkA fusion protein.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more of O1, O2, O3, O5 K. pneumoniae OPS , wherein the K. pneumoniae OPS is conjugated to one or more polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more second polymers; And

(c) FlaBD2-MrkA fusion protein.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers and one or more second polymers. In some embodiments, one or more of the first polymers and one or more of the second polymers are the same. In some embodiments, one or more of the first polymers and one or more of the second polymers are different. In some embodiments, one or more of the first polymers comprise a capsular polysaccharide from a first pathogen. In some embodiments, the one or more second polymers comprise a capsular polysaccharide from a second pathogen. In some embodiments, the first pathogen and the second pathogen are different bacteria. In some embodiments, the first pathogen and the second pathogen are the same bacteria of the same serotype. In some embodiments, the first pathogen and the second pathogen are the same bacteria of different serotypes. In some embodiments, one or more first polymers include PLL. In some embodiments, one or more of the second polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more polymers; And

(b) FlaBD2-PcrV fusion protein.

In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is conjugated to one or more first polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more second polymers; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers and one or more second polymers. In some embodiments, one or more of the first polymers and one or more of the second polymers are the same. In some embodiments, one or more of the first polymers and one or more of the second polymers are different. In some embodiments, one or more of the first polymers comprise a capsular polysaccharide from a first pathogen. In some embodiments, the one or more second polymers comprise a capsular polysaccharide from a second pathogen. In some embodiments, the first pathogen and the second pathogen are different bacteria. In some embodiments, the first pathogen and the second pathogen are the same bacteria of the same serotype. In some embodiments, the first pathogen and the second pathogen are the same bacteria of different serotypes. In some embodiments, one or more first polymers include PLL. In some embodiments, one or more of the second polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more of O1, O2, O3, O5 K. pneumoniae OPS , wherein the K. pneumoniae OPS is conjugated to one or more polymers;

(b) FlaBD2-MrkA fusion protein; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some OPS embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more polymers;

(b) FlaBD2-MrkA fusion protein; And

(c) FlaBD2-PcrV fusion protein.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-MrkA fusion protein are conjugated to one or more polymers. In some embodiments, the OPS and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some OPS embodiments, the FlaBD2-MrkA fusion protein and the FlaBD2-PcrV fusion protein are conjugated to one or more polymers. In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is conjugated to one or more first polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is conjugated to one or more second polymers;

(c) FlaBD2-MrkA fusion protein; And

(d) FlaBD2-PcrV fusion protein.

In some embodiments, one or more of the first polymers and one or more of the second polymers are the same. In some embodiments, one or more of the first polymers and one or more of the second polymers are different. In some embodiments, one or more of the first polymers comprise a capsular polysaccharide from a first pathogen. In some embodiments, the one or more second polymers comprise a capsular polysaccharide from a second pathogen. In some embodiments, the first pathogen and the second pathogen are different bacteria. In some embodiments, the first pathogen and the second pathogen are the same bacteria of the same serotype. In some embodiments, the first pathogen and the second pathogen are the same bacteria of different serotypes. In some embodiments, one or more first polymers include PLL. In some embodiments, one or more of the second polymers include PLL. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers and the FlaBD2-PcrV fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers and the FlaBD2-MrkA fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers and one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers and one or more second polymers.

In some embodiments, the implementation of the vaccine composition contains two or more types of immunogenic compositions selected from

(a) an immunogenic composition comprising a KP O1 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(a') an immunogenic composition comprising a KP O1 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(b) an immunogenic composition comprising a KP O2 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(b') an immunogenic composition comprising a KP O2 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(c) an immunogenic composition comprising a KP O3 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(c') an immunogenic composition comprising a KP O3 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(d) an immunogenic composition comprising a KP O5 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(d') an immunogenic composition comprising a KP O5 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(e) an immunogenic composition comprising a PA O1 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(e') an immunogenic composition comprising a PA O1 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(f) an immunogenic composition comprising a PA O2 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(f') an immunogenic composition comprising a PA O2 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(g) an immunogenic composition comprising a PA O3 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(g') an immunogenic composition comprising a PA O3 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(h) an immunogenic composition comprising a PA O4 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(h') an immunogenic composition comprising a PA O4 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(i) an immunogenic composition comprising a PA O5 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(i') an immunogenic composition comprising a PA O5 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(j) an immunogenic composition comprising a PA O6 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(j') an immunogenic composition comprising a PA O6 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(k) an immunogenic composition comprising a PA O10 OPS covalently linked to a FlaBD2-MrkA fusion protein;

(k') an immunogenic composition comprising PA O10 OPS covalently linked to a FlaBD2-PcrV fusion protein;

(l) an immunogenic composition comprising PA O11 OPS covalently linked to a FlaBD2-MrkA fusion protein; And

(l') an immunogenic composition containing PA O11 OPS covalently linked to a FlaBD2-PcrV fusion protein.

In some embodiments, the implementation of the vaccine composition contains two or more types of immunogenic compositions selected from

(a) an immunogenic composition comprising a polymer, KP O1 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O1 OPS;

(a') an immunogenic composition comprising a polymer, KP O1 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O1 OPS;

(b) an immunogenic composition comprising a polymer, KP O2 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O2 OPS;

(b') an immunogenic composition comprising a polymer, KP O2 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O2 OPS;

(c) an immunogenic composition comprising a polymer, KP O3 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O3 OPS;

(c') an immunogenic composition comprising a polymer, KP O3 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O3 OPS;

(d) an immunogenic composition comprising a polymer, KP O5 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O5 OPS;

(d') an immunogenic composition comprising a polymer, KP O5 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or KP O5 OPS;

(e) an immunogenic composition comprising a polymer, PA O1 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or the PA O1 OPS;

(e') an immunogenic composition comprising a polymer, PA O1 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or the PA O1 OPS;

(f) an immunogenic composition comprising a polymer, PA O2 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or the PA O2 OPS;

(f') an immunogenic composition comprising a polymer, PA O2 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O2 OPS;

(g) an immunogenic composition comprising a polymer, PA O3 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O3 OPS;

(g') an immunogenic composition comprising a polymer, PA O3 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O3 OPS;

(h) an immunogenic composition comprising a polymer, PA O4 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O4 OPS;

(h') an immunogenic composition comprising a polymer, PA O4 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O4 OPS;

(i) an immunogenic composition comprising a polymer, PA O5 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O5 OPS;

(i') an immunogenic composition comprising a polymer, PA O5 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O5 OPS;

(j) an immunogenic composition comprising a polymer, PA O6 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O6 OPS;

(j') an immunogenic composition comprising a polymer, PA O6 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O6 OPS;

(k) an immunogenic composition comprising a polymer, PA O10 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O10 OPS;

(k') an immunogenic composition comprising a polymer, PA O10 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O10 OPS;

(l) an immunogenic composition comprising a polymer, PA O11 OPS, and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O11 OPS; And

(l') an immunogenic composition comprising a polymer, PA O11 OPS, and a FlaBD2-PcrV fusion protein, wherein the fusion protein is covalently linked to the polymer and/or PA O11 OPS.

In some embodiments, at least one polymer comprises a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the vaccine composition comprises an immunogenic composition selected from

(a) an immunogenic composition comprising polymer, combination of OPS types O1, O2, O3, O5K. pneumoniae and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to a polymer and/or at least one of the OPS types; And

(b) an immunogenic composition comprising polymer, combination of OPS types O1, O2, O3, O4, O5, O6, O10, O11P. aeruginosa and a FlaBD2-MrkA fusion protein, wherein the fusion protein is covalently linked to a polymer and/or at least one of the OPS types.

In some embodiments, at least one polymer comprises a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, the implementation of the polymer includes PLL.

In some embodiments, the vaccine composition comprises an immunogenic composition selected from

(a) an immunogenic composition comprising one or more first polymers, a combination of K. pneumoniae type O1, O2, O3, O5 OPS, a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more first polymers and/or at least one of the OPS types, and a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more first polymers and/or at least one of the OPS types; And

(b) an immunogenic composition comprising one or more second polymers, a combination of OPS type O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa , a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked with at least a portion of one or more second polymers and/or at least one of the OPS types, and a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more second polymers and/or at least one of the OPS types.

In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers and the FlaBD2-PcrV fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers and the FlaBD2-MrkA fusion protein is conjugated to one or more second polymers. In some embodiments, the FlaBD2-MrkA fusion protein is conjugated to one or more first polymers and one or more second polymers. In some embodiments, the FlaBD2-PcrV fusion protein is conjugated to one or more first polymers and one or more second polymers. In some embodiments, one or more of the first polymers and one or more of the second polymers are the same. In some embodiments, one or more of the first polymers and one or more of the second polymers are different. In some embodiments, one or more of the first polymers comprise a capsular polysaccharide from a first pathogen. In some embodiments, the one or more second polymers comprise a capsular polysaccharide from a second pathogen. In some embodiments, the first pathogen and the second pathogen are different bacteria. In some embodiments, the first pathogen and the second pathogen are the same bacteria of the same serotype. In some embodiments, the first pathogen and the second pathogen are the same bacteria of different serotypes. In some embodiments, one or more first polymers include PLL. In some embodiments, one or more second polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers;

(b) one or more of O1, O2, O3, O5 K. pneumoniae OPS; And

(c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12, or 16-26, wherein the polypeptide is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12, or 16-26, wherein the polypeptide is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of O1, O2, O3, O5 K. pneumoniae OPS;

(c) one or more of OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(d) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12, or 16-26, wherein the polypeptide is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of O1, O2, O3, O5 K. pneumoniae OPS; And

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of O1, O2, O3, O5 K. pneumoniae OPS;

(c) one or more of OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(d) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of K. pneumoniae type O1, O2, O3, O5 OPS; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of K. pneumoniae type O1, O2, O3, O5 OPS;

(c) one or more of OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(d) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of K. pneumoniae type O1, O2, O3, O5 OPS;

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(d) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(d) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of K. pneumoniae type O1, O2, O3, O5 OPS;

(c) one or more of OPS types O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa ; And

(d) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(e) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers; And

(b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12 or 16-26, wherein the polypeptide is covalently linked to at least a portion of the polymer and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12, or 16-26, wherein the polypeptide is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with one or more of the sequences of SEQ ID NO: 1-3, 6-12, or 16-26, wherein the polypeptide is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers; And

(b) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(b) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more polymers,

(b) one or more of K. pneumoniae type O1, O2, O3, O5 OPS; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(b) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers;

(b) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers;

(b) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(c) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains

(a) one or more K. pneumoniae type O1, O2, O3, O5 OPS, wherein the K. pneumoniae OPS is covalently linked to one or more polymers;

(b) one or more P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11 OPS, wherein the P. aeruginosa OPS is covalently linked to one or more polymers;

(c) a FlaBD2-MrkA fusion protein, wherein the FlaBD2-MrkA fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types; And

(d) a FlaBD2-PcrV fusion protein, wherein the FlaBD2-PcrV fusion protein is covalently linked to at least a portion of one or more polymers and/or at least one of the OPS types.

In some embodiments, one or more polymers include a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

In some embodiments, the implementation of the vaccine composition contains:

(a) a first immunogenic composition comprising a polymer, KP O1 OPS, conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer;

(b) a second immunogenic composition comprising a polymer, KP O2 OPS, conjugated to a polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(c) a third immunogenic composition comprising a polymer, KP O3 OPS, conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer;

(d) a fourth immunogenic composition comprising a polymer, KP O5 OPS, conjugated to a polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(e) a fifth immunogenic composition comprising a polymer, PA O1 OPS conjugated to a polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(f) a sixth immunogenic composition comprising a polymer, PA O2 OPS conjugated to a polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(g) a seventh immunogenic composition comprising a polymer, PA O3 OPS conjugated to a polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(h) an eighth immunogenic composition comprising a polymer, PA O4 OPS conjugated to the polymer, and a FlaBD2-PcrV fusion protein covalently linked to the polymer;

(i) a ninth immunogenic composition comprising a polymer, PA O5 OPS conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer;

(j) a tenth immunogenic composition comprising a polymer, PA O6 OPS conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer;

(k) an eleventh immunogenic composition comprising a polymer, PA O10 OPS conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer; And

(l) a twelfth immunogenic composition comprising a polymer, PA O11 OPS conjugated to a polymer, and a FlaBD2-MrkA fusion protein covalently linked to the polymer.

In some embodiments, at least one of the polymers includes a capsular polysaccharide. In some embodiments, the capsular polysaccharide is a Klebsiella spp. capsular polysaccharide. K19. In some embodiments, one or more polymers include PLL.

The optimal amounts of components for a particular vaccine can be established in standard studies, including the observation of appropriate immune responses in patients. After the initial vaccination, patients may receive one or more booster immunizations at appropriate time intervals.

The immunogenic complexes described in the present invention and/or preparations thereof can be formulated in unit dosage form for ease of administration and uniformity of doses. The specific therapeutically effective dose level for any particular patient or microorganism may depend on various factors, including the severity or risk of infection; the activity of the particular vaccine or vaccine composition used; other characteristics of the particular vaccine or vaccine composition used; age, body weight, general health, gender of the patient, diet of the patient, pharmacokinetic state of the patient, time of administration (eg, in relation to other activities of the patient such as eating, sleeping, taking other medications, including doses of other vaccines, and etc.), route of administration, rate of excretion of the specific vaccine or vaccine composition used; vaccines used in combination or in parallel with the used vaccine composition; and similar factors well known in the medical field.

Immunogenic complexes for use in the present invention may be formulated into compositions (eg, pharmaceutical compositions) by known methods. The vaccine formulation is described in general terms in Vaccine Design (Powell and Newman, 1995). For example, an immunogenic amount of a vaccine preparation may be formulated with one or more organic or inorganic, liquid or solid, pharmaceutically acceptable carrier materials. The production of pneumococcal polysaccharide and conjugate vaccines is described, for example, in USSN 11/395593, issued March 31, 2006, the contents of which are incorporated herein by reference.

Typically, the pharmaceutically acceptable carrier(s) includes solvents, dispersion media, and the like that are compatible with pharmaceutical administration. For example, materials that serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, dextrose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powder tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; polyols such as glycerol, propylene glycol and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, in addition, preservatives and antioxidants may also be present in the composition, in accordance with the decision of the formulation specialist (Martin, 1975).

Vaccines can be formulated by combining one or more immunogenic complexes disclosed in the present invention with carriers and/or other optional components by any available means, including, for example, conventional mixing, granulation, dissolution, lyophilization, or similar methods.

Vaccine compositions that can be used in the proposed methods can be in a freeze-dried state up to about the time they are used, at which point they can be reconstituted with a solvent just prior to administration. In some embodiments, the vaccine components or compositions are lyophilized in the presence of one or more other components (eg, adjuvants) and reconstituted with saline just prior to administration. Alternatively, individual components, or kits of components, can be separately lyophilized and/or stored (eg, in a vaccination kit); the components are reconstituted and either mixed prior to use or administered separately to the patient.

Lyophilization can contribute to greater stability of the composition (for example, by preventing or reducing the destruction of polysaccharide antigens). Lyophilization of vaccines or vaccine components is well known in the art. Typically, a liquid vaccine, or vaccine component, is lyophilized, often in the presence of an anti-caking agent (such as, for example, a sugar, such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10-200 mg/mL. Lyophilization is usually carried out in a series of steps, for example, the cycle starts at -69°C, gradually brought to -24°C over 3 hours, then maintained at this temperature for 18 hours, after which it is gradually brought to -16°C. C for 1 h, then maintained at this temperature for 6 h, after which it was gradually brought to +34°C for 3 h, and finally maintained at this temperature for 9 h.

Vaccines, or vaccine components, for use in the present invention may be incorporated into liposomes, cochleates, biodegradable polymers such as polylactide, polyglycolide, and lactide-glycolide polymers, or immunostimulatory complexes (ISCOMS).

In some situations, it may be desirable to prolong the action of the vaccine used according to the present invention, for example, by slowing down the absorption of one or more vaccine components. Such delay in absorption can be accomplished, for example, by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the product depends on the rate of its dissolution, which, in turn, may depend on the size and shape. Alternatively or additionally, slowing absorption can be accomplished by dissolving or suspending one or more vaccine components in an oil medium. Depot injectables can also be used to delay absorption. Such depot forms can be obtained by forming microcapsular matrices from one or more vaccine components in a network of biodegradable polymers. Depending on the ratio of polymer to vaccine component, as well as the nature of the particular polymer(s) used, the release rate can be controlled.

Examples of biodegradable polymers that can be used in the present invention include, for example, poly(orthoesters) and poly(anhydrides). One specific illustrative polymer is polylactide-polyglycolide.

Depot injectables can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

Polymeric delivery systems can also be used in non-depot formulations, including, for example, oral formulations. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and so on, can be used in oral preparations. Polysaccharide antigens or conjugates can be formulated with such polymers, for example, to form particles, microparticles, extrudates, solid dispersions, mixtures, or other combinations, to facilitate the preparation of useful preparations (eg, oral preparations).

Vaccines for use in the present invention include immunogenic compositions and, in addition, may include one or more additional active agents (ie, agents that have a biological effect - not inert ingredients). It should be understood that such additional agents may be formulated together with one or more other vaccine components, or may be stored separately and combined at or about the time of administration. In some embodiments, such additional components can be administered separately from some, or all, other vaccine components, in an appropriate time window to achieve the appropriate effect.

For example, in the manufacture of vaccines, it is common to include one or more adjuvants. Adjuvants are generally agents that enhance the immune response to an antigen. In some embodiments, adjuvants are used that enhance the Th1-type immune response.

In some embodiments, adjuvants are used that enhance a Th17-type immune response.

Adjuvants

In some embodiments, the immunogenic complexes described herein are formulated and/or administered in combination with an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of aluminum hydroxide, phosphated aluminum hydroxide, aluminum phosphate, TLR agonist, TLR2 agonist (e.g., saponin (e.g., QS21, etc.), porin, etc.), TLR3 agonist (e.g., dsRNA, polyI:polyC, poly-ICLC, ^Hiltonol® , etc.), TLR4 agonist (eg, monophosphoryl lipid A (MPL A)), TLR5 agonist (eg, flagellin); a TLR 7, 8, or 9 agonist (eg, a CpG oligonucleotide, etc.).

In some embodiments, adjuvants suitable for use in the present invention include, but are not limited to:

(1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and so on;

(2) oil-in-water emulsion formulations (with or without the addition of other specific immunostimulatory agents such as muramyl peptides (described below) or bacterial cell wall components such as, for example,

(a) MF59 (PCT Publication No. WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE (see below, though not required) ) composed into submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics, Newton, MA),

(b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic block copolymer L121 and thr-MDP (see below), either microfluidized into a submicron emulsion or vortexed to produce an emulsion with coarser particles, and

(c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deacyl-monophospholipid A (MPL™) described in US Pat. No. 4,912,094 (Corixa), trehalose dimycolate (TDM) and cell wall backbone (CWS), preferably MPL+CWS (Detox™);

(3) saponin-based adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (US Pat. No. 5,057,540) can be used, or particles derived therefrom, such as ISCOM (Immunostimulatory Complexes);

(4) bacterial lipopolysaccharides, synthetic lipid A analogues such as aminoalkylglucosamine phosphate (AGP) compounds, or derivatives or analogues thereof, available from Corixa and described in US Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl-2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, which is also known as 529 (formerly RC529), which is formulated in aqueous or stable emulsion form, synthetic polynucleotides such as oligonucleotides containing CpG motif(s) (US Pat. No. 6,207,646); TLR3 agonists (synthetic dsRNA, polyI:polyC, Hiltonol ^® available from InvivoGen);

(5) cytokines such as interleukins (eg IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18 and so on) , interferons (eg, interferon gamma), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), co-stimulatory molecules B7-1 and B7-2, and so on;

(6) inactivated mutants of bacterial ADP-ribosylating toxin, such as cholera toxin (CT), either wild-type or mutated, for example, in which the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably histidine, according to a published international patent application number WO 00/18434 (see also WO 02/098368 and WO 02/098369), pertussis toxin (PT) or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72 and the new mutant LT, designated LT(R192G/L211A) or dmLT can elicit Th17 responses to vaccine antigens (Andreasen, 2009; Norton, 2011; Norton, 2012; Leach, 2012; Toprani, 2017), CT-S109, PT-K9/G129 ( see for example WO 93/13302 and WO 92/19265); And

(7) other substances that act as immunostimulatory agents, increasing the effectiveness of the composition, for example, delta-inulin (Advax ^® ), Matrix M.

Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2'-dipalmitoyl- sn -glycero -3-hydroxyphosphoryloxy)ethylamine (MTP-PE) and so on.

Vaccines for use in the present invention may include, or be administered simultaneously with, other antimicrobial therapeutic agents. For example, such vaccines may include, or be administered with, one or more agents that kill or slow the growth of the pathogen . Such agents include, for example, penicillin, vancomycin, erythromycin, azithromycin and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.

Alternatively or additionally, vaccines for use in the present invention may include, or be administered with, one or more other vaccines or therapy options. For example, one or more non-pneumococcal antigens may be included, or administered with vaccines.

Introduction

In some embodiments, the immunogenic complexes are administered to a patient at risk of developing a disease, such as hospitalized patients. It should be understood that an individual may be considered at risk of developing a disease without being diagnosed with any symptoms of the disease. For example, if an individual is known to have been, or plans to be, in situations of relatively high risk of exposure to an infection, that individual would be considered to be at high risk of developing the disease (e.g. hospitalized patient, elderly patient living in a nursing home, etc.). Further).

Effective routes of administration can be used, such as, for example, oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, intradermal, rectal, vaginal, topical, intraocular, pulmonary, or contact application. In some embodiments, the vaccine compositions can be administered by injection (eg, intramuscular, intraperitoneal, intradermal, and/or subcutaneous); or delivered via a mucosal (eg, oral/alimentary, respiratory and/or urogenital tract). Intranasal vaccines may be particularly useful in some contexts, such as for the treatment of pneumonia or otitis media (because nasopharyngeal carriage of pneumococci may be more effectively prevented, resulting in attenuation of infection at a very early stage). In some embodiments of the invention, it may be desirable to administer different doses of the vaccine in different ways; in some embodiments, it may be desirable to administer different components of a single dose via different routes.

In some embodiments of the present invention, pharmaceutical compositions (eg, vaccines) are administered intradermally. A common intradermal injection technique, the "mantoux procedure", involves the steps of clearing the skin, followed by extension of one arm and insertion of a small gauge needle (26-31 gauge), with the needle beveled upward at a 10-15° angle. After insertion of the beveled part of the needle, the needle shaft is lowered and pushed forward, providing slight pressure to lift it under the skin. The fluid is then injected very slowly, creating a bubble or swelling on the surface of the skin, and then the needle is slowly withdrawn.

Devices specially designed for the administration of liquid agents into or through the skin are described in, for example, WO 99/34850 and EP 1092444, in addition devices for jet injection are described in, for example, WO 01/13977; U.S. Patent No. 5,480,381 U.S. Patent No. 5,599,302 U.S. Patent No. 5,334,144 U.S. Patent No. 5,993,412 U.S. Patent No. 5,649,912 U.S. Patent No. 5,569,189 U.S. Patent No. 5,704,911 US Pat. No. 5,339,163, US Pat. No. 5,312,335, US Pat. No. 5,503,627, US Pat. WO 97/13537. Other methods of intradermal administration of vaccine preparations may include the use of conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961) or transdermal patches (WO 97/48440; WO 98/28037); or application to the surface of the skin (transdermal or transdermal delivery, WO 98/20734; WO 98/28037).

As described above, pharmaceutical compositions (eg, vaccines) can be administered as a single dose or multiple doses. It should be understood that administration is a single "dose" administration, provided that all relevant components are administered to the patient within the time window; it is not necessary that each component be present in one composition. For example, administration of two different immunogenic compositions within less than 24 hours is considered to be a single dose administration. By way of example, immunogenic compositions having different antigenic components may be administered in different compositions but as part of a single dose. As noted above, such separate compositions may be administered by different routes or by the same route. Alternatively or additionally, in embodiments where the vaccine comprises a combination of immunogenic compositions and additional types of active agents, the immunogenic compositions may be administered by one route and the second active agent may be administered by the same route or by another route.

Pharmaceutical compositions (eg, vaccines) are administered in such amounts and for such periods of time as are necessary to achieve the desired result. In specific embodiments, implementation of the present invention, the vaccine composition contains a therapeutically effective amount of at least one immunogenic composition. The exact amount necessary to achieve a therapeutically effective amount may vary depending on the immunogenic composition, as well as for different patients, depending on the species, age and general health of the patient, stage of the disease, particular pharmaceutical mixture, route of administration, and the like.

The amount of polysaccharide antigen(s) or conjugate(s) in each dose of a pharmaceutical composition (e.g., vaccine) is chosen such that the vaccine, when administered as described herein, can induce an appropriate protective immune response without significant adverse side effects. As used herein, the term "protective immune" or "immunoprotective" response means an immune response sufficient to protect an immunized patient from effective infection with the specific pathogen or pathogens against which the vaccine is directed (eg, K. pneumoniae infections). Such amounts may vary depending on the particular immunogenic composition or compositions used and on its presentation.

In some embodiments, a pharmaceutical composition comprising an immunogenic complex described herein induces a Th1 and/or Th17 cellular response upon administration to a patient. In some embodiments, a pharmaceutical composition comprising an immunogenic complex described herein elicits an opsonic/bactericidal response against one or more of K. pneumoniae , P. aeruginosa , and E. coli upon administration to a patient . In some embodiments, a pharmaceutical composition comprising an immunogenic complex of the present invention results in a reduction in the rate of transmission of infection and/or colonization of the mucosal surfaces of one or more of K. pneumoniae , P. aeruginosa , and E. coli after administration to a patient.

In some embodiments, a pharmaceutical composition comprising an immunogenic complex described herein results in a reduction in the rate of transmission of infection and/or colonization of the GI tract by one or more of K. pneumoniae , P. aeruginosa , and E. coli upon infection.

Some embodiments relate to a method of immunizing a patient against an infection caused by K. pneumoniae , comprising administering to the patient an effective amount of the immunogenic complex described in the present invention. Some embodiments relate to a method of immunizing a patient against an infection caused by K. pneumoniae , comprising administering to the patient an effective amount of a pharmaceutical composition described in the present invention.

Some embodiments relate to a method of immunizing a patient against an infection caused by P. aeruginosa , comprising administering to the patient an effective amount of the immunogenic complex described in the present invention. Some embodiments relate to a method of immunizing a patient against a P. aeruginosa infection, comprising administering to the patient an effective amount of a pharmaceutical composition.

Some embodiments relate to a method for immunizing a patient against an infection caused byE. coli, comprising administering to the patient an effective amount of the immunogenic complex described in the present invention. Some embodiments relate to a method for immunizing a patient against an infection caused byE. coli, comprising administering to the patient an effective amount of the pharmaceutical composition.

Dosing

In accordance with the present invention, the administration of a pharmaceutical composition (eg, a vaccine) may include the delivery of only one dose, or, alternatively, may include the administration of an initial dose, followed by the administration of one or more additional immunizing doses at appropriate time intervals. An immunization regimen is a program for administering one or more specific doses of one or more specific pneumococcal vaccines, by one or more specific routes of administration, to a patient of one or more specific ages.

The present invention relates to methods of immunization comprising administering at least one dose of vaccine to a young patient. In some embodiments, the young patient is 18 years of age or less. In some embodiments, the young patient has previously received one or more doses of a pneumococcal polysaccharide conjugate vaccine; in other embodiments, the young patient has not previously received pneumococcal vaccines. In some embodiments, the young patient has previously been infected with or exposed to a Klebsiella, Pseudomonas , and/or E. coli infection.

The present invention relates to methods of immunization comprising administering at least one dose of a vaccine to an adult patient. In some embodiments, the adult patient is greater than about 50 years of age. In some embodiments, the adult patient is greater than about 65 years of age. In some embodiments, the adult patient has previously received one or more doses of a pneumococcal polysaccharide conjugate vaccine; in other embodiments, the adult patient has not previously received pneumococcal vaccines. In some embodiments, the adult patient has previously been infected with or exposed to a Klebsiella, Pseudomonas , and/or E. coli infection.

Immunization regimens The present disclosure relates to eliciting or inducing an immune response (e.g., a protective immune response) in a patient sufficient to reduce at least one indicator selected from the group consisting of incidence, prevalence, incidence, and/or severity of at least at least one infection, disease, or disorder, and/or at least one surrogate marker for infection, disease, or disorder, in a patient population and/or subpopulation. A supplementary immunization regimen is one whose effect is related to the effect of the standard regimen it supplements. An additional regimen may require additional administrations and/or superimmunogenic doses of the immunogenic compositions described in the present invention, which take place in a standard regimen, or administration of vaccines that are not part of a standard regimen. The complete immunization regimen of the present invention may include both a standard regimen and a supplementary regimen. Illustrative examples of vaccination schedules are provided for illustrative purposes. The detailed description of the methods for assessing the immunogenic response given in the present invention allows the development of alternatives to exemplary immunization regimens without undue experimentation.

In one embodiment of the present invention, the first pneumococcal vaccine administration is typically given when the patient is over about 50 years old, over about 55 years old, over about 60 years old, over about 65 years old, or over about 70 years old.

In some embodiments, a single vaccine administration is used. It is possible that the objectives of the present invention can be achieved using a single administration, especially when one or more of the vaccine polysaccharides and/or conjugates used, or combinations thereof, is/are potent(s), and in such a situation, the regimen with the introduction of a single dose is sufficient to induce a long-term protective immune response.

In specific embodiments, it is desirable to administer two or more doses of a vaccine for greater efficacy and coverage of a protective immune response. Thus, in some embodiments, the number of doses is at least two, at least three, or more doses. The maximum number of doses is not defined, but in clinical practice it is recommended not to immunize more often than necessary to achieve the desired effect.

Without wishing to be bound by a particular theory, the first dose of a vaccine administered in accordance with the invention may be considered a "priming" dose. In specific embodiments, the immunization schedule includes more than one dose. In such a situation, the subsequent dose can be considered a "booster" dose.

A priming dose can be administered to a previously untreated patient (a patient who has never previously received a polysaccharide vaccine). In some embodiments, a tame dose may be administered to a patient who has previously received a polysaccharide vaccine at least five or more years prior to the initial dose of the vaccine of the invention. In other embodiments, a priming dose may be administered to a patient who previously received a conjugate vaccine at least twenty years or more prior to the priming dose of a vaccine of the invention.

If the immunization schedule requires the administration of two or more separate doses, decide on the interval between doses. The interval between two consecutive doses may be the same throughout the entire immunization schedule or may be changed as the age of the patient increases. In the immunization schedules of the present invention, following the first dose of vaccine, a first interval is maintained until the next dose is given. The first interval is typically at least about 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 12 months or more. If more than one dose is subsequently administered, second (or more) intervals may be maintained between such subsequent doses. In some embodiments, all intervals between successive doses are of the same length; in other embodiments, the implementation of the second intervals may differ in duration. In some embodiments, the interval between successive doses may be at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, or at least about 2 years. In specific embodiments, the interval between doses can be up to 3 years, up to about 4 years, or up to about 5 years or 10 years, or more. In specific embodiments, the implementation of the intervals between subsequent doses may decrease as the age of the patient increases.

Those skilled in the art will appreciate that various possible combinations and subcombinations of different time conditions for the first administration, the shortest interval, the longest interval, and the total number of administrations (in absolute terms or within a specified period) can be used, and all such combinations and subcombinations should be considered as provided by the inventors, even if they are not specifically listed in the present invention.

Assays to determine the immunogenic response

In some embodiments, a method for evaluating the immunogenicity and/or efficacy of an immunogenic complex (and/or a composition comprising the immunogenic complex) comprises evaluating an immune response to the immunogenic composition. In some embodiments, the evaluation is carried out by conducting one or more of a series of in vitro assays. In some embodiments, the immune response being assessed is a B-cell or T-cell response.

In some embodiments, the in vitro assay is selected from one or more of ELISA, flow cytometry, Th1/Th17 cell response assessment, measurement of cytokine and functional antibody levels by PFU, serum kill bacteria (SBA), agglutination, motility, cytotoxicity, adhesion, agglutination.

In some embodiments, the evaluation is carried out by conducting one or more of a series of in vitro assays. In some embodiments, an animal model of nosocomial disease is used for the in vivo assay. In some embodiments, the animal model is a model of one or more of pneumonia, sepsis, burn wounds, GI infection, or surgical site infection. In some embodiments, the in vivo assay determines one or more of bacterial clearance, colonization, or mortality, with passive protection after infection with one or more target pathogens, or active protection after infection with one or more target pathogens. In some embodiments, the target pathogen is a nosocomial pathogen.

In general, it may be desirable to evaluate humoral responses, cellular responses and/or the interaction between them . When assessing humoral responses, antibody titers and/or types (e.g., total IgG, IgG1, IgG2, IgM, IgA, etc.) to specific pathogen serotypes can be determined, for example, before and/or after administration of an initial or booster dose of vaccine (and/ or compared with antibody levels in the absence of antigen challenge). Cellular responses can be assessed by monitoring responses such as delayed type hypersensitivity, and so on, to the carrier protein. Cellular responses can also be determined directly by assessing the response of peripheral blood mononuclear cell (PBMC) monocytes to stimulation with antigens of interest. Populations of B-cell progenitors and memory cells can be assessed in cellular enzyme-linked immunosorbent assays (ELISpot) using CPS and/or OPS of specific pathogens.

Any of a variety of assays can be used to determine serum antibody levels and/or activity in a patient. Suitable assays include, for example, ligand binding assays such as radioimmunoassays (RIAs), ELISAs, and multiplex assays (Luminex, Bioplex); functional assays, opsonophagocytic activity (OPA) assays; and in vivo protection assays (protection assays in neonatal rats and in adult mouse lung colonization and rodent GI tract and mortality assays).

Type-specific antibodies are detected in RIA by incubating serum with radiolabeled type-specific polysaccharides in suspension (eg, Schiffiman et al., 1980). The antigen-antibody complexes are then precipitated with ammonium sulfate and the radioactively labeled precipitates are analyzed by counts per minute (ppm).

In the ELISA detection method, serotype-specific antibodies from the sera of vaccinated patients are quantified by incubation with serotype-specific polysaccharides that have been adsorbed onto a solid support (e.g., Koskela and Leinonen (1981); Kojimaet al., 1990; Conception and Frasch, 2001). Bound antibody is detected using enzyme-conjugated secondary detection antibodies. ELISA also allows the determination of the isotype and subclass of immune response antibodies (i.e., IgM versus IgG or IgG1 versus IgG2) using isotype- or subclass-specific secondary antibodies, and this assay can be adapted to assess the avidity of serotype-specific antibodies (Anttila,et al., 1998; Romero Steiner,et al., 2005). Multiplex analyzes (for example, Luminex, Bioplex) allow simultaneous detection of antibodies to several serotypes. Serotype-specific CPS and/or OPS conjugated with spectrally different microspheres, which are mixed and incubated with diluted serum. Bound antibody is detected with a phycoerythrin-conjugated secondary antibody and quantified on a specialized flow cytometer that uses one laser to identify the bead type (serotype) and a second laser to quantify the bound secondary antibody (Pickering,et al., 2002; lol,et al. 2005).

An approach for assessing functional antibody in serum is OFA, which only quantifies antibodies that are capable of opsonizing bacteria, resulting in engulfment and killing of the bacteria. The standard assay uses a human phagocytic effector cell, complement source, bacteria, and diluted serum. The assay measure is the final serum titer at which ≥50% kill occurs compared to bacteria incubated with complement and human cells alone (Romero-Steiner, et al., 1997). This kill OFA can also be multiplexed by using targeted pathogen strains that carry different antibiotic resistance markers (Kim, et al., 2003). A final titer of 1:8 or more is considered a positive result in these kill ROAs. Another type of multiplex opsonophagocytic activity assay is the no-kill assay, in which uptake by phagocytic effector cells of a fluorescently stained encapsulated pathogen or fluorescent microspheres conjugated to antigenic CPS and/or OPS from a target pathogen in the presence of diluted serum plus a complement source is assessed by the PC method (Martinez, et al . al., 1999). Opsonic activity of serum antibody plus complement can also be assessed by measuring the oxidative response of phagocytic human effector cells to an ingested pathogen (Munro, et al. 1985; Ojo-Amaize, et al. 1995).

Several in vivo model systems can be used to evaluate the protection afforded by serum antibodies elicited by the vaccines of the present invention. In such passive defense systems, mice or rats are injected with a dilute serum pathogen and a final serum titer is determined that provides protection against bacteremia, organ or tissue colonization, or mortality (Stack, et al. 1998; Saeland, et al. 2000).

Antibody compositions

Some embodiments relate to an antibody composition comprising antibodies produced in a mammal immunized with an immunogenic complex of the invention. In some embodiments, the antibody comprises at least one antibody selected from the group consisting of mAbs and anti-idiotypic antibodies. In some embodiments, the antibody composition contains an isolated fraction of gamma globulin.

Sets

The present invention also relates to kits for preparing the immunogenic complex described in the present invention, which allow the researcher to create an immunogenic complex with preferred antigens, for example, for research purposes to evaluate the effect of an antigen, or combination of antigens, on an immune response. Such kits can be prepared from readily available materials and reagents. For example, such kits may include any one or more of the following: a container containing a backbone polymer crosslinked with a plurality of first affinity molecules; and a container containing a complementary affinity molecule that binds to the first affinity molecule, wherein the complementary affinity molecule binds to an antigen.

In another embodiment, the kit may include a container containing a polymer, such as a polysaccharide, a container containing a plurality of first affinity molecules, and a container containing a crosslinker for crosslinking the first affinity molecules to the backbone polymer.

In some embodiments, the kit also includes means for attaching a complementary affinity molecule to an antigen, which attachment can be by a crosslinker or by some intermediate fusion protein. In some embodiments, the kit may include at least one costimulatory factor that may be added to the polymer. In some embodiments, the kit includes a crosslinking reagent, for example, but not limited to, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodium cyanoborohydride; cyanogen bromide; ammonium bicarbonate/ioacetic acid, to bind the cofactor to the polymer.

Various kits and components can be created for use in the methods described in the present invention, depending on the intended use of the kit, the specific target antigen and the needs of the user.

EXAMPLES

For a more complete understanding of the invention described in the present invention, the following examples are given. Representative examples include information, exemplary explanation, and guidance that can be adapted to practice the present invention in its various embodiments and equivalents. It should be understood that these examples are for illustrative purposes only and should not be taken as limiting the present invention in any way.

Example 1: Bacterial strains

Reagent strains were subcultured three times on media free of animal products to remove any possible animal product impurities. The characteristics of the strains were compared before passage, during passage and after passage. For Pseudomonas : Gram stain, colony morphology, APS, oxidase test, growth on McConkey agar, IATS serotyping, growth kinetics. For Klebsiella : Gram stain, colony morphology, API, molecular serotyping by polymerase chain reaction (PCR), confirmation of mutations by PCR and sequencing (as required), K-typing (by PCR), growth kinetics.

Table 2: Strains Klebsiella and reagent strains used in the study

View Strains O-type K-type Reagent strain ^a K. pneumoniae B5055 1 2 ∆guaBA
Δwzabc K. pneumoniae 7380 2 Without capsule wild type K. pneumoniae 390 3 eleven wild type K. pneumoniae 4425/51 5 57 Δwzabc K. oxytoca 160011 1 19 wild type ^{b a} ΔguaBA=guanine biosynthetic mutant (attenuated); Δwzabc=capsule mutant
^b Klebsiella oxytoca

The guaBA and wzabc genes were deleted using Lambda Red recombination. Briefly, the DNA upstream and downstream of the guaBA and wzabc genes was fused to the kanamycin resistance cassette, and the linear DNA was introduced by transformation into K. pneumoniae strains that also contained a helper plasmid capable of expressing the lambda phage recombinase genes. Upon induction, these genes stimulate homologous recombination, which allows the replacement of the guaBA and wzabc genes with a kanamycin resistance cassette. The kanamycin resistance cassette was subsequently removed using the Flippase enzyme, which stimulates recombination between FRT sites flanking the cassette. The mutants were confirmed to be susceptible to kanamycin and the deletion was confirmed by PCR and deletion sequencing.

Table 3: Strains Pseudomonas aeruginosa (PA) used in the study

Reagent Strains Final reagent strain O1 IATS-O1 wild type O2 IATS-O2 wild type O3 IATS-O3 wild type O4 IATS-O4 wild type O5 IATS-O5 wild type O6 IATS-O6 wild type O10 IATS-O10 wild type O11 IATS-O11 wild type

Creation of a PA strain that overproduces PsL exopolysaccharide

Exemplary genetic approaches to reconstruct a PsL-responsive P. aeruginosa strain PAO1 Δpsl /pBAD- psl using 1886-bp synthetic DNA from Genscript, Piscataway, NJ, are presented in Figure 9B.

The araC-pBAD promoter was introduced upstream of the P. aeruginosa PAO1 pslA gene by homologous recombination. In fact, DNA upstream of pslA was amplified from P. aeruginosa PAO1 and ligated upstream of the gentamicin resistance cassette. A construct consisting of synthetic DNA containing the araC-pBAD promoter and the pslA gene was ligated downstream of the gentamicin resistance cassette. This entire construct was inserted into the pEX100T plasmid for Pseudomonas mutagenesis. Mutagenesis was performed as previously described (Schweizer et al., 1992). The gentamicin resistance gene was subsequently deleted and the insertion of the araC-pBAD promoter was confirmed by PCR and sequencing.

Example 2 Preparation of O- and K-Polysaccharide Antigens

Growing strains Klebsiella spp. And P. aeruginosa

All Klebsiella and PA reagent strains were subcultured three times on media free of animal products to remove any possible animal product contaminants. Research Cell Banks (RPBs) were grown in shake flasks in Hy- ^Soy® broth and aliquoted aseptically into 15% glycerol vials . Bacteria were grown in an 8 L BioFlo 415 fermenter in chemically defined medium (CDM) containing monobasic potassium phosphate, ammonium phosphate, citric acid, monohydrate, magnesium sulfate (MgSO ₄ ), polypropylene glycol (PPG) (antifoam), 3% glycerol as a source of carbon, vitamins and trace elements, as well as individual amino acids, as needed. Dissolved oxygen was maintained at 30% with cascade set to a minimum stirring speed of 200 rpm and the pH was adjusted to 7 with ammonium hydroxide. During fermentation, 50% glycerol, MgSO ₄ , trace amounts of vitamins and microelements were added to the medium in portions, as needed. Bacteria were grown for 12-18 hours to stationary phase, after which cell cultures were harvested to obtain the polysaccharide.

Purification of O-polysaccharide KP and core O-polysaccharide PA

The sequence diagram of the subsequent purification process of OPS from KP and PA is presented in Figure 8A. Briefly, after fermentation was complete, the entire fermentation culture was treated with either 1% acetic acid at 100°C for 4 h in the case of a P. aeruginosa cell culture, or with sodium nitrite in acetic acid at 4°C in the case of a K. pneumoniae cell culture, for 12 hours. After clarification by centrifugation and depth filtration (0.45-μm), the clarified solution containing hydrolyzed and released OPS was subjected to flow filtration along the flow (TFF) of UV-DF with a 10 kDa or 30 kDa NMWCO membrane, diafiltration against 1M NaCl and then Tris buffer. The retentate was then passed through a strong anion exchange membrane for ion exchange chromatography (IOC) in Tris buffer. Negatively charged PA OPS were captured on the membrane (Figure 8A) and then eluted with sodium chloride. Neutral KP OPS and neutral or slightly acidic PA OPS were obtained in a flow phase (FP) through an IOC membrane. The flow phase or eluate after the IOC was then precipitated with ammonium sulfate to remove residual protein. KP OPS in the ammonium sulfate supernatant was then subjected to two consecutive TFF filtrations starting with a 300 kDa NMWCO membrane, after which the PF was concentrated and diafiltered using a 5 or 10 kDa NMWCO membrane. The retentate of this later PF TFF contained purified neutral KP OPS (Figure 8A). Purified neutral and acidic PA OPS in ammonium sulfate (NH ₄ SO ₄ ) supernatant were subjected to TFF with a 5-10 kDa membrane (Figure 8A), after which they were in retentate. The final OPS product had an average MW measured by EC-Multi-Angle Laser Light Scattering (EC-MALLS) in the range of 10-30 kDa depending on the O-type, and was low in endotoxin, protein, and nucleic acids.

Purification of the capsular polysaccharide Klebsiella oxytoca K19 (KP K19 CPS)

An exemplary flow diagram of the subsequent purification process KP K19 CPS is shown in Figure 9A. Briefly, after formaldehyde inactivation and clarification by intermittent centrifugation and surface filtration (0.45 μm), the clarified solution containing CPS was concentrated and TFF diafiltered with a 30 kDa NMWCO membrane. The 30 kDa retentate was precipitated with CTAB. The precipitate was redissolved with calcium chloride and precipitated with 20% ethanol to remove nucleic acid and protein impurities. After centrifugation, the collected supernatant was precipitated with 80% ethanol. The CPS-containing pellet was redissolved in Tris buffer and captured on a weak anion exchange resin. K19 CPS eluted from the resin was further treated (decontaminated) with 0.1 M NaOH in 90% ethanol to remove any residual terminal lipid involved in its aggregation and endotoxin (as determined by horseshoe crab amoebocyte lysate (LAL) activity) (Figure 9A ). The final polysaccharide product K19 had an average MW of 220 kDa as measured by SEC-MALLS and was low in endotoxin, protein and nucleic acids.

Purification of the exopolysaccharide PsL PA

The sequence diagram of the subsequent purification process of the PsL exopolysaccharide is shown in Figure 9C. Briefly, the PA bacterial culture was clarified by centrifugation at 10,000 x g for 40 minutes at RT and the supernatant was filtered through a 0.5-0.3 μm depth filter. The filtered supernatant was diafiltered with TFF using a 100 kDa MWCO PES membrane with 10 diavolumes of water for injection (WFI), the permeate was collected and concentrated using a 2 kDa Hydrosart 10-20x UV membrane, diafiltered with 10 diavolumes of 50 mM Tris HCl, 50 mM NaCl , pH 7.5. The concentrated eluate was then subjected to strong anion exchange resin (IEX) chromatography using GE Q-Sepharose Fast Flow and the flow phase was collected by washing with another column volume of eluent. The eluate was concentrated and diafiltered with WFI using a 2 kDa Hydrosart membrane and then precipitated with 80% ethanol at RT for 18 h. and lyophilized. The final purified polysaccharide material was analyzed by HPAEC/PAD (high performance anion exchange chromatography with pulsed electrochemical detection) using a Dionex ICS-4000 capillary instrument and a CarboPac PA10 column, as well as commercially available monosaccharide standards, to determine the monosaccharide composition, residual protein was determined by the Bradford method, residual nucleic acids were determined using the Q-bit Life Technologies quant-IT kit, endotoxin was determined in the LAL assay, and total carbohydrates were determined in the anthrone reagent assay. The final material was also analyzed by high resolution H-NMR to confirm its structure and by SEC-HPLC to estimate the molecular weight.

Analytical methods for characterizing O- and K-antigens

The purity and nature of the OPS and CPS obtained by the methods described above was determined as follows. Residual protein concentration was determined by the Bradford and/or BCA method; nucleic acid concentration was determined in pico green assay or based on absorbance at 260 nm; endotoxin levels were determined using LAL assay; and the sugar composition of the polysaccharides was determined by acid hydrolysis followed by separation of individual monosaccharides by HPAEC-PAD (Dionex) as described above. The physical integrity of the polysaccharides was determined by high resolution 1H-NMR at 500, 800 or 950 MHz using a Bruker spectrophotometer to record spectra and compare the resulting spectra with spectra published in the literature. The nature of the polysaccharides and the integrity of the epitopes on the purified native and chemically derivatized polysaccharides in the process and in the final products were confirmed by ELISA using specific monoclonal antibodies to OPS and CPS, as well as polyclonal rabbit immune sera against heat-killed type-specific bacteria. The presence of O-acetyl groups on the polysaccharides was determined by Hestrin colorimetric analysis and 1H-NMR. The size of the polysaccharides was determined by SEC-HPLC (Figures 8B and 8C) and SEC-MALLS using appropriately sized columns. The concentration of polysaccharides was determined in the analysis with the anthrone reagent.

Example 3 General Procedure for Biotinylation of OPS and CPS

Biotinylation of polysaccharides (PS) containing hydroxyl groups was carried out using CDAP as an activating reagent. The polysaccharides were dissolved in LPS-free water (cell culture category; HyClone) to a final concentration of 1-5 mg/mL. At time 0, a volume of CDAP (freshly prepared at 100 mg/mL in acetonitrile) was added at a final ratio of 1 mg CDAP per 1 mg PS with vortex mixing. After 30 seconds, a volume of 0.2 M triethylamine (TEA; Sigma-Aldrich) was added to raise the pH to 8 (in the case of neutral PS, the volume of TEA is equal to that of CDAP; in the case of acidic PS, the volume of TEA is doubled). After 2.5 minutes, a volume of biotin derivative (Pierce EZ-Link amine-PEG3-biotin, 20 mg/ml in water) was added to a final ratio of 1 mg biotin per 1 mg PS, followed by incubation at room temperature for 1-4 h. For more dilute samples (<1 mg/ml) overnight incubation was used. The reaction was stopped by adding 25 mm glycine, and excess biotin was removed by intensive dialysis against PBS.

In the case of backbone polymer BP-1.2, biotinylation of polymer K19 CPS was continued by activating the carboxylate groups of uronic acid residues of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Pierce) and N -hydroxysuccinimide (NHS) to increase efficiency or create stable dry (amino-reactive) intermediates. EDC couples NHS to carboxyls to form an NHS ester that is significantly more stable than the O-acylisourea intermediate while allowing efficient conjugation to primary amines at physiological pH. An amino derivative of biotin (Pierce EZ-Link amine-PEG3-biotin) was then used to biotinylate NHS ester intermediates K19 CPS.

Total sugar concentration was determined either in an anthrone assay for OPS/CPS Klebsiella or in a resorcinol assay for PA OPS.

Example 4: Construction of Recombinant Rizavidin and Rizavidin Fusion Protein Antigens

Design and construction of rizavidin fusion protein expression constructs from K. pneumoniae And P. aeruginosa

In the case of Pseudomonas flagellins (FliC) A (FlaA) and B (FlaB), the in silico flagellar fiber design based on Pseudomonas FliC packaging in crystal indicates that domain 2 (D2) would be exposed to solution and could play an important role. in immunogenicity (Song, 2014). In addition, Pseudomonas FliC activates innate immune responses through its recognition of TLR5, which likely results in reactivity, therefore, the inventors created and evaluated some of the Pseudomonas flagellins in which the TLR5-binding domain and adjacent regions were removed, and only domain 2 was left. The resulting FlaA2-Domain2 and FlaB-Domain2 sequences lacking the TLR5-binding domain were expressed either as a fusion protein with rizavidin alone or with rizavidin and MrkA or PcrV Klebsiella . Schematic representation of the generated and expressed variants of rizavidin-antigen fusion proteins derived from Pseudomonas (FlaB; FlaA1; FlaA2; PcrV; FlaB-PcrV; FlaB-D2; FlaA2-D2; FlaB-D2-PcrV) and rizavidin-derived fusion proteins from both Klebsiella and Pseudomonas (FlaB-D2-MrkA; PcrV-MrkA) are shown in Figure 12A.

In the case of P. aeruginosa , flagellin was chosen as the likely carrier protein and protective antigen because it is a major component of the flagella; in addition, anti-flagellin antibodies are known to inhibit the motility required for disseminated infection and have been shown to be protective in an animal model of infection. In the case of flagellin, serotype A2 is highly conserved, but differs slightly from A1 and significantly in domain 2 from serotype B. To assess the activity of these variants, serotype A1 (FlaA1), A2 (FlaA2), and B (FlaB) flagellins were cloned and expressed as full length proteins. Domain 2 only from flagellins A2 and B was also cloned and expressed because this domain is on the surface of the assembled flagella and is likely to be a target for neutralizing antibodies. This made it possible to separately evaluate the contribution of the TLR5-binding domain to the adaptive immune response. Selected sequences were synthesized and cloned into plasmids for direct expression in E. coli with an amino-terminal rizavidin fusion protein and a six-residue histidine tag for initial affinity purification (Figure 12A).

In the case of P. aeruginosa , PcrV was also chosen because it encodes a type III secretory terminal protein. It is also known as a protective antigen, since antibodies specific for PcrV are able to protect against invasive infection. The sequence is highly conserved, with amino acid changes occurring at only 4 positions out of 294 amino acids in 18 different strains of P. aeruginosa . The sequence was synthesized and cloned into an E. coli direct expression plasmid with an amino-terminal rizavidin fusion protein and a six-residue histidine tag for initial affinity purification (Figure 12A).

In the case of K. pneumoniae , MrkA was chosen because it encodes the major pili fimbriae subunit type 3 and the immunologically dominant part of the fimbriae. Type 3 fimbriae are required for biofilm formation and MrkA plays a major role in biofilm formation and adhesion, suggesting that this antigen may confer protection in a vaccine formulation. MrkA was found to be highly conserved across 9 different K. pneumoniae genotypes, with only 1 amino acid change among 180 amino acids. One additional genotype has a slightly higher number of changes, at 9 positions among 180 amino acids (Table 4). For other type 3 pili fimbrial subunits, a strategy has been developed to stabilize the monomer during expression, called the donor chain complementation method (Walczak, 2014). According to this strategy, the amino-terminal sequence is repeated at the carboxyl end so that the sequence can be folded back and linked in the beta chain orientation normally provided by the adjacent subunit in the structure. The inventors attempted to apply this strategy to the MrkA subunit of pili type 3 K. pneumoniae by removing the secretion signal sequence first, as they planned to concentrate on the cytoplasmic expression of their rizavidin fusion proteins. The Langmuir model for the putative secretion signal is given in Chan et al. 2012, based on alignment with E. coli FimA amino acids 1-24. The inventors then added a linker of six glycine residues to the carboxyl end, followed by a repeat of the first 20 amino acids from the N-terminus of MrkA (no signal sequence) to ensure complementation of the donor strand. The sequence was synthesized and cloned into an E. coli expression control plasmid, with an amino-terminal rizavidin fusion protein and a six-residue histidine tag for initial affinity purification (Figure 12A).

Table 4: Multiple alignment of MrkA sequences K. pneumoniae using Clustal 0 (1.2.1)

For the K. pneumoniae MrkA protein, which is highly conserved, the AEO27492 sequence (in bold) in Table 4 was used as the starting sequence to generate the synthetic gene.

The recombinant rhavidin (rRhavi) used in these studies includes amino acids 45-179 of the genome-encoded protein because the predicted signal sequences (amino acids 1-44) are not included. To optimize the expression level of rRhavi in E. coli , the sequence of the gene encoding the rizavidin polypeptide (45-179; SEQ ID NO: 14) was modified with codons preferred for expression in E. coli and synthesized. The codon-optimized gene was cloned into the pET21b vector to control protein expression. In order to facilitate proper folding and disulfide bonding in rizavidin, an E. coli strain that promotes cytoplasmic disulfide bonding, T7 SHuffle express (NEB), was chosen for expression.

To construct the rhizavidin-antigen fusion proteins, the DNA sequence encoding the seven amino acid flexible linker region (GGGGSSS; SEQ ID NO: 15) was directly inserted into the 3' end of the synthetic rRhavi gene to ensure separation from rRhavi and promote proper folding of the resulting fusion. squirrel. Genes encoding candidate antigens (full-length or desired fragments) were synthesized and inserted into the rRhavi expression vector immediately after the linker region. To facilitate purification, a label of six histidine residues was added at the end of the construct.

DNA-containing plasmids driving the expression of rRhavi fusion proteins were introduced by transformation into E. coli T7 shuffle express according to the manufacturer's instructions. Cultivation was started with a single colony, which was inoculated into 30 ml of Luria-Bertani (LB) medium containing ampicillin (Amp+) and cultured overnight at 30°C. On day 2, 5 ml of the original culture was inoculated into 1 liter of LB/Amp+ medium and grown at 30° C. until an OD ₆₀₀ of ~1.2-1.6 was reached. After the culture was cooled to 16°C, IPTG was added to a final concentration of 0.1 mM. The induced culture was incubated at 16°C with shaking for 16-20 hours. Bacteria were collected by centrifugation at 5000 xg for 20 min and the pellet was frozen at -20°C.

Exemplary fusion proteins

Exemplary rizavidin fusion proteins of the present invention are shown in the following sequence listing. For convenience, the amino acid sequences of the fusion proteins in the one-letter code are given below:

Rhavi-PcrV-His, SEQ ID NO: 16

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH

Rhavi-MrkA-donor strand complementation-His, SEQ ID NO: 17

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSGSGHHHHHH

Rhavi-FlaA1-His, SEQ ID NO: 18

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFKADGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFSDGDTISYVSKAGKDGSGAITSAVSGVVIADTGSTGVGTAAGVTPSATAFAKTNDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLRGSGHHHHHH

Rhavi-FlaA2-His, SEQ ID NO: 19

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLRGSGHHHHHH

Rhavi-FlaB-His, SEQ ID NO: 20

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLRGSGHHHHHH

Rhavi-FlaB-Domain2-His, SEQ ID NO: 21

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSGSGHHHHHH

Rhavi-FlaA2-Domain2-His, SEQ ID NO: 22

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMNGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTGSGHHHHHH

Rhavi-FlaB-PcrV-His, SEQ ID NO: 23

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLRAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH

Rhavi-FlaB-Domain2-MrkA-donor strand complementation-His, SEQ ID NO: 24

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSAAAAMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSGSGHHHHHH

Rhavi-MrkA-donor strand complementation-PcrV-His, SEQ ID NO: 25

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH

Rhavi-FlaB-Domain2-PcrV-His, SEQ ID NO: 26

MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH

Purification of fusion proteins

Purification was started by resuspension of bacterial pellets in 4 ml chilled 20 mM Tris, pH 8.0, containing 500 mM NaCl, 20 mM imidazole, 10 mM MgCl ₂ and 2X Halt protease inhibitor cocktail (Thermo Fisher), per gram of cell pellet. Bacteria were destroyed by sonication, followed by the addition of DNase I to a final concentration of 25 μg/ml. Insoluble detritus was removed by centrifugation at 5000 xg for 20 min. The clarified lysate was diluted with an equivalent volume of 20 mM Tris, pH 8.0 containing 500 mM NaCl to bring the final buffer concentration to 20 mM Tris, 500 mM NaCl, 10 mM imidazole, 5 mM MgCl ₂ and 1x protease inhibitor cocktail. Proteins were purified by binding to a nickel affinity resin, washed with 20 mM Tris containing 500 mM NaCl, 20 mM imidazole, and the bound protein was eluted with 20 mM Tris containing 500 mM NaCl, 500 mM imidazole. Fractions corresponding to the elution peaks of the expected target protein dimer were pooled and concentrated. Proteins eluting from these columns were further purified by gel filtration and/or SEC. These proteins were then analyzed by SDS-PAGE, Western blotting and SEC. All fusion proteins migrated to SDS-PAGE according to the expected MW. Protein concentration in each sample was measured using a BCA protein assay kit (BioRad). Purified proteins were aliquoted, flash frozen in liquid nitrogen and stored at -80°C until future use.

A summary of the physical characteristics of exemplary rizavidin fusion proteins and variants of the invention is provided in Table 5. All rizavidin fusion proteins of the invention were expressed and shown to be dimeric proteins based on size exclusion chromatography (SEC) results (Table 5).

Table 5: Physical characteristics of the expressed in E. coli fusion proteins of rizavidin according to the invention

Protein bacteria MM monomer (kDa) The presence of a dimer, EC Presence of a TLR5 binding domain Rhavi-PcrV-his P. aeruginosa 48 Yes N/A Rhavi-FlaB-his P. aeruginosa 65 Yes Yes Rhavi-FlaA1-his P. aeruginosa 56 Yes Yes Rhavi-FlaA2-his P. aeruginosa 55 Yes Yes Rhavi-MrkA-his K. pneumoniae 37 Yes N/A Rhavi-FlaBD2-his P. aeruginosa 37 Yes No Rhavi-FlaA2D2-his P. aeruginosa 27 Yes No Rhavi-FlaBD2-MrkA-his P. aeruginosa
K. pneumoniae 58 Yes No Rhavi-FlaB-PcrV-his P. aeruginosa 97 Yes Yes Rhavi-PcrV-MrkA-his P. aeruginosa
K. pneumoniae 69 Yes N/A Rhavi-FlaBD2-PcrV-his P. aeruginosa 70 Yes No

Assessing activity against TLR5 flagellin fusion proteins

To evaluate the correct folding of the fusion proteins, HEK293-NFkB:Luc cells were stimulated by incubation for 4 hours with each of the tested proteins, or complexes, titrated as shown in Figure 12B. HEK293-NFkB: Luc cells, as previously described, respond to flagellin but not to LPS or flagellaless Salmonella (Simon and Samuel, 2007) . After cell lysis, luciferase activity was determined using firefly luciferase from Promega. The assay measures NF-kB activation (Simon and Samuel, 2007).

The results of the determination of biological activity against TLR5 in the assay using HEK293-NFkB: Luc cells, as well as native Pseudomonas flagellins and Rhavi fusion proteins, alone or complexed with MAPS with PnPS 6B, are shown in Figure 12B. These results clearly show that the rhizavidin and flagellin constructs FlaB, FlaA1, FlaA2 stimulated TLR5 activity on their own or as a MAPS complex with PnPS 6B and therefore were correctly folded.

Immunogenicity of exemplary fusion proteins

Serums with high titers to variants of the fusion proteins were obtained to assess the in vitro activity of protein-specific antibodies. ELISA-determined IgG titers to the respective immunogen for each of the following fusion proteins (FPs): Rhavi-FlaB-D2; Rhavi-FlaA2-D2; Rhavi-FlaB-PcrV; Rhavi-FlaB-D2-MrkA and Rhavi-PcrV-MrkA are shown in Figure 14. All SBs are highly immunogenic and produce high titers of SB-specific IgG antibodies.

The results of the determination of cross-reactivity for FlaB and FlaA1 of rabbit serum obtained against the FlaB-containing rizavidin fusion proteins are shown in Table 6.

Table 6: Cross-reactivity of sera from rabbits immunized with FlaB-containing rhizavidin fusion proteins for native FlaB purified from PA PAO1 or FlaA1 purified from PA PAK

Rabbit No. FlaA1 ELISA FlaB ELISA P0 P2 P3 P0 P2 P3 Rhavi-FlaB-D2-His AFV791 121 356 744 22 344903 279209 AFV792 397 442 627 309 126159 154104 AFV793 162 379 763 155 434194 423514 Rhavi-FlaB-PcrV-His AFV797 263 23995 31.497 278 317789 318117 AFV798 126 38872 40.911 128 250535 230702 AFV799 178 27108 41.566 139 299454 499941 Rhavi-FlaB-D2-MrkA-His AFV800 213 217 1916 45 17602 35651 AFV801 54 165 944 48 21320 53477 AFV802 440 161 291 298 24500 34790

Both anti-Rhavi-FlaB-D2 and anti-Rhavi-FlaB-PcrV rabbit sera had high titers of FlaB-specific antibody, but anti-Rhavi-FlaB-D2-MrkA sera had relatively low titers of FlaB-specific antibody. The anti-Rhavi-FlaB-PcrV sera had significant cross-reactivity with the FlaA1-specific antibody, indicating that the FlaB component of the fusion protein is correctly folded and that the cross-reactive FlaA1 epitopes are presumably outside flagellin domain 2, in common conserved D0 domains. and D1 (Song and Yoon, 2014), which are not expressed in either Rhavi-FlaB-D2 or FlaB-D2-MrkA. Thus, due to its significant cross-reactivity with FlaA1, Rhavi-FlaB-PcrV is an excellent candidate fusion protein that can confer broad flagellin-based immunity against Pseudomonas infection.

Example 5 Preparation of Immunogenic Complexes

Obtaining framework polymers from native OPS

Native OPS

O-polysaccharides (OPS) of K. pneumoniae (KP OPS) of serotypes O1, O2, O3 and O5 and core OPS of P. aeruginosa (PA OPS) of serotypes O1, O2, O3, O4, O5, O6, O10, O11 were isolated from the fermentation biomass of microorganisms grown in CDM after treatment either by boiling at 100° C. in 1% acetic acid in the case of PA OPS, or either in acetic acid and sodium nitrite (NaNO ₂ ) in the case of KP OPS. In the case of PA LPS, treatment with acetic acid resulted in cleavage of the KDO bond between the LPS inner core and lipid A (Figure 1A) and a KDO moiety at the reducing end of the COPS. In the case of LPS KP, deamination with nitrous acid results in cleavage of the bond between the inner core glucosamine residue and the rest of the inner core lipid A of the LPS to produce a reducing 2,5-anhydromannose residue with an aldehyde at carbon 1 (Figure 1C). The structures of PA OPS (Knirel et al., 2006) and KP OPS (Vinogradov et al., 2002) are presented in Figures 1B and 1D, respectively. The post purification process of PA and KP OPS is shown in Figure 8A. These purification methods result in high amounts (>50-100 mg/l purified material) of highly pure OPS with low levels of residual protein, nucleic acids and endotoxin. The chemical and immunochemical nature of OPS was confirmed by TFA depolymerization and Dionex HPAEC-PAD, comparison of high-resolution 1H-NMR spectra (chemical bonds and interaction constants) with data published in the literature, and ELISA determination of immunological reactivity with monoclonal antibodies specific for KP OPS and PA OPS types or immune sera obtained by immunization with heat-killed whole bacteria (Figure 6C). The average MM OPS as measured by SEC and SEC-MALLS ranged from approximately 10 kDa to 30 kDa, depending on the type, with PA OPS generally larger than KP OPS. The combined results of the HPAEC-PAD (Dionex) analysis of the monosaccharide composition of purified KP OPS and PA OPS are shown in Table 7.

Table 7. HPAEC-PAD Analysis of the Monosaccharide Composition of Purified KP OPS and PA COPS

bacteria O-polysaccharide HPAEC-PAD analysis of monosaccharides ^a Expected OPS replay lineup ^b K. pneumoniae O1 Galactose Galactose K. pneumoniae O2a Galactose Galactose K. pneumoniae O3 Mannose Mannose K. pneumoniae O5 Mannose Mannose P. aeruginosa IATS O1 Fucosamine, Quinovosamine, Galactosamine NAc-fucosamine, NAc-galactosamine, NAc-quinovosamine, 2,3-diamino-2,3-dideoxy-NAc-glucuronic acid P. aeruginosa IATS O2 Fucosamine 2,3-diamino-2,3-dideoxy-NAc-mannuronic acid, 2,3-diamino-2,3-dideoxy-NAc-glucuronic acid, NAc-fucosamine P. aeruginosa IATS O3 Rhamnose, Glucosamine Rhamnose, NAc-glucosamine, NAc-galacturonic acid, P. aeruginosa IATS O4 Fucosamine, Quinovosamine, Rhamnose NAc-Fucosamine, Rhamnose, NAc-Quinosamine P. aeruginosa IATS O5 Fucosamine NAc-NAc-mannuronic acid, NAc-NAm-mannuronic acid, NAc-fucosamine P. aeruginosa IATS O6 Quinovosamine, Rhamnose ^c Rhamnose, NAc-Ac-galacturonic acid, Nfo-galacturonic acid, NAc-quinosamine P. aeruginosa IATS O10 Quinovosamine, Rhamnose Ac-rhamnose, NAc-galacturonic acid, NAc-quinosamine P. aeruginosa IATS O11 Fucosamine, Glucose NAc-fucosamine, Glucose ^a The instrument detects only neutral saccharides, not uronic acid; polysaccharides depolymerized in 2M TFA/100°C/4 hours unless otherwise noted
^b Knirel et al. 2006
^c determined after 4M TFA/100°C/18 hours

Frame resin OPS

Figure 1E shows the chemical structure of the capsular polysaccharide repeating unit (CPS)K. oxytoca(KO) K19 which is identical to CPSK. pneumoniae K19. Figure 1F shows the H-NMR spectrum at 950 MHz of purified KP K19 CPS. Natural KP OPS and PA OPS with an average molecular weight of 10-30 kDa were covalently linked to the larger K19 CPS (average MM of 218 kDa) to increase both their effective MM and their epitope valency. The OPS framework polymers were biotinylated after, or during, the enlargement step, either selectively on the K19 CPS framework polymer to give BP-1, BP-1.2, or BP-1.3 (Figures 2A-2C), or randomly on both OPS and and on CPS, to obtain BP-2a (Figure 3), or selectively on OPS, to obtain BP-2b (Figure 4).

The framework polymers elute significantly earlier on the size exclusion chromatography column than the individual K19 CPS or OPS, indicating that they are significantly enlarged compared to the individual components and show a decrease in free OPS compared to the amount in the starting material, which indicates its inclusion in the framework polymer.

This upscaling process using CPS, e.g. K19 CPS, as the backbone polymer is a versatile process that can be applied to any CPS/OPS or low molecular weight bacterial polysaccharides and can result in a well-defined and reproducible product while creating minimal, or does not create changes in OPS/CPS epitopes. As shown in Figure 6C, this was clearly demonstrated after antigenicity assays by ELISA with native PA OPS O1, O2, O3, O6, O10 and O11 and the corresponding constructs PA OPS: K19 BP-1, using hyperimmune rabbit sera after immunization with PA CN (Figure 6C). In these experiments, no difference in antigenicity was observed between native OPS PA and the corresponding OPS backbone polymer, indicating that OPS epitopes were conserved during enlargement.

Biotinylation of backbone polymers (BP)

BP-1

First, a short spacer containing a primary amine at each end (eg ADH) was added at the reducing end of the OPS by reductive amination with NaBH ₃ CN. Reactive aldehydes on KP OPS were placed at the 2,5-anhydromannose (2,5-anMan) terminal residues during the extraction procedure by treating whole LPS with sodium nitrite (Figure 8A), while reactive ketones on PA OPS were placed on reducing terminal residues KDO of the PA OPS inner core by treating the LPS with acetic acid as shown in Figure 8A. ADH-derivatized OPS were subsequently mixed with a partially oxidized periodate backbone KP 19 CPS (illustrative SEC-HPLC chromatograms of these derivatives are shown in Figures 6A and 6B) and biotin containing a primary amine (such as amine-PEG3-biotin) and the mixture was reductively aminated , resulting in an OPS backbone polymer biotinylated only on the polysaccharide backbone, which the inventors called backbone polymer 1 (BP-1) (Figure 2A).

BP-1.2

Alternatively, the reactive carboxylate groups of the KP 19 CPS backbone can first be biotinylated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and biotin containing a primary amine (such as amine-PEG- biotin), then oxidized with periodate, blended with ADH-derivatized OPS, and reductively aminated the mixture to produce an OPS backbone polymer biotinylated on the polysaccharide backbone only, which the inventors named BP-1.2 (Figure 2B). Alternatively, the ADH step can be omitted in the case of PA OPS since they contain an alanine amino acid substituent with a free α-amino group on carbon 2 of their outer core galactosamine residues (Figure 1A). Thus, these available amino groups can be easily used for direct coupling by reductive amination to periodate-oxidized CPS polymer K19 together with amine-containing biotin to produce biotinylated BP-1 (Figure 2A) or BP-1.2 (Figure 2B).

Alternatively, OPS containing an aldehyde or ketone at their reducing end are derivatized with an azido-amine compound in the presence of a reducing agent to give OPS with an azido group at their end. The polysaccharide backbone is then provided with alkyne groups via polysaccharide carboxylate groups and EDC. The backbone polysaccharide is then biotinylated using CDAP and a biotin hydrazide compound to produce a biotinylated and alkynylated polysaccharide. The derivatized azido-OPS and biotinylated alkyne-CPS are then coupled by copper catalyzed click chemistry to give (OPS) _n -CPS BP-1 in which the backbone polymer is biotinylated and OPS is not biotinylated (Figure 5B; Scheme 1) . Using this coupling in a click chemistry reaction, OPS BP-1 can also be prepared by first introducing alkyne groups into CPS using carboxylate groups and EDC, and then aminating using amino PEG azide and copper sulfate catalyst, biotinylating CPS using CDAP and biotin hydrazide; biotinylated CPS containing primary amino groups is mixed with native OPS (containing aldehyde or ketone groups at their reducing ends) and reductively aminated with a reducing reagent to give OPS/CPS BP-1 (Figure 5B; Scheme 2). BP-2b can be generated by such a click chemistry reaction by first selectively introducing biotin residues into OPS derivatized with azido groups at their terminus, and then biotinylating using CDAP chemistry and biotin hydrazide; and a second click chemistry coupling of the biotinylated azido-derivatized OPS to the alkynylated CPS as shown in Figure 5B; scheme 3.

BP-2a

Alternatively, ADH-derivatized OPS or non-derivatized OPS is mixed with a periodate-partially oxidized CPS KP strain 19 backbone and reductively aminated to form an OPS backbone polymer; the framework polymer is then derivatized using CDAP and biotin-PEG-amine to produce a framework polymer randomly biotinylated with biotin on both the framework polysaccharide and OPS, which the inventors named BP-2a (BP-2a) (Figure 3) .

BP-2b

Alternatively, OPS is first biotinylated using CDAP and amine-PEG-biotin; the resulting biotinylated OPS is partially oxidized with periodate to introduce aldehydes into its KDO fragments or inner core heptoses and reductive amination is carried out using ADH; the biotinylated and aminated OPS is then mixed with a partially oxidized periodate backbone KP 19 CPS and reductively aminated to produce an OPS backbone polymer selectively biotinylated on its OPS; the inventors named it BP-2b (Figure 4 and Figure 5B, scheme 3).

BP-3

Alternatively, reductive amination of the ADH-derivatized OPS with ε-NH ₂ with PLL backbone groups is first carried out to form an OPS-PLL backbone polymer. The resulting OPS-PLL is treated with an acylating agent such as acetic anhydride to block the unreacted amino groups (ε-NH ₂ ) of the PLL backbone to form an OPS-poly-N-acetyl-lysine (OPS-PNAcLL) backbone polymer. This OPS-PNAcLL backbone polymer is then biotinylated on its OPS by first being treated with CDAP and then with amine-PEG-biotin. This backbone polymer, selectively biotinylated on its OPS, is referred to as BP-3 (Figure 5A).

Unreacted OPS, biotin, CPS and residual conjugation reagents were removed by FLC-SEC with Superdex 200 or 50-300 kD TFF UV-DF.

Preparation and Purification of Exemplary Immunogenic Complexes

MAPS or variant MAPS complexes were generated by adding a candidate rRhavi-antigen fusion protein to a biotinylated polysaccharide in 20 mM Tris, pH 8.0, 150 mM NaCl, in a selected ratio (typically 3:1, protein:PS, by weight). ), with 0.01% thimerosal added to inhibit microbial growth. The sample was incubated with inversion at 25°C overnight, followed by centrifugation at 10,000 xg for 5 min to remove any insoluble material (Zhang et al., 2013). Soluble material was collected and MAPS or variant MAPS complexes were purified by size exclusion chromatography on a Superdex 75 column (Native OPS PS MAPS) or a Superdex 200 column (CPS MAPS and backbone polymer-PS variants of MAPS) with 2 mM Tris, pH 8.0, 150 mM NaCl (Zhang et al., 2013). Peak-corresponding fractions were analyzed for protein content by SDS-PAGE of the reconstituted samples without heating and visualized by staining for total protein. Fractions containing MAPS or variant MAPS complexes were identified in the gel as stained proteins retained in the gel in high molecular weight complexes, rather than as proteins expected to migrate in the gel according to the size of the protein dimer (Zhang et al., 2013). Fractions containing MAPS complexes were pooled and the protein/polysaccharide ratio in MAPS or MAPS variant was determined using the BCA protein assay kit and in the anthrone polysaccharide assay (Zhang et al., 2013). The integrity of MAPS or variant MAPS complexes was assessed by SDS-PAGE of reconstituted samples without heating, visualized by total protein staining (Zhang et al., 2013). The content of free protein in MAPS or variant MAPS complexes was quantified by SDS-PAGE densitometry of the content of protein dimers in the SDS-PAGE of MAPS complexes or variant MAPS complexes without heating in comparison with the standard amount of control protein dimer (Zhang et al., 2013). The molecular weight of protein incorporated into MAPS or variant MAPS complexes was analyzed by SDS-PAGE of reconstituted, boiled samples, visualized by staining for total protein and using standard molecular weight marker proteins.

KP/PA OPS were prepared as different forms of OPS backbone polymers (1, 2a, 2b) using K19 CPS as the backbone polymer, and variant MAPS complexes were made with the following fusion products of E. coli recombinantly produced rizavidin with proteins from the KP genome and PA: Rhavi-PcrV; Rhavi-MrkA; Rhavi-FlaA1; Rhavi-FlaA2; Rhavi FlaB; Rhavi-FlaB-Domain2; Rhavi-FlaA2-Domain2; Rhavi-FlaB-PcrV; Rhavi-FlaB-Domain2-MrkA; Rhavi-MrkA-PcrV; Rhavi-FlaB-Domen2-PcrV having SEQ ID NOs: 16-26 and using PRO-CN as a control protein.

Exemplary characteristics of some MAPS lots prepared with the rhizavidin fusion protein of the invention and complexed with biotinylated PS types 6B, 7F and 19A pneumococcal polysaccharides are shown in Table 8. Exemplary purification results for variant MAPS complexes are shown in Figures 10 and 11. Variant MAPS complexes was purified from unreacted protein by SEC and gel-eluted fractions were analyzed by SDS-PAGE, a typical analysis is shown in Figure 10 where free protein is shown gel-eluting in late fractions and can be separated from the variant MAPS complex as originally described for of MAPS complexes in Zhang et al., 2013. Figures 11A-11C show the results of SDS-PAGE analysis of purified K19-PA O1 OPS BP-1 MAPS complexes, K19-PA O2 OPS BP-1 MAPS complexes, and K19-PA complexes O10 OPS BP-1 MAPS with PRO-CN (Figure 11A); Rhavi-FlaB-PcrV (Figure 11B); Rhavi-FlaB-D2-MrkA (Figure 11C). Each of the purified samples of the variant MAPS was separately treated with and without heating. The MAPS variant complexes were not destroyed without heating, and the protein in the MAPS variant complexes remained at the very top of the gel (Zhang et al., 2013). Upon boiling, the MAPS variant complexes were destroyed, the protein was released, and the protein dimer formed due to the intermolecular interaction of rhizavidin also dissociated (Zhang et al., 2013). The protein content of the variant MAPS was then determined in the BCA assay, and the polysaccharide content was determined in the anthrone reagent assay. The protein to polysaccharide ratio was calculated as a ratio by weight and as a molar ratio.

All final purified MAPS and variant MAPS were amenable to filter sterilization through a 0.22 µm filter (Table 8 and data not shown).

Table 8: Characteristics of MAPS Purified Rhizavidin Fusion Proteins

PS MM PS (kDa) Protein Protein MM (kDa) PS, mg/ml (anthrone) Protein, mg/ml (BCA) Attitude
protein:PS (by weight) Molar.protein ratio:PS IUD complexes in SDS-PAGE (no boiling) Appropriate sized protein in SDS-PAGE (boil) % free protein (densitometry, SDS / PAAG, boiled vs. not boiled. Filtration through 0.22-µm PES 6B 1092 PRO-CN 82 0.16 0.37 2.2:1 33:1 Yes Yes 4 Yes 6B 1092 Rhavi-PcrV-his 48 0.13 0.21 1.7:1 38:1 Yes Yes 7 Yes 6B 1092 Rhavi-FlaB-his 65 0.14 0.37 2.6:1 44:1 Yes Yes <1 Yes 6B 1092 Rhavi-FlaA1-his 56 0.13 0.21 1.6:1 31:1 Yes Yes 5 Yes 6B 1092 Rhavi-FlaA2-his 56 0.12 0.12 1.0:1 20:1 Yes Yes <1 Yes 6B 1092 Rhavi-MrkA-his 37 0.14 0.20 1.4:1 42:1 Yes Yes <1 Yes 7F 901 PRO-CN 82 0.18 0.68 3.8:1 41:1 Yes Yes <1 Yes 7F 901 Rhavi-PcrV-his 48 0.14 0.19 1.4:1 25:1 Yes Yes <1 Yes 7F 901 Rhavi-FlaB-his 65 0.13 0.29 2.2:1 31:1 Yes Yes 3 Yes 7F 901 Rhavi-FlaA1-his 56 0.12 0.14 1.2:1 19:1 Yes Yes <1 Yes 7F 901 Rhavi-FlaA2-his 56 0.13 0.11 0.8:1 14:1 Yes Yes <1 Yes 7F 901 Rhavi-MrkA-his 37 0.16 0.17 1.1:1 26:1 Yes Yes <1 Yes 19A 157 PRO-CN 82 0.23 0.89 3.9:1 7:1 Yes Yes <1 Yes 19A 157 Rhavi-PcrV-his 48 0.09 0.19 2.1:1 7:1 Yes Yes 4 Yes 19A 157 Rhavi-FlaB-his 65 0.14 0.39 2.8:1 7:1 Yes Yes <1 Yes 19A 157 Rhavi-FlaA1-his 56 0.10 0.26 2.6:1 7:1 Yes Yes 6 Yes 19A 157 Rhavi-FlaA2-his 56 0.11 0.15 1.4:1 4:1 Yes Yes <1 Yes 19A 157 Rhavi-MrkA-his 37 0.08 0.19 2.4:1 10:1 Yes Yes <1 Yes

Example 6 Immunization Studies

Animal Immunization

Prior to immunization of rabbits, MAPS or variant MAPS or PS alone was formulated with an adjuvant approximately 48 hours prior to injection. MAPS or variant MAPS were adsorbed to adjuvant at a final concentration of 10 μg/ml PS of each serotype, either alone or as a multivalent mixture, with 40 mM histidine, pH 5.5, 150 mM NaCl and 0.25 mg/ml aluminum phosphate gel , with stirring by turning over night at 4°C. This formulated mixture was used directly for immunizations in a volume of 0.5 ml for a dose of 5 μg PS. For lower doses of PS, an appropriate dilution was performed with 40 mM histidine, pH 5.5, 150 mM NaCl. Two IM immunizations (0.5 ml) were performed 2 or 4 weeks apart in 4 month old New Zealand White rabbits (Cocalico Biologicals, n=8-10 per group). Blood samples were collected 2 weeks after the first and second immunizations with a 2 week dosing interval. Samples were collected 2 and 4 weeks after the first immunization and 2 weeks after the second immunization with a 4 week dosing interval.

To obtain a higher antibody titer against candidate fusion protein carriers, complete Freund's adjuvant was used for the first immunization and incomplete Freund's adjuvant for the second and third immunizations, respectively. Each carrier protein (0.1 mg) was separately mixed with Freund's adjuvant to a final volume of 1 ml, 0.2 ml was injected subcutaneously at 4 different sites, and 0.2 ml was injected intramuscularly into each rabbit at each immunization. Immunizations were given to 4-month-old New Zealand White rabbits (Cocalico Biologicals), (n=3 per group) on day 0, day 14 and day 21, and blood samples were collected 2 weeks after the second and third immunizations.

Measurement of antibody levels

Assays for detection of antibodies to capsular polysaccharides (CPS) or OPS, or various protein antigens, were performed in medium binding Immulon 2 HB or Greneir Bio-one (Thermo Scientific Waltham Mass) 96-well plates coated with (1 μg CPS or OPS, or PS conjugated to HSA/ml PBS) or protein antigens (1 µg protein/ml PBS). The plates were blocked with 1% BSA in PBS. Antibodies diluted in PBS-T were added and incubated at room temperature for 2 hours. The plates were washed with PBS-T, a secondary HRP-conjugated anti-rabbit immunoglobulin G antibody (from Sigma) was added and incubated at room temperature for 1 hour. The plates were washed and treated to develop color with SureBlue TMB Microwell peroxidase substrate (KPL, Gaithersburg, MD).

The titer of rabbit IgG in serum samples specific for different protein antigens was determined by ELISA and a standard curve was built based on the results for polyclonal serum using arbitrary units. Protein antigens were coated overnight at room temperature in Immulon 2 HB 96-well plates (Thermo Scientific) using 0.5-1 μg protein/ml PBS and 100 μl per well. The protein solution was removed, the plates were blocked with PBS+1.0% BSA for 1 h at room temperature and washed with Dulbecco's phosphate-buffered saline with 0.05% Tween-20 (PBS-T). Rabbit serum was diluted in PBS-T, added to the wells of the plate and incubated at room temperature for 2 hours. The plates were washed with PBS-T, donkey anti-rabbit IgG secondary antibodies conjugated with HRP (Santa Cruz Biotechnology), diluted 1 to 20000 in PBS-T, 100 μl per well and incubated at room temperature for 1 hour. The plates were washed and treated for color development with SureBlue TMB Microwell peroxidase substrate (KPL, Gaithersburg, MD). The reaction was stopped with an equal volume of 1 N HCl and absorbance was determined at 450 nm on a Spectramax plate reader (Molecular Devices). A VU value of 12500 VU/ml was assigned to the standard polyclonal serum used in each plate, and a 4-parameter curve equation was used to determine the VU value for each diluted serum sample, based on absorbance at 450 nm and corrected for the dilution factor. Acceptance criteria for the polyclonal serum standard curve (AFV160 specific for PRO-CN, rizavidin and 6-histidine tag) were an ^R2 value for the 4-parameter curve of 0.99 or more and less than 10% CV between duplicates. An internal control serum (AFV151 specific for PRO-CN, rizavidin and 6-histidine tag) was also used in each plate and the CV for the assigned value had to be equal to or less than 20% between plates for data acceptability.

Carrier function evaluation

The ability of rhizavidin carrier fusion proteins to elicit an increased immune response to the polysaccharides in the MAPS complexes was evaluated and compared to rhizavidin alone and a control carrier protein (PRO-CN) that had previously elicited strong immune responses to the complexed polysaccharides. Various resulting fusion proteins were complexed with polysaccharides of known immunogenicity ( S. pneumoniae 6B, 7F and 19A) (Zhang et al., 2013) and compared with a control strong functional carrier protein (PRO-CN). All fusion proteins were capable of complexing with biotinylated PS and forming high molecular weight complexes that could be purified and subsequently used to immunize rabbits and evaluate vehicle function.

Rabbits were immunized with MAPS complexes formed by pneumococcal polysaccharides (PnPS) 6B and 19A with various rhizavidin fusion proteins. Sera were collected 2 weeks after the first immunization (P1) and the second immunization (P2). The levels of rabbit IgG to PnPS 19A and 6B in each sample were determined by ELISA and expressed in VU, which was determined from a standard curve constructed for polyclonal serum, which was assigned a VU value of 12500 U/ml. Figures 13A and 13B show that Pseudomonas flagellin B (FlaB) as a fusion protein with rizavidin (Rhavi-FlaB) and Pseudomonas FlaB fused with Pseudomonas PcrV as a fusion protein with rizavidin (Rhavi-FlaB-PcrV) were able to induce production of IgG to pneumococcal polysaccharides (PnPS) 6B and 19A with titers similar to those of the previously identified carrier protein, PRO-CN. Pseudomonas FlaA1 and FlaA2 fusion proteins with rizavidin (Rhavi-FlaA1 and Rhavi-FlaA2) and Pseudomonas FlaB-Domain2 fused with MrkA Klebsiella as a fusion protein with rizavidin (Rhavi-FlaBD2-MrkA) showed titers that were similar to those or within 2-fold excess, titers in the case of Rhavi-FlaB and PRO-CN. MrkA Klebsiella and Pseudomonas PcrV showed very low to undetectable IgG titers for 19A PS administered and low titers for 6B and 7F at the 0.044 µg dose. To overcome the weak functioning of MrkA as a carrier and, at the same time, to generate antibodies directed to MrkA, it was fused with a strong carrier protein FlaB-Domain2 in order to preserve the function of the carrier (Rhavi-FlaBD2-MrkA).

Table 9 summarizes the characteristics of carriers of exemplary rizavidin fusion proteins of the invention. The carrier function of each fusion protein was expressed as a percentage of the function of the PRO-CN carrier protein and compared to the single protein rizavidin, which resulted in only a 4% immune response to 6B and a 5% immune response to 19A compared to PRO-CN. Rhavi-FlaB-PcrV, a protein with dual pathogenic specificity (a chimeric protein from two Pseudomonas antigens, FlaB and PcrV, fused to rizavidin), was shown to be a strong vehicle in inducing a strong PnPS-specific immune response in rabbits to type 6B (62% ) and 19A (102%), compared to PRO-CN (100%). Rhavi-FlaB also showed strong carrier function, with strong immune responses to both 6B (66%) and 19A (49%), compared to PRO-CN. Rhavi-MrkA had a weaker function as a carrier protein for 6B (27%) and 19A (2%) compared to PRO-CN, respectively. Rhavi-FlaBD2 had a strong function as a carrier for 6B (66%), and a weaker function as a carrier for 19A (20%). Chimeric Rhavi-FlaBD2-MrkA, containing both Pseudomonas (FlaBD2) and Klebsiella (MrkA) antigens, provided a carrier function for both 6B (34%) and 19A (45%), which was approximately 2-fold weaker than the function of the PRO-CN carrier protein and exceeded that of a single rizavidin. Since an immune response was induced to both PnPS that exceeded 30% of the response in the case of PRO-CN, and since a large number of pathogen-specific proteins, both Rhavi-FlaB-PcrV and Rhavi-FlaBD2-MrkA, were presented, the variants were selected in as potential carrier proteins for complexation in variant MAPS with the OPS backbone polymer of the invention. In addition to the demonstrated carrier function, anti-carrier protein IgGs specific for the highly conserved Pseudomonas PcrV and, to some extent, FlaB (i.e., approximately 45 percent of Pseudomonas strains express FlaB), as well as for the conserved MrkA Klebsiella , are raised against these SBs. , may provide significant additional broad protective coverage against disease caused by these 2 pathogens.

Table 9: Carrier protein characteristics of exemplary rizavidin fusion proteins and variants

Protein bacteria Presence of a TLR5 binding domain Carrier function ^* % of PRO-CN geometric mean for 6B % of PRO-CN geometric mean for 19A PRO-CN S. pneumoniae N/A ++++ 100 100 Rhavi-PcrV-his P. aeruginosa N/A ++ 38 7 Rhavi-FlaB-his P. aeruginosa Yes ++++ 66 49 Rhavi-FlaA1-his P. aeruginosa Yes +++ 40 9 Rhavi-FlaA2-his P. aeruginosa Yes +++ 42 16 Rhavi-MrkA-his K. pneumoniae N/A + 27 2 Rhavi his S. pneumoniae N/A + 4 5 Rhavi-FlaBD2-his P. aeruginosa No +++ 66 20 Rhavi-FlaA2D2-his P. aeruginosa No + eleven 5 Rhavi-FlaBD2-MrkA-his P. aeruginosa
K. pneumoniae No +++ 34 45 Rhavi-FlaB-PcrV-his P. aeruginosa Yes ++++ 62 102 Rhavi-PcrV-MrkA-his P. aeruginosa
K. pneumoniae N/A ++ thirty 23 *The ability of the fusion protein to induce the production of antibodies to pneumococcal PS (PnPS) type 19A and 6B, compared to the reference PRO-CN fusion protein (100 percent (++++)).

Assessment of immunogenicity

The results of a comparative study of immunogenicity in rabbits of different preparations of native PA O6 and O10 OPS in the form of MAPS complexes with the rizavidin PRO-CN fusion protein, or rizavidin, are presented in Figure 15. OPS-specific IgG titers in the ELISA assay are presented in ELISA units. OPS-specific IgG antibody titers greater than 100 times P0 titers were not achieved with any of these drugs after 2 immunizations (P2). This suggests that the molecular size of the native PA OPS (10-30 kDa) was too small, the epitope valency was too low, or the molecular structure was not suitable to elicit a response even when MAPS was complexed with the carrier protein, PRO-CN.

An immunogenicity study comparing different variants of the KP-O1 OPS backbone polymer as variant MAPS complexes was performed in rabbits. BP-1, BP-2a and BP-2b variants of KP O1 OPS associated with Klebsiella K19 capsular polysaccharide (CPS) were evaluated for immunogenicity. The BP-1 variant of KP O5 associated with K19 was also evaluated. The immunogenicity of four mixtures of variant MAPS complexes was compared; 2-valent KP-O1 and -O5 OPS BP-1 (schematic representation of which is shown in Figure 2A); KP-O1 BP-2a (Figure 3) and KP-O1 BP-2b (Figure 4). All of these polysaccharides were incorporated into MAPS variant complexes with the PRO-CN carrier protein and formulated with alum prior to injection. Anti-KP-O1 OPS IgG titers are shown in ELISA units for pre-immune sera (P0), sera after first (P1) or 2 immunizations (P2) in Figure 16. BP-2a OPS (12,000 EIA units) resulted in O1 OPS- specific IgG, more than 100 times higher than P0 titers. O1 OPS BP-1 and O5 OPS BP-1 resulted in titers 10 times greater than those of P0.

A similar immunogenicity study in rabbits compared different preparations of PA-O6 and -O10 OPS BP-1: 2-valent PA-O6, -O10 OPS BP-1 PRO-CN MAPS with alum; 2-valent PA-O6,-O10 OPS BP-1 PRO-CN MAPS without adjuvant (no alum); 2-valent PA-O6,-O10 OPS BP-1 polysaccharide only (alum, no protein); 2-valent PA-O6,-O10 OPS BP-1 mixed with PRO-CN without complex formation (not biotinylated; alum; without variant MAPS formation); and monovalent PA-IATS O6 OPS BP-1 PRO-CN MAPS with alum (additional source of PA O6 strain OPS). Anti-PA-O6 and PA-O10 OPS IgG titers are shown in ELISA units for pre-immune sera (P0), sera after one (P1) or 2 immunizations (P2) in Figure 17. Titers of PA-O6 and PA-O10 OPS-specific IgG greater than 100-fold P0 titers could only be achieved (5000-11000 range in ELISA units) when the OPS backbone polymer was present in the variant MAPS complex with alum.

The size of KP OPS obtained using standardized laboratory growth conditions in a medium with a certain chemical composition is in the range of 10-15 kDa, relatively smaller than the size of PA OPS (20-30 kDa) obtained under similar conditions, these size differences can , in part, to explain why, in these comparative immunogenicity studies, PA OPS BP-1 were more immunogenic than KP OPS BP-1. The KP OPS BP-2b constructs, on the other hand, were largely immunogenic (Figure 16). Other structural differences in OPS repeating units could also have an effect, such as charge; the repeat units of both PA-O6 and -O10 are negatively charged, while KP-O1 OPS is neutral.

Example 7 Functional Antibody Assays

Opsonophagocytic killing assay

Opsonophagocytic killing (OPK) assays with HL-60: Assays were performed as previously described with modifications (Ramachandran et al., 2016). Briefly, assays were performed in 96-well round bottom plates using P. aeruginosa and K. pneumoniae strains from log phase cultures diluted to 3×10 ⁵ cells/ml; rabbit complement (BRC); PMA-differentiated HL-60 cells (2×10 ⁷ cells/ml) as a source of polymorphonuclear leukocytes; and rabbit preimmune/immune serum in several dilutions. After a 45 min incubation at 37°C, aliquots were removed from the wells and plated on bacteriological agar plates (5% Hy-Yeast extract [Kerry Bio-Science], 10% soyton without animal products [Teknova], 5% NaCl [Americanbio] and 1.4% agar [Americanbio], hereinafter referred to as HySoy agar or HSA). After overnight incubation on HSA, CFUs were counted. A positive OFV response was obtained when the CFU for bacteria treated with immune serum was reduced compared to treatment with pre-immune serum at the same dilution.

Figure 18A compares CFU of Klebsiella strain KP 12-0581 (O1: K62) with HL-60 cells and rabbit immune serum to: KP-O1 OPS BP-1 PRO-CN MAPS before immunization and after the second immunization (left), and also KP-O1 OPS BP-2a/rizavidin PRO-CN MAPS before immunization and after the second immunization (right). Significant killing of KP was observed with anti-BP-2a immune sera (P2) compared to pre-immune sera (P0) from the same rabbit. There was no significant killing in the case of anti-BP-1 immune sera, which was consistent with the O1-OPS-specific IgG titers obtained using these constructs, ie BP-2a > BP-1 in relation to the respective titers.

The results of PFU of Pseudomonas strain PA IATS O6 with HL-60 cells and rabbit immune sera to bivalent PA O6, O10-OPS BP-1 PRO-CN MAPS and monovalent PA IATSO6-OPS BP-1 PRO-CN MAPS are shown in Figure 18B. 2 separate rabbit sera P2 (AFV 624; AFV 625) to PA O6, O10-BP-1 MAPS resulted in significant RFU of PA IATSO6 (left panel), rabbit immune sera (P2) against PA IATSO6-BP-1 MAPS also resulted in significant OFU PA IATS O6, with AFV643 (P2) serum producing a prozone effect indicative of a strong anti-OPS antibody titer.

Four separate rabbit immune sera (P2) prepared using the 2-valent PA-O6, -O10 OPS BP-1-PRO-CN-MAPS vaccine elicited reliable OFU of Pseudomonas O10 bacteria in HL-60 cells (Figure 18C).

The RFU of the target P. aeruginosa strain PAO1 (O5: FlaB expressing) with pooled rabbit anti-Rhavi-FlaB-His serum and HL-60 cells is shown in Figure 22. In this experiment, there was some RFU (1/100 dilution) caused by anti-Rhavi-FlaB-His serum. Severe OFU has been noted with human IV IG induced by Pseudomonas vaccine and anti- Pseudomonas FlaB sera. Control (-) serum did not induce OFU.

CFU assays with human PMN

Analyzes were performed as previously described, with modifications (Cross et al., 1986). Briefly, assays were performed in 96-well plates using polymorphonuclear leukocytes (PMN) freshly isolated from the blood of healthy human donors by dextran precipitation and Ficoll-Hypaque density gradient centrifugation. The PMN stock suspension was adjusted to 23×10 ⁶ cells/ml in HBSS with calcium/magnesium. Sixty µl of PMN stock suspension was added to a well of the plate along with 10-20 µl of either rabbit complement or fresh normal human serum (NHS), 10 µl of bacteria (~10 ⁶ cfu for ~1:1 MOI) in the presence or absence of 10 µl heat-inactivated pre- or post-immune serum in various dilutions. At time 0, a sample was taken, diluted and plated on HySoy plates, then the 96-well plate was incubated for 2 hours at 37° C. with shaking. After 2 hours, samples were taken again, diluted and plated. After overnight incubation at 37° C., cells were counted in dishes. The number of cells at time 2 h for each well was divided by the number of cells at time 0, and the % kill was calculated using the formula 1.0-T _2h CFU/T ₀ CFU. Alternatively, we converted the cfu for each well to log cfu, subtracted the log cfu for the 2h sample from the log cfu for time 0, and expressed the difference in kill as "delta log cfu". The inventors then compared kills in wells containing pre-immune and immune sera with control without antibody.

Binding assays by flow cytometry

P. aeruginosa and K. pneumoniae strains in the middle of the logarithmic growth phase were concentrated in PBS to an OD ₆₀₀ of 0.8. For polysaccharide antigens, bacteria were fixed with 1% formaldehyde before antibody addition. In the case of protein antigens, the bacteria were not fixed with formaldehyde prior to the addition of the antibody in order to preserve the cell surface antigens. Bacteria were incubated with rabbit pre-immune/immune serum in various dilutions for 1 h at room temperature. Unbound antibody was removed by pelleting the bacteria by centrifugation and washing with PBS, washed cells were incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (Invitrogen) for 1 hour at room temperature. Unbound secondary antibody was removed by pelleting the bacteria by centrifugation and washing with PBS. Immunologically stained bacteria were fixed in 1% formaldehyde to kill any viable microorganisms. Samples were injected into an LSR II flow cytometer (BD) and analyzed using FACS Diva (v. 6.1.3; BD) and FlowJo (v. 9.2; Tree Star).

The results of binding of Klebsiella strain B5055 (O1:K2) with rabbit serum to K-19:O1- and O5-OPS BP-1 and BP-2a PRO-CN MAPS determined by the PC method are shown in Figure 19A. The results of binding of whole bacteria Klebsiella strain B5055 with individual rabbit sera to 2-valent KP O1, O5 OPS BP-1 (#651-655) (left) and KP O1 OPS BP-2a (#666-670) are shown by PC method ( on right).

These QC results for whole bacteria correlate well with the corresponding ELISA titers of IgG to purified KP O1 OPS shown in Table 10, i.e. a low titer corresponds to a low level of binding as determined by QC, a small number of double positive events indicates poor antibody binding, or limited access for anti-OPS antibody on the surface of KP B5055.

Table 10: Anti-KP O1-OPS IgG anti-O1-OPS backbone polymer ELISA titers in BP-1 MAPS and BP-2a MAPS constructs

Rabbit immune serum EIA credits P0 P1 P2 KP 2-in O1, O5 BP-1
№651-655 5729 13025 53731 KP O1 BP-2a
№666-670 5428 411496 119731

Figure 19B compares the scatterplots of the data PC for three strainsKlebsiella O1 with rabbit immune serum (AFV667) induced by KP complex O1 OPS BP-2a PRO-CN MAPS; top panels show weak binding to KP B5055 (left), weak binding toK. caroli (center) and strong binding to non-encapsulated strain KP CVD 3001 (right) as determined by the PC method. The lower panel (Figure 19B) compares the total binding events determined by the QC method with the same 3 strains.Klebsiella O1 for sera from rabbit vaccinated with 2-valent KP O1, O5 OPS BP-1 (AFV651) PRO-CN MAPS, and rabbit sera (AFV667) induced with univalent KP O1 OPS BP-2a PRO-CN MAPS. Of note, there is significantly more antibody binding to the O1 OPS BP-2a construct than to the corresponding BP-1 OPS construct as determined by the QC method, and these data appear to correlate with anti-O1 OPS IgG titers, i.e. , KP O1 OPS BP-2a is more immunogenic than its equivalent BP-1. These binding data also suggest that the amount of CPS that affects the OPS exposure of the antibody may be dependent on growth conditions.

Comparison of PC-Determined Antibody Binding to Whole BacteriaPseudomonas strain PAK (O6:FlaA1) shown in Figure 20: with rabbit serum (pooled serum from 3 rabbits, after the second immunization) to 2-valent PA-O6, -O10 native OPS-PRO-CN-MAPS (left); rabbit serum (pooled serum from 3 rabbits, after the second immunization) to 2-valent PA O6, O10-OPS BP-1-PRO-CN MAPS (center); rabbit serum (pooled serum from 3 rabbits, after the second immunization) to monovalent PAO6-IATS-OPS BP-1-PRO-CN MAPS; a stronger PC-determined binding of the antibody to the PAK strain was observed in the case of PAO6 OPS BP-1 PRO-CN MAPS when compared with the native form PA O6 OPS MAPS, indicating that there is a minimum OPS size that is critical for its immunogenicity, i.e. , the size of native PA OPS is too small and must be increased by chemical bonding to the backbone polysaccharide to increase its epitope valence as well as its size.

Binding of Pseudomonas strain PAO1 (O5 expressing FlaB) to individual rabbit anti-FlaB antibodies induced by various rizavidin SB variants containing FlaB epitopes was studied by CV and the results are shown in Table 11. Binding, in percent above threshold, and high bindings of pre-immune sera (P0) versus sera after the third immunization (P3) are shown for 3 separate rabbit sera to fusion protein antigens. These binding data demonstrate that FlaB-D2 and FlaB-PcrV fusion proteins with rizavidin can induce strong binding and antibody recognition of whole Pseudomonas strain PAO1 expressing FlaB flagella, suggesting that these 2 proteins are produced in a properly folded manner and are able to induce production of anti-FlaB antibodies with specificity for natural B flagella epitopes on whole Pseudomonas bacteria. Serum against the RhaviFlaB-D2-MrkA fusion protein does not significantly (or at best only weakly recognize) flagella on intact Pseudomonas strain PAO1, indicating that the FlaB portion of the rhizavidin construct is either misfolded or not is sufficiently immunogenic in the context of a fusion protein with MrkA, as assessed by immunization with one protein with Freund's adjuvant.

Scatterplots The QCs for some of the individual rabbit sera (#792, #798, and #800) shown in Table 11, also shown in Figure 21B (top panel), show strong binding by the QC method for the individual rabbit serum #792 (sample after P3) with rizavidin-FlaB-D2; (center panel) intermediate binding for rabbit serum #798 (sample after P3) with rizavidin-FlaB-PcrV; and (bottom) weak binding for separate rabbit serum no. 800 (sample after P3) to rizavidin-FlaB-D2-MrkA. In Figure 21A pooled rabbit sera from rabbits immunized with the rizavidin-FlaB-Domain2 fusion protein (lacking the TL5 binding region) (P3) shows a high percentage of labeled populationPseudomonas PAO1 strain (scatterplot, right panel) versus pre-immune serum (P0).

Table 11: Binding results of rabbit anti-FlaB antibodies to Pseudomonas strain PAO1 (O5 expressing FlaB)

Rhiz-Fusion Proteins Rabbit No. Percentage Above Threshold Percentage of high degree of binding P0 P3 P0 P3 FlaB-D2 791 2.3 2.1 0 0.1 792 2.3 44.2 0.1 14.8 793 1 31.8 0 11.4 FlaB-PcrV 797 3.3 28.5 0.1 7.7 798 3.6 13 0 2.2 799 2 11.7 0.1 2.2 FlaB-D2-MrkA 800 0.3 3.7 0 0.1 801 0.3 2.2 0 0.1 802 1.9 3 0 0

These ELISA and PC binding data suggest that FlaB-containing rizavidin fusion proteins, such as rizavidin-FlaB-Domain2 and rizavidin-FlaB-PcrV, can be properly folded and induce the production of functional antibodies capable of recognizing natural B flagella on whole Pseudomonas microorganisms.

Motility and Motility Inhibition Assays

P. aeruginosa strain PAO1 (O5: expressing FlaB) was grown to mid-log phase in HySoy broth. The bacteria were pelleted by centrifugation and suspended in PBS to an OD ₆₀₀ of 0.3, then diluted 100-fold in PBS. Soft agar was poured into 24-well plates in the presence or absence of immune serum to flagellin in different dilutions. Dilutions of immune serum were repeated three times in the wells. The flame-fired needle was immersed in the bacterial suspension, then used to inoculate the center of the agar plug. The plates were incubated at 30° C. for 14 hours or longer, or until the bacteria spread from the inoculation site over the surface of the agar. The plates were imaged using a digital camera to record the diameter of the bacterial colony. Motility inhibition test results are positive when colony size on anti-flagellin serum agar is significantly reduced compared to pre-immune serum.

ELISA results and corresponding motility-inhibiting titers of sera from rabbits vaccinated with the rhavidin-FlaB constructs: Rhavi-FlaB-D2; Rhavi-FlaB-PcrV and Rhavi-FlaB-D2-MrkA are shown in Table 12. In this ELISA table, the titers for FlaA1 and FlaB are shown for immune sera after the third immunization (P3), and the motility-inhibiting titers for the corresponding P3 immune sera are also shown. using P. aeruginosa strain PAO1 (O5: expressing FlaB). There is a strong correlation between anti-FlaB antibody levels (ELISA titers) and corresponding motility-inhibiting titers, i.e. both anti-Rhavi-FlaB-D2 and Rhavi-FlaB-PcrV immune sera with high specific anti-FlaB antibody titers resulted in high levels inhibition of PAO1 mobility, as opposed to serum, to the Rhavi-FlaB-D2-MrkA fusion protein construct, presumably due to low levels of anti-FlaB antibody, likely either due to either misfolding of the FlaB protein component in the construct or lack of immune response in the context of the fusion protein with MrkA when evaluating immunization with one protein with Freund's adjuvant.

Table 12: ELISA titers and motility inhibition titers for various constructs containing rizavidin-FlaB

Rizavidin fusion protein construct Rabbit No. Serum after the third vaccination FlaA1
ELISA FlaB
ELISA Motility inhibition titer FlaB-D2 AFV791 744 279209 1250 AFV791 627 154104 1250 AFV793 763 423514 250 FlaB-PcrV AFV797 31497 318117 1250 AFV798 40911 230702 1250 AFV799 41566 499941 1250 FlaB-D2 MrkA AFV800 1916 35651 10 AFV801 944 53477 50 AFV802 291 34790 10

Cytotoxicity assays (for constructs with PcrV protein Pseudomonas )

The analyzes were carried out as follows. Briefly, rabbit immune sera raised against the PcrV protein of the bacterial secretory system was added to A549 cells seeded in flat-bottomed 96-well plates (Corning Costar). All dilutions of immune serum and bacterial cultures were performed in RPMI medium without antibiotic. A P. aeruginosa strain of PAK capable of expressing ExoU in logarithmic growth phase was added at an MOI of approximately 10 and incubated for 2 hours at 37°/5% CO ₂ , during which time the A549 cells were intoxicated and became non-viable. The number of viable cells in each well was estimated based on cell uptake of the intravital dye neutral red ( Pseudomonas also metabolizes alamar blue and cannot be used). The percentage of viable cells in each well was assessed by measuring the absorbance of neutral red and comparing these values with the values for cells that were not infected with bacteria. The results of this assay are positive when PcrV immune sera results in an increase in viable cells (ie, cells capable of absorbing neutral red) compared to equivalent dilution preimmune sera.

The Pseudomonas cellular cytotoxicity prevention model (Warrener et al., 2014) was used to test the efficacy of anti-Rhavi-FlaB-PcrV BP rabbit antibody and the results are shown in Figure 24A. Protection of anti-PcrV antibody of A549 cells (lung carcinoma) against Pseudomonas strain PAK (O6:FlaA1) intoxication was assessed as follows. A549 monolayers were infected with Pseudomonas strain PAK (O6:FlaA1) for 4 hours in the presence of 10% sera from rabbits (pooled 3 sera, after third immunization, P3) vaccinated with the Rhavi-FlaB-PcrV fusion protein. Anti-Rhavi-FlaB-PcrV serum protected cells from Pseudomonas strain PAK intoxication compared to pre-immune serum, as evidenced by fewer rounded and detached cells. In the following experiment (Figure 24B), a monolayer of A549 cells was infected with Pseudomonas strain PAK for 4 h in the presence of anti-Rhavi-FlaB-PcrV sera (pooled 3 sera, P3) at various dilutions. Cells were washed with PBS and incubated for 1 h with neutral red. Cells were lysed and dye uptake was recorded in each well. Anti-Rhavi-FlaB-PcrV (P3) serum protected cells from PAK intoxication compared to pre-immune serum at a dilution of 1:40.

Without wishing to be bound by theory, the polyclonal nature of the Rhavi-FlaB-PcrV response, as well as its configuration in the fusion protein, may account for the increased affinity of the antibody for the PcrV component of the fusion protein. Several strains of Pseudomonas were tested in this cytotoxicity assay using A549 cells and anti-FlaB-PcrV rabbit serum (after the third immunization; P3) (Figure 25): in the case of highly toxic PA IATS O1 (closed diamonds), there was no serum protection against cytotoxicity; in the case of the cytotoxic strain PA M2 O5 (closed circles), serum protection was also absent; in the case of non-cytotoxic PA IATS O6 (closed triangles), there was no toxicity; in the case of cytotoxic PA 17002 (O10) (closed triangles), serum protection was clearly observed; these strain-dependent differences in serum protection may reflect the expression and level of target PcrV protein on Pseudomonas strains.

Cell adhesion assay

Klebsiella adhesion to A549 cells was analyzed as described (Clements et al., 2008), with some modifications. Rabbit immune sera prepared against MrkA fimbrial protein were diluted in RPMI buffer (no additives) and added to A549 cells seeded in 96-well plates. An equal volume of K. pneumoniae O5 strain 6997 (10 ⁴ CFU/ml) was mixed with immune serum in various dilutions. Infected cells were incubated for 1 h at 37°C and 5% CO ₂ after which the wells were washed 6 times with sterile PBS to remove unbound bacteria. Cells were lysed with 0.1% Triton X-100 in PBS and an aliquot of the solution was applied to HSA. The plates were incubated overnight and the CFU counted. Assay results are positive when the number of adherent Klebsiella is reduced by using anti-MrkA immune sera compared to pre-immune sera at the same dilution.

Blocking the adhesion of Klebsiella KP O5 6997 (origin South Africa) to A549 cells with anti-Rhavi-FlaBD2-MrkA rabbit serum is shown in Figure 23. High titer Rhavi-FlaBD2-MrkA specific serum (after third immunization, P3) significantly inhibited binding KP strain 6997 with A549 cells at a dilution up to 1/1000, in contrast to pre-immune serum (P0). This indicated that the MrkA protein component in the Rhavi-FlaBD2-MrkA SB was correctly folded and was able to induce the production of functional blocking antibodies specific for native MrkA on the surface of KP bacteria. A second experiment was performed using separate anti-MrkA rabbit immune sera obtained by immunization with two MrkA-rhizavidin fusion proteins: Rhavi-FlaB-D2-MrkA and Rhavi-PcrV-MrkA, the results of the experiment are shown in Table 13. All rabbit anti-SB sera MrkA (with the exception of one #804) were able to reduce the adhesion of Klebsiella O5 strain 6997 to A549 cells, indicating that these 2 RBs of rizavidin had the potential to induce the production of functional MrkA-specific antibodies.

Table 13: Serum Inhibition of Anti-MrkA Binding Klebsiella O5 strain 6997 with A549 cells

rabbit serum Dilution at which P3 reduced adhesion No. 800 - Rhavi FlaBD2 MrkA 1 to 150 No. 801 - Rhavi FlaBD2 MrkA 1 to 2400 No. 802 - Rhavi FlaBD2 MrkA 1 to 75 No. 803 - Rhavi PcrV MrkA 1 to 300 No. 804 - Rhavi PcrV MrkA 0 reduction No. 805 - Rhavi PcrV MrkA 1 to 75

Anti-MrkA antibody production assay

The anti-MrkA antibody agglutinates Klebsiella expressing MrkA/fimbria type 3, leading to the formation of clusters of bacteria that fall out of solution. Preimmune or immune sera were evaluated from rabbits that had been inoculated with Rhavi-MrkA-his, Rhavi-FlaB-D2-MrkA-his or Rhavi-PcrV-MrkA-his carrier proteins. Klebsiella strain 4425 (O5: K57) was incubated with different dilutions of sera in PBS at 25°C for 1 hour in 96-well round bottom titration plates. The plates were gently shaken to suspend non-agglutinated bacteria. The supernatant from each well was carefully removed and transferred to a new titration plate and the light scattering at 600 nm was measured to assess the turbidity of the solution. The result is positive when the absorbance is reduced compared to the control well without antibody, indicating a decrease in the number of bacteria in the supernatant due to agglutination caused by the antibody. The assay result was recorded as the highest antibody dilution (lowest concentration) at which K. pneumoniae agglutinated due to immune serum more than due to preimmune serum when measuring the difference in absorbance at 600 nm.

The results of the assessment of agglutination of Klebsiella pneumoniae O5K57, strain 4425, and O1 K22, strain 170381, due to rabbit immune serum to different constructs of the MrkA-rhizavidin fusion protein: Rhavi-FlaB-D2-MrkA and Rhavi-PcrV-MrkA are shown in Table 14. Titers for 3 separate rabbit immune sera specific for each construct, after the second (P2) and third (P3) immunizations represent the maximum dilution of serum with detectable agglutination. In this experiment, the agglutinated clumps are allowed to settle and the number of remaining (non-agglutinated) bacteria is measured based on optical density (OD) at 600 nm. MrkA-specific antibodies induced by the 2 constructs, Rhavi-FlaB-D2-MrkA and Rhavi-PcrV-MrkA, recognized MrkA expressed on both strains of Klebsiella , indicating that the MrkA component of these 2 fusion proteins is correctly folded and causes response in the form of production of functional antibodies.

Table 14: Agglutination of bacteria klebsiella, caused by antibodies specific for MrkA SB of rizavidin

Protein construct Rabbit No. Highest serum dilution with detectable agglutination KP 4425 O5 K57 KP 170381 O1 K22 P2 P3 P2 P3 Rhavi-FlaB-D2-MrkA-His AFV800 10 ³ 10 ⁵ 10 ² 10 ³ AFV801 10 ⁴ 10 ⁵ 10 ³ 10 ⁵ AFV802 10 ⁴ 10 ⁴ 10 ² 10 ⁴ Rhavi-PcrV-MrkA-His AFV803 10 ³ 10 ⁴ 10 ² 10 ⁴ AFV804 10 ⁴ 10 ⁵ 10 ² 10 ⁵ AFV805 10 ⁴ 10 ⁴ 10 ² 10 ⁴

Example 8: Mouse Models of Thermal Burn and Bacteremia Caused by P. aeruginosa

Thermal burn and bacteremia models were created as described, with modifications (Neely et al., 2002). For thermal burn, 11-week-old outbred CD-1 mice were shaved at the back and anaesthetized with ketamine and xylazine prior to application of a 1 x 1.5 inch heat resistant polymer template. This platform has been designed to create a thermal burn of 12-15% of the body surface. Ethanol (0.5 ml, 100%) was evenly distributed over the surface of the back defined by the window, ignited and left to burn for exactly 10 seconds, after which the flame was extinguished. Immediately after the burn, mice received 0.5 ml of sterile normal saline for hydration. P. aeruginosa was then injected sc under the burn wound. The wound was left uncovered. Animals were closely monitored for 14 days post-infection for mortality and organ burden (i.e., distant spread of infection).

A mouse model of burn wound sepsis was used to evaluate the efficacy of rabbit antibodies against Pseudomonas FlaB (Cryz et al., 1984). In this model, CD1 mice were burned as described above and 28 cfu of P. aeruginosa strain M2 (O5:FlaB) (burn wound infection LD ₅₀ <1x10 ² ) was injected into the burn wound. CD-1 mice were injected ip with PBS, pre-immune or anti-FlaB serum prior to infection of the burn wound. As shown in the Kaplan-Meier survival plots in Figure 26, mice were protected by anti-FlaB for 10 days or more after wound infection with FlaB-expressing Pseudomonas strain M-2, while all mice from other groups died due to infection. wounds.

In a bacteremia model, 7-week-old BALB/c mice were treated with control IgG or vaccine-inducible antibody by ip injection 24 h prior to iv challenge with K. pneumoniae or P. aeruginosa . Depending on the microorganism, the animals were bled at regular intervals for 1-4 hours to control clearance from the blood, followed by euthanasia of mice and collection of the liver and spleen 1-24 hours after infection.

Example 9 Mice Defense Assays

Mouse Model of Organ Clearance and Loading

The ability of the KP OPS-specific antibody to prevent Klebsiella infection was tested in a mouse model of organ clearance and loading. CD1 mice (groups of 3) were injected ip with rabbit anti-KPO1 BP-2a OPS-PRO-CN MAPS serum followed by (iv) Klebsiella KP B5055 O1 (9×10 ⁴ cfu). The number of viable KP in the spleen was measured 14 hours after infection. A significant decrease in the number of viable KPs was observed in the spleen of mice treated with immune serum (P2) compared to the spleens of mice treated with pre-immune serum (P0) (Figure 27). These data indicate that the O1 OPS-specific antibody induced by the OPS BP-2a MAPS vaccine was able to induce clearance of microorganisms from the spleens of mice, thus being protective against Klebsiella infection.

KP(O1:K2) grown in sodium salicylate

Klebsiella pneumoniae strain B5055 (O1:K2) was grown overnight in HySoy broth containing 2.5 mM sodium salicylate (Sigma) to reduce CPS expression (Domenico, 1989). 12 ml HySoy broth with 2.5 mM sodium salicylate was inoculated with a 5% overnight culture which was then grown to mid-log phase growth (37° C. with shaking). Bacteria were pelleted by centrifugation, then resuspended in sterile PBS to a concentration of approximately 1×10 ⁸ cfu/ml. The concentrated bacteria were diluted in PBS to 2×10 ⁵ cfu/ml. 1 hour after i.p. administration of 0.1 ml of rabbit serum, female CD-1 mice aged 8-12 weeks were injected i.p. with 2×10 ⁴ cfu of B5055 in 0.1 ml. Mice were monitored for mortality for 7 days.

In experiment 1, CD1 mice were injected with 0.1 ml of rabbit immune serum against KP O1 OPS BP-1; BP-2a PRO-CN MAPS or UN KP O1: K2 (1/10 dilution) ip 1 hour before ip administration of 2×10 ⁴ KP O1 B5055 bacteria. The results of the experiment are shown in Table 15.

Table 15: Passive protection of mice against KP O1:K2 organisms grown in sodium salicylate

Serum Mice Survival ELISA titer Experiment 1 Experiment 2 PBS 0 out of 5 0 out of 5 0 P0 BP-1 1 of 4 0 out of 5 57 P2 BP-1 0 out of 5 2 out of 5 537 P0 BP-2a 0 out of 5 2 out of 5 54 P2 BP-2a 5 out of 5 4 out of 5 11973 UN, control* 5 out of 5 3 out of 5 27343 *Rabbit immune sera against the KP O1 microorganism NR as a positive control was introduced at a dilution of 1/10

In Experiment 1, KP O1 anti-UN antiserum as a positive control and anti-KP O1 OPS BP-2 PRO-CN MAPS anti-KP O1 antiserum provided complete protection against KP O1:K2 (B5055) challenge, while PBS control and anti-KP O1 anti-serum against KP O1 OPS BP-1 PRO-CN MAPS did not protect mice.

In Experiment 2, CD1 mice (5/group) were injected ip with 0.1 ml pooled rabbit immune or pre-immune serum, followed by ip bacterial challenge with 2×10 ⁴ KP O1 B5055 bacteria. Mice received PBS and anti-UN serum KP O1 (1/10 dilution) as negative and positive controls, respectively. All sera against KP O1 OPS PRO-CN MAPS were able to exert a protective effect in this model, although mice that received BP-2a (4 of 5) survived significantly better when challenged. Serum against KP O1 OPS BP-1 PRO-CN MAPS had some protective effect, with 2 out of 5 mice surviving. None of the mice treated with PBS survived the challenge. These survival results are in good agreement and correlate well with anti-KP O1 OPS-specific IgG levels as determined by ELISA titers (i.e., higher protection was observed at higher antibody levels).

Passive protection against microorganism Pseudomonas in mouse model

The ability of the PA OPS-specific antibody to prevent Pseudomonas infection was tested in a passive defense survival mouse model using CD1 mice challenged ip with PA strain 17002 (O10) along with D-galactosamine to sensitize the animals to the lethal effects of endotoxin (Galanos et al., 1979) and to make them more susceptible to infection. Mice received rabbit antibodies against 2-valent PA O6/O10 BP-1 PRO-CN MAPS (rabbit AFV624 or AFV629, i.p.) 1 hour before challenge (1×10 ⁶ cfu) and D-galactosamine. The results are shown in Table 16. In this protection study, anti-PA O6/O10 2-b BP-1 PRO-CN MAPS immune sera (AFV624; P2) protected 40% more mice against PA O10 strain 17002 compared to pre-immune sera ( AFV624 P0). Similarly, PA O6/O10 2-in BP-1 PRO-CN MAPS immune sera (AFV629; P2) protected 40% more mice from PA O10 strain 17002 compared to pre-immune sera (AFV629 P0). High titer rabbit immune serum prepared against heat-killed PA O10 bacteria as a positive control had a significant protective effect compared to the PBS control. Pre-immune serum from some rabbits may have provided some protection compared to PBS (AFV624 P0). There appeared to be some correlation between levels of protection (survival) and vaccine-induced PA O10 OPS-specific IgG antibody levels as measured by ELISA (Table 16).

Table 16: Passive protection in mice treated with PA O10 strain 17002 and D-galactosamine in the group with antibodies against 2-valent PA O6/O10 BP-1 PRO-CN MAPS.

Group Survival ELISA TITER,
PA O10 OPS (ELISA units/ml) PBS control 0/5 Against heat-killed O10 4/5 7745537 Rabbit AFV624, P0 2/5 12.5 Rabbit AFV624, P2 4/5 15246 Rabbit AFV629, P0 0/5 12.5 Rabbit AFV629, P2 2/5 3955

Figure 28 is a Kaplan-Meier plot of survival when mice are passively protected with administered rabbit anti-KPO1 BP-1 OPS-PRO-CN MAPS serum prior to infection with Klebsiella strain B5055 (O1:K2) grown in salicylate to reduce capsule expression. CD1 mice (5/group) were injected ip with 0.1 ml of pooled rabbit immune (P2) or pre-immune (P0) serum, followed by ip bacterial challenge with 2x10 ⁴ CFU of Klebsiella strain B5055 (O1:K2). Shown are Kaplan-Meier plots of survival for mice treated with P0 or P2 serum against KPO1 BP-1 OPS-PRO-CN MAPS. Serum P2 protected animals in the model, with mice treated with serum against KPO1 BP-1 OPS-PRO-CN MAPS surviving in greater numbers and for a longer time than mice treated with serum P0.

Example 10 Exemplary Multivalent Immunogenic Compositions

Illustrative 4-valent KP vaccine

Separately prepared 4 different immunogenic complexes, and then formulated together, receiving an exemplary 4-valent KP vaccine.

Each complex included a BP-1 polymer obtained by conjugation of OPS from one KP bacterial serotype with an oxidized K19 polysaccharide, followed by biotinylation of the K19 polysaccharide. They were then combined in complex with the rizavidin carrier fusion protein. The components of each of these complexes are shown in Table 17.

Table 17: Components of complexes included in an exemplary 4-valent KP vaccine.

Complex BP-1 CPS pathogen Serotype carrier protein 1 KP-K19 Klebsiella O1 Rhavi-FlaBD2-MrkA 2 KP-K19 Klebsiella O2 Rhavi-FlaBD2-MrkA 3 KP-K19 Klebsiella O3 Rhavi-FlaBD2-MrkA 4 KP-K19 Klebsiella O5 Rhavi-FlaBD2-MrkA

These four complexes were co-formulated with an adjuvant as described in Table 21 below.

Illustrative 8-valent PA vaccine

Separately prepared 8 different immunogenic complexes, and then formulated together, receiving an exemplary 8-valent PA vaccine.

Each complex included a BP-1 polymer obtained by conjugation of OPS from one bacterial serotype PA with an oxidized K19 polysaccharide, followed by biotinylation of the K19 polysaccharide. They were then combined in complex with the rizavidin carrier fusion protein. The components of each of these complexes are shown in Table 18.

Table 18: Components of complexes included in an exemplary 8-valent PA vaccine.

Complex BP-1 CPS OPS Rizavidin carrier fusion protein 1 KP-K19 PAO1 Rhavi-FlaBD2-MrkA 2 KP-K19 PAO2 Rhavi-FlaBD2-MrkA 3 KP-K19 PAO3 Rhavi-FlaBD2-MrkA 4 KP-K19 PAO4 Rhavi-FlaBD2-MrkA 5 KP-K19 PAO5 Rhavi-FlaBD2-MrkA 6 KP-K19 PAO6 Rhavi-FlaBD2-MrkA 7 KP-K19 PAO10 Rhavi-FlaBD2-MrkA 8 KP-K19 PAO11 Rhavi-FlaBD2-MrkA

These eight complexes were co-formulated with an adjuvant as described in Table 21 below.

Illustrative 12-valent KP/PA vaccine 1

Separately, 12 different immunogenic complexes were prepared and then co-formulated to give an exemplary 12-valent KP/PA vaccine 1.

Each complex included a BP-1 polymer obtained by conjugation of OPS with an oxidized K19 polysaccharide, followed by biotinylation of the K19 polysaccharide. They were then combined in complex with the rizavidin carrier fusion protein. The components of each of these complexes are listed in Table 19.

Table 19: Components of the complexes included in the exemplary 12-valent KP/PA vaccine 1.

Complex BP-1 CPS OPS Rizavidin carrier fusion protein 1 KP-K19 KPO1 Rhavi-FlaBD2-MrkA 2 KP-K19 KPO2 Rhavi-FlaBD2-PcrV 3 KP-K19 KPO3 Rhavi-FlaBD2-MrkA 4 KP-K19 KPO5 Rhavi-FlaBD2-PcrV 5 KP-K19 PAO1 Rhavi-FlaBD2-PcrV 6 KP-K19 PAO2 Rhavi-FlaBD2-PcrV 7 KP-K19 PAO3 Rhavi-FlaBD2-PcrV 8 KP-K19 PAO4 Rhavi-FlaBD2-PcrV 9 KP-K19 PAO5 Rhavi-FlaBD2-MrkA 10 KP-K19 PAO6 Rhavi-FlaBD2-MrkA eleven KP-K19 PAO10 Rhavi-FlaBD2-MrkA 12 KP-K19 PAO11 Rhavi-FlaBD2-MrkA

These twelve complexes were co-formulated with an adjuvant as described in Table 21 below.

Illustrative 12-valent KP/PA vaccine 2

Separately, 12 different immunogenic complexes were prepared and then co-formulated to give an exemplary 12-valent KP/PA vaccine 2.

Each complex included a BP-1 polymer obtained by conjugation of OPS with an oxidized K19 polysaccharide, followed by biotinylation of the K19 polysaccharide. They were then combined in complex with the rizavidin carrier fusion protein. The components of each of these complexes are shown in Table 20.

Table 20: Components of complexes included in an exemplary 12-valent KP/PA vaccine 2.

Complex BP-1 CPS OPS Rizavidin carrier fusion protein 1 KP-K19 KPO1 Rhavi-FlaBD2-MrkA 2 KP-K19 KPO2 Rhavi-FlaBD2-MrkA 3 KP-K19 KPO3 Rhavi-FlaBD2-MrkA 4 KP-K19 KPO5 Rhavi-FlaBD2-MrkA 5 KP-K19 PAO1 Rhavi-FlaBD2-MrkA 6 KP-K19 PAO2 Rhavi-FlaBD2-MrkA 7 KP-K19 PAO3 Rhavi-FlaBD2-MrkA 8 KP-K19 PAO4 Rhavi-FlaBD2-MrkA 9 KP-K19 PAO5 Rhavi-FlaBD2-MrkA 10 KP-K19 PAO6 Rhavi-FlaBD2-MrkA eleven KP-K19 PAO10 Rhavi-FlaBD2-MrkA 12 KP-K19 PAO11 Rhavi-FlaBD2-MrkA

These twelve complexes were co-formulated with an adjuvant as described in Table 21 below.

Example 11 Immunization studies of rabbits with a 12-valent vaccine against P. aeruginosa/K. pneumoniae

Animal Immunization

Prior to immunization of rabbits, exemplary 4-valent KP vaccine, exemplary 8-valent PA vaccine, exemplary 12-valent KP/PA vaccine 1, and exemplary 12-valent KP/PA vaccine 2 were formulated with an adjuvant approximately 48 hours prior to injection. The complexes were adsorbed to the adjuvant at a final concentration of 10 μg/ml for the 5 μg PS dose, and 2 μg/ml for the 1 μg PS dose, of each BP-1 serotype, either alone or as a multivalent mixture, with 40 mM histidine, pH 5 ,5, 150 mM NaCl and 1.25 mg/ml aluminum phosphate gel, with stirring by inversion overnight at 4°C. These formulated mixtures were used directly for immunization in a volume of 0.5 ml per dose. A summary of the composition of illustrative 4-valent KP vaccine, illustrative 8-valent PA vaccine, illustrative 12-valent KP/PA vaccine 1 and illustrative 12-valent KP/PA vaccine 2 are shown in Table 21.

Table 21: Summary of vaccine preparations used in the NCB012 study in rabbits.

Group Vaccine pathogen carrier protein Serotype Dose PS (total) Protein:PS ratio (total protein) Adjuvant rabbits 1 12-valent KP/PA vaccine 2
5 µg PS per BP Pseudomonas Rhavi-FlaBD2-MrkA-his O1, O2, O3, O4, O5, O6, O10, O11 5 mcg
(60 mcg) 3:1
(180 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O1, O2, O3, O5 2 12-valent KP/PA vaccine 2
1 µg PS per BP Pseudomonas Rhavi-FlaBD2-MrkA-his O1, O2, O3, O4, O5, O6, O10, O11 1 mcg
(12 mcg) 3:1
(36 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O1, O2, O3, O5 3 8-valent, PA Pseudomonas Rhavi-FlaBD2-MrkA-his O1, O2, O3, O4, O5, O6, O10, O11 5 mcg
(40 mcg) 3:1
(120 mcg) 625 µg AlPO ₄ 10 4 4-valent, KP Klebsiella Rhavi-FlaBD2-MrkA-his O1, O2, O3, O5 5 mcg
(20 mcg) 3:1
(60 mcg) 625 µg AlPO ₄ 10 5 12-valent KP/PA vaccine 1
5 µg PS per BP Pseudomonas Rhavi-FlaBD2-MrkA-his O5, O6, O10, O11 5 mcg
(60 mcg) 3:1
(180 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O1, O3 Pseudomonas Rhavi-FlaBD2-PcrV-his O1, O2, O3, O4 Klebsiella Rhavi-FlaBD2-PcrV-his O2, O5 6 12-valent KP/PA vaccine 1
1 µg PS per BP Pseudomonas Rhavi-FlaBD2-MrkA-his O5, O6, O10, O11 1 mcg
(12 mcg) 3:1
(36 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O1, O3 Pseudomonas Rhavi-FlaBD2-PcrV-his O1, O2, O3, O4 Klebsiella Rhavi-FlaBD2-PcrV-his O2, O5

12-valent KP/PA vaccine 1 was administered to experimental groups 5 and 6 (with FlaBD2-MrkA and FlaBD2-PcrV as carrier proteins), and 12-valent KP/PA vaccine 2 was administered to experimental groups 1 and 2 (with FlaBD2-MrkA as a carrier protein). For comparison, two additional experimental groups were included in this study, which received an 8-valent PA vaccine or a 4-valent KP vaccine. Experimental group 3 received the 8-valent PA vaccine and experimental group 4 received the 4-valent KP vaccine. Groups 1, 3, 4 and 5 received 5 μg of each polysaccharide (PS) in the vaccine, and experimental groups 2 and 6 received a lower dose of 1 μg of each PS in the vaccine. All vaccines contained the same amount of aluminum phosphate adjuvant (625 µg).

Two IM immunizations (0.5 ml) were given 4 weeks apart to 4 month old New Zealand White rabbits (Cocalico Biologicals, n=10 per group). Blood samples were collected 4 weeks after the first immunization and 2 weeks after the second immunization.

Immune responses to OPS

Immune responses to KP OPS in 12-valent KP/PA vaccine 1 versus 4-valent KP vaccine

Figure 29 shows the results of the final ELISA titers of anti-KP OPS IgG in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 (dose 5 μg, treatment group 5) compared to 4-valent KP vaccine (dose 5 μg, experimental group 4).

A strong IgG response was demonstrated to all KP OPS vaccine serotypes (as well as to KP19 CPS), with no significant differences in KP OPS titers of each type in the 4-valent KP vaccine, and in IgG titers for each of the respective KP OPS in 12-valent KP/PA vaccine 1. There was a trend towards a slightly stronger response to KP OPS induced by 12-valent KP/PA vaccine 1. These results also indicate that there is no negative interference in immune responses to each from KP OPS in the 4-valent KP vaccine when combined with the additional 8 PA OPS that are included in the 12-valent KP/PA vaccine 1 (all manufactured as MAPS BP-1).

Immune responses to PA OPS in 12-valent KP/PA vaccine 1 versus 8-valent PA vaccine

Figure 30 shows the results of the final ELISA titers of anti-PA OPS IgG in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 (dose 5 μg, treatment group 5) compared with 8-valent PA vaccine (dose 5 μg, experimental group 3).

As observed in response to KP OPS, strong IgG responses were demonstrated to all serotypes of the PA OPS vaccine, with no significant differences in titers for each type of PA OPS in the 8-valent PA MAPS vaccine, and in IgG titers for each of the respective PA OPS vaccines. in the 12-valent KP/PA vaccine 1. These results also indicate that there is no negative interference in immune responses to each of the PA OPS of the 8-valent vaccine when combined with an additional 4 KP OPS in the 12-valent KP/ PA vaccine 1 (all manufactured as BP-1 MAPS). Strong IgG responses were also demonstrated to each of the PA OPS after one immunization (P1) without a significant increase after the second immunization (P2).

Immune responses to 12-valent KP/PA vaccine 1 at two different dose levels

Figure 31 shows ELISA results of end-point anti-PA OPS IgG titers in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 at 5 μg PS from each BP type at vaccine dose (experimental group 5) compared with 12-valent KP/PA vaccine 1 at a dose of 1 µg PS for each type of BP in the vaccine (experimental group 6).

No significant differences in IgG titers to PA and KP OPS were observed for the 12-valent KP/PA vaccine 1 when comparing experimental groups treated with a dose of 5 µg and a dose of 1 µg, after one (P1) or after two immunizations (P2), with the exception of KP O3 where an improved IgG response was observed after the second immunization.

Separate ELISA results of OPS-specific IgG titers for 12-valent KP/PA vaccine 1 28 days after the first immunization (P1) and 14 days after the second immunization (P2) are shown in Figure 32 and Figure 33. Strong IgG responses to all 12 OPS vaccine serotypes, as well as on KP K19 CPS, are demonstrated for the 12-valent KP/PA vaccine 1 at a dose of 5 μg (Figure 32) and at a dose of 1 μg (Figure 33). However, although the responses were strong, the intensity of the IgG response to OPS KPO2 and KPO3 was not as strong as the response to KPO1 and KPO5, especially after the first immunization. The strongest response was observed for KO K19.

Comparison of individual x-fold induction of anti-OPS IgG using 12-valent KP/PA vaccine 1 was performed for the experimental group with a dose of 5 μg. The results are shown in Figure 34. With the exception of two outliers, all animals showed at least 4-fold induction of ELISA titers from baseline (P2/P0) for each of the OPS serotypes. On average, a 20-fold induction was exceeded for all OPS serotypes with the 12-valent KP/PA vaccine 1. The highest induction was observed for KO 19.

Figure 35 compares the individual x-fold induction of anti-OPS IgG, as determined by ELISA titers, versus baseline (P2/P0) for 12-valent KP/PA vaccine 1 at a dose of 5 μg and a dose of 1 μg. Interestingly, all animals in the 1 μg experimental group showed at least a 4-fold increase from baseline, as already described for the 5 μg experimental group. The mean x-fold increase in IgG titers for all OPS serotypes included in the 12-valent KP/PA vaccine 1 was greater than the 20-fold increase from baseline for all OPS serotypes at the 5 µg dose as well as the 1 µg dose administered to animals. The highest induction folds were observed for KO K19 at both dose levels, and the lowest x-fold inductions were observed for KP O2 and KP O3.

Immune responses to 12-valent KP/PA vaccine 2 at two different dose levels

Figure 36 shows the results of a comparison of responses to KP/PA OPS in pooled sera 28 days after the first vaccination (P1) and 14 days after the second vaccination (P2) from rabbits immunized with 12-valent KP/PA vaccine 2. Notably, in the experimental group with a dose of 5 μg, the vaccine induced a strong IgG response to all OPS serotypes after the first immunization, with no significant increase in titers after the second immunization, with the exception of KP O2 and KP O3 OPS. For these OPS serotypes, the second immunization resulted in a significant increase in titers. IgG responses to OPS were equivalent between the 5 µg and 1 µg dose groups, with the exception of KP O3 OPS where a smaller immune response was observed at the lower dose.

Individual ELISA-derived OPS-specific IgG titers for the 12-valent KP/PA vaccine 2 28 days after the first immunization (P1) and 14 days after the second immunization (P2) are shown in Figure 37 and Figure 38. Strong IgG responses to all 12 OPS vaccine serotypes, as well as on KP K19 CPS, were demonstrated for the 12-valent KP/PA vaccine 2 at 5 μg (Figure 37) and at 1 μg (Figure 38). Titers for each of the OPS serotypes increased further 14 days after the second vaccination. However, the magnitude of this increase was negligible, with the exception of KP O3. As observed in the case of 12-valent KP/PA vaccine 1, the difference in immune response between experimental groups with a dose of 5 μg and a dose of 1 μg was minimal. The highest titers were observed for the OPS KO K19 serotype, and the lowest titers were observed for KP O2 and KP O3.

Summary of OPS Immune Response Results for 12-valent KP/PA Vaccine 1 and 12-V KP/PA Vaccine 2

Anti-OPS immune responses to all PA OPS of the 12-valent KP/PA vaccine 1 in pooled rabbit serum were equivalent to those observed with the 8-valent PA MAPS vaccine.

Anti-OPS immune responses to all KP OPS of the 12-valent KP/PA vaccine 1 in pooled rabbit serum were similar to or slightly better than those of the 4-valent KP MAPS vaccine.

An approximately 200-fold increase from baseline was observed for anti-PA OPS antibodies, and an approximately 50-fold increase from baseline was observed for anti-KP OPS antibodies.

Antibodies in the experimental groups with 5 μg PS per dose of BP and 1 μg PS per dose of BP in pooled sera were similar for 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2, except for KP O3.

Analysis of individual sera in experimental groups with 5 μg PS per dose of BP and 1 μg PS per dose of BP in the case of 12-valent KP/PA vaccine 1 showed a strong response to all KP and PA OPS after one immunization, with a very small increase after the second immunization , with the exception of the response to KP O2 and KP O3 OPS, where the second immunization resulted in a significant increase in IgG antibody titers.

Analysis of individual sera in experimental groups with 5 μg PS per dose of BP and 1 μg PS per dose of BP in the case of the 12-valent KP/PA vaccine 2 also showed a strong response to all KP and PA OPS after one immunization, with a very small increase after the second immunizations, with the exception of the response to KP O3 OPS, where the second immunization resulted in a significant increase in IgG titers.

Carrier protein efficiency

Antibody response to carrier proteins

Carrier protein antibody responses induced by 12-valent KP/PA vaccine 1 with FlaBD2-MrkA and FlaBD2-PcrV as carrier proteins and 12-valent KP/PA vaccine 2 with FlaBD2-MrkA alone were assessed. carrier protein. Responses to carrier proteins in individual rabbits were expressed as ELISA units of IgG for pre-immune (P0) levels and levels reached after one (P1) and two immunizations (P2). ELISA analysis of IgG in individual rabbits for carrier proteins was determined using the respective individual recombinant proteins as coat antigens (ie, FlaBD2, MrkA and PcrV). In each assay, responses to carrier protein components were compared for vaccines 1 and 2 with responses to the 8-valent PA FlaBD2-MrkA vaccine and the 4-valent KP FlaBD2-MrkA vaccine. Information on vaccine doses and treatment groups is shown in Table 21.

(a) ELISA results for various carrier proteins

ELISA results for anti-FlaB-D2 IgG titers are shown in Figure 39. A strong increase in IgG titers was observed after the first immunization, followed by a second increase in titers after the second immunization, in all compared groups. Antibody levels after each vaccination were similar across all groups, two different dose groups (1 µg or 5 µg total PS from each type of complex present in the vaccine formulation, e.g. total 12 µg or 60 µg PS in 12-valent KP/ PA vaccine 1 and vaccine 2) for 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2, as well as groups of 8-valent PA vaccine and 4-valent KP vaccine (each with 5 μg of each PS in the vaccine, for example, just 40 µg or 20 µg of PS in a vaccine, respectively).

ELISA results for anti-MrkA IgG titers are shown in Figure 40. As with FlaBD2, a strong increase in IgG titers was observed after the first immunization in all groups, with a similar increase in each group, between the two dose groups (1 μg or 5 μg total PS of each type of complex present in the vaccine) for the 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2, as well as the 8-valent PA and 4-valent KP vaccine groups (each with 5 µg of PS from the complex each type in the vaccine).

Figure 41 shows the results of the anti-PcrV antibody response following immunization with 1 μg 12-valent KP/PA vaccine 1 and 5 μg of each PS in the vaccine. The IgG response was strong after the first immunization, with an additional increase in titers after the second vaccination. However, responses were similar across the two dose groups.

Carrier protein responses induced by 12-valent KP/PA vaccine 1 with FlaBD2-MrkA and FlaBD2-PcrV as carrier proteins, 12-valent KP/PA vaccine 2 with FlaBD2-MrkA as carrier protein, 8-valent PA vaccine (FlaBD2-MrkA) and 4-valent KP vaccine (FlaBD2-MrkA) expressed as x-fold change in ELISA titers relative to pre-immune (P0) levels after one (P1) and two (P2) immunizations shown in Figure 42.

Strong responses were demonstrated to each of the FlaBD2, MrkA and PcrV carrier proteins in the 12-valent KP/PA vaccine 1, with over a 30-fold change after the first immunization and a 500-fold change after the second immunization. No significant differences in the immunogenicity of the three proteins were found between the 5 μg and 1 μg dose groups.

A strong IgG response to each of the two protein components was observed already after one immunization, with an increase exceeding 30-fold for the two components of the FlaBD2 and MrkA fusion protein in the 12-valent KP/PA vaccine 2. An additional significant increase was observed after the second immunization, which exceeded 1000-fold increase. No significant differences were observed in x-fold changes for FlaBD2 and MrkA between the 5 μg and 1 μg dose groups.

No significant differences were observed in the immunogenicity of the two components FlaBD2 and MrkA between the groups that received the 8-valent PA vaccine or the 4-valent KP vaccine. However, these responses were slightly lower in the 4-valent vaccine group compared to the responses in the 12-valent KP/PA vaccine 2 group and the 8-valent PA vaccine group.

(b) Comparison of x-fold change in IgG responses to different carrier proteins in three studies

Comparison of the x-fold change in IgG responses to FlaB was performed for the results obtained in three immunogenicity studies in rabbits: NCB007, NCB008 and NCB012. In NCB007, a dose of 5 μg of 6-valent PA FlaBD2-MrkA (containing PA O1, O2, O3, O6, O10 and O11 OPS) was compared with an equivalent dose of 6-valent PA FlaB-PcrV, in NCB008 a dose of 5 μg was compared between 4-valent (O1, O2, O3, O5) KP FlaBD2-MrkA and 4-valent KP FlaB-PcrV, and in NCB012, a dose of 5 µg was compared between 12-valent KP/PA vaccine 2 with FlaBD2-MrkA and 12-valent KP/PA vaccine 1 with FlaBD2-MrkA and FlaBD2-PcrV. All vaccines had a BP-1 type scaffold. The results as x-fold changes are presented in Figure 43, Figure 44 and Figure 45.

Figure 43 shows x-fold change in IgG responses to FlaB, with a maximum increase in response to 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2. A good response was observed already after the first immunization, with an additional increase after the second immunization. Examination of responses to 4-valent KP FlaBD2-MrkA and 4-valent FlaB-PcrV complexes in NCB008 showed a good response after the first immunization, with a slight additional increase after the second immunization, to FlaBD-2-MrkA, and almost no response after any immunization. on FlaB-PcrV. A weak response to FlaB was also observed for the 6-valent FlaBD2-MrkA PA complex in NCB007 after the first immunization, but a significant increase occurred after the second vaccination. The response to FlaB-PcrV in NCB007 was strong after the first immunization, with no additional increase after the second immunization.

Figure 44 shows responses to MrkA, with maximum change in response from baseline for 12-valent KP/PA vaccine 2 and 12-valent KP/PA vaccine 1 in NCB012, compared with responses to 4-valent KP FlaBD2-MrkA in NCB008 and 6-valent PA FlaBD2-MrkA in NCB007. Almost no response was observed for MrkA in NCB008 after the first and second vaccinations, while a strong response to MrkA was observed with 6-valent PA FlaBD2-MrkA after the first immunization and an additional increase in response after the second immunization.

Figure 45 shows the x-fold change in IgG response to PcrV, with a maximum increase for the 12-valent KP/PA vaccine 1 in the NCB012 study after the first and second immunizations. Almost no response to PcrV was observed with 4-valent KP FlaB-PcrV in study NCB008. A strong response to PcrV was observed after the first and second immunizations in study NCB007.

(c) Summary of results

- A strong IgG antibody response to all three carrier proteins was demonstrated with 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2.

- Responses to carrier proteins were similar in the 1 µg and 5 µg s dose groups.

- x-fold change obtained for each of the three carrier proteins was approximately 500 to 1000-fold change in the case of the 12-valent KP/PA vaccine 1.

- There was a persistent increase in response between the first and second doses in naive animals. Without wishing to be bound by any theory, in previously exposed adults, approximately maximal responses are expected after a single dose and within 7-10 days.

- The previous problem with low responses to MrkA and PcrV in animals immunized with 4-valent KP vaccine was not present in animals immunized with 12-valent KP/PA vaccine 1 or 12-valent KP/PA vaccine 2.

Lack of TLR5 agonist biological activity in carrier proteins containing only FlaB2

Figure 12B shows the TLR5 bioactivity of MAPS fusion proteins and complexes containing FlaA and FlaB proteins using HEK293 cells expressing the NF-kB driven luciferase reporter. Activation of TLR5 by flagellin causes an increase in reporter expression.

MAPS KP/PA carrier proteins containing only FlaB domain 2 (FlaBD2) were found to lack TLR5 agonist biological activity as seen in the luciferase assay results for FlaBD2-MrkA and FlaBD2-PcrV shown in Figure 46. FlaBD2-MrkA and FlaBD2-PcrV are the two carrier proteins in the 12-valent KP/PA vaccine 1.

Control FlaB and the Rhavi-FlaB-his fusion protein showed strong TLR5 agonist biological activity, while FlaBD2, FlaBD2-MrkA and FlaBD2-PcrV, all containing only FlaB domain 2 (FlaBD2), showed no activity in this assay.

FACS Binding Spectrum Width and Agglutination for Non-OPS Vaccine Strains

Non-OPS vaccine serotypes KP and PA were tested by flow cytometry and agglutination assays to study the coverage of antibodies to the 12-valent KP/PA vaccine 1.

Table 22 shows the results of FACS binding and bacterial agglutination of non-OPS KP strains obtained with pooled sera (AFV1502-1511) from rabbits immunized with 12-valent KP/PA vaccine 1 at a dose of 5 μg of each BP-1. Twelve of the 14 strains tested showed clear differences in sera recognition of P0 and P2 in an agglutination assay and in increased bacterial binding as measured by flow cytometry at a 1:100 dilution of serum.

Table 22: Response to 12-valent KP/PA Vaccine 1 - Binding and Agglutination of Non-OPS KP Vaccine Strains

Strain Country of origin O-type Bacterial agglutination Bacterial binding, flow cytometry The presence of the MrkA gene (PCR) 70721 USA NOT (O1,O2,O3,O5) No No No GN05275 USA NOT (O1,O2,O3,O5) Yes Yes Yes NUH5575 Japan NOT (O1,O2,O3,O5) Yes Yes Yes 170241 USA NOT (O1,O2,O3,O5) Yes Yes Yes 60111 USA NOT (O1,O2,O3,O5) Yes Yes Yes 5205 South Africa NOT (O1,O2,O3,O5) Yes Yes Yes 7075 South Africa NOT (O1,O2,O3,O5) Yes Yes Yes 7069 South Africa NOT (O1,O2,O3,O5) Yes Yes Yes 11818/3 Mali NOT (O1,O2,O3,O5) No No No 9536/15 Mali NOT (O1,O2,O3,O5) Yes Yes Yes 12287/3 Mali NOT (O1,O2,O3,O5) Yes Yes Yes KP1015 Denmark O4 Yes Yes Yes NUH5218 Japan O4 Yes Yes Yes EC1793 Singapore O4 Yes Yes Yes

Table 23 shows the results of FACS binding as well as bacterial agglutination of non-OPS PA strains obtained with pooled sera (AFV1502-1511) from rabbits immunized with 12-valent KP/PA vaccine 1 at a dose of 5 µg of each BP-1. In the case of flagellin B, agglutination of 6 out of 7 strains occurred, and for 7 out of 7, antibody binding was observed by flow cytometry. In the case of flagellin A1, antibody binding was observed for 3 out of 4 strains by flow cytometry. In the case of flagellin A2, there were no signs of antibody recognition for 4 strains.

Table 23: Response to 12-valent KP/PA Vaccine 1 - Binding and Agglutination of Non-OPS PA Vaccine Strains

Strain Country of origin O-serotype (Medimabs) flagellin type Bacterial agglutination Bacterial binding, flow cytometry 12-01831 USA O8 A2 No No 379/2014 Greece O8 B Yes Yes 12-03320 USA O9 A1 No Maybe 425/2015 Greece O9 A2 No No PA0814 Denmark O9 A1 No Yes NUH6346 Japan O9 A2 No No CJLSG533 Japan O9 B Yes Yes 197/2012 Greece O12 B Yes Yes 12-04889 USA O14 B No Yes 12-01852 USA O15 B Yes Yes 12-00232 USA O16 B Yes Yes 10024 South Africa O16 A1 No No 10-04727 USA O17 B Yes Yes 11-04251 USA O17 A1 No Yes PA0002 Singapore Type not defined A2 No No

Fourteen KP strains and 15 PA strains other than OPS vaccine serotypes were tested to determine the breadth of antibody coverage obtained in rabbits to 12-valent KP/PA vaccine 1. Summary:

- Generated antibodies bind and agglutinate 12 out of 14 non-OPS KP strains

- Serotypes that do not contain the MrkA gene do not bind antibodies

- Binding of these strains is probably due to antibodies directed to MrkA

- Developed antibodies bind 10 out of 15 non-OPS vaccine serotypes of PA strains:

- Binding occurs for FlaB strains and some FlaA1 strains

- Binding is most likely due to antibodies directed to FlaB and due to cross-reactivity with FlaA1

- Antibodies directed to carrier proteins (FlaBD2 and MrkA) have coverage extending to non-vaccine OPS serotypes.

Passive protection in animals by antibodies to carrier proteins

The potential ability of carrier proteins to confer passive protection in mice was evaluated in a Klebsiella KP B5055 (O1:K2) burn wound infection model using anti-O1 sera and anti-MrkA sera as indicated in Table 24.

The results suggest that there is a trend towards a protective role for MrkA (FlaBD2-MrkA) in preventing the death of animals infected with the encapsulated KP strain. On day 6 post challenge, 60% (3/5) of the animals were still alive. Anti-OPS O1 antibodies did not provide protection against infection in this experiment.

Table 24: Protection against KP B5055 (O1:K2) burn wound infection using anti-O1 and anti-MrkA sera

Group Infection* Impact Survivability (D6) 1 B5055
(12 cfu/mouse) PBS 33% (1/3) 2 Anti-KP O1 P0 serum 50% (2/4) 3 Anti-KP O1 P2 serum 20% (1/5) 4 Anti-MrkA P0 serum 25% (1/4) 5 Anti-MrkA P2 Serum 60% (3/5) 6 Anti-KP O1+MrkA P0 serum 25% (1/4) 7 Anti-KP O1+MrkA P2 serum 40% (2/5)

*Mice were passively transferred twice with 200 µl of serum, infected sc with 12 CFU B5055 after burn wound.

KPO1 antibody: Pool of 3 highest titer sera from NCB008 with FlaB-PcrV 4-valent KP MAPS

MrkA antibody: Pool of 10 sera from NCB010 with FlaBD2-MrkA 1-in PA MAPS.

Summary of the results of the study of the effectiveness of two carrier proteins in a vaccine preparation

- The carrier proteins FlaBD2-MrkA and FlaBD2-PcrV in 12-valent KP/PA vaccine 1 and 12-valent KP/PA vaccine 2 have been shown to be strong carriers for KP/PA OPS.

- A strong protein response was observed for FlaBD2-MrkA and FlaBD2-PcrV in 12-valent KP/PA vaccine 1, as well as for FlaBD2-MrkA in 12-valent KP/PA vaccine 2.

- The breadth of coverage for antibodies directed to carrier proteins in the 12-valent KP/PA vaccine 1 extended to non-vaccine OPS serotypes.

- Following an experimental design of FlaB containing only FlaB domain 2 (FlaBD2), complete abolition of TLR5 agonist activity was demonstrated for FlaBD2 carrier fusion proteins.

- The first results of infection in a mouse model of a burn wound infected with Klebsiella showed a trend towards a protective role of anti-MrkA antibodies.

Example 12 Functional Assays for 12-valent P. aeruginosa/K. pneumoniae vaccines

Opsonophagocytic kill assays (OPAs)

CFU analyzes with human neutrophils

An opsonophagocytic killing (OPK) assay with human neutrophils was used to assess the functional response of pooled rabbit serum (P2) raised against 12-valent KP/PA vaccine 1 at a dose of 5 µg (NCB012, Table 21), using the O2-free KP serotype (KP O2 K-) strain 7380. The results of the CFU analysis are shown in Figure 47.

The immune serum provided 100% killing of KP O2 bacteria at a 1/10 dilution after 24 hours of incubation, while the serum at a 1/100 dilution had no bacterial killing activity. The killing activity of the serum could be related to either KP O2 OPS or MrkA or a combination of the two vaccine components.

Analysis of CFU with macrophage cell line J774

In this RFU assay, the J774 macrophage cell line was used to evaluate functional activity in the presence or absence of the antibiotic gentamicin. Some antibiotics cannot enter eukaryotic cells. Thus, these antibiotics cannot harm bacteria that are already internalized. The use of such antibiotics makes it possible to distinguish between bacteria that were able to enter eukaryotic cells and those that could not. The use of such an antibiotic in a culture of eukaryotic cells infected with bacteria would kill bacteria that remain outside the cells, but would not affect the bacteria that entered the cells. The antibiotic of choice for this assay was the aminoglycoside gentamicin.

Pooled rabbit sera from rabbits in experimental group 5 that received a 5 µg dose of 12-valent KP/PA vaccine 1 in NCB012 and had the highest anti-OPS titers (1502, 1506, 1508, 1511) were used in the PFU assay with the J774 macrophage cell line to assess the effect of serum on the phagocytic uptake of Klebsiella strains KP O1, KP O2, KP O3 and KP O5.

Macrophages of the J774 cell line were seeded in 96-well plates 24 hours before the experiment. Klebsiella strains of different O-types were grown to mid-log growth phase in broth and pooled sera were used in a kill assay. Approximately 1×10 ⁵ cfu of bacteria were incubated with pooled pre-immune (P0) or immune (P2) sera for 30 minutes. The opsonized bacteria were incubated with J774 cells for 15 minutes at room temperature, sufficient time for antibody-mediated phagocytosis to occur. Unbound bacteria were removed from the cells and fresh buffer ±50 μg/ml gentamicin was added for another 20 minutes. Cells were washed twice with PBS, then lysed by adding pure water. Ten ml from each well were plated in duplicate on plates and colony forming units (CFU) were counted the next day.

The results of this study are presented in Figures 48A and 48B. The results of phagocytic uptake by J774 cells of Klebsiella bacteria opsonized with immune or pre-immune serum at 1/20, 1/100 and 1/500 dilutions showed increased uptake of KP O1, KP O2, KP O3 and KP O5 in the presence of immune serum and in the presence of gentamicin ( Figure 48A). Maximum absorption was observed at a 1/100 dilution for KP O1, KP O2 and KP O3. In the case of KP O5, the absorbance was similar at 1/20 and 1/100 dilutions.

In the absence of gentamicin, significant uptake by J774 cells of KP O1, KP O3, and KP O5 was also observed with P2 serum, compared to pre-immune serum, which had less bacterial uptake. Results for KP O2 are not shown because the number of CFUs was too high to count (Figure 48B). For KP O1 and KP O3, absorbance decreased at higher dilution, and for KP O5, maximum absorbance was observed at 1/100 dilution, slightly less absorbance at 1/20 dilution, and minimum absorbance at 1/500 dilution.

Pooled sera from rabbits dosed with 5 µg of 12-valent KP/PA vaccine 1 in NCB012 and having the highest anti-OPS titers (1502, 1506, 1508, 1511) were also used in the PFU assay with the J774 macrophage cell line to assess the effect of serum on phagocytic uptake of both Pseudomonas PA (O5:FlaB and O6:FlaA1) and Klebsiella strain KP O5 (strain 6997). The assay was performed as described above for testing the KP O1, KP O2, KP O3 and KP O5 strains KP O1, KP O2, KP O3 and KP O5 in the J774 CFU assay with Klebsiella .

The results of this assay are shown in Figures 49, 50 and 51. Immune serum (P0) mediated the uptake of each of these bacterial strains by phagocytosis as shown in Figure 49. Uptake of the bacteria by J774 macrophage-like cells was also demonstrated by microscopy using GFP-labeled PA (PA O1, PA O5 FlaB) as illustrated in Figure 50. PA exposed to immune serum was actively taken up by the cells. Several P2-treated PA isolates in each J774 cell are shown enlarged in Figure 50. Figure 51 shows that a higher percentage of cells (left panel) and a higher number of bacteria per cell (right panel) were observed for cells treated with immune serum compared to cells treated with pre-immune serum.

CFU analysis with HL-60

In this assay, KP O2 strain 7380 bacteria were first opsonized with serum and then HL-60 cells were added to the mixture. After the mixture was incubated for 45 minutes, viable bacteria were counted. The OFU titer was defined as the titer at which less than 50% of the bacteria survived compared to the negative control (bacteria+HL-60 cells only).

Figure 52 and Table 25 show the results of HL60 PFU analysis using KP O2 strain 7380 and pooled sera from rabbits immunized with a 5 μg dose of 12-valent KP/PA vaccine 1. Rabbit serum against heat-killed (HK) KP O2 to and served as a positive control after immunization. The analysis was performed using sera in dilutions from 1/200 to 1/204800.

Sera from rabbits immunized with the 12-valent KP/PA vaccine 1 showed significant PFU activity against KP O2 strain 7380, while the corresponding pre-immune sera showed a significantly weaker bacterial killing effect. The killing activity of the serum could be associated with antibodies to either KP O2 OPS or MrkA, or both. Information on OFU titers for sera before and after immunization, as well as for positive controls, is shown in Table 25. There was a significant difference (>15-fold increase) in killing activity between sera before (1/800) and after immunization (1 /12800).

Table 25: OFU titer for KP O2 strain 7380 in HL-60 assay

Serum Time point Titer NCB012 (combined) P0 1/800 P2 1/12800 Heat Killed Bacteria (Combined) Before Immunization <1/200 After immunization >1/204800

A similar assay was performed using KP O5 strain 6997. Bacteria were mixed with either preimmune (P0) or immune (P2) pooled sera from the NCB012 study and added to HL-60 cells in the presence of complement (Table 26). A 50 percent shorter survival was observed with immune serum up to a titer of 1:1280, while pooled preimmune serum resulted in a modest reduction in survival at a serum dilution of 1:40.

Table 26: Percent Survival KP O5 of 6997 Strain in HL-60 Cells Using Pooled Serum

Breeding 1:40 1:160 1:320 1:1280 NCB012 P0 83 >100 >100 >100 NCB012 P2 23 50 48 50

Findings for different analyzes of FFU

Three different OFU assays were used to assess the efficacy of antibodies induced in rabbits following two administrations of the 12-valent KP/PA vaccine 1. Significant OFU activity could be demonstrated against KP serotypes O1, O2, O3 and O5 in assays using either human PMN, macrophage J774 cells, or differentiated HL60 cells. OFU activity could be associated with either OPS-specific antibodies and/or antibodies to the MrkA carrier protein. The uptake of KP and PA by phagocytic cells stimulated by 12-valent immune sera was probably related to the anti-OPS antibody.

Protection against cellular toxicity

Following the protocol described in Example 7, (Cytotoxicity Assays for Pseudomonas PcrV Protein Constructs ), Pseudomonas strain PAK (O6 FlaA1) was grown overnight and used to infect human lung epithelial A549 cell monolayers in the presence of different concentrations of serum (10 %, 5%, 2.5%, or none) from rabbits immunized with 12-valent KP/PA vaccine 1 containing an anti-PcrV antibody generated by an immune response to the MAPS carrier protein or in the presence of preimmune serum from the same rabbits in the same range of serum concentrations. Pseudomonas- induced cytotoxicity for A549 is characterized by cell rounding as the cells become apoptotic and detach from the surface of the tissue culture plate. Anti-PcrV antibody-mediated protection against cytotoxicity is indicated by a decrease in the number of rounded (apoptotic) cells in the microscope field, compared with cells treated with pre-immune serum at a similar concentration. The maximum difference in the number of rounded (apoptotic) cells between samples treated with immune and pre-immune sera was observed at a serum concentration of 2.5% (Figure 53, left panel). Photomicrographs of representative PAK-infected A549 cells treated with 2.5% serum demonstrate these differences as a decrease in the number of round cells with high refraction in the sample treated with immune serum (Figure 53, right panel). Similar images were used to estimate the number of affected cells at each serum concentration (Figure 53, left panel).

Decrease in P. aeruginosa motility due to anti-FlaB antibodies

As shown in Figure 54, in cells treated with pooled sera from rabbits immunized with 5 μg of each PS (e.g. total 60 μg of PS in the vaccine; 5 μg of PS from each BP type in the vaccine) in a 12-valent KP/PA vaccine 1 , observed reduced motility of the PA O5 FlaB strain by the anti-FlaB antibody compared to pre-immune serum treatment.

Aggregation P. aeruginosa due to antibodies to FlaB

P. aeruginosa strain PAO1 (O5 FlaB) was grown overnight and suspended in PBS to a concentration of 10 ⁷ CFU/ml, then incubated with 1% serum from rabbits vaccinated with a 4-valent KP vaccine, or with preimmune serum from the same rabbits, for 1 hour at room temperature. Anti-FlaB antibody in sera resulting from an immune response to the Rhavi-FlaBD2-MrkA-His carrier protein was able to agglutinate FlaB-expressing Pseudomonas as evidenced by bacterial clusters that are visible in black rod micrographs (Figure 55 , right panel) labeled "aggregation", while such aggregates were absent in bacteria treated with pre-immune serum (Figure 55, left panel). FlaBD2 was the only Pseudomonas antigen in this vaccine preparation and the presence of cross-linked bacteria indicates that the carrier protein causes the production of a functional antibody capable of recognizing the native epitope in bacterial flagellin. Sera from rabbits immunized with the 12-valent KP/PA MAPS 1 vaccine was able to agglutinate O-type FlaB-expressing Pseudomonas absent from the vaccine (data not shown), strongly suggesting that the FlaBD2-containing carrier protein caused production of a functional anti-FlaB antibody when using a 12-valent preparation, although a contribution to the observed agglutination that cross-reactivity between Pseudomonas O-types cannot be ruled out.

Adhesion inhibition assay

In this assay, pooled sera from the NCB012 study were used to evaluate adhesion inhibition by antibodies raised against various exemplary vaccines. Pili type 3 expressing bacterial cells KP O5 strain 6997 were mixed with preimmune and immune serum (10%) and incubated with A549 epithelial cells. Non-attached bacteria were washed off the cells, followed by cell lysis and determination of the number of attached bacteria. Inhibition of adhesion resulted in fewer CFUs per plate. Figure 56 shows pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1, pooled sera from rabbits immunized with 8-valent PA vaccine (MrkA is the only KP antigen in the 8-valent PA vaccine), pooled sera from rabbits , immunized with a 4-valent KP vaccine, blocked the adhesion of KP O5 strain 6997 to A549 cells. Without wishing to be bound by any theory, since pooled sera from rabbits immunized with the 8-valent PA OPS vaccine alone also inhibited adhesion, this suggested that the anti-MrkA antibody induced by the carrier protein had a protective effect.

Example 13: Mice Protection Analysis

Passive immunization studies were performed in mice using immune sera obtained from rabbits immunized with KP/PA vaccines. Table 21 provides information on the vaccine formulations used to immunize rabbits. Preimmune and immune rabbit sera (as pooled sera) were used to passively immunize mice as described for other models. Information on the different strains used for challenge is also provided for each study.

Study of passive protection of mice against lethal PA infection using sera from rabbits immunized with 12-valent KP/PA vaccine 1

A passive protection study in mice was performed using pooled sera from rabbits immunized with 5 µg of each PS (e.g. total 60 µg PS in vaccine; 5 µg PS from each BP type in vaccine) in 12-valent KP/PA vaccine 1 (Table 21 , group 5) in study NCB012. Mice were pre-injected with either pre-immune or immune serum (pooled rabbit serum) and challenged ip with different strains of PA.

At lethal doses of PA challenge, immune sera protected mice from infection with PA O5 and PA O6. There was a trend towards a protective effect when challenged with PA O10 and O11, however, the marked difference in survival of mice treated with pre-immune serum and challenged with 1×10 ⁸ CFU and only 3 times lower dose shows how steep the dose-response curve can be for many gram-negative bacteria. This often creates problems in conducting such analyses. Information on survival rates is shown in Table 27.

Table 27: Survival rates of mice in a lethal PA challenge study

bacteria Dose Survival Preimmune immune Pseudomonas O5 FlaB strain M2 1.3x 10 ⁷ CFU 1/5 4/5 Pseudomonas O5 FlaB strain M2 1.1x 10 ⁸ CFU 0/5 4/5 Pseudomonas O6 FlaA2 strain SBI-N 1x 10 ⁸ cfu 0/5 5/5 Pseudomonas O10 FlaB strain 17002 1x 10 ⁷ CFU 2/5 5/5 Pseudomonas O11 FlaB strain 1071 1x 10 ⁸ cfu 0/5 3/5 Pseudomonas O11 FlaB strain 1071 3x 10 ⁷ CFU 4/5 5/5

Study of the passive defense of mice against Klebsiella KP O3 using sera from rabbits immunized with 12-valent KP/PA vaccine 1

Serum from the four rabbits with the highest anti-KP-O3 titers (rabbits 1502, 1506, 1508, and 1511; Table 28) immunized with 12-valent KP/PA vaccine 1 at 5 µg of each PS (e.g., a total of 60 µg of PS per vaccine; 5 μg PS from each type of BP in the vaccine) (group 5) in the NCB012 study, pooled and administered by ip injection (pre-immune or immune serum) 20 hours and 2 hours before the iv challenge of KP O3 strain 700603 at a dose of 50000 CFU in 0.1 ml.

Table 28: Titres of preimmune and immune rabbit sera included in pooled samples

Rabbit number/
serum sample Pre-immune serum titer against O3 Immune serum titer against O3 1502 5 1017 1506 10 2306 1508 19 4024 1511 5 704

Four hours after infection, mice were euthanized, blood, liver and spleen were collected, and the number of colonies was determined. The results are shown in Table 29. Mice treated with pooled immune sera showed clearance from the blood and fewer bacteria in the liver and spleen compared to mice treated with pre-immune sera from the same rabbits. These results were similar to those for bacterial clearance using equivalent titer sera from rabbits immunized with 4-valent MAPS KP in study NCB008.

Table 29: Geometric Mean Number of CFU for Clearance from Blood and Viable Bacteria in Organs after Infection Klebsiella KP O3

Serum sample Blood Liver Spleen NCB008 Preimmune 5235 12590 7353290 NCB008 Immune without bacteria 3302 89159 NCB012 Preimmune 10020 27180 2635845 NCB012 Immune without bacteria 2952 195421

Study of the passive defense of mice against Klebsiella KP O1 strain B5055 using sera from rabbits immunized with 12-valent KP/PA vaccine 1

In this study, three mice per group were injected with pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 at a dose of 5 µg of each PS (e.g., total 60 µg of PS in the vaccine; 5 µg of PS from each BP type in the vaccine) ( Table 21, group 5) in the NCB012 study (pre-immune and immune serum), followed by intravenous (IV) infection with KP O1 strain B5055 at a dose of 5×10 ⁶ . The results of the analysis of clearance from the blood at 1 and 4 hours after infection are shown in Table 30 as the geometric mean value of CFU/ml of blood, and are presented in Figure 57 for individual mice.

Four hours after challenge, mice in the immune serum group showed a significant decrease in CFU/ml in the blood, while CFU/ml in mice treated with pre-immune serum remained elevated.

Table 30: Results of analysis of blood clearance as geometric mean cfu/ml in mice after challenge with KP O1 strain B5055

Serum sample Dose Blood 1 hour 4 hours Preimmune 5x 10 ⁶ cfu 1362840 4449118 immune 5x 10 ⁶ cfu 1593725 91308

The results of determining the load of organs in the spleen and liver of these mice are shown in Table 31 as the geometric mean of CFU/g of tissue for three mice. Figure 58 shows the results of determining the load of organs in the spleen and liver of three mice 20 hours after infection.

Two of the three mice treated with immune serum showed a decrease in geometric mean CFU in the liver compared to mice treated with preimmune serum. The results for the spleen show a similar pattern in mice pre-treated with immune sera, indicating a significant decrease in the score at 20 hours post-challenge compared to mice treated with pre-immune sera, which had a significantly higher bacterial count.

Table 31: Organ loading in mice as geometric mean cfu/g tissue after infection with KP O1 strain B5055

Dose Spleen Liver Preimmune 5x 10 ⁶ cfu 672204849 21447778 immune 5x 10 ⁶ cfu 12867923 3308911

Study of the passive defense of mice against Klebsiella KP O5 strain 6997 using sera from rabbits immunized with 12-valent KP/PA vaccine 1

In this study, three mice per group were injected with pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 at a dose of 5 µg of each PS (e.g., total 60 µg of PS in the vaccine; 5 µg of PS from each type of BP in the vaccine) per study NCB012 (Table 21, group 5, preimmune and immune serum), followed by intravenous infection with KP O5 strain 6997 at a dose of 5×10 ⁶ . The results of the analysis of clearance from the blood at 1 and 4 hours after infection are shown in Table 32 as the average value of CFU/ml of blood, and are presented in Figure 59 for individual mice.

Four hours after challenge, the blood of 2 of 3 mice in the immune serum group was cleared of bacteria, with a significant decrease in CFU load in the third mouse, while the number of CFU in mice treated with preimmune serum remained elevated (in 2 of 3 mice) four hours after infection.

Table 32: Results of analysis of blood clearance as geometric mean cfu/ml in mice after challenge with KP O5 strain 6997

Dose Blood 1 hour 4 hours Preimmune 5x 10 ⁶ cfu 1910 458 immune 5x 10 ⁶ cfu 2340 42

The spleen organ loading results of these mice are shown in Table 33 as geometric mean cfu/g tissue for three mice, and Figure 60 shows the spleen organ loading results of three mice 20 hours post infection.

It was found that the spleen of mice treated with immune serum had much less bacteria 20 hours after infection than mice treated with pre-immune serum.

Table 33: Organ loading in mice as geometric mean cfu/g tissue after infection with KP O5 strain 6997

Dose Spleen Preimmune 5x 10 ⁶ cfu 93750 immune 5x 10 ⁶ cfu 20432

Study of the passive defense of mice against Klebsiella KP O2 (strain KPN 12) using sera from rabbits immunized with 12-valent KP/PA vaccine 1

In this study, CD-1 mice (3/group) were injected with 0.2 ml of pooled pre-immune or immune serum (pooled serum from 10 rabbits, study NCB012) 24 hours and 2 hours prior to 2 x 10 ⁵ cfu IV administration. KP O2 strain KPN-12. Spleens were harvested 72 hours after infection and the bacterial load per gram of tissue was determined. Mice treated with immune serum had fewer viable bacteria compared to mice treated with pre-immune serum (7978 vs 114687, mean cfu; Figure 61). The pooled immune serum stimulated the clearance of KP O2 (strain KPN-12) from the circulatory system.

Several experiments were performed with pre-immune and immune sera from the NCB012 study to develop and optimize a passive protection protocol in a Klebsiella KP O2 lethal challenge model. Similar experiments can be performed to develop and optimize a passive protection protocol for other organisms, other vaccines or immunogenic compositions, and/or other pathogens/strains. Variables such as dose of bacteria for challenge, delivery of the inoculum (eg, opsonization of bacteria prior to injection), and the amount, time of administration, and serum concentration for passive protection (eg, serum obtained with a polysaccharide dose of either 5 µg or 1 µg) were studied. ). Exemplary test parameters and results are shown in Figure 62.

Th17 response and colonization in CD-1 mice - upon challenge

This experiment was designed to determine the ability of KP O1 MAPS vaccine with FlaBD2 and MrkA at three doses administered to mice, with each dose administered fourteen days after the previous dose, to reduce bacterial colonization following bacterial challenge two weeks after the last immunization. Two groups of CD-1 mice were to undergo bacterial challenge; in one group, animals were immunized with KPO1/FlaBD2-MrkA MAPS vaccine, and in the other group, animals were immunized with buffer alone (PBS).

Five mice per group were injected with 3-5×10 ³ CFU of KP B5055 (O1:K2) intratracheally (i/t). 48 hours after bacterial infection, the lungs of the animals were collected and weighed, the homogenates were plated for bacteria count, and then centrifuged. Supernatants were evaluated for IL-17a levels. In parallel, spleen cells were co-cultured with PBS, FlaBD2 and MrkA as described above and supernatants were assessed for IL-17a levels three days later.

Figure 63 shows the induction of IL-17a in stimulated spleen cells after bacterial challenge. Significant induction was observed for FlaBD2-stimulated spleen cells (p=0.02).

Effect of immunization on colonization of the GI tract P. aeruginosa

The following experimental protocol was developed to study the effect of immunization with a monovalent PA O6 MAPS vaccine on colonization of the gastrointestinal (GI) tract by P. aeruginosa .

Eight Sprague-Dawley rats were immunized twice with 0.5 ml sc at two-week intervals with a monovalent MAPS vaccine, MA6-93-PAO6 ( Klebsiella pneumoniae K19: Pseudomonas aeruginosa O6/Rhavi-FlaBD2-MrkA-his BP-1). Seven rats were immunized with buffer alone. Fourteen days after the second immunization, rats were injected with ceftriaxone (50 mg/kg IM) and continued to receive once daily antibiotic doses for nine days. Rats were treated with antibiotics to overcome resistance to colonization in the GI tract by opportunistic bacteria such as PA and to mimic the clinical situation. After four days of antibiotic use alone, rats were administered P. aeruginosa (PA, IATS O6) at a dose of 10 ⁸ CFU by gavage for a total of five days. After five daily doses of PA and nine daily doses of ceftriaxone, rats were euthanized, the caecum was removed, weighed, and then cultured. Blood was collected to assess antibody levels prior to antibiotic administration, bacteria were injected, and stool samples were obtained for PA culture one day before and two days after the first dose of PA. A detailed description can be found at the top of Figure 64.

The results of a colonization study in rats using two doses of the PA O6 FlaBD2-MrkA MAPS vaccine are shown at the bottom of Figure 64. The rats that received the PA O6 vaccine had an elevated IgG titer as determined by the ELISA method compared to the rats that received PBS (lower left of the figure). ). Vaccinated rats also showed, on average, less than 10 ³ CFU/g of bacteria compared to control rats, which had, on average, >10 ⁴ CFU/g (bottom right of the figure). The results of the study show a reduction in caecal PA O6 colonization after two immunizations with the monovalent PA O6 MAPS vaccine.

Example 14 Anti-OPS Antibody to Carbapenem-Resistant KP Strains (CRKP) Expressing the Gal-III Epitope

Unlike other classes of bacterial carbapenemases (eg, oxa-48 or metallo-beta-lactamases), distribution of KPC ( K. pneumoniae carbapenemase) appears to be at least partially clonal. A particular lineage, termed sequence type (ST) 258, has been shown to be responsible for the majority of KPC-producing Klebsiella infections that are endemic in several geographic regions. Currently, >30% of all nosocomial K. pneumoniae infections in the northeastern United States are due to CRKP, and approximately 70% of these belong to the KPC-producing ST258 clone. Similarly, this clone (including single-locus ST variants) causes epidemics in other parts of the world, including Israel, Poland, Italy, Greece, South America, and China. Recently, a study of the molecular evolution of ST258 has identified two clades that are associated with the expression of different capsular polysaccharides (Chen 2014a, Chen 2014b, DeLeo 2014). Klebsiella pneumoniae ST258 is a globally widespread, highly drug-resistant nosocomial clone with limited treatment options.

The KP O1 and KP O2 antigens are composed of galactose homopolymers, that is, galactans. The KP O2 antigen, called d-galactan-I (gal-I), is composed of disaccharide repeat units of galactose. In the case of KP O1, gal-I is covered by repeating units of an antigenically distinct galactose disaccharide called d-galactan-II (gal-II), which determines serotype specificity. The rare serotype KP O2ac has a similar structure, i.e. the repeats of the gal-I subunits are covered by a polymer from other, in this case non-galactan, repeat units. Accordingly, all KP O1, KP O2, and KP O2ac strains have a common gal-I antigen; however, if in KP O2 this is the only O-antigenic determinant, in the case of KP O1 and KP O2ac, gal-I is shielded by external repeating units. In addition, gal-I disaccharide repeat units can have stoichiometric and non-stoichiometric additions of O-acetyl or galactosyl groups, which create subtypes in the KP O2 serogroup (Figure 65). A clear majority of ST258 isolates express the modified lipopolysaccharide O antigen d-galactan-I with galactosyl group-containing Gal-I, hereinafter referred to as d-galactan-III (Szijártó V. et al., 2016) .

KP O1 OPS consists of a short D-Gal-I or D-Gal-III "primer" region followed by a long D-Gal-II region. KP O2 OPS consists only of D-Gal-I or D-Gal-III (Figure 65). The Gal-II epitope is characteristic only for the KP O1 serotype.

B5055 OPS, which is the source of the KP O1 antigen in the MAPS vaccine, has a short DGal-III region followed by a long D-Gal-II polymer. KP 7380 (source of vaccine antigen KP O2) has only D-Gal-I.

Whether the KP/PA MAPS vaccine was able to induce antibodies to the Gal-III epitope and thus have the potential to prevent infections caused by clonal strains of KPCR ST 258 was investigated. Pooled sera (P2) from rabbits immunized with 4-valent KP PRO -CN MAPS vaccine at a dose of 5 μg was used in a flow cytometric assay to demonstrate the ability to bind to bacteria expressing KP D-Gal-III and KP D-Gal-I epitopes. In this case, the only adequate response would be the recognition of D-Gal-I O2 and D-Gal-III O2 OPS, D-Gal-II O1 OPS. The results showed increased binding, compared to the corresponding pre-immune serum (P0), for both KP O1 and KP O2, to D-Gal III and D-Gal I OPS (Figure 66). Thus, it is likely that antibodies have been developed against all three types of KP D-Gal.

The results of the flow cytometry analysis shown in Table 34 and Figure 67 show that antibodies in pooled sera from rabbits immunized with 12-valent KP/PA vaccine 1 bound two of the three ST258 strains from the bacteremic isolates. Immune sera prepared for the 12-valent KP/PA vaccine 1 bound three of the four serotype strains not included in the vaccine, including strains with MrkA alleles with slight sequence variations (Figure 67). Without being bound by theory, these data suggest that most KP strains, not just those with serotypes of the OPS components included in the 12-valent KP/PA vaccine 1, will be recognized by vaccine-induced antibodies by component of the MrkA fusion protein.

Table 34: Results of determination by flow cytometry of the binding of serum induced by 12-valent vaccine 1 to strains Klebsiella serotypes not included in the vaccine and having mutant MrkA alleles

Strain A country Serotype ST K-type MrkA alleles Binding by flow cytometry 1405472 USA O1 258 Wzi154 Unknown Yes 1105463 USA O2 258 Wzi154 Unknown No 1103113 USA O2 258 Wzi154 Conservative Yes NUH5575 Japan Not O1, O2, O3 or O5 Unknown Wzi41 T23N Yes 7075 South Africa Not O1, O2, O3 or O5 Unknown Wzi212 A9V, T12S Yes 170241 USA Not O1, O2, O3 or O5 Unknown K24 Conservative Yes 118181/3 DRC Not O1, O2, O3 or O5 Unknown Wzi52 Without MrkA No

Results of determining the binding ability of the 4-valent KP MAPS vaccine to KPs expressing D-Gal-I and D-Gal-III epitopes indicate that the 12-valent KP/PA vaccine has the potential to generate antibodies to the Gal-III epitope. and to the prevention of infection caused by KPCR ST 258 clonal strains.

Example 15: Receive BP-1.2

The beneficial properties of BP-1 are the presence of biotin residues located exclusively on the CPS aspect of the BP, which may be advantageous in terms of product characterization and minimal disruption of OPS epitopes. A variant of the method for obtaining such a scaffold was developed, which is based on the binding of biotin residues to CPS in an orthogonal region, using carboxylates of polysaccharide uronic acid residues instead of aldehyde. This reaction, which includes the use of carbodiimide (EDC) and N-hydroxysuccinimide (NHS), is highly controlled and provides a constant level of biotinylation in CPS and final BP. The BP obtained in this way is called BP-1.2. A diagram of an exemplary process for obtaining BP-1.2 is presented in Figure 2B.

Some of the advantages of this method include providing reproducible biotinylation with better control over the level of biotin derivatization and better product reproducibility since all BPs come from the same batch of biotinylated CPS, resulting in a stable biotinylated intermediate that can be stored in large batches and the ability to achieving a higher OPS/CPS ratio in the BP as more aldehyde groups are available on the CPS resulting in less competition for the same binding sites.

Example 16 Immunization Studies with BP-1 and BP-1.2 Immunogenic Complexes

Quadruple vaccines were prepared as either MAPS BP-1 or MAPS BP-1.2 and formulated according to Table 35. These formulations were administered to rabbits using the protocols described above, for example, in Examples 6 and 11.

Table 35: Aggregate data on vaccine formulations used in the comparison study for BP-1 and BP-1.2.

Group Vaccine pathogen carrier protein Serotype Dose PS (total) Protein:PS ratio (total protein) Adjuvant rabbits 1 4-valent KP/PA vaccine
BP-1
5 μg per dose PS Pseudomonas Rhavi-FlaBD2-MrkA-his O6, O11 5 mcg
(20 mcg) 3:1
(60 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O3, O5 2 4-valent KP/PA vaccine
BP-1.2
5 μg per dose PS Pseudomonas Rhavi-FlaBD2-MrkA-his O6, O11 5 mcg
(20 mcg) 3:1
(60 mcg) 625 µg AlPO ₄ 10 Klebsiella Rhavi-FlaBD2-MrkA-his O3, O5

A strong response in the form of production of IgG antibodies to KP and PA OPS was observed in both groups BP-1 and BP-1.2. Individual IgG titers for OPS and CPS K19 for group 1 and group 2 are shown in Figure 68. Anti-OPS immune responses to both PA O6 and PA O11, OPS were equivalent for the BP-1 and BP-1.2 MAPS constructs. Similarly, equivalent responses were observed for KP O3 and KP O5 OPS, although significantly higher titers for KP O3 with the BP-1.2 MAPS construct (approximately 5-6 times higher geometric mean titers). Anti-K19 CPS IgG titers were equivalent for both BP-1 and BP-1.2 MAPS constructs. A strong response in the form of IgG antibodies to both FlaB-D2 and MrkA carrier proteins was also observed in both BP-1 and BP-1.2 groups. Antibody responses (pooled serum) to both carrier proteins were comparable in both BP-1 and BP-1.2 groups (Figure 69). Summary results are shown in Table 36.

Table 36: Comparison of the immunogenicity of BP-1 MAPS (Group 1) and BP-1.2 MAPS (Group 2): IgG response to OPS

Individual IgG titers for OPS and CPS K19 for group 1 and group 2 are shown in Figure 68. Anti-OPS immune responses to both PA O6 and PA O11, OPS were equivalent for the BP-1 and BP-1.2 MAPS constructs. Similarly, equivalent responses were observed for KP O3 and KP O5 OPS, although significantly higher titers for KP O3 with the BP-1.2 MAPS construct (approximately 5-6 times higher geometric mean titers). Anti-K19 CPS IgG titers were equivalent for both BP-1 and BP-1.2 MAPS constructs.

Study NCB013 compared 2-valent KP/PA vaccines with BP-1 (framework 1) and BP-1.2 (framework 1.2) in rabbits (see also Table 35 for information on vaccine preparations). Figure 70 and Figure 71 show individual antibody responses to the two carrier proteins FlaBD2 and MrkA for BP-1 and BP-1.2 (KP/PA, 4-valent), respectively. A significant increase in antibody titers was observed after the first immunization for both carrier proteins and both scaffolds. Antibody titers increased further after the second immunization. A strong response in the form of antibody production in the groups for both FlaBD2 and MrkA proteins, and both scaffolds BP-1 and BP-1.2 was observed, and the production of antibodies to both proteins was comparable in groups BP-1 and BP-1.2.

Sequences

SEQ ID Description Subsequence SEQ ID NO: 1 K. pneumoniae, type I fimbrial protein mkiktlamivvsalslsstaaladtttvnggtvhfkgevvnaacavdagsidqtvqlgqvrsaklatagstssavgfniqlddcdttvatkasvafagtaidssnttvlalqnsaagsatnvgvqildntgtplaldgatfsaattlndgpniipfqaryyatgaatagianadatfkvqye SEQ ID NO: 2 K. pneumoniae, type III conserved fimbrial protein MrkA MKKVLLSAAMATAFFGMTAAHAADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYQ SEQ ID NO: 3 K. pneumoniae, type III stabilized fimbrial protein MrkA ADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYVSQGGGGGADTTVGK SEQ ID NO: 4 Sequence in PsL-binding mAb Cam-003 QVRLQQSGPGLVKPSETLSLTCTVSGGSTSPYFWSWLRQPPGKGLEWIGYIHSNGGTNYNPSLKSRLTISGDTSKNQFSLNLSFVTAADTALYYCARTDYDVYGPAFDIWGQGTMVTV SEQ ID NO: 5 Sequence in PsL-binding mAb Cam-003 SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL SEQ ID NO: 6 P. aeruginosa, FliC flagellin subtype A1 MALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFKADGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFSDGDTISYVSKAGKDGSGAITSAVSGVVIADTGSTGVGTAAGVTPSATAFAKTNDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLR SEQ ID NO: 7 P. aeruginosa, FliC flagellin subtype B MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLR SEQ ID NO: 8 P. aeruginosa, type III secretory system PcrV virulence factor (TTSS) MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI SEQ ID NO: 9 P. aeruginosa, flagellin FlaA2 MALTVNTNIASLNTQRNLNNSSAS LNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLR SEQ ID NO: 10 P. aeruginosa, D2 domain of flagellin FlaB lacking TLR5 binding motif GSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSTGTQAAK SEQ ID NO: 11 P. aeruginosa, FlaA1 flagellin D2 domain lacking TLR5-binding motif NGTYFKADGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFSDGDTISYVSKAGKDGSGAITSAAVSGVVIADTGSTGVGTAAGVTPSATAFAKTNDT SEQ ID NO: 12 P. aeruginosa, FlaA2 flagellin D2 domain lacking TLR5-binding motif NGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDT SEQ ID NO: 13 rizavidin MIITSLYATFGTIADGRRTSGGKTMIRTNAVAALVFAVATSALAFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD SEQ ID NO: 14 rizavidin without signal sequences FDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKD SEQ ID NO: 15 GGGGSSS linker GGGGSSS SEQ ID NO: 16 Rhavi-PcrV-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH SEQ ID NO: 17 Rhavi-MrkA-donor strand complementation-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSGSGHHHHHH SEQ ID NO: 18 Rhavi-FlaA1-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFKADGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFSDGDTISYVSKAGKDGSGAITSAVSGVVIADTGSTGVGTAAGVTPSATAFAKTNDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLRGSGHHHHHH SEQ ID NO: 19 Rhavi-FlaA2-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNNSSASLNTSLQRLSTGSRINSAKDDAAGLQIANRLTSQVNGLNVATKNANDGISLAQTAEGALQQSTNILQRMRDLSLQSANGSNSDSERTALNGEVKQLQKELDRISNTTTFGGRKLLDGSFGVASFQVGSAANEIISVGIDEMSAESLNGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTVAKIDISTAKGAQSAVLVIDEAIKQIDAQRADLGAVQNRFDNTINNLKNIGENVSAARGRIEDTDFAAETANLTKNQVLQQAGTAILAQANQLPQSVLSLLRGSGHHHHHH SEQ ID NO: 20 Rhavi-FlaB-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLRGSGHHHHHH SEQ ID NO: 21 rhavi-flab-domain2-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSGSGHHHHHH SEQ ID NO: 22 rhavi-flaa2-domain2-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMNGTYFTATGGGAVTAATASGTVDIAIGITGGSAVNVKVDMKGNETAEQAAAKIAAAVNDANVGIGAFTDGAQISYVSKASADGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTGSGHHHHHH SEQ ID NO: 23 Rhavi-FlaB-PcrV-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISGLNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQAELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLPQAVLSLLRAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH SEQ ID NO: 24 Rhavi-FlaB-Domain2-MrkA-donor strand complementation-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSAAAAMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSGSGHHHHHH SEQ ID NO: 25 Rhavi-MrkA-donor strand complementation-PcrV-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMADTTVGGGQVNFFGKVTDVSCTVSVNGQGSDANVYLSPVTLTEVKAAAADTYLKPKSFTIDVSNCQAADGTKQDDVSKLGVNWTGGNLLAGATSKQQGYLANTEASGAQNIQLVLSTDNATALTNKIIPGDSTQPKAKGDASAVADGARFTYYVGYATSAPTTVTTGVVNSYATYEITYQGGGGGGADTTVGGGQVNFFGKVTDVSAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH SEQ ID NO: 26 Rhavi-FlaB-Domain2-PcrV-his MFDASNFKDFSSIASASSSWQNQSGSTMIIQVDSFGNVSGQYVNRAQGTGCQNSPYPLTGRVNGTFIAFSVGWNNSTENCNSATGWTGYAQVNGNNTEIVTSWNLAYEGGSGPAIEQGQDTFQYVPTTENKSLLKDGGGGSSSMGSYQVGSNGAGTVASVAGTATASGIASGTVNLVGGGQVKNIAIAAGDSAKAIAEKMDGAIPNLSARARTVFTADVSGVTGGSLNFDVTVGSNTVSLAGVTSTQDLADQLNSNSSKLGITASINDKGVLTITSATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSSAAAAMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGSGHHHHHH

Literature

Agodi A, Voulgari E, Barchitta M, Politi L, Koumaki V, et al. Containment of an outbreak of KPC-3-producing Klebsiella pneumoniae in Italy. J Clin Microbiol. 2011 Nov;49(11):3986-9.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10.

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389-402.

Andreasen C., Powell DA., Carbonetti N. Pertussis Toxin Stimulates IL-17 Production in Response to Bordetella pertussis Infection in Mice. PloS One 2009 4:Issue 9 e7079.

Anttila M, Eskola J, Ahman H, Käyhty H. Avidity of IgG for Streptococcus pneumoniae type 6B and 23F polysaccharides in infants primed with pneumococcal conjugates and boosted with polysaccharide or conjugate vaccines. J Infect Dis. 1998 Jun;177(6):1614-21.

Baer M, Sawa T, Flynn P, Luehrsen K, Martinez D, et al. An engineered human antibody fab fragment specific for Pseudomonas aeruginosa PcrV antigen has potent antibacterial activity. Infect Immun. 2009 Mar;77(3):1083-90.

Baraniak, A., Grabowska, A., Izdebski, R., Fiett, J., Herda, M., Bojarska, K., Zabicka, D., Kania-Pudlo, M., Mlynarczyk, G., Zak-Pulawska , Z., Hryniewicz, W., Gniadkowski, M., 2011. Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008-2009. Antimicrob. Agents Chemother. 55, 5493-5499.

Barnea Y, Carmeli Y, Gur E, Kuzmenko B, Gat A, et al. Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa flagellin for treating infections in a murine burn wound model. Plast Reconstr Surg. 2006 Jun;117(7):2284-91.

Barnea Y, Carmeli Y, Neville LF, Kahel-Reifer H, Eren R, et al. Therapy with anti-flagellin A monoclonal antibody limits Pseudomonas aeruginosa invasiveness in a mouse burn wound sepsis model. Burns. 2009 May;35(3):390-6.

Baumgartner JD. Monoclonal anti-endotoxin antibodies for the treatment of gram-negative bacteremia and septic shock. Eur J Clin Microbiol Infect Dis. 1990 Oct;9(10):711-6.

Baxevanis AD and Ouellette BF, Eds., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. New York, NY: Wiley-Interscience, 1998.

Bergogne-Bérézin E. [Nosocomial infections: new agents, incidence, prevention]. Press Med. 1995 Jan 14;24(2):89-97. review. French.

Brisse S. et al., wzi Gene Sequencing, a Rapid Method for Determination of Capsular Type for Klebsiella Strains. J Clin Microbiol. 2013 51(12):4073-4078.

Bryan CS, Reynolds KL, Brenner ER. Analysis of 1,186 episodes of gram-negative bacteremia in non-university hospitals: the effects of antimicrobial therapy. Rev Infect Dis. 1983 Jul-Aug;5(4):629-38.

Burmølle M, Bahl MI, Jensen LB, Sørensen SJ, Hansen LH. Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains. microbiology. 2008 Jan;154(Pt 1):187-95.

Campodónico VL, Llosa NJ, Grout M, Döring G, Maira-Litrán T, et al. Evaluation of flagella and flagellin of Pseudomonas aeruginosa as vaccines. Infect Immun. 2010 Feb;78(2):746-55.

Campodónico VL, Llosa NJ, Bentancor LV, Maira-Litran T, Pier GB. Efficacy of a conjugate vaccine containing polymannuronic acid and flagellin against experimental Pseudomonas aeruginosa lung infection in mice. Infect Immun. 2011 Aug;79(8):3455-64.

Centers for Disease Control and Prevention (CDC). Antibiotic Resistance Threats in the United States, 2013. http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf. 2013.

Chaloupka I, Schuler A, Marschall M, Meier-Ewert H. Comparative analysis of six European influenza vaccines. Eur J Clin Microbiol Infect Dis. 1996 Feb;15(2):121-7.

Chang HH, Cohen T, Grad YH, Hanage WP, O'Brien TF, et al. Origin and proliferation of multiple-drug resistance in bacterial pathogens. Microbiol Mol Biol Rev. 2015 Mar;79(1):101-16.

Chen K, McAleer JP, Lin Y, Paterson DL, Zheng M, et al. Th17 cells mediate clade-specific, serotype-independent mucosal immunity. immunity. 2011 Dec 23;35(6):997-1009.

Chen, L., Chavda, KD, Findlay, J., Peirano, G., Hopkins, K., Pitout, JD, Bonomo, RA, Woodford, N., DeLeo, FR, Kreiswirth, BN, 2014a. Multiplex PCR for identification of two capsular types in epidemic KPC-producing Klebsiella pneumoniae sequence Type 258 strains. Antimicrob. Agents Chemother. 58, 4196-4199.

Chen, L., Mathema, B., Pitout, J.D., DeLeo, F.R., Kreiswirth, B.N., 2014b. Epidemic Klebsiella pneumoniae ST258 is a hybrid strain. mBio 5, e01355-14.

Clegg S, Gerlach G F. Enterobacterial fimbriae. J Bacteriol. 1987;169:934-938.

Clements A, Gaboriaud F, Duval JF, Farn JL, Jenney AW, et al. The major surface-associated saccharides of Klebsiella pneumoniae contribute to host cell association. PLOS One. 2008;3(11):e3817.

Collignon P. Resistant Escherichia coli --we are what we eat. Clin Infect Dis. 2009 Jul 15;49(2):202-4.

Conception NF, Frasch CE. Pneumococcal type 22f polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 2001 Mar;8(2):266-72.

Cross AS, Zollinger W, Mandrell R, Gemski P, Sadoff J. Evaluation of immunotherapeutic approaches for the potential treatment of infections caused by K1-positive Escherichia coli . J Infect Dis. 1983 Jan;147(1):68-76.

Cross AS, Gemski P, Sadoff JC, Orskov F, Orskov I. The importance of the K1 capsule in invasive infections caused by Escherichia coli . J Infect Dis. 1984 Feb;149(2):184-93.

Cross AS, Kim KS, Wright DC, Sadoff JC, Gemski P. Role of lipopolysaccharide and capsule in the serum resistance of bacteremic strains of Escherichia coli. J Infect Dis. 1986 Sep; 154(3):497-503.

Cross A, Artenstein A, Que J, Fredeking T, Furer E, et al. Safety and immunogenicity of a polyvalent Escherichia coli vaccine in human volunteers. J Infect Dis. 1994 Oct;170(4):834-40.

Crowe BA, Enzersberger O, Schober-Bendixen S, Mitterer A, Mundt W, et al. The first clinical trial of immuno's experimental Pseudomonas aeruginosa flagellar vaccines. Antibiot Chemother (1971). 1991;44:143-56.

Cryz SJ Jr, Fürer E, Germanier R. Protection against fatal Pseudomonas aeruginosa burn wound sepsis by immunization with lipopolysaccharide and high-molecular-weight polysaccharide. Infect Immun. 1984 Mar;43(3):795-9.

Cryz SJ, Jr, Fürer E, Germanier R. Safety and immunogenicity of Klebsiella pneumoniae K1 capsular polysaccharide vaccine in humans. J Infect Dis. 1985;151:665-671.

Cryz SJ Jr, Mortimer P, Cross AS, Fürer E, Germanier R. Safety and immunogenicity of a polyvalent Klebsiella capsular polysaccharide vaccine in humans. Vaccine. 1986 Mar; 4(1):15-20.

Cryz SJ Jr, Mortimer PM, Mansfield V, Germanier R. Seroepidemiology of Klebsiella bacteremic isolates and implications for vaccine development. J Clin Microbiol. 1986 Apr; 23(4):687-90.

Cryz SJ Jr, Wedgwood J, Lang AB, Ruedeberg A, Que JU, et al. Immunization of noncolonized cystic fibrosis patients against Pseudomonas aeruginosa . J Infect Dis. May 1994; 169(5):1159-62.

DeLeo, FR, Chen, L., Porcella, SF, Martens, CA, Kobayashi, SD, Porter, AR, Chavda, KD, Jacobs, MR, Mathema, B., Olsen, RJ, Bonomo, RA, Musser, JM, Kreiswirth, BN, 2014. Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae . Proc. Natl. Acad. sci. US A 111, 4988-4993.

Deng JC, Moore TA, Newstead MW, Zeng X, Krieg AM, et al. CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection. J Immunol. 2004 Oct 15;173(8):5148-55.

DiGiandomenico A, Sellman BR. Antibacterial monoclonal antibodies: the next generation?. Curr Opin Microbiol. 2015 Oct; 27:78-85.

DiGiandomenico A, Rao J, Goldberg JB. Oral vaccination of BALB/c mice with Salmonella enterica serovar Typhimurium expressing Pseudomonas aeruginosa O antigen promotes increased survival in an acute fatal pneumonia model. Infect Immun. 2004 Dec;72(12):7012-21.

DiGiandomenico A, Rao J, Harcher K, Zaidi TS, Gardner J, et al. Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections. Proc Natl Acad Sci US A. 2007 Mar 13;104(11):4624-9.

DiGiandomenico A, Warrener P, Hamilton M, Guillard S, Ravn P, et al. Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening. J Exp Med. 2012 Jul 2;209(7):1273-87.

DiGiandomenico A, Keller AE, Gao C, Rainey GJ, Warrener P, et al. A multifunctional bispecific antibody protects against Pseudomonas aeruginosa . Sci Transl Med. 2014 Nov 12;6(262):262ra155.

Domenico P, Schwartz S, Cunha BA. Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect Immun. 1989 Dec;57(12):3778-82.

Döring G, Dorner F. A multicenter vaccine trial using the Pseudomonas aeruginosa flagella vaccine IMMUNO in patients with cystic fibrosis. Behring Inst Mitt. Feb 1997;

Döring G, Pier GB. Vaccines and immunotherapy against Pseudomonas aeruginosa . Vaccine. 2008 Feb 20;26(8):1011-24.

Döring G, Pfeiffer C, Weber U, Mohr-Pennert A, Dorner F. Parenteral application of a Pseudomonas aeruginosa flagella vaccine elicits specific anti-flagella antibodies in the airways of healthy individuals. Am J Respir Crit Care Med. 1995 Apr;151(4):983-5.

Döring G, Meisner C, Stern M. A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Sci US A. 2007 Jun 26;104(26):11020-5.

Du H, Chen L, Tang YW, Kreiswirth BN. Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. Lancet Infect Dis. Mar 2016;16(3):287-8.

Edelman R, Taylor DN, Wasserman SS, McClain JB, Cross AS, et al. Phase 1 trial of a 24-valent Klebsiella capsular polysaccharide vaccine and an eight-valent Pseudomonas O-polysaccharide conjugate vaccine administered simultaneously. Vaccine. 1994 Nov;12(14):1288-94.

Faezi S, Sattari M, Mahdavi M, Roudkenar MH. Passive immunization against Pseudomonas aeruginosa recombinant flagellin in an experimental model of burn wound sepsis. Burns. 2011 Aug; 37(5):865-72.

Faezi S, Safarloo M, Amirmozafari N, Nikokar I, Siadat SD, et al. Protective efficacy of Pseudomonas aeruginosa type-A flagellin in the murine burn wound model of infection. APMIS. 2014 Feb;122(2):115-27.

Feldman MF, Wacker M, Hernandez M, Hitchen PG, Marolda CL, et al. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli . Proc Natl Acad Sci US A. 2005 Feb 22;102(8):3016-21.

fine PE. Herd immunity: history, theory, practice. Epidemiol Rev. 1993;15(2):265-302.

Foglia G, Shah S, Luxemburger C, Pietrobon PJ. Clostridium difficile : development of a novel candidate vaccine. Vaccine. 2012 Jun 19;30(29):4307-9.

Fowler VG Jr, Proctor RA. Where does a Staphylococcus aureus vaccine stand?. Clin Microbiol Infect. May 2014; 20 Suppl 5:66-75.

B, Luyt CE, Dugard A, Wolff M, Diehl JL, et al. Safety and pharmacokinetics of an anti-PcrV PEGylated monoclonal antibody fragment in mechanically ventilated patients colonized with Pseudomonas aeruginosa: a randomized, double-blind, placebo-controlled trial. Crit Care Med. 2012 Aug; 40(8):2320-6.

B, Luyt CE, Dugard A, Wolff M, Diehl JL, et al. Safety and pharmacokinetics of an anti-PcrV PEGylated monoclonal antibody fragment in mechanically ventilated patients colonized with Pseudomonas aeruginosa : a randomized, double-blind, placebo-controlled trial. Crit Care Med. 2012 Aug; 40(8):2320-6.

Frank DW, Vallis A, Wiener-Kronish JP, Roy-Burman A, Spack EG, et al. Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV. J Infect Dis. 2002 Jul 1;186(1):64-73.

Galanos C, Freudenberg MA, Reutter W. Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5939-43.

Gerding DN, Meyer T, Lee C, Cohen SH, Murthy UK, et al. Administration of spores of nontoxigenic Clostridium difficile strain M3 for prevention of recurrent C difficile infection: a randomized clinical trial. JAMA. 2015 May 5;313(17):1719-27.

Gerlach GF, Clegg S, Allen BL. Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae . J Bacteriol. 1989 Mar;171(3):1262-70.

Giakkoupi, P., Papagiannitsis, CC, Miriagou, V., Pappa, O., Polemis, M., Tryfinopoulou, K., Tzouvelekis, LS, Vatopoulos, AC, 2011. An update of the evolving epidemic of blaKPC-2- carrying Klebsiella pneumoniae in Greece (2009-10). J. Antimicrob. Chemother. 66, 1510-1513.

Giani, T., Pini, B., Arena, F., Conte, V., Bracco, S., Migliavacca, R., Pantosti, A., Pagani, L., Luzzaro, F., Rossolini, GM, 2013 Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey, 15 May to 30 June 2011. Eurosurveillance, 18.

Gransden WR, Eykyn SJ, Phillips I, Rowe B. Bacteremia due to Escherichia coli : a study of 861 episodes. Rev Infect Dis. 1990 Nov-Dec; 12(6):1008-18.

Greenberger MJ, Kunkel SL, Strieter RM, Lukacs NW, Bramson J, et al. IL-12 gene therapy protects mice in lethal Klebsiella pneumonia . J Immunol. 1996 Oct 1;157(7):3006-12.

Hansen DS, Mestre F, Alberti S, Hernández-Allés S, Alvarez D, et al. Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries. J Clin Microbiol. 1999 Jan; 37(1):56-62.

Henriques-Normark B, Normark S. Bacterial vaccines and antibiotic resistance. Ups J Med Sci. May 2014;119(2):205-8.

Holder IA, Naglich JG. Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa : immunization using divalent flagella preparations. J Trauma. 1986 Feb;26(2):118-22.

Holder IA, Wheeler R, Montie TC. Flagellar preparations from Pseudomonas aeruginosa : animal protection studies. Infect Immun. 1982 Jan;35(1):276-80.

Holder IA, Neely AN, Frank DW. PcrV immunization enhances survival of burned Pseudomonas aeruginosa -infected mice. Infect Immun. 2001 Sep;69(9):5908-10.

Holliger P, Prospero T, Winter G. "Diabodies": small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8.

Horan T, Culver D, Jarvis W, Emori G, Banerjee S, Martone W, Thornsberry C. Pathogens Causing Nosocomial Infections. Antimicrobial Newsl. 1988 Sep;5(9):65-68.

Hornick DB, Allen BL, Horn MA, Clegg S. Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products. Infect Immun. 1992 Apr;60(4):1577-88.

Hu R. et al., Outer Membrane Protein A (OmpA) Conferred Immunoprotection against Enterobacteriaceae Infection in Mice. Israel Journal of Veterinary Medicine. 2103. Vol. 68(1):48-55.

Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: " Staphyloccocus aureus vaccines: problems and prospects". Vaccine. 2013 Jun 7;31(25):2723-30.

Jones RJ, Roe EA, Lowbury EJ, Miler JJ, Spilsbury JF. A new Pseudomonas vaccine: preliminary trial on human volunteers. J Hyg (Lond). 1976 Jun;76(3):429-39.

Jones RJ, Roe EA, Gupta JL. Low mortality in burned patients in a Pseudomonas vaccine trial. Lancet. 1978 Aug 19;2(8086):401-3.

Jones RJ, Roe EA, Gupta JL. Controlled trials of a polyvalent pseudomonas vaccine in burns. Lancet. 1979 Nov 10;2(8150):977-82.

Jones RJ, Roe EA, Gupta JL. Controlled trial of Pseudomonas immunoglobulin and vaccine in burn patients. Lancet. 1980 Dec 13;2(8207):1263-5.

Kaijser B, Ahlstedt S. Protective capacity of antibodies against Escherichia coli and K antigens. Infect Immun. 1977 Aug; 17(2):286-9.

Kelly, RF et al., 1995. Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related D-galactan O-antigens in Klebsiella spp. J. Endotoxin Res. 2:131-140.

Kim KH, Yu J, Nahm MH. Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies. Clin Diagn Lab Immunol. 2003 Jul; 10(4):616-21.

Knirel YA, Bystrova OV, Kocharova NA, Zahringer U, Pier GB. Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa . J Endotoxin Res. 2006;12(6):324-36.

Kojima K, Ishizaka A, Oshika E, Taguchi Y, Tomizawa K, et al. Quantitation of IgG subclass antibodies to pneumococcal capsular polysaccharides by ELISA, using Pneumovax-specific antibodies as a reference. Tohoku J Exp Med. 1990 Jul;161(3):209-15.

Kol, O., Wieruszeski, JM, Strecker, G., Fournet, B., Zalisz, R., Smets, P., 1992. Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation. Carbohydr. Res. 236, 339-344.

Koskela M, Leinonen M. Comparison of ELISA and RIA for measurement of pneumococcal antibodies before and after vaccination with 14-valent pneumococcal capsular polysaccharide vaccine. J Clin Pathol. 1981 Jan;34(1):93-8.

Kreger BE, Craven DE, Carling PC, McCabe WR. Gram-negative bacteremia III Reassessment of etiology, epidemiology and ecology in 612 patients. Am J Med. 1980 Mar; 68(3):332-43.

Lal G, Balmer P, Stanford E, Martin S, Warrington R, et al. Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes. J Immunol Methods. 2005 Jan;296(1-2):135-47.

Landman, D., Babu, E., Shah, N., Kelly, P., Olawole, O., Backer, M., Bratu, S., Quale, J., 2012. Transmission of carbapenem-resistant pathogens in New York City hospitals: progress and frustration. J. Antimicrob. Chemother. 67, 1427-1431.

Lang AB, Schaad UB, Rüdeberg A, Wedgwood J, Que JU, et al. Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis. J Pediatr. Nov 1995; 127(5):711-7.

Langford DT, Hiller J. Prospective, controlled study of a polyvalent pseudomonas vaccine in cystic fibrosis--three year results. Arch DisChild. 1984 Dec;59(12):1131-4.

Langstraat J, Bohse M, Clegg S. Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae , but not the fimbrial adhesin (MrkD), facilitates biofilm formation. Infect Immun. 2001 Sep; 69(9):5805-12.

Leach S, Clements JD, Kaim J, Lundgren A. The adjuvant double mutant Escherichia coli heat labile toxin enhances IL-17A production in human T cells specific for bacterial vaccine antigens. PLOS One. 2012;7(12):e51718. doi: 10.1371.

Levin BR, Cornejo OE. The population and evolutionary dynamics of homologous gene recombination in bacterial populations. PLoS Genet. 2009 Aug; 5(8):e1000601.

Levin BR, Stewart FM, Rice VA. The kinetics of conjugative plasmid transmission: fit of a simple mass action model. Plasmid. 1979 Apr;2(2):247-60.

Lipsitch M, Siber GR. How Can Vaccines Contribute to Solving the Antimicrobial Resistance Problem?. MBio. 2016 Jun 7;7(3)

Liu Y, Filler S.G. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryots Cell. 2011 Feb;10(2):168-73.

Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016 Feb;16(2):161-8.

Ma L, Jackson KD, Landry RM, Parsek MR, Wozniak DJ. Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure post attachment. J Bacteriol. 2006 Dec;188(23):8213-21.

Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, et al. Multistate point-prevalence survey of health care-associated infections. N Engl J Med. 2014 Mar 27;370(13):1198-208.

Mandine E, Salles MF, Zalisz R, Guenounou M, Smets P. Murine monoclonal antibodies to Klebsiella pneumoniae protect against lethal endotoxemia and experimental infection with capsulated K pneumoniae. Infect Immun. 1990 Sep;58(9):2828-33.

Martin, EW, Ed. Remington's Pharmaceutical Sciences. 15th ed. Easton, PA: Mack Publishing Company, 1975.

Martinez JE, Romero-Steiner S, Pilishvili T, Barnard S, Schinsky J, et al. A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after vaccination with the 23-valent pneumococcal polysaccharide vaccine. Clin Diagn Lab Immunol. 1999 Jul;6(4):581-6.

McCabe WR, Kaijser B, Olling S, Uwaydah M, Hanson LA. Escherichia coli in bacteremia: K and O antigens and serum sensitivity of strains from adults and neonates. J Infect Dis. 1978 Jul;138(1):33-41.

McGann P, Chahine S, Okafor D, Ong AC, Maybank R, et al. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin. J Clin Microbiol. Jan 2016; 54(1):208-11.

Meir A, Helppolainen SH, Podoly E, Nordlund HR, Hytönen VP, et al. Crystal structure of rhizavidin: insights into the enigmatic high-affinity interaction of an innate biotin-binding protein dimer. J Mol Biol. 2009 Feb 20;386(2):379-90.

Milla CE, Chmiel JF, Accurso FJ, VanDevanter DR, Konstan MW, et al. Anti-PcrV antibody in cystic fibrosis: a novel approach targeting Pseudomonas aeruginosa airway infection. Pediatric Pulmonol. 2014 Jul;49(7):650-8.

Misener S and Krawetz SA, Eds., Bioinformatics: Methods and Protocols (Methods in Molecular Biology, Volume 132). Totowa, NJ: Humana Press, 1999.

Montie TC, Drake D, Sellin H, Slater O, Edmonds S. Motility, virulence, and protection with a flagella vaccine against Pseudomonas aeruginosa infection. Antibiot Chemother (1971). 1987;39:233-48.

Moore TA, Perry ML, Getsoian AG, Newstead MW, Standiford TJ. Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection. Infect Immun. 2002 Nov;70(11):6310-8.

Moriel DG, Bertoldi I, Spagnuolo A, Marchi S, Rosini R, et al. Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli . Proc Natl Acad Sci US A. 2010 May 18;107(20):9072-7.

Munro CS, Stanley PJ, Cole PJ. Assessment of biological activity of immunoglobulin preparations by using opsonized micro-organisms to stimulate neutrophil chemiluminescence. ClinExp Immunol. 1985 Jul;61(1):183-8.

Murphy CN, Clegg S. Klebsiella pneumoniae and type 3 fimbriae: nosocomial infection, regulation and biofilm formation. Future Microbiol. 2012 Aug;7(8):991-1002.

Navon-Venezia, S., Leavitt, A., Schwaber, MJ, Rasheed, JK, Srinivasan, A., Patel, JB, Carmeli, Y., 2009. First report on a hyperepidemic clone of KPC-3-producing Klebsiella pneumoniae in Israel genetically related to a strain causing outbreaks in the United States. Antimicrob. Agents Chemother. 53, 818-820.

Neely AN, Bhattacharjee AK, Babcock GF, Holder IA, Cross AS. Differential effects of two different routes of immunization on protection against gram-negative sepsis by a detoxified Escherichia coli J5 lipopolysaccharide group B meningococcal outer membrane protein complex vaccine in a burned mouse model. J Burn Care Rehabil. 2002 Sep-Oct;23(5):333-40.

Nesta B, Spraggon G, Alteri C, Moriel DG, Rosini R, et al. FdeC, a novel broadly conserved Escherichia coli adhesin eliciting protection against urinary tract infections. MBio. 2012;3(2)

Norton EB et al., Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant. Clin Vaccine Immunol. 2011 18:546-551.

Norton EB et al., The A Subunit of Escherichia coli Heat-Labile Enterotoxin Functions as a Mucosal Adjuvant and Promotes IgG2a, IgA, and Th17 Responses to Vaccine Antigens. Infection and Immunity 2012, 80:2426-2435.

Ojo-Amaize EA, Church JA, Barka NE, Agopian MS, Peter JB. A rapid and sensitive chemiluminescence assay for evaluation of functional opsonic activity of Haemophilus influenzae type b-specific antibodies. Clin Diagn Lab Immunol. 1995 May;2(3):286-90.

Old DC, Adegbola RA. Antigenic relationships among type-3 fimbriae of Enterobacterioscopy revealed by immunoelectronmicr. J Med Microbiol. 1985 Aug; 20(1):113-21.

Pereira, PS, de Araujo, CF, Seki, LM, Zahner, V., Carvalho-Assef, AP, Asensi, MD, 2013. Update of the molecular epidemiology of KPC-2-producing Klebsiella pneumoniae in Brazil: spread of clonal complex 11 (ST11 ST437 and ST340). J. Antimicrob. Chemother. 68, 312-316.

Pickering JW, Martins TB, Greer RW, Schroder MC, Astill ME, et al. A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides. Am J Clin Pathol. 2002 Apr; 117(4):589-96.

Podschun R, Ullmann U. Klebsiella spp as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998 Oct;11(4):589-603.

Poljak RJ. Production and structure of diabodies. structure. 1994 Dec 15;2(12):1121-3.

Poolman JT, Wacker M. Extraintestinal Pathogenic Escherichia coli , a Common Human Pathogen: Challenges for Vaccine Development and Progress in the Field. J Infect Dis. 2016 Jan 1;213(1):6-13.

Powell M.F. and Newman M.J., Eds. Vaccine Design: The Subunit and Adjuvant Approach. New York, NY: Plenum Press, 1995.

Priebe GP, Walsh RL, Cederroth TA, Kamei A, Coutinho-Sledge YS, et al. IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa . J Immunol. 2008 Oct 1;181(7):4965-75.

Qi, Y., Wei, Z., Ji, S., Du, X., Shen, P., Yu, Y., 2011. ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China. J. Antimicrob. Chemother. 66, 307-312.

Ramachandran G, Boyd MA, MacSwords J, Higginson EE, Simon R, et al. Opsonophagocytic Assay To Evaluate Immunogenicity of Nontyphoidal Salmonella Vaccines. Clin Vaccine Immunol. 2016 Jun;23(6):520-3.

Rappuoli R. Reverse vaccinology. Curr Opin Microbiol. 2000 Oct;3(5):445-50.

Rayner BL, Willcox PA. Community-acquired bacteremia; a prospective survey of 239 cases. Q J Med. 1988 Nov;69(259):907-19.

Robbins JB, McCracken GH Jr, Gotschlich EC, Orskov F, Orskov I, et al. Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis. N Engl J Med. 1974 May 30;290(22):1216-20.

Romero-Steiner S, Libutti D, Pais LB, Dykes J, Anderson P, et al. Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells. Clin Diagn Lab Immunol. 1997 Jul;4(4):415-22.

Romero-Steiner S, Holder PF, Gomez de Leon P, Spear W, Hennessy TW, et al. Avidity determinations for Haemophilus influenzae Type b anti-polyribosylribitol phosphate antibodies. Clin Diagn Lab Immunol. 2005 Sep;12(9):1029-35.

Ross PJ, Sutton CE, Higgins S, Allen AC, Walsh K, et al. Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis : towards the rational design of an improved acellular pertussis vaccine. PLoS Pathog. 2013;9(4):e1003264.

Saeland E, Vidarsson G, Jonsdottir I. Pneumococcal pneumonia and bacteremia model in mice for the analysis of protective antibodies. microbe pathogen. 2000 Aug;29(2):81-91.

Saha S, Takeshita F, Matsuda T, Jounai N, Kobiyama K, et al. Blocking of the TLR5 activation domain hampers protective potential of flagellin DNA vaccine. J Immunol. 2007 Jul 15;179(2):1147-54.

Sambrook J, Maniatis T and Fritsch EF. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.

Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, et al. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat Med. 1999 Apr;5(4):392-8.

Schaad UB, Lang AB, Wedgwood J, Ruedeberg A, Que JU, et al. Safety and immunogenicity of Pseudomonas aeruginosa conjugate A vaccine in cystic fibrosis. Lancet. 1991 Nov 16;338(8777):1236-7.

Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbial etiology of nosocomial infection. Am J Med. 1991 Sep 16;91(3B):72S-75S.

Schmidt AC, McAuliffe JM, Murphy BR, Collins PL. Recombinant bovine/human parainfluenza virus type 3 (B/HPIV3) expressing the respiratory syncytial virus (RSV) G and F proteins can be used to achieve simultaneous mucosal immunization against RSV and HPIV3. J Virol. 2001 May;75(10):4594-603.

Schmidt CS, White CJ, Ibrahim AS, Filler SG, Fu Y, et al. NDV-3, a recombinant alum-adjuvanted vaccine for Candida and Staphylococcus aureus , is safe and immunogenic in healthy adults. Vaccine. 2012 Dec 14;30(52):7594-600.

Schweizer HP. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195-204.

Scully IL, Liberator PA, Jansen KU, Anderson AS. Covering all the Bases: Preclinical Development of an Effective Staphylococcus aureus Vaccine. Front Immunol. 2014 Mar 24;5:109.

Shime N, Sawa T, Fujimoto J, Faure K, Allmond LR, et al. Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa . J Immunol. 2001 Nov 15;167(10):5880-6.

Simon R, Samuel C.E. Activation of NF-kappaB-dependent gene expression by Salmonella flagellins FliC and FljB. Biochem Biophys Res Commun. 2007 Mar 30;355(1):280-5.

Song W.S., Yoon S.I. Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament. Biochem Biophys Res Commun. 2014 Feb 7;444(2):109-15.

Stack AM, Malley R, Thompson CM, Kobzik L, Siber GR, et al. Minimum protective serum concentrations of pneumococcal anti-capsular antibodies in infant rats. J Infect Dis. 1998 Apr;177(4):986-90.

Szijártó V. et al., 2016. Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type. International Journal of Medical Microbiology 306:89-98.

Tarkkanen AM, Virkola R, Clegg S, Korhonen TK. Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect Immun. 1997 Apr;65(4):1546-9.

Tarkkanen AM, Allen BL, Westerlund B, Holthöfer H, Kuusela P, et al. Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles. Mol Microbiol. 1990 Aug;4(8):1353-61.

Thammavongsa V, Kim HK, Missiakas D, Schneewind O. Staphylococcal manipulation of host immune responses. Nat Rev Microbiol. 2015 Sep;13(9):529-43.

Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, et al. New Guidelines to Evaluate the Response to Treatment in Solid Tumors. J Natl Cancer Inst. 2000 Feb 2;92(3):205-216.

Tomas JM, Camprubi S, Williams P. Surface exposure of the O-antigen in Klebsiella pneumoniae O1:K1 serotype strains. microbe pathogen. 1988 Aug;5(2):141-7.

Toprani VS. et al., Development of a candidate stabilizing formulation for bulk storage of a double mutant heat labile toxin (dmLT) protein based adjuvant. Vaccine. 2017. 35:5471-548.

Trautmann, M., et al., 1997. O-Antigen Seroepidemiology of Klebsiella Clinical Isolates and Implications for Immunoprophylaxis of Klebsiella Infections. Clinical and Diagnostic Laboratory Immunology 4:550-555.

Westritschnig K, Hochreiter R, Wallner G, Firbas C, Schwameis M, Jilma B. 2014. A randomized, placebo-controlled phase I study assessing the safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein OprF/I vaccine (IC43) in healthy volunteers. Hum Vaccin Immunother 10:170-183.

Ullmann U. [Bacterial infection agents in hospitalized patients]. Zentralbl Bakteriol Mikrobiol Hyg B. 1986 Dec;183(2-3):103-13.

Vaudaux P and Waldvogel F. A. (1979). Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrobial Agents Chemother. 16(6): 743-749.

Vinogradov E, Frirdich E, MacLean LL, Perry MB, Petersen BO, et al. Structures of lipopolysaccharides from Klebsiella pneumoniae Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains. J Biol Chem. 2002 Jul 12;277(28):25070-81.

Wang Q, Chang CS, Pennini M, Pelletier M, Rajan S, et al. Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit. J Infect Dis. 2016 Jun 1;213(11):1800-8.

Walczak MJ, Puorger C, Glockshuber R, Wider G. Intramolecular donor strand complementation in the E. coli type 1 pilus subunit FimA explains the existence of FimA monomers as off-pathway products of pilus assembly that inhibit host cell apoptosis. J Mol Biol. 2014 Feb 6;426(3):542-9.

Warfel JM, Zimmerman LI, Merkel TJ. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc Natl Acad Sci U S A. 2014 Jan 14;111(2):787-92.

Warrener P, Varkey R, Bonnell JC, DiGiandomenico A, Camara M, et al. A novel anti-PcrV antibody providing enhanced protection against Pseudomonas aeruginosa in multiple animal infection models. Antimicrobial Agents Chemother. 2014 Aug;58(8):4384-91.

Welch WD, Martin WJ, Stevens P, Young LS. Relative opsonic and protective activities of antibodies against K1, O and lipid A antigens of Escherichia coli . Scand J Infect Dis. 1979;11(4):291-301.

Whitfield, C., Perry, M.B., MacLean, L.L., Yu, S.H., 1992. Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a) O2(2a, 2b), and O2(2a , 2c). J. Bacteriol. 174, 4913-4919.

Whitfield, C., Richards, JC, Perry, MB, Clarke, BR, MacLean, LL, 1991. Expression of two structurally distinct d-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1. J. Bacteriol. 173, 1420-1431.

Woodford N, Turton JF, Livermore DM. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev. 2011 Sep;35(5):736-55.

World Health Organization (WHO). Antimicrobial Resistance - Global Report on Surveillance. 2014.

Wu W, Huang J, Duan B, Traficante DC, Hong H, et al. Th17-stimulating protein vaccines confer protection against Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med. 2012 Sep 1;186(5):420-7.

Würker M, Beuth J, Ko HL, Przondo-Mordarska A, Pulverer G. Type of fimbriation determines adherence of Klebsiella bacteria to human epithelial cells. Zentralbl Bakteriol. 1990 Nov;274(2):239-45

Zhang F, Lu YJ, Malley R. Multiple antigen-presenting system (MAPS) to induce comprehensive B- and T-cell immunity. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13564-9.

Equivalents

Those skilled in the art will be able to discover, or develop using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present invention. The scope of the present invention is not to be limited by the above description, but only by the following claims.

Claims (143)

1. An immunogenic composition containing (i) a backbone polymer comprising a polymer and one or more antigenic polysaccharides conjugated to the polymer; and (ii) one or more polypeptide antigens in a non-covalent complex with a polymer and/or antigenic polysaccharide; in which the polymer is a capsular polysaccharide Klebsiella spp. K19; And wherein each of the one or more antigenic polysaccharides is an O-antigenic polysaccharide (OPS) of K. pneumoniae and/or P. aeruginosa ; And wherein each of the one or more polypeptide antigens is independently selected from: P. aeruginosa flagellin, P. aeruginosa PcrV, K. pneumoniae MrkA, antigenic fragments thereof, and combinations thereof; And wherein the immunogenic composition is characterized in that, upon administration to a subject, it induces an immune response against Klebsiella and/or Pseudomonas . 2. An immunogenic composition according to claim 1, in which one or more polypeptide antigens are in a non-covalent complex with a polymer and/or one or more antigenic polysaccharides due to at least one pair of complementary affinity molecules, including (i) the first affinity molecule and ( ii) a second affinity molecule complementary to the first molecule, wherein the first affinity molecule is associated with a polymer and/or one or more antigenic polysaccharides and the second affinity molecule is associated with one or more polypeptide antigens. 3. The immunogenic composition according to claim 2, wherein the pair of affinity molecules is selected from the group consisting of: biotin/biotin-binding protein, antibody/antigen, enzyme/substrate, receptor/ligand, metal/metal-binding protein, carbohydrate/carbohydrate-binding protein, lipid/ a lipid-binding protein; and a His-tag/His-tag-binding molecule. 4. An immunogenic composition according to claim 2 or 3, wherein the first affinity molecule is biotin or a derivative thereof and the second affinity molecule is a biotin-binding protein or a biotin-binding domain thereof. 5. An immunogenic composition according to claim 4, wherein the biotin-binding protein is rizavidin or its biotin-binding domain. 6. Immunogenic composition according to any one of paragraphs. 2-5, wherein the first affinity molecule is cross-linked or covalently linked to a polymer and/or one or more antigenic polysaccharides. 7. Immunogenic composition according to any one of paragraphs. 2-6, wherein the one or more polypeptide antigens comprise one or more P. aeruginosa proteins or variants, and wherein the second affinity molecule comprises a biotin-binding protein or a biotin-binding domain thereof. 8. Immunogenic composition according to any one of paragraphs. 2-7, wherein the one or more polypeptide antigens comprise at least two polypeptide antigens. 9. Immunogenic composition according to any one of paragraphs. 2-8, wherein one or more polypeptide antigens bind to a second affinity molecule to form a fusion protein. 10. An immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 (D2 domain of subtype B of P. aeruginosa flagellin (FlaBD2) lacking a TLR5 binding motif) or an immunogenic fragment thereof, or (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 ( P. aeruginosa flagellin (FlaA1) subtype A1 D2 domain lacking a TLR5-binding motif), or an immunogenic fragment thereof or (iii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 12 ( the D2 domain of subtype A2 of P. aeruginosa flagellin (FlaA2) lacking the TLR5-binding motif), or an immunogenic fragment thereof. 11. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 10 (D2 domain of subtype B of P. aeruginosa flagellin (FlaBD2) without TLR5 binding motif), or an immunogenic fragment thereof. 12. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof. 13. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 14. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 7 ( P. aeruginosa FliC subtype B flagellin (FlaB)), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 15. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 (D2 domain of flagellin FlaB lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 16. The immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 7 ( P. aeruginosa FliC subtype B flagellin (FlaB)), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% identity with the sequence of SEQ with ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or its immunogenic fragment. 17. An immunogenic composition according to claim 9, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 (subtype B D2 domain of P. aeruginosa flagellin (FlaBD2) lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 18. Immunogenic composition according to any one of paragraphs. 11-17, characterized in that when administered to a patient, the immunogenic composition causes the production of antibodies that recognize native MrkA on K. pneumoniae expressing MrkA. 19. Immunogenic composition according to any one of paragraphs. 1-18, in which one or more antigenic polysaccharides contain OPS type O1, O2, O3, O5 K. pneumoniae or a combination thereof. 20. Immunogenic composition according to any one of paragraphs. 1-19, in which one or more antigenic polysaccharides contain P. aeruginosa type O1, O2, O3, O4, O5, O6, O10, O11, O12 OPS , or a combination thereof. 21. Immunogenic composition according to any one of paragraphs. 1-20, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 2 (MrkA K. pneumoniae ), and/or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 3, or its immunogenic fragment. 22. Immunogenic composition according to any one of paragraphs. 1-21, wherein at least one of the polypeptide antigens is P. aeruginosa subtype A flagellin, P. aeruginosa subtype B flagellin, or an immunogenic fragment thereof. 23. Immunogenic composition according to any one of paragraphs. 1-22, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof. 24. Immunogenic composition according to any one of paragraphs. 1-23, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 7 ( P. aeruginosa FliC subtype B flagellin (FlaB)), or an immunogenic fragment thereof. 25. Immunogenic composition according to any one of paragraphs. 1-24, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 10 ( P. aeruginosa flagellin subtype B D2 domain (FlaBD2) lacking a TLR5 binding motif), or an immunogenic fragment thereof. 26. Immunogenic composition containing (a) a backbone polymer comprising (i) a polymer containing a Klebsiella spp. capsular polysaccharide. K19; And (ii) one or more antigenic polysaccharides containing an O-antigenic polysaccharide (OPS)Klebsiella and/orPseudomonas, conjugated with capsular polysaccharideKlebsiella spp. K19; And (b) one or more polypeptide antigens in a non-covalent complex with a polymer through at least one pair of affinity molecules, including (i) a first affinity molecule associated with a polymer; And (ii) a second affinity molecule complementary to the first molecule and associated with one or more polypeptide antigens, where each of the one or more polypeptide antigens is independently selected from: P. aeruginosa flagellin, P. aeruginosa PcrV, K. pneumoniae MrkA, antigenic fragments thereof, or their combinations; And wherein the immunogenic composition is characterized in that, upon administration to a subject, it induces an immune response against Klebsiella and/or Pseudomonas . 27. The immunogenic composition of claim 26, wherein the one or more antigenic polysaccharides comprise K. pneumoniae type O1, O2, O3, O5 OPS , or a combination thereof. 28. The immunogenic composition of claim 26 or 27, wherein the one or more antigenic polysaccharides comprise O1, O2, O3, O4, O5, O6, O10, O11 P. aeruginosa OPS, or a combination thereof. 29. Immunogenic composition according to any one of paragraphs. 26-28, wherein the first affinity molecule is biotin or a derivative thereof and the second affinity molecule is a biotin-binding protein or its biotin-binding domain. 30. The immunogenic composition of claim 29, wherein the biotin-binding protein is rizavidin or its biotin-binding domain. 31. Immunogenic composition according to any one of paragraphs. 26-30, wherein one or more polypeptide antigens are selected from the D2 domain of P. aeruginosa subtype B flagellin (FlaBD2) lacking a TLR5 binding motif, P. aeruginosa PcrV, K. pneumoniae MrkA, antigenic fragments thereof, or combinations thereof. 32. Immunogenic composition according to any one of paragraphs. 26-31, wherein the one or more polypeptide antigens comprise one or more P. aeruginosa proteins or variants, and wherein the second affinity molecule comprises a biotin-binding protein or a biotin-binding domain thereof. 33. Immunogenic composition according to any one of paragraphs. 26-32, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 7 (flagellin subtype B (FlaB) P. aeruginosa ), or an immunogenic fragment thereof. 34. Immunogenic composition according to any one of paragraphs. 26-33, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 10 ( P. aeruginosa flagellin subtype B D2 domain (FlaBD2) devoid of TLR5 binding motif), or an immunogenic fragment thereof. 35. Immunogenic composition according to any one of paragraphs. 26-34, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 8 (PcrV P. aeruginosa ), or an immunogenic fragment thereof. 36. Immunogenic composition according to any one of paragraphs. 26-35, in which at least one of the polypeptide antigens contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 37. Immunogenic composition according to any one of paragraphs. 26-36, wherein the one or more polypeptide antigens comprise at least two polypeptide antigens. 38. Immunogenic composition according to any one of paragraphs. 26-37, wherein one or more polypeptide antigens bind to a second affinity molecule to form a fusion protein. 39. The composition of claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 (subtype B D2 domain of P. aeruginosa flagellin (FlaBD2) lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 40. The immunogenic composition according to claim 38, in which the fusion protein contains a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 24 (Rhavi-FlaB-Domain2-MrkA-His). 41. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 ( P. aeruginosa subtype B flagellin (FlaBD2) D2 domain lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 42. The immunogenic composition according to claim 38, in which the fusion protein contains a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 26. 43. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 10 (D2 domain of subtype B of P. aeruginosa flagellin (FlaBD2) lacking a TLR5 binding motif), or an immunogenic fragment thereof, or (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 ( P. aeruginosa flagellin (FlaA1) subtype A1 D2 domain lacking a TLR5-binding motif), or its immunogenic a fragment, or (iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 12 (the D2 domain of subtype A2 of P. aeruginosa flagellin (FlaA2) lacking a TLR5-binding motif), or an immunogenic fragment thereof. 44. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 10 (D2 domain of subtype B of P. aeruginosa flagellin (FlaBD2) without TLR5 binding motif), or an immunogenic fragment thereof. 45. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ) or an immunogenic fragment thereof. 46. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 47. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 7 ( P. aeruginosa FliC subtype B flagellin (FlaB)), or an immunogenic fragment thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 48. The immunogenic composition according to claim 38, in which the fusion protein contains one or more polypeptide antigens and a second affinity molecule, where the second affinity molecule is a biotin-binding protein or its biotin-binding domain, and where one or more polypeptide antigens contain (i) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the SEQ sequence ID NO: 7 ( P. aeruginosa subtype B flagellin (FlaB)), or an immunogenic fragment thereof, and (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or an immunogenic fragment thereof. 49. Immunogenic composition according to any one of paragraphs. 39-48, characterized in that, when administered to a patient, the immunogenic composition causes the production of antibodies that recognize native MrkA on K. pneumoniae expressing MrkA. 50. Vaccine composition containing: (a) the first immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) a second immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) a third immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And (d) a fourth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer, wherein the vaccine composition is characterized in that, when administered to a patient, it induces an immune response against Klebsiella . 51. Vaccine composition containing: (a) the first immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) a second immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) a third immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) a fourth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) the fifth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) the sixth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) the seventh immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And (h) the eighth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; wherein the vaccine composition is characterized in that, when administered to a patient, it elicits an immune response against Pseudomonas . 52. Vaccine composition containing: (a) the first immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O1 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (b) a second immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (c) a third immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (d) a fourth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, KP O5 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (e) the fifth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O1 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (f) the sixth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O2 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (g) the seventh immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O3 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (h) the eighth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O4 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and a Rhavi-FlaBD2-PcrV fusion protein in a non-covalent complex with a biotinylated backbone polymer; (i) the ninth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide Klebsiella spp. K19, PA O5 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (j) the tenth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O6 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; (k) the eleventh immunogenic immunogenic composition according to claim 1, containing a framework polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O10 OPS conjugated to Klebsiella spp. capsular polysaccharide. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; And (l) the twelfth immunogenic composition according to claim 1, containing a backbone polymer, including a polymer that is a biotinylated capsular polysaccharide of Klebsiella spp. K19, PA O11 OPS conjugated to the capsular polysaccharide of Klebsiella spp. K19, and the Rhavi-FlaBD2-MrkA fusion protein in a non-covalent complex with a biotinylated backbone polymer; wherein the vaccine composition is characterized in that, when administered to a patient, it induces an immune response against Klebsiella and/or Pseudomonas . 53. Vaccine composition according to any one of paragraphs. 50-52, wherein the Rhavi-FlaBD2-MrkA fusion protein and/or the Rhavi-FlaBD2-PcrV fusion protein contains (i) a biotin-binding protein or biotin-binding domain thereof, and (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10 ( P. aeruginosa flagellin (FlaBD2) subtype B D2 domain without TLR5 binding motif), or its immunogenic fragment. 54. Vaccine composition according to any one of paragraphs. 50-52, wherein the Rhavi-FlaBD2-PcrV fusion protein comprises (i) a biotin-binding protein or biotin-binding domain thereof, and (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 55. Vaccine composition according to any one of paragraphs. 50-52, in which the Rhavi-FlaBD2-PcrV protein contains (i) a biotin-binding protein or a biotin-binding domain thereof, (ii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 10 (D2 domain of flagellin FlaB lacking a TLR5 binding motif), or an immunogenic fragment thereof, and (iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence SEQ ID NO: 8 (PcrV P. aeruginosa ), or its immunogenic fragment. 56. Vaccine composition according to any one of paragraphs. 50-52, wherein the Rhavi-FlaBD2-MrkA fusion protein comprises (i) a biotin-binding protein or biotin-binding domain thereof, (ii) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 10 (flab flagellin D2 domain devoid of a TLR5 binding motif), or an immunogenic fragment thereof, and (iii) a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 3 (MrkA K. pneumoniae ), or its immunogenic fragment. 57. Vaccine composition according to any one of paragraphs. 50-52, characterized in that, when administered to a patient, the vaccine composition causes the production of antibodies that recognize native MrkA on K. pneumoniae expressing MrkA. 58. Vaccine composition according to any one of paragraphs. 50-52, wherein the Rhavi-FlaBD2-MrkA fusion protein contains a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 24. 59. Vaccine composition according to any one of paragraphs. 50-52, wherein the Rhavi-FlaBD2-PcrV fusion protein comprises a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 26. 60. Immunogenic composition or vaccine composition according to any one of paragraphs. 1-59, wherein the immune response is antibody production or a B cell response. 61. Immunogenic composition or vaccine composition according to any one of paragraphs. 1-59, wherein the immune response is a CD4+ T cell response, including a Th1, Th2, or Th17 response, or a CD8+ T cell response, or a CD4+/CD8+ T cell response. 62. Immunogenic composition or vaccine composition according to any one of paragraphs. 1-59, wherein the immune response is: antibody or B-cell response; And T cell response. 63. Pharmaceutical composition containing one or more immunogenic compositions or vaccine compositions according to paragraphs. 1-62 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is characterized in that, when administered to a patient, it induces an immune response against Klebsiella and/or Pseudomonas . 64. Pharmaceutical composition according to claim 63, additionally containing aluminum phosphate as an adjuvant. 65. Pharmaceutical composition according to claim 63 or 64 formulated for injection. 66. Pharmaceutical composition according to any one of paragraphs. 63-65, characterized in that after administration to the patient, the pharmaceutical composition causes a Th1 and/or Th17 cell response. 67. Pharmaceutical composition according to any one of paragraphs. 63-66, characterized in that after administration to the patient, the pharmaceutical composition causes an opsonic/bactericidal response against one or more of Klebsiella and Pseudomonas . 68. Pharmaceutical composition according to any one of paragraphs. 63-67, characterized in that after administration to the patient, the pharmaceutical composition leads to a decrease in the rate of infection transmission and/or colonization of mucous surfaces by one or more of Klebsiella and Pseudomonas. 69. Pharmaceutical composition according to any one of paragraphs. 63-68, characterized in that after administration to the patient, the pharmaceutical composition leads to a decrease in the rate of transmission of infection and / or colonization of the gastrointestinal tract by one or more of Klebsiella and Pseudomonas . 70. A method of immunizing a patient against an infection caused by Klebsiella , comprising administering to the patient (i) an effective amount of an immunogenic composition or vaccine composition according to any one of paragraphs. 1-62 or (ii) an effective amount of a pharmaceutical composition according to any one of paragraphs. 63-69. 71. A method of immunizing a patient against an infection caused by P. aeruginosa , comprising administering to the patient (i) an effective amount of an immunogenic composition or vaccine composition according to any one of paragraphs. 1-62 or (ii) an effective amount of a pharmaceutical composition according to any one of paragraphs. 63-69.

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