WO2025038896A1

WO2025038896A1 - Broad spectrum conjugate vaccine to prevent klebsiella pneumoniae and pseudomonas aeruginosa infections

Info

Publication number: WO2025038896A1
Application number: PCT/US2024/042585
Authority: WO
Inventors: Alan S. Cross; Sharon M. TENNANT
Original assignee: University of Maryland Baltimore; University of Maryland College Park
Current assignee: University of Maryland Baltimore; University of Maryland College Park
Priority date: 2023-08-16
Filing date: 2024-08-15
Publication date: 2025-02-20
Anticipated expiration: 2026-02-16

Abstract

The present invention is drawn to conjugates comprising a glycosylated native FlaA flagellin protein of Pseudomonas and Klebsiella surface polysaccharide antigens, such as Klebsiella pneumoniae O polysaccharides from serovars O1, O2a, O2a,c, O3, O4, O5, O7, O8 and O12. The present invention also provides pharmaceutical compositions comprising the same and methods of inducing an immune response in subjects by administering the compositions.

Description

Broad Spectrum Conjugate Vaccine to Prevent Klebsiella pneumoniae and Pseudomonas aeruginosa Infections

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. No.: 63/532,963, filed August 16, 2023. The content of the aforementioned application is relied upon and is incorporated by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND

DEVELOPMENT

This invention was made with government support under Contract Number All 42725 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 94,873 Byte xml file named “seqjisting.xml,” created on August 15, 2024.

FIELD OF THE INVENTION

The field of the invention generally relates at least to the fields of medicine, immunology, molecular biology and infectious diseases. In particular, the field of the invention relates to vaccines for treating or preventing invasive blood infections, urinary tract infections, respiratory infections (including cystic fibrosis), wound infections, central nervous system infections and burn infections as well as nosocomial and community acquired infections caused by Klebsiella and Pseudomonas bacteria and septic shock.

BACKGROUND OF THE INVENTION

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are leading causes of both community-onset and healthcare-associated infections (Magill et al., N Engl J Med, (2014), 27;370: 1198-208). With their dramatic increase in antimicrobial resistance and the collapse of the antibiotic development pipeline, the CDC has classified these pathogens as “urgent” and “serious” threats respectively (CDC. Antibiotic resistance threats in the United States 2019). Consequently, there has been a renewed interest in the development of vaccines for the prevention of these infections which might also reduce the need for antibiotics as well as decrease their transmissibility. The O-poly saccharides (OPS) of both of these bacterial genera have been shown to be targets for vaccine development. (Cryz etal., ] Clin Investig, (1987), 80:51-56; Hegerle et al., PLoS One, (2018), 13:e0203143). However, since the T cellindependent polysaccharide antigens are poor immunogens, they have been covalently linked to carrier proteins which enables the recruitment of T cell help and improves polysaccharide immunogenicity (Rappuoli R, Sci Transl Med, (2018), 29;10:eaat4615. doi: 10.1 126/scitranslmed.aat4615. PMID: 30158151 ). Many currently licensed bacterial polysaccharide vaccines are formulated as glycoconjugates.

A limited number of proteins, such as tetanus toxoid or a mutant of diphtheria toxoid, CRM197, have typically been used for many glycoconjugate vaccines; however, these proteins function solely as carriers to provide T cell help and are irrelevant to the infections targeted by the polysaccharides (Rappuoli R, Sci Transl Med, (2018), 29;10:eaat4615. doi: 10.1126/scitranslmed.aat4615. PMID: 30158151). A quadrivalent glycoconjugate vaccine was developed that targets both Klebsiella and Pseudomonas infections by conjugation of four O-polysaccharides of Klebsiella to a Psezdsfomonas-relevant protein, the flagellar (Fla) proteins, which are essential virulence factors of PA (Hegerle et al., PLoS One, (2018), 13:e0203143). Nearly 80% of Klebsiella infections are caused by these four O-polysaccharides (Trautmann et al. , Vaccine, (2004), 22:818-821; Choi M, et al., Frontiers in Microbiology, (2020), 11 :1249). Nearly 100% of invasive PA isolates express flagellar proteins A (having two subtypes) or B. In a recent survey of 386 invasive PA isolates, 59% expressed either FlaAl (28%) or FlaA2 (31%) (Nasrin et al., BMC Microbiology, (2022), 22:13).

The Fla proteins are excellent carriers for the KP OPS as demonstrated by the marked increase in anti-KP OPS antibodies when conjugated to the KP OPS (Hegerle el al., PLoS One, (2018), 13:e0203143). In contrast, when simply admixed with the KP OPS (i.e. not conjugated) there was little KP OPS antibody formation. Significantly, PA flagellin is a potent TLR5 agonist that when administered to human subjects is highly reactogenic (Turley et al., Vaccine, (2011), 29:5145-5152). However, there was a loss of TLR5 signaling when recombinant FlaA (rFlaA) or recombinant FlaB (rFlaB) underwent conjugation to the Klebsiella polysaccharides (Hegerle et al., PLoS One, (2018), 13:e0203143). The rFlaA and rFlaB proteins used in this vaccine elicited robust antibody responses, which in the case of rFlaB-induced antibodies were protective against experimental infection and reduced PA motility in vitro. However, while the rFlaA elicited a strong immune response, these antibodies lacked functional activity against FlaA-bearing Pseudomonas strains (Hegerle et al. , PLoS One, (2018), 13:e0203143).

Further confirmation was established when it was found that antisera generated to the KP OPS was highly protective when given passively in a murine intravenous challenge model (Hegerle et al., PLoS One, (2018), 13:e0203143). The rFlaA and rFlaB proteins used in this vaccine elicited robust antibody responses, which in the case of rFlaB-induced antibodies were protective in a burned wound model of infection with FlaB-bearing Pseudomonas and reduced PA motility in vitro. However, while the rFlaA elicited a strong immune response, these antibodies lacked functional activity against FlaA-bearing Pseudomonas strains. Since nearly 60% of invasive PA clinical isolates express FlaA, a successful PA flagellar vaccine must elicit functionally active antibodies to FlaA as well as to FlaB.

Klebsiella and Pseudomonas bacteria are known to cause a wide variety of infections in human subjects including but not limited to wound infections, burn infections, urinary tract infections, respiratory infections, central nervous system infections, abscess formations, cystic fibrosis, in-dwelling catheter infections, invasive bacteremia, and septic shock.

What is needed are new and effective vaccines to treat or prevent infections caused by Klebsiella and/or Pseudomonas. This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be constmed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary, and thus do not restrict the scope of the embodiments. According to non-limiting example embodiments, in one aspect, the invention is directed to a conjugate for preventing bacterial infections in a subject caused by Klebsiella and Pseudomonas bacteria, wherein the conjugate comprises a glycosylated native FlaA flagellin protein of Pseudomonas and surface polysaccharide antigens and/or the core oligosaccharides of Klebsiella.

In another aspect, the invention is directed to a pharmaceutical composition for preventing bacterial infections in a subject caused by Klebsiella and Pseudomonas bacteria comprising a first conjugate and a second conjugate, wherein the first conjugate comprises a glycosylated native FlaA flagellin protein of Pseudomonas and surface polysaccharide antigens and/or the core oligosaccharides of Klebsiella, and the second conjugate comprises a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof and surface polysaccharide antigens and/or the core oligosaccharides of Klebsiella. In some embodiments, the FlaB flagellin protein is recombinantly produced and is not glycosylated.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a first conjugate and a second conjugate, wherein the first conjugate comprises a glycosylated native FlaA flagellin protein of Pseudomonas and O polysaccharide antigens and/or the core oligosaccharides derived from Klebsiella, and the second conjugate comprises a recombinant FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof and O polysaccharide antigens and/or the core oligosaccharides derived from Klebsiella.

In another embodiment the present invention relates to a pharmaceutical composition comprising one or more conjugates, wherein the one or more conjugates individually or collectively comprise six individual antigens selected from two flagellin proteins or fragments or derivatives thereof derived from Pseudomonas and nine O polysaccharide (OPS) antigens selected from Klebsiella species, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. In some embodiments, the second flagellin protein comprises a recombinant FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof.

In another embodiment the present invention relates to a conjugate comprising two Pseudomonas flagellins or fragments or derivatives thereof as carriers for four Klebsiella O polysaccharide antigens, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas . In some embodiments, the second flagellin protein comprises a recombinant FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof.

In another embodiment the present invention relates to a method for preparing a conjugate for preventing Pseudomonas and Klebsiella bacterial infections comprising linking OPS antigens and flagellin proteins or fragments or derivatives thereof using a chemical crosslinking agent, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. In some embodiments, the second flagellin protein comprises a recombinant FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof.

In another embodiment the present invention relates to a passive immunization method for treating a subject with a Pseudomonas or Klebsiella bacterial infection with an immunologically effective amount of an intravenous immunoglobulin preparation (IVIG) prepared from a host which has been immunized with a one or more conjugates comprising O polysaccharides or core oligosaccharides from Klebsiella and flagellin proteins or fragments or derivatives thereof, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. In some embodiments, the second flagellin protein comprises a recombinant FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof.

In another embodiment the present invention relates to a method for eliciting a passive immune response in a subject comprising administering to the subject in need thereof an immunologically effective amount of an intravenous immunoglobulin preparation prepared by administering to a subject one or more conjugates comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a flagellin protein or fragment or a derivative thereof from a Pseudomonas, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. In some embodiments, the subject is administered a first and second conjugate, the first conjugate comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of Pseudomonas and the second conjugate comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof is recombinant.

In another embodiment the present invention relates to a method for eliciting an active immune response and antibody production in a subject comprising administering to the subject in need thereof an immunologically effective amount of a conjugate comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of a Pseudomonas.

In another embodiment the present invention relates to a method for eliciting an active immune response and antibody production in a subject comprising administering to the subject in need thereof an immunologically effective amount of i) a first conjugate comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of a Pseudomonas and ii) a second conjugate comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a FlaB flagellin protein of a Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof is recombinant.

In another embodiment the present invention relates to a method for inducing an immune response in a mammal comprising administering to the subject in need thereof one or more conjugates comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a flagellin protein or fragment or a derivative thereof from a Pseudomonas, wherein the dosage of conjugate is about 5 to about 50 micrograms, wherein one conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of a Pseudomonas. In some embodiments, a second conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin or fragments or derivatives thereof is recombinant.

In another embodiment the present invention relates to a method for inducing an immune response in a mammal comprising administering to the subject in need thereof one or more conjugates comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a flagellin protein or fragment or a derivative thereof from a Pseudomonas, wherein one conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of a Pseudomonas, wherein the route of administration is subcutaneous, intravenous, intradermal, intramuscular or intranasal. In some embodiments, a second conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin or fragments or derivatives thereof is recombinant.

In another embodiment the present invention relates to a method for inducing an immune response in a mammal comprising administering to the subject in need thereof a composition comprising one or more conjugates comprising an O polysaccharide (OPS) from a Klebsiella covalently linked to a flagellin protein or fragment or a derivative thereof from a Pseudomonas along with an adjuvant selected from the group comprising or consisting of alum, a PRR ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand, TLR6 ligand, TLR7/8 ligand, TLR9 ligand, N0D2 ligand, and lipid A and analogues thereof, wherein one conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a glycosylated native FlaA flagellin protein of a Pseudomonas. In some embodiments, a second conjugate comprises an O polysaccharide (OPS) from a Klebsiella covalently linked to a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin or fragments or derivatives thereof is recombinant.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1. Chemical deglycosylation of native FlaA and native FlaB. Both native flagellin preparations were treated with TMFS as described in the Examples (methods to remove the attached O-glycans). The preparations were then subjected to SDS- PAGE and stained with Coomassie blue. Lane 1 -Protein molecular weight markers, lane 2- native FlaA, lane 3- deglycosylated FlaA, lane 4- recombinant FlaA-His tag, lane 5-empty, lane 6- native FlaB, lane 7- deglycosylated FlaB, lane 8- recombinant FlaB-His tag.

FIG. 2. Immune response in mice to native FlaA (nFlaA), deglycosylated native FlaA (d nFlaA), recombinant FlaA (rFlaA), and placebo (PBS). Anti-nFlaA (panel A), anti-PAO6 (panel B), and anti-PAO2 titers were measured by ELISA. Anti- PA02 titer was measured by either coating PA02 COPS (panel C) or P. aeruginosa strain NUH5446 lysate (panel D). For positive control in the PA02 COPS ELISA, sera from rabbits immunized with heat inactivated PA IATS PA02 strain was used. ELISA units represent the absorbance multiplied by serum dilution just above 0.2.

FIG. 3. Motility Inhibition of P. aeruginosa strain PAK (IAIS 06 FlaAl). Pooled mice sera were assessed for the ability to inhibit motility of FlaA-i- Pseudomonas strain PAK. A. The diameter of the motility halo (in mm) was measured in duplicate for each group. B. Relative motility (i.e. percentage of motility inhibition) was calculated after motility was normalized to that of the PBS group (pooled sera from mice immunized with saline). C. The diameter of the halo depicts the extent of the motility. The smaller the halo, the lesser the motility. The positive control used in this assay is anti-native FlaA sera. In the "No sera control", the bacteria were added to the agar well containing PBS instead of sera.

FIG. 4. TLR4 and TLR5 bioactivity. Pseudomonas LPS, native flagellin (nFlaA, nFlaB), recombinant flagellin (rFlaA, rFlaB) and deglycoslylated flagellin (dFlaA, dFlaB) were added to human TLR4 and TLR5 reporter cells. (A) FlaA formulations and LPS were added to humanTLR4 reporter cells. (B) FlaB formulations and LPS were added to human TLR4 reporter cells. (C) FlaA formulations were added to human TLR5 reporter cells; (D) FlaB formulations were added to the TLR5 reporter cells.

FIG. 5. Immunization of mice with native FlaA (nFlaA) protects mice against lethal infection with FlaA-bearing strains of Pseudomonas. (A) Mice were immunized with either rFlaA or nFlaA prepared from a PA06 strain (or given an equal volume of PBS) and challenged with PA 06 FlaAl. (B) Mice were immunizaed with either nFlaA or deglycosylated FlaA and challenged with FlaA-bearing Pseudomonas with a different IATS 0 type (PA 02/16) to ensure that the protection was not due to any residual LPS. (A) Immunization with the nFlaA but not the rFlaA protected the mice from lethal infection. (B) Immunization with nFlaA, but not with deglycosylated FlaA protected against lethal infection with a FlaA-bearing strain but of a different O serotype.

FIG. 6. Conjugation of KP O polysaccharide to nFlaA. Two separate lots of nFlaA conjugate vaccines (conjugates 1 and 2) were prepared that differed in molecular weight as shown on SDS-PAGE (A), Western Blot ( B) and size-exclusion chromatography (C).Lane 1 -molecular weight marker; lane 2-KP01 -nFlaA conjugate 1 (2.5 pg); Lane 3- KPOl-nFlaA conjugate 1 (5 pg); lane 4-empty; lane 5 KP01- nFlaA conjugate 2 (1.13 pg); lane 6 -KPOl-nFlaA conjugate 2 (1.13 pg); lane 7- empty; lane 8. nFlaA (1 pg).(C) UPLC-SEC Profiles of KP-nFlaA Final Conjugates. Lot U 1 -C VD210917-01 (black trace) is higher molecular weight compared to Lot U1 - CVD210917-02 (blue trace). The SDS-PAGE gel shows high molecular weight conjugates with no free nFlaA. Anti-FlaA sera was used for the Western blot.

FIG. 7. Differing protective efficacy in a mouse burn wound infecton model of two batches of Kp Ol mFlaA vaccines differing in nFlaA content. 8-10 week old CD-I mice received 3 doses of either lOug KPOl-nFlaA-Ol or KPO 1 -nFlaA-02 conjugate vaccine at 2 week intervals by subcutaneous administration. (A) KPOl- nFlaA conjugate 1 elicited a more robust IgG antibody response to FlaA than KPOl- nFlaA conjugate 2; (B) The protection of KPOl-nFlaA-Lot 01 conjugate vaccine in a mouse bum model. The mice were challenged with 6.4xlO^A5 CFU of PA UNH5446 PA 02 (i.e. non-06) immediately post-bum by subcutaneous administration. The survival rate of vaccine group was 80%, while the survival rate of control group was 10%. The difference was significant (p=0.0006). (C) The protection of KPOl-nFlaA- Lot 02 conjugate vaccine in a mouse bum model. The mice were challenged with 6.4xlO^A5 CFU of PA UNH5446 (i.e. non-06) immediately post-burn by subcutaneous administration. The survival rate of the vaccine group was 20%, while the survival rate of control group was 10%.

FIG. 8. Antibodies elicited by KP Ol-nFlaA-01 were better able to reduce the motility of a FlaA-i- Pseudomonas strain than KP 01 -nFlaA conjugate 2. 8-10 week old CD-I mice received 3 doses of either lOug KPOl-nFlaA-Ol or KPO1- nFlaA-02 conjugate vaccine at 2 week intervals by subcutaneous administration, and sera harvested. The sera were diluted 1:50 and added to 0.3% soft tryptone agar and pre-incubated in a 24 well plate (in duplicate). Pseudomonas strains PAK (FlaA+) and PAO1 (FlaB+) were grown to to log phase, normalized to OD 1.0 and diluted 1 : 1000. Using a sterile toothpick, they were then stabbed centrally into the agar and incubated at 30° C with a wet towel. KPO 1 -nFlaA-01 sera inhibited the motility of PAK but not PAO1 . KPO2-nFlaA-02 sera did not inhibit the motility of either PAK or PAO1 .

FIG. 9. Native FlaA retains it carrier protein function when conjugated to KP O1 O polysaccharide. Both KPOl-nFlaA conjugate vaccines 01 and 02 were able to enhance the immunogenicity of KP: 01 OPS in mice following immunization subcutaneously with 10 ug mcg OPS on days 0, 14 and 28. Sera was obtained at day 35 (7 days after the final vaccine dose) and KPO1 antibody levels were measured by ELISA.

FIG. 10. Enzymatic deglycosylation of native FlaA using OglyZOR enzymes. (Enzyme mix composed of endoglycosidase OglyZOR and SialEXO).

FIG. 11. ELISA: KPO1 OPS-nFlaA conjugate vaccine mouse sera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved conjugates for generating immune responses in subjects against Klebsiella and Pseudomonas antigens, and for use as vaccines to prevent or treat against Klebsiella and Pseudomonas infections in subjects. Further investigation of the failure of existing Klebsiella and Pseudomonas conjugates resulted in an evaluation of the glycosylation properties of the Fla proteins. Native FlaA and FlaB are glycosylated with different glycans, but neither rFlaA nor rFlaB, used as carrier proteins in the KP quadrivalent vaccine, are glycosylated. The present inventors have surprisingly discovered that whereas antisera to the rFlaA that lacks glycosylation is not protective in murine challenge studies, antisera to the fully glycosylated native FlaA (nFlaA) is highly protective when given either alone or as part of a glycoconjugate vaccine. These findings support that unlike the case for FlaB, the FlaA glycan is part of the protective epitope and should be conserved.

In some embodiments, the present invention relates to a pharmaceutical composition, such as a vaccine product, which encompasses a first and second conjugate, the first conjugate comprising four different OPS polysaccharide antigens from Klebsiella pneumoniae serovars conjugated with a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa, and the second conjugate comprising four different OPS polysaccharide antigens from Klebsiella pneumoniae serovars conjugated with a FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, the FlaB flagellin protein is recombinantly produced and is not glycosylated. In some embodiments, the vaccine can have efficacy for therapeutic use to treat or prevent Pseudomonas and Klebsiella bacterial infections.

At present, there is no simple and broadly effective vaccine which is effective against both Klebsiella and Pseudomonas. In some aspects, the invention described herein is a novel conjugate vaccine which comprises antigens from both bacterial types and can be manufactured in a large scalable fashion. Moreover, in some embodiments, the vaccine could also be used to generate therapeutic immunoglobulin (IVIG) preparations for passive protection against acute infections.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.j; The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471 186341); and other similar technical references.

For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of "or" means "and/or" unless stated otherwise. The use of “a” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and/or “consisting of.”

Conjugate

In one aspect, the present invention is directed to a conjugate comprising a Klebsiella surface polysaccharide antigen and a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa. In particular aspects of the invention, the surface polysaccharide antigen and the flagellin are covalently linked optionally via a linker. In some embodiments, the linker is sulfo-GMBS (N-y-maleimidobutyryl- oxy sulfosuccinimide ester) (ThermoFisher Scientific).

In some embodiments, the conjugate further comprises one or more flagellins of Pseudomonas aeruginosa or fragments or derivatives thereof. In some embodiments, the one or more flagellins comprise FlaB flagellin protein of Pseudomonas or fragments or derivatives thereof. In some embodiments, FlaB is recombinantly produced.

The Klebsiella surface polysaccharide antigen can be any known Klebsiella surface polysaccharide antigen or a derivative or antigenic fragment thereof. In some embodiments, the surface polysaccharide is from one or more Klebsiella pneumoniae serovars. In some aspects of the invention, the Klebsiella surface polysaccharide antigen can be an O polysaccharide (OPS), a core oligosaccharide and an O polysaccharide (COPS), a capsule polysaccharide or combinations thereof.

As used herein, “OPS” is a polysaccharide in which the lipid A moiety from lipopolysaccharide (LPS) and core oligosaccharide have been removed. In some embodiments of the invention, the surface polysaccharide antigen is an OPS. In some embodiments, the surface polysaccharide antigen is from epidemiologically relevant Klebsiella O serovars such as Klebsiella pneumoniae serovar 01, 02 (including any subtypes such as 02a, 02ac, 02c, 02ae, 02aeh, and 02afg), 03 and 05. In some embodiments, the surface polysaccharide antigen is an OPS derived from Klebsiella pneumoniae serovars 01, 02a, 02ac, 02c, 02ae, 02aeh, 02afg, 03, 04, 05, 07, 08 and 012. In some embodiments, the surface polysaccharide antigen is an OPS derived from Klebsiella pneumoniae serov ars 01, 02a, 03 and 05.

As provided herein, one conjugate comprises a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa. In some embodiments, the conjugate further comprises one or more additional flagellins from Pseudomonas or fragments or derivatives thereof. The one or more additional flagellins can be any known Pseudomonas flagellin. In some embodiments, the one or more additional flagellins are native and can be glycosylated. In some embodiments, the one or more additional flagellins are recombinant. As used herein, the term “flagellin” encompasses flagellin, fragments of flagellin and derivatives thereof. A “glycosylated native” flagellin from Pseudomonas refers to a Pseudomonas flagellin that is isolated from its Pseudomonas cell and exhibits a normal glycosylation pattern. In particular aspects of the invention, the Pseudomonas flagellin is a Pseudomonas aeruginosa (PA) flagellin. It is believed that all pathogenic Pseudomonas aeruginosa express a single polar flagellum that extends from the cell surface to enable motility, that is comprised chiefly by polymers of either type A or B flagellin proteins. In some aspects of the invention, the one or more additional flagellin is a Pseudomonas aeruginosa (PA) flagellin type B (FlaB) or an antigenic fragment or derivative thereof.

In some embodiments, the Pseudomonas aeruginosa flagellin type A (FlaA) comprises SEQ ID NO: 1. In some embodiments, the Pseudomonas aeruginosa flagellin type A amino acid sequence is encoded by a nucleotide sequence comprising SEQ ID N0:3. Pseudomonas aeruginosa flagellin type B (FlaB) comprises SEQ ID N0:2 or an antigenic fragment or derivative thereof.

In some embodiments, the conjugate comprises i) a glycosylated native Pseudomonas aeruginosa flagellin type A (FlaA) and ii) OPS from Klebsiella pneumoniae selected from Klebsiella pneumoniae serovars 01 , 02a, 03, 05 or combinations thereof. In some embodiments, the conjugate further comprises a Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or derivative thereof. In some embodiments, the FlaB or antigenic fragment or derivative thereof is recombinant. In some embodiments, the Pseudomonas flagellin can be covalently linked to one or more OPS from a single Klebsiella pneumoniae serovar type or may be linked to OPS from multiple Klebsiella pneumoniae serovar types.

The ratio or stoichiometry of surface polysaccharide to flagellin is not limiting. In some embodiments, the Pseudomonas flagellin (or an antigenic fragment or derivative thereof) can be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more surface polysaccharides, such as OPS, from the same Klebsiella or from mixtures of Klebsiella serovar types.

In some embodiments, the Pseudomonas flagellin (or an antigenic fragment or derivative thereof) is linked to one to four OPS from the same serovar type. In another embodiment, the Pseudomonas flagellin (or an antigenic fragment or derivative thereof) is linked to one to four OPS from at least two different serovar types. In another embodiment, the flagellin (or an antigenic fragment or derivative thereof) is linked to one to four OPS, each from different serovar types. In some embodiments, the Klebsiella serovars comprise Klebsiella pneumoniae serovar 01, 02a, 03, and 05.

In one embodiment, the conjugate comprises a glycosylated native Pseudomonas aeruginosa flagellin type A (FlaA) comprising a sequence of SEQ ID NO:1 and a surface polysaccharide from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof, including all of 01, 02a, 03, and 05. In some embodiments, the surface polysaccharide is OPS.

In another embodiment, the conjugate further comprises SEQ ID N0:2 or an antigenic fragment or derivative thereof that can also be conjugated to surface polysaccharide from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof. In some embodiments, the surface polysaccharide is OPS.

Examples of fragments or derivatives of Pseudomonas flagellin can include fragments of the natural protein, including internal sequence fragments of the protein that retain their ability to elicit protective antibodies against a desired bacteria. The derivatives are also intended to include variants of the natural protein such as proteins having changes in amino acid sequence but that retain the ability to elicit an immunogenic, biological, or antigenic property as exhibited by the natural molecule.

By derivative is further meant an amino acid sequence that is not identical to the wild type amino acid sequence, but rather contains at least one or more amino acid changes (deletion, substitutions, inversion, insertions, etc.) that do not essentially affect the immunogenicity or protective antibody responses induced by the modified protein as compared to a similar activity of the wild type amino acid sequence, when used for the desired purpose. In some embodiments, a derivative amino acid sequence contains at least 85-99% homology at the amino acid level to the specific amino acid sequence. In further embodiments, the derivative has at least 90% homology at the amino acid level. In other embodiments, the derivative has at least 95% homology.

In the case of FlaB, the flagellin may be a peptide encoding the native amino acid sequence or it may be a derivative or antigenic fragment of the native amino acid sequence.

In some embodiments, the surface polysaccharide antigen of a Klebsiella is covalently linked to the glycosylated native Pseudomonas flagellin FlaA or FlaB protein or an antigenic fragment or a derivative thereof either directly or with a linker. In some embodiments, the linker or linking chemical is selected from sulfo-GMBS (N-y-maleimidobutyryl-oxysulfosuccinimide ester), l-cyano-4- dimethylaminopyridinium tetrafluoroborate (CDAP), adipic acid dihydrazide, e- aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p- nitrophenylethyl amine. In a particular embodiment, the linking chemical is CDAP.

In some embodiments, the surface polysaccharide, such as OPS, is conjugated with flagellin at a weight ratio of from about 1 : 1 (OPS: flagellin) to about 20:1 (OPS:flagellin). In some embodiments, the weight ratio is about 2:1 to about 6: 1 (OPS:flagellin).

Compositions

In some embodiments, the invention provides compositions comprising effective amounts of conjugates of the invention.

In one embodiment, the compositions comprise an effective amount of a conjugate comprising glycosylated native Pseudomonas aeruginosa flagellin type A (FlaA) conjugated to surface polysaccharide from Klebsiella pneumoniae, such as from serovars 01, 02a, 03, 05 or combinations thereof.

In some embodiments, the compositions further comprise an effective amount of one or more additional conjugates. In some embodiments, the one or more additional conjugates comprise a FlaB flagellin protein of Pseudomonas aeruginosa or fragments or derivatives thereof and surface polysaccharide antigens from Klebsiella pneumoniae, such as from serovars 01, 02a, 03, 05 or combinations thereof. In some embodiments, the FlaB flagellin protein is recombinantly produced and is not glycosylated. In some embodiments, the surface polysaccharide is OPS.

In some embodiments, the compositions are pharmaceutical or vaccine compositions which provide protective immunity against one or more Klebsiella and/or Pseudomonas pathogens and which comprise one or more of the above- mentioned conjugates. In some embodiments, effective amounts of one or more unconjugated Pseudomonas flagellin can be added to the compositions of the invention. In some embodiments, adding one or more unconjugated flagellin to compositions comprising one or more conjugates can enhance the immune response to the flagellin epitopes. In some embodiments, the one or more unconjugated Pseudomonas flagellin is selected from flagellin comprising SEQ ID NO:1, SEQ ID N0:2, antigenic fragments and derivatives thereof and combinations thereof.

In some embodiments, the vaccine composition is a multivalent conjugate vaccine comprising an effective amount of one or more Pseudomonas flagellins linked to one or more Klebsiella surface polysaccharides, such as O polysaccharides (OPS), wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. For example, the composition can be a multivalent conjugate vaccine comprising an effective amount of two different Pseudomonas flagellin proteins or antigenic fragments or derivatives thereof covalently linked to one or more Klebsiella O polysaccharides (OPS). In some embodiments, the multivalent conjugate vaccine comprises an effective amount of OPS antigens from one or more of Klebsiella pneumoniae serovars 01, 02 (including any subtypes such as 02a, 02ac, 02c, 02ae, 02aeh, and 02afg), 03, 04, 05, 07, 08 and 012. In some embodiments, the multivalent conjugate vaccine comprises an effective amount of four different OPS antigens from Klebsiella pneumoniae serovars 01, 02a, 03, and 05. In some embodiments, the Pseudomonas is Pseudomonas aeruginosa.

In some embodiments, the composition comprises an effective amount of one or more conjugates comprising a Pseudomonas flagellin protein or an antigenic fragment or derivative thereof and a surface polysaccharide from Klebsiella, wherein one conjugate comprises a glycosylated native FlaA flagellin protein of Pseudomonas. In one embodiment, the composition comprises a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa and an OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof.

In some embodiments, the composition comprises a multivalent conjugate vaccine comprising an effective amount of a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa having a sequence comprising SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae; and ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO: 1 and one or more OPS from Klebsiella pneumoniae; and ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof; and ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof; and ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovars 01, 02a, 03, 05 or combinations thereof.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 01; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 02a; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 03; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 05. In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO: 1 and one or more OPS from Klebsiella pneumoniae serovar 01 ; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovar 02a; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 03; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 05.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 01; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 03; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 02a; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 05.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovar 01 ; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovar 03; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID NO:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 02a; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID NO:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 05.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 01; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and one or more OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar O2a; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 03.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovar 01; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and one or more OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 02a; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and one or more OPS from Klebsiella pneumoniae serovar 03.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 02a; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 03.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 02a; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 03.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 03 ; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 02a.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 03; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 05; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 02a.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 02a; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and OPS from Klebsiella pneumoniae serovar 03; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 05.

In some embodiments, the composition comprises: i) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 02a; ii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein according to SEQ ID NO:1 and OPS from Klebsiella pneumoniae serovar 03; iii) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 01 ; and iv) an effective amount of a conjugate comprising a Pseudomonas aeruginosa FlaB flagellin protein according to SEQ ID N0:2 or an antigenic fragment or derivative thereof and OPS from Klebsiella pneumoniae serovar 05.

In some embodiments, the invention provides a composition comprising an effective amount of sera from a subject administered one or more conjugates or pharmaceutical compositions comprising conjugates herein. In some embodiments, the invention provides a composition comprising an effective amount of purified or enriched immunoglobulins from a subject administered one or more conjugates or pharmaceutical compositions comprising conjugates herein. In some embodiments, the composition comprising sera or the immunoglobulins can be administered to a subject in immunotherapy applications.

In some embodiments, the compositions are pharmaceutical compositions comprising one or more conjugates and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. Adjuvants are substances that can be used to specifically augment a specific immune response. In some embodiments, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based upon their composition. These groups include oil adjuvants (for example, Freund's complete and incomplete), mineral salts (for example, A1K(SO4)2, AlNa(SO4h, AINH4 (SO4), silica, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella. Adjuvants are described by Warren et al. (Ann. Rev. Biochem., 4:369-388, 1986), the entire disclosure of which is hereby incorporated by reference. Further adjuvants suitable for use in the present invention include alum, a PRR ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand, TLR6 ligand, TLR7/8 ligand, TLR9 ligand, N0D2 ligand, and lipid A and analogues thereof.

In some embodiments use of a flagellin protein or antigenic fragment or derivative thereof provides an inherent adjuvant boost, and stimulates a robust immune response without the addition of further adjuvant. Thus, in some embodiments, the flagellin protein antigenic fragment or derivative thereof acts an adjuvant which stimulates innate immunity through TLR5 to improve the immunogenicity of surface polysaccharide antigen (e.g., OPS) within the composition. In some embodiments, the carrier is a flagellin antigenic fragment or derivative thereof which has a diminished capability to stimulate innate immunity through TLR5. In some embodiments, an adjuvant is added to the compositions while in other embodiments, no adjuvant is added. In some embodiments, conventional adjuvants can be administered. Among those substances that can be included are the saponins such as, for example, Quil A. (Superfos A/S, Denmark). In some embodiments, immunogenicity of the conjugates in both mice and rabbits is enhanced by the use of monophosphoryl lipid A plus trehalose dimycolate (Ribi-700; Ribi Immunochemical Research, Hamilton, Mont.) as an adjuvant. Alum, a PRR ligand, TLR3 ligand, TLR 4 ligand, TLR5 ligand, TLR6 ligand, TLR7/8 ligand, TLR9 ligand, N0D2 ligand, and lipid A and analogues thereof may separately or in combination may also be used as adjuvants. Examples of materials suitable for use in vaccine compositions are provided in Remington's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp. 1324- 1341 (1980), which disclosure is incorporated herein by reference).

In some embodiments, the pharmaceutical or vaccine composition can be formulated into liquid preparations for, e.g., nasal, rectal, buccal, vaginal, peroral, intragastric, mucosal, perlinqual, alveolar, gingival, olfactory, or respiratory mucosa administration. Suitable forms for such administration include solutions, suspensions, emulsions, syrups, and elixirs. The pharmaceutical or vaccine composition can also be formulated for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration, injectable administration, sustained release from implants, or administration by eye drops. Suitable forms for such administration include sterile suspensions and emulsions. Such pharmaceutical or vaccine composition can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, and the like. The pharmaceutical or vaccine composition can also be lyophilized. The pharmaceutical or vaccine composition can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Texts, such as Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) and Remington's Pharmaceutical Sciences, Mack Pub. Co.; 18^th and 19^th editions (December 1985, and June 1990, respectively), incorporated herein by reference in their entirety, can be consulted to prepare suitable preparations. Such preparations can include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components can influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

In some embodiments, the pharmaceutical or vaccine composition of the invention is administered parenterally. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. In some embodiments, the pharmaceutical or vaccine composition for parenteral administration may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Suspensions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or solvent, such as a solution in 1,3 -butanediol. Suitable diluents include, for example, water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectable preparations.

Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

In some embodiments, the pharmaceutical or vaccine composition is provided as a liquid suspension or as a freeze-dried product (or freeze-dried hyperimmune globulin for oral administration). Suitable liquid preparations include, e.g., isotonic aqueous solutions, suspensions, emulsions, or viscous compositions that are buffered to a selected pH. Transdermal preparations include lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size, as discussed below.

In some embodiments, when in the form of solutions, suspensions and gels, in some embodiments, the composition contains a major amount of water (preferably purified endotoxin-free water) in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers, dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, colors, and the like can also be present.

In some embodiments, the compositions are preferably isotonic with the blood or other body fluid of the recipient. The isotonicity of the compositions can be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is particularly preferred. Buffering agents can be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. In some embodiments of the invention, phosphate buffered saline is used for suspension.

In some embodiments, the viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent. In some embodiments, methylcellulose is used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. In some embodiments, viscous compositions are prepared from solutions by the addition of such thickening agents.

In some embodiments, a pharmaceutically acceptable preservative can be employed to increase the shelf life of the compositions. Benzyl alcohol can be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride can also be employed. A suitable concentration of the preservative can be from 0.02% to 2% based on the total weight although there can be appreciable variation depending upon the agent selected.

In some embodiments, pulmonary delivery of the composition can also be employed. In some embodiments, the composition is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for the dispensing of the conjugate. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

In embodiments where the compositions are prepared for pulmonary delivery in particulate form, it has an average particle size of from 0.1 pm or less to 10 pm or more. In some embodiments, it has an average particle size of from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 pm to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 pm for pulmonary delivery. Pharmaceutically acceptable carriers for pulmonary delivery of the insufflation include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations can include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be used, including polyethylene glycol and dextrans, such as cyclodextran and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids can also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers can also be employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise the composition dissolved or suspended in water at a concentration of about 0.01 or less to 100 mg or more of conjugate per mL of solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg of conjugate per mL of solution. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant, to reduce or prevent surface induced aggregation of the conjugate or composition caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the vaccine composition suspended in a propellant with the aid of a surfactant. The propellant can include conventional propellants, such chlorofluorocarbon, a hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons, such as trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1, 1,2- tetrafluoroethane, and combinations thereof. Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing the vaccine composition, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.

In some embodiments, the invention is directed to kits comprising one or more pharmaceutical or vaccine compositions of the invention. Such kits can be provided to an administering physician or other health care professional.

In some embodiments, the kit is a package that houses one or more containers which comprises one or more vaccine compositions and instructions for administering the vaccine composition to a subject. In some embodiments, the kit can also comprise one or more other therapeutic agents. The kit can optionally contain one or more diagnostic tools and instructions for use.

In some embodiments, the kit comprises an immunization schedule. In some embodiments, a pharmaceutical or vaccine cocktail containing two or more conjugates or compositions can be included, or separate pharmaceutical compositions containing different conjugates or therapeutic agents. The kit can also contain separate doses of the pharmaceutical or vaccine composition for serial or sequential administration.

In some embodiments, the kit further comprises suitable delivery devices, e.g. , syringes, inhalation devices, and the like, along with instructions for administrating the therapeutic agents. The kit can optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits can include a plurality of containers reflecting the number of administrations to be given to a subject. If the kit contains a first and second container, then a plurality of these can be present.

Methods of Treatment

In another embodiment, the invention is directed to a method of inducing an immune response in a subject, comprising administering to a subject in need thereof an effective amount of the above-mentioned conjugates or compositions. In some embodiments, the surface polysaccharide antigen is an O polysaccharide (OPS), a core oligosaccharide and an O polysaccharide (COPS), a capsule polysaccharide, or combinations thereof. In some embodiments of the invention, the surface polysaccharide antigen is an O polysaccharide antigen (OPS). In some embodiments, the surface polysaccharide antigen and the flagellin can be covalently linked.

In some embodiments, method comprises administering multiple conjugates comprising one or more Pseudomonas flagellins or antigenic fragments or derivatives thereof covalently linked to one or more Klebsiella O polysaccharides (OPS) to induce an immune response, wherein one flagellin protein comprises a glycosylated native FlaA flagellin protein of Pseudomonas. The multiple conjugates can comprise two different Pseudomonas flagellin covalently linked to one or more Klebsiella O polysaccharides (OPS). The two different Pseudomonas flagellins can be a Pseudomonas aeruginosa glycosylated native flagellin type A (FlaA) and a Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or derivative thereof.

In some embodiments, the multiple conjugates can comprise at least four different OPS antigens from Klebsiella pneumoniae. For example, in some embodiments, the four different OPS can be derived from Klebsiella pneumoniae serovars 01, 02a, 03 and 05. Further, the two different Pseudomonas flagellins can be a glycosylated native flagellin type A (FlaA) of Pseudomonas aeruginosa and Pseudomonas aeruginosa flagellin type B (FlaB) and/or the four Klebsiella OPS can be from Klebsiella pneumoniae serovars 01, 02a, 03 and 05. In some embodiments, the Pseudomonas flagellin can be covalently linked to one or more OPS from a single Klebsiella pneumoniae serovar type or can be covalently linked to OPS from multiple Klebsiella pneumoniae serovar types. The glycosylated native flagellin type A (FlaA) of Pseudomonas aeruginosa can comprise SEQ ID NO: 1 and/or the Pseudomonas aeruginosa flagellin type B (FlaB) can comprise SEQ ID N0:2.

In some embodiments, the conjugate or composition is administered multiple times to the subject. The conjugate or composition may also be administered a single time to the subject. The term "subject" as used herein, refers to animals, such as mammals. For example, mammals contemplated include humans, primates, dogs, cats, sheep, cattle, goats, pigs, horses, chickens, mice, rats, rabbits, guinea pigs, and the like. The terms "subject", "patient", and "host" are used interchangeably.

Human subjects are not limiting and can include deployed soldiers, hospital workers, patients and residents of chronic care facilities. In some embodiments, the patient is in a hospital or in a skilled nursing facility. In some embodiments, the subject is administered the conjugate or composition prior to, during, or after a surgery. The surgery is not limiting and can be, for example, colon surgery, hip arthroplasty, or small-bowel surgery. Further, the conjugate or composition can be administered prior to, during, or after a procedure selected from central venous catheterization, urinary tract catheterization, and intubation with a ventilator tube.

As used herein, an “immune response” is the physiological response of the subject’s immune system to an immunizing composition. An immune response may include an innate immune response, an adaptive immune response, or both. In some embodiments of the present invention, the immune response is a protective immune response. A protective immune response confers immunological cellular memory upon the subject, with the effect that a secondary exposure to the same or a similar antigen is characterized by one or more of the following characteristics: shorter lag phase than the lag phase resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; production of antibody which continues for a longer period than production of antibody resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a change in the type and quality of antibody produced in comparison to the type and quality of antibody produced upon exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a shift in class response, with IgG antibodies appearing in higher concentrations and with greater persistence than IgM, than occurs in response to exposure to the selected antigen in the absence of prior exposure to the immunizing composition; an increased average affinity (binding constant) of the antibodies for the antigen in comparison with the average affinity of antibodies for the antigen resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; and/or other characteristics known in the art to characterize a secondary immune response.

In some embodiments, the immunogenicity of the conjugates and compositions of the invention are greater than the immunogenicity of at least one of the surface polysaccharide antigen or flagellin protein or an antigenic fragment or a derivative thereof alone. Methods of measuring immunogenicity are well known to those in the art and primarily include measurement of serum antibody including measurement of amount, avidity, and isotype distribution at various times after injection of the conjugate vaccine. Greater immunogenicity may be reflected by a higher titer and/or increased life span of the antibodies. Immunogenicity may also be measured by the ability to induce protection to challenge with noxious substances or virulent organisms. Immunogenicity may also be measured by the ability to immunize neonatal and/or immune deficient mice. Immunogenicity may be measured in the patient population to be treated or in a population that mimics the immune response of the patient population.

In some embodiments, the immune response that is generated by the conjugates and compositions of the invention is a protective immune response against infection by one or more Klebsiella and/or Pseudomonas serovars, including those serovars described herein.

In some embodiments, the conjugates and compositions of the invention are administered alone in a single dose or administered in sequential doses. In other aspects of the invention, the conjugates and compositions of the invention are administered as a component of a homologous or heterologous prime/boost regimen in conjunction with one or more vaccines. In some embodiments of the invention, a single boost is used. In some embodiments of the invention, multiple boost immunizations are performed. In particular aspects of the invention drawn to a heterologous prime/boost, a mucosal bacterial prime/parenteral conjugate boost immunization strategy is used. In some embodiments, one or more (or all) of the live (or killed) attenuated Salmonella enterica serovars used as a reagent strain to express a Pseudomonas FlaB flagellin as taught herein can be administered orally to a subject and the subject can be subsequently boosted parenterally with a conjugate(s) and compositions of the invention as described herein. In some embodiments, one or more (or all) of the live (or killed) attenuated Klebsiella used as a reagent strain to isolate surface polysaccharide as taught herein can be administered orally to a subject and the subject can be subsequently boosted parenterally with a conjugates and compositions of the invention as described herein.

In practicing immunization protocols for treatment and/or prevention, an immunologically-effective amount of conjugates and compositions of the invention are administered to a subject. As used herein, the term "immunologically-effective amount" means the total amount of therapeutic agent (e.g., conjugate or composition) or other active component that is sufficient to show an enhanced immune response in the subject. When "immunologically-effective amount" is applied to an individual therapeutic agent administered alone, the term refers to that therapeutic agent alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously, and regardless of order of administration.

The particular dosage depends upon the age, weight, sex and medical condition of the subject to be treated, as well as on the method of administration. Suitable doses can be readily determined by those of skill in the art.

The conjugates and compositions of the invention can be administered by either single or multiple dosages of an effective amount. In some embodiments, an effective amount of the compositions of the invention can vary from 0.01-5,000 pg/ml per dose. In other embodiments, an effective amount of the conjugate or composition of the invention can vary from 0.1-500 pg/ml per dose, and in other embodiments, it can vary from 10-300 pg/ml per dose. In one embodiment, the dosage of the conjugate or composition administered will range from about 10 pg to about 1000 pg. In another embodiment, the amount administered will be between about 20 pg and about 500 pg. In some embodiments, the amount administered will be between about 75 pg and 250 pg. Greater doses may be administered on the basis of body weight. The exact dosage can be determined by routine dose/response protocols known to one of ordinary skill in the art.

In some embodiments, the amount of conjugates and compositions of the invention that provide an immunologically-effective amount for vaccination against Klebsiella and/or Pseudomonas infections is from about 1 pg or less to about 100 pg or more. In some embodiments, it is from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pg to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 pg per kg body weight. In some embodiments, the immunologically-effective amount for vaccination against Klebsiella and/or Pseudomonas infection is from 0.01 pg to 10 pg.

The conjugates and compositions of the invention may confer resistance to Klebsiella and/or Pseudomonas infections by either passive immunization or active immunization. In one embodiment of passive immunization, the conjugate or composition is provided to a subject (i.e. a human or mammal), and the elicited antisera is recovered and directly provided to a recipient suspected of having an infection caused by Klebsiella and/or Pseudomonas.

In some embodiments, the present invention provides a means for preventing or attenuating infection by Klebsiella and/or Pseudomonas or by organisms which have antigens that can be recognized and bound by antisera to the polysaccharide and/or protein of the conjugate or composition.

The administration of the conjugate or composition (or the antisera which it elicits) may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the conjugate or composition is provided in advance of any symptom of Klebsiella and/or Pseudomonas infection. The prophylactic administration of the conjugate or composition serves to prevent or attenuate any subsequent infection. When provided therapeutically, the conjugate or composition is provided upon the detection of a symptom of actual infection. The therapeutic administration of the conjugate or composition serves to attenuate any actual infection. The conjugate or composition of the invention may, thus, be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.

The conjugate or composition of the invention may be administered to warmblooded mammals of any age. The conjugate or composition can be administered as a single dose or in a series including one or more boosters. In some embodiments, the immunization schedule would involve a primary series of three immunizations with a spacing of 1-2 months between the doses. Tn some settings a booster dose could be administered - 6-12 months later. For example, an infant can receive three doses at 6, 10 and 14 weeks of age (schedule for infants in sub-Saharan Africa) or at 2, 4, and 6 months of life (schedule for U.S. infants). In some embodiments, U.S. infants might receive a booster at 12-18 months of age. Another target population would be U.S. elderly who would likely receive 2-3 doses spaced 1 -2 months apart. A further target population would be patients upon admission to a hospital.

Methods of making the conjugates

The methods that can be used to make the conjugates of the invention are not limiting. Methods useful for producing conjugate vaccines have been previously described and disclosed in U.S. Pat. No. 4,673,574, U.S. Pat. No. 4,789,735, U.S. Pat. No. 4,619,828, U.S. Pat. No. 4,284,537, U.S. Pat. No. 5,370,872, U.S. Pat. No. 5,302,386, U.S. Pat. No. 5,576,002, and U.S. Patent Application Pub. No. 2011/0274714, all of which disclosures are incorporated herein by reference.

In one embodiment, the invention is directed towards a method of making the conjugates described herein comprising binding a Klebsiella surface polysaccharide antigen and a Pseudomonas aeruginosa glycosylated native FlaA flagellin protein and/or a Pseudomonas flagellin FlaB protein or an antigenic fragment or a derivative thereof. In some embodiments, the binding is covalent. In some embodiments, the surface polysaccharide antigen is an O polysaccharide (OPS). Further embodiments include covalently bonding Pseudomonas aeruginosa glycosylated native FlaA and/or Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or variant thereof to at least one OPS from Klebsiella pneumoniae serov ars 01 , O2a, 03 and 05 to arrive at the conjugates described herein. In some embodiments, the surface polysaccharide antigen is isolated from a Klebsiella pneumoniae serovar having one or more mutations. For example, the Klebsiella pneumoniae may have an attenuating mutation in the guaBA locus and/or a mutation in the wza-wzc locus.

In some embodiments, the glycosylated native FlaA flagellin protein is isolated from a wild-type Pseudomonas aeruginosa strain. In some embodiments, the glycosylated native FlaA flagellin protein is isolated from an attenuated Pseudomonas aeruginosa strain.

In some embodiments, the glycosylated native FlaA flagellin protein from Pseudomonas aeruginosa can be prepared as described in Montie et al. , Infect Immun, (1982), 35: 281-8). Preferably endotoxin contaminant is removed from the preparation. See Example 1, infra, hi some embodiments, the glycosylated native FlaA flagellin protein from Pseudomonas aeruginosa can be purified and isolated using conventional techniques and methods. Such methods can include mechanical shearing, removal at low pH, heating or purification from bacterial supernatants. Methods of purification of a flagellin protein from whole flagella are known in the art or can be readily modified by one of ordinary skill in the art using methods known in the art. For example, by modifying the method of Ibrahim et al., purification of flagella is achieved; below pH 3.0, flagella dissociate into flagellin subunits (Ibrahim et al. J. Clin. Microbiol. 1985; 22:1040-4). Further methods for purification include adaptation of the mechanical shearing, and sequential centrifugation steps for purification of flagellin in flagella from bacterial cells.

In some embodiments, the Pseudomonas FlaB flagellin protein is isolated from a heterologous Gram-negative bacteria (GNB) expression system, including Salmonella and Escherichia coli. In some embodiments, the FlaB flagellin protein is isolated from a Salmonella enterica serovar strain engineered to express Pseudomonas aeruginosa FlaB flagellin protein. In some embodiments, the Salmonella enterica serovar is Enteritidis. In some embodiments, the Salmonella enterica serovar strain may have an attenuating mutation, for example, in the guaBA locus. In some embodiments, the FlaB flagellin is purified from the bacterial supernatant of the Salmonella enterica serovar reagent strains described herein by chromatographic methods. In some embodiments, COPS and OPS can be isolated by methods including, but not limited to mild acid hydrolysis removal of lipid A from LPS. Other embodiments may include use of hydrazine as an agent for COPS or OPS preparation. Preparation of LPS can be accomplished by known methods in the art. In some embodiments, LPS is prepared according to methods of Darveau et al. J. Bacterial., 155(2) : 831 -838 (1983), or Westphal et al. Methods in Carbohydrate Chemistry. 5:83- 91 (1965) which are incorporated by reference herein.

In some embodiments, the LPS is purified by a modification of the methods of Darveau et al., supra, followed by mild acid hydrolysis.

The surface polysaccharide antigen and flagellin can be conjugated using known techniques and methods. For example, techniques to conjugate surface polysaccharide antigen and flagellin can include, in part, coupling through available functional groups (such as amino, carboxyl, thiol and aldehyde groups). See, e.g., Hermanson, Bioconjugate Techniques (Academic Press; 1992); Aslam and Dent, eds. Bioconjugation: Protein coupling Techniques for the Biomedical Sciences (MacMillan: 1998); S. S. Wong, Chemistry of Protein Conjugate and Crosslinking CRC Press (1991), and Brenkeley et al., Brief Survey of Methods for Preparing Protein Conjugates With Dyes, Haptens and Cross-Linking Agents, Bioconjugate Chemistry 3 #1 (Jan. 1992).

In some embodiments of the present invention, the surface polysaccharide antigen and flagellin or fragments or derivatives thereof, can include functional groups or, alternatively, can be chemically manipulated to bear functional groups. In some embodiments, the presence of functional groups can facilitate covalent conjugation. Such functional groups can include amino groups, carboxyl groups, aldehydes, hydrazides, epoxides, and thiols, for example. Functional amino and sulfhydryl groups can be incorporated therein by conventional chemistry. Primary amino groups can be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent.

Flagellin may contain amino acid side chains such as amino, carbonyl, hydroxyl, or sulfhydryl groups or aromatic rings that can serve as sites for conjugation. Residues that have such functional groups can be added to either the surface polysaccharide antigen or flagellin. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, for example.

Surface polysaccharide antigen and flagellin can be chemically conjugated using conventional crosslinking agents such as carbodiimides. Examples of carbodiimides are l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1- ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC), and l-ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide.

Examples of other crosslinking agents are cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homobifunctional agents including a homobifunctional aldehyde, a homobifunctional epoxide, a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl halide, a homobifunctional hydrazide, a homobifunctional diazonium derivative or a homobifunctional photoreactive compound can be used. Also included are heterobifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group, and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.

Specific examples of homobifunctional crosslinking agents include the bifunctional N-hydroxysuccinimide esters dithiobis (succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imidoesters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1 ,4-di-[3'-(2'-pyridyldithio) propion- amido]butane, bismaleimidohexane, and bis-N-maleimido-l,8-octane; the bifunctional aryl halides l,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'- dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4- azidosalicylamide)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adiphaldehyde; a bifunctional epoxied such as 1 ,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides N 1 N'-ethylene-bis(iodoacetamide), N 1 N’-hexamethylene- bis(iodoacetamide), NlN'-undecamethylene-bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala'-diiodo-p-xylene sulfonic acid and tri(2- chloroethyl)amine, respectively.

Examples of other common heterobifunctional crosslinking agents that may be used include, but are not limited to, SMCC (succinimidyl-4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate), MBS (m-maleimidobenzoyl-N- hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodacteyl) aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS (N-(- maleimidobutyryloxy)succinimide ester), MPHB (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl)cyclohexane-l -carboxylhydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate). For example, crosslinking may be accomplished by coupling a carbonyl group to an amine group or to a hydrazide group by reductive amination.

In another aspect of the invention, the surface polysaccharide antigen and flagellin can be conjugated through polymers, such as PEG, poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, immunoglobulins, and copolymers of D-lysine and D- glutamic acid. Conjugation of the surface polysaccharide antigen and flagellin may be achieved in any number of ways, including involving one or more crosslinking agents and functional groups on the surface polysaccharide antigen and/or flagellin. The polymer can be derivatized to contain functional groups if it does not already possess appropriate functional groups.

In some embodiments, l-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) conjugation chemistry is used to achieve efficient synthesis of the surface polysaccharide antigen and flagellin conjugates. In some embodiments, l-cyano-4- dimethylaminopyridinium tetrafluoroborate (CDAP) is used to conjugate OPS-FlaA conjugates and OPS-FlaB conjugates.

In some embodiments, the surface polysaccharide antigen or flagellin is conjugated to a linker prior to conjugation. In some embodiments, the linker is adipic acid dihydrazide (ADH). The present invention contemplates the use of any linker capable of conjugating the surface polysaccharide antigen to flagellin. In some embodiments, the presence of a linker promotes optimum immunogenicity of the conjugate and composition and more efficient coupling. In some embodiments, the linkers separate the two or more antigenic components by chains whose length and flexibility can be adjusted as desired. Between the bifunctional sites, the chains can contain a variety of structural features, including heteroatoms and cleavage sites. In some embodiments, linkers also permit corresponding increases in translational and rotational characteristics of the antigens, increasing access of the binding sites to soluble antibodies. Besides ADH, suitable linkers include, for example, heterodifunctional linkers such as e-aminohexanoic acid, chlorohexanol dimethyl acetal, D -glucuronolactone and p-nitrophenyl amine. Coupling reagents contemplated for use in the present invention include hydroxysuccinimides and carbodiimides. Many other linkers and coupling reagents known to those of ordinary skill in the art are also suitable for use in the invention. Such compounds are discussed in detail by Dick et al., Conjugate Vaccines, J. M. Cruse and R. E. Lewis, Jr., eds., Karger, New York, pp. 48-114, hereby incorporated by reference.

In some embodiments, ADH is used as the linker. In some embodiments, the molar ratio of ADH to surface polysaccharide antigen such as OPS in the reaction mixture is typically between about 10:1 and about 250:1. In some embodiments, a molar excess of ADH is used to ensure more efficient coupling and to limit OPS-OPS coupling. In some embodiments, the molar ratio is between about 50:1 and about 150: 1. In other embodiments, the molar ratio is about 100:1. Similar ratios of AH- OPS to the flagellin in the reaction mixture are also contemplated. In some embodiments, one ADH per OPS is present in the AH-OPS conjugate.

Other linkers are available and can be used to link two aldehyde moieties, two carboxylic acid moieties, or mixtures thereof. Such linkers include (Ci-Ce) alkylene dihydrazides, (Ci-Cc) alkylene or arylene diamines, -aminoalkanoic acids, alkylene diols or oxy alkene diols or dithiols, cyclic amides and anhydrides and the like. For examples, see U.S. Pat. No. 5,739,313, incorporated herein in its entirety.

In some embodiments, conjugation is conducted at a temperature of from about 0° C to about 5° C for about 36 to about 48 hours. In one embodiment, conjugation is conducted at about 4°C for about 36 hours, followed by about an additional 18 to 24 hours at a temperature of from about 20° C to about 25° C. In another embodiment, conjugation is conducted for about 18 hours at about 20 to 24° C, such that the residual cyanate groups react with water and decompose. Longer or shorter conjugation times and/or higher or lower conjugation temperatures can be employed, as desired. In some embodiments, it is desirable, however, to conduct the conjugation reaction, at least initially, at low temperatures, for example, from about 0° C to about 5° C, such as about 4° C, so as to reduce the degree of precipitation of the conjugate.

In some embodiments of the invention, conjugation of the surface polysaccharide antigen and flagellin protein is on the terminal amino group of lysine residues. In some embodiments of the invention, conjugation is to cysteine groups. In some embodiments of the invention, conjugation of the surface polysaccharide antigen is to N-terminal serine groups. In some embodiments of the invention, conjugation of the surface polysaccharide antigen to the flagellin is directed towards the C-terminal carboxylic acid group. In some embodiments of the invention, conjugation is to naturally occurring amino acid groups. In other embodiments of the invention, conjugation is to engineered amino acid sequences and residues within the flagellin protein.

In some embodiments of the invention, conjugation of the surface polysaccharide antigen and flagellin is on random free hydroxyl groups on the OPS polysaccharide chain. In some embodiments of the invention, conjugation of the flagellin to the surface polysaccharide antigen and is at the terminal end of the polysaccharide chain.

In some embodiments of the invention, the surface polysaccharide antigen and flagellin reactants contain multiple reactive groups per molecule. In some embodiments, an activated surface polysaccharide antigen molecule can react with and form more than one linkage to more than one flagellin. Likewise, an activated flagellin can react with and form more than one linkage to more than one activated surface polysaccharide antigen. Therefore, in some embodiments, the conjugate product is a mixture of various cross-linked matrix-type lattice structures. For example, a single linkage can be present, or 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, or 30 or more linkages can be present. The average number of linkages between a surface polysaccharide and flagellin antigen can be adjusted, as desired. In some embodiments, the average number of linkages can depend upon the type of OPS polysaccharide, the type of flagellin protein, the conjugation method, the reaction conditions, and the like.

In some embodiments, purification processes such as column chromatography and/or ammonium sulfate precipitation of the conjugate from unconjugated polysaccharide may not be necessary. However, in certain embodiments it can be desirable to conduct one or more purification steps. In some embodiments, after conjugation, the conjugate can be purified by any suitable method. Purification can be employed to remove unreacted polysaccharide, protein, or small molecule reaction byproducts. Purification methods include ultrafiltration, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, ammonium sulfate fractionation, ion exchange chromatography, ligand exchange chromatography, immuno-affinity chromatography, polymyxin-b chromatography, and the like, as are known in the art. In some embodiments, the conjugation reactions proceed with higher yield, and generate fewer undesirable small molecule reaction byproducts. Accordingly, in some embodiments no purification may be necessary, or only a minor degree of purification can be desirable.

The conjugate or composition of the invention can be concentrated or diluted, or processed into any suitable form for use in pharmaceutical compositions, as desired. Genetically engineered strains

In some embodiments, a modified Klebsiella is useful for isolating the surface polysaccharide antigen for use in making the conjugates of the invention. In some embodiments, the modified Klebsiella is a modified Klebsiella pneumonia. In some embodiments, the modified Klebsiella comprises one or more attenuating mutations. In some embodiments, the modified Klebsiella has an attenuating mutation in the guaBA locus. In some embodiments, the Klebsiella comprises one or more mutations in the wza-wzc locus. In some embodiments, the Klebsiella pneumoniae serovar can be 01, 02 (including any subtypes such as 02a, 02ac, 02c, 02ae, 02aeh, and 02afg), 03, and/or 05. In some embodiments, the Klebsiella is Klebsiella pneumoniae serovar 01 , 02 (including any subtypes such as 02a, 02ac, 02c, 02ae, 02aeh, and 02afg), 03, or 05 having an attenuating mutation in the guaBA locus and a mutation in the wza-wzc locus. In some embodiments the guaA gene (NCBI-ProteinlD: AB R78243 NCBI- GI: 152971364 NCBI-GenelD: 5339904 UniProt: A6TCC2) of Klebsiella pneumoniae comprises SEQ ID NO:5, and encodes guanosine monophosphate synthase.

In some embodiments the guaB gene (NCBI-ProteinlD: ABR78244 NCBI-GI: 152971365 NCBI-GenelD: 5339905 UniProt: A6TCC3) of Klebsiella pneumoniae comprises SEQ ID NO:6, and encodes inosine 5 ’-monophosphate dehydrogenase.

In some embodiments the wza gene (NCBI-ProteinlD: ABR77930 NCBI-GI: 152971051 NCBI-GenelD: 5340218 UniProt: A6TBF9) of Klebsiella pneumoniae comprises SEQ ID NO:7, and encodes capsule export-outer membrane protein.

In some embodiments the wzb gene (NCBI-ProteinlD: ABR77929 NCBI-GI: 152971050 NCBI-GenelD: 5340217 UniProt: A6TBF8) of Klebsiella pneumoniae comprises SEQ ID NO: 8, and encodes protein tyrosine phosphatase.

In some embodiments the K2-wzc gene (NCBI-ProteinlD: ABR77928 NCBI- GI: 152971049 NCBI-GenelD: 5340932 UniProt: A6TBF7) of Klebsiella pneumoniae comprises SEQ ID NO:9, and encodes tyrosine autokinase.

In another embodiment, a modified Gram-negative bacteria (GNB) engineered to express Pseudomonas FlaB flagellin can be used to isolate and prepare conjugates. In some embodiments, the Gram-negative bacteria is Escherichia coli. In some embodiments, the Gram-negative bacteria is a Salmonella such as a Salmonella enterica serovar strain. In some embodiments, the Salmonella enterica serovar is selected from Enteritidis, Typhimurium, and Paratyphi A. In some embodiments, the Salmonella enterica serovar is Enteritidis.

In some embodiments, the Gram-negative bacteria expressing Pseudomonas FlaB flagellin has one or more mutations. In some embodiments, the Gram-negative bacteria has one or more mutations in the guaBA locus, the guaB gene, the guaA gene, the clpP gene, the clpX gene and/or the clpPX locus. In some embodiments, the Gramnegative bacteria expressing Pseudomonas FlaB flagellin has one or more codon optimized Pseudomonas fliC genes. In some embodiments, the Gram-negative bacteria expressing Pseudomonas FlaB flagellin encodes a excretion signal for flagellin. In some embodiments, the Gram-negative bacteria, such as Salmonella enterica, has at least one attenuating mutation in the guaBA locus and/or the clpPX locus. In some embodiments, one or more of guaBA, clpPX and/Z/ are mutated to create highly attenuated strains that hyper-secrete FlaB flagellin monomers into the supernatant. A guaBA mutation (involved in guanosine nucleotide synthesis (Samant S et al., PLoS Pathog. 2008; 4(2):e37)) is highly attenuating in several Gram negative pathogens (e.g., Shigella (Kotloff KL et al., Hum Vaccin. 2007; 3(6):268-275), Salmonella (Tennant SM et al., Infect Imnuin. 201 1 ; 79( 10):4175-4185; Gat O et al., PLoS Negl Trop Dis. 2011 ; 5(1 l):el373), Francisella (Santiago AE et al., Vaccine. 2009; 27(18):2426-2436)). When either clpP or clpX (that form the ClpPX protease) is deleted, the master flagella regulator complex FlhD/FlhC is not degraded and large amounts of flagella are produced. Deletion of clpPX is also independently attenuating (Tennant SM et al., Infect Immun. 2011 ; 79(10):4175-4185; Tomoyasu T et al., J Bacteriol. 2002; 184(3):645-653). Deletion of the gene for the flagella capping protein FliD causes flagellin monomers to be exported into the supernatant, and engineered Salmonella mutants deficient in clpPX and fliD produce and export large amounts of flagellin into the culture supernatant. These recombinant strains are considered as safe from an occupational health and safety perspective and enable conjugate vaccine carrier proteins to be expressed at high levels, thus lowering the overall cost of manufacture.

Growth conditions in fully chemically defined minimal media for attenuated S. Enteritidis and S. Typhimurium strains have been established, whereby an optical density at 600 nm (ODeoo ) of 15-18 is consistently attained at 20 L fermentation scale. Prototype attenuated S. Enteritidis reagent strain CVD 1943 AguaBA \clpP AfliD was constructed from wild-type strain S. Enteritidis R1 1 (a Malian clinical isolate) (Richmond P, J Infect Dis. 2000; 181 (2):761 -764).

In some embodiments, the Gram negative bacteria has an inactivating mutation in JliC such as a deletion in JliC. Such strain may further have an inserted (either in the chromosome or on a plasmid) heterologous fliC such as fliC from Pseudomonas aeruginosa or a bacteria producing flagellin with cross-reactivity o fliC from Pseudomonas aeruginosa. In some embodiments, the Gram negative bacteria is Salmonella enterica having a mutation in fliC and having a plasmid encoding Pseudomonas aeruginosa Type B flagellin (FlaB). In some embodiments, the amino acid sequence of FlaB comprises SEQ ID NO:2 and the nucleotide sequence comprises SEQ ID NO:4. In some embodiments, the Salmonella enterica expressing Pseudomonas FlaB flagellin has one or more codon optimized Pseudomonas fliC genes. In some embodiments, the Salmonella enterica expressing Pseudomonas FlaB flagellin encodes a Salmonella enterica Enteritidis fliC excretion signal.

In some embodiments, the Gram negative bacteria hyper-secretes Pseudomonas FlaB flagellin. In some embodiments, the Gram negative bacteria comprises a clpP or clpX (that form the ClpPX protease) mutation causing the master flagella regulator complex FlhD/FlhC to not be degraded, thereby causing the production of large amounts of flagella.

In some embodiments, using modified strains with attenuating mutations can simplify purification. Attenuated Salmonella strains are considered as safe from an occupational health and safety perspective. As used herein, attenuated strains are those that have a reduced, decreased, or suppressed ability to cause disease in a subject, or those completely lacking in the ability to cause disease in a subject. Attenuated strains may exhibit reduced or no expression of one or more genes, may express one or more proteins with reduced or no activity, may exhibit a reduced ability to grow and divide, or a combination of two or more of these characteristics.

In some embodiments, the attenuated strains producing Pseudomonas flagellin of the invention have a mutation in one or more of the guaBA locus, the guaB gene, the guaA gene, the clpP gene, the clpX gene and the clpPX locus. For example, the attenuated strains can have a mutation (i) in the guaB gene and the clpP gene, (ii) in the guaA gene and the clpP gene, (iii) in the guaBA locus, and the clpP gene, (iv) in the guaB gene and the clpX gene, (v) in the guaA gene and the clpX gene, (vi) in the guaBA locus, and the clpX gene, (vii) in the guaB gene and the clpPX locus, (viii) in the guaA gene and the clpPX locus, or (ix) in both the guaBA locus and the clpPX locus.

In some embodiments, attenuated strains are prepared having inactivating mutations (such as chromosomal deletions) in both the guaBA locus (encoding enzymes involved in guanine nucleotide biosynthesis) and the clpPX locus (encoding an important metabolic ATPase) genes. In some embodiments, one or more of the attenuated strains also have fliD and fliC mutations.

The mutations of the loci and genes described herein can be any mutation, such as one or more nucleic acid deletions, insertions or substitutions. The mutations can be any deletion, insertion or substitution of the loci or genes that results in a reduction or absence of expression from the loci or genes, or a reduction or absence of activity of a polypeptide encoded by the loci or genes. The mutations may be in the coding or non-coding regions of the loci or genes.

In some embodiments, the chromosomal genome of the Gram negative bacteria or Klebsiella is modified by removing or otherwise modifying the guaBA locus, and thus blocking the de novo biosynthesis of guanine nucleotides. In some embodiments, a mutation in the guaBA locus inactivates the purine metabolic pathway enzymes IMP dehydrogenase (encoded by guaB) and GMP synthetase (encoded by guaA). In some embodiments, the strains are unable to de novo synthesize GMP, and consequently GDP and GTP nucleotides, which severely limits bacterial growth in mammalian tissues. The AguaBA mutants of the present invention are unable to grow in minimal medium unless supplemented with guanine.

In some embodiments, the guaA gene of .S'. Enteritidis, which encodes GMP synthetase, is 1578 bp in size (GenBank Accession Number NC_011294. 1 (2623838- 2625415) (SEQ ID NO: 10). In some embodiments, the guaA gene of S. Typhimurium, is 1578 bp in size (GenBank Accession Number NC_003197.1 (2622805..2624382, complement) (SEQ ID NO: 11). In some embodiments, the guaA gene of 5. Typhi, is 1578 bp in size (GenBank Accession Number NC_004631.1 (415601..417178) (SEQ ID NO: 12). In some embodiments, the guaA gene of S. Paratyphi A, is 1578 bp in size (GenBank Accession Number NC_006511.1 (421828..423405) (SEQ ID NO:13). In some embodiments, the guaA gene of 5. Paratyphi B is 1578 bp in size (GenBank Accession Number NC_010102.1 (418694..420271) (SEQ ID NO: 14).

Deletion mutants can be produced by eliminating portions of the coding region of the guaA gene so that proper folding or activity of GuaA is prevented. For example, about 25 to about 1500 bp, about 75 to about 1400 bp, about 100 to about 1300 bp, or all of the coding region can be deleted. Alternatively, the deletion mutants can be produced by eliminating, for example, a 1 to 100 bp fragment of the guaA gene so that the proper reading frame of the gene is shifted. In the latter instance, a nonsense polypeptide may be produced or polypeptide synthesis may be aborted due to a frameshift-induced stop codon. The preferred size of the deletion removes both guaB and guaA, from the ATG start codon of guaB to the stop codon of guaA.

In some embodiments, the guaB gene of S. Enteritidis which encodes IMP dehydrogenase, is 1467 bp in size (GenBank Accession Number NC_01 1294.1 (2625485-2626951, complement) (SEQ ID NO:15). In some embodiments, the guaB gene of 5. Typhimurium is 1467 bp in size (GenBank Accession Number NC_003197.1 (2624452..2625918, complement) (SEQ ID NO: 16). In some embodiments, the guaB gene of S. Paratyphi A is 1467 bp in size (GenBank Accession Number NC_006511.1 (420292..421758) (SEQ ID NO:17). Deletion mutants can be produced by eliminating portions of the coding region of the guaB gene so that proper folding or activity of GuaB is prevented. For example, about 25 to about 1400 bp, about 75 to about 1300 bp, about 100 to about 1200 bp, or all of the coding region can be deleted. Alternatively, the deletion mutants can be produced by eliminating, for example, a 1 to 100 bp fragment of the guaB gene so that the proper reading frame of the gene is shifted. In the latter instance, a nonsense polypeptide may be produced or polypeptide synthesis may be aborted due to a frame-shift-induced stop codon. The preferred size of the deletion removes both guaB and guaA, from the ATG start codon of guaB to the stop codon of guaA.

In some embodiments, the clpP gene of S. Enteritidis, which encodes a serine- protease, is 624 bp in size (GenBank Accession Number NC_011294.1 (482580- 483203) (SEQ ID NO: 18). In some embodiments, the clpP gene of S. Typhimurium is 624 bp in size (GenBank Accession Number NC_003197.1 (503210..503833) (SEQ ID NO: 19). In some embodiments, the clpP gene of S. Paratyphi A is 624 bp in size (GenBank Accession Number NC_006511.1 (2369275. 2369898, complement) (SEQ ID NO:20).

Deletion mutants can be produced by eliminating portions of the coding region of the clpP gene so that proper folding or activity of ClpP is prevented. For example, about 25 to about 600 bp, about 75 to about 500 bp, about 100 to about 400 bp, or all of the coding region can be deleted. Alternatively, the deletion mutants can be produced by eliminating, for example, a 1 to 100 bp fragment of the clpP gene so that the proper reading frame of the gene is shifted. In the latter instance, a nonsense polypeptide may be produced or polypeptide synthesis may be aborted due to a frameshift-induced stop codon. clpP forms an operon with clpX; the preferred size of the deletion encompasses only the downstream clpX gene and extends from the ATG start codon to the stop codon, inclusive.

In some embodiments, the clpX gene of S. Enteritidis, which encodes a chaperone ATPase, is 1272 bp in size (GenBank Accession Number NC_011294.1 (483455-484726) (SEQ ID NO:21). In some embodiments, the clpX gene of S. Typhimurium is 1272 bp in size (GenBank Accession Number NC_003197.1 (504085..505356) (SEQ ID NO:22). In some embodiments, the clpX gene of S. Paratyphi A is 1272 bp in size (GenBank Accession Number NC_006511.1 (2367752..2369023, complement) (SEQ ID NO:23).

Deletion mutants can be produced by eliminating portions of the coding region of the clpX gene so that proper folding or activity of ClpX is prevented. For example, about 25 to about 1200 bp, about 75 to about 1100 bp, about 100 to about 1000 bp, or all of the coding region can be deleted. Alternatively, the deletion mutants can be produced by eliminating, for example, a 1 to 100 bp fragment of the clpX gene so that the proper reading frame of the gene is shifted. In the latter instance, a nonsense polypeptide may be produced or polypeptide synthesis may be aborted due to a frame- shift-induced stop codon. clpP forms an operon with clpX; the preferred size of the deletion encompasses only the downstream clpX gene and extends from the ATG start codon to the stop codon, inclusive.

The fliC gene can be mutated using conventional techniques known in the art. The /7/C gene encodes a flagellin protein. In some embodiments, the fliC gene from S. Enteritidis is 1518 bp in size (GenBank Accession Number NC_011294.1 (1146600..1148117) (SEQ ID NO:24). In some embodiments, the fliC gene of S. Typhimurium is 1488 bp in size (GenBank Accession Number NC_003197.1 (2047658..2049145, complement) (SEQ ID NO:25). In some embodiments, the fliC gene of S. Paratyphi A, is 1488 bp in size (GenBank Accession Number NC_00651 1.1 (989787..991274) (SEQ ID NO:26). In some embodiments, deletions can be made in any of the loci or genes included herein by using convenient restriction sites located within the loci or genes, or by site-directed mutagenesis with oligonucleotides (Sambrook et al. , Molecular Cloning, A Laboratory Manual, Eds., Cold Spring Harbor Publications (1989)).

In some embodiments, inactivation of the loci or genes can also be carried out by an insertion of foreign DNA using any convenient restriction site, or by site- directed mutagenesis with oligonucleotides (Sambrook et al., supra) so as to interrupt the correct transcription of the loci or genes. The typical size of an insertion that can inactivate the loci or genes is from 1 base pair to 100 kbp, although insertions smaller than 100 kbp are preferable. In some embodiments, the insertion can be made anywhere inside the loci or gene coding regions or between the coding regions and the promoters. In some embodiments, the bacterial loci and genes are mutated using Lambda Red-mediated mutagenesis (see, e.g., Datsenko and Wanner, PNAS USA 97:6640-6645 (2000)).

While the invention has been described with reference to certain particular examples and embodiments herein, those skilled in the art will appreciate that various examples and embodiments can be combined for the purpose of complying with all relevant patent laws e.g., methods described in specific examples can be used to describe particular aspects of the invention and its operation even though such are not explicitly set forth in reference thereto).

EXAMPLES

Example 1. Preparation and testing of Pseudomonas aeruginosa and Klebsiella pneumoniae conjugate vaccines.

Materials and Methods

Purification of Native Flagellin FlaA

Flagella were prepared as previously described. (Montie et al. , Infect Immun, (1982), 35: 281-8 ). Briefly, Pseudomonas aeruginosa strain PAK (IATS 06, FlaAi) was grown in 2 L of Hy-Soy media at 37 °C and 80 rpm for 24 h. Bacteria were harvested by pelleting the culture at 8,000 rpm at 4 °C for 20 min. The pellet was resuspended in 40 mL of cold PBS pH 7.4. Flagella filaments were sheared by blending in Waring blender for 2 min at 4 °C. Sheared flagellin filaments were pelleted by ultracentrifugation at 100,000 X g at 4 °C for 4 h and dissolved in PBS pH 7.4. Flagellin filaments were monomerized by lowering the pH to 2.0 by adding 5 M HC1 and stirring at room temperature for 30 min. Purified nFlaA was collected by final ultracentrifugation at 100,000 X g at 4 °C for 4 h. The pH was increased to pH 7.0 and sterile filtered with 0.22 pm filter (Millipore, MA) and stored at -20 °C. Purified nFlaA was analyzed by 4-20 % Tris-Glycine SDS-PAGE (Invitrogen) with Coomassie staining and Western Blot with mouse anti-FlaA antibody. The protein concentration was determined by the BCA method. Endotoxin levels were assessed with the Endosafe PTS and nexgen-PTS systems with the use of Endosafe PTS chromogenic Limulus amebocyte lysate assay cartridges (Charles River, MA).

Removal of Endotoxin contaminant from purified nFlaA

LPS contaminants from purified nFlaA were removed by batch binding with polymyxin B resin (Sigma Cat No. P1411). Pure nFlaA devoid of LPS was eluted in PBS pH 7.4. Protein- containing fractions were confirmed by SDS-PAGE with Coomassie blue staining. Endotoxin levels were assessed as described above.

Chemical Deglycosylation of Flagellin

Purified flagellin was desalted by dialysis in milli-Q (Millipore) water, thoroughly dried by lyophilization, and transferred to glass vials. Deglycosylation was carried out by using the GlycoFree Chemical Deglycosylation kit according to the manufacturer’s protocol (PROzyme). Briefly, 50 pl of trifluoromethanesulfonic (TFMS) acid/toluene mixture was added slowly to the protein samples in each glass vial placed in a dry ice-ethanol bath and incubated at -20 °C for 4 h. After 4 h, 150 pl of pyridine solution (pyridine/methanol/water in a 3: 1 : 1 ratio) was added to each glass vial placed in a dry ice-ethanol bath for 5 min and transferred to wet ice for a further 15 min. The reaction mixture was neutralized by adding 400 pl of neutralization solution (0.5 % w/v ammonium bicarbonate) and mixed briefly. Deglycosylated protein was recovered by centrifugation. Briefly, the sample was cooled to 4 °C for 30 min and centrifuged at high speed 10,000 rpm for 15 min. Pelleted protein was dissolved in 8 M urea and step-dialyzed in PBS buffer containing decreasing concentration of urea (6 M, 4 M, 2 M, and 0 M) using Slide- A-Lyzer dialysis cassette with 10000 Da MWCO (Thermo Scientific). The sample was run on 4-25 % Tris- Glycine SDS-PAGE with Coomassie staining to assess the deglycosylation. The final protein concentration was determined by BCA protein assay. Endotoxin levels were determined as described above.

Preparation of nFlaA/KP OPS conjugate vaccine

Klebsiella 01 polysaccharide was fermented, purified, and prepared for conjugation from strain Klebsiella B5055 as previously described (Hegerle et al., PLoS One, (2018), 13:e0203143). Native FlaA was purified as described above and labeled with sulfo-GMBS. Protein labeling and the remaining conjugation steps were also performed as previously detailed (Hegerle et al., PLoS One, (2018), 13:e0203143). Specifically, labeled nFlaA was purified and diafiltered into appropriate buffer with a 10 kDa tangential flow filtration (TFF) membrane. The purified GMBS-nFlaA was conjugated to the labeled OPS in a ratio of 6: 1 wt:wt of OPS to nFlaA and subsequently purified over a Superdex 200 16/600 column run on an AKTA chromatography system. The polysaccharide and protein content in the purified conjugates were assessed by resorcinol and BCA assays with the polysaccharide and unconjugated protein standards respectively. Residual endotoxin was assessed by Limulus amebocyte lysate assay as described above. Size was evaluated by ultra-performance liquid chromatography with size exclusion chromatography (UPLC-SEC) with a BioZen 1.8 pm SEC-3 column (Phenomenex, CA) run at 0.3 mL/min on an Acquity H-Class Plus Bio System (Waters, MA) in PBS, 0.02 % sodium azide pH 7.4 with detection by UV280.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to measure serum IgG levels before and after immunization. For anti-FlaA titration, clear flat-bottom MicroIon medium binding plates (Greiner bio-one) were coated with 2 pg/ml of FlaA in 0.05 M sodium carbonate buffer pH 9.6 for 3 h at 37 °C. For PAO6 COPS titration, clear flat-bottom MicroIon medium binding plates (Greiner bio-one) were coated with 10 pg/ml of PAK (IATS 06, FlaA 1) COPS in sodium carbonate buffer pH 9.6 for 3 h at 37 °C. For P. aeruginosa strain NUH5446 (IATS 02/16) COPS ELISA, plates were coated with either 10 pg/rnl of 02/16 COPS or 10 pg/ml of P. aeruginosa strain NUH5446 crude LPS lysate. Following coating, the plates were washed with PBS, pH 7.4 + 0.05 % Tween- 20 (PBS-T) 6 times with 2 minutes soak in between. The plates were blocked with 10 % non-fat dry milk Omniblok (American Bio) in PBS, pH 7.4 at 4 °C overnight. After washing the plates as described above, mouse serum samples were diluted in PBS-T + 10 % non-fat dry milk Omniblok, added in duplicates to the plates, and incubated for 1 h at 37 °C. Following washes, the bound mouse IgG was detected by HRP- labelled Goat anti-Mouse IgG (Invitrogen) diluted to 1:2000 in PBS-T + 10% non-fat dry milk at 37 °C for 1 h. After washes, substrate 3,3’,5,5’-tetramethylbenzidine (Thermo Scientific) was added and incubated at ambient temperature for 15 min in darkness. The reaction was stopped by adding 2 N Sulfuric acid (MACRON Fine Chemicals) and the absorbance at 450 nm was recorded using Spectra Max Plus reader (Molecular Devices) and SoftMax Pro software. End point titers were reported as ELISA units, which represents the absorbance multiplied by serum dilution just above 0.2 OD450 reading.

Motility Inhibition Assay

P. aeruginosa strain PAK was grown overnight at 37 °C without shaking, in Hy-Soy medium to stationary phase. The cells were pelleted at 3500 rpm for 20 min at 4 °C, washed the twice with PBS, pH 7.4, resuspended in PBS, and normalized to an ODeoo of 1.0. Normalized cells were diluted to 1 :1000 in PBS. 0.3 % Tryptone soft agar (1 % tryptone, 0.5 % NaCl, and 0.3 % agar) was autoclaved for 20 min, cooled at 56 °C for 30 min, and then at room temperature for 15 min. 1 mL of cooled agar was poured into the 24- well plate containing pre-immune or post- immune sera at 1 : 30 dilution. Control wells contained no sera. Diluted P. aeruginosa cells were stabbed to the center of the agar well using a sterile toothpick and incubated at 30 °C for 19 hours in a humidified chamber. The motility halo originating from the center of inoculation was captured with a ChemiDocMP system (BIO-RAD) and the diameter of the motility was measured using ImageJ software (NIH).

Toll-like receptor (TLR) 4 and 5 activity assays

TLR4 and TLR5 activity assays were performed as described previously with minor modifications (Caballero et al., Sci Rep, (2017), 7:40981; Gregg et al., mBio, (2017), 8(3)). Briefly, HEK-Blue™-hTLR4 cell and HEK- Blue™ -hTLR5 cells carrying a secreted embryonic alkaline phosphatase (SEAP) reporter construct were obtained from InvivoGen (San Diego, CA). Cells were maintained in DMEM supplemented with 10 % FBS, 0.5 % penicillin/streptomycin with and without 0.2 % Normocin™ (InvivoGen, CA) for TLR5 cells and TLR4 cells at 37 °C with 5 % CO2, respectively. Monolayers of IxlO⁵ cells per well in a 96-well plate were incubated with media alone, PBS, and FlaA and FlaB proteins at different concentrations ranging from 10 pg/mL to 10 pg/mL for 24 h. E. coli lipopolysaccharide O111 :B4 (LPS; List Biological laboratories. Inc, CA) was added to HEK-Blue™-hTLR4 cell as a positive control. Twenty pl of cell supernatant was added to QuantiBlue substrate (InvivoGen, CA) according to the manufacturer’s instructions. SEAP activity was measured as optical density at 620 nm. Curve-fitting was performed to estimate the half maximal effective concentration (EC50) using a dose-response curve of GraphPad Prism v6.0 (GraphPad Software Inc., CA). The strong correlations were evaluated according to the correlation coefficient R that is greater than 0.9.

Animal studies

Mice immunization

Six-to seven- week-old female Crl:CD-l mice (Charles River Laboratories, MA) were immunized with FlaA and FlaB proteins by intramuscular administration. Briefly, mice were immunized with 5 pg of proteins or PBS (negative control) on days

O, 14, 28 (Hegerle et al. , PLoS One, (2018), 13:e0203143). Sera were obtained prior to the first dose and 14 days post the last dose and stored at -20 °C.

P. aeruginosa burn wound infections

Mice were infected with two P. aeruginosa isolates expressing FlaA; flagellin protein as described previously with minor modifications (Stieritz et al. , J Infect Dis, (1975), 131 : 688-691 ). Briefly, mice were clipped a day before a burn procedure under anesthesia with 5 % isoflurane for 5 mins. On the following day, they were anesthetized and then ignited flame was induced on the shaved back for 10 seconds to allow a nonlethal thermal injury on 10 % of the body surface. Mice were challenged with 100 pl of 4X10⁶ CFU PAK (06, FlaAl type) on the burned site subcutaneously. In order to rule out any possible protection against P. aeruginosa due to immune responses to impurified flagellin proteins with LPS, 6xlO⁵ CFU of non-06 type of

P. aeruginosa, NUH5446 clinical isolate (IATS 02/16, FlaAl type), was used in this model. Mice were given 500 pl of the 0.9 % sodium chloride (Baxter, IL) for rehydration. Mortality was recorded for seven days after the infection.

Statistics All statistical analyses were performed with GraphPad Prism v6.0. Survival analyses for Kaplan-Meier curves were conducted by log-rank test. P-values less than 0.05 were considered as a statistical significance between groups.

Results

Chemical Deglycosylation of Flagellin

In order to explain the differences between FlaA and FlaB in eliciting functionally active antibodies, it was hypothesized that the FlaA glycan may play an important role. Both FlaA and FlaB are O-glycosylated; however, the O-glycan on the FlaA is considerably larger and more complex than the O-glycan on FlaB (Schirm et al., J Bacteriol, (2004), 186: 2523-2531 ; Verma et al. , J Bacteriol, (2006), 188:4395- 4403; Arora et al., Proc Natl Acad Sci USA, (2001), 98:9342-7). Native FlaA has a complex O-glycan attached via rhamnose to two amino acid sites (tyrosine 189 and serine 260), whereas native FlaB has a simple glycan without rhamnose and is attached via deoxyhexosamine (Schirm et al., J Bacteriol, (2004), 186: 2523-2531; Verma et al., J Bacteriol, (2006), 188:4395-4403; Arora et al. , Proc Natl Acad Sci USA, (2001), 98:9342-7)). While N-glycans are readily removed from proteins by the enzyme PNGaseF, enzymatic removal of O-glycans has not been possible. Consequently, we used TFMS acid, a “super acid”, to chemically remove the O- glycans (Brimer et al., J Bacteriol, (1998), 180:3209-3217). Native FlaA from P. aeruginosa strain PAK and native FlaB from Pseudomonas strain PAO1 (IATS 05 FlaB) were deglycosylated. After deglycosylation the proteins were run on SDS- PAGE and stained with Coomassie blue to evaluate the effect of deglycosylation on molecular weight (MW). Treatment of native FlaA with TFMS resulted in a decreased size shift in MW of the nFlaA, but not of the nFlaB (Figure 1). This difference may reflect the fact that in contrast to the nFlaA, removal of the considerably smaller glycan from nFlaB might not have caused as great a shift in MW of FlaB.

Immune response in mice measured by ELISA

In order to determine the immunogenicity, mice were immunized with native FlaA (nFlaA), deglycosylated nFlaA (dnFlaA), recombinant FlaA (rFlaA), and PBS. Mice immunized with all the three antigens generated very high levels of anti-FlaA titer after three immunizations (Figure 2A). Only mice immunized with native FlaA had high levels of anti-PAO6 COPS compared to deglycosylated native FlaA (Figure 2B). This result is expected, as the endotoxin levels are high in native HaA but decrease significantly after deglycosylation.(Table 1). None of the mouse groups had detectable anti-PAO2 levels when measured by with either PAO2 COPS-coated or NUH5446 lysate-coated ELISA plate (Figure 2C and 2D).

Table 1: Endotoxin and Beta-D-Glucan levels

Motility inhibition Assay

Sera from mice immunized with either nFlaA, deglycosylated native FlaA (d nFlaA), or rFlaA were compared to determine the ability to inhibit the swimming motility of PA FlaA strain PAK. Sera from mice immunized with native FlaA (nFlaA) inhibited the motility of PAK as expected (Figure 3). However, sera from the deglycosylated native FlaA (d nFlaA) group did not inhibit PAK motility, suggesting that the deglycosylation might have altered a protective epitope of FlaA. As before, sera from rFlaA also did not inhibit PAK motility.

Endotoxin levels

Since antibodies to Pseudomonas O-polysaccharides are protective in murine models of protection (3), before we tested the protective efficacy of antibodies to native HaA we assessed the endotoxin levels in the native FlaA preparations from which the FlaA was purified. The endotoxin level was drastically reduced after deglycosylation (Table 1). Pseudomonas strains inherently have high levels of betaglucan which can interfere with the endotoxin detection assay, giving false positives. We did indeed detect high levels of glucan in nFlaA samples, which could contribute to the high level of endotoxin observed in native FlaA sample. The flagellar protein remained intact during the TFMS treatment, as evidenced by the protein migrating as a single band (Figure 1). The presence of LPS in native flagellin was assessed by measuring SEAP secreted from hTLR4 reporter cells

Since impurified native flagellin with endotoxin were identified in Table 1, we assessed the TLR4 reporter assay to determine the comparative amount of LPS in each protein. The mTLR4 cells were strongly responsive to LPS (purple circles) which is its agonist (Figure 4A). The maximal TLR4 activation was achieved when cells were incubated with LPS and nFlaA, (blue squares) each at the highest concentration used in the assay ( 10 pg/ml). The EC50 values of LPS and nFlaA were 0.075 and 1 195 ng/ml, respectively. However, the EC50 values for rFlaA (red circles) and dFlaA (green diamonds) proteins were not determined due to weak TLR4 activation. Similar to nFlaA, 10 pg/ml of nFlaB protein (blue squares) stimulated maximal TLR4 activation (Figure 4B). The EC50 value of nFlaB was 295.5 ng/ml while that of rFlaB (red circles) and d nFlaB (green diamonds) was not estimated due to poor TLR4 activity.

Recombinant flagellin proteins stimulated TLR5 activation

In order to see whether flagellin proteins without glycan can be recognized by immune system, we performed the TLR5 reporter assay with each of the native flagellin (i.e. glycosylated form), recombinant (non-glycosylated form), and chemically deglycosylated proteins. Among FlaA proteins, nFlaA showed the highest potency in mTLR5 reporter cells (Figure 4C). Compared with the estimated EC50 of nFlaA at 2.1 ng/ml, those of rFlaA and d nFlaA were higher. The EC50 of rFlaA and d nFlaA proteins were estimated at 41.5 and 291.5 ng/ml, respectively. In Figure 4D, nFlaB induced strong TLR5 activation with the EC50 value of 44.4 ng/ml. Unlike the 19.8-fold decreased potency of rFlaA compared with nFlaA, rFlaB showed 2.3-fold reduced potency of TLR5 activity (EC50 =104.3). d nFlaA protein showed the highest EC50 value of 442.2 ng/ml with TLR5 activation.

Mice immunized with native FlaA, but not rFlaA were protected against PA To determine whether the immunization with FlaA proteins without glycans would protect mice with bum injury against FlaA 1 -expressing P. aeruginosa, mice vaccinated with nFlaA and rFlaA were challenged with a sub-lethal dose of PAK 06, FlaAl) and the NUH5446 (02/16, FlaAl) isolate, the latter a FlaA-i- PA strain of a different O serotype from which the nFlaA was prepared. Of 15 mice that received PBS, 20% of those who were infected with PAK survived (Figure 5A). All the mice immunized with nFlaA survived, while only 26.7 % of those immunized with rFlaA were alive after PAK infection. When mice with bum wound injury were infected with NUH5446 (02/16 type), the survival rate in non-immunized mice was 10 % (Figure 5B). Interestingly, 90 % of those immunized with nFlaA survived whereas 20 % of mice that received dFlaA were alive. In this rodent model, vaccination with nFlaA improved survival against two FlaA+ P. aeruginosa isolates significantly.

Preparation of a KP OPS/nFlaA conjugate vaccine. Based on the protective efficacy of nFlaA, a KP 01 OPSmFlaA conjugate vaccine was prepared to determine if this formulation retained the ability to induce functionally active antibodies to FlaA- bearing PA following conjugation. The KP 01 OPSmFlaA conjugate vaccine was made as described above. It was observed after S200 purification using SDS-PAGE (Figure 6, Panel A) and Western blot (Panel B) that there were two different sized conjugates present (Figure 6, lanes 2 and 3 vs. lanes 5 and 6). The S200 fractions were split into two separate lots. By size exclusion chromatography, the first lot had a slightly higher molecular weight compared to the second lot, which confirms the difference in sizes noted by SDS-PAGE (Figure 6C). In the first lot, KPOl-nFlaA- 01 -01, the final conjugate had a nFlaA concentration of 0.16 mg/mL, an OPSmFlaA ratio of 0.625: 1, and endotoxin levels of 2.53 EU/mg OPS. In contrast, the second lot, KPOl-nFlaA-01 -02, had an increased OPSmFlaA ratio of 2.2:1, a lower nFlaA concentration of 0.05 mg/m, and lower endotoxin level (Table 2).

Table 2. Data Summary of KP-nFlaA Final Conjugates

Immune response to the two KP 01 OPS/nFlaA vaccine lots. Two groups of mice were immunized with the different lots of KP Ol FlaA conjugate vaccine at days 0. 14 and 28 prior to subjecting them to burn wound infection (Figure 7). Mice immunized with the 01 lot of conjugate vaccine had significantly higher anti-nFlaA antibody levels than those immunized with the 02 lot of vaccine (Figure 7A). Two weeks later they were challenged with a non-IATS 06 strain of PA and followed for survival. 80% of mice that were immunized with the lot 01 conjugate (Figure 7B) survived compared to 20% of mice immunized with the lot 02 vaccine (Figure 7 C). The difference in efficacy could be attributed either to the greater amounts of nFlaA in lot 01 or perhaps the shielding of the nFlaA by the greater amount of KP 01 OPS in lot 02.

The lot 01 conjugate also was better able to reduce the motility of a FlaA- bearing Pseudomonas (strain PAK) than the 02 conjugate (Figure 8). Both lots of KP 01 OPS FlaA conjugates retained their carrier function in enhancing the anti-KP 01 OPS antibody response (Figure 9).

Discussion

Our studies show that unlike the case for FlaB, the FlaA glycan is critical for the induction of functionally active antibodies against FlaA-bearing Pseudomonas strains. Further, this epitope is retained when native FlaA is conjugated to the Klebsiella O polysaccharide.

The single, unipolar flagella, an essential virulence factor of Pseudomonas aeruginosa, is comprised of repeating units of flagellin protein monomers, products of the fliC gene (Brimer et al., J Bacteriol, (1998), 180:3209-3217). It is required for Pseudomonas motility and chemotaxis that enable the systemic spread of Pseudomonas from the site of infection. Nonmotile variants are markedly attenuated in virulence (Totten et al., J Bacteriol, (1990), 172:7188-7199). The two major flagellar types, A and B, are distinguished on the basis of their molecular weight and reactivity with type-specific antisera. Al and A2 subtypes have different amino acid sequences, while the amino acid sequence of FlaB is more conserved and has an invariant molecular mass of -53 kDa. Each Pseudomonas strain makes only one type of Fla and there is no switching between types A and B. We recently reported that Pseudomonas flagellin proteins not only induce functionally active antibodies, but also serve as carrier proteins for Klebsiella O- polysaccharide antigens in glycoconjugate vaccines (Hegerle et al., PLoS One, (2018), 13:e0203143). Four Klebsiella O-polysaccharides were conjugated to either rFlaB or rFlaA, both recombinant proteins being non-glycosylated. The conjugated vaccines induced a robust antibody response to the Klebsiella O-polysaccharides which was not the case when the OPS and flagellin proteins were admixed. This indicates that the Fla proteins may serve as carrier proteins in glycoconjugate vaccines. Both flagellin proteins elicited a potent antibody response whether they were conjugated to the OPS or in the admixture. However, whereas the rFlaB -induced antibodies protected against lethal infection with FlaB -bearing Pseudomonas and inhibited motility, this was not the case for the rFlaA-induced antibodies.

In our current study we hypothesized that the difference in the O-glycans attached to the two flagellar proteins might explain the difference in the functional antibody responses. Since administration of the non-glycosylated rFlaB protected mice against lethal infection we concluded that the FlaB-associated O-glycan was not part of a critical vaccine epitope. In contrast, FlaA required the presence of its O- glycan to induce functionally active antibodies. We show that a glycoconjugate vaccine that used the glycosylated, native FlaA flagellin induced antibodies that both inhibited motility and had protective efficacy against a FlaA-bearing Pseudomonas, while retaining its carrier function for the Klebsiella polysaccharide. Conjugation of flagella to the O polysaccharide markedly reduced its TLR5 reactivity (Hegerle et al. , PLoS One, (2018), 13:e0203143).

In a series of studies it was reported that Pseudomonas FlaA and FlaB were differentially O-glycosylated (Schirm et al., J Bacteriol, (2004), 186: 2523-2531; Verma et al., J Bacteriol, (2006), 188:4395-4403; Arora et al., Proc Natl Acad Sci USA, (2001), 98:9342-7). FlaA has a heterogeneous glycan comprising of up to 11 monosaccharide units O-linked to the protein through rhamnose residues on the flagelln backbones (Schirm et al., J Bacteriol, (2004), 186: 2523-2531). FlaA-bearing Pseudomonas (PAK 06) have a unique genomic island that contains a cluster of 14 genes involved in the synthesis, activation or polymerization of sugars necessary for FlaA glycosylation. (Verma et al., J Bacteriol, (2006), 188:4395-4403). The glycans on HaA are localized to the central, surface-exposed domain of the monomer in the assembled filament. The TLR5 recognition sequences are localized in the conserved DI domain of flagellin. (Verma et al. , Infect Immun, (2005), 73:8237-46). In contrast, the glycan attached to the FlaB monomers is less heterogeneous than the glycan of the (PAK) FlaA. The glycan, O-linked to FlaB at threonine and serine resides, is 709 D and is less heterogeneous than the glycan of the PAK FlaA (Verma et al., J Bacteriol, (2006), 188:4395-4403). Our studies now show that these differences have different functional consequences with the O-glycan in FlaA being a critical functional antibody epitope.

In order to study the role of the glycans in flagellar functional activity, we had to remove the O-linked glycans. While N-linked glycans can be removed from proteins enzymatically (PNGaseF), until recently no such enzymes have been available to remove the O-linked glycans following the manufacturer’s instructions, we tried unsuccessfully to remove the O glycans with Ogly-ZORR (Genovis, Inc., Cambridge, MA, an enzyme that removes mucin-type core 1 O glycans )(See Figure 10). Consequently, we used the “superacid” TFMS to remove the glycan from native FlaA, as previously described (Brimer et al., J Bacteriol, (1998), 180:3209-3217). Surprisingly such treatment had no observable effect of the FlaA protein backbone (Figure 1) while reducing endotoxin levels in the preparation.

There have been a great many Pseudomonas vaccine candidates, including flagellin, but to date there are none licensed (for review, see Doring/Pier-20). Pseudomonas flagellin has been proposed not only as a vaccine (21, 22 23) but also as a carrier protein (24, 25) and adjuvant (26, 27, 8) (Campodonico et al., Infect Immun, (2010), 78:746-755; Doring G et al., Proc Natl Acad Sci USA, (2007), 104: 11020-25; Montie et al., United States Patent Number 4,831,121 (May 16, 1989); Campodonico et al., Infect Immun, (2011), 79:3455-64; Simon R et al., PLoS One, (2013), 8:e64680; Cui B et al., Expert Rev Vaccines, (2018), 17:335-349; Georgel et al., Antiviral Res, (2019), 168:28-35; Nasrin el al. , BMC Microbiology, (2022), 22: 13) however, most of these studies have used the native, glycosylated, not recombinant flagellin. Pseudomonas flagellins are readily expressed and purified from heterologous Gram-negative bacterial expression systems, including Salmonella and Escherichia coli (Campodonico et al., Infect Immun. (2010), 78(2):746-55). The FliD capping protein is essential for polymerization of secreted flagellin monomers into flagella polymers. Campodononico and Pier reported that polymeric flagellin proteins (i.e., flagella) were superior to monomeric flagellin for generating an immune response to Pseudomonas, but the flagellin monomer was a more potent activator of TLR5 activity than flagella. However, they concluded that the flagellar antigens alone would not induce solid immunity to Pseudomonas (Campodonico et al., Infect Immun. (2010), 78(2):746-55).

Experimental studies with divalent, native (i.e. glycosylated) flagella preparations demonstrated flagella- specific protection independent of the O antigen in murine burn injury models. (Holder et al. , J. Trauma, (1986), 26: 118-22; Monte et al., Antibiot Chemother, (1987), 39:233-48) and later in respiratory infection models that in addition inhibited Pseudomonas motility (Feldman et al., Infect Immun, (1998), 66:43-51 ; Ramphal et al., J Immunol, (2008), 181 :586-592). Clinical trials were conducted with bivalent Pseudomonas flagella by Immuno AG, first in 1971 and then in 2007 with the latter Phase III, double-blind, randomized placebo-controlled study showing a decreased risk of infection in the 381 cystic fibrosis patients studied(Crowe et al., Antibiot Chemother, (1991), 44: 143-56; Doring G et al., Proc Natl Acad Sci USA, (2007), 104:11020-25). These studies employed native flagellar proteins. Consequently, the role of flagellar glycans in the induction of functional antibodies had not been directly addressed. Few reports, however, have used recombinant flagellar proteins as vaccines. Faezi and colleagues reported that immunization with rFlaA produced in E. coli provided 83% protection against bum wound infection with a FlaA-expressing PA and surprisingly, protected 25% of mice infected with a FlaB-bearing Pseudomonas (Faezi et al., APMIS, (2014), 122:115-27 ). The vaccine itself was not characterized in this study, and specifically did not consider whether any LPS contamination may have contributed to the protection. Pseudomonas flagella also induce a potent TLR5- signaling response that have limited their use as flagellar vaccines or as adjuvants (Turley el al. , Vaccine, (2011), 29:5145- 5152).

Many non-Pseudomonas recombinant flagellin-based vaccines have been tested in mice and to a lesser extent in non-human primates. Salmonella flagellin proteins (FliC) have been not only good carriers for OPS-based conjugate vaccines, but anti-flagellin antibodies mediate protective immunity against lethal infection with non-typhoidal Salmonella (Simon R et al., PLoS One, (2013), 8:e64680; Ramachandran et al., PLoS One, (2016), 11 :e0151875 (25,36).

Since the re-introduction of glycoconjugate vaccines for bacterial infections, many licensed vaccines have used a limited number of protein carriers, particularly tetanus and diphtheria toxoids. These proteins induce potent antibodies. However, as more glycoconjugate vaccines use a limited number of protein carriers, there is the danger of the carrier-induced antibodies causing carrier-induced suppression as we have seen in a previous study (Cryz et al., J Clin Investig, (1987), 80:51-56). Further, the antibodies to these carrier proteins do not contribute to the host defenses against the bacteria to which the glycoconjugate vaccines are directed. In our Klebsiella O- po 1 y saccharide/ Pseudomonas flagellar vaccine we introduced the concept of “pathogen-relevant” carrier proteins whereby the protein carrier chosen might also contribute to the protection against bacterial infection targeted by the vaccine (Hegerle etal., PLoS One, (2018), 13:e0203143). Since flagellin is an essential virulence factor for Pseudomonas and >95% of clinically -relevant Pseudomonas isolates carry either FlaA or FlaB, this KP OPS/Pseudomonas flagellin vaccine targets two pathogens that are often resistant to multiple antimicrobial agents and which the CDC has labeled “urgent” and “serious” threats respectively.

It was originally thought that prokaryotes had a limited capacity to glycosylate their proteins, (i.e. it was eukaryotic-specific), but there have been increased reports of bacterial glycosylation since the discovery in the mid-1970s of surface layer glycosylation on the cell envelope in archaea and hyperthermophies. These glycans were mostly O-linked (Hayakawa et al.,. InTech, Chapter 6). In addition to the role of FlaA-associated glycans in inducing a protective antibody response, Castric and colleagues described a second glycan-related pathway in Pseudomonas which induces a protective antibody response (Allison TM et al., Microbiol 201; 161: 1780- 1789; Smedley el al., Infect Immun, (2005), 73:7922-31). They demonstrated that the glycosylated product of PilA, type IV pili, in Pseudomonas strain 12.4.4 (PA IATS 08) induced protective antibodies against infection with the homologous O type from which the pili were isolated. Glycosylation of 12.4.4 pilin required the presence of the pilO gene, a component of an operon containing pilA, the pilin structural gene. PilO is an oligosaccharide transferase that catalyzes O glycosylation of PA 12.4.4 pilin by adding a single O-antigen repeating unit to the C terminal residue. They later showed structural similarity between the pilin glycan and the O antigen of PA 12.4.4, which suggested that the pilin glycan of 12.4.4 is a product of the O antigen biosynthetic pathway (Miller et al., J Biol Chem, (2008), 283:3507-3518; DiGiandomenico et al. , Molecular Microbiology, (2002), 2:519-530). The wbpM or wbpL genes, essential to early steps of O-antigen biosynthesis, were able to produce both O-antigen and glycosylated pilin. Miller et al. reported that flagellin glycosylation of the PAK (FlaA+) strain of Pseudomonas required the O-antigen biosynthesis enzyme WbpO. but not WbpP. Moreover, this enzyme was able to use O-antigen genes from other Pseudomonas O types or even from other Gram-negative bacteria which were then co-expressed on the individual pili with the O-antigens from the homologous PAK strain (DiGiandomenico et al., Molecular Microbiology, (2002), 2:519-530). The structural diversity of the O antigens used by the 12.4.4 pilin glycosylation apparatus indicates that the glycan substrate specificity of the reaction is non-selective with regard to O-antigen structure. It even can add E coli O antigen. This also suggests that pilin glycosylation does not occur through sequential attachment of O antigen sugars to pilin. Since the pilin glycan stimulates a protective response that targets the O antigen (Horzempa et al. , Clin Vaccin Immunol, (2008), 15:590-7), they propose that this system may serve as the basis for a bioconjugate vaccine. These observations may explain the mechanism by which the passive administration of rabbit rPilA (type IV pili) IgG protected in mouse burn wound model (Mousavi M et al. , Microb Pathog, (2016), 101 :83-88).

Another potential explanation for the role of the O-glycan of FlaA is the fact that, unlike FlaB, its glycan has a rhamnose. Anti-rhamnose antibodies are among the most abundant circulating natural antibodies (Hossain et al., ACS Chem Biol, (2018), 13:2130-2142). It has been proposed that antigens containing a rhamnose sugar may be recognized by the anti-rhamnose antibodies and be more efficiently presented to antigen presenting cells through antigen uptake by the Fc receptors. If this were the case with the FlaA glycan, the antigen may be processed differently.

Example 2. Generation of reagent strains. Reagent strain to purify heterologous flagellins - We have created a recombinant reagent strain that can be used to purify large amounts of heterologous flagellin by deleting fliC from the .S'. Enteritidis reagent strain CVD 1943. The new reagent strain .S'. Enteritidis R11 AguaBA AclpP AfliD AfliC is designated CVD 1947. Heterologous fliC genes can subsequently be cloned into pGEN206 (Stokes MG et al., Infection and Immunity, 2007; 75(4): 1827-1834), a low copy number highly stable plasmid and introduced into CVD 1947.

Development of scalable upstream and downstream bioprocesses for obtaining purified flagellins and OPS- Robust, scalable, high yield and generalized purification methods have been developed to purify OPS and flagellins. We have developed and confirmed broadly applicable and scalable downstream manufacturing processes to purify secreted flagellins from culture supernatants, and OPS from bacterial cells using common bioprocess methods and equipment. We have also confirmed performance at 20 L scale for two different Salmonella serovar (Typhimurium and Enteritidis) where we can reliably purify to near homogeneity > 150 mg of flagellin/L of supernatant, and ~ 3 mg of COPS/g wet cell paste. By using fully chemically defined medium that does not contain any exogenous biological material (e.g., peptides, proteins), all biological components originate from the bacterial strain, thus further simplifying flagellin purification. Notably, we have found that secreted flagellin represents the major (> 90%) detectable protein species in fermentation culture supernatant. For flagellin purification, protein can be purified by an initial capture directly from fermentation supernatants onto cation exchange membranes. A secondary anion exchange purification step, coupled with a final tangential flow filtration step for buffer exchange and size selection, are sufficient to yield highly pure FliC (> 500 mg/L from fermentation culture) with very low endotoxin levels (< 0.1 EU/pg), and no detectable residual nucleic acid. COPS extraction can be accomplished by a series of organic extraction steps coupled with ion exchange chromatography, TFF and ammonium sulfate precipitation steps, and purified to near homogeneity at a yield of ~3 mg COPS/g wet cell paste. We have successfully used these bioprocess schemes to purify FliC flagellins from Salmonella serovars Typhimurium, Enteritidis and Typhi, and COPS from .S'. Typhimurium and S. Enteritidis. Development of methods to conjugate OPS with flagellin- We have developed several methods that can be used to simply and reliably conjugate OPS with carrier proteins, and generate different types of conjugates. Salmonella COPS was successfully conjugated directly to the s-amino groups of flagellin lysines or to carboxylic acid groups after modification with hydrazides, at random COPS hydroxyl groups along the polysaccharide chain using l-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), generating a lattice-type conjugate. End-linked sun-type conjugates have also been generated by conjugating at the carbonyl group present in the COPS ketocidic terminus with amino-oxime thioether chemistry to Sulfo-GMBS (N-[y-maleimidobutyryloxy]sulfosuccinimide ester) modified protein lysines. Removal of unconjugated components and conjugation reagents can be accomplished by a 2-step purification approach developed at the Center for Vaccine Development (CVD), separating first by size with size-exclusion chromatography (SEC) and then by charge using ion-exchange chromatography membranes. These conjugation methods have all been used successfully for the homologous COPS and flagellins from S. Enteritidis and S. Typhimurium.

Engineering bacteria so that large amounts of PA flagellin and O polysaccharides (OPS) can be purified safely and economically- Large-scale fermentation using wildtype pathogenic KP bacteria to manufacture COPS constitutes a significant occupational health hazard. The use of attenuated and avirulent strains from which to purify polysaccharide vaccine antigens markedly decreases these risks, and such a strategy is already being implemented for new generation S. Typhi Vi polysaccharide based vaccines (Micoli F et al., Vaccine. 2012; 30(5):853-861 ). Precise deletions in select metabolic and virulence genes of several GNB pathogens have resulted in attenuated strains useful as live oral vaccines (Tennant SM et al., Infect Immun. 2011 ; 79(10):4175-4185; Tacket CO, Levine MM et al., Clin Infect Dis. 2007; 45 Suppl LS20-23). We have experience in constructing such attenuated vaccine strains and in demonstrating their clinical acceptability, safety and immunogenicity in animal models and in human clinical trials (Inaba S et al., Biopolymers. 2013; 99( l):63-72; Kotloff KL et al., Hum Vaccin. 2007; 3(6):268-275). We have had success using a guaBA mutation (Samant S et al., PLoS Pathog. 2008; 4(2):e37) as the primary attenuating mutation in live attenuated Shigella vaccines where safety has been documented in clinical trials (Kotloff KL et al., Hum Vaccin. 2007; 3(6):268-275). A Phase 1 clinical trial conducted at the CVD has also shown that 5. Paratyphi A CVD 1902 (which possesses deletions in guaBA and clpX) was safe and well-tolerated in human volunteers including at the highest dosage levels tested (IO¹⁰ CFU)(Levine MM., Paper presented at: 8th International conference on typhoid fever and other invasive Salmonelloses 2013; Dhaka, Bangladesh). Genetically engineered attenuated strains can improve the safety of large-scale manufacture of Klebsiella pneumoniae OPS and can provide a means for enhanced Pseudomonas aeruginosa flagellin expression. Thus, we have created recombinant reagent strains that can be used to purify large amounts of Klebsiella pneumoniae OPS and PA flagellin.

Research Design for KP and PA strains- Genetically engineered Klebsiella pneumoniae reagent strains are created to improve occupational safety for large scale fermentation, and simplify and enhance OPS purification and yields. GuaBA from K. pneumoniae 01, 02, 03 and 05 strains is deleted using lambda red recombination (Datsenko KA, Wanner BL., Proc Natl Acad Sci U S A. Jun 6 2000; 97(12):6640- 6645). Capsule synthesis (cps) gene cluster is deleted from the four guaBA mutants. CPS mutation serves two purposes: 1) It is a secondary independently attenuating mutation that safeguards against the possibility of reversion to virulence; and 2) purification of core-0 polysaccharide will be simpler as there will be no contaminating capsular polysaccharide.

The gene encoding PA flagellins FlaB is cloned into pSEClO, a highly stable low copy number plasmid, and then transform the plasmids into our S. Enteritidis reagent strain CVD 1947. The reagent strains grow in chemically defined minimal media and secrete large amounts of PA flagellin is confirmed by performing SDS- PAGE and western blots of culture supernatant.

Reagents strains are grown in 5 L fermentation culture, as optimization at this scale is generally translatable to larger volumes (e.g., 50 L - 1,000 L). KP reagent strain fermentation is optimzed with rich media to supply an optimal environment for growth, making use of animal product free formulation to comply with FDA regulations for biologies. PA-Fla CVD 1947 expression vectors is grown in fully chemically defined minimal media to reduce the contaminant background, as the PA- Fla product will be in the supernatant. KP OPS and PA- Fla purification is conducted with previously optimized biochemical purification protocols that we developed for Salmonella COPS and FliC. Products are tracked through the process using standardized assays, and are verified to meet the following release parameters (TABLE 3):

Construction of K. pneumoniae reagent strains- We genetically engineered Klebsiella pneumoniae reagent strains to improve occupational safety for large scale fermentation, and simplify and enhance COPS purification and yields. We deleted guaBA from K pneumoniae 01, 02, 03 and 05 strains using lambda red recombination. We also deleted the capsule synthesis (cps~) gene cluster from the four guaBA mutants. CPS mutation will serve two purposes: 1) It is a secondary independently attenuating mutation that safeguards against the possibility of reversion to virulence; and 2) purification of core-0 polysaccharide will be simpler as there will be no contaminating capsular polysaccharide.

We used lambda red recombination to delete guaBA (for attenuation) and the capsule ops) gene cluster from the following KP strains: B5055 (01 :K2), 7380 (02ab:K-), 390 (O3:K11) and 4425/51 (O5:K7). We have genetically engineered the B5O55 (01) and 7380 (O2ab) Klebsiella strains and have deleted guaBA and cps genes, as necessary. We have also created the 390 (03) AguaBA mutant. See Table 4.

The primers used for the genetic engineering are shown in Table 5:

Deletion of guaBA from K. pneumoniae B5O55- DNA was first purified from B5055 with the Qiagen DNEasy Blood and Tissue kit according to the manufacturer’s protocol. DNA upstream of guaA was amplified using the following primers that produce a 688 bp DNA fragment (KP_guamut_F: 5’- GGTCGACGGATCCCCGGAATGGAGTAATCCCCGGCGTTAG-3’ (SEQ ID NO:31); KP guaBA_688_R: 5’-TGATTGGTCTGACTGGACGC-3’ (SEQ ID NO:28)). DNA downstream of guaB was amplified using primers that produce a 676bp DNA fragment (KP guaBA_676_F: 5’-GGGTAGATGATCACCGGCAG-3’ (SEQ ID NO:27); KP_guamut_R: 5’-

GAAGCAGCTCCAGCCTACACGGGCAATATCTCGACCAGGG-3’ (SEQ ID NO:32)). PCR amplification of the giiaAlguaB flanking regions was conducted using Vent polymerase. PCR products were electrophoresed on a 1% agarose gel and extracted and purified with a Qiagen Gel extraction kit according to the manufacturer’s protocol. The PCR products were combined in an overlapping PCR reaction using a Kan cassette amplified from pKD13 as described by Datsenko and Wanner. The PCR product of ~2.4 kb was gel extracted and amplified with guaBA_676_F/guaBA_688_R before transformation. Electrocompetent B5055 cells were transformed by electroporation with pKD46. Electrocompetent cells of K. pneumoniae B5055 expressing lambda red recombinase were prepared and electroporated with the 2.4 kb PCR product. Kanamycin resistant colonies were selected and screened for integration of the Kanamycin resistance cassette. The Kanamycin resistance cassette was subsequently deleted using pCP20 that removes the cassette via the FRT sites present in the sequence. To remove pCP20, cells were grown at 42°C and tested after each passage for loss of Carbenicillin or Chloramphenicol resistance.

Deletion of capsule genes from K. pneumoniae B5055- The genes encoding capsule synthesis in K. pneumoniae B5055 were also deleted using lambda red recombination. DNA downstream of wza was amplified using the following primers that produce a 600 bp DNA fragment (wza_F: 5’-GAGCCGACTCTAGGGTGGC-3’ (SEQ ID NO:37); wza_R: 5’-

GAAGCAGCTCCAGCC7ACACTAATGTCACATCATCAGTAAAT CAAAATTTG-3’ (SEQ ID NO:38)). Primers for the other flank amplify a region inside wzc itself since it is specific for the capsule type while the surrounding regions are highly variable between different capsule types. The primers (K2_wzc_F: 5’- GGTCGA

CGGA TCCCCGGAA TGT A AT AG AT ATGTT AT A G AGTTTGG A GGGG AG- 3 ’ (SEQ ID NO:39); K2_wzc_R: 5’-

TATTTAATTTCCCTCTTTCATCCTGTAATGTT-3’ (SEQ ID NO:40)) produced a 600 bp fragment. The same procedure as used for the guaBA mutagenesis were used.

The capsule deletion was assessed by India Ink staining and microscopic observation of the parental and mutant strain. The K. pneumoniae B5055 AguaBA Awzabc strain showed no evidence of capsule whereas the wild-type strain was capsule positive.

We have confirmed the guanine auxotrophy phenotype by growing the recombinant strains on minimal media containing or lacking guanine. We have shown that guanine must be supplied for growth of the KP AguaBA mutants.

Verification of attenuation- KP 01 :K2 strains are highly virulent for mice but most other serotypes that are human pathogens have been found to be avirulent in mice. To confirm that the CVD 3001 reagent strain (B5055 AguaBA Awzabc) is attenuated, we tested this mutant in mice and showed that the intraperitoneal 50% lethal dose is higher than the wild- type parental strain (Table 7). LD50 analysis was conducted using 5 CD-I mice per group injected IP with 10-fold dilutions of wild-type KP and the candidate engineered attenuated derivative.

Table 6. Verification of attenuation of KP reagent strain in vivo.

Construction of recombinant S, Enteritidis that express Type A and B flagella from P. aeruginosa- We cloned flaA from P. aeruginosa PAK which encodes Type A flagella (40 kDa) into pSEClO, a low copy number highly stable plasmid. We also cloned/ZaB from P. aeruginosa PAO1 which encodes Type B flagella (52 kDa) into pSEClO. The recombinant plasmids were transformed separately into CVD 1947 (5. Enteritidis R11 AguaBA AclpP AfliD AfliC) to create reagent strains capable of expressing large amounts of Type A or B flagellin. Mutagenesis was verified by PCR and sequencing upstream and downstream of the deletion. Secretion of Type A or B flagella was verified by SDS-PAGE.

The fliC gene was amplified from P. aeruginosa PAK using primers PAK_fliC_F and PAK_fliC_R and cloned into pSEClO so that it is expressed using the PompC promoter. Likewise, the fliC gene was amplified from P. aeruginosa PAO1 using primers PAOl_fliC_F and PAOl_fliC_R and cloned into pSEClO so that it is expressed using the PompC promoter. Primers used for cloning are shown in Table 4. Schematic diagrams of the resultant plasmids pSECl O-flaA and pSEC lO- flaB are shown in Figures 16 and 17, respectively.

The primers used for the genetic engineering are shown in Table 7. Table 7. Primers used for cloning of P. aeruginosa fliC genes in pSEClO.

Construction of the reagent strain S. Enteritidis CVD1947- We previously used Salmonella Enteritidis CVD 1943 (Rl l \guaBA \clpP AfliD) to express large amounts of flagellin into the supernatant. We genetically engineered this strain so that it no longer expresses native fliC. The objective is to use this strain to express exogenous fliC from a plasmid and which is secreted into the supernatant. We used lambda red recombination to delete the fliC gene. To ensure transcription of downstream genes after deletion of fliC in CVD 1943, the kanamycin cassette from pKD4 was used since it allows conservation of multiple promoter sites in the scar region after removing the kanamycin cassette from the genome. The primers shown in Table 5 were used to create a construct by overlapping PCR containing the Kanamycin cassette flanked by DNA upstream and downstream of fliC. Primers R1 l_fliC_up_F3 and R1 l_all_up_R3 amplify a 259 bp fragment upstream of fliC. R1 l_fliC_dwn_F3 and R1 l_fliC_dwn_R3 amplify a 301 bp fragment downstream of fliC. The fliC gene was subsequently deleted using lambda red recombination.

Table 8. Primers used for mutagenesis of Salmonella Enteritidis CVD1947.

We verified the deletion oifliC in CVD 1947 by sequencing the deletion. The entire fliC gene was deleted.

The pSEClO-flaB plasmid was transformed into S. Enteritidis CVD 1947. We confirmed that CVD 1947 (pSECl O-flaB) can express FlaB in the supernatant where they demonstrated the approximate predicted molecular weight of ~ 50 kDa by SDS- PAGE and coomassie analysis.

Purification and characterization of Klebsiella pneumoniae 01 O-polysaccharide (OPS)- Recombinant K. pneumoniae strain CVD3001 was grown to stationary phase by overnight growth in shaking culture at 37 °C in fully chemically defined media supplemented with guanine. OPS was extracted from the bulk growth culture by two different methods. In the first method, OPS was released from the core PS KDO by reduction of the culture pH to -3.7 with acetic acid and incubation at 100 °C for 4 hours. In the second method, the culture was brought to pH -3.7 with acetic acid and incubated with 200 mg/L sodium nitrite for 6 hours at 4 °C to release the OPS by nitrous acid deamination. Following OPS release, cells and insoluble debris were removed by centrifugation and clarification through a 0.45 um filter. Extraction by either method yielded OPS molecules of similar size that could he distinguished from residual contaminants in the post-hydrolysis supernatant by high-performance liquid size-exclusion chromatography (HPLC-SEC) analysis with detection by refractive index (RI). The OPS was purified from residual soluble contaminants by sequential steps involving 30 kDa molecular weight cutoff (MWCO) tangential flow filtration (TFF), anion-exchange chromatography, and ammonium sulfate precipitation. The purified material was concentrated and diafiltered into water by 10 kDa MWCO TFF. Analysis of the final-purified and in-process material by HPLC-SEC/RI demonstrated a single major molecular weight OPS species that was retained throughout the purification process.

The identity of the final purified 01 OPS was accomplished by depolymerization with 2M Trifluoroacetic acid and analysis of the monosaccharide constituents by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Monosaccharide composition analyses revealed that the OPS was comprised primarily of galactose with a minor N-acetyl- glucosamine peak detected. This is consistent with the published chemical structure of 01 OPS that is comprised entirely of galactose with a terminal N-acetyl- glucosamine residue present at the reducing end adjacent to the KDO, that is the expected site of hydrolysis by our extraction method (Vinogradov et al., J Biol. Chem. 2002; 277:25070-25081).

While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Claims

WHAT IS CLAIMED IS:

1. A conjugate comprising a Klebsiella surface polysaccharide antigen and a glycosylated native FlaA flagellin protein of Pseudomonas aeruginosa.

2. The conjugate of claim 1, wherein the surface polysaccharide antigen comprises a polysaccharide antigen selected from the group consisting of an O polysaccharide (OPS), a core oligosaccharide and an O polysaccharide (COPS), a capsule polysaccharide, and combinations thereof.

3. The conjugate of claim 1 or 2, wherein the surface polysaccharide antigen and the flagellin are covalently linked.

4. The conjugate of any one of claims 1-3, wherein the surface polysaccharide antigen is an O polysaccharide antigen (OPS).

5. The conjugate of any one of claims 2-4, wherein the Klebsiella OPS is from a Klebsiella pneumoniae serovar selected from the group consisting of Klebsiella pneumoniae serovars 01, 02a, 03, 05, and combinations thereof.

6. The conjugate of any one of claims 1-5, wherein the conjugate comprises i) a glycosylated native FlaA flagellin of Pseudomonas aeruginosa and ii) OPS from Klebsiella pneumoniae serovars 01, 02a, 03, and 05.

7. The conjugate of claim 5, wherein the glycosylated native FlaA flagellin is covalently linked to one or more OPS from a single Klebsiella pneumoniae serovar type.

8. The conjugate of any of claims 1-7, wherein the glycosylated native FlaA flagellin of Pseudomonas aeruginosa comprises SEQ ID NO: 1.

9. The conjugate of any of claims 1-8, wherein the conjugate further comprises a Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or variant thereof.

10. A pharmaceutical composition comprising an effective amount of the conjugate of any of claims 1-9.

11. The pharmaceutical composition of claim 10, wherein the composition further comprises an effective amount of a second conjugate comprising a Klebsiella surface polysaccharide antigen and a Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or variant thereof.

12. The pharmaceutical composition of claim 11, wherein the surface polysaccharide antigen of the second conjugate comprises a polysaccharide antigen selected from the group consisting of an O polysaccharide (OPS), a core oligosaccharide and an O polysaccharide (COPS), a capsule polysaccharide, and combinations thereof.

13. The pharmaceutical composition of claim 11 or 12, wherein the surface polysaccharide antigen and the flagellin of the second conjugate are covalently linked.

14. The pharmaceutical composition of any one of claims 11-13, wherein the surface polysaccharide antigen of the second conjugate is an O polysaccharide antigen (OPS).

15. The pharmaceutical composition of any one of claims 11-14, wherein the Klebsiella OPS is from a Klebsiella pneumoniae serovar selected from the group consisting of Klebsiella pneumoniae serovars 01, 02a, 03, 05, and combinations thereof.

16. The pharmaceutical composition of any one of claims 11-15, wherein the second conjugate comprises i) a Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or variant thereof and ii) OPS from Klebsiella pneumoniae serovars 01, 02a, 03, and 05.

17. The pharmaceutical composition of claim 15, wherein the Pseudomonas aeruginosa flagellin type B (FlaB) or an antigenic fragment or variant thereof is covalently linked to one or more OPS from a single Klebsiella pneumoniae serovar type.

18. The pharmaceutical composition of any one of claims 11-17, wherein the Pseudomonas aeruginosa flagellin type B (FlaB) comprises SEQ ID NO: 2.

19. A method of inducing an immune response in a subject, comprising administering to the subject an immunologically-effective amount of a pharmaceutical composition according to any of claims 10-18.