Fusion proteins generating protective antibodies and protective th17 responses for pseudomonas aeruginosa

Abstract

Technologies for the prevention and / or treatment of Pseudomonas aeruginosa infections. Disclosed herein are fusion proteins comprising a PcrV and PopB Pseudomonas aeruginosa polypeptide fused to a biotin-binding protein. Aspects of the technology also relate to compositions comprising the fusion protein, methods of production of the fusion protein, nucleic acids encoding the fusion protein and cells comprising the same; and methods of use of the fusion protein to induce an immune response to Pseudomonas aeruginosa infections, including prevention and / or treatment of Pseudomonas aeruginosa infections.

Description

FUSION PROTEINS GENERATING PROTECTIVE ANTIBODIES AND PROTECTIVE TH17 RESPONSES FOR PSEUDOMONAS AERUGINOSACROSS-REFERENCED APPEICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 521,635 filed June 16, 2023, the contents of which are hereby incorporated herein in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 13, 2024, is named 701039-000121WOPT_SL.xml and is 148,643 bytes in size.BACKGROUND

[0003] The Gram-negative bacterial pathogen Pseudomonas aeruginosa (Psi) causes a wide range of infections, mostly in hospitalized and immunocompromised patients, those with bums or combat-related wounds, and in people with cystic fibrosis (CF). There are no currently available vaccines for P. aeruginosa that are approved for human use. Moreover, Pseudomonas aeruginosa continues to be a significant opportunistic pathogen, with growing antibiotic resistance and no available vaccine. Previous work showed that the P. aeruginosa type III secretion system (T3SS) protein PopB elicits a protective Thl7 response after intranasal immunization using the Thl7 adjuvant curdlan, but the protection is of low potency. In those studies, PopB was co-purified with its chaperone PcrH.

[0004] Populations at risk for acute Pa infections include those at risk for hospitalization due to trauma, bums, major surgery, or cancer. These span groups such as police, firefighters, military service members, preoperative patients scheduled for major surgery, and cancer patients at diagnosis. CF-related infection is not an initial target of our vaccine approach since the pathogenesis of chronic Pa lung infection in people with CF and a strain types are very different from acute Pa infections. Moreover, vaccines stimulating Th 17 cells carry risk of exacerbating lung inflammation given the high numbers of Th 17 cells found in the CF lung after infection is established.1Prevention of initial infection by a vaccine is expected to be safe, therefore the Pa vaccine described herein can also be used for preventing infection in CF patient population. Active over passive immunization / immunotherapy strategy described herein using the Pa vaccines centers on the fact that passively administered antibodies function in the absence of adaptive Thl7 responses and lung TRM (tissue resident memory) Thl7 cells and thus are unlikely to be as broadly or potently protective. Monoclonal antibody approaches also suffer from ease of escape by point mutations, especially in a highly mutable pathogen such as Pa. Accordingly, there is a need for an active vaccine, particularly in the era of COVID- 19 and the need to protect those at risk for a hospital-acquired infections.SUMMARY OF THE INVENTION

[0005] The present disclosure addresses the lack of suitable technologies for the prevention and / or treatment of Pseudomonas aeruginosa infection. Among other things, preparing vaccines for the bacterial pathogen Pseudomonas aeruginosa. These immunogenic compositions and vaccines disclosed herein induce protective T cell responses to proteins and protective antibody responses directed at the protein antigens, including PopB and PcrV. Herein, the inventors demonstrated that fusing PopB with PcrV, a T3SS protein known to elicit protective antibodies, enhances protective efficacy of PopB and also demonstrated that adding the Th 17 antigen (PopB) antigen to PcrV surprisingly improved mucosal IgA responses to PcrV.

[0006] In particular, the technology disclosed herein is related to a fusion protein comprised of a rhizavidin (Rhavi) protein fused to two Pseudomonas aeruginosa proteins PcrV and PopB, where the called fusion protein is referred to herein generically as “RVB” fusion protein. In some embodiments, the fusion protein can comprise a rhizavidin protein and PcrV (referred to herein as Rhavi -PcrV -His or “RV- His” fusion protein). In some embodiments, the fusion protein can comprise a rhizavidin protein and PopB (referred to herein as Rhavi-PopB-His or “RB-His” fusion protein).

[0007] In some embodiments, a fusion protein described herein, when administered to a subject, can induce a higher Thl7 response by at least 25% or more including, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more, as compared to that induced by individual antigenic components of the fusion protein. In some embodiments, a fusion protein described herein, when administered to a subject, can induce a higher Th 17 response by at least 1.1-fold or more, including, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 10-fold, or higher, as compared to that induced by individual antigenic components of the fusion protein.

[0008] In some embodiments, a RSV fusion protein described herein, when administered to a subject, can induce an immune response to one or more representative non-vaccine Pseudomonas aeruginosa serotype(s) that is / are not included in a commercially-available vaccine. In some embodiments, an RVB fusion protein described herein, when administered to a subject, can induce an immune response to multiple Pseudomonas aeruginosa serotypes.Brief Description of the Drawings

[0009] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0010] FIG. 1 shows that human MHC / T cells can recognize PopB. Heparinized whole blood from a mechanically ventilated child with prior Pa respiratory infection was incubated with PopB / PcrHor PcrH for 7 days and then tested for IL-17 in the supernatant Bars are means of 4 wells; error bars SEM. P values by ANOVA with Tukey’s multiple comparison tests. *P<0.01 vs. medium control;#P<0.05 vs. PcrH at same concentration. G.K. PAO1, gentamicin-killed Pa strain PAO1.

[0011] FIG. 2 is a schematic of a composition of RVB fusion protein (without His-tag) (90 kDa), with a SDS-PAGE (right) showing RVB fusion protein isolated without PopB’s chaperone PcrH (20 kDa).

[0012] FIGS. 3A-3C shows intranasal (IN) immunization of mice with a Rhavi-PcrV -PopB fusion protein (RVB) elicits Th 17 responses to PopB, IgA to PcrV, and protection against high-dose challenge. FIG. 3A shows a RVB fusion protein (30 ug) as a vaccine composition elicits higher Thl7 responses in splenocytes to PopB / PcrH as compared to immunization with PopB / PcrH (30 ug). Splenocytes were stimulated for 7 days with the indicated antigens. IL- 17 in the supernatant was measured by ELISA. Bars are means of 3 wells; error bars are SD. *P<0.01 by ANOVA with Tukey’s multiple comparison test compared to PopB / PcrH alone at 0. 1 ug / mL. FIG. 3B shows the RVB fusion protein as a vaccine composition elicits higher IgA titers to PcrV in BALF compared to immunization with Rhavi-PcrV (RV) or PcrV (30 ug). Points are means of duplicate wells from BALF pooled from 4 mice / group. FIG. 3C shows protection after high-dose challenge with P. aeruginosa strain N13 (IxlO7CFU / mouse). *P<0.05, **P<0.01, ***P<0.001 by logrank test vs. curdlan.

[0013] FIG. 4A-4B shows optimization of production of RVB fusion protein with and without His-tag. FIG. 4A is a schematic showing RVB that lacks a His tag can be purified by being pulled down by binding to its His-tagged PcrH chaperone (upper portion). However, SDS-PAGE shows minimal binding to the biotinylated type 1 pneumococcal polysaccharide CPS1 (B-CPS1, also called PneumoPS in other figures, MW -1000 kDa). FIG. 4B is a schematic showing RVB-His composition (upper portion). SDS-PAGE shows RVB-His [MW -90 kDa] alone (lanes 1&2) and RVB-His mixed with biotinylated pneumococcal capsular polysaccharide [B-CPS1, also called PneumoPS in other figures, MW -1000 kDa] lanes 3&4, where lanes 1&3 were boiled, 2&4 not boiled, with lane 4 showing disappearance of the RVB-His band when not boiled, indicating MAPS complex formation via biotin- rhizavidin binding.

[0014] FIGS. 5A-5C show subcutaneous (SC) vaccination of mice with Rhavi-PopB-His (RB- His) alone or an exemplary RB-His MAPS vaccine, comprising Rhavi-PopB-His (RB-His) fusion protein complexed with biotinylated CPS 1 (PneumoPS), with Alum as adjuvant, is protective against pneumonia and induces both Th 17 responses and anti -PS responses. FIG. 5A shows SDS-PAGE of MAPS complex (lane 4) of biotinylated pneumococcal capsular polysaccharide [B-CPS1, also called PneumoPS in other figures, MW -1000 kDa] and Rhavi-PopB-His (lanes 1&3 boiled, 2&4 not boiled). FIG. 5B shows RB- His MAPS vaccine is protective against pneumonia with a strain N13 (2xl06CFU) (n=8 / group). FIG. 5C shows RB-His MAPS vaccine elicits the highest splenic Thl7 response. FIG. 5D shows PneumoPS- based MAPS vaccine with RB-His as carrier protein elicits high IgG to PneumoPS while vaccinationwith unbiotinylated PneumoPS mixed with RB-His does not. P values in FIG. 5A by logrank test. *P<0.05, **P<0.01 vs. Alum, and#P<0.01 vs. PneumoPS+Rhavi-PopB by ANOVA with Sidak’s multiple comparisons test.

[0015] FIG 6A-6D show subcutaneous (SC) immunization of mice with Rhavi-PcrV-PopB-His (RVB-His) alone inhibits T3SS cytotoxicity and induces protective Thl7 and antibody responses. FIG. 6A shows protection after challenge with P. aeruginosa strain N 13 (3xl06CFU / mouse, n=7-8 mice / group). P value by logrank test. FIG. 6B shows RVB-His elicits the significant splenic Thl7 recall responses after stimulation with either RVB-His or PopB / PcrH. Splenocytes were pooled from n=4 mice / group; bars are means of 5 replicates, error bars SD. *P<0.05, ****P<0.0001 by ANOVA with Sidak’s multiple comparisons test. FIG. 6C shows that RVB-His immunization alone (not in a MAPS complex) elicits high IgG titers in serum to RVB-His by ELISA. FIG. 4F shows antisera to RVB-His inhibits PcrV-mediated cytotoxicity while antisera to Alum does not. For E and F, antisera were pooled from 4 mice / group.

[0016] FIGS. 7A-7B show discovery of an exemplary PopB peptide epitope. FIG. 7A shows pooled splenocytes obtained from C57BL / 6 mice (n=4) immunized intranasally (IN) with PopB / PcrH+curdlan were stimulated with PopB / PcrH or the indicated peptides (selected from in silico and in vitro (Prolmmune) screens for mouse and human class II MHC binding). IL-17 measured at day 7 in supernatant by ELISA. PopB residues 171-185 (SEQ ID NO: 79) were identified as potential regions of PopB T-cell epitope. Bars are means of triplicates and error bars SD. ****P<0.0001 by ANOVA with Sidak’s multiple comparisons test. FIG. 7B shows pooled unstimulated splenocytes from C57BL / 6 mice (n=4) previously immunized IN with curdlan or PopB / PcrH+curdlan were stained with APC-conjugated mouse class II (I-Ab) tetramer loaded with PopBni-iss peptide (SEQ ID NO: 79) (2ug / mL) and then incubated with Fc-block followed by CD3-FITC, CD4-PE, and Live / Dead stain. Gated on CD3+cells.Detailed Description of Certain Embodiments

[0017] The present disclosure relates, generally, to novel immunogenic fusion proteins of Pseudomonas aeruginosa that can be used, e.g. , to induce and / or increase an immunoprotective response, or to reduce Pseudomonas aeruginosa infection or colonization in subjects at risk of or suffering from Pseudomonas aeruginosa infection.

[0018] The presently disclosed novel immunogenic proteins represent a substantial advance over the currently available options for immunizing patients against Pseudomonas aeruginosa infection. Such immunogenic proteins can be used, e.g. , to induce and / or increase an immunoprotective response or to reduce Pseudomonas aeruginosa colonization in subjects, such as those at risk of or suffering from Pseudomonas aeruginosa infection.Fusion Proteins

[0019] The present disclosure describes novel immunogenic fusion proteins of Pseudomonas aeruginosa. Fusion proteins described and / or utilized herein provide improved immunogenicity and IL- 17 response to protein stimulation, as well as further reduction of Pseudomonas aeruginosa colonization and protection from invasive diseases.

[0020] A fusion protein includes one, two, or more polypeptides that elicit (e.g., primarily elicit) a T cell response, or that elicit both a T cell and a B cell response. In some embodiments, the fusion protein comprises one or more of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises two of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises three of the polypeptides listed in Table 1. In some embodiments, the fusion protein comprises one or more of polypeptides encoded by one or more of the genes listed in Table 1. In some embodiments, the fusion protein comprises two of polypeptides encoded by two or more of the genes listed in Table 1. In some embodiments, the fusion protein comprises three polypeptides encoded by three of the genes listed in Table 1.Table 1. Exemplary Polypeptide Components of Fusion Proteins

[0021] In some embodiments, a fusion protein comprises one or more antigenic polypeptides of Pseudomonas aeruginosa having an amino acid sequence comprising any of SEQ ID NOs: 3 or 6, or antigenic fragments thereof. In some embodiments, a fusion protein comprises two antigenic polypeptides having an amino acid sequence comprising any of SEQ ID NOs: 3 or 6, or antigenic fragments thereof. In some embodiments, a fusion protein comprises (i) two antigenic polypeptides having an amino acid sequence comprising any of SEQ ID NOs: 3 or 6, or antigenic fragments thereof, and (i) a biotin-binding moiety comprising SEQ ID NO: 1 or 2, or biotin-binding fragments thereof. In some such embodiments, at least one antigenic polypeptide is or comprises an PcrV polypeptide (e.g. , SEQ ID NO: 3). In some such embodiments, at least one antigenic polypeptide is or comprises an PopB polypeptide (e.g , SEQ ID NO: 6).

[0022] In some embodiments, a fusion protein comprises one or more polypeptides homologous to the Pseudomonas aeruginosa polypeptides listed in Table 1, e.g., an PcrV polypeptide or an PopBpolypeptide isolated from different serotypes of Pseudomonas aeruginosa. Individual serotypes of Pseudomonas aeruginosa contain numerous mutations relative to each other, and some of these result in different protein sequences between the different serotypes. One of skill in the art may readily substitute an amino acid sequence, or a portion thereof with the homologous amino acid sequence from a different Pseudomonas aeruginosa serotype. In some embodiments, antigenic polypeptides have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptides listed in Table 1, or antigenic fragments thereof. Serotypic variation may be used to design such variants of the polypeptides listed in Table 1.

[0023] In some embodiments, fusion proteins described herein comprise one or more fragments of polypeptides listed in Table 1, e.g , biotin-binding fragments of rhizavidin, antigenic fragments ofa PcrV polypeptide with or without a signal sequence, or antigenic fragments of a PopB polypeptide with or without a signal sequence. In some embodiments, fusion proteins described herein comprise truncation mutants that are close in size to the polypeptides listed in Table 1. For example, they may lack at most one, two, three, four, five, ten, or twenty amino acids from one or both termini (referring to component polypeptides in a fusion protein). In some embodiments, a fragment is a truncated fragment of any of SEQ ID NOs: 1-3, 6 or 26 lacking 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both, of any one of SEQ ID NOs: 1-3, 6 or 26. In some embodiments, a fragment is a truncated fragment of any of SEQ ID NOs: 1-3, 6 or 26 lacking 1-10 amino acid residues from the N-terminus, C- terminus, or both, of any one of SEQ ID Nos: 1-3, 6 or 26. For instance, a fragment may lack 10 amino acid residues at both the N-terminus and C-terminus of any one of SEQ ID NOs: 1-3, 6 or 26, resulting in a protein lacking 20 amino acid residues. Internal deletions, e.g, of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.

[0024] In some embodiments, a fusion protein comprises an N-terminal polypeptide and a C- terminal polypeptide. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide is an antigenic polypeptide, for example, a polypeptide having an amino acid sequence comprising one or more of SEQ ID NOs: 3 or 6, or an antigenic fragment or variant thereof. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide is a biotinbinding moiety, for example a polypeptide having an amino acid sequence comprising SEQ ID NO: 1 or 2, or a biotin-binding fragment thereof. In some embodiments, one of the N-terminal polypeptide or the C- terminal polypeptide is a biotin-binding moiety, for example a polypeptide having an amino acid sequence comprising SEQ ID NO: 1 or 2, or a biotin-binding fragment thereof, and the other terminal polypeptide is an antigenic polypeptide, for example, a polypeptide having an amino acid sequence comprising one or more of SEQ ID NOs: 3 or 6, or an antigenic fragment or variant thereof.

[0025] In some embodiments, the N-terminal polypeptide and the C-terminal polypeptide are directly bound to each other. In some embodiments, the N-terminal polypeptide and the C-terminal polypeptide are linked via a linker peptide. The length and / or amino acids of a linker, when present, canbe adjusted to obtain a more flexible, semi-rigid, or rigid linker. Exemplary flexible peptide linkers are shown as SEQ ID NOs:37-40. A linker can generally be from 1-40, such as 3-10 or 10-30 and specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some embodiments, the fusion protein comprises one linker. In some embodiments, the fusion protein comprises two linkers. In some embodiments, the one or two linkers are selected from GS, or SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises an amino acid sequence AAA (SEQ ID NO: 38) residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37 (GGGGSSS) and an amino acid sequence AAA (SEQ ID NO: 38) residual from a Not I restriction site.

[0026] Exemplary fusion proteins are shown in Table 2.Table 2. Exemplary Fusion Proteins

[0027] In some embodiments, the present disclosure provides fusion proteins with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a fusion protein listed in Table 2. In some embodiments, a fusion protein is or includes an amino acid sequence having at least80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 14, 17-26. In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:23. In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to RVB comprising an amino acid sequence of SEQ ID NO: 23.

[0028] In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:25. In some embodiments, a fusion protein is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to RVB-His comprising an amino acid sequence of SEQ ID NO: 25.

[0029] In some embodiments, a fusion protein described herein comprises an antigenic fragment of a fusion protein shown in Table 2. In some embodiments, a fusion protein is or includes an antigenic fragment of any of SEQ ID NOs: 14, 17-26. For example, a fusion protein may lack at most one, two three, four, five, ten, or twenty amino acids from the N-terminus, C-terminus, or both, of any one of SEQ ID NOs: 14, 17-26. In some embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N- terminus compared to the C-terminus. In some embodiments, a fusion protein is or includes an antigenic fragment of SEQ ID NO:23. In some embodiments, a fusion protein is or includes an antigenic fragment of RVB.

[0030] In some embodiments, a fusion protein is or includes an antigenic fragment of SEQ ID NO:25. In some embodiments, a fusion protein is or includes an antigenic fragment of RVB-His.

[0031] In some embodiments, a fusion protein described herein comprises a biotin-binding moiety. In some embodiments, the fusion protein comprises a biotin-binding moiety, and one or more polypeptide antigens. In some embodiments, the fusion protein comprises a biotin-binding moiety and two or more polypeptide antigens. As used herein, a “biotin-binding moiety” refers to a biotin-binding polypeptide or protein, a biotin-binding fragment thereof, or a biotin-binding domain thereof. In some embodiments, the biotin-binding moiety of the fusion protein comprises rhizavidin or a biotin-binding fragment thereof, as further described in WO 2012 / 155053, the contents of which are herein incorporated by reference in their entirety.

[0032] In some embodiments, a fusion protein described herein comprises a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 2 (amino acids 45-179 of rhizavidin, denoted Rhavi), or biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3 (Pseudomonas aeruginosa PcrV polypeptide), or an antigenic fragment thereof. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6 (Pseudomonas aeruginosa PopB polypeptide), or an antigenic fragment thereof.

[0033] In some embodiments, a fusion protein described herein comprises each of: (a) a biotinbinding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 1 (rhizavidin), or biotin-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3 (Pseudomonas aeruginosa PcrV polypeptide), or an antigenic fragment thereof; and (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:6 (Pseudomonas aeruginosa PopB polypeptide) or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from GS, or SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises an amino acid sequence AAA (SEQ ID NO: 38) residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37 (GGGGSSS) and an amino acid sequence AAA (SEQ ID NO: 38) residual from a Not I restriction site.

[0034] In some embodiments, a fusion protein described herein comprises each of: (a) a biotinbinding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:2 (amino acids 45-179 of rhizavidin, denoted Rhavi), or biotin-binding fragment thereof; (b) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO:3 (Pseudomonas aeruginosa PcrV polypeptide) or an antigenic fragment thereof; and (c) a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 6 (Pseudomonas aeruginosa PopB polypeptide) or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises an amino acid sequence AAA (SEQ ID NO: 38) residual from a Not I restriction site. In some embodiments, the fusion protein comprises a linker of SEQ ID NO:37 (GGGGSSS) and an amino acid sequence GS or AAA (SEQ ID NO: 38) residual from a Not I restriction site. In some embodiments, a fusion protein described herein comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence SEQ ID NO: 23 or SEQ ID NO: 25. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO:23. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO:23 (RVB).

[0035] In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO:25. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO:25 (RVB-His).

[0036] In some embodiments, a fusion protein described herein includes a variant or fragment of a polypeptide listed in Table 1. In some embodiments, a fusion protein described herein includes a polypeptide encoded by a variant or fragment of a gene listed in Table 1. In some embodiments, a fragment included in a fusion protein described herein is close in size to a full-length polypeptide or a polypeptide listed in Table 1. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini. In some embodiments, the fragment is 25-50 amino acids in length, or 50-100, or 100-150, or 150-200, or 200-250, or 250-300, or 300-350 amino acids in length. In some embodiments, the fragments result from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g. , the baculovirus expression system), or a mammalian (e.g. , human or Chinese Hamster Ovary) cell line. The fragments described above or subfragments thereof (e.g, fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the biological activities described below, such as increasing the amount of IL-17 released by at least 1.5 fold or 2 fold or more (e.g, either as an absolute measure or relative to a control protein).

[0037] The DNA and protein sequence of each gene and polypeptide may be identified by searching for the Locus Tag in a publicly available database, e.g., Entrez Gene (on the NCBI NIH web site on the World Wide Web, at www.ncbi.nlm.nih. gov / sites / entrez?db=gene ), in the Streptococcus pneumoniae TIGR4 genome, and the indicated sequences are also included within the scope of the present disclosure.

[0038] Certain polypeptides of Table 1, variants thereof, and additional exemplary polypeptides and linkers which constitute components of various embodiments of the fusion proteins are described in greater detail below.PcrV Polypeptides and Variants Thereof

[0039] PcrV is a conserved Pseudomonas aeruginosa protein. In some embodiments, a PcrV polypeptide is an type III secretion system needle tip protein PcrV conserved across Pseudomonas aeruginosa strains. PcrV is conserved and is a T3SS protein and is a serotype-independent antigen. It was found in 902 of the 913 isolates (99%) in a study that contained a pcrV gene.58Of the isolates with amino acid variants in PcrV, most were near the N terminus, far from the critical T3SS-inhibiting epitope of PcrV at amino acids 144-257,68but 31% had an S225R SNP and 13% had an S225K SNP,58in similar proportions previously seen in 90 clinical isolates reported by Lynch and co-workers68

[0040] In some embodiments, a PcrV polypeptide is or comprises a full-length PcrV polypeptide. For example, in some embodiments, a full-length PcrV polypeptide has 394 amino acids and is represented by the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, a fusionprotein comprises a PcrV polypeptide of Pseudomonas aeruginosa. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 380 consecutive amino acids of a PcrV polypeptide.

[0041] In some embodiments, a PcrV polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 380 consecutive amino acids of the sequence shown in SEQ ID NO: 3. In some embodiments, a PcrV polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367, or 399 consecutive amino acids of the sequence shown in SEQ ID NO: 3. In some embodiments, the PcrV polypeptide does not include a signal sequence. In some embodiments, the PcrV polypeptide does include a signal sequence at the N-terminus of the amino acid sequence of SEQ ID NO: 3.

[0042] Sequence variation occurs at the protein level between different Pseudomonas aeruginosa serotypes, and a consensus sequence illustrating combinations of PcrV sequences from different Pseudomonas aeruginosa serotypes. Accordingly, the amino acid sequence of SEQ ID NO: 3 refers to the consensus sequence across a range of subtypes of Pseudomonas aeruginosa. An exemplary nucleotide sequence encoding a PcrV polypeptide is provided herein as SEQ ID NO: 11.PopB Polypeptides and Variants Thereof

[0043] In some embodiments, a PopB polypeptide is a Type III secretion system translocon subunit PopB polypeptide conserved across Pseudomonas aeruginosa strains. In some embodiments, a PopB polypeptide is or comprises a full-length PopB polypeptide. For example, in some embodiments, a full- length PopB polypeptide has 390 amino acids and is represented by the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, a fusion protein comprises a PopB polypeptide of Pseudomonas aeruginosa. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 252, or 390 consecutive amino acids of a PopB polypeptide.

[0044] In some embodiments, a PopB polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 252, or 398 consecutive amino acids of the sequence shown in SEQ ID NO:6. In some embodiments, a PopB polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 252, or 350 consecutive amino acids of the sequence shown in SEQ ID NO:6. In some embodiments, the PopB polypeptide does not include a signal sequence. In some embodiments, the PopB polypeptide does include a signal sequence at the N-terminus of the amino acid sequence of SEQ ID NO: 3.

[0045] In some embodiments, a fusion protein as disclosed herein comprises a fragment of PopB polypeptide that comprises a MHC class II binding site, or strong binding to a MHC class II molecule. For example, in some embodiments, a fusion protein as disclosed herein can comprise a fragment of PopB polypeptide where the fragment of PopB comprises a peptide is selected from any of those in Table 3 or 4 herein, or comprises a peptide having an amino acid sequence of SEQ ID NO: 65-94. In a particular embodiment, a fusion protein as disclosed herein that comprises a fragment of PopB polypeptide comprises at least one or more peptides selected from: SEQ ID NO: 65, 66 or 79. In some embodiments, a fusion protein as disclosed herein that comprises at least the amino acid sequence of SEQ ID NO: 79.

[0046] Sequence variation occurs at the protein level between different Pseudomonas aeruginosa serotypes, and a consensus sequence illustrating combinations of PopB sequences from different Pseudomonas aeruginosa serotypes can be used. In some embodiments, a PopB polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO: 6 [consensus]. In some embodiments, a PopB polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO:6 [consensus].

[0047] An exemplary nucleotide sequence encoding a PopB polypeptide is provided herein as SEQ ID NO: 13.Rhizavidin

[0048] Rhizavidin is a naturally occurring dimeric protein in the avidin protein family, was first discovered in Rhizobium etli, a symbiotic bacterium of the common bean. Rhizavidin has only a 22% amino acid identity with chicken avidin, a protein commonly found in eggs, but with high conservation of amino acid residues involved in biotin binding [Helppolainen et al, 2007], In some embodiments, the nucleotide sequence of rhizavidin is set forth in SEQ ID NON. In some embodiments, the amino acid sequence of rhizavidin is set forth in SEQ ID NO: 1. Amino acids 1-44 of SEQ ID NO: 1 are predicted to be a signal sequence(s) of rhizavidin (amino acids 1-44 of the full-length protein). In some embodiments, a fusion protein comprises rhizavidin. In some embodiments, a fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of a rhizavidin polypeptide.

[0049] In some embodiments, a rhizavidin polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of the sequence shown in SEQ ID NO: 1 [full-length]. In some embodiments, a rhizavidin polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or 179 consecutive amino acids of the sequence shown in SEQ ID NO: I [full-length],

[0050] In some embodiments, a rhizavidin polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 135 consecutive amino acids of the sequence shown in SEQ ID NO:2 [minus signal sequence]. In some embodiments, a rhizavidin polypeptide of the fusion protein comprises an amino acid sequence that is at least 60% or more (including, e.g.„ at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 135 consecutive amino acids of the sequence shown in SEQ ID NO:2 [minus signal sequence].

[0051] In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 50-179 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 55-179 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 60-179 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 65-179 of SEQ ID NO: 1.

[0052] In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 45-175 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 45-171 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 45-167 of SEQ ID NO: 1. In some embodiments, a rhizavidin polypeptide of the fusion protein is or comprises amino acids 45-163 of SEQ ID NO: 1.Linker or Spacer

[0053] In some embodiments, a fusion protein comprises one or more linkers. In some embodiments, a linker is or comprises one or more amino acids. In some embodiments, a fusion protein comprises an antigenic polypeptide joined to a biotin-binding moiety by a linker. In some embodiments, a fusion protein comprises a first antigenic polypeptide, a second antigenic polypeptide, a biotin-binding moiety, and at least one linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are joined by a linker. In some embodiments, the first antigenic polypeptide or the second antigenic polypeptide are joined to the biotin-binding moiety by a linker. In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are joined by a first linker; and the first antigenic polypeptide or the second antigenic polypeptide are joined to the biotin-binding moiety by asecond linker.

[0054] In some embodiments, a linker interposes a structure between two protein moieties. In some embodiments, the structure is or comprises an a-helix. In some embodiments the structure is or comprises a P-strand. In some embodiments, the structure is or comprises a coil / bend. In some embodiments, the structure is or comprises a turn. In some embodiments, a linker decreases steric hindrance between two protein moieties joined by the linker. In some embodiments, a linker decreases unfavorable interactions between two protein moieties joined by the linker. In some embodiments, a linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline, and / or alanine residues. In some embodiment a linker is hydrophilic. In some embodiments a linker is hydrophobic. In some embodiments a linker increases the stability of the fusion protein containing the linker.

[0055] In some embodiments, a linker does not interfere with the folding of an antigenic polypeptide to which it is joined. In some embodiments, a linker does not interfere with the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments, a linker does not reduce the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments, a linker does not eliminate the antigenicity of an antigenic polypeptide to which it is joined. In some embodiments the effect of the linker is determined by comparing the polypeptide with the polypeptide joined to the linker.

[0056] In some embodiments, a linker does not interfere with the folding of a biotin-binding moiety to which it is joined. In some embodiments, a linker does not interfere with the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments, a linker does not reduce the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments, a linker does not eliminate the biotin-binding ability of a biotin-binding moiety to which it is joined. In some embodiments the effect of the linker is determined by comparing the biotin-binding moiety with the biotin-binding moiety joined to the linker.

[0057] In some embodiments, a linker is not antigenic. In some embodiments, a linker does not elicit a T cell response. In some embodiments, a linker does not elicit a B cell response. In some embodiments, a linker does not induce a T cell or a B cell response.

[0058] In some embodiments, a linker comprises two or more amino acids. In some embodiments, a linker may be 3-100, 5-100, 10-100, 20-100 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, or 2-3 amino acids in length. In some embodiments, a linker comprises between 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0059] In some embodiments, a linker is a flexible linker. Flexible linkers may be useful forjoining domains that require a certain degree of movement or interaction and may include small, nonpolar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce unfavorable interactions between the linker and the protein moieties. In some embodiments a linker comprises small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. In some embodiments, a linker is a Gly-Ser linker.

[0060] In some embodiments, a linker is or comprises an amino acid sequence of GS or GGGGSSS (SEQ ID NO:37). In some embodiments, a linker is or comprises a sequence of (GGGGS)n(SEQ ID NO:39), where n represents the number of repeating GGGGS (SEQ ID NO: 62) units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a polypeptide linker may have an amino acid sequence that is or comprises GGGGSGGGGSGGGGS (SEQ ID NO:41) (z.e., (GGGGS)3(SEQ ID NO: 41)) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:42) (z.e., (GGGGS)e (SEQ ID NO: 42)). In some embodiments, a linker comprises one or more of Gly, Ser, Thr, Ala, Lys, and Glu. In some embodiments, a linker is or comprises KESGSVSSEQLAQFRSLD (SEQ ID NO:43). In some embodiments, a linker is or comprises EGKSSGSGSESKST (SEQ ID NO:44). In some embodiments, a linker is or comprises (Gly)n(SEQ ID NO:45) where n represents the number of repeating Gly residues and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments a linker is or comprises GS or GGG. In some embodiments, a linker is or comprises (Gly)e (SEQ ID NO:40). In some embodiments, a linker is or comprises (Gly)s (SEQ ID NO:46). In some embodiments, a linker is or comprises GSAGSAAGSGEF (SEQ ID NO:47). In some embodiments, a linker is or comprises an amino acid sequence of AAA (SEQ ID NO:38).

[0061] In some embodiments, a linker is a rigid linker. Rigid linkers are useful to keep a fixed distance between domains and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the fusion. In some embodiments, a linker is or comprises (EAAAK)n(SEQ ID NO:48) where n represents the number of repeating EAAAK (SEQ ID NO: 63) units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 ormore. In some embodiments, a linker is or comprises A(EAAAK)nA, (SEQ ID NO:49) where n represents the number of repeating EAAAK (SEQ ID NO: 63) units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, a linker is or comprises A(EAAAK)nA (SEQ ID NO: 108), where n represents the number of repeating EAAAK (SEQ ID NO: 63) units and is 2, 3, 4, or 5. In some embodiments, a linker is or comprises A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:50). In some embodiments, a linker is or comprises [A(EAAAK)nA]m, (SEQ ID NO:51) wherein n is 2, 3, or 4 and m is 1 or 2. In some embodiments, a linker is or comprises AEAAAKEAAAKA (SEQ ID NO:52).

[0062] In some embodiments a linker is or comprises (X-Pro)n(SEQ ID NO:53) , with Xdesignating any amino acid, where n represents the number of repeating X-Pro units and is 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments a linker is or comprises (Ala-Pro)n(SEQ ID NO:54), where n represents the number of repeating Ala-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments a linker is or comprises (Ala-Pro)n(SEQ ID NO: 109), where n represents the number of repeating Ala-Pro units and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.

[0063] In some embodiments a linker is or comprises (Lys-Pro)n(SEQ ID NO:55), where n represents the number of repeating Lys-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments a linker is or comprises (Gln-Pro)n(SEQ ID NO:56), where n represents the number of repeating Gin-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 ormore. In some embodiments, a linker is or comprises (Ala-Pro)? (SEQ ID NO:57).

[0064] In some embodiments a linker is or comprises GAPGGGGGAAAAAGGGGGGAP (GAG linker, SEQ ID NO:58). In some embodiments a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 linker, SEQ ID NO: 59). In some embodiments a linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GA G3 linker, SEQ ID NO:60).

[0065] Suitable linkers or spacers also include those having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the above exemplary linkers.

[0066] Additional linkers suitable for use with some embodiments may be found in U.S. Patent Publication No. 2012 / 0232021, filed on March 2, 2012, and [Chen, 2013] the disclosures of which is hereby incorporated by reference in their entireties.

[0067] In some embodiments, a fusion protein described herein may comprise a tag. A tag may be N-terminal or C-terminal. For instance, tags may be added to a polypeptide (via additions or modifications on the encoding DNA sequence) to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein. In some embodiments a tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification.

[0068] In some embodiments, a tag is, comprises, or is derived from one or more of polyhistidine (His), Glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose binding protein (MBP), vesicular Stomatitis viral glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), Calmodulin, Nus, S tags, lipoprotein D, and galactosidase.

[0069] In some embodiments, a His tag is or comprises an amino acid sequence of Hn, wherein n is an integer between 2 and 10 (SEQ ID NO: 64). Exemplary His tags include HHHHHH (SEQ ID NO: 15) and MSYYHHHHHH (SEQ ID NO: 16). As disclosed herein in the Examples and in FIG. 4B, a C- terminal His tag on the RVB fusion protein resulted in improved folding of the fusion protein, resulting in improved binding of the rhizavidin polypeptide of the RVB fusion protein to biotinylated pneumoPS. Moreover, as shown in FIG. 6A-6B, the RVB-His fusion protein was demonstrated to be protective and induce an IL17 response, as well as inhibit PcrV-mediated cytotoxicity.

[0070] Accordingly, in some embodiments, a fusion protein as disclosed herein., e.g., a fusion protein disclosed in Table 2 comprises a His tag, which is not removed prior to administration to a subject in a vaccine composition.

[0071] In other embodiments, the fusion protein is free of tags such as protein purification tags and is purified by a method not relying on affinity for a purification tag. In some embodiments, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of a polypeptide of Table 1 or fusion protein of Table 2. In some embodiments, as disclosed in FIG. 4B, a fusion protein as disclosed herein for use in an immunogenic composition, e.g., a RVB fusion protein for use in a immunogenic composition lacks aN-terminal signal sequence. In some embodiments, a RVB fusion protein as disclosed herein is produced by expression from an expression vector, where a signal sequence is not present. In some embodiments, a RVB fusion protein as disclosed herein comprises a C- terminal His tag and is produced from in a cell by expression from an expression vector, where a signal sequence is not present.

[0072] In some embodiments, a fusion protein described herein may contain a membrane translocating sequence (MTS), to facilitate introduction of the fusion protein into a mammalian cell and subsequent stimulation of the cell-mediated immune response. Exemplary membrane translocating sequences include the hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of a synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin 3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in U.S. Pat. Nos. 6,248, 558, 6,432,680 and 6,248,558.Nucleic Acids

[0073] In some embodiments, the present disclosure provides nucleic acids, e.g., DNA, RNA, or analogs thereof, encoding one or more of the polypeptides and / or fusion proteins described herein. An underlying DNA sequence for the polypeptides described herein may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention. In some embodiments, a DNA sequence may be codon-optimized to improve expression in a host such as a bacterial cell line, e.g. , E. coll, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g. , human or Chinese Hamster Ovary) cell line. In some embodiments, a fusion protein disclosed herein is produced by a cell-free methodology.

[0074] In some embodiments, the present disclosure provides nucleic acids, e.g., DNA, RNA, or analogs thereof, that are at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identical to a nucleic acid sequence provide in Table 1, Table 2, or a variant or portion thereof. In some embodiments, the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, 1000-2000, 2000-3000, or 3000-4000 nucleotides in length. In some embodiments, a nucleic acid may be used for recombinant production of a polypeptide or fusion protein of Table 1 or Table 2, or antigenic fragments thereof. In some embodiments, a nucleic acid may be used as a vaccine.

[0075] Nucleic acid sequences encoding variants of PcrV (SEQ ID NO: 3) are provided as SEQ ID NOs: 11. Nucleic acid sequences encoding variants of PopB (SEQ ID NO:6) are provided as SEQ ID NO: 13. Nucleic acid sequences encoding different fusion proteins (SEQ ID NOs: 14, 17-26) are provided as SEQ ID NOs: 12, 27-36. In all cases, due to degeneracy in the genetic code, other DNA sequences (including multiple codon-optimized sequences) could be contemplated by those of ordinary skill to encode such polypeptides and fusion proteins.

[0076] Nucleic acids encoding polypeptides or fusion proteins of Table 1 or Table 2, or fragments thereof, can be cloned into any of a variety of expression vectors, under the control of a variety of regulatory elements, and fusions can be created with other sequences of interest. Methods of cloning nucleic acids are routine and conventional in the art. For general references describing methods of molecular biology which are mentioned in this application, e.g., isolating, cloning, modifying, labeling, manipulating, sequencing and otherwise treating or analyzing nucleic acids and / or proteins, see, e.g., Sambrook et al, 1989; Ausubel et al, 1995; Davis et al, 1986; Hames et al, 1985; Dracopoli et al, 2018; and Coligan et al, 2018.Uses of Fusion Proteins

[0077] In some embodiments, a fusion protein described herein does not have, or has minimal, hemolytic activity. For example, in some embodiments, the hemolytic activity of a fusion protein described herein can be established by turbidimetry (OD420) after incubation of the fusion protein at different dilutions with red blood cells (e.g., sheep erythrocytes), to determine the protein concentration at which 50% of the red blood cells are lysed. In some such embodiments, the hemolytic activity of a fusion protein described herein can be characterized by an OD42oof less than 0.4 or lower, including, e.g., less than 0.3, less than 0.25, less than 0.2, or lower, for a given protein concentration.

[0078] In some embodiments, polypeptides of Pseudomonas aeruginosa and fusion proteins described herein, and fragments and variants thereof, are immunogenic. These polypeptides and fusion proteins may be immunogenic in mammals, for example mice, rats, guinea pigs, or humans. An antigenic polypeptide or fusion protein is typically one capable of raising a significant immune response in an assay or in a subject. The immune response may be innate, humoral, cell-mediated, or mucosal (combining elements of innate, humoral and cell-mediated immunity). For instance, an antigenic polypeptide orfusion protein may increase the amount of IL-17 produced by T cells. Alternatively or additionally, an antigenic polypeptide or fusion protein may (i) induce production of antibodies, e.g., neutralizing antibodies, that bind to the polypeptide and / or the whole bacteria, (ii) induce Th 17 immunity, (iii) activate the CD4+ T cell response, for example by increasing the number of CD4+ T cells and / or increasing localization of CD4+ T cells to the site of infection or reinfection, (iv) activate the CD8+ T cell response, for example by increasing the number of CD8+ T cells and / or increasing localization of CD8+ T cells to the site of infection or reinfection, (v) activate both the CD4+ and the CD 8+ response, (vi) activate CD4- / CD8- immunity, (vii) induce Thl immunity, (viii) induce anti-microbial peptides, (ix) activate innate immunity, or any combination of the foregoing. In some embodiments, an antigenic polypeptide or fusion protein elicits production of a detectable amount of antibody specific to that antigen.

[0079] In some embodiments, a fusion protein described herein is an antigen or has antigenic properties. In some embodiments, a fusion protein described herein is a carrier protein or has carrier properties. In some embodiments, a fusion protein described herein is both an antigen and a carrier protein. In some embodiments, a fusion protein described herein has both carrier properties and antigenic properties.

[0080] In some embodiments, a fusion protein described herein can be formulated to be an antigen in a multi-component immunogenic complex. An exemplary multi-component immunogenic complex is a Multiple Antigen Presenting System (MAPS) complex as described in WO 2012 / 155007, the entire contents of which are incorporated herein by reference for the purposes indicated herein). In some embodiments, a fusion protein described herein is a carrier protein of an immunogenic complex. In some embodiments, a fusion protein described herein is both a carrier protein and an antigen of an immunogenic complex.

[0081] In some embodiments, polypeptides of the fusion proteins described herein have less than 20%, 30%, 40%, 50%, 60% or 70% identity to human auto-antigens and / or gut commensal bacteria (e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus , Bifidobacterium, Escherichia, and Lactobacillus species). Examples of human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.

[0082] A polypeptide included in a fusion protein described herein may comprise one or more immunogenic portions and one or more non-immunogenic portions. The immunogenic portions may be identified by various methods, including protein microarrays, ELISPOT / ELISA techniques, and / or specific assays on different deletion mutants (e.g., fragments) of the polypeptide in question.Immunogenic portions may also be identified by computer algorithms. Some such algorithms, like EpiMatrix (produced by EpiVax), use a computational matrix approach. Other computational tools for identifying antigenic epitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted by Dana Farber Cancer Institute on the world wide web at immunax.dfci.harvard.edu / PEPVAC), MHCPred (whichuses a partial least squares approach and is hosted by The Jenner Institute on the world wide web at www.jenner. ac.uk / MHCPred), and Immune Epitope Database algorithms on the World Wide Web at tools.immuneepitope.org. An antigenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by algorithm (for example, the SYFPEITHI algorithm found at www.syfpeithi.de).Immunogenic and Vaccine Compositions

[0083] The present disclosure also provides immunogenic compositions (e.g, vaccine compositions) of, or comprising, one or more fusion proteins described herein. In some embodiments, the immunogenic composition comprises one or more fusion proteins with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a fusion protein listed in Table 2. In some embodiments, the immunogenic composition comprises a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 14, 17-26. In some embodiments, the immunogenic composition comprises a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 23 or 24 or SEQ ID NO: 25. In some embodiments, the immunogenic composition comprises a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to RVB (SEQ ID NO: 23) or RVB-His (SEQ ID NO: 25).

[0084] In some embodiments, an immunogenic composition may also comprise portions of fusion proteins described herein, for example internal deletion mutants, truncation mutants, and fragments. In some embodiments, the portions of said fusion proteins are immunogenic. The immunogenicity of a portion of a fusion protein is readily determined using the same assays that are used to determine immunogenicity of the full-length fusion protein. In some embodiments, the portion of the fusion protein has substantially the same immunogenicity as the full-length fusion protein. In some embodiments, the immunogenicity is no less than 10%, 20%, 30%, 40%, or 50% that of the fusion proteins of Table 2.RVB fusion proteins for use in multi-component Immunogenic and Vaccine Compositions

[0085] In some embodiments, an immunogenic composition described herein (e.g., a vaccine composition) comprises, or consist essentially of one or more fusion proteins as described herein. In some embodiments, the fusion protein, e.g., for example but not limited to RVB and RVB-His are present in an immunogenic composition and are not associated with a biotinylated polysaccharide. In some embodiments, an immunogenic composition described herein (e.g., a vaccine composition) includes a fusion protein as described herein and additionally one or more, or two or more vaccine components to form a multi-component immunogenic composition. In some embodiments, the additional vaccine components can be, e.g., additional antigens, including but not limited to Pseudomonas aeruginosaantigens or .S'. pneumoniae antigens. An exemplary .S'. pneumoniae antigen is disclosed in US patent No., 11,576,958 and US application US2023 / 0089151, each of which is incorporated herein in its entirety by reference. In some instances, a known Pseudomonas aeruginosa antigen is an antibody target. In some instances, a known Pseudomonas aeruginosa antigens is a polysaccharide. In some instances, a known Pseudomonas aeruginosa antigen protect from Pseudomonas aeruginosa colonization, or from Pseudomonas aeruginosa-m uccA sepsis, pneumonia, meningitis, otitis media, sinusitis, or infection of other sites or organs by Pseudomonas aeruginosa.

[0086] In some embodiments, an immunogenic composition (e.g , a vaccine composition) contains one or more fusion proteins described herein in combination with one or more polypeptides from Table 1, or antigenic fragments or variants thereof, in a mixture. In some embodiments, the mixture contains both full-length polypeptides and fragments resulting from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g. , the baculovirus expression system), or a mammalian cell line (e.g. , human or Chinese Hamster Ovary).

[0087] In some embodiments, an immunogenic composition contains one or more fusion proteins of any of SEQ ID NOs: 14, 17-26 in the absence of any other antigens. In some embodiments, an immunogenic composition contains a fusion protein of SEQ ID NO:23 (RVB) or SEQ ID NO: 24 or SEQ ID NO: 25 (RVB-His) in the absence of any other antigens. In some embodiments, an immunogenic composition contains one or more fusion proteins of any of SEQ ID NOs: 14, 17-26 in combination with one or more additional proteins of any of SEQ ID NOs: 1-6, in the absence of other antigens. In some embodiments, an immunogenic composition contains a fusion protein of SEQ ID NO:23 or SEQ ID NO: 24 or SEQ ID NO: 25 in combination with one or more additional proteins of any of SEQ ID NOs: 1-6, in the absence of any other antigens.

[0088] In some embodiments, fusion proteins as described herein are useful for combining, or mixing with, additional components to form a multi-component immunogenic composition. For example, but not limited to, a fusion protein as disclosed herein, e.g., a fusion comprising SEQ ID NO: 23 or SEQ ID NO: 25 or a functional fragment thereof, may be present in a multi-component complex comprising a biotinylated polysaccharide, for example, a .S', pneumoniae polysaccharides. The .S', pneumoniae polysaccharides may be, for example, as described in U.S. Pat. No. 5,623,057, U.S. Pat. No. 5,371,197, or PCT / US2011 / 023526. The non-covalent complexes may be, for example, those of the Multiple Antigen Presenting System (MAPS), as described in PCT / US2012 / 037412, PCT / US2012 / 037541, and Zhang et al, 2013. S. pneumoniae polysaccharides are exemplary polysaccharides as disclosed herein in the Examples, however it is envisioned that a fusion protein disclosed herein, e.g., but not limited to, a fusion protein comprising SEQ ID NO: 23 or SEQ ID NO: 25 or a functional fragment thereof, may be present in a multi-component complex comprising a biotinylated polysaccharide from other bacteria, including but not limited to a polysaccharide from Pseudomonas aeruginosa. In some embodiments, a fusion protein as disclosed herein can be produced and expressed according to the methods disclosed herein and combinedwith a biotinylated Pseudomonas aeruginosa polysaccharide. In some embodiments, the polysaccharide is a purified biotinylated and lipidated Psi Pseudomonas aeruginosa oligosaccharide.

[0089] In some embodiments, a fusion protein as described herein is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioavailability, or immunogenicity of the fusion protein. Molecules that may be covalently bound to the fusion protein include a carbohydrate, biotin, polyethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, ranging from molecular weights of below 300 g / mol to over 10,000,000 g / mol. PEG chains can be linear, branched, or with comb or star geometries. In some embodiments, the fusion protein is covalently bound to a moeity that stimulates the immune system. An example of such a moeity is a lipid moeity. In some instances, lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innate immune system. In some embodiments the Rhizavidin of the fusion protein is lipidated (i.e., a lipidated-Rhaviadin in a RVB fusion protein). Methods for lapidating Rhizavidin are disclosed in U.S. Patent 9,499,593, which is incorporated herein in its entirety by reference.

[0090] In some embodiments, a fusion protein and one or more additional components described herein are mixed together using known methods to form a multi-component immunogenic composition. In some embodiments, a fusion protein and one or more additional components described herein are nanoencapsulated using known methods. In some embodiments, a fusion protein and one or more additional components described herein are molded into nano- or micro- particles using known methods. In some embodiments, a fusion protein and one or more additional components described herein are conjugated through a covalent bond using known methods to form a multi-component immunogenic composition. In some embodiments, a fusion protein and one or more additional components described herein are joined non-covalently using known methods to form a multi- component immunogenic composition. Additional methods of combining a fusion protein and one or more additional components are described in, e.g., PCT / US20I2 / 374I2 and PCT / US2009 / 44956.

[0091] In some embodiments, an immunogenic composition comprising a fusion protein disclosed herein is a multi-component immunogenic composition comprising one or more additional components described herein, e.g., OprF / I as disclosed in the Examples.

[0092] A fusion protein as disclosed herein can be formulated into a multi-component vaccine or immunogenic composition by combining one or more fusion proteins described herein with one or more additional antigens, or carriers and / or other optional components by any available means including, for example, conventional mixing, granulating, dissolving, lyophilizing, or similar processes. In some embodiments, the multi-component vaccine or immunogenic composition contains both full-length fusion polypeptides as disclosed herein, or fragments resulting from processing, or partial processing, or removal of signal sequences or tags (e.g., His-tags) by an expression host, e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian cell line (e.g., human or Chinese Hamster Ovary). Insome embodiment, the a multi-component vaccine or immunogenic composition comprises a fusion polypeptides as disclosed herein, where the His-tag remains present on the fusion protein following expression from an expression host.Nucleic Acid-based Immunogenic Compositions and Vaccines

[0093] The present disclosure also provides immunogenic compositions (e.g., vaccine compositions) of, or comprising, one or more nucleic acids encoding a fusion protein as described herein. In some embodiments, the immunogenic composition comprises one or more nucleic acids encoding fusion proteins with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a fusion protein listed in Table 2. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 14, 17-26. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to RVB.

[0094] In some embodiments, the immunogenic composition comprises one or more nucleic acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 12, 27-36. In some embodiments, the immunogenic composition comprises a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO:33. In all cases, due to degeneracy in the genetic code, other DNA sequences (including multiple codon- optimized sequences) could encode such fusion proteins. In some embodiments, these nucleic acids are expressed in the immunized individual, resulting in production of the encoded Pseudomonas aeruginosa fusion proteins, and the Pseudomonas aeruginosa fusion proteins so produced have an immunostimulatory or immunoprotective effect in the immunized individual.

[0095] Such a nucleic acid-containing immunostimulatory composition may comprise, for example, an origin of replication, and / or a promoter that drives expression of one or more nucleic acids encoding one or more fusion proteins of SEQ ID NOs: 13, 27-36. Such a composition may also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more fusion proteins of SEQ ID NOs: 14, 17-26, and a polyadenylation / transcriptional termination sequence. In some instances, the nucleic acid is DNA. In some instances, the nucleic acid is RNA.Uses of Immunogenic and Vaccine Compositions

[0096] In some embodiments, an immunogenic composition or vaccine that includes one or more fusion proteins described herein is characterized in that one or more of the opsonization potential or immune responses to one or more fusion proteins is increased relative to a pre-determined level, as measured by ELISA and / or by a functional antibody assay. In some embodiments, one or more of the opsonization potential or immune response to the one or more fusion proteins is increased by at least 30% or more, including, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, relative to a predetermined level, as measured by ELISA and / or by a functional antibody assay. In some embodiments, one or more of the opsonization potential or immune responses to the one or more fusion proteins is increased at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold relative to a pre-determined level, as measured by ELISA and / or by a functional antibody assay. In some embodiments, the predetermined level is a pre-immune level (e.g., a level observed when a subject is not immunized, or is immunized in the absence of one or more fusion proteins described herein).

[0097] In some embodiments, an immunogenic composition or vaccine that includes one or more fusion proteins described herein, upon administration to a subject, induces an immune response against Pseudomonas aeruginosa. In some embodiments, the immunogenic composition or vaccine, upon administration to a subject, induces an immune response against one or more serotypes of Pseudomonas aeruginosa. In some embodiments, the immunogenic composition or vaccine, upon administration to a subject, induces a protective immune response against one or more serotypes of Pseudomonas aeruginosa. In some embodiments, the immune response is an antibody or B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including Thl, Th2, or Thl7 response, or a CD8+ T cell response, or a CD4+ and a CD8+ T cell response, or a CD4- / CD8- T cell response. In some embodiments, the immune response is an antibody or B cell response and a T cell response. In some embodiments, the immune response is an antibody or B cell response, a T cell response, and an innate immune response.

[0098] In some embodiments, an immunogenic composition or vaccine that includes one or more fusion proteins described herein may be used for prophylactic and / or therapeutic treatment of Pseudomonas aeruginosa. Accordingly, the present disclosure provides a method for immunizing a subject suffering from or susceptible to Pseudomonas aeruginosa infection, comprising administering an immunologically effective amount of any immunogenic composition or vaccine that includes one or more fusion proteins described herein. The subject receiving the immunization may be a male or a female, and may be an infant, child, adolescent, or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal.

[0099] In some embodiments, upon administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein treats or prevents infection by Pseudomonasaeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents an infection caused by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents bacteremia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents sepsis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents organ damage due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents meningitis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents pneumonia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents otitis media due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine treats or prevents sinusitis due to infection by Pseudomonas aeruginosa.[000100] In some embodiments, upon administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein inhibits or reduces the rate of occurrence of infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of Invasive Pseudomonas aeruginosa Disease (IPD) due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of bacteremia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of sepsis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of organ damage due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of meningitis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of pneumonia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of otitis media due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits or reduces the rate of occurrence of sinusitis due to infection by Pseudomonas aeruginosa.[000101] In some embodiments, upon administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein reduces the severity of infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of Invasive Pseudomonas aeruginosa Disease (IPD) due to infection byPseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of bacteremia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of sepsis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of organ damage due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of meningitis due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of pneumonia due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of otitis media due to infection by Pseudomonas aeruginosa. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine reduces the severity of sinusitis due to infection by Pseudomonas aeruginosa.[000102] In some embodiments, upon administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein inhibits transmission of Pseudomonas aeruginosa from the subject to another subject. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits colonization by Pseudomonas aeruginosa in the subject. In some embodiments, upon administration to a subject, the immunogenic composition or vaccine inhibits colonization by Pseudomonas aeruginosa in the nasopharynx of the subject.[000103] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein, upon administration to a subject, induces an immune response against Pseudomonas aeruginosa in the subject at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine, upon administration to a subject, induces an immune response against one or more serotypes of Pseudomonas aeruginosa at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.[000104] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein, upon administration to a subject, induces an immune response that can help protect against the establishment of Pseudomonas aeruginosa at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine protects against colonization at a level greater than a control composition. In some embodiments, the immunogenic composition or vaccine inhibits infection by Pseudomonas aeruginosa in a non-colonized or uninfected subject at a level greaterthan a control composition. In some embodiments, the immunogenic composition or vaccine reduces the duration of colonization by Pseudomonas aeruginosa in a subject who is already colonized at a level greater than a control composition. In some embodiments, the level greater is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the control composition.Antibody Compositions[000105] Some embodiments provide for an antibody composition comprising antibodies raised in a mammal immunized with an immunogenic composition or vaccine comprising a fusion protein described herein. In some embodiments, an antibody comprises at least one antibody selected from the group consisting of monoclonal Abs (mAbs) and anti -idiotype antibodies. In some embodiments, an antibody composition comprises an isolated gamma globulin fraction. In some embodiments, an antibody composition comprises polyclonal antibodies. In some embodiments, the antibody composition is administered to a subject.Vaccine Formulations[000106] Optimal amounts of components for a particular vaccine comprising a fusion protein described herein can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial immunization, subjects can receive one or several booster immunizations adequately spaced in time.[000107] The immunogenic composition or vaccine comprising a fusion protein described herein, and / or preparations thereof, may be formulated in a unit dosage form for ease of administration and uniformity of dosage. The specific therapeutically effective dose level for any particular subject or organism may depend upon a variety of factors including the severity or degree of risk of infection; the activity of the specific vaccine or vaccine composition employed; other characteristics of the specific vaccine or vaccine composition employed; the age, body weight, general health, sex of the subject, diet of the subject, pharmacokinetic condition of the subject, the time of administration (e.g. , with regard to other activities of the subject such as eating, sleeping, receiving other medicines including other vaccine doses, etc.), route of administration, rate of excretion of the specific vaccine or vaccine composition employed; vaccines used in combination or coincidental with the vaccine composition employed; and like factors well known in the medical arts.[000108] An immunogenic composition or vaccine comprising a fusion protein described herein for use in accordance with the present disclosure may be formulated into compositions (e.g., pharmaceutical compositions) according to known techniques. Vaccine preparation is generally described in Vaccine Design (Powell and Newman, 1995). For example, an immunologically amount of a vaccine product canbe formulated together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials.[000109] In general, pharmaceutically acceptable carriers) include solvents, dispersion media, and the like, which are compatible with pharmaceutical administration. For example, materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose, dextrose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; polyols such as glycerol, propylene glycol, and liquid polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator (Martin, 1975).[000110] Vaccines may be formulated by combining one or more fusion proteins described herein with carriers and / or other optional components by any available means including, for example, conventional mixing, granulating, dissolving, lyophilizing, or similar processes.[000111] Vaccines comprising one or more fusion proteins described herein may be lyophilized up until they are about to be used, at which point they are extemporaneously reconstituted with diluent. In some embodiments, vaccine components or compositions are lyophilized in the presence of one or more other components (e.g., adjuvants), and are extemporaneously reconstituted with saline solution. Alternatively, individual components, or sets of components may be separately lyophilized and / or stored (e.g. , in a vaccination kit), the components being reconstituted and either mixed prior to use or administered separately to the subject.[000112] Lyophilization can produce a more stable composition (for instance by preventing or reducing breakdown of polysaccharide antigens). Lyophilizing of vaccines or vaccine components is well known in the art. Typically, a liquid vaccine or vaccine component is freeze dried, often in the presence of an anti-caking agent (such as, for example, sugars such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10-200 mg / ml. Lyophilization typically occurs over a series of steps, for instance a cycle starting at -69° C, gradually adjusting to -24°C over 3 h, then retaining this temperature for 18 h, then gradually adjusting to -16°C over 1 h, then retaining this temperature for 6 h, then gradually adjusting to +34°C over 3 h, and finally retaining this temperature over 9 h.[000113] In some embodiments, a vaccine comprising a fusion protein described herein is a liquid. In some embodiments the liquid is a reconstituted lyophylate. In some embodiments a vaccine has a pH of about 5, about 6, about 7, or about 8. In some embodiments a vaccine has a pH between about 5 andabout 7.5. In some embodiments a vaccine has a pH between 5 and 7.5. In some embodiments a vaccine has a pH between about 5.3 and about 6.3. In some embodiments a vaccine has a pH between 5.3 and 6.3. In some embodiments a vaccine has a pH of about 5.0, about 5. 1, about 5.2, about 5.3, about 5.4, about5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.[000114] Vaccines or vaccine components for use in accordance with the present disclosure may be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or immune -stimulating complexes (ISCOMS).[000115] In certain situations, it may be desirable to prolong the effect or release of a vaccine for use in accordance with the present invention, for example, by slowing the absorption of one or more vaccine components. Such delay of absorption may be accomplished, for example, by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the product then depends upon its rate of dissolution, which in turn, may depend upon size and form. Alternatively, or additionally, delayed absorption may be accomplished by dissolving or suspending one or more vaccine components in an oil vehicle. Injectable depot forms can also be employed to delay absorption. Such depot forms can be prepared by forming microcapsule matrices of one or more vaccine components a biodegradable polymer network. Depending upon the ratio of polymer to vaccine component, and the nature of the particular polymer(s) employed, the rate of release can be controlled.[000116] Examples of biodegradable polymers that can be employed in accordance with the present disclosure include, for example, poly(orthoesters) and poly(anhydrides). One particular exemplary polymer is polylactide -polyglycolide.[000117] Depot injectable formulations may also be prepared by entrapping the product in liposomes or microemulsions, which are compatible with body tissues.[000118] Polymeric delivery systems can also be employed in non-depot formulations including, for example, oral formulations. For example, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, etc., can be used in oral formulations. Polysaccharide antigens or conjugates may be formulated with such polymers, for example to prepare particles, microparticles, extrudates, solid dispersions, admixtures, or other combinations in order to facilitate preparation of useful formulations (e.g , oral).[000119] Vaccines comprising one or more fusion proteins described herein for use in accordance with the present disclosure include immunogenic compositions, and may additionally include one or more additional active agents (i. e. , agents that exert a biological effect - not inert ingredients). For example, it is common in vaccine preparation to include one or more adjuvants. It will be appreciated that such additional agents may be formulated together with one or more other vaccine components, or may be maintained separately and combined at or near the time of administration. In some embodiments, suchadditional components may be administered separately from some or all of the other vaccine components, within an appropriate time window for the relevant effect to be achieved.Adjuvants[000120] The vaccine formulations and immunogenic compositions comprising a fusion protein described herein may include an adjuvant. Adjuvants, generally, are agents that enhance the immune response to an antigen. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g, Singh et al, 2003). In most vaccine formulations, the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen. Vaccine delivery systems are often particulate formulations, e.g. , emulsions, microparticles, immune -stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and / or matrices, and liposomes. In contrast, immunostimulatory adjuvants are sometimes from or derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), or CpG-containing DNA, which activate cells of the innate immune system.[000121] Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. Non-limiting examples of organic adjuvants include cholera toxin / toxoids, other enterotoxins / toxoids or labile toxins / toxoids of Gram-negative bacteria, interleukins (e.g. , IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g. , gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M- CSF), and tumor necrosis factor (TNF).[000122] Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the generation, proliferation, or activation of Thl cells or Th2 cells. In other embodiments, the adjuvant induces the generation, proliferation, or activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.[000123] In some embodiments, the adjuvant induces the generation, proliferation, or activation of Thl7 cells. The adjuvant may promote the CD4+ or CD8+ T cells to secrete IL-17. In some embodiments, an adjuvant that induces the generation, proliferation, or activation of Th 17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL- 17 levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce Thl7 generation, proliferation, or activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the generation, proliferation, or activation ofTh 17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL- 17 than the cells of population (2). IL- 17 may be measured, for example, by ELISA or ELI SPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E coll, or ETEC), activate a Th 17 response. Thus, in some embodiments, the adjuvant is a toxin or toxoid. Cholera toxin was successfully used in a mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1. One form of labile toxin is produced by Intercell. Mutant derivates of labile toxin (toxoids) that are active as adjuvants but significantly less toxic can be used as well. Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al, 1995) and LTK63 (Williams et al, 2004), LT-G192 (Douce et al, 1999), and LTR72 (Giuliani et al, 1998).[000124] In some embodiments, the adjuvant comprises a VLP (vims-like particle). One such adjuvant platform, Alphavirus replicons, induces the activation of Th 17 cells using alphavirus and is produced by Alphavax. In some embodiments of the Alphavirus replicon system, alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL- 17 or a cytokine that stimulates IL- 17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, a vaccine formulation is administered to a subject in combination with a nucleic acid encoding a cytokine.[000125] Certain classes of adjuvants activate toll -like receptors (TLRs) in order to activate a Th 17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram-negative bacteria, such as .S', minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans et al, 2003. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka et al, 2007). Various TLR agonists (including TLR-4 agonists) have been produced and / or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).[000126] In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponinextract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.[000127] In some embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.[000128] Adjuvants may be covalently or non-covalently bound to antigens. In some embodiments, the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen- presenting cells (APCs). In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (e.g, see Wu et al, 2005).[000129] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein is formulated and / or administered in combination with an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, and phosphate aluminum hydroxide. In some embodiments, the adjuvant comprises aluminum phosphate. In some embodiments, the adjuvant is aluminum phosphate.[000130] Typically, the same adjuvant or mixture of adjuvants is present in each dose of a vaccine. Optionally, however, an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (z.e., booster shots). Alternatively, a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human subjects, non-human animals, or both.[000131] Vaccines for use in accordance with the present disclosure may include, or be administered concurrently with, antimicrobial therapy. For example, such vaccines may include or be administered with one or more agents that kills or retards growth of a pathogen Such agents include, for example, penicillin, vancomycin, erythromycin, azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.[000132] Alternatively or additionally, vaccines for use in accordance with the present invention may include, or be administered with, one or more other vaccines or therapies. For example, one or more non- Pseudomonas aeruginosa antigens may be included in or administered with the vaccines.Additional Components and Excipients in a vaccine composition comprising a fusion protein disclosed herein[000133] In addition to the fusion proteins described herein and the adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components.[000134] In some embodiments, the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.[000135] In some embodiments, the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.[000136] In some embodiments, the vaccine formulation or immunogenic composition includes one or more surfactants, for example, but not limited to, polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20), Polyethylene glycol p-(l, l,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100), and 4- (l,l,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or nonionic.[000137] In some embodiments, the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.[000138] In some embodiments, a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multidose vaccine vials, and is less often needed in single-dose vaccine vials. In some embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and / or sorbic acid.Methods of Administration[000139] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein is administered to a subject at risk of developing Pseudomonas aeruginosa disease, e.g. an infant, a toddler, a juvenile, or an older adult. In some embodiments, the immunogenic composition or vaccine is administered to a subject at elevated risk of developing Pseudomonas aeruginosa disease, e.g. , immunocompromised subjects, subjects having sickle cell disease or other hemoglobinopathies, congenital or acquired asplenia, splenic dysfunction, chronic renal failure or nephrotic syndrome, diseases associated with treatment with immunosuppressive drugs or radiation therapy, including malignant neoplasm, leukemia, lymphomas, Hodgkin's disease, or solid organ transplantation, congenital or acquired immunodeficiency, HIV infection, cerebrospinal fluid leaks, cochlear implant(s), chronic heart disease, chronic lung disease, diabetes mellitus, alcoholism, chronic liver disease, cigarette smoking, asthma,generalized malignancy, multiple myeloma, or solid organ transplantation. It will be appreciated that a subject can be considered at risk for developing a disease without having been diagnosed with any symptoms of the disease. For example, if the subject is known to have been, or to be intended to be, in situations with relatively high risk of infection, that subject will be considered at risk for developing the disease.[000140] Any effective route of administration may be utilized such as, for example, oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, transdermal, intradermal, rectal, vaginal, topical, ocular, pulmonary, or by contact application. In some embodiments, the immunogenic composition or vaccine may be injected (e.g., via intramuscular, intraperitoneal, intradermal and / or subcutaneous routes); or delivered via the mucosa (e.g , to the oral / alimentary, respiratory, and / or genitourinary tracts). Intranasal administration may be particularly useful in some contexts. In some embodiments, it may be desirable to administer different doses of the immunogenic composition or vaccine by different routes; in some embodiments, it may be desirable to administer different components of one dose via different routes.[000141] In some embodiments, pharmaceutical compositions (e.g, immunogenic compositions or vaccines) are administered intradermally. Conventional technique of intradermal injection, the "Mantoux procedure", comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26-31 gauge) facing upwards the needle is inserted at an angle of between 10-15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced while providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.[000142] Devices that are specifically designed to administer liquid agents into or across the skin have been described, for example the devices described in WO 99 / 34850 and EP 1092444, also the jet injection devices described for example in WO 01 / 13977; US Patent No. 5,480,381, US Patent No. 5,599,302, US Patent No. 5,334,144, US Patent No. 5,993,412, US Patent No. 5,649,912, US Patent No. 5,569,189, US Patent No. 5,704,911, US Patent No. 5,383,851, US Patent No. 5,893,397, US Patent No. 5,466,220, US Patent No. 5,339,163, US Patent No. 5,312,335, US Patent No. 5,503,627, US Patent No. 5,064,413, US Patent No. 5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US PatentNo. 4,941,880, US PatentNo. 4,940,460, WO 97 / 37705 and WO 97 / 13537. Other methods of intradermal administration of the immunogenic compositions or vaccines may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99 / 27961), or transdermal patches (W O 97 / 48440; WO 98 / 28037); or applied to the surface of the skin (transdermal or transcutaneous delivery WO 98 / 20734; WO 98 / 28037).[000143] As described above, pharmaceutical compositions (e.g , immunogenic compositions or vaccines) may be administered as a single dose or as multiple doses. It will be appreciated that an administration is a single “dose” so long as all relevant components are administered to a subject within awindow of time; it is not necessary that every component be present in a single composition. For example, administration of two different immunogenic compositions or vaccines, within a period of less than 24 h, is considered a single dose. To give but one example, immunogenic compositions or vaccines having different antigenic components may be administered in separate compositions, but as part of a single dose. As noted above, such separate compositions may be administered via different routes or via the same route. Alternatively or additionally, in embodiments wherein an immunogenic composition or vaccine is combined with additional types of active agents, the immunogenic composition or vaccine may be administered via one route, and a second active agent may be administered by the same route or by a different route.[000144] Pharmaceutical compositions (e.g, immunogenic compositions or vaccines) are administered in such amounts and for such time as is necessary to achieve a desired result. In some embodiments of the present invention, the immunogenic composition or vaccine comprises an immunologically effective amount of at least immunogenic composition. The exact amount required to achieve an immunologically effective amount may vary, depending on the immunogenic composition, and from subject to subject, depending on the species, age, and general condition of the subject, the stage of the disease, the particular pharmaceutical mixture, its mode of administration, and the like.[000145] The amount of fusion protein(s) described hereinin each pharmaceutical composition (e.g., immunogenic composition or vaccine) dose is selected to allow the vaccine, when administered as described herein, to induce an appropriate immunoprotective response without significant adverse side effects.[000146] In some embodiments, a pharmaceutical composition comprising a fusion protein described herein induces a Thl and / or Thl7 cell response upon administration to a subject. In some embodiments, the pharmaceutical composition induces an opsonic / bactericidal response against Pseudomonas aeruginosa upon administration to a subject In some embodiments, the pharmaceutical composition comprising a fusion protein disclosed herein reduces rate of transmission and / or colonization of the mucosal surfaces by Streptococcus pneumoniae upon administration to a subject. In some embodiments, the pharmaceutical composition reduces rate of transmission and / or colonization of the nasopharynx or the lungs by Pseudomonas aeruginosa upon transmission.[000147] Some embodiments provide for a method of immunizing a subject against Pseudomonas aeruginosa infection comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising a fusion protein described herein. Some embodiments provide for a method of immunizing a subject against Pseudomonas aeruginosa infection comprising administering to the subject an immunologically effective amount of a vaccine composition comprising a fusion protein described herein. Some embodiments provide for a method of immunizing a subject against Pseudomonas aeruginosa infection comprising administering to the subject an immunologically effective amount of a pharmaceutical composition comprising a fusion protein described herein.Combination Prophylaxis or Combination Therapy[000148] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein may be administered in combination with another agent. In some embodiments, the agent is or comprises PCV13. In some embodiments, the agent is or comprises PPSV23. In some embodiments, the agent is or comprises an antibiotic.Dosing[000149] In some embodiments, administration of an immunogenic composition or vaccine comprising a fusion protein described herein may involve the delivery of a single dose. In some embodiments, administration may involve an initial dose followed by one or several additional immunization doses, adequately spaced. An immunization schedule is a program for the administration of one or more specified doses of one or more specified Pa vaccines, by one or more specified routes of administration, at one or more specified ages of a subject.[000150] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to an infant subject. In some embodiments, the infant subject is 18 months old or younger. In some embodiments, the infant subject is 12 months old or younger. In some embodiments, the infant subject has previously received one or more doses of a conjugated Pseudomonas aeruginosa polysaccharide vaccine; in other embodiments, the infant subject is naive to Pa vaccines. In some embodiments, the infant subject has previously been infected with, or exposed to infection by Pseudomonas aeruginosa.[000151] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to a toddler subject. In some embodiments, the toddler subject is 5 years old or younger. In some embodiments, the toddler subject is 4 years old or younger. In some embodiments, the toddler subject has previously received one or more doses of a conjugated Pseudomonas aeruginosa polysaccharide vaccine; in other embodiments, the toddler subject is naive to Pa vaccines. In some embodiments, the toddler subject has previously been infected with, or exposed to infection by Pseudomonas aeruginosa.[000152] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to a juvenile subject. In some embodiments, the juvenile subject is 18 years old or younger. In some embodiments, the juvenile subject is 15 years old or younger. In some embodiments, the juvenile subject has previously received one or more doses of a conjugated Pseudomonas aeruginosa polysaccharide vaccine; in other embodiments, the juvenile subject is naive to Pa vaccines. In some embodiments, the juvenile subject has previously been infected with, or exposed to infection by Pseudomonas aeruginosa.[000153] The present disclosure provides immunization methods that involve administering at least one dose of a vaccine to an adult subject. In some embodiments, the adult subject is older than about 50years of age. In some embodiments, the adult subject is older than about 65 years of age. In some embodiments, the adult subject has previously received one or more doses of a conjugated Pseudomonas aeruginosa polysaccharide vaccine; in other embodiments, the adult subject is naive to Pa vaccines. In some embodiments, the adult subject has previously been infected with, or exposed to infection by Pseudomonas aeruginosa. In some embodiments, the subject has cystic fibrosis (CF). In some embodiments, the subject has previously had a lung infection.[000154] Immunization schedules of the present disclosure are provided to induce an immune response (e.g., an immunoprotective response) in a subject sufficient to reduce at least one measure selected from the group consisting of incidence, prevalence, frequency, and / or severity of at least one infection, disease, or disorder, and / or at least one surrogate marker of the infection, disease, or disorder, in a population and / or subpopulation of the subject(s). A supplemental immunization schedule is one which has this effect relative to the standard schedule which it supplements. A supplemental schedule may call for additional administrations and / or supra-immunogenic doses of the immunogenic compositions or vaccines disclosed herein, found in the standard schedule, or for the administration of immunogenic compositions or vaccines not part of the standard schedule. A full immunization schedule of the present invention may comprise both a standard schedule and a supplemental schedule. Exemplary sample immunization schedules are provided for illustrative purposes. Detailed descriptions of methods to assess immunogenic response discussed herein allow one to develop alterations to the sample immunization schedules without undue experimentation.[000155] In one embodiment of the present disclosure, a first administration of a Pa vaccine usually occurs when a subject is more than about 2 weeks old, more than about 5 weeks old, more than about 1 year old, more than about 2 years old, more than about 15 years old, or more than about 18 years old. [000156] In one embodiment of the present disclosure, a first administration of a Pa vaccine usually occurs when a subject is more than about 50 years old, more than about 55 years old, more than about 60 years old, more than about 65 years old, or more than about 70 years old.[000157] In some embodiments of the disclosure, a single administration of vaccine is employed. It is possible that the purposes of the present invention can be served with a single administration, especially when one or more utilized vaccine polypeptides, polysaccharide(s) and / or conjugate(s) or combinations thereof is / are strong, and in such a situation a single dose schedule is sufficient to induce a lasting immune-protective response.[000158] In some embodiments, it is desirable to administer two or more doses of vaccine, for greater immune-protective efficacy and coverage. Thus, in some embodiments, a number of doses is at least two, at least three or more doses. There is no set maximum number of doses, however it is good clinical practice not to immunize more often than necessary to achieve the desired effect.[000159] Without being bound by theory, a first dose of vaccine administered according to the disclosure may be considered a “priming” dose. In some embodiments, more than one dose is included in an immunization schedule. In such a scenario, a subsequent dose may be considered a “boosting” dose. [000160] A priming dose may be administered to a naive subject (a subject who has never previously received a conjugated polysaccharide vaccine). In some embodiments, a priming dose may be administered to a subject who has previously received conjugated polysaccharide vaccine at least five or more years previous to administration of an initial vaccine dose according to the invention. In other embodiments, a priming dose may be administered to a subject who has previously received a conjugated polysaccharide vaccine at least twenty or more years previous to administration of a priming vaccine according to the invention.[000161] When an immunization schedule calls for two or more separate doses, the interval between doses is considered. The interval between two successive doses may be the same throughout an immunization schedule, or it may change as the subject ages. In immunization schedules of the present invention, once a first vaccine dose has been administered, there is a first interval before administration of a subsequent dose. A first interval is generally at least about 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 12 months, or longer. Where more than one subsequent dose(s) are administered, second (or higher) intervals may be provided between such subsequent doses. In some embodiments, all intervals between subsequent doses are of the same length; in other embodiments, second intervals may vary in length. In some embodiments, the interval between subsequent doses may be at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months or at least about 2 years. In some embodiments, the interval between doses may be up to 3 years, up to about 4 years, or up to about 5 years or 10 years or more. In some embodiments, intervals between subsequent doses may decrease as the subject ages.[000162] It will be appreciated by those skilled in the art that a variety of possible combinations and sub-combinations of the various conditions of timing of the first administration, shortest interval, largest interval and total number of administrations (in absolute terms, or within a stated period) exist, and all of these combinations and sub-combinations should be considered to be within the inventor's contemplation though not explicitly enumerated here.Assays for Determinins Immune Response[000163] In some embodiments, a method of assessing the immunogenicity of a pharmaceutical composition, immunogenic composition, or vaccine comprising a fusion protein described herein comprises evaluating, measuring, and / or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, Thl / Thl7 cell response, cytokine level measurement and functional antibody levels as measured by OPK, serum bactericidal killing (SBA), agglutination, motility,cytotoxicity, or adherence; and in vivo assays in animal models of Pseudomonas aeruginosa disease (e.g pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization). Parameters of in vivo assays include bacterial clearance from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with the Pseudomonas aeruginosa pathogens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition.[000164] In some embodiments, a method of assessing the potency of a pharmaceutical composition, immunogenic composition, or vaccine comprising a fusion protein described herein comprises evaluating, measuring, and / or comparing an immune response using one or more in vitro bioassays, including B cell and T cell responses such as antibody levels by ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, Thl / Thl7 cell response, cytokine level measurement and functional antibody levels as measured by OPK, serum bactericidal killing (SBA), internalization, activity neutralization, agglutination, motility, cytotoxicity, or adherence; and in vivo assays in animal models of Pseudomonas aeruginosa disease (e.g. pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization). Parameters include bacterial clearance or reduction from mucosal surfaces or bloodstream, reduction or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of colonization of the nasopharynx, reduction of mortality, and passive and active protection following challenge with the Pseudomonas aeruginosa pathogens that are the targets of the immunogenic composition. In some embodiments, the immune response is compared to a control composition.[000165] Generally speaking, it may be desirable to assess humoral responses, cellular responses, and / or interactions between the two. Where humoral responses are being assessed, antibody titers and / or types (e.g, total IgG, IgGl, IgG2, IgM, IgA, etc.) to specific pathogen antigens (e.g., polypeptides or polysaccharides, either serotype-specific or conserved across two or more serotypes) may be determined, for example before and / or after administration of an initial or a boosting dose of vaccine (and / or as compared with antibody levels in the absence of antigenic stimulation). Cellular responses may be assessed by monitoring reactions such as delayed type hypersensitivity responses, etc. to the antigens. Cellular responses can also be measured directly by evaluating the response of peripheral blood mononuclear cells (PBMCs) monocytes to stimulation with the antigens of interest. Precursor and memory B cell populations may be assessed in enzyme-linked immunospot (ELISpot) assays directed against specific pathogen antigens.[000166] The RIA method detects specific antibodies through incubation of sera with radio-labeled polysaccharides or polypeptides in suspension (e.g., Schiffiman et al, 1980). The antigen-antibody complexes are then precipitated with ammonium sulfate and the radiolabeled pellets assayed for counts per minute (cpm).[000167] In the ELISA detection method, specific antibodies from the sera of vaccinated subjects are quantitated by incubation with antigens (e.g., polypeptides or polysaccharides, either serotype-specific or conserved across two or more serotypes) which have been adsorbed to a solid support (e.g, Koskela and Leinonen (1981); Kojima et al, 1990; Concepcion and Frasch, 2001). The bound antibody is detected using enzyme -conjugated secondary detection antibodies. The ELISA also allows isotyping and subclassing of the immune response (z.e., IgM vs. IgG or IgGl vs. IgG2) by using isotype- or subclassspecific secondary antibodies and can be adapted to evaluate the avidity of the antibodies (Anttila et al, 1998; Romero-Steiner et al, 2005). Multiplex assays (e.g., Luminex) facilitate simultaneous detection of antibodies to multiple antigens. Antigens are conjugated to spectrally distinct microspheres that are mixed and incubated with serum. The antibodies bound to the antigens on the coated microspheres are detected using a secondary antibody (e.g , R-Phycoerythrin-conjugated goat anti-human IgG).[000168] An approach for assessing functional antibody in serum is the opsonophagocytic assay (OPA) which quantitates only the antibodies that can opsonize the bacteria, leading to ingestion and killing of the bacteria. The standard assay utilizes a human phagocytic effector cell, a source of complement, bacteria, and diluted sera. The assay readout is the serum endpoint titer at which there is >50% killing compared to bacteria incubated with complement and human cells alone (Romero-Steiner et al, 1997). This killing OPA can also be multiplexed by utilizing target strains of pathogen that carry different antibiotic resistance markers (Kim et al, 2003). Another type of multiplex opsonic assay is a nonkilling assay in which the uptake by phagocytic effector cells of fluorescent stained encapsulated pathogen or fluorescent microspheres conjugated with antigens from a target pathogen in the presence of diluted sera plus a complement source is evaluated by FC (Martinez et al, 1999). Opsonic activity of serum antibody plus complement can also be evaluated by measuring the oxidative response of phagocytic human effector cells to ingested pathogen (Munro et al. 1985; Ojo-Amaize et al. 1995).[000169] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by immunogenic compositions or vaccines comprising a fusion protein described herein. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which provides protection against pneumonia, bacteremia, colonization of organs or tissues, or mortality is determined (Stack et al. 1998; Saeland et al. 2000). [000170] In some embodiments, efficacy of immunization may be determined by assaying one or more cytokine levels by stimulating T cells from a subject after immunization. The one or more cytokine levels may be compared to the one or more cytokine levels in the same subject before immunization. Increased levels of the one or more cytokine, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase over pre -immunization cytokine levels, would indicate an increased response to the immunogenic composition or vaccine. In some embodiments, the one or more cytokines are selected from GM-CSP; IL-Ia; IL-lp; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F or other members of the IL-17 family; IL-22; IL-23; IFN-a; IFN- ; IFN-y; MIP-la; MIP-1 ; TGF-P; TNFa,or TNF-p. In a non-limiting example, efficacy of immunization may be determined by assaying IL- 17 levels (particularly IL-17A) by stimulating T cells from a subject after immunization. The IL- 17 levels may be compared to IL-17 levels in the same subject before immunization. Increased IL-17 (e.g. , IL- 17A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the immunogenic composition or vaccine.[000171] In some embodiments, one may assay neutrophils in the presence of T cells or antibodies from the patient for Pseudomonas aeruginosa killing. Increased Pseudomonas aeruginosa killing, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the immunogenic composition or vaccine. For example, one may measure Th 17 cell activation, where increased Th 17 cell activation, such as a 1.5 fold, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the immunogenic composition or vaccine. In another non-limiting example, one may measure Th I cell activation, where increased Thl cell activation, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the immunogenic composition or vaccine. One may also measure levels of an antibody specific to the immunogenic composition or vaccine, where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased efficacy. In some embodiments, two or more of these assays are used. For example, one may measure IL-17 levels and the levels of immunogenic composition- or vaccine-specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of Pseudomonas aeruginosa infection in vaccinated individuals compared to unvaccinated individuals.[000172] Immunogenic composition or vaccine efficacy may also be assayed in various model systems such as the mouse challenge model. For instance, BALB / c or C57BL / 6 strains of mice may be used. After administering the test immunogenic composition or vaccine to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of Pseudomonas aeruginosa. In some cases, a challenge dose administered intranasally is sufficient to cause Pseudomonas aeruginosa colonization (especially nasal colonization) in an unvaccinated animal, and in some cases a challenge dose administered via aspiration is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intraperitoneal injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. In some cases, a challenge dose administered via intravenous injection is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals. [000173] Certain in vivo model systems can be used to evaluate the protection afforded by serum antibodies induced by vaccines of the present invention. In such passive protection systems, mice or rats are challenged with the pathogen plus diluted sera, and the endpoint titer of the sera which providesprotection against bacteremia, colonization of organs or tissues, or mortality is determined (Stack et al. 1998; Saeland et al. 2000).Certain Definitions[000174] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.[000175] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.[000176] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g, by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastrical, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g , intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g, by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g, a plurality of doses separated in time) and / or periodic (e.g. , individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g, perfusion) for at least a selected period of time.[000177] Agent: In general, the term “agent”, as used herein, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context willmake clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.[000178] Amino acid: In its broadest sense, the term “amino acid”, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g. , through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N- C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D- amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g. , of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.[000179] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kDa tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kDa each) and two identical light chain polypeptides (about 25 kDa each) that associate with each other into what iscommonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g. , 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes ofFc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in some embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- ormulti- specific antibodies (e.g , Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g, shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g, Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPs1 1): single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g. , attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g, attachment of a glycan, a payload [e.g, a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g, poly-ethylene glycol, etc.]). [000180] Antigen: The term “antigen”, as used herein, refers to (i) an agent that induces an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g. , when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen induces a humoral response (e.g. , including production of antigen-specific antibodies); in some embodiments, an antigen induces a cellular response (e.g. , involving T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen induces a humoral response and a cellular response. In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer (e.g. , other than a nucleic acid or amino acid polymer)), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a polysaccharide. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g. , together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen. In some embodiments, an antigen is a polypeptide or a polysaccharide that, upon administration to a subject, induces a specific and / or clinically relevant immune response to such polypeptide or polysaccharide. In some embodiments, an antigen is selected to induce a specific and / or clinically relevant immune response to such polypeptide or polysaccharide.[000181] Associated with: Two entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one anotherare not covalently linked to one another but are non-covalently associated, for example by means of affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.[000182] Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g, while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).[000183] Carrier protein: As used herein, the term “carrier protein” refers to a protein or peptide that is coupled, complexed, or otherwise associated with a hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g., a polysaccharide) and that induces or improves an immune response to such a coupled, or complexed, or otherwise associated hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g. , a polysaccharide). In some embodiments, such an immune response is or comprises a response to a hapten or less immunogenic antigen that is coupled, complexed, or otherwise associated with such a carrier protein. In some embodiments, such an immune response is or comprises a response to both a carrier protein and a hapten or less immunogenic antigen that is coupled, complexed, or otherwise associated with such a carrier protein. In some embodiments, no significant immune response to a carrier protein itself occurs. In some embodiments, immune response to a carrier protein may be detected; in some such embodiments, immune response to such a carrier protein is strong. In some embodiments, a carrier protein is coupled, complexed, or otherwise associated with one or more other molecules.[000184] Colonization: As used herein, the term “colonization” generally refers to the ability of a microbe to grow at a target site or surface. For example, the term “colonization” refers to the ability of a microbe (e.g., a bacterium) to grow at an anatomical site (e.g., a mucosal membrane, gastrointestinal tract, injury site, organ, etc.) of a host.[000185] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g. , all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may beadministered together in a combination composition, or even in a combination compound (e.g, as part of a single chemical complex or covalent entity).[000186] Derivative: As used herein, the term “derivative”, or grammatical equivalents thereof, refers to a structural analogue of a reference substance. That is, a “derivative” is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. Such a substance would be said to be “derived from” said reference substance. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g. , sharing a plurality of steps with) one that generates the reference substance.[000187] Domain: The term “domain” as used herein refers to a section or portion of an entity. In some embodiments, a “domain” is associated with a particular structural and / or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and / or functional feature. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, a-helix character, p-sheet character, coiled-coil character, random coil character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).[000188] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g. , a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population ( / . e. , with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.[000189] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosingregimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (z.e., is a therapeutic dosing regimen).[000190] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment includes a discrete portion of the whole which discrete portion shares one or more functional characteristics found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment of a polymer, e.g., a polypeptide or polysaccharide, comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g, residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.[000191] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g, DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.[000192] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g, DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g, gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g. , nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.[000193] Improve, increase, inhibit or reduce: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (e.g. , prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.[000194] Multi-component immunogenic composition: As used herein, a “multi-component immunogenic composition” is a composition that comprises an immunologically effective amount of a fusion protein disclosed herein in combination with one or more additional components. In some embodiments, the additional components can be, e.g., other components in immunologically effectiveamount that elicit an immunoprotective response or protective response. In some embodiments, the one or more additional components can be, but are not limited to, other polypeptide antigens from Pa or other pathogens, and / or a polysaccharide antigen. In some embodiments, an additional component that is a polypeptide antigen and / or polysaccharide antigen can be biotinylated, and in some embodiments, a fusion protein as disclosed herein (e.g., RVB or RVB-His) can be non-covalently associated, via rhizavidin with a biotin on the polypeptide antigen and / or polysaccharide antigen to form a complex. Exemplary other additional components include, but are not limited to OprF / I, adjuvants, stabilizers, surfactants, components for intranasal delivery, stabilizers, buffers, surfactants, salts or preservatives.[000195] Immunologically effective amount or immunologically effective dose: As used herein, “immunologically effective amount” or “immunologically effective dose” refers to an amount of an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, which when administered to a subject, either in a single dose or as part of a series of doses, that is sufficient to enhance a subject’s own immune response against a subsequent exposure to a pathogen. In some embodiments, the pathogen is P. aeruginosa. In some embodiments, the immune response is against one or more different serotypes of P. aeruginosa. In some embodiments, the immune response is against two or more different serotypes of P. aeruginosa. In some embodiments, the immune response is against nine or more different serotypes ofP. aeruginosa. In some embodiments, the immune response is against thirteen or more different serotypes of P. aeruginosa. In some embodiments, the immune response is against fifteen or more different serotypes of P. aeruginosa. In some embodiments, the immune response is against twenty-three or more different serotypes of P. aeruginosa. In some embodiments, the immune response is against twenty-four or more different serotypes of P. aeruginosa. An immunologically effective amount may vary based on the subject to be treated, the species of the subject, the degree of immune response desired to induce, etc. In some embodiments, an immunologically effective amount is sufficient for treatment or protection of a subject having or at risk of having disease. In some embodiments, an immunologically effective amount refers to a non-toxic but sufficient amount that can be an amount to treat, attenuate, or prevent infection and / or disease (e.g., bacterial infection, Pseudomonas aeruginosa infection, bacterial colonization, Pseudomonas aeruginosa colonization, complications associated with bacterial infection, complications associated with Pseudomonas aeruginosa infection, etc.) in any subject. In some embodiments, an immunologically effective amount is sufficient to induce an immunoprotective response upon administration to a subject.[000196] Immunoprotective response or protective response: As used herein, “immunoprotective response” or “protective response” refers to an immune response that mediates antigen or immunogen- induced immunological memory. In some embodiments, an immunoprotective response is induced by the administration of a substance, e.g., an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition to a subject. In some embodiments, immunoprotection involves one or more of active immune surveillance, a more rapid and effectiveresponse upon immune activation as compared to a response observed in a naive subject, efficient clearance of the activating agent or pathogen, followed by rapid resolution of inflammation. In some embodiments, an immunoprotective response is an adaptive immune response. In some embodiments, an immunoprotective response is sufficient to protect an immunized subject from productive infection by a particular pathogen or pathogens to which a vaccine is directed (e.g., P. aeruginosa infection).[000197] Immunization: As used herein, “immunization”, or grammatical equivalents thereof, refers to a process of inducing an immune response to an infectious organism or agent in a subject (“active immunization”), or alternatively, providing immune system components against an infectious organism or agent to a subject (“passive immunization”). In some embodiments, immunization involves the administration of one or more antigens, immunogens, immunogenic complexes, vaccines, immune molecules such as antibodies, immune sera, immune cells such as T cells or B cells, or pharmaceutical compositions to a subject. In some embodiments, immunization is performed by administering an immunologically effective amount of a substance, e.g, an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, immune molecule such as an antibody, immune serum, immune cell such as a T cell or B cell, or pharmaceutical composition to a subject. In some embodiments, immunization results in an immunoprotective response in the subject. In some embodiments, active immunization is performed by administering to a subject an antigenic or immunogenic substance, e.g., an antigen, immunogen, immunogenic complex, vaccine, or pharmaceutical composition. In some embodiments, passive immunization is performed by administering to a subject an immune system component, e.g., an immune molecule such as an antibody, immune serum, or immune cell such as a T cell or B cell.[000198] Isolated: As used herein, the term “isolated”, or grammatical equivalents thereof, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g. , buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polysaccharide that occurs in nature is considered to be "isolated" when, a) by virtue of itsorigin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide or polysaccharide. Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide or polysaccharide to the extent that it has been separated from other components a) with which it is associated in nature; and / or b) with which it was associated when initially produced.[000199] Linker: As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form Sl- L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g, fusion polypeptides) are known in the art (Holliger et al, 1993; Poljak, 1994).[000200] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g. , those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.[000201] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.[000202] Polysaccharide: The term “polysaccharide” as used herein refers to a polymeric carbohydrate molecule composed of long chains of monosaccharide units bound together by glycosidic, phosphodiester, or other linkages, and on hydrolysis give the constituent monosaccharides or oligosaccharides. Polysaccharides range in structure from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, structural polysaccharides such as cellulose and chitin and microbial polysaccharides, and antigenic polysaccharides found in microorganisms including, but not limited to, capsular polysaccharides (CPS), O polysaccharides (OPS), core O polysaccharides (COPS), and lipopolysaccharides (LPS).[000203] Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids, e.g., linked to each other by peptide bonds. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (z.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L- amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.[000204] Prevention: The term “prevent” or “prevention”, as used herein in connection with a disease, disorder, and / or medical condition, refers to reducing the risk of developing the disease, disorder and / or condition, and / or a delay of onset, and / or reduction in frequency and / or severity of one or more characteristics or symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder or condition is observed in a populationsusceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. [000205] Protein: As used herein, the term “protein” encompasses a polypeptide. Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain 1-amino acids, d-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g. , terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.[000206] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g. , from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).[000207] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, subject, population, sample, sequence or value of interest is compared with a reference or control agent, animal, subject, population, sample, sequence or value. In some embodiments, a reference or control is testedand / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.[000208] Response: As used herein, a “response” to treatment may refer to any beneficial alteration in a subject’s condition that occurs as a result of or correlates with treatment. Such alteration may include stabilization of the condition (e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and / or improvement in the prospects for cure of the condition, etc. It may refer to a subject’s response or to a tumor’s response. Subject or tumor response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of biomarkers in a sample obtained from a subject, cytology, and / or histology. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of subjects and / or tumors, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.[000209] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular subject will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments a reference sample or group of reference samples are from subjects comparable to a particular subject. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.[000210] Serotype: As used herein, the term “serotype”, also referred to as a serovar, refers to a distinct variation within a species of bacteria or virus or among immune cells of different subjects. These microorganisms, viruses, or cells are classified together based on their cell surface antigens, allowing the epidemiologic classification of organisms to the sub-species level. A group of serovars with common antigens may be referred to as a serogroup or sometimes serocomplex.[000211] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g, a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms orcharacteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is a subject to whom diagnosis and / or therapy is and / or has been administered.[000212] Susceptible to: A subject who is “susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, a subject who is susceptible to a disease, disorder, or condition is a subject who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of subjects suffering from the disease, disorder, or condition).[000213] Symptoms are reduced: As used herein, “symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency, e.g. , to a statistically and / or clinically significant or relevant level. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.[000214] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.[000215] Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent. For the purposes of the present invention, vaccination can be administered before, during, and / or after exposure to a disease-causing agent, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition. In some embodiments, vaccination initiates immunization.[000216] In some embodiments, the present invention may be defined in any of the following numbered paragraphs'.1. A fusion protein comprising, in any order, (i) a biotin-binding moiety, (ii) a PcrV polypeptide or a fragment thereof, and (iii) a PopB polypeptide or a fragment thereof.2. A fusion protein comprising, in any order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof; and(iii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof.3. The fusion protein of any of paragraphs 1-2 comprising, in the following order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical toSEQ ID NO: 6 or an antigenic portion thereof; and(iii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof.4. The fusion protein of any of paragraphs 1-2 comprising, in the following order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical toSEQ ID NO: 3 or an antigenic portion thereof, and(iii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof.5. The fusion protein of any of paragraphs 1-2 comprising, in the following order:(i) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof;(ii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof; and(iii) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof.6. The fusion protein of any of paragraphs 1-2 comprising, in the following order:(i) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof; and(iii) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof.7. The fusion protein of any one of paragraphs 1-6, further comprising (i) a first linker positioned between the biotin-binding moiety and the PopB or PcrV polypeptide or a fragment thereof; and / or (ii) a second linker positioned between the PopB polypeptide or a fragment thereof and the PcrV polypeptide or a fragment thereof.8. A fusion protein comprising, in any order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 2 or a biotin binding portion thereof;(ii) a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof;(iii) a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof; and(iv) a first linker positioned between the biotin-binding moiety and the first polypeptide and / or a second linker positioned between the first polypeptide and the second polypeptide.9. The fusion protein of any of paragraphs 1-8, wherein the first linker comprises the amino acid sequence GGGGSSS (SEQ ID NO: 37).10. The fusion protein of any one of paragraphs 1-9, wherein the second linker comprises the amino acid sequence AAA (SEQ ID NO: 38) or GS.11. The fusion protein of any one of paragraphs 1-10, comprising in order from N terminus to C terminus: (i) the biotin-binding moiety; and (ii) the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof.12. The fusion protein of any one of paragraphs 1-10, comprising in order from N terminus to C terminus: (i) the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof; and (ii) the biotin-binding moiety.13. The fusion protein of paragraph 11, comprising:(A) in order from N terminus to C terminus: (i) the biotin-binding moiety; (ii) the PopB polypeptide or a fragment thereof; and (iii) the PcrV polypeptide or a fragment thereof; or(B) in order from N terminus to C terminus: (i) the biotin-binding moiety; (ii) the PcrV polypeptide or a fragment thereof; and (iii) the PopB polypeptide or a fragment thereof.14. The fusion protein of paragraph 12, comprising:(A) in order from N terminus to C terminus: (i) the PopB polypeptide or a fragment thereof; (ii) the PcrV polypeptide or a fragment thereof; and (iii) the biotin-binding moiety; or(B) in order from N terminus to C terminus: (i) the PcrV polypeptide or a fragment thereof; (ii) the PopB polypeptide or a fragment thereof; and (iii) the biotin-binding moiety.15. The fusion protein of any one of paragraphs 1-14, wherein the biotin-binding moiety comprises the amino acid sequence of SEQ ID NO:2, or an amino acid having at least 80% sequence identity to SEQ ID NO: 2.16. The fusion protein of any one of paragraphs 1-15, wherein the PopB polypeptide of fragment thereof comprises the amino acid sequence of SEQ ID NO:6, or an amino acid having at least 80% sequence identity to SEQ ID NO: 6.17. The fusion protein of any one of paragraphs 1-16, wherein the PcrV polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid having at least 80% sequence identity to SEQ ID NO: 3.18. The fusion protein of any one of paragraphs 1-17, comprising, or consisting essentially of an amino acid sequence that is at least 80%, 85%, 90% identical to SEQ ID NO:23 or SEQ ID NO: 24 or SEQ ID NO: 25.19. The fusion protein of any one of paragraphs 1-18, wherein the fusion protein comprises aN-terminal and / or C-terminal His Tag.20. The fusion protein of any one of paragraphs 1-19, wherein the fusion protein has the amino acid sequence of any of the fusion proteins in Table 2, or the amino acid sequence of any of the fusion proteins of SEQ ID NO: 14, 17-25, or an amino acid sequence that is a least 80%, 85%, 90% identical to any of SEQ ID NO: 17-25.21. A nucleic acid sequence encoding a fusion protein of any of paragraphs 1-20.The nucleic acid sequence of paragraph 21, wherein the nucleotide sequence comprises, in any order;(i) a nucleotide sequence encoding a biotin-binding moiety;(ii) a nucleotide sequence encoding the PopB polypeptide or a fragment thereof; and(iii) a nucleotide sequence encoding the PcrV polypeptide or a fragment thereof. The nucleic acid sequence of paragraph 21 or 22, wherein the nucleotide sequence encoding the biotin-binding moiety is at least 80%, 85%, 90% identical to SEQ ID NO: 10. The nucleic acid sequence of paragraph 21 or 22, wherein the nucleotide sequence encoding the PopB polypeptide or a fragment thereof is at least 80%, 85%, 90% identical to SEQ ID NO: 13. The nucleic acid sequence of paragraph 21 or 22, wherein the nucleotide sequence encoding the PcrV polypeptide or a fragment thereof is at least 80%, 85%, 90% identical to SEQ ID NO: 11. The nucleic acid sequence of any of paragraphs 21-25, wherein the nucleic acid comprises in the following order: a. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; b. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; c. a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11, and a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; or d. a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10. The nucleic acid sequence of any of paragraphs 21-26, wherein the nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12.The nucleic acid sequence of any of paragraphs 21-27, wherein the nucleic acid sequence is SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12. A nucleic acid sequence comprising a promoter, operatively linked to a heterologous nucleic acid sequence comprising, in any order, a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 11. The nucleic acid sequence of paragraph 29, wherein the heterologous nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12. The nucleic acid sequence of paragraph 29, wherein the heterologous nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12. The nucleic acid sequence of paragraph 29, wherein the heterologous nucleic acid further comprises a nucleic acid that encodes at least one linker, or a nucleic acid that encodes an expression tag, or both. The nucleic acid sequence of paragraph 32, wherein the nucleic acid sequence encoding at least one linker is located between SEQ ID NO: 10 and SEQ ID NO: 13, or SEQ ID NO: 13 and SEQ ID NO: 11, or SEQ ID NO: 10 and SEQ ID NO: 11. The nucleic acid sequence of paragraph 32, wherein the nucleic acid sequence encoding at least one linker encodes a linker having an amino acid sequence selected from any of: GGGGSSS (SEQ ID NO: 37) or AAA (SEQ ID NO: 38) or GS, and any of: SEQ ID NOS: 39-60 and SEQ ID NO: 62, 63, 108, 109. The nucleic acid sequence of paragraph 29, wherein the heterologous nucleic acid comprises in the following order: a. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; b. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; c. a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or anucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11, and a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; or d. a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10.36. A cell transfected with the nucleic acid sequence of any of paragraphs 21-35.37. The cell of paragraph 36, wherein the cell is transfected with the nucleic acid sequence of any of paragraphs 26 or paragraph 35.38. The cell of paragraph 36, wherein the cell is transfected with the nucleic acid sequence of any of paragraphs 27, 28, 30 or 31.39. The cell of paragraph 36, wherein the cell is an expression host cell or a stable cell line.40. The cell of paragraph 39, wherein the expression host cell is selected from the group consisting of: a prokaryotic cell line, an insect cell line or a mammalian cell line.41. The cell of paragraph 40, wherein the prokaryotic cell line is an E. Coli cell line.42. The cell of paragraph 40, wherein the insect cell line is baculovirus expression system.43. The cell of paragraph 40, wherein the mammalian cell line is a human cell line or ChineseHamster ovary (CHO) cell line.44. The cell of any of paragraphs 36-43, wherein the heterologous nucleic acid sequence is codon- optimized to improve expression in the host cell.45. The cell of any of paragraphs 36-44, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding one or more of: polyadenylation sequence or termination sequence, a signal sequence.46. An expression vector comprising the nucleic acid sequence of any of paragraphs 21-35.47. The expression vector of paragraph 46, comprising the nucleic acid sequence of paragraphs 26 or 35.48. The expression vector of paragraph 46, comprising the nucleic acid sequence of paragraph 27, 28, 30 or 3I.49. The cell of paragraph 36, comprising the expression vector of paragraph 46.50. The cell of paragraph 36, comprising the expression vector of paragraph 47.51. The cell of paragraph 36, comprising the expression vector of paragraph 48.52. An immunogenic composition comprising the fusion protein of any of paragraphs 1-20.53. A vaccine composition comprising the fusion protein of any of paragraphs 1-20.54. A vaccine composition comprising a nucleic acid sequence of any of paragraphs 21-3555. A method for producing or manufacturing a component of a vaccine composition or pharmaceutical composition comprising a fusion protein of any of paragraphs 1-20, by expressing the nucleic acid sequence of any of paragraphs 21-35.56. A method for producing or manufacturing a component of a vaccine composition, or pharmaceutical composition comprising a fusion protein of any of paragraphs 1-21, by using a cell of any of paragraphs 36-45 or 49-51.57. A method comprising expressing a protein from a heterologous nucleic acid sequence, the heterologous nucleic acid sequence comprising, in any order, a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 11.58. A method comprising growing the cell of paragraph 46 in conditions sufficient for the production of a protein for use in a multi-component immunogenic composition.59. A method comprising transfecting a cell with the expression vector of paragraph 46 and expressing the protein from the expression vector.60. A method comprising obtaining a protein expressed from the nucleic acid of paragraph 29 and combining the protein with one or more additional components to form a multi-component immunogenic composition.61. The method of paragraph 60, further comprising purifying the protein prior to combining with one of more components.62. The method of paragraph 60, further comprising lyophilizing the multi-component immunogenic composition.63. The method of paragraph 62, wherein the purified protein is lysophilized prior to combining with other components to form the multi-component immunogenic composition.64. The method of paragraph 60, wherein the one or more additional components are selected from the group consisting of: adjuvant, stabilizers, buffers, surfactants, salts or preservatives.65. The method of paragraph 60, wherein the one or more additional components are suitable for formulating the multi-component immunogenic composition for nasal administration to a subject.66. The method of paragraph 61, wherein the purification comprises a first purification step and a second purification step.67. The method of paragraph 61, wherein the purification results in separating the protein from about 85%, 86%, 87%, 89%, or 90% or more than 90% of the components of the cell from which it was expressed.68. The fusion protein of any of paragraphs 1-20, or vaccine composition of any of paragraphs 53-54, or immunogenic composition of paragraph 52, wherein any one or more of the fusion protein, immunogenic composition, or vaccine composition is lyophilized.69. A method for inducing an immune response in a subject, the method comprising administering to a subject any one or more of: the fusion protein according to any of paragraphs 1-20, the immunogenic composition of paragraph 52, or the vaccine composition of any of paragraphs 53-54.70. The method of paragraph 69, wherein the subject is a human subject.71. The method of paragraph 69, wherein the immune response is a Th 1 and / or Th 17 cell response to at least one of: the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof, or both.72. The method of paragraph 69, wherein the immune response is a B cell, or T cell response, or both a T-cell or B-cell response, to at least one of: the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof, or both.Exemplification[000217] The Examples herein demonstrate production and use of a RVB fusion protein as disclosed herein, e.g., for use in a vaccine, e.g., a multi-component immunogenic vaccine composition for treatment and / or prevention of Pseudomonas aeruginosa (Pa) infection. Herein, the inventors assess the mechanisms of protection of a fusion protein comprising Rhazavadin, PopB and PcrV for use as a Pa vaccine and for the contribution of tissue resident memory T cells (TRM) elicited by different routes of immunization, and also its ability to form a multi-component vaccine with a biotinylated polysaccharide, and if such association would provide protective efficacy against pneumonia. The RVB fusion protein as disclosed herein can be used alone, or it can be used in a multi-component immunogenic composition, e.g., a biotinylated polysaccharide comprising one or more biotin-binding proteins, including but not limited to a RBV fusion protein as disclosed herein. For exemplary purposes only, to demonstrate that RVB fusion protein is expressed properly and can (i) be associated with a biotinylated polysaccharide, and (ii) able to elicit a IL-17 immune response to PopB or PcrV and / or Pseudomonas aeruginosa infection, the RVB fusion protein as disclosed herein (e.g., not His tagged, or comprising a His-tag) was associated a biotinylated .S', pneumococcal polysaccharide to form a multi-component immunogenic composition. It is envisioned that a RVB fusion protein as disclosed herein (e.g., not His tagged, or comprising a His-tag) can be used alone in a vaccine composition, or used in a multi-component MAPS immunogenic complex with biotinylated polysaccharides other than a .S', pneumococcal polysaccharide, e.g., Pseudomonas aeruginosa polysaccharide.[000218] Recently published studies2suggest that for Klebsiella pneumoniae, an OmpX+LTAl IN vaccine provides optimal protection from pneumonia through the generation of IL-17+TRM cells that control lung bacterial burden and BALF IgA antibodies that control bacterial dissemination. Thus, the role of pulmonary TRM and IgA in the disclosed Pa vaccine and challenge model will be assessed. Although the role of vaccine-induced IgA in protection against a is controversial,3in part because Pa elastase can degrade IgA,4,5increased susceptibility to Pa pneumonia has been reported in patients with antibiotic-induced secondary IgA deficiency.6Furthermore, lung IgA responses to a streptococcal vaccine given SC / IN to mice were dependent on lung Th 17 TRM cells.7EXAMPLE 1: Recognition ofPopB by human cells[000219] Pseudomonas aeruginosa continues to be a significant opportunistic pathogen, with growing antibiotic resistance and no available vaccine. Previous work showed that the P. aeruginosa type III secretion system (T3SS) protein PopB elicits a protective Th 17 response after intranasal immunization using the Thl7 adjuvant curdlan, but the protection is of low potency. In those studies, PopB was copurified with its chaperone PcrH.[000220] To assess the human translational potential of the PopB / PcrH vaccine, PopB / PcrH or PcrH was incubated with whole blood obtained from mechanically ventilated children recovering from Pa lung infections and it was discovered that the 3 patients who showed ConA -stimulated IL- 17 secretion by whole blood cells (so were not anergic) had IL- 17 production after stimulation by PopB / PcrH but not by PcrH (FIG. 1, results of a representative patient). These findings demonstrate that PopB is at least recognized by human cells / MHC.[000221] Combining PopB with OprF / I. Although the PopB vaccine can elicit Thl7-mediated, antibody-independent protection, the potency of the protection is relatively low - despite a significantly higher protection as compared to controls against challenge doses up to IxlO6CFU / mouse with Thl7- mediated protection, only 40-60% of mice survive. In contrast, there was a 100% survival with doses of bacteria up to 8xl08CFU when high levels of OPK antibodies are present. Thus, in an attempt to improve the PopB vaccine, the inventors combined it with OprF / I, a serotype-independent antigen that induces OPK and anti-virulence antibodies.[000222] Next, the inventors prepared the OprF / I fusion protein vaccine (OprF190.342-OprI21.83) based on published methods.8Immunizing mice by either the IN or SC route, the inventors discovered that PopB / PcrH induced robust Th 17 responses in the spleen, while OprF / I did not.9After challenge by IN inoculation with Pa strain N13, a serotype 06 clinical isolate, there was much better protection from the PopB / PcrH+OprF / I combination vaccine compared to either vaccine alone, fitting well with our hypothesis that combining Th 17 responses (PopB) with functional antibody responses (OprF / I) will improve protective efficacy. These studies also demonstrated that PopB-based vaccines can protect against multiple serotypes since our prior work was with PAO1 -based strains. It was discovered that high serum IgG responses in mice immunized either IN or SC with the combination of PopB / PcrH and OprF / I (30 ug each), with no decrease in titer to OprF / I upon adding PopB / PcrH. These antisera to OprF / I inhibited the binding of OprF to IFN-gamma, a key anti-virulence property described for prior OprF / I vaccines, demonstrating a mechanism for better protection by the combination vaccine.EXAMPLE 2: Production ofRVB fusion protein[000223] As disclosed herein, the inventors tested whether fusing or mixing PopB with PcrV, a T3SS protein known to elicit protective antibodies, would enhance protective efficacy. The inventors also assessed if adding a Th 17 antigen (PopB) to PcrV would improve mucosal IgA responses to PcrV.[000224] Fusion proteins to the avidin homolog rhizavidin (Rhavi) were constructed to achieve biotin-mediated dimer formation in vivo and for future coupling to biotinylated polysaccharides. A fusion protein of Rhavi to full-length PcrV and PopB (herein denoted RVB) was purified from E. coli using a pACYCDuet-1 vector co-expressing His-PcrH, where untagged RVB bound to His-PcrH was pulled down by nickel affinity resin (see FIG. 2), followed by ion exchange chromatography and then negative affinity chromatography with nickel resin using elution with 0.1% LDAO to dissociate PcrH from RVB. Recombinant Rhavi-PcrV-His, PcrV, and PopB / PcrH were prepared by published methods. Mice were immunized intranasally using curdlan as adjuvant. IgA titers in bronchoalveolar lavage fluid (BALF) and IgG titers in sera were assayed by ELISA, and Thl7 responses were assessed by measuring IL-17 (ELISA) in supernatants of splenocytes stimulated with antigens in vitro. Protective efficacy was evaluated in a murine acute pneumonia model using P. aeruginosa strain N13. (FIG. 3A).[000225] The inventors demonstrated that there was significantly higher IgA titers to PcrV in BALF of mice immunized with RVB when compared to mice immunized with either PcrV or Rhavi-PcrV-His (RV-His), where IgA titers were undetectable (FIG. 3B). Th 17 responses to PopB were significantly higher in RVB-immunized mice compared to mice immunized with PopB / PcrH (FIG. 3A). In the P. aeruginosa acute pneumonia model, RVB-immunized mice displayed 100% survival, whereas PcrV- and Rhavi-PcrV-His (RV-His)-immunized mice had approximately 60% survival (logrank P=0.06 compared to RVB), all significantly higher than curdlan-immunized mice (0% survival). (FIG. 3C)[000226] Therefore, these results demonstrate that integrating PopB with PcrV into the same rhizavidin fusion protein elicits improved IgA titers to PcrV while maintaining a robust Th 17 response to PopB, ultimately conferring broad and potent protection against P. aeruginosa lung infection.EXAMPLE 3: Optimization of production of RVB fusion protein[000227] The inventors herein demonstrate using specific purification methods13 14that a recombinant fusion protein containing PopB and PcrV without PcrH elicits high Th 17 responses and mucosal IgA and is protective in mice.[000228] Herein, the inventors constructed expression vectors for preparing rhizavidin (Rhavi) fusion proteins containing PcrV and PopB (referred to herein as “RVB”). Although initial efforts attempted to combine PcrV and OprF / I into a single rhizavidin fusion protein, all constructs showed low expression and / or proteins were insoluble or had very low yields (data not shown), prompting alternative approaches.[000229] In an initial production of RBV fusion protein, the inventors used a pACYCDuet-1 plasmid13for expression of a His-tagged version of PopB’s chaperone PcrH that is used to pull-down the Rhavi -PcrV-PopB fusion protein via binding of PcrH to PopB (see FIG. 2). Subsequently, the Rhavi-PcrV-PopB fusion protein is released from PcrH by incubation with the zwitterionic surfactant LDAO (lauryldimethylamine oxide), which serves to stabilize PopB in the absence of PcrH.[000230] When the inventors tested for binding of the expressed RVB fusion protein to biotinylated type 1 pneumococcal polysaccharide CPS 1 (referred to herein as “PneumoPS”), there was minimal binding to the biotinylated polysaccharide (FIG. 4A). Similar issues were encountered with a Rhavi-PcrV fusion protein (not shown). Despite multiple protein treatments with re-dox steps, EDTA, urea, LDAO, and their combinations, the inventors did not observe significant MAPS complex formation, indicating that misfolding of RVB and Rhavi-PcrV disrupts the conformation of rhizavidin enough to prevent binding to biotin. These results highlight the challenges of using membrane proteins such as PopB to make fusion proteins with Rhavi.[000231] Thus, the inventors optimized and changed the methods for expression of the rhizavidin fusion proteins that comprise either PopB alone (e.g., RB) or PcrV-PopB (e.g., RVB) using a Duet plasmid that lacks a rhizavidin signal sequence) and different expression host, with vectors having a His tag on the C terminus of PopB. As shown in FIG. 4B, these methods were successful in preparing Rhavi- PopB-His (RB-His) and Rhavi-PcrV-PopB-His (RVB-His) with high purity and ability to bind biotinylated polysaccharides.EXAMPLE 4[000232] Immune responses and protection studies after active immunization (IN) with rhizavidin fusion proteins RVB and RV alone (without biotinylated polysaccharide, e.g., not in a MAPS complex). The inventors next assessed whether active immunization of mice via the IN route with the non-His- tagged version of RVB alone (without a biotinylated polysaccharide) would induce protective immunity. Here, the inventors compared with OprF / I alone, PcrV alone, Rhavi-PcrV -His (RV-His) (made by a CRO Genescript), where all vaccines were mixed with the Thl7 adjuvant curdlan, and curdlan alone as control. Using a high dose of P. aeruginosa strain N 13 for challenge in the pneumonia model, a robust protection in mice immunized with either RVB or RVB plus OprF / I was detected (FIG. 3C). Notably, fusing PopB to PcrV improved the protection from about 60% to 100% (FIG. 3C). The inventors surprisingly discovered that splenic Th 17 responses to PopB were higher after immunization with RVB compared to immunization with PopB / PcrH (FIG. 3A), and BALF IgA titers to PcrV were higher after immunization with RVB as compared to immunization to RV or PcrV (FIG. 3B). These IgA results are particularly compelling evidence that PopB is eliciting pulmonary Th 17 responses.EXAMPLE 5[000233] Immune responses and protection studies after active immunization (SC) with Rhavi-PopB-His (RB-His) alone or in a MAPS immunogenic complex comprising biotinylated pneumococcal polysaccharide.[000234] After the production of the Pseudomonas rhizavidin fusion proteins Rhavi-PopB-His (RB- His) and Rhavi-PcrV-PopB-His (RVB-His) and showing good binding to biotinylated pneumococcal polysaccharide CPS1, also called PneumoPS (FIG. 4B), the inventors next assessed the immune responses in mice after SC immunization as well as the protective efficacy in mice against pneumonia of these model PneumoPS-based MAPS vaccines. As shown in FIG. 5A-5D, SC immunization of mice with RVB-His present in a MAPS immunogenic complex (e.g., the Rhavi-PopB-His MAPS vaccine complexed with biotinylated PneumoPS) was demonstrated to be protective against acute lethal pneumonia, which was significant as compared to minimal protection after immunization with Rhavi-PopB-His mixed with non-biotinylated PneumoPS (FIG. 5B). (Vaccine doses were 10 ug protein for each group).[000235] Importantly, the MAPS vaccine comprising RB-His and PneumoPS, induced good Th 17 responses in the spleen to Rhavi-PopB-His but not to Rhavi alone after SC immunization (FIG. 5C). IgG responses to PopB were high and not different between the RB-His:PneumoPS MAPS vaccine and the mixture of Rhavi-PopB (RB-His) and non-biotinylated PneumoPS (not shown). The RB-His:PneumoPS MAPS vaccine elicited IgG responses to the pneumococcal polysaccharide PneumoPS, which was significant as compared to the mixture of PneumoPS + Rhavi-PopB-His did not (FIG. 5D), indicating that Rhavi-PopB-His can serve as a good carrier protein for induction of polysaccharide -specific antibodies (even though, in this experiment, these IgG are not expected to be protective since they are directed against a pneumococcal polysaccharide).EXAMPLE 6[000236] Immune responses and protection studies after active immunization (SC) with Rhavi- PcrV-PopB-His (RVB-His) alone.[000237] Next, the inventors also assessed SC immunization of mice with MAPS vaccines comprising the Rhavi-PcrV -PopB-His (RVB-His) fusion protein depicted in FIG. 4B above, again using 10 ug protein per vaccine dose, as done with Rhavi-PopB-His (see FIGS. 5A-5C).[000238] As shown in FIG. 6A, a vaccine comprising the fusion protein RVB-His alone (i.e., not in a MAP complex with biotinylated pneumococcal polysaccharide (PneumoPS) elicited robust protection (100% survival). Th 17 responses were also detected with the use of the RVB-His fusion protein (FIG. 6B), and would be expected to be higher when RVB-His is in a MAPS complex with PneumoPS (data not shown). IgG titers to PopB / PcrH and to RVB-His were high (FIG. 6C) and were not different between the MAPS vaccine and the mixture of RVB-His and non-biotinylated PneumoPS (not shown), indicating that RVB-His can ve used to induce an immune response by itself, or alternatively, function as a good carrier protein for anti-PS antibody responses. Importantly, antisera produced by immunization with a vaccine comprising RVB-His (not in a MAPS complex) inhibited PcrV -mediated (T3SS-mediated) cytotoxicity of Pa strain PA 14 incubated with A549 epithelial cells2 (FIG. 6D), demonstrating RVB-His alone is useful for inducing an immune response in a subject for treatment and / or protection against P. aeruginosa infection.EXAMPLE 7[000239] In Silico analysis of PopB for optimal selection of antigens for protection against P. aeruginosa lung infection.[000240] To determine small peptides of PopB that can be used as antigens in a RVB fusion protein, in silico analyses was performed for MCH class II binding predictions for both the H-2 lAb allele (of C57BL / 6 mice) and the H-2 lAq allele (of FVB / N mice). While most of the prior work with the PopB / PcrH vaccine was with FVB / N mice, the inventors also demonstrated that C57BL / 6 mice are also protected by the PopB / PcrH vaccine. As shown in Table 3 below, PopB peptides predicted in silico to bind murine class II MHC were in the N terminus, while the single peptide predicted to bind a common human class II MHC allele (DRB1) was in the C terminus.[000241] Table 3: In silico predicted PopB peptides with a strong binding to MHC class II. These predictions were done using the NetMHCIIpan server (http: / / www.cbs.dtu.dk / services / _NetMHCIIpan / ). Peptides shown are predicted to have strong binding (rank <0.5%).[000242] Class II HLA peptide binding assays was also used (offered by Prohnmune’s “REVEAL Rapid Epitope Discovery System”) to assess the binding of an overlapping PopB 15-mer peptide library to mouse class II MHC (the H-2 lAb allele of C57BL / 6 mice). The Prolmmune assay uses an antibody to detect the presence or absence of the native conformation of the MHC-peptide complex. It compares the binding of each peptide to that of a positive control high-affinity known T cell epitope, and each peptide is given a relative binding score. Of note, the H-2 lAq allele of FVB / N mice is not available for the Prolmmune REVEAL assay. As shown in FIG. 7A, the results of the C terminus fragments of PopB, was assessed as the inventors prior experiments had shown that the N terminus did not stimulate IL-17 production from FVB / N immunized mice (data not shown). Herein, the inventors determined that the PopB_35-39 peptide (SEQ ID NO: 66) that was predicted in silico was demonstrated to be able to bind both murine class II alleles (see FIG. 7A). The Prolmmune assays (Table 4) showed several peptides inthe C terminal fragment with high binding to class II MHC but no significant binding of the N terminal peptide, and overall lower binding to murine MHC class II.[000243] Table 4 : Prolmmune in vitro assay for MHC class II binding using a single murine allele (H- 2 lAb) and a common human allele (about 12% of North American and global populations) with synthetic overlapping peptides spanning PopB positions 171-390 and two selected peptides within positions 1-171. Peptides with scores below 5 are not shown since they are considered weak binders. Peptide PopB_35-49 (SEQ ID NO: 66) showed a score <5 in this assay.[000244] Selected peptide epitopes discovered in this screen were tested by incubation with splenocytes from C57BL / 6 mice previously immunized with the PopB / PcrH vaccine, with IL- 17 secretion in the supernatant as readout (FIG. 7A). These assays showed a weak signal for one of the peptides in the N terminus (PopB_34-48) (SEQ ID NO: 65) and a strong response to a peptide in the C terminus (PopB_171-185) (SEQ ID NO: 79).[000245] Accordingly, the inventors have demonstrated that PopB fragments across the range of the protein could elicit T cell activation across human MHC haplotypes, and suggest that most, or the entire PopB protein is likely needed to elicit and immune response and see activity in both murine and human systems.[000246] Materials and Methods. Vaccine composition and immunogenic compositions used in the Examples comprising a RVB fusion protein were formulated with whichever adjuvant is found to give the best route-specific protection (e.g., dmLT vs. Alum for SC; LTA1 vs. curdlan for IN). For exemplary purposes only, the RVB fusion protein was combined in multi-component immunogenic composition comprising a 5 pneumoniae polysaccharide.[000247] Summary: The inventors have demonstrated that the Pa protein PopB stimulates T helper cells called Th 17 cells to protect against pneumonia in murine models. The inventors demonstrate the generation of vaccines combining PopB with antigens that induce protective antibodies (Psi, PcrV, and OprF / I). These vaccines described herein can be used for host protection as a vaccine for Pa.[000248] References — The references disclosed in the specification and Examples are each incorporated herein in their entirety.1. Tan HL, Regamey N, Brown S, Bush A, Lloyd CM, Davies JC. The Th 17 pathway in cystic fibrosis lung disease. Am J Respir Crit Care Med 2011 ; 184:252-8.2. Iwanaga N, Chen K, Yang H, Lu S, Hoffmann JP, Wanek A, McCombs JE, Song K, Rangel- Moreno J, Norton EB, Kolls JK. Vaccine -driven lung TRM cells provide immunity against Klebsiella via fibroblast IL-17R signaling. Sci Immunol 2021;6:eabfl 198.3. DiGiandomenico A, Rao J, Harcher K, Zaidi TS, Gardner J, Neely AN, Pier GB, Goldberg JB . Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections. Proc Natl Acad Sci U S A 2007;104:4624-9.4. Diebel LN, Liberati DM, Amin PB, Diglio CA. Cleavage of SIgA by gram negative respiratory pathogens enhance neutrophil inflammatory potential. J Trauma 2009;66: 1336-42; discussion 42.5. Heck LW, Alarcon PG, Kulhavy RM, Morihara K, Russell MW, Mestecky JF. Degradation of IgA proteins by Pseudomonas aeruginosa elastase. J Immunol 1990;144:2253-7.6. Robak OH, Heimesaat MM, Kruglov AA, Prepens S, Ninnemann J, Gutbier B, Reppe K, Hochrein H, Suter M, Kirschning CJ, Marathe V, Buer J, Homef MW, Schnare M, Schneider P, Witzenrath M, Bereswill S, Steinhoff U, Suttorp N, Sander LE, Chaput C, Opitz B. Antibiotic treatment-induced secondary IgA deficiency enhances susceptibility to Pseudomonas aeruginosa pneumonia. J Clin Invest 2018;128:3535-45.7. Christensen D, Mortensen R, Rosenkrands I, Dietrich J, Andersen P. Vaccine-induced Thl7 cells are established as resident memory cells in the lung and promote local IgA responses. Mucosal Immunol 2017;10:260-70.8. Westritschnig K, Hochreiter R, Wallner G, Firbas C, Schwameis M, Jilma B. A randomized, placebo-controlled phase I study assessing the safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein OprF / I vaccine (IC43) in healthy volunteers. Hum Vaccin Immunother 2014;10: 170-83.9. Shaikh MOF, Schaefers MM, Merakou C, DiBlasi M, Bonney S, Liao T, Zurakowski D, Kehl M, Tabor DE, DiGiandomenico A, Priebe GP. Multicomponent Pseudomonas aeruginosa Vaccines Eliciting Th 17 Cells and Functional Antibody Responses Confer Enhanced Protection against Experimental Acute Pneumonia in Mice. Infect hnmun 2022;90:e0020322.10. Das S, Howlader DR, Zheng Q, Ratnakaram SSK, Whittier SK, Lu T, Keith JD, Picking WD, Birket SE, Picking WL. Development of a Broadly Protective, Self-Adjuvanting Subunit Vaccine to Prevent Infections by Pseudomonas aeruginosa. Front Immunol 2020;l 1:583008.11. Howlader DR, Das S, Lu T, Hu G, Varisco DJ, Dietz ZK, Walton SP, Ratnakaram SSK, Gardner FM, Ernst RK, Picking WD, Picking WL. Effect of Two Unique Nanoparticle Formulations on the Efficacy of a Broadly Protective Vaccine Against Pseudomonas aeruginosa. Front Pharmacol 2021;12:706157.Wu W, Huang J, Duan B, Traficante DC, Hong H, et al. Thl7-stimulating protein vaccines confer protection against Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med. 2012 Sep l;186(5):420-7.Zhang F, Lu YJ, Malley R. Multiple antigen-presenting system (MAPS) to induce comprehensiveB- and T-cell immunity. Proc Natl Acad Sci U S A. 2013 ; 110: 13564-9.US Patent 10,766,932US patent 11,576,958US Patent 9,499,593W02012 / 155007WO2012 / 155053W02020 / 056127Equivalents[000249] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

Claims:

1. A fusion protein comprising, in any order,(i) a biotin-binding moiety,(ii) a PcrV polypeptide or a fragment thereof, and(iii) a PopB polypeptide or a fragment thereof.

2. A fusion protein comprising, in any order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof; and(iii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof.

3. The fusion protein of any of claims 1-2 comprising, in the following order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof; and(iii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof.

4. The fusion protein of any of claims 1-2 comprising, in the following order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof;(ii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof, and(iii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof.

5. The fusion protein of any of claims 1-2 comprising, in the following order:(i) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof;(ii) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof; and(iii) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof.

6. The fusion protein of any of claims 1-2 comprising, in the following order:(i) a PcrV polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof;(ii) a PopB polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof; and(iii) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO:2 or a biotin binding portion thereof.

7. The fusion protein of any one of claims 1-6, further comprising (i) a first linker positioned between the biotin-binding moiety and the PopB or PcrV polypeptide or a fragment thereof; and / or (ii) a second linker positioned between the PopB polypeptide or a fragment thereof and the PcrV polypeptide or a fragment thereof.

8. A fusion protein comprising, in any order:(i) a biotin-binding moiety comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 2 or a biotin binding portion thereof;(ii) a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 6 or an antigenic portion thereof;(iii) a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90% identical to SEQ ID NO: 3 or an antigenic portion thereof; and(iv) a first linker positioned between the biotin-binding moiety and the first polypeptide and / or a second linker positioned between the first polypeptide and the second polypeptide.

9. The fusion protein of any of claims 1-8, wherein the first linker comprises the amino acid sequence GGGGSSS (SEQ ID NO: 37).

10. The fusion protein of any one of claims 1-9, wherein the second linker comprises the amino acid sequence AAA (SEQ ID NO: 38) or GS.

11. The fusion protein of any one of claims 1-10, comprising in order from N terminus to C terminus: (i) the biotin-binding moiety; and (ii) the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof.

12. The fusion protein of any one of claims 1-10, comprising in order from N terminus to C terminus: (i) the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof; and (ii) the biotin-binding moiety.

13. The fusion protein of claim 11, comprising:(A) in order from N terminus to C terminus: (i) the biotin-binding moiety; (ii) the PopB polypeptide or a fragment thereof; and (iii) the PcrV polypeptide or a fragment thereof; or(B) in order from N terminus to C terminus: (i) the biotin-binding moiety; (ii) the PcrV polypeptide or a fragment thereof; and (iii) the PopB polypeptide or a fragment thereof.

14. The fusion protein of claim 12, comprising:(A) in order from N terminus to C terminus: (i) the PopB polypeptide or a fragment thereof; (ii) the PcrV polypeptide or a fragment thereof; and (iii) the biotin-binding moiety; or(B) in order from N terminus to C terminus: (i) the PcrV polypeptide or a fragment thereof; (ii) the PopB polypeptide or a fragment thereof; and (iii) the biotin-binding moiety.

15. The fusion protein of any one of claims 1-14, wherein the biotin-binding moiety comprises the amino acid sequence of SEQ ID NO:2, or an amino acid having at least 80% sequence identity to SEQ ID NO: 2.

16. The fusion protein of any one of claims 1-15, wherein the PopB polypeptide of fragment thereof comprises the amino acid sequence of SEQ ID NO:6, or an amino acid having at least 80% sequence identity to SEQ ID NO: 6.

17. The fusion protein of any one of claims 1-16, wherein the PcrV polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid having at least 80% sequence identity to SEQ ID NO: 3.

18. The fusion protein of any one of claims 1-17, comprising, or consisting essentially of an amino acid sequence that is at least 80%, 85%, 90% identical to SEQ ID NO:23 or SEQ ID NO: 24 or SEQ ID NO: 25.

19. The fusion protein of any one of claims 1-18, wherein the fusion protein comprises a C-terminal and / or N-terminal His Tag.

20. The fusion protein of any one of claims 1-19, wherein the fusion protein has the amino acid sequence of any of the fusion proteins in Table 2, or the amino acid sequence of any of the fusion proteins of SEQ ID NO: 14, 17-25, or an amino acid sequence that is a least 80%, 85%, 90% identical to any of SEQ ID NO: 17-25.

21. A nucleic acid sequence encoding a fusion protein of any of claims 1-20.

22. The nucleic acid sequence of claim 21, wherein the nucleotide sequence comprises, in any order;(i) a nucleotide sequence encoding a biotin-binding moiety;(ii) a nucleotide sequence encoding the PopB polypeptide or a fragment thereof; and(iii) a nucleotide sequence encoding the PcrV polypeptide or a fragment thereof.

23. The nucleic acid sequence of claim 21 or 22, wherein the nucleotide sequence encoding the biotinbinding moiety is at least 80%, 85%, 90% identical to SEQ ID NOTO.

24. The nucleic acid sequence of claim 21 or 22, wherein the nucleotide sequence encoding the PopB polypeptide or a fragment thereof is at least 80%, 85%, 90% identical to SEQ ID NO: 13.

25. The nucleic acid sequence of claim 21 or 22, wherein the nucleotide sequence encoding the PcrV polypeptide or a fragment thereof is at least 80%, 85%, 90% identical to SEQ ID NO: 11.

26. The nucleic acid sequence of any of claims 21-25, wherein the nucleic acid comprises in the following order: a. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; b. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; c. a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11, and a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; or d. a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10.

27. The nucleic acid sequence of any of claims 21-26, wherein the nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12.

28. The nucleic acid sequence of any of claims 21-27, wherein the nucleic acid sequence is SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12.

29. A nucleic acid sequence comprising a promoter, operatively linked to a heterologous nucleic acid sequence comprising, in any order, a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 11.

30. The nucleic acid sequence of claim 29, wherein the heterologous nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12.

31. The nucleic acid sequence of claim 29, wherein the heterologous nucleic acid sequence comprises SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12, or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 12.

32. The nucleic acid sequence of claim 29, wherein the heterologous nucleic acid further comprises a nucleic acid that encodes at least one linker, or a nucleic acid that encodes an expression tag, or both.

33. The nucleic acid sequence of claim 32, wherein the nucleic acid sequence encoding at least one linker is located between SEQ ID NO: 10 and SEQ ID NO: 13, or SEQ ID NO: 13 and SEQ ID NO: 11, or SEQ ID NO: 10 and SEQ ID NO: 11.

34. The nucleic acid sequence of claim 32, wherein the nucleic acid sequence encoding at least one linker encodes a linker having an amino acid sequence selected from any of: GGGGSSS (SEQ ID NO: 37) or AAA (SEQ ID NO: 38) or GS, and any of: SEQ ID NOS: 39-60 and SEQ ID NO: 62, 63, 108, 109.

35. The nucleic acid sequence of claim 29, wherein the heterologous nucleic acid comprises in the following order: a. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; b. a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13;c. a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11, and a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10; or d. a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 11; and a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 13; a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% sequence identity to SEQ ID NO: 10.

36. A cell transfected with the nucleic acid sequence of any of claims 21-35.

37. The cell of claim 36, wherein the cell is transfected with the nucleic acid sequence of any of claims 26 or claim 35.

38. The cell of claim 36, wherein the cell is transfected with the nucleic acid sequence of any of claims 27, 28, 30 or 31.

39. The cell of claim 36, wherein the cell is an expression host cell or a stable cell line.

40. The cell of claim 39, wherein the expression host cell is selected from the group consisting of: a prokaryotic cell line, an insect cell line or a mammalian cell line.

41. The cell of claim 40, wherein the prokaryotic cell line is an E. Coli cell line.

42. The cell of claim 40, wherein the insect cell line is baculovirus expression system.

43. The cell of claim 40, wherein the mammalian cell line is a human cell line or Chinese Hamster ovary (CHO) cell line.

44. The cell of any of claims 36-43, wherein the heterologous nucleic acid sequence is codon- optimized to improve expression in the host cell.

45. The cell of any of claims 36-44, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding one or more of: polyadenylation sequence or termination sequence, a signal sequence.

46. An expression vector comprising the nucleic acid sequence of any of claims 21-35.

47. The expression vector of claim 46, comprising the nucleic acid sequence of claims 26 or 35.

48. The expression vector of claim 46, comprising the nucleic acid sequence of claim 27, 28, 30 or31.

49. The cell of claim 36, comprising the expression vector of claim 46.

50. The cell of claim 36, comprising the expression vector of claim 47.

51. The cell of claim 36, comprising the expression vector of claim 48.

52. An immunogenic composition comprising the fusion protein of any of claims 1-20.

53. A vaccine composition comprising the fusion protein of any of claims 1-20.

54. A vaccine composition comprising a nucleic acid sequence of any of claims 21-3555. A method for producing or manufacturing a component of a vaccine composition or pharmaceutical composition comprising a fusion protein of any of claims 1-20, by expressing the nucleic acid sequence of any of claims 21-35.

56. A method for producing or manufacturing a component of a vaccine composition, or pharmaceutical composition comprising a fusion protein of any of claims 1-21, by using a cell of any of claims 36-45 or 49-51.

57. A method comprising expressing a protein from a heterologous nucleic acid sequence, the heterologous nucleic acid sequence comprising, in any order, a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 10; a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 13; and a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 11.

58. A method comprising growing the cell of claim 46 in conditions sufficient for the production of a protein for use in a multi-component immunogenic composition.

59. A method comprising transfecting a cell with the expression vector of claim 46 and expressing the protein from the expression vector.

60. A method comprising obtaining a protein expressed from the nucleic acid of claim 29 and combining the protein with one or more additional components to form a multi-component immunogenic composition.

61. The method of claim 60, further comprising purifying the protein prior to combining with one of more components.

62. The method of claim 60, further comprising lyophilizing the multi-component immunogenic composition.

63. The method of claim 62, wherein the purified protein is lysophilized prior to combining with other components to form the multi-component immunogenic composition.

64. The method of claim 60, wherein the one or more additional components are selected from the group consisting of: adjuvant, stabilizers, buffers, surfactants, salts or preservatives.

65. The method of claim 60, wherein the one or more additional components are suitable for formulating the multi-component immunogenic composition for nasal administration to a subject.

66. The method of claim 61, wherein the purification comprises a first purification step and a second purification step.

67. The method of claim 61, wherein the purification results in separating the protein from about 85%, 86%, 87%, 89%, or 90% or more than 90% of the components of the cell from which it was expressed.

68. The fusion protein of any of claims 1-20, or vaccine composition of any of claims 53-54, or immunogenic composition of claim 52, wherein any one or more of the fusion protein, immunogenic composition, or vaccine composition is lyophilized.

69. A method for inducing an immune response in a subject, the method comprising administering to a subject any one or more of: the fusion protein according to any of claims 1-20, the immunogenic composition of claim 52, or the vaccine composition of any of claims 53-54.

70. The method of claim 69, wherein the subject is a human subject.

71. The method of claim 69, wherein the immune response is a Thl and / or Thl7 cell response to at least one of: the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof, or both.

72. The method of claim 69, wherein the immune response is a B cell, or T cell response, or both a T- cell or B-cell response, to at least one of: the PopB polypeptide or a fragment thereof, or the PcrV polypeptide or a fragment thereof, or both.

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