Immunogenic compositions and vaccines derived from bacterial surface receptor proteins

Abstract

The present disclosure provides immunogenic compositions comprising a polypeptide comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative bacterial species. The HIBP surface receptor protein has been modified in such a way that it cannot bind to host iron-binding proteins. Methods of generating and using these immunogenic compositions to prepare animal and human vaccines are also provided.

Description

[0001] Related Applications

[0002] This PCT application claims the benefit of U.S. Provisional Patent Application No. 61 / 910,817, filed Dec. 2, 2013, and U.S. Provisional Patent Application No. 62 / 007,068, filed Jun. 3, 2014, which are hereby incorporated by reference in their entireties.

[0003] Field of Disclosure

[0004] The present disclosure relates to immunogenic compositions and vaccines and methods for their production and evaluation. More particularly, the present disclosure relates to immunogenic compositions derived from bacterial surface receptor proteins and vaccines against Gram-negative bacterial organisms, including but not limited to bacterial organisms belonging to the families Pasteurellaceae, Neisseriaceae, and Moraxellaceae.

[0005] Background of the Disclosure

[0006] The following paragraphs are intended to introduce the reader to the more detailed description that follows and are not intended to define or limit the claimed subject matter of the present disclosure.

[0007] Vaccines capable of mediating an effective immune response are crucial in health strategies aimed at combating diseases caused by microbial pathogens. Two basic strategies for inducing an effective immune response in a host involve administering a 'live' agent capable of replicating in the host, or administering a material or substance that cannot replicate in the host. Administration of live vaccines can represent a safety risk for immunocompromised individuals if the agent or contaminating organism replicates and adversely affects the immunized subject. These risks are not associated with vaccines based on inactivated whole pathogens, extracts from pathogens, or purified components, which are referred to as subunit vaccines. Subunit vaccines avoid the safety issues associated with live vaccines, but the purified components may not themselves deliver the desired protective effect against infectious organisms in the subject and require appropriate components (referred to as adjuvants) to enhance the immune response.

[0008] Methods for the design of vaccines against Gram-negative bacterial organisms have generally focused on the use of proteins that are naturally associated with the outer membrane of the bacteria and are exposed on the surface of the bacterial cell. Particularly attractive targets for vaccination are proteins that are presumably essential for survival in the host, as they cannot be lost or drastically altered to avoid the host immune response. In this regard, bacterial surface receptor proteins that are able to interact with and bind to host iron-binding proteins (transferrin and lactoferrin) have for some time been considered suitable components for the preparation of vaccines (1-3). This group of surface receptor proteins (hereinafter referred to as "HIBP" (host iron-binding protein) surface receptor proteins) is present in pathogens belonging to the bacterial families Pasteurellaceae, Moraxellaceae, and Neisseriaceae in humans and animals (4). Thus, these proteins have been recognized as potential targets for the development of vaccines against a variety of different pathogens in humans and food-producing animals (5)(6-10).

[0009] The HIBP surface receptors typically comprise two proteins: a surface lipoprotein, namely transferrin-binding B (TbpB) or lactoferrin-binding protein B (LbpB), and a TonB-dependent integral membrane protein, namely transferrin-binding protein A (TbpA) or lactoferrin-binding protein A (LbpA) (11). Recently, the detailed three-dimensional structures of TbpB from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Neisseria meningitidis have been determined at high resolution (12-14). The intrinsic properties of the TbpB or LbpB proteins are extremely different from those of the integral outer membrane proteins TbpA and LbpA and substantially affect the strategies for vaccine development. For example, it is possible to produce and purify TbpB or LbpB in relatively high yields from the Escherichia coli cytoplasm for the production of subunit vaccines. However, these proteins are absent or lacking, particularly in outer membrane vesicle (OMV) vaccines prepared by selective detergent extraction, due to their removal during the extraction process. In contrast, functional TbpA or LbpA can only be produced in the outer membrane, presenting a limitation for the high-yield production of purified proteins to be used in subunit vaccines. Alternative methods of producing misfolded proteins that aggregate into large inclusion bodies and subsequently attempting to refold the proteins from enriched inclusion body preparations are also problematic for commercial production. Thus, most strategies for TbpA- or LbpA-based vaccines generally involve the design of the production of OMVs or the development of attenuated strains.

[0010] Alternative methods that have been successfully used for invasive bacterial pathogens are to use extracellular capsular polysaccharides as the primary antigen and conjugate them to a protein carrier to induce T cell help. These conjugate capsular vaccines have proven highly effective in providing protection from infection by strains expressing the specific capsular polysaccharide, but do not provide cross-protection against other capsular types. Although conjugate capsular vaccines were initially developed against the human pathogens Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae to prevent invasive infections, post-marketing carriage studies have demonstrated that the systemically administered vaccines eliminate colonization by the pathogens expressing the specific targeted polysaccharide (15-17). This has the added benefit of providing herd immunity, thereby providing protection to non-immune individuals due to the reduced carriage frequency within the population. Although not the initial intent of these early vaccines, conferring herd immunity has likely become an important criterion for evaluating new and upcoming bacterial vaccines. However, determining or predicting whether a new vaccine will be able to affect or prevent colonization (carriage) is an important challenge (18).

[0011] Evaluating the ability of a protein antigen to ultimately provide broad protection against a diverse collection of disease isolates is also a challenge worthy of consideration (19). Initial work on testing this ability typically involves immunizing other animal species (mice, rabbits) and then analyzing the cross-reactivity and cross-protection properties of the resulting sera. For surface antigens that can be readily produced in soluble form, such as surface lipoproteins, the first step is usually to generate and purify a collection of variant proteins and use them in a standard ELISA (enzyme-linked immunosorbent assay) to evaluate the ability of the antiserum to recognize the variant proteins. This is rather labor-intensive, making the analysis of a broad collection of variants an expensive endeavor and relying on the assumption that the binding of the antigen to the ELISA plate is random, such that all protein surfaces are detectable.

[0012] Given the limitations in the various assays used to evaluate and predict the cross-protection and cross-reactivity properties of antisera generated against antigens, the selection and design of new and improved protein-based vaccines should be carried out in conjunction with the development of improved assays, such that the optimization and improvement of the developed vaccines can be undertaken on a rational basis.

[0013] Even though a great deal of work has been done over the years since its initial discovery (20, 21), it remains unclear whether and how the HIBP surface receptor protein can be used to prepare effective vaccines against Gram-negative bacterial pathogens, and specifically whether and how a broadly protective vaccine can be developed. Accordingly, there is a need in the art for improved immunogenic compositions and vaccines based on the HIBP surface receptor protein against Gram-negative bacterial organisms.

[0014] Disclosure Overview

[0015] The present disclosure provides novel immunogenic compositions and, in particular, immunogenic compositions based on HIBP surface receptor proteins from Gram-negative pathogenic bacterial species.

[0016] Accordingly, the present disclosure provides, in at least one embodiment, an immunogenic composition comprising an antigen derived from a HIBP surface receptor protein from a Gram-negative pathogenic bacterial species, wherein the protein derived from the HIBP surface receptor protein has been modified in such a way that it cannot substantially bind to host iron-binding proteins.

[0017] The present disclosure provides, in at least one embodiment, an immunogenic composition comprising a polypeptide comprising the C-terminal half domain or the N-terminal half domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative bacterial species, wherein the polypeptide cannot substantially bind to host iron-binding proteins. In a preferred embodiment, the present disclosure provides an immunogenic composition comprising the C-terminal half domain of a HIBP surface receptor polypeptide, wherein the polypeptide cannot substantially bind to host iron-binding proteins. In a further preferred embodiment, the present disclosure provides an immunogenic composition comprising a mixture of at least two polypeptides, each polypeptide comprising a C-terminal half domain, wherein the C-terminal half domain is obtainable from or obtained from at least two Gram-negative bacterial species or at least two Gram-negative strains. In a further preferred embodiment, the C-terminal half domain is obtained from a HIBP surface receptor protein that is antigenically divergent.

[0018] In other embodiments, the present disclosure further provides an immunogenic composition comprising a polypeptide comprising the N-terminal half domain and / or the C-terminal half domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal half domain or the C-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains of the N-terminal half domain or the C-terminal half domain has been modified, and wherein the polypeptide cannot substantially bind to host iron-binding proteins. In a preferred embodiment, the modification comprises the modification of at least one amino acid residue within a loop domain.

[0019] In other embodiments, at least two of the plurality of loop domains within the C-terminal half domain and / or the N-terminal half domain of the HIBP surface receptor protein have been modified.

[0020] In other embodiments, the present disclosure provides (i) a first polypeptide comprising an N-terminal or C-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal or C-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains of the N-terminal or C-terminal domain has been modified, the first polypeptide being linked to (ii) a second polypeptide comprising a HIBP surface receptor protein or a portion thereof obtainable from a Gram-negative bacterial species, and wherein the linked polypeptides are not capable of substantially binding to a host iron-binding protein. In a preferred embodiment, the portion of the H1BP surface protein is an N-terminal or C-terminal domain. In a further preferred embodiment, the portion of the HIBP surface protein is an N-terminal or C-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal or C-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains of the N-terminal or C-terminal domain has been modified.

[0021] In other embodiments, the C-terminal or N-terminal domain is the C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a bacterial species belonging to the family Pasteurellaceae, Moraxellaceae or Neisseriaceae, and in a further preferred embodiment, the C-terminal or N-terminal domain is the C-terminal or N-terminal domain of a HIBP surface receptor polypeptide obtainable from or obtained from a bacterial species belonging to the genus Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella or Moraxella.

[0022] In a further preferred embodiment, the HIBP surface receptor protein is modified in such a way that the N-terminal anchor polypeptide or a portion thereof of the HIBP surface receptor protein is removed, and wherein the polypeptide is not capable of substantially binding to a host iron-binding protein.

[0023] In other aspects, the present disclosure provides methods for preparing immunogenic compositions. Accordingly, the present disclosure provides a method for preparing an immunogenic composition, the method comprising:

[0024] (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components:

[0025] (i) A nucleic acid sequence encoding a polypeptide comprising a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; and

[0026] (ii) A nucleic acid sequence capable of controlling expression in a recombinant host cell;

[0027] (b) Introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the polypeptide comprising the C-terminal or N-terminal domain;

[0028] (c) Recovering the polypeptide comprising the C-terminal or N-terminal domain from the host cell; and

[0029] (d) Preparing an immunogenic composition.

[0030] In other embodiments, the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, wherein at least one of the plurality of loop domains has been modified.

[0031] In other aspects, methods for eliciting an immune response in a vertebrate subject are provided. Accordingly, the present disclosure further provides a method for eliciting an immune response in a vertebrate subject, the method comprising administering to the subject:

[0032] (a) An immunogen comprising a polypeptide comprising a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; or

[0033] (b) An expression vector comprising a polynucleotide encoding an immunogen comprising a polypeptide comprising a C-terminal or N-terminal domain of a HIBP surface receptor polypeptide obtainable from a Gram-negative bacterial species; and wherein the immunogen is administered or expressed in an amount sufficient to elicit an immune response in the vertebrate subject.

[0034] In a preferred embodiment, the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, wherein at least one of the plurality of loop domains has been modified.

[0035] The present disclosure further includes an immunogen comprising a C-terminal or N-terminal domain of a HIBP surface receptor polypeptide for use as a medicament, wherein the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified.

[0036] The present disclosure further includes an immunogen comprising the C-terminal or N-terminal domain of a HIBP surface receptor, which is used for preventing an infection (e.g., by preventing colonization) or a disease caused by an infectious Gram-negative bacterium, including bacteria belonging to the genera Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella, or Moraxella. In a preferred embodiment, the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, wherein at least one of the plurality of loop domains has been modified.

[0037] The present disclosure further includes an immunogen comprising the C-terminal and / or N-terminal domain of a HIBP surface receptor polypeptide, which is used for manufacturing a vaccine for preventing an infection or a disease caused by an infectious Gram-negative bacterium, including bacteria belonging to the genera Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella, or Moraxella. In a preferred embodiment, the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, wherein at least one of the plurality of loop domains has been modified.

[0038] The immunogenic composition of the present disclosure can be used to prepare a vaccine. Accordingly, the present disclosure further provides a vaccine composition comprising an antigen derived from a HIBP surface receptor protein from a Gram-negative pathogenic bacterial species, wherein the protein derived from the HIBP surface receptor protein has been modified in such a way that it cannot substantially bind to a host iron-binding protein.

[0039] In other embodiments, the present disclosure further includes a vaccine composition comprising a polypeptide comprising the C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species, wherein the polypeptide cannot substantially bind to a host iron-binding protein.

[0040] In other embodiments, the vaccine composition comprises a polypeptide comprising the C-terminal or N-terminal domain of a HIBP surface receptor polypeptide, wherein the C-terminal or N-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified and wherein the polypeptide cannot substantially bind to a host iron-binding protein.

[0041] The present disclosure further provides a method for administering a vaccine to a vertebrate subject, the method comprising administering to the subject a vaccine comprising a polypeptide comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species, wherein the vaccine is administered in an amount sufficient to prevent or treat a disease caused by a Gram-negative bacterial species.

[0042] The present disclosure further includes a vaccine comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor for preventing an infection or disease caused by an infectious Gram-negative bacterium, the bacterium including a bacterium belonging to the genus Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella, or Moraxella.

[0043] In a preferred embodiment, the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and at least one of the plurality of loop domains has been modified.

[0044] Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description, while indicating preferred embodiments of the present disclosure, is given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the detailed description. Brief Description of the Drawings

[0046] The present disclosure is described with respect to its drawings in the paragraphs provided below. The drawings provided herein are for illustrative purposes and are not intended to limit the present disclosure. Brief Description of the Drawings

[0048] Figure 1 Depicting the alignment of the polypeptide sequences of several TbpBs from the porcine TbpB pathogen and the structural models of the proteins (pdb 3HOL, 3PQS, and 3PQU, respectively), the polypeptide sequences being in particular ApH49 TbpB (SEQ.ID NO: 2), Actinobacillus pleuropneumoniae ApH87 TbpB (SEQ.ID NO: 12), and AsH57 TbpB (SEQ.ID NO: 28). These three proteins provide a good representation of the sequence diversity in TbpB from porcine pathogens ( Figure 4, large black arrows). The upper panel illustrates the polypeptide sequence alignment, while the lower panel illustrates the structural model. In the sequence alignment, the domain structures are distinguished by background shading and labeled accordingly. The secondary structure elements are illustrated and the nomenclature and numbering for the β-strands ("β1" - "β31") and loops (L1 - L32) are shown directly above the aligned sequences. Sub-regions of loop 8 (8a; 8b; and 8c) in this figure are mentioned due to the large size of loop 8 and the differences between the TbpB variants and the secondary structure elements in some of the loop variants. The C-terminal and N-terminal half-cap regions, labeled "C-terminal half-cap region" and "N-terminal half-cap region" respectively, and the C-terminal and N-terminal half-handle regions, labeled "C-terminal half-handle" and "N-terminal half-handle" regions respectively, are further indicated. The structural models for the three TbpBs aligned in the upper panel are depicted in the bottom panel and the domains are labeled for the third structural model (AsH57TbpB), which is depicted in an orientation consistent with the other two models.

[0049] Figure 2 A schematic diagram depicting certain secondary structure features of the amino acid sequence of a TbpB polypeptide with the nomenclature recommended for such proteins. The N-terminus and C-terminus of the polypeptide are indicated and labeled "N" and "C" respectively. The β-strands are indicated by arrows and labeled sequentially from the N-terminus "β1" to "β31". The loop domains are indicated and labeled "L1" to "L32". The loop sequences of the TbpB polypeptide H49 TbpB (SEQ.ID NO: 2) from Actinobacillus pleuropneumoniae strain are included in this patent application (SEQ.ID NO: 41 to SEQ.ID NO: 106). The C-terminal and N-terminal half-cap regions, labeled "C-terminal half-cap region" and "N-terminal half-cap region" respectively, and the C-terminal and N-terminal half-handle regions, labeled "C-terminal half-handle" and "N-terminal half-handle" regions respectively, are further indicated.

[0050] Figure 3A Comparison of the magnitude of the antibody response against the full-length TbpB protein from the human pathogen Neisseria meningitidis (strain B16B6 - SEQ.ID NO: 117) or the full-length TbpB from the porcine pathogen Actinobacillus pleuropneumoniae (strain H49 - SEQ.ID NO: 2) using 33% Emulsigen D as an adjuvant in different host species (mouse, rabbit or pig). The antibody titers (gray bars) against TbpB from Actinobacillus pleuropneumoniae in mice and rabbits are slightly higher than those against TbpB from Neisseria meningitidis (black bars), but are generally lower than in pigs. These results imply that the binding of host transferrin can affect the development of the antibody response.

[0051] Figure 3BDepicts a comparison of the magnitudes of antibody responses against full-length TbpB from the bovine pathogen Mannheimia haemolytica (strain H196 - SEQ.ID NO: 206) or full-length TbpB (SEQ.ID NO: 2), the N-terminal half of TbpB (SEQ.ID NO: 8), or the C-terminal half of TbpB (SEQ.ID NO: 6) from the porcine pathogen Actinobacillus pleuropneumoniae (strain H49) in pigs. The bars represent serum samples taken from individual pigs immunized at day 0 (before the first immunization), day 21 (after the first immunization), day 42 (after the second immunization), and day 56 (after the third immunization). Serum from pigs immunized with full-length TbpB from Mannheimia haemolytica (Mh full; strain H196 - SEQ.ID NO: 206) was tested with full-length Mannheimia haemolytica TbpB bound to an ELISA plate. Serum from pigs immunized with Actinobacillus pleuropneumoniae TbpB (Ap full), the N-terminal half of TbpB (Ap N-terminal half), or the C-terminal half of TbpB (Ap C-terminal half) was analyzed with full-length Actinobacillus pleuropneumoniae TbpB bound to an ELISA plate.

[0052] Figure 3C Depicts a comparison of the cross-reactivity of antisera against the C-terminal half polypeptide domain (SEQ.ID NO: 6), the N-terminal half polypeptide domain (SEQ.ID NO: 8), and full-length TbpB (SEQ.ID NO: 2) of TbpB from the porcine pathogen Actinobacillus pleuropneumoniae (strain H49). The bars represent the reactivity of sera against full-length TbpB from three different porcine pathogens; Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 2, black bar), Haemophilus parasuis strain HP5 (SEQ.ID NO: 115, dark gray bar), and Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 12, light gray bar) were selected to represent antigenically diverse TbpB( Figure 4 ). The results illustrate the reactivity of sera immunized with full-length TbpB (the first bar labeled "full" on the left), the N-terminal half of TbpB (the second bar labeled "N-terminal half" on the left), the C-terminal half of TbpB (the third bar labeled "C-terminal half" on the left), or a mixture of the N-terminal and C-terminal halves (the fourth bar labeled "N + C-terminal half" on the left). Error bars showing the standard error of the mean (SEM) are shown. Statistics were performed via ANOVA, with a Tukey HSD (true significant difference) test as a post hoc test. Asterisks shown in the schema indicate specific immune / protein pairs that are significantly different from the C-terminal half or N + C-terminal half tested against H49.

[0053] Figure 4Depict the sequence diversity of TbpB from Actinobacillus pleuropneumoniae strains, Actinobacillus suis, and Haemophilus parasuis strains isolated from pigs from North America, Europe, and Asia. The maximum likelihood phylogenetic tree illustrates the relationships among 56 TbpBs based on sequences from our collection of clinical isolates or obtained from public databases. The TbpB sequences clustered into 3 major groups with representative isolates indicated by arrows (SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28 and SEQ.ID NO: 107 to SEQ.ID NO: 115). Strains expressing TbpB variants used in the ELISA assay illustrated in Figure 3 are indicated by large black stars; Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 12), Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 2), and Haemophilus parasuis strain HP5 (SEQ.ID NO: 115). The large black arrows depict the three TbpBs used for the Figure 1 alignment illustrated in

[0054] Figure 5 Depict the non-random binding of the N-terminal half of TbpB from Actinobacillus pleuropneumoniae H49 (SEQ.ID NO: 8) to ELISA plates, as illustrated by a substantial decrease in the binding of labeled transferrin when using purified TbpB protein to coat the ELISA plates rather than the precursor of the fusion protein (i.e., maltose-binding protein (Mbp) fused to the N-terminal half of TbpB) (left portions of Figures A and B). The schema also illustrates how the use of N-terminal biotinylated peptides overcomes random binding (right portions of Figures A and B). The left portion of Figure A illustrates the results of an assay using labeled transferrin (Tf) to measure the binding of purified Mbp-TbpB N-terminal half or TbpB N-terminal half to regular ELISA plates. The right portion illustrates the results when the recombinant protein contains a biotinylated N-terminal peptide tag for attachment to streptavidin-coated ELISA plates. Figure B is a cartoon illustrating what is thought to be present in different ELISA wells, with the corresponding results shown directly above in Figure A. *** indicates statistical significance p < 0.001 between the MBP N half and the N-terminal half, ns - not statistically significant.

[0055] Figure 6Describe the design and generation of multimers depicting the C-terminal half of TbpB from three different porcine pathogens. Panel A shows the DNA and protein sequences (in that order) of the trimers (SEQ.ID NO: 39; SEQ.ID NO: 40) of the C-terminal halves (SEQ.ID NO: 6) from Actinobacillus pleuropneumoniae strain H49, Actinobacillus suis strain H57, and Actinobacillus pleuropneumoniae strain H87. Underlines indicate the linker peptide sequences between individual C-terminal halves or in front of the first C-terminal half. Panel B illustrates SDS-PAGE analysis of preparations of the C-terminal half trimers compared to preparations of the N-terminal and C-terminal halves from Neisseria meningitidis strain M982. 1 μl of sample was applied to lanes 1, 4, and 7, 5 μl to lanes 2, 5, and 8 and 10 μl to lanes 3, 6, and 9. The protein molecular weight standards (MWS) observed on these gels were 93, 70, 63, 41, 30, and 22.

[0056] Figure 7 Depict the immunoreactivity against the C-terminal half of TbpB from Actinobacillus pleuropneumoniae (H49 C-terminal half, SEQ.ID NO: 6), compared to the immunoreactivity against the trimer of the C-terminal halves of TbpB containing the C-terminal halves from Actinobacillus pleuropneumoniae H49, Actinobacillus suis H57, and Actinobacillus pleuropneumoniae H87 (C-terminal half trimer, SEQ.ID NO: 40). The bars on the left side of the schema represent the immunoreactivity against the H49 C-terminal half, while the bars on the left side of the schema represent the immunoreactivity against the C-terminal half trimer. Black bars represent the immunoreactivity against TbpB from Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 2), dark gray bars represent the immunoreactivity against TbpB from Haemophilus parasuis strain HP5 (SEQ.ID NO: 115) and light gray bars represent the immunoreactivity against TbpB from Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 22).

[0057] Figure 8Depict the loop regions of the N-terminal half of TbpB of Actinobacillus pleuropneumoniae strain H49 targeted for loop reduction (labeled "Loop 1", SEQ.ID NO: 42; "Loop 5", SEQ.ID NO: 50; "Loop 8a" and "Loop 8c", SEQ.ID NO: 55; and "Loop 12", SEQ.ID NO: 63) respectively, and the sequences of the initial and modified loops. Loop 8a and Loop 8c refer to the Loop 8 portion present in TbpB from strain H49. Figure A is a structural model of the N-terminal half of Actinobacillus pleuropneumoniae TbpB viewed from the side (relative to the predicted major orientation at the cell surface), with the targeted regions labeled. Figure B is the same structural model viewed from the top to illustrate the association of Loops 1 and 5 with the handle domain and Loops 8a, 8c, and 12 that associate with the barrel domain. Figure C is an alignment of native TbpB and TbpB with reduced targeted loop regions. The sequence regions encoding the loops are highlighted in gray and labeled with loop numbers.

[0058] Figure 9 Demonstrate that engineering loop reduction in the N-terminal half of TbpB from Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 10), Actinobacillus suis strain H57 (SEQ.ID NO: 38), and Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 26) does not adversely affect its production or stability, but eliminates the binding of porcine Tf. The upper panel illustrates the production of full-length H49 TbpB (SEQ.ID NO: 2), native h49 TbpB N-terminal half (SEQ.ID NO: 8), and engineered TbpB N-terminal halves from Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 10), Actinobacillus suis strain H57 (SEQ.ID NO: 38), or Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 26). Their expression is as a fusion protein with an N-terminal maltose-binding protein with a polyhistidine tag and is captured on Ni-NTA resin. The bound protein is released in SDS-PAGE buffer and analyzed on a 10% SDS-PAGE gel. The middle panel represents the same preparations captured with an affinity resin consisting of porcine transferrin conjugated to agarose (pTf-agarose) and eluted in SDS-PAGE buffer. The bottom panel illustrates a dot blot analysis, where the material from the upper panel is eluted from the Ni-NTA resin and spotted on nitrocellulose resin, blocked, and exposed to horseradish peroxidase-conjugated porcine transferrin (HRP-pTf) in the blocking solution, and the bound HRP is detected by incubation in the HRP substrate.

[0059] Figure 10 depicts the sequence diversity of TbpB from the human pathogen Neisseria meningitidis. From the http: / / pubmlst.org / software / database / bigsdb / -A subset of the tbpB gene sequenced from over 100 strains assembled from a large collection of sequences obtained from the Bacterial Isolate Genome Sequence Database is represented in this figure. The collection of sequences represents a global collection of isolates over a relatively long period of nearly 50 years and is thus a fairly comprehensive representation of overall TbpB diversity. Figure 10A Illustrate the sequence diversity of full-length TbpB. Sequences of TbpB from strains indicated by arrows, double arrows, or lines (SEQ.ID NO: 117; SEQ.ID NO: 124; SEQ.ID NO: 132 to SEQ.ID NO: 147; SEQ.ID NO: 177; and SEQ.ID NO: 178) are attached to provide representative sequences for the identified groups. Two major clades represented by Group 1 and by Group 2 - 4 are identified within this dendrogram corresponding to the type I and type II Tbp B lineages (22). Support values for the major branches are depicted, and "*" identifies branches with 100% support. Antigen derived from TbpB (SEQ.ID NO: 117, black arrow) from strain B16B6 was used to generate Figure 11 the antiserum analyzed in Figure 5 and screened for reactivity against TbpB (SEQ.ID NO: 123; and SEQ.ID NO: 132 to SEQ.ID NO: 139) from the strain indicated by the grey arrow using our custom ELISA assay ( Figure 10B Depict the sequence diversity of the C-terminal half of TbpB derived from the TbpB sequences. Sequences of the C-terminal half of TbpB from strains indicated by arrows or lines (SEQ.ID NO: 119; SEQ.ID NO: 125; and SEQ.ID NO: 179 to SEQ.ID NO: 195) are attached to provide representative sequences. Two strains indicated by double-headed arrows are included to provide a more comprehensive representation of C-terminal half diversity but are not available for Figure 11 analysis of the antiserum described in

[0060] Figure 11 Depict the reactivity of antisera against truncated full-length TbpB (SEQ.ID NO: 148, aa 43 - 575) and the C-terminal half of TbpB (SEQ.ID NO: 119 aa 342 - 575) derived from B16B6 (a representative type I strain of Neisseria meningitidis). The antiserum was analyzed in our custom ELISA assay ( Figure 5) was tested against a panel of TbpB that represents the overall sequence diversity of TbpB in Neisseria meningitidis (arrows, Figure 10). The panel of TbpB was from Neisseria meningitidis strains B16B6 (SEQ.ID NO: 117), H44 / 76 (SEQ.ID NO: 133), S3131 (SEQ.ID NO: 132), M990 (SEQ.ID NO: 134), M978 (SEQ.ID NO: 135), M992 (SEQ.ID NO: 138), P3006 (SEQ.ID NO: 139), 120M (SEQ.ID NO: 137), MC58 (SEQ.ID NO: 136), and M982 (SEQ.ID NO: 123). The results demonstrated that the C-terminal half antiserum had a higher titer than the TbpB antisera against all TbpB except those from B16B6 and H44 / 76.

[0061] Figure 12 Depicts the design and generation of a dimer (SEQ.ID NO: 118) comprising the C-terminal half of TbpB from two different strains of the human pathogen Neisseria meningitidis. Panel A shows the DNA and protein sequences (in that order) of the dimer of the C-terminal halves from Neisseria meningitidis strains B16B6 (SEQ.ID NO: 118; and SEQ.ID NO: 119) and M982 (SEQ.ID NOS: 124; and SEQ.ID NO: 125). The underlines indicate the DNA sequences of the peptide regions that link the individual C-terminal halves. Panel B illustrates the SDS-PAGE analysis of the preparation of the C-terminal half dimer compared to the preparations of the individual C-terminal halves from Neisseria meningitidis strains M982 and B16B6.

[0062] Figure 13 Depicts the analysis of the immunoreactivity against a dimer (SEQ.ID NO: 150) comprising the C-terminal half of TbpB from two different strains of the human pathogen Neisseria meningitidis. The paired bars represent sera from rabbits immunized with either adjuvant alone (native), with the B16B6 C-terminal half (SEQ.ID NO: 119), with the M982 C-terminal half (SEQ.ID NO: 125), or Figure 12 the dimer (SEQ.ID NO: 150) of the B16B6 and M982 C-terminal halves as described in. The white bars represent the results of a novel custom ELISA assay using immobilized full-length M982 TbpB (SEQ.ID NO: 123) and the black bars represent the results using immobilized full-length B16B6 protein (SEQ.ID NO: 117).

[0063] Figure 14Depiction of the reduction of the loop domain of the C-terminal half of the TbpB polypeptide of Neisseria meningitidis M982. In panel A, structural models of the native C-terminal half (SEQ.ID NO: 125) and the modified C-terminal half (SEQ.ID NO: 129) are shown to illustrate the reduction of four loops (L18, L21, L23, and L27). In the left-hand model (SEQ.ID NO: 125), the loops targeted for reduction are indicated by black dashed lines. The middle model (SEQ.ID NO: 129) illustrates the modified loop domain. In the right-hand model, the two previous structures are overlaid to show how the large variable loops have been removed without affecting the overall protein structure. Panel B is a polypeptide sequence alignment comparing the sequences of the native C-terminal half, an engineered C-terminal half in which a single loop has been modified, and a C-terminal half in which all four loops (L18, L21, L23, and L27) have been modified (the sequence labeled "no loops"). The sequence regions encompassing the targeted loops are highlighted in gray and the loop numbers are indicated in gray font.

[0064] Figure 15 Depiction Figure 14 Microbial production of the modified C-terminal half of Neisseria meningitidis M982 described in. The wild-type (WT) C-terminal half (SEQ.ID NO: 125) corresponds to the left-hand model in panel A of Figure 14 Other samples represent truncated proteins with loops L18, L21, L23, and L27 and a protein in which all four loops have been removed (all loops). The structural model for this protein (SEQ.ID NO: 129) is illustrated in the middle of panel A in Figure 14 The protein molecular weight standards (MWS) observed on this gel were 93, 70, and 41 kDa.

[0065] Figure 16Describe the immunogenicity of the modified C-terminal half of Neisseria meningitidis strain M982 relative to the native C-terminal half from strains M982 and B16B6. The endpoint titers of mouse antisera were determined using our custom ELISA assay. Mice were immunized with the C-terminal half from strain M982 (SEQ.ID NO: 125, first), the C-terminal half TbpB from strain B16B6 (SEQ.ID NO: 119, second), or the 'acycled' M982 C-terminal half (SEQ.ID NO: 129, last two). Sera were tested against immobilized full-length TbpB from strain M982 (SEQ.ID NO: 123) (first and third) or strain B16B6 (SEQ.ID NO: 117) (second and fourth). Results showed that the modified C-terminal half was more immunogenic as it generated higher titers against full-length TbpB from strain M982 than the parental C-terminal half protein (compare bars 3 and 1). Unexpectedly, the modified C-terminal half even generated a degree of reactivity against heterologous B16B6 TbpB similar to that of the C-terminal half from the said strain (compare bars 4 and 2).

[0066] Figure 17 Describe the design of a hybrid protein presenting the TbpA region on the TbpB C-terminal half backbone. Panel A is a structural model of TbpA (SEQ.ID NO: 152), highlighting the regions selected for 'transplantation' onto the TbpB C-terminal half. TbpA loop 3 helix, loop 10, loop 11, and the plug loop are shown as space-filling regions. Panel B shows the alignment of the native C-terminal half (C-terminal half) (SEQ.ID NO: 125), the acycled C-terminal half backbone (acycled C) (SEQ.ID NO: 129), and the hybrid protein presenting all TbpA regions (SEQ.ID NO: 131). In the hybrid protein, TbpA loop 3 helix replaces loop 18 of the TbpB C-terminal half, TbpA loop 10 replaces loop 21 of the TbpB C-terminal half, TbpA loop 11 replaces loop 23 of the TbpB C-terminal half, and the TbpA plug loop replaces loop 27 of the TbpB C-terminal half.

[0067] Figure 18 Describe the use of Figure 17Microbial production of the hybrid TbpA-TbpB C-terminal half generated by the strategy described in . Figure A illustrates the production of recombinant fusion proteins with an N-terminal maltose-binding protein (Mbp) fusion partner and Figure B illustrates the protein after cleavage with TEV protease. The wild-type (WT) protein is the native M982 C-terminal half (SEQ.ID NO: 125) and the negative loop is the C-terminal half with all four loops removed (SEQ.ID NO: 129), which effectively acts as a scaffold for presenting the TbpA region. Loop 10 refers to the protein with the extracellular loop region of TbpA inserted into loop 21 of the TbpB C-terminal half (SEQ.ID NO: 154). Loop 11 refers to the protein with the extracellular loop region of TbpA inserted into loop 23 of the TbpB C-terminal half (SEQ.ID NO: 156). Helix 3 refers to the extracellular loop 3 region of TbpA inserted into loop 18 of the TbpB C-terminal half (SEQ.ID NO: 158). The plug loop refers to the region from the TbpA plug domain inserted into loop 27 of the TbpB C-terminal half (SEQ.ID NO: 160). The protein molecular weight standards (MWS) observed on these gels were 93, 70, 53, 41, and 22.

[0068] Figure 19 Depicts the immunogenicity of the modified C-terminal half of Neisseria meningitidis strain M982 compared to the modified C-terminal half with foreign loop regions from TbpA spliced into modified loop sites. The endpoint titers of mouse antisera were determined using our custom ELISA assay. Mice were immunized with: (i) the 'loopless' C-terminal half with all four loops removed (SEQ.ID NO: 129), (ii) the 'loopless' C-terminal half with TbpA loop 10 inserted into loop 21 of the TbpB C-terminal half (SEQ.ID NO: 154), (iii) the 'loopless' C-terminal half with TbpA loop 11 inserted into loop 23 of the TbpB C-terminal half (SEQ.ID NO: 156), (iv) the 'loopless' C-terminal half with TbpA loop helix 3 inserted into loop 18 of the TbpB C-terminal half (SEQ.ID NO: 158), or (v) the 'loopless' C-terminal half with the TbpA plug loop inserted into loop 27 of the TbpB C-terminal half (SEQ.ID NO: 160). The sera were tested against the hybrid TbpA-TbpB antigen (SEQ.ID NO: 131) in which the 'loopless' C-terminal half had all four TbpA loops inserted. The results show that all hybrid antigens are immunogenic, at least as immunogenic as the 'loopless' C-terminal half.

[0069] Figure 20Depiction of the design and generation of a hybrid protein presenting the LbpA region (SEQ.ID NO: 162) on the C-terminal half-skeleton of TbpB. Figure A is a structural model of LbpA, highlighting the regions selected for 'transplantation' onto the C-terminal half of TbpB. LbpA loop 3 helix is colored in a darker gray and loop 2 is colored black. Figure B shows the alignment of the native C-terminal half (C-terminal half, SEQ.ID NO: 125), the acyclic C-terminal half-skeleton (acyclic C, SEQ.ID NO: 129), and the hybrid protein in which the LbpA region is presented. In the hybrid protein, LbpA loop 2 replaces loop 21 (SEQ.ID NO: 164) of the TbpB C-terminal half, LbpA loop 3 helix replaces loop 18 (SEQ.ID NO: 166) of the TbpB C-terminal half and. The protein molecular weight standards (MWS) observed on these gels are 100, 75, 63, and 48.

[0070] Figure 21 Depiction of the 'conjugation loop' in the C-terminal half of TbpB from the human pathogen Haemophilus influenzae. Figure A shows the DNA and protein sequences for the gene encoding the hybrid gene, which has a DNA region encoding the conjugation loop shown in larger font (SEQ.ID NO: 167). Amino acids are shown in single-letter code, where lysine is indicated by the letter K, and where there are 42 in the conjugation loop compared to 24 in the full C-terminal half (SEQ.ID NO: 168). Figure B illustrates the structural model of the Haemophilus influenzae TbpB C-terminal half, indicating the insertion position of the conjugation loop. As illustrated, the conjugation loop is inserted into the handle domain of the C-terminal half, replacing loop L23 of the C-terminal half (using the loop nomenclature used in this disclosure)( Figure 2 ). It should be noted that for illustrative purposes, the model was generated with an 11-amino acid conjugation loop rather than the 91 in the actual protein.

[0071] Figure 22Characterize the transferrin-binding properties of site-directed mutant TbpB proteins depicting recombinant truncated TbpB proteins derived from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis. The recombinant truncated TbpB proteins are expressed as fusion proteins and tested for binding activity. The recombinant fusion proteins are initially screened for transferrin binding by solid-phase binding assays and affinity capture assays. The purified mutant proteins (23-25) are then evaluated for binding to pTf by isothermal calorimetry, surface plasmon resonance, or biolayer interferometry. Several mutations cause a ≥100-fold increase in the affinity constant (Kd), such as the F171A mutation in TbpB from Actinobacillus pleuropneumoniae strain H49, the Y174A mutation in TbpB from Actinobacillus pleuropneumoniae strain H87, or the Y167A mutation in TbpB from Actinobacillus suis strain HP5. Interestingly, it is noted that all of these mutants map to loop 8.

[0072] Figure 23 Characterize the enhanced ability of site-directed mutant proteins to induce a protective immune response in the natural host. In this figure, the ability of site-directed Y167A TbpB (SEQ.ID NO: 174) from Haemophilus parasuis strain HP5 is compared to wild-type TbpB (SEQ.ID NO: 115) and control groups including a commercial vaccine (Porcillis Glasser) and adjuvant alone. The pigs are challenged by intratracheal inoculation with 108 colony-forming units (cfu) of the Hp5 (Nagasaki) strain and monitored for clinical signs and symptoms for the duration of the experiment. Animals with severe symptoms are euthanized before the end of the experiment. The figure shows survival curves at 12-h increments from 24 to 108 h and then connected to the final time point at 14 days.

[0073] Figure 24 Characterize the cellular immune responses induced by native and mutant TbpB antigens. Figures A and B illustrate B-cell responses on the day of challenge (after two IM immunizations) and 4 days (96 h) after challenge, respectively. Figures C and D illustrate T-helper cell responses on the day of challenge and 4 days after challenge. Diamonds, triangles, and squares represent pigs immunized with native TbpB, Y167A mutant TbpB, and Porcilis (PG) vaccine, respectively. The number of samples for native TbpB and PG vaccine-treated pigs decreased on day 4 after challenge. The analysis was performed by FACS analysis of peripheral blood mononuclear cells. Significant differences between groups: *p < 0.05, ***p < 0.001.

[0074] Figure 25Depiction of immunization with recombinant truncated TbpB, recombinant truncated N-terminal half of TbpB, or recombinant C-terminal half of TbpB from Neisseria meningitidis provides protection from colonization in a humanized transgenic mouse model. In the experiment illustrated in Panel A, transgenic C57BL mice expressing the human CEACAM1 receptor were immunized on days 1 and 21 with recombinant truncated TbpB from Neisseria meningitidis strain M982 or with adjuvant alone. Mice were inoculated intranasally on day 35 with approximately 1×107 CFU of Neisseria meningitidis strain M982. Squares and circles represent the CFU recovered from individual mice 3 days after challenge (day 38). Panel B illustrates a follow-up experiment in which mice were immunized on days 1 and 21 with recombinant truncated TbpB, recombinant truncated N-terminal half of TbpB, recombinant C-terminal half of TbpB, recombinant factor H binding protein, or adjuvant alone. As in Panel A, the number of CFU of Neisseria meningitidis strain M982 recovered 3 days after challenge in individual mice is plotted in this figure.

[0075] Figure 26 depicts the sequence diversity of TbpB and the C-terminal half of TbpB from the human pathogen Neisseria gonorrhoeae. Panel A ( Figure 26A ) illustrates the sequence diversity of full-length TbpB and Panel A ( Figure 26B ) depicts the sequence diversity of the C-terminal half of TbpB. Sequences for representative Neisseria meningitidis TbpB and the C-terminal half of TbpB (Figure 10, arrows and double arrows) were included in this analysis to determine the extent to which the sequences for TbpB and the C-terminal half of TbpB from Neisseria gonorrhoeae are a subset of the sequence diversity of the sequences present in Neisseria meningitidis. As for Figure 10, the representative Neisseria gonorrhoeae TbpB sequences (SEQ ID NOs: 207 to 212) and C-terminal half of TbpB sequences (SEQ ID NOs: 213 to 218) indicated by arrows are included in the Appendix to provide a representation of the overall sequence diversity. As illustrated in Panel A, there are two clusters of Neisseria gonorrhoeae TbpB that are sub-branches of meningococcal serogroup 2 TbpB. There is a larger cluster most closely related to TbpB from Neisseria meningitidis strain H44 / 76 and a smaller cluster most closely related to TbpB from strain P3306. The C-terminal half dendrogram in Panel B reveals that the C-terminal half of Neisseria gonorrhoeae TbpB forms a cluster distinct from the C-terminal half of meningococcal TbpB most closely related to meningococcal serogroup 2 TbpB.

[0076] Figure 27 depicts the sequence diversity of TbpB and the C-terminal half of TbpB from the human pathogen Haemophilus influenzae. Panel A ( Figure 27A)Describe the sequence diversity of full-length TbpB and Figure B( Figure 26B )represents the sequence diversity of the C-terminal half of TbpB. Representative Haemophilus influenzae TbpB sequences (SEQ ID NO: 196 - 204), indicated by arrows, are included in the appendix to provide a representation of the overall sequence diversity. As illustrated in Figure A, there are three major clusters (groups) of Haemophilus influenzae TbpB, which include a mixture of type b and non-typeable Haemophilus influenzae strains, indicating that TbpB diversity is not associated with any other property, such as the presence of a capsule. There are also three major groups of C-terminal half diversity.

[0077] Figure 28 Depict the sequence diversity of TbpB in the ruminant pathogens Mannheimia haemolytica, Mannheimia glucosida, and Pasteurella trehalosi (also known in the art as Pasteurella haemolytica and Pasteurella trehalosi, respectively). Arrows indicate the representative sequences included in the SEQ.ID NO: list.

[0078] Figure 29 Depict the sequence diversity of TbpB from Moraxella catarrhalis. Arrows indicate the representative sequences of TbpB from three major clusters included in the SEQ.ID NO: list.

[0079] Figure 30 Depict an illustrative phylogenetic tree.

[0080] Tables 1 - 3 represent combinations of 1, 2, or 3 loop domains selected from the loop domains L1 - L32 of the HIBP polypeptide, which can be modified according to the present disclosure.

[0081] Represents loop domains or combinations of loop domains that can be modified according to certain embodiments of the present disclosure.

[0082] Represents non-permissible combinations of loop domains according to certain embodiments of the present disclosure.

[0083] □ represents permissible combinations of loop domains, however presented

[0084] Elsewhere in the same table, according to certain embodiments of the present disclosure.

[0085] Disclosed in detail

[0086] Various compositions and methods will be described below to provide examples of embodiments of each claimed subject matter. None of the embodiments described below limit any claimed subject matter and any claimed subject matter may cover methods, processes, compositions, or systems different from those described below. The claimed subject matter is not limited to compositions or methods having all of the features of any of the compositions, methods, systems, or processes described below, or to features common to multiple or all of the compositions, systems, or methods described below. It is possible that the compositions, systems, methods, or processes described below are not embodiments of any claimed subject matter. Any subject matter disclosed in the compositions, systems, methods, or processes described below that is not claimed in this document may be the subject of another protective document (such as a continuation patent application), and the applicant, inventor, or owner does not intend to disclaim, waive, or dedicate to the public any such subject matter by the disclosure of any such subject matter in this document.

[0087] It should be noted that degree terms such as "substantially", "essentially", "about", and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These degree terms should be construed to include deviations of the modified term as long as such deviations do not negate the meaning of the term being modified.

[0088] As used herein, the phrase "and / or" is intended to mean an inclusive - or. That is, "X and / or Y" is intended to mean, for example, X or Y or both. As another example, "X, Y, and / or Z" is intended to mean X or Y or Z or any combination thereof.

[0089] All publications, patents, and patent applications are hereby incorporated by reference in their entirety as if each individual publication were incorporated.

[0090] As mentioned above, the present disclosure provides novel immunogenic compositions and specifically immunogenic compositions based on HIBP surface receptor proteins from Gram - negative pathogenic bacterial species such as Neisseria meningitidis. The immunogenic compositions of the present disclosure are applicable because they can be used to prepare novel vaccine formulations to protect humans and animals against infectious pathogenic Gram - negative bacterial species. According to the present disclosure, the HIBP surface receptor proteins of the present disclosure are modified in such a way that they cannot substantially bind to host iron - binding proteins. The modified HIBP surface receptor proteins exhibit unexpectedly strong immunogenic properties. In addition, the immunogenic compositions of the present disclosure are substantially stable polypeptides and can thus be easily manufactured. Moreover, and importantly, the immunogenic compositions of the present disclosure are unexpectedly effective, for example, by inducing cross - reactive immune responses, thus allowing protection against multiple pathogenic microorganisms by administering a single effective vaccination compound. Vaccines prepared according to the present disclosure do not contain live organisms or crude extracts, thereby representing an extremely limited health risk.

[0091] Accordingly, in at least one embodiment, the present disclosure provides an immunogenic composition comprising a HIBP surface receptor protein from a Gram-negative pathogenic bacterial species, wherein the HIBP surface receptor protein has been modified in such a way that it cannot substantially bind to a host iron-binding protein.

[0092] The present disclosure further provides an immunogenic composition comprising a polypeptide comprising the C-terminal half domain or the N-terminal half domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species, wherein the polypeptide cannot substantially bind to a host iron-binding protein. In certain embodiments, the N-terminal half domain or the C-terminal half domain of the HIBP surface receptor protein comprises a plurality of β-strands connected by a plurality of loop domains, and one or more of the plurality of loop domains have been modified.

[0093] Terms and Definitions

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Wherever permitted, all patents and patent applications and other publications (including nucleic acid and polypeptide sequences from GenBank, SwissPro, and other databases) cited herein (above or below) are hereby incorporated by reference in their entirety. It should be further noted that, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "an immunogen" includes a mixture of two or more of such agents, a reference to "a polypeptide" includes a reference to a mixture of two or more polypeptides, a reference to "a cell" includes two or more of such cells, and so on.

[0095] The terms "immunogen" and "immunogenic composition", which are used interchangeably herein, are used in their broadest sense to refer to a molecule containing one or more epitopes that will stimulate an immune response in a host organism to generate a cell-mediated immunogen-specific immune response and / or a humoral antibody response. Immunogens include nucleic acids, proteins, polypeptides, peptides, and immunogenic protein fragments.

[0096] The terms "vaccine" and "vaccine composition", which are used interchangeably herein, refer to any pharmaceutical composition containing an immunogen that can be used to prevent or treat a disease or condition in a subject. The term thus encompasses subunit vaccines, i.e., vaccine compositions containing an immunogen that is isolated and separated from the intact organism with which it is actually associated.

[0097] The term "vertebrate subject" refers to any member of the subphylum Chordata, particularly mammals, including but not limited to humans and other primates. The term does not denote a specific age. Thus, neonates, infants, children, and adult individuals are intended to be encompassed.

[0098] The terms "HIBP surface receptor protein", "HIBP surface receptor polypeptide", "host iron-binding protein surface receptor protein", or "host iron-binding protein surface polypeptide", which may be used interchangeably herein, refer to any membrane-anchored protein or polypeptide obtainable from a Gram-negative bacterial species that is capable of interacting with a host iron-binding protein. The term includes any TbpB and LbpB proteins. The HIBP surface receptor protein, when folded in its native three-dimensional structure, contains a bipartite structure that includes an N-terminal half-domain and a C-terminal half-domain, each of which contains multiple β-strands assembled in a β-barrel and multiple β-strands assembled in a β-sheet structure adjacent to the β-barrel (referred to as the handle domain), where the β-strands are connected by multiple loop domains (as further described in Figure 1 ). The term further refers to any and all HIBP surface receptor polypeptide sequences, including all bacterial HIBP surface receptor polypeptides, including but not limited to those shown in SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28; SEQ.ID NO: 107 to SEQ.ID NO: 115; SEQ ID NO: 117; SEQ.ID NO: 123; SEQ.IDNO: 131 to SEQ.ID NO: 147; SEQ.ID NO: 177; SEQ.ID NO: 178; SEQ.ID NO: 196 to SEQ.ID NO: 204; SEQ.ID NO: 206 to SEQ.ID NO: 212; and those shown in SEQ.ID NO: 219 to SEQ.ID NO: 228, and those of sequences containing the following amino acid residues that (i) are substantially identical to the amino acid sequences constituting any HIBP surface receptor protein shown herein; (ii) are encoded by a nucleic acid sequence capable of hybridizing to any nucleic acid sequence encoding any HIBP surface receptor protein shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, capable of hybridizing to any nucleic acid sequence encoding any HIBP surface receptor protein shown herein under at least moderately stringent conditions. The term further includes any HIBP surface receptor protein precursor polypeptide; or (iii) when submitted to a structure modeling server, will use a transferrin-binding protein, lactoferrin-binding protein, or a subdomain thereof as a template, such as Phyre2 ( http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) or Swiss-Model ( http: / / swissmodel.expasy.org / ), the latter selecting the automated mode. The term further includes mature TbpB polypeptides and any HIBP surface receptor polypeptide precursors, including any pre-HIBP surface receptor polypeptide precursors or HIBP surface receptor polypeptide precursors that contain an N-terminal or other signal sequence.

[0099] The terms "integral outer membrane protein" and "IOM protein" as used interchangeably herein refer to any integral outer membrane protein from a Gram-negative bacterial species, including any protein belonging to the TonB-dependent subclass of proteins that, when folded in its native three-dimensional structure, contains a 22-stranded C-terminal beta-barrel domain and an N-terminal plug or cork domain capable of filling the channel in the C-terminal beta-barrel domain. The term includes, without limitation, TbpA and LbpA proteins. The term further refers to any and all IOM polypeptide sequences, including all of the following, including those shown in SEQ.ID NO: 152 and SEQ.ID NO: 162 and those of sequences containing the following amino acid residues that (i) are substantially identical to the amino acid sequence constituting any IOM protein shown herein; (ii) are encoded by a nucleic acid sequence capable of hybridizing to any nucleic acid sequence encoding any IOM protein shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, capable of hybridizing to any nucleic acid sequence encoding any IOM protein shown herein under at least moderately stringent conditions. The term further includes any IOM protein precursor polypeptide; or (iii) will use 3V8X or its subdomains as a template when submitted to a structure modeling server such as Phyre2( http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) or Swiss-Model( http: / / swissmodel.expasy.org / ), the latter selecting the automated mode.

[0100] The term "N-terminal half domain" as used herein refers to the N-terminal portion of a HIBP surface receptor protein that contains multiple beta-strands connected by multiple loop domains, some of which beta-strands are configured to form a beta-barrel and an adjacent beta-sheet structure (referred to as the handle domain) (see: Figure 1 ; amino acid residues 46 to amino acid residue 342). The term N-terminal half domain further includes, without limitation, all polypeptides having the sequences shown in SEQ.ID NO: 8; SEQ.ID NO: 10; SEQ.ID NO: 24; SEQ.ID NO: 26; SEQ.ID NO: 36; SEQ.ID NO: 38; SEQ.ID NO: 121; SEQ.ID NO: 127; SEQ.ID NO: 229; SEQ.ID NO: 231; and SEQ.ID NO: 233.

[0101] As used herein, the term "C-terminal half domain" refers to the C-terminal portion of the HIBP surface receptor protein, which comprises a plurality of β-strands connected by a plurality of loop domains, some of which are configured to form a β-barrel and an adjacent β-sheet structure (referred to as the handle domain). Further reference is made to Figure 1 and Figure 2 , the C-terminal half handle domain is a continuous polypeptide domain starting from β-strand 16 and extending to and including β-strand 23, which in the case of the ApH49, ApH57 and ApH87 TbpB polypeptides depicted in Figure 2 is composed of amino acid residues 344 to 431, and in SEQ.ID.NO:2 from amino acid residue 314 to amino acid residue 401 (ApH 49), in SEQ.ID.NO:27 from amino acid residue 363 to amino acid residue 450 (ApH 57), and in SEQ.ID.NO:12 from amino acid residue 315 to amino acid residue 401 (ApH 87). The C-terminal half β-barrel domain is a continuous polypeptide domain starting from β-strand 23 and extending to the C-terminal of the polypeptide, which in the case of the ApH49, ApH57 and ApH87 TbpB polypeptides depicted in Figure 2 is the polypeptide chain starting from amino acid residue 443 and extending to the C-terminal, and in SEQ.ID.NO:2 starting from amino acid residue 413 (ApH49), in SEQ.ID.NO:27 starting from amino acid residue 462 (ApH 57), and in SEQ.ID.NO:12 starting from amino acid residue 413. It should be noted that the C-terminal half handle domain and the C-terminal half β-barrel domain may be connected by short loops ( Figure 1 and Figure 2 represented as "L24" in). It should be further noted that the term C-terminal half domain as used herein is specifically intended to include not only the C-terminal half β-barrel domain, but also the handle domain that forms the β-sheet structure, which typically contains about 90 or more amino acid residues and is located N-terminal to the C-terminal half β-barrel. The term C-terminal half domain as used herein further includes, but is not limited to, all polypeptides shown in SEQ.ID NO:5; SEQ.ID NO:6; SEQ.ID NO:22; SEQ.ID NO:33; SEQ.ID NO:34; SEQ.ID NO:119; SEQ.ID NO:125; SEQ.ID NO:179 to SEQ.ID NO:195; SEQ.ID NO:213 to SEQ.ID NO:218; SEQ.ID NO:230; SEQ.ID NO:232; SEQ.ID NO:234 to SEQ.ID NO:278; and SEQ.ID NO:288 to SEQ.ID NO:292.

[0102] The term "loop domain" refers to the polypeptide sequences in the HIBP surface receptor protein that connect two β-strands. The lengths of these polypeptide sequences can vary significantly from several amino acid residues to 150 or more amino acid residues.

[0103] The terms "TbpB", "TbpB protein", "TbpB polypeptide", as used interchangeably herein, refer to any and all transferrin-binding protein B sequences, including all bacterial TbpB polypeptides and polypeptides comprising the amino acid residues that (i) are substantially identical to the amino acid sequence of any TbpB polypeptide shown herein, including but not limited to SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28; SEQ.ID NO: 107 to SEQ.ID NO: 115; SEQ ID NO: 117; SEQ.ID NO: 123; SEQ.ID NO: 131 to SEQ.ID NO: 147; SEQ.ID NO: 177; SEQ.ID NO: 178; SEQ.ID NO: 196 to SEQ.ID NO: 204; SEQ.ID NO: 206 to SEQ.ID NO: 212; and SEQ.ID NO: 219 to SEQ.ID NO: 228, or (ii) are encoded by a nucleic acid sequence capable of hybridizing to any nucleic acid sequence encoding any TbpB polypeptide shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, capable of hybridizing to any nucleic acid sequence encoding any TbpB polypeptide shown herein under at least moderately stringent conditions. The term further includes mature TbpB polypeptides and any TbpB precursors, including any pre-TbpB or TbpB comprising an N-terminal or other signal sequence.

[0104] The terms "LbpB", "LbpB protein", "LbpB polypeptide", as used interchangeably herein, refer to any and all lactoferrin-binding protein B sequences, including all bacterial LbpB polypeptides and polypeptides comprising the amino acid residues that (i) are substantially identical to the amino acid sequence of any LbpB polypeptide shown herein, including but not limited to SEQ.ID NO: 285, or (ii) are encoded by a nucleic acid sequence capable of hybridizing to any nucleic acid sequence encoding any LbpB polypeptide shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, capable of hybridizing to any nucleic acid sequence encoding any LbpB polypeptide shown herein under at least moderately stringent conditions. The term further includes any LbpB precursors, including pre-LbpB.

[0105] The terms "TbpA", "TbpA protein", "TbpA polypeptide", which may be used interchangeably herein, refer to any and all transferrin-binding protein A sequences, including all bacterial TbpA polypeptides and polypeptides comprising a sequence of amino acid residues that (i) are substantially identical to the amino acid sequence of any TbpA polypeptide shown herein, including but not limited to SEQ.ID NO: 152, or (ii) are encoded by a nucleic acid sequence that is capable of hybridizing to any nucleic acid sequence encoding any TbpA polypeptide shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, is capable of hybridizing to any nucleic acid sequence encoding any TbpA polypeptide shown herein under at least moderately stringent conditions. The term further includes any TbpA precursor, including pro-TbpA.

[0106] The terms "LbpA", "LbpA protein", "LbpA polypeptide", which may be used interchangeably herein, refer to any and all lactoferrin-binding protein A sequences, including all bacterial LbpA polypeptides and polypeptides comprising a sequence of amino acid residues that (i) are substantially identical to the amino acid sequence of any LbpA polypeptide shown herein, including but not limited to SEQ.ID NO: 162, or (ii) are encoded by a nucleic acid sequence that is capable of hybridizing to any nucleic acid sequence encoding any LbpA polypeptide shown herein under at least moderately stringent conditions or, except for the use of synonymous codons, is capable of hybridizing to any nucleic acid sequence encoding any LbpA polypeptide shown herein under at least moderately stringent conditions. The term further includes any LbpA precursor, including pro-LbpA.

[0107] The term "nucleic acid sequence" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include azido and deazido adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine.

[0108] The terms "nucleic acid sequence encoding a HIBP surface receptor protein" and "nucleic acid sequence encoding a HIBP surface receptor polypeptide", which are used interchangeably herein, refer to any and all nucleic acid sequences encoding a HIBP surface receptor protein, including any HIBP surface receptor protein and any nucleic acid sequence encoding a HIBP surface receptor protein precursor, including but not limited to those shown in SEQ.ID NO:1; SEQ.ID NO:11; SEQ.ID NO:27; SEQ.ID NO:116; SEQ.ID NO:122; and SEQ.ID NO:173. As used herein, a "HIBP surface receptor protein precursor" refers to a HIBP surface receptor protein molecule that additionally contains an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding a HIBP surface receptor protein further includes any and all of the following nucleic acid sequences: (i) encoding a polypeptide that is substantially identical to the HIBP surface receptor protein sequence shown herein; or (ii) hybridizing to any of the HIBP surface receptor protein nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or, except for the use of synonymous codons, would hybridize thereto under at least moderately stringent conditions.

[0109] The terms "nucleic acid sequence encoding an IOM protein" and "nucleic acid sequence encoding an IOM polypeptide", which are used interchangeably herein, refer to any and all nucleic acid sequences encoding an IOM protein, including any IOM protein and any nucleic acid sequence encoding an IOM protein precursor, including but not limited to those shown in SEQ.ID NO:151 and SEQ.ID NO:161. As used herein, an "IOM protein precursor" refers to an IOM protein molecule that additionally contains an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding an IOM protein further includes any and all of the following nucleic acid sequences: (i) encoding a polypeptide that is substantially identical to the IOM protein sequence shown herein; or (ii) hybridizing to any of the IOM protein nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or, except for the use of synonymous codons, would hybridize thereto under at least moderately stringent conditions.

[0110] As used interchangeably herein, the terms "nucleic acid sequence encoding TbpB" and "nucleic acid sequence encoding a TbpB polypeptide" refer to any and all nucleic acid sequences encoding a TbpB polypeptide, including any TbpB polypeptide, including but not limited to those shown in SEQ.ID NO:1; SEQ.ID NO:11; SEQ.ID NO:27; SEQ.ID NO:116; SEQ.ID NO:122; and SEQ.ID NO:173, and further including any nucleic acid sequence encoding a TbpB precursor. As used herein, a "TbpB precursor" refers to a TbpB molecule that further includes an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding a TbpB polypeptide further includes any and all of the following nucleic acid sequences that (i) encode a polypeptide that is substantially identical to the TbpB polypeptide sequences shown herein; or (ii) hybridize to any of the TbpB nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or would hybridize thereto under at least moderately stringent conditions except for the use of synonymous codons.

[0111] As used interchangeably herein, the terms "nucleic acid sequence encoding LbpB" and "nucleic acid sequence encoding an LbpB polypeptide" refer to any and all nucleic acid sequences encoding an LbpB polypeptide, including any LbpB polypeptide (including but not limited to the sequence shown in SEQ.ID NO:284) and any nucleic acid sequence encoding an LbpB precursor. As used herein, an "LbpB precursor" refers to an LbpB molecule that further includes an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding an LbpB polypeptide further includes any and all of the following nucleic acid sequences that (i) encode a polypeptide that is substantially identical to the LbpB polypeptide sequences shown herein; or (ii) hybridize to any of the LbpB nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or would hybridize thereto under at least moderately stringent conditions except for the use of synonymous codons.

[0112] As used interchangeably herein, the terms "nucleic acid sequence encoding TbpA" and "nucleic acid sequence encoding a TbpA polypeptide" refer to any and all nucleic acid sequences encoding a TbpA polypeptide, including any TbpA polypeptide (including, but not limited to, the nucleic acid sequence shown in SEQ.ID NO: 151) and any nucleic acid sequence encoding a TbpA precursor. As used herein, "TbpA precursor" refers to a TbpA molecule further comprising an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding a TbpA polypeptide further includes any and all of the following nucleic acid sequences: (i) encoding a polypeptide substantially identical to the TbpA polypeptide sequence shown herein; or (ii) hybridizing to any of the TbpA nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or, except for the use of synonymous codons, would hybridize to it under at least moderately stringent conditions.

[0113] As used interchangeably herein, the terms "nucleic acid sequence encoding LbpA" and "nucleic acid sequence encoding an LbpA polypeptide" refer to any and all nucleic acid sequences encoding an LbpA polypeptide, including any LbpA polypeptide (including, but not limited to, the nucleic acid sequence shown in SEQ.ID NO: 161) and any nucleic acid sequence encoding an LbpA precursor. As used herein, "LbpA precursor" refers to an LbpA molecule further comprising an N-terminal signal sequence that facilitates the export of the polypeptide chain across the cytoplasmic membrane. The nucleic acid sequence encoding an LbpA polypeptide further includes any and all of the following nucleic acid sequences: (i) encoding a polypeptide substantially identical to the LbpA polypeptide sequence shown herein; or (ii) hybridizing to any of the LbpA nucleic acid sequences shown herein under at least moderately stringent hybridization conditions or, except for the use of synonymous codons, would hybridize to it under at least moderately stringent conditions.

[0114] The term "substantially identical" means that two polypeptide sequences are preferably at least 50% identical, more preferably at least 85% identical and most preferably at least 95% identical, such as 96%, 97%, 98% or 99% identical. To determine the percentage of identity between two polypeptide sequences, an alignment method such as that of Needleman and Wunsch (26) is used to align the amino acid sequences of the two sequences, as modified by Smith and Waterman (27), such that a maximum order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. A preferred, widely applicable method for accurately aligning two polypeptides involves the Clustal W algorithm (28) used in conjunction with the BLOSUM 62 scoring matrix (29) with a gap opening penalty of 10 and a gap extension penalty of 0.1. This enables the identification of a high-scoring alignment between the two sequences, where at least 50% of the total length of one of the two sequences is involved in the alignment. Methods for calculating the percentage of identity between two aligned amino acid sequences are generally recognized in the art and include, for example, those described by Carillo and Lipton (30) and those described in Computational Molecular Biology, Lesk ed Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. In general, computer programs will be used for the calculation. Computer programs that can be used in this regard include, but are not limited to, GCG (31) BLASTP, BLASTN and FASTA (32).

[0115] "At least moderately stringent hybridization conditions" means conditions selected to promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by the Tm, which in a sodium-containing buffer is a function of the sodium ion concentration and temperature (Tm = 81.5 °C - 16.6 (Log10[Na+]) + 0.41 (%(G+C) - 600 / l), or a similar equation). Thus, the parameters that determine hybrid stability in the wash conditions are the sodium ion concentration and temperature. To identify a molecule that is similar but not identical to a known nucleic acid molecule, a 1% mismatch can be assumed to result in an approximate 1 °C decrease in Tm. For example, if a nucleic acid molecule with >95% identity is being sought, the final wash temperature will be decreased by approximately 5 °C. Based on these considerations, those skilled in the art will be able to readily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. For example, the following conditions can be used to achieve stringent hybridization: hybridize at Tm (based on the above equation) - 5 °C in 5× sodium chloride / sodium citrate (SSC) / 5× Denhardt's solution / 1.0% SDS, followed by washing at 60 °C in 0.2× SSC / 0.1% SDS. Moderately stringent hybridization conditions include a wash step in 3× SSC at 42 °C. However, it should be understood that alternative buffers, salts, and temperatures can be used to achieve equivalent stringency. Additional guidance on hybridization conditions can be found in: Green and Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012 (33).

[0116] As used herein in the context of nucleic acid sequences, the term "chimeric" refers to at least two joined nucleic acid sequences that are not naturally joined. Chimeric nucleic acid sequences include nucleic acid sequences joined from different natural origins. For example, a nucleic acid sequence of a bacterial promoter that forms part of a nucleic acid sequence linked to a TbpB polypeptide or HIBP surface receptor protein is considered chimeric, and a nucleic acid sequence encoding a TbpB polypeptide in which some portions have been removed and replaced with portions of a TbpA polypeptide is considered chimeric. Chimeric nucleic acid sequences can also contain nucleic acid sequences of the same natural origin, provided they are not naturally joined. For example, a nucleic acid sequence that forms a promoter obtained from a particular cell type can be linked to a nucleic acid sequence encoding a polypeptide obtained from the same cell type but that is not normally linked to the nucleic acid sequence that forms the promoter. Chimeric nucleic acid sequences also include nucleic acid sequences that contain any naturally occurring nucleic acid sequence linked to any non-naturally occurring nucleic acid sequence.

[0117] The term "substantially unable to bind host iron-binding proteins" means that the ability of host iron-binding proteins to bind to the HIBP surface receptor protein is attenuated in such a way that the binding constant (Kd) or dissociation constant value of the binding interaction between the native host iron-binding protein (i.e., the host iron-binding protein present in the host organism) and the modified HIBP surface receptor protein is at least 10-fold that of the binding constant value of the binding interaction between the native host iron-binding protein and its complementary native HIBP surface receptor protein. In other words, the modified protein has 10-fold lower affinity for binding the native host iron-binding protein than the native receptor protein. In a preferred embodiment, the relative affinity of the modified protein for binding the native host iron-binding protein is 30-fold lower than the affinity of the native HIBP surface receptor protein, and in the most preferred embodiment, the relative affinity of the modified protein for binding the native host iron-binding protein is 100-fold lower than the affinity of the native HIBP surface receptor protein. In a further preferred embodiment, the binding constant between the modified HIBP surface receptor protein and the native host iron-binding protein is at least 300 nM. Preferably, the binding constant is at least 500 nM, and most preferably at least 1 μM.

[0118] As used herein, the term "substantially free of" is a term of degree and means that the composition does not contain a substantial amount of the compound that the composition is said to be substantially free of. When a composition is substantially free of a compound, such as substantially free of the N-terminal half domain, the composition preferably contains less than 5.0% of the compound, more preferably less than 1.0% of the compound, and most preferably less than 0.1% of the compound.

[0119] Immunogenic Composition

[0120] As mentioned above, the present disclosure provides, in at least one embodiment, an immunogenic composition comprising a HIBP surface receptor protein from a Gram-negative pathogenic bacterial species, wherein the HIBP surface receptor protein has been modified in such a way that it cannot substantially bind to a host iron-binding protein. The term "modified" in connection with the HIBP surface receptor protein is intended to refer to a non-native HIBP surface receptor protein in which at least one amino acid residue has been removed or in which at least one amino acid residue has been replaced by another residue, or a HIBP surface receptor protein that has been fragmented into two or more independent polypeptides. Thus, modified HIBP surface receptor proteins include, but are not limited to, truncated HIBP surface receptor proteins (e.g., the N-terminal or C-terminal half-domain of a HIBP surface receptor protein); HIBP surface receptor proteins in which one or more amino acid residues have been removed (e.g., HIBP surface receptor proteins in which one or more amino acids within a loop domain in the N-terminal or C-terminal half-domain have been removed); HIBP surface receptor proteins in which additional amino acids have been inserted (e.g., HIBP surface receptor proteins in which one or more amino acids have been added to a loop domain within the N-terminal or C-terminal half-domain); multimeric or extended HIBP polypeptides (e.g., dimers and trimers and N-terminal or C-terminal half-domain dimers and trimers); HIBP surface receptor proteins that have been modified by site-directed mutagenesis to alter one or more amino acids; and mixtures of two or more HIBP surface receptor protein polypeptides (e.g., mixtures comprising the independent N-terminal and C-terminal half-domains of a HIBP surface receptor protein). The modified HIBP surface receptor proteins of the present disclosure cannot substantially bind to native host iron-binding proteins.

[0121] As mentioned above, the present disclosure provides, in one aspect, an immunogenic composition comprising or consisting of a polypeptide that comprises the C-terminal or N-terminal half-domain of a HIBP surface receptor protein obtainable or obtained from a Gram-negative bacterial species, wherein the polypeptide cannot substantially bind to a host iron-binding protein. In other aspects, the present disclosure provides a polypeptide that comprises the C-terminal or N-terminal half-domain of a HIBP surface receptor polypeptide, wherein the C-terminal or N-terminal half-domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified, and wherein the polypeptide cannot substantially bind to a host iron-binding protein.

[0122] According to the present disclosure, any polypeptide comprising or consisting of the C-terminal or N-terminal half-domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species or a nucleic acid sequence encoding such a polypeptide can be used.

[0123] In embodiments of the present disclosure in which the native C-terminal half domain is used, the polypeptide comprising the native C-terminal half domain does not comprise the native N-terminal half domain of the HIBP surface receptor protein and is not chemically linked via a peptide bond to the native N-terminal half domain, and is thus a separated native C-terminal half domain, i.e., a native C-terminal half domain separated from the native N-terminal half domain. Accordingly, in certain embodiments, there is provided a preparation comprising a C-terminal half domain of a HIBP surface receptor polypeptide that is free or substantially free of the N-terminal half domain or a portion thereof of the HIBP surface receptor polypeptide. In embodiments of the present disclosure in which the native N-terminal half domain is used, the polypeptide comprising the native N-terminal half domain does not comprise the native C-terminal half domain of the HIBP surface receptor protein and is not chemically linked via a peptide bond to the native C-terminal half domain, and is thus a separated native N-terminal half domain, i.e., an N-terminal half domain separated from the native C-terminal half domain. Accordingly, in certain embodiments, there is provided a preparation comprising an N-terminal half domain of a HIBP surface receptor polypeptide that is free or substantially free of the C-terminal half domain or a portion thereof of the HIBP surface receptor polypeptide. However, in certain embodiments, a mixture of the native C-terminal half domain or a portion thereof and the native N-terminal half domain or a portion thereof may be used, provided that the N-terminal half domain and the C-terminal half domain are not chemically linked, i.e., they are not chemically linked via a peptide bond. Accordingly, the present disclosure includes an immunogenic composition comprising a mixture of a polypeptide comprising an N-terminal half domain and a polypeptide comprising a C-terminal half domain, wherein the N-terminal half domain and the C-terminal half domain are not physically linked.

[0124] To obtain the polypeptides of the present disclosure, any HIBP surface receptor protein or TbpB polypeptide obtainable from or derived from any Gram-negative bacterial species can be used, said bacterial species including but not limited to any pathogenic bacterial species or strain and including but not limited to any bacterial species belonging to the bacterial families Pasteurellaceae, Moraxellaceae or Neisseriaceae and bacterial species belonging to the bacterial genera Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella or Moraxella. The polypeptides further include any polypeptide obtainable from or derived from any HIBP surface receptor protein or any polypeptide obtainable from or derived from any TbpB polypeptide obtainable from or derived from the following bacterial species: Actinobacillus pleuropneumoniae (12, 34), Actinobacillus suis, Haemophilus influenzae (35, 36), Haemophilus parasuis (37), Haemophilus somnus (also known in the art as Histophilus somni) (38), Mannheimia haemolytica (also known in the art as Pasteurella haemolytica) (39), Moraxella catarrhalis (40), Moraxella bovis, Neisseria gonorrhoeae, Neisseria meningitidis (41, 42), Mannheimia glucosida (also known in the art as Pasteurella haemolytica) and Pasteurella trehalosi (also known in the art as Pasteurella trehalosi).

[0125] Exemplary C-terminal and N-terminal domain polypeptides that can be used according to the present disclosure further include SEQ.ID NO: 5; SEQ.ID NO: 6; SEQ.ID NO: 22; SEQ.ID NO: 33; SEQ.ID NO: 34; SEQ.ID NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 179 to SEQ.ID NO: 195; SEQ.ID NO: 213 to SEQ.ID NO: 218; SEQ.ID NO: 230; SEQ.ID NO: 232; SEQ.ID NO: 234 to SEQ.ID NO: 278; and any C-terminal domain shown in SEQ.ID NO: 288 to SEQ.ID NO: 292, and SEQ.ID NO: 8; SEQ.ID NO: 10; SEQ.ID NO: 24; SEQ.ID NO: 26; SEQ.ID NO: 36; SEQ.ID NO: 38; SEQ.ID NO: 121; SEQ.ID NO: 127; SEQ.ID NO: 229; SEQ.ID NO: 231; and any N-terminal domain shown in SEQ.ID NO: 233, and further include any C-terminal or N-terminal domain that can be prepared from HIBP polypeptide or TbpB polypeptide or by using nucleic acid sequences encoded by SEQ.ID NO: 1; SEQ.ID NO: 11; SEQ.ID NO: 27; SEQ.ID NO: 116; SEQ.ID NO: 122; and SEQ.ID NO: 173, the HIBP polypeptide or TbpB polypeptide including but not limited to SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28; SEQ.ID NO: 107 to SEQ.ID NO: 115; SEQ ID NO: 117; SEQ.ID NO: 123; SEQ.ID NO: 131 to SEQ.ID NO: 147; SEQ.ID NO: 177; SEQ.ID NO: 178; SEQ.ID NO: 196 to SEQ.ID NO: 204; SEQ.ID NO: 206 to SEQ.ID NO: 212; and SEQ.ID NO: 219 to SEQ.ID NO: 228. Using these nucleic acid sequences and polypeptide sequences, additional novel HIBP surface receptor proteins and TbpB sequences and C-terminal or N-terminal domains can be readily identified by those skilled in the art. For example, expression libraries, cDNA libraries, and genomic libraries can be screened and databases containing sequence information can be searched for similar sequences.

[0126] The immunogenic preparation of the present disclosure elicits an immune response in a vertebrate subject after administration thereof to the vertebrate subject, in the form of stimulating the vertebrate subject to produce antibodies. Accordingly, the antibodies are reactive against at least one Gram-negative strain. However, preferably the antibodies are cross-reactive and / or cross-protective against multiple strains or bacterial species, and preferably the cross-reactivity and / or cross-protection is obtained in a host expressing one or more host iron-binding proteins such as transferrin or lactoferrin. As used herein, the term "cross-reactivity" refers to the ability of an immune response induced by an immunogenic composition obtained from one strain to stimulate the production of antibodies that are additionally reactive with a different strain or species. As used herein, the term "cross-protection" refers to the ability of an immune response induced by an immunogenic composition obtained from one strain to prevent or attenuate an infection or disease caused by at least one additional strain or bacterial species. In a preferred embodiment of the present disclosure, the immunogenic composition of the present disclosure is cross-reactive and / or cross-protective against multiple strains or bacterial species, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 bacterial species or strains. Cross-reactivity is considered an indicator of cross-protection. Those skilled in the art will readily appreciate that the foregoing aspects of the present disclosure facilitate vaccine manufacture by allowing the production of one immunogenic compound (i.e., an immunogenic compound obtainable from a HIBP surface receptor protein) that provides protection against multiple infectious strains or bacterial species.

[0127] The immunogenic preparation of the present disclosure produces an unexpectedly effective immune response in a vertebrate subject and particularly in a vertebrate subject expressing host iron-binding proteins such as transferrin and lactoferrin, beyond the effectiveness of the immune response produced when using an immunizing preparation utilizing the native HIBP protein. One aspect of the effective immune response is the magnitude of the immune response. Preferably, the antibody titer obtained using the immunogenic composition of the present disclosure exceeds the antibody titer of the native HIBP protein by at least 2-fold, more preferably at least 5-fold, and most preferably at least 10-fold.

[0128] In a preferred embodiment, a mixture comprising at least two polypeptides is used, each polypeptide comprising a C-terminal half domain or consisting of a C-terminal half domain; or a mixture comprising at least two polypeptides is used, each polypeptide comprising an N-terminal half domain or consisting of an N-terminal half domain; or a mixture comprising at least three polypeptides is used, the polypeptides comprising at least two C-terminal half domains and at least one N-terminal half domain or consisting of said domains; or a mixture comprising at least three polypeptides is used, the polypeptides comprising at least two N-terminal half domains and at least one C-terminal half domain or consisting of said domains. In a preferred embodiment, the at least two polypeptides are obtained from or are obtainable from a Gram-negative bacterial genus or species capable of infecting the same vertebrate species. Thus, the at least two polypeptides will be selected from, for example, two C-terminal half domains of a TbpB polypeptide, wherein the two C-terminal half domains are obtained from or are obtainable from a TbpB polypeptide, the TbpB polypeptide being obtained from or obtainable from an Actinobacillus strain capable of infecting pigs; or, for example, two C-terminal half domains of a TbpB polypeptide, wherein the two C-terminal half domains are obtained from or are obtainable from a TbpB polypeptide, the TbpB polypeptide being obtained from or obtainable from a Haemophilus strain capable of infecting cattle. In embodiments where at least two C-terminal half domains are used, the mixture preferably does not contain or substantially does not contain an N-terminal half domain or a portion thereof. In embodiments where at least two N-terminal half domains are used, the mixture preferably does not contain or substantially does not contain a C-terminal half domain or a portion thereof.

[0129] In a particularly preferred embodiment, according to the present disclosure, a mixture of at least two polypeptides is used, each polypeptide comprising or consisting of the C-terminal half domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative bacterial species. In said embodiment, the mixture preferably does not contain the N-terminal half domain or a portion thereof. In a particularly preferred embodiment, any two C-terminal half domains of the HIBP surface receptor polypeptides shown in SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28; SEQ.ID NO: 107 to SEQ.ID NO: 115; SEQ ID NO: 117; SEQ.ID NO: 123; SEQ.ID NO: 131 to SEQ.ID NO: 147; SEQ.ID NO: 177; SEQ.ID NO: 178; SEQ.ID NO: 196 to SEQ.ID NO: 204; SEQ.ID NO: 206 to SEQ.ID NO: 212; and SEQ.ID NO: 219 to SEQ.ID NO: 228 are used; or any two C-terminal half domains of the nucleic acid sequences shown in SEQ.ID NO: 1; SEQ.ID NO: 11; SEQ.ID NO: 27; SEQ.ID NO: 116; SEQ.ID NO: 122; and SEQ.ID NO: 173 are used. In a further preferred embodiment, any two C-terminal half domains selected from SEQ.ID NO: 5; SEQ.ID NO: 6; SEQ.ID NO: 22; SEQ.ID NO: 33; SEQ.ID NO: 34; SEQ.ID NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 179 to SEQ.ID NO: 195; and SEQ.ID NO: 213 to SEQ.ID NO: 218; SEQ.ID NO: 230; SEQ.ID NO: 232; SEQ.ID NO: 234 to SEQ.ID NO: 278; and SEQ.ID NO: 288 to SEQ.ID NO: 292 are used.

[0130] In a further particularly preferred embodiment, at least two C-terminal half domains of a HIBP surface receptor protein are used, wherein at least one C-terminal half domain is obtainable from or obtained from a bacterial species belonging to the genera Actinobacillus, Haemophilus, Histophilus, Mannheimia, Moraxella, Neisseria, Pasteurella, and Bibersteinia.

[0131] In a further particularly preferred embodiment, at least two C-terminal half domains are used, wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Actinobacillus pleuropneumoniae, preferably the C-terminal half domain shown in SEQ.ID.NO: 6 or SEQ.ID NO: 22, or wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Actinobacillus suis, preferably the C-terminal half domain shown in SEQ.ID NO: 34.

[0132] In a further particularly preferred embodiment, at least two C-terminal half domains are used, wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Mannheimia haemolytica, preferably the C-terminal half domain shown in SEQ.ID.NO: 232 or SEQ.ID NO: 234, or wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Mannheimia glucosida.

[0133] In a further particularly preferred embodiment, at least two C-terminal half domains are used, wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Neisseria gonorrhoeae, preferably one of the C-terminal half domains shown in SEQ.ID.NO: 213 to SEQ.ID NO:, 218, or wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, said HIBP surface receptor protein being obtainable from or obtained from Neisseria meningitidis, preferably SEQ.ID.NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 128; SEQ.IDNO: 129; SEQ.ID NO: 130; SEQ.ID NO: 131; SEQ.ID NO: 152; SEQ.ID NO: 154; SEQ.ID NO: 156; SEQ.ID NO: 158; SEQ.ID NO: 160; SEQ.ID NO: 164; SEQ.ID NO: 166; SEQ.ID NO: 168; SEQ.IDNO: 179 to SEQ.ID NO: 195; and one of the C-terminal half domains shown in SEQ.ID NO: 235 to SEQ.ID NO: 278.

[0134] In a further particularly preferred embodiment, at least two C-terminal half domains are used, wherein at least one C-terminal half domain is obtainable from or obtained from a HIBP surface receptor protein, and the HIBP surface receptor protein is obtainable from or obtained from Pasteurella trehalosi, preferably the C-terminal half domain shown in SEQ.ID.NO: 292.

[0135] In a further preferred embodiment, at least two C-terminal half domains are used, which are obtainable from or obtained from a HIBP surface receptor polypeptide, and the two C-terminal half domains are both obtainable from or obtained from two bacterial species, and the bacterial species are selected from Actinobacillus pleuropneumoniae, preferably the C-terminal half domain shown in SEQ.ID NO: 6 or SEQ.ID NO: 22; Actinobacillus suis, preferably the C-terminal half domain shown in SEQ.ID NO: 34; and Haemophilus parasuis, preferably the C-terminal half domain shown in SEQ.IDNO: 294.

[0136] In a further preferred embodiment, at least two C-terminal half domains are used, which are obtainable from or obtained from a HIBP surface receptor polypeptide, and one of the two C-terminal half domains is obtainable from or obtained from Neisseria gonorrhoeae, preferably one of the C-terminal half domains shown in SEQ.ID.NO: 213 to SEQ.ID NO:, 218, and the other C-terminal half domain is obtainable from Neisseria meningitidis, preferably SEQ.ID.NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 128; SEQ.ID NO: 129; SEQ.ID NO: 130; SEQ.ID NO: 131; SEQ.ID NO: 152; SEQ.ID NO: 154; SEQ.IDNO: 156; SEQ.ID NO: 158; SEQ.ID NO: 160; SEQ.ID NO: 164; SEQ.ID NO: 166; SEQ.ID NO: 168; SEQ.ID NO: 179 to SEQ.ID NO: 195; and one of the C-terminal half domains shown in SEQ.ID NO: 235 to SEQ.ID NO: 278.

[0137] In a further preferred embodiment, at least two C-terminal half domains are used, which are obtainable from or obtained from a HIBP surface receptor polypeptide, and one of the two C-terminal half domains is obtainable from or obtained from Mannheimia haemolytica, preferably the C-terminal half domain shown in SEQ.ID.NO: 232; or SEQ.ID NO: 234, and the other C-terminal half is obtainable from or obtained from Pasteurella trehalosi, preferably the C-terminal half domain shown in SEQ.ID NO: 292.

[0138] In a further preferred embodiment, at least two C-terminal half domain structures obtainable from or derived from a TbpB polypeptide are used, wherein the two C-terminal half domain structures are each obtainable from or derived from one bacterial species or two bacterial species, and wherein the TbpB polypeptide or the C-terminal half domain structure derived therefrom is antigenically divergent. The TbpB or C-terminal half domain structure is preferably obtained from a bacterial species or strain capable of exchanging TbpB variants. As used herein, the term "antigenically divergent" with respect to two TbpB polypeptides or C-terminal half domain structures of a TbpB polypeptide means that the two TbpB polypeptides or C-terminal half domain structures of a TbpB polypeptide belong to divergent branches or groups of a phylogenetic tree when used to construct a phylogenetic tree using a representative number of TbpB polypeptides or C-terminal half domain structure polypeptides. Accordingly, a phylogenetic tree can be constructed with any amount of TbpB or C-terminal half domain structure polypeptides, however, it is preferably constructed using at least 25 TbpB polypeptides or C-terminal half domain structure polypeptides, more preferably at least 30, at least 40 or at least 50 TbpB polypeptides or C-terminal half domain structure polypeptides, and preferably the phylogenetic tree is constructed in such a way that it contains at least 2 nodes above the root level, more preferably the phylogenetic tree contains at least 3, 4 or 5 nodes above the root level, most preferably at least 6, 7, 8, 9 or 10 nodes above the root level (as hereinafter and Figure 30Further explained in). Antigenically divergent TbpB polypeptides or C-terminal half domain polypeptides preferably belong to different clades that (i) diverge at a node at least 2 steps below the highest order node of the phylogenetic tree (e.g., if the highest order node of the phylogenetic tree is the 9th order node, then the antigenically divergent polypeptides are those that diverge at the 7th order node or lower (i.e., the 6th, 5th, 4th, 3rd, 2nd, or 1st order node)); and / or (ii) diverge at the 1st, 2nd, or 3rd step of the phylogenetic tree. Several computer programs can be used to facilitate the construction of phylogenetic trees using TbpB polypeptides or C-terminal half domain polypeptides, including: (i) computer programs that perform sequence alignment, such as programs that use the M-Coffee alignment algorithm implemented on the T-Coffee server site (http: / / www.tcoffee.org / ) (43); (ii) computer programs that edit alignments, such as Geneious Pro (44); (iii) computer programs that automatically clean alignments, such as GBlocks (45); (iv) computer programs that select an evolutionary model compatible with the alignment, such as ProtTest v3.2 (Darriba et al., 2011); and (iv) computer programs that generate phylogenetic trees, such as programs that use the maximum likelihood method PhyML (46), which runs on the general time reversible (GTR) model (47)(48, 49) or other models (such as the JTT+I+G+F model or the WAG+G=F model), or programs such as PHYLIP and PAUP (University of Washington). Each of these programs is preferably configured such that tree branches are considered statistically significant. However, it should be noted that more distal branches may have lower statistical significance, and thus it is preferred to select strains belonging to groups based on the lowest order nodes.

[0139] Now refer to Figure 30, for illustrative purposes, a phylogenetic tree 100 is shown having a root 120, intermediate branches (illustrated by 130, 131, 140, 141, 142, and 143), and a total of 38 distal branches (illustrated by distal branches 150, 151, 152, 153, 154, and 155), with each distal branch representing a related polypeptide obtained from one of 38 strains (strains 1 - 38 (110)). Each of the shown branches originates from a node (illustrated by nodes 161, 171, 172, 181, 182, 183, and 184). Thus, for example, branch 130 originates from node 161 and branch 143 originates from node 172. The node (161) closest to the tree root (120) is more specifically referred to as the first - order node 161; nodes 171 and 172 are more specifically referred to as second - order nodes 171 and 172; nodes 181, 182, 183, and 184 are more specifically referred to as third - order nodes 181, 182, 183, and 184; and other nodes (mutatis mutandis) can be referred to as fourth, fifth, sixth, seventh, etc. - order nodes. Figure 30 shows four other groups (group 1 (105), polypeptides of strains (110) 1 - 17; group 2 (106), polypeptides of strains (110) 18 - 24; group 3 (107), polypeptides of strains (110) 25 - 30; and group 4 (108), polypeptides of strains (110) 26 - 38). Polypeptides of strains belonging to group 1 (105) or group 2 (106) all belong to a branch (131) that diverges at the first - order node (161) of the phylogenetic tree (100). Similarly, polypeptides of strains belonging to group 3 (105) or group 4 (106) all belong to a branch (131) that diverges at the first - order node (161) of the phylogenetic tree (100). Thus, all polypeptides of strains belonging to group 1 (105) or group 2 (106) are antigenically divergent from all polypeptides of strains belonging to group 3 (107) or group 4 (108). Polypeptides of strains belonging to group 1 (105) or group 2 (106) belong to groups that diverge at the second - order node (172) of the phylogenetic tree (100). Polypeptides of strains belonging to group 1 (105) are thus also antigenically divergent from polypeptides of strains belonging to group 2 (106). It should be noted that the phylogenetic tree can be represented in different formats, such as in a rectangular format (e.g., Figure 30 as shown) or in a circular format (as in FIG. 10). Exemplary phylogenetic trees constructed using the TbpB polypeptide or the C - terminal half or the TbpB polypeptide are provided in Figure 4 (containing Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis strains), FIG. 10 (containing Neisseria meningitidis strains), FIG. 26 (containing Neisseria meningitidis and Neisseria gonorrhoeae strains), FIG. 27 (containing Haemophilus influenzae strains), Figure 28 (containing Mannheimia haemolytica and Pasteurella trehalosi strains) and Figure 29 (containing Moraxella catarrhalis strains).

[0140] In a further preferred embodiment, at least two C-terminal half domains obtained from or derived from the TbpB polypeptide are used, wherein the two C-terminal half domains are each obtained from or derived from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis. See Figure 4 the phylogenetic tree shown in Figure 4 In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any one of the C-terminal half domains of Actinobacillus pleuropneumoniae, Actinobacillus suis, or Haemophilus parasuis strains belonging to Figure 4 phylogenetic group 1, phylogenetic group 2, or phylogenetic group 3 shown in Figure 4 and the second C-terminal half domain is selected from any one of the C-terminal half domains of Actinobacillus pleuropneumoniae, Actinobacillus suis, or Haemophilus parasuis strains belonging to Figure 4 a phylogenetic group (other than the phylogenetic group from which the first C-terminal half domain was selected) shown in Figure 4 In a further preferred embodiment, at least three C-terminal half domains are used, wherein a first C-terminal half domain of Actinobacillus pleuropneumoniae, Actinobacillus suis, or Haemophilus parasuis strains belonging to Figure 4 phylogenetic group 1 shown in Figure 4 is used, a second C-terminal half domain of Actinobacillus pleuropneumoniae, Actinobacillus suis, or Haemophilus parasuis strains belonging to Figure 4 phylogenetic group 2 shown in

[0141] In a further preferred embodiment, at least two C-terminal half domains obtained from or derived from the TbpB protein are used, wherein the two C-terminal half domains are each obtained from or derived from Neisseria meningitidis. See Figure 10A the phylogenetic tree shown in Figure 10A In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any one of the C-terminal half domains of Neisseria meningitidis strains belonging to Figure 10AAny of the Neisseria meningitidis strains of the phylogenetic group shown (the phylogenetic group that does not select the first C-terminal half domain). Thus, by way of example only, the C-terminal half domain of TbpB obtained from the Neisseria meningitidis strain B16B6 (phylogenetic group 1; Figure 10A , black arrow) can be combined with the C-terminal half domain of TbpB from the strain M982 (phylogenetic group 4; Figure 10A , black arrow). In a further preferred embodiment, at least three C-terminal half domains are used, wherein the C-terminal half domains are selected from strains belonging to Figure 10A the three different groups shown (e.g., C-terminal half domains selected from phylogenetic group 1, phylogenetic group 2, and phylogenetic group 3, respectively). In a further preferred embodiment, at least four C-terminal half domains are used, wherein the first C-terminal half domain from a Neisseria meningitidis strain belonging to group 1 shown in Figure 10A is used, the second C-terminal half domain from a Neisseria meningitidis strain belonging to group 2 shown in Figure 10A is used, and the third C-terminal half domain from a Neisseria meningitidis strain of phylogenetic group 3 shown in Figure 10A is used and the fourth C-terminal half domain from a Neisseria meningitidis strain of phylogenetic group 4 shown in Figure 10A is used. Thus, by way of specific example only, the C-terminal half domains of TbpB from the Neisseria meningitidis strains B16B6 (phylogenetic group 1; Figure 10A , black arrow), BZ169 (phylogenetic group 2; Figure 10A , black arrow), S3131 (phylogenetic group 3; Figure 10A , black arrow), and M982 (phylogenetic group 4; Figure 10A , black arrow) can be selected.

[0142] In a further preferred embodiment, at least two C-terminal half domains obtainable from or obtained from a TbpB polypeptide are used, wherein one of the two C-terminal half domains is obtainable from or obtained from Neisseria meningitidis and the other C-terminal half is obtainable from or obtained from Neisseria gonorrhoeae. Referring to the phylogenetic tree shown in Figure 26B , in a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is obtained from any of the C-terminal half domains of Neisseria gonorrhoeae strains belonging to phylogenetic group 3 shown in Figure 26B , and wherein the second C-terminal half domain is obtained from any of the C-terminal half domains of Neisseria gonorrhoeae strains belonging to Figure 26BAny of the C-terminal half domains of Neisseria meningitidis strains of phylogenetic group 1 or group 2 as shown. More preferably, at least three C-terminal half domains obtainable from or obtained from the TbpB polypeptide are used, wherein two C-terminal half domains are obtainable from or obtained from Neisseria meningitidis and the other C-terminal half is obtainable from or obtained from Neisseria gonorrhoeae. Preferably, the C-terminal half domain is selected from those belonging to Figure 26B Neisseria gonorrhoeae strains of phylogenetic group 3 as shown, and the second C-terminal half domain is selected from any of the C-terminal half domains of Neisseria meningitidis strains of phylogenetic group 2 as shown in Figure 26B and the third C-terminal half domain is selected from any of the C-terminal half domains of Neisseria meningitidis strains of phylogenetic group 1 as shown in Figure 26B In a further preferred embodiment, at least four C-terminal half domains obtainable from or obtained from the TbpB polypeptide are used, wherein three C-terminal half domains are obtainable from or obtained from Neisseria meningitidis and the other C-terminal half is obtainable from or obtained from Neisseria gonorrhoeae. Preferably, the C-terminal half domain is selected from those belonging to Figure 26B Neisseria gonorrhoeae strains of phylogenetic group 3 as shown, and the second and third C-terminal half domains are selected from any of the C-terminal half domains of Neisseria meningitidis strains of two different subgroups (e.g., phylogenetic subgroup 2.1 and phylogenetic subgroup 2.2) of phylogenetic group 2 as shown in Figure 26B and the fourth C-terminal half domain is selected from any of the C-terminal half domains of Neisseria meningitidis strains of phylogenetic group 1 as shown in Figure 26B In a further preferred embodiment, at least four C-terminal half domains obtainable from or obtained from the TbpB polypeptide are used, wherein two C-terminal half domains are obtainable from or obtained from Neisseria meningitidis and the other two C-terminal halves are obtainable from or obtained from Neisseria gonorrhoeae. Preferably, the two C-terminal half domains selected from Neisseria gonorrhoeae strains belong to Figure 26B two different phylogenetic subgroups (e.g., subgroup 3.1 and subgroup 3.2) of phylogenetic group 3 as shown, and the third and fourth C-terminal half domains are obtained from Neisseria meningitidis strains belonging to phylogenetic group 1 and phylogenetic group 2, respectively. In another embodiment, at least five C-terminal half domains obtainable from or obtained from the TbpB polypeptide are used, wherein three C-terminal half domains are obtainable from or obtained from Neisseria meningitidis and the other two C-terminal halves are obtainable from or obtained from Neisseria gonorrhoeae. Preferably, the two C-terminal half domains selected from Neisseria gonorrhoeae strains belong to Figure 26B two different phylogenetic subgroups (e.g., subgroup 3.1 and subgroup 3.2) of phylogenetic group 3 as shown, and the third and fourth C-terminal half domains are obtained from those belonging to Figure 26BNeisseria meningitidis strains of two different phylogenetic subgroups of phylogenetic group 2 shown therein (e.g., subgroup 2.1 and subgroup 2.2), and the fifth C-terminal half domain belongs to Figure 26B Neisseria meningitidis strains of phylogenetic group 1 shown therein. In a further preferred embodiment, at least six C-terminal half domains obtained from or obtainable from the TbpB polypeptide are used. In this embodiment, at least one C-terminal half domain is obtained from or obtainable from Neisseria meningitidis and at least one C-terminal half domain is obtained from or obtainable from Neisseria gonorrhoeae, and the other C-terminal halves are obtained from or obtainable from according to Figure 26B antigenically divergent strains. In a preferred embodiment, three C-terminal half domains are obtained from or obtainable from Neisseria meningitidis and the other three C-terminal halves are obtained from or obtainable from Neisseria gonorrhoeae. See Figure 26B , preferably the three C-terminal half domains obtained from Neisseria gonorrhoeae strains belong to three antigenically divergent groups, and the three C-terminal half domains obtained from Neisseria meningitidis belong to three antigenically divergent strains.

[0143] In a further preferred embodiment, at least two C-terminal half domains obtained from or obtainable from the TbpB polypeptide are used, wherein one of the two C-terminal half domains is obtained from or obtainable from Neisseria meningitidis and the other C-terminal half is obtained from or obtainable from Neisseria gonorrhoeae. See Figure 26A the phylogenetic tree shown therein. In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any one of the C-terminal half domains of Neisseria gonorrhoeae strains belonging to Figure 26A phylogenetic group 3 or phylogenetic group 1 shown therein, and wherein the second C-terminal half domain is selected from any one of the C-terminal half domains of Neisseria meningitidis strains belonging to Figure 26A phylogenetic group 2, group 4 or group 5 shown therein.

[0144] In a further preferred embodiment, at least two C-terminal half domains obtained from or obtainable from the TbpB protein are used, wherein both of the two C-terminal half domains are obtained from or obtainable from Haemophilus influenzae. See Figure 27A and Figure 27B the phylogenetic tree shown therein. In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any one of the C-terminal half domains of Haemophilus influenzae strains belonging to Figure 27A or Figure 27B phylogenetic group 1, phylogenetic group 2 or phylogenetic group 3 shown therein, and wherein the second C-terminal half domain is obtained from a Haemophilus influenzae strain belonging to Figure 27A or Figure 27BAny of the Haemophilus influenzae strains of the phylogenetic group shown (the phylogenetic group that does not select the first C-terminal half domain). Thus, by way of example only, the C-terminal half domain of TbpB obtained from Haemophilus influenzae strain H216 (phylogenetic group 3; Figure 27B , black arrow) can be combined with the C-terminal half domain of TbpB from strain H214 (phylogenetic group 1; Figure 27B , black arrow). In a further preferred embodiment, at least three C-terminal half domains are used, wherein the C-terminal half domains are selected from strains belonging to Figure 27A and Figure 27B shown in the three different groups (i.e., C-terminal half domains selected from phylogenetic group 1, phylogenetic group 2, and phylogenetic group 3, respectively). Thus, by way of specific example, the C-terminal half domains of TbpB from Haemophilus influenzae strains H216 (phylogenetic group 3; Figure 27B , black arrow), H214 (phylogenetic group 1; Figure 27B , black arrow), and H011 (phylogenetic group 2; Figure 27B , black arrow) can be selected.

[0145] In a further preferred embodiment, at least two C-terminal half domains obtainable from or obtained from the TbpB protein are used, wherein both of the two C-terminal half domains are obtainable from or obtained from Mannheimia haemolytica. See the phylogenetic tree shown in Figure 28 . In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any of the C-terminal half domains of Mannheimia haemolytica strains belonging to phylogenetic group 1 shown in Figure 28 , and wherein the second C-terminal half domain is obtained from any of the Mannheimia haemolytica strains belonging to phylogenetic group 3.

[0146] In a further preferred embodiment, at least two C-terminal half domains obtainable from or obtained from the TbpB polypeptide are used, wherein one of the two C-terminal half domains is obtainable from or obtained from Pasteurella trehalosi and the other C-terminal half is obtainable from or obtained from Mannheimia haemolytica. See Figure 28 . In a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any of the C-terminal half domains of Pasteurella trehalosi strains belonging to phylogenetic group 2 shown in Figure 28 , and wherein the second C-terminal half domain is selected from any of the C-terminal half domains of Mannheimia haemolytica strains belonging to phylogenetic group 1 or group 3 shown in Figure 28 .

[0147] In a further preferred embodiment, at least two C-terminal half domains obtainable from or derived from the TbpB protein are used, wherein the two C-terminal half domains are both obtainable from or derived from Moraxella catarrhalis. See Figure 29 the phylogenetic tree shown in Figure 29 , in a preferred embodiment, at least two C-terminal half domains are used, wherein the first C-terminal half domain is selected from any one of the C-terminal half domains of Moraxella catarrhalis strains belonging to Figure 29 phylogenetic group 1, phylogenetic group 2 or phylogenetic group 3 shown in Figure 29 , and wherein the second C-terminal half domain is derived from any one of Moraxella catarrhalis strains belonging to Figure 29 the phylogenetic group (other than the phylogenetic group from which the first C-terminal half domain is selected) shown in Figure 29 . Thus, by way of example only, the TbpB C-terminal half domain obtained from Moraxella catarrhalis strain AAC34279.1 (phylogenetic group 3; Figure 29 , black arrow) can be combined with the TbpB C-terminal half domain from strain AAD12263.1 (phylogenetic group 1; Figure 29 , black arrow). In a further preferred embodiment, at least three C-terminal half domains are used, wherein the C-terminal half domains are selected from strains belonging to Figure 29 three different groups shown in Figure 29 (i.e., C-terminal half domains selected from phylogenetic group 1, phylogenetic group 2 and phylogenetic group 3 respectively). Thus, by way of specific example only, the TbpB C-terminal half domains from Moraxella catarrhalis strains AAC34279.1 (phylogenetic group 3; Figure 29 , black arrow), AAD12263.1 (phylogenetic group 1; Figure 29 , black arrow) and 003664398.1 (phylogenetic group 2; Figure 29 , black arrow) can be selected.

[0148] In a particularly preferred embodiment, the aforementioned mixture of polypeptides comprising or consisting of a C-terminal half domain is a C-terminal half domain obtainable from or derived from a TbpB polypeptide, including but not limited to the C-terminal half domains shown in SEQ.ID NO: 5; SEQ.ID NO: 6; SEQ.ID NO: 22; SEQ.ID NO: 33; SEQ.ID NO: 34; SEQ.ID NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 179 to SEQ.ID NO: 195; and SEQ.ID NO: 213 to SEQ.ID NO: 218; SEQ.ID NO: 230; SEQ.ID NO: 232; and SEQ.ID NO: 234 to SEQ.ID NO: 278.

[0149] The aforementioned mixture of C-terminal half domain can be prepared by mixing preparations containing individual C-terminal half domains or by recombinantly producing a fusion polypeptide containing two or more C-terminal half domains.

[0150] As mentioned above, the immunogenic preparations of the present disclosure are preferably cross-reactive and / or cross-protective. Although, as mentioned above, preparations containing a single C-terminal half domain can be cross-reactive and / or cross-protective, a mixture of C-terminal half domains is particularly preferred because it can be used to prepare immunogenic preparations that generally broaden cross-reactivity and / or cross-protection against a wider range of strains and / or bacterial species, and allows the preparation of vaccine preparations that provide protection from infections or diseases transmitted by multiple bacterial species or strains.

[0151] According to other embodiments, a polypeptide containing the C-terminal half domain and / or N-terminal half domain of the HIBP surface receptor polypeptide is prepared in such a way that the loop domain connecting two β-strands within the C-terminal half domain or the N-terminal half domain is modified and the polypeptide cannot substantially bind to the host iron-binding protein. As used herein, the term "modified" when used in connection with a loop domain refers to a loop in which at least one amino acid residue has been removed or replaced. Thus, the resulting loop within the C-terminal half domain or the N-terminal half domain can be truncated, or in other embodiments, the amino acid residue can be replaced with one or more alternative amino residues. Figure 1 and Figure 2 Showing the loop domain of an exemplary HIBP surface receptor protein. Figure 8 and Figure 14 Examples of loop reduction in the N-terminal half domain and C-terminal half domain are provided respectively. Accordingly, at least one loop domain connecting two β-strands within the C-terminal half domain or N-terminal half domain of the HIBP-binding membrane receptor protein is modified to remove at least one amino acid residue from the loop domain, and the resulting polypeptide contains a modified N-terminal half domain or C-terminal half domain and cannot substantially bind to the host iron-binding protein. In other embodiments, multiple amino acid residues are removed, such as removing at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acid residues from the loop domain. In other embodiments, the entire loop domain is removed. Any of the loop domains can be selected for modification according to the present disclosure, as long as the modification results in a polypeptide that cannot substantially bind to the host iron-binding protein. Thus, see Figure 1Exemplary porcine TbpB polypeptides, in embodiments of the present disclosure in which one of the loop domains is modified, the loop domain can be selected as any one of loop domains L1-L32 (such as exemplified by Actinobacillus pleuropneumoniae loop L1-L32 polypeptide sequences: SEQ.ID NO: NO42; SEQ.ID: NO 44; SEQ.ID: NO 46; SEQ.ID: NO 48; SEQ.ID: NO 50; SEQ.ID: NO 52; SEQ.ID: NO54; SEQ.ID: NO 56; SEQ.ID: NO 58; SEQ.ID: NO 60; SEQ.ID: NO 62; SEQ.ID: NO 64; SEQ.ID: NO66; SEQ.ID: NO 68; SEQ.ID: NO 70; SEQ.ID: NO 72; SEQ.ID: NO 74; SEQ.ID: NO 76; SEQ.ID: NO78; SEQ.ID: NO 80; SEQ.ID: NO 82; SEQ.ID: NO 84; SEQ.ID: NO 86; SEQ.ID: NO 88; SEQ.ID: NO90; SEQ.ID: NO 92; SEQ.ID: NO 94; SEQ.ID: NO 96; SEQ.ID: NO 98; SEQ.ID: NO 100; SEQ.ID: NO 102; SEQ.ID: NO 104; and SEQ.ID: NO 106, and encoded by nucleic acid sequences SEQ.ID: NO 41; SEQ.ID: NO 43; SEQ.ID: NO 45; SEQ.ID: NO 47; SEQ.ID: NO 49; SEQ.ID: NO 51; SEQ.ID: NO 53; SEQ.ID: NO 55; SEQ.ID: NO 57; SEQ.ID: NO 59; SEQ.ID: NO 61; SEQ.ID: NO 63; SEQ.ID: NO65; SEQ.ID: NO 67; SEQ.ID: NO 69; SEQ.ID: NO 71; SEQ.ID: NO 73; SEQ.ID: NO 75; SEQ.ID: NO77; SEQ.ID: NO 79; SEQ.ID: NO 81; SEQ.ID: NO 83; SEQ.ID: NO 85; SEQ.ID: NO 87; SEQ.ID: NO89; SEQ.ID: NO 91; SEQ.ID: NO 93; SEQ.ID: NO 95; SEQ.ID: NO 97; SEQ.ID: NO 99; SEQ.ID: NO101; SEQ.ID: NO 103; and SEQ.ID: NO 105), as further shown in Table 1.In embodiments of the present disclosure in which two loop domains are modified, the two loop domains can be any two loop domains selected from the loop domains L1-L32 domains (see again. Figure 1 exemplary TbpB polypeptides), as further shown in Table 2. In embodiments of the present disclosure in which three loop domains are modified, the three loop domains can be any three loop domains selected from the loop domains L1-L32 domains (see again Figure 1 exemplary TbpB polypeptides), as further shown in Table 3. In embodiments of the present disclosure in which four loop domains are modified, the four loop domains can be any three loop domains selected from the combinations of loops shown in Table 3 plus an additional loop domain selected from the loop domains L1-L32 (see again Figure 1exemplary TbpB polypeptides). In other embodiments, a total of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 loop domains may be modified. Those skilled in the art will appreciate that in each of these embodiments of the present disclosure, the exact number of modified loop domains may vary and may be selected from any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 loop domains of loops L1-L32 in a manner similar to the selection of loop domains described for embodiments in which 2 or 3 loop domains have been modified. In particularly preferred embodiments, one or all of loops L18, L21, L23, and L27 of the C-terminal half domain (such as exemplified by Actinobacillus pleuropneumoniae SEQ.ID NO: 76; SEQ.ID NO: 82; SEQ.ID NO: 86; and SEQ.ID NO: 96, respectively) are modified. In further particularly preferred embodiments, one or all of loops L1, L5, L8, and L12 of the N-terminal half domain (such as exemplified by Actinobacillus pleuropneumoniae SEQ.ID NO: 42; SEQ.ID NO: 50; SEQ.IDNO: 56; SEQ.ID NO: 64, respectively) are modified. The loop domains that can be modified are loop domains that connect two β-strands assembled within a β-barrel or handle domain β-sheet of the C-terminal half domain or the N-terminal half domain, or loop domains that connect two assembled β-strands, or combinations of the foregoing loop domains. To truncate the loop domains within the C-terminal half domain or the N-terminal half domain, the polypeptide can be prepared in such a way that the loop domain is removed in its entirety and optionally replaced with one or more linked amino acids, thus creating a more or less direct connection between the two β-strands, or in such a way that a part or parts of the loop domain are removed. Accordingly, it is preferred to remove at least half of the amino acid residues from at least one loop domain of the C-terminal half domain or the N-terminal half domain. In a further preferred embodiment, at least half of all the amino acid residues of the loop domain are removed. Thus, in embodiments where the loop domain contains, for example, 40 amino residues, at least 20 amino acid residues of the loop domain will be removed. In other embodiments, the loop domain is modified in such a way that at least 60%, 70%, 80%, or 90% of the amino acid residues of the loop domain are removed.In other embodiments, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the loop domain are retained after truncation, and in other embodiments, up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the loop domain is retained after truncation. In embodiments in which the loop domain is modified in such a way that only a portion of the loop domain is removed, the amino acid residues removed can be located at the N-terminus of the loop domain, the C-terminus of the loop domain, or between the N-terminus and the C-terminus. In embodiments in which multiple loop domains of the C-terminal half domain or the N-terminal half domain are modified, the loop reduction can involve removing the same number of amino acid residues from each loop, for example, 10 amino acid residues can be removed from each loop within the C-terminal half domain or the N-terminal half domain, or the loop reduction can involve removing different amounts of amino acid residues from each loop, for example, 10 residues in one loop and 20 residues in another loop.

[0152] In a further preferred embodiment, the amino acid residues within the loop domain of the C-terminal half domain and / or the N-terminal half domain are replaced by other groups, for example, by site-directed mutagenesis, and the resulting polypeptide comprises a modified C-terminal half domain or N-terminal half domain and cannot substantially bind to the host iron-binding protein. Figure 22 Examples of residue substitutions in the loop regions of the N-terminal half domains of various TbpB polypeptides are provided and Figure 23The reduction in Tf binding due to replacement of these residues is illustrated. Thus, for example, one or more amino acid residues may be replaced in any of loops L1-L32. In certain preferred embodiments, one or more amino acid residues in loop domains L1, L3, L5, or L8 (as exemplified by Actinobacillus pleuropneumoniae SEQ. ID NO: 42; SEQ. ID NO: 46; SEQ. ID NO: 50; and SEQ. ID NO: 56, respectively) of the N-terminal half domain are replaced. In preferred embodiments, aromatic amino acids (phenylalanine, tyrosine, and tryptophan) within loop domain L8 are replaced with aliphatic amino acids (glycine, valine, leucine, isoleucine). These aromatic amino acids are aromatic amino acid residues that are readily accessible to the surface in surface regions that are otherwise generally cationic. In a particularly preferred embodiment, a Haemophilus parasuis TbpB polypeptide is selected and one or more of the following mutations are made in the TbpB polypeptide to obtain a modified TbpB polypeptide: Y93A (SEQ.ID NO: 170; SEQ.ID NO: 171); Y117A (SEQ.ID NO: 172; SEQ.ID NO: 173); Y167A (SEQ.ID NO: 174; SEQ.ID NO: 175;) or W176A (SEQ.ID NO: 176; SEQ.ID NO: 177), and in a further preferred embodiment, an Actinobacillus pleuropneumoniae TbpB polypeptide is selected and one or more of the following mutations are made in the polypeptide to obtain a modified TbpB polypeptide: F1 71A (SEQ.ID NO: 3; SEQ.ID NO: 4); Y95A (SEQ.ID NO: 13; SEQ.ID NO: 14); Y121A (SEQ.ID NO: 15; SEQ.ID NO: 16; SEQ.ID NO: 177). NO: 16); Y174A (SEQ.ID NO: 17; SEQ.ID NO: 18); or R179E (SEQ.ID NO: 19; SEQ.ID NO: 20), and in a further preferred embodiment, the Actinobacillus suis TbpB polypeptide is selected and one or more of the following mutations are made in the polypeptide to obtain a modified TbpB polypeptide: F63A (SEQ.ID NO: 29; SEQ.ID NO: 30) or F152A (SEQ.ID NO: 31; SEQ.ID NO: 32). The present disclosure includes each of the aforementioned modified polypeptides and nucleic acid sequences encoding these polypeptides, as well as immunogenic compositions and vaccine compositions comprising these polypeptides.

[0153] The reduction in size of one or more loop domains of the HIBP polypeptide according to the present disclosure, or the modification of amino acids in the loop domains, is preferably carried out in such a way that the resulting polypeptide conformation is stable. The term "conformationally stable" means that the conformational state or conformation of the polypeptide remains substantially the same after modification of the size of the loop or substitution of amino acid residues. The conformational state of the modified loop domain may be more or less altered. Determinants of the conformational state or conformation of a polypeptide include: the primary structure of the polypeptide as reflected in its amino acid sequence, the secondary structure of the polypeptide (e.g., α-helix, β-sheet, etc.), the tertiary structure of the polypeptide (i.e., the three-dimensional folding of the polypeptide chain), and the quaternary structure (i.e., the interaction of the polypeptide with other protein subunits). Protein conformation can be further influenced by environmental factors such as pH, osmolarity, ionic strength, and salt concentration. The design of loop reduction can be learned through the alignment and comparison of multiple heterologous sequences, the comparison of three-dimensional conformational structures of various heterologous polypeptides known in the art, and the use of conservative amino acid substitutions (e.g., combinations such as gly, ala; val, ile; leu, met; asp, glu; asn, gln; ser, thr; lys, arg; cys, met; and phe, trp, tyr). In addition, the conformational state of a protein can be analyzed by functional assays (e.g., binding to a host iron-binding protein) or by physical methods such as X-ray crystallography or nuclear magnetic resonance (NMR).

[0154] In other embodiments, at least one loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein containing the longest loop is selected for modification. In embodiments where the entire loop is modified, this will generally involve the removal of at least 25 amino acid residues and can result in the removal of 150 amino acid residues or more residues.

[0155] In a preferred embodiment, at least one loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the longest loop (i.e., contains the most amino acid residues) of the C-terminal half domain or the N-terminal half domain is modified. In a further preferred embodiment, at least one loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the longest loop is modified, and the second loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the second longest loop is selected for modification. In a further preferred embodiment, at least one loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the longest loop and the second loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the second longest loop are selected for modification and the third loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the third longest loop is selected for modification. In a further preferred embodiment, at least one loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the longest loop and the second loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the second longest loop are selected for modification and the third loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the third longest loop is selected for modification, and the fourth loop domain of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein that contains the fourth longest loop is selected for modification. The foregoing embodiments are further detailed in Examples 3 and 4 in this regard.

[0156] Surprisingly, it has now been found that polypeptides consisting essentially of the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein can be readily produced, for example in a microbial production system (when one or more loop domains are modified), and the modified polypeptides are substantially conformationally stable.

[0157] Accordingly, the modified C-terminal or N-terminal domain polypeptides can be used as immunogens per se, or the polypeptides can be engineered to include further modifications. Modifications that can be made to the modified C-terminal or N-terminal domain polypeptides accordingly include the preparation of N-terminal or C-terminal polypeptide extensions of the native or modified C-terminal or N-terminal domain polypeptides. The N-terminal and C-terminal polypeptide extensions include adding a second full-length C-terminal domain polypeptide to the C-terminal domain, thus providing a C-terminal domain dimer; adding a second full-length N-terminal domain polypeptide to the N-terminal domain, thus providing an N-terminal domain dimer; or including the addition of a portion of a C-terminal domain polypeptide or a portion of an N-terminal domain polypeptide. The multimer can be assembled using the same monomeric polypeptide (i.e., homodimers, homotrimers, etc.), or it can be assembled using different polypeptides, such as C-terminal or N-terminal domains obtained from different variants (i.e., heterodimers, heterotrimers, etc.). In a preferred embodiment, the assembly represents a heteromultimeric protein of different pathogens or strains. Thus, in one preferred embodiment, a heteromultimeric polypeptide is prepared that comprises a C-terminal or N-terminal domain selected from the group consisting of C-terminal or N-terminal domains of Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis. In a particularly preferred embodiment, the C-terminal or N-terminal domain is selected from the group consisting of the C-terminal or N-terminal domains of Actinobacillus pleuropneumoniae H49, Actinobacillus suis H57, and Actinobacillus pleuropneumoniae H87. In a further preferred embodiment, a heteromultimeric polypeptide is prepared that comprises a C-terminal domain selected from at least two TbpB C-terminal domains or an N-terminal domain selected from Neisseria meningitidis strains. In a particularly preferred embodiment, the strain is selected from Neisseria meningitidis M982 or Neisseria meningitidis B16B6. The heteromultimeric protein can convey immunogenicity to different pathogens. In a further preferred embodiment, the present disclosure provides (i) a first polypeptide that comprises an N-terminal or C-terminal domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal or C-terminal domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains of the N-terminal or C-terminal domain has been modified, and the first polypeptide is linked to (ii) a second polypeptide that comprises a HIBP surface receptor protein or a portion thereof obtainable from a Gram-negative bacterial species. In a preferred embodiment, the portion of the HIBP surface receptor protein is an N-terminal or C-terminal domain.In a further preferred embodiment, the portion of the HIBP surface protein is the N-terminal half domain or the C-terminal half domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal half domain or the C-terminal half domain comprises a plurality of beta strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains of the N-terminal half domain or the C-terminal half domain has been modified.

[0158] In other embodiments, a multimeric polypeptide is prepared, the multimeric protein comprising multiple N-terminal and C-terminal extensions, including adding a second, third, fourth, fifth, sixth, or seventh full-length C-terminal half-domain polypeptide to the C-terminal half-domain, thus providing a C-terminal half-domain multimer; or adding a second, third, fourth, fifth, sixth, or seventh full-length N-terminal half-domain polypeptide to the N-terminal half-domain, thus providing an N-terminal half-domain dimer; or including the addition of a portion of a C-terminal half-domain polypeptide or a portion of an N-terminal half-domain polypeptide. Thus, for example, in one embodiment, the C-terminal half-domain is at least two or at least three C-terminal half-domains of the TbpB polypeptide obtainable from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis. According to the embodiment, the nucleic acid sequences encoding the C-terminal half-domains of TbpB from Actinobacillus pleuropneumoniae H49 (SEQ.ID NO: 5), Actinobacillus suis H57 (SEQ.ID NO: 33), and Actinobacillus pleuropneumoniae H87 (SEQ.ID NO: 21) can be ligated to form a chimeric nucleic acid sequence (SEQ.ID NO: 39) encoding a single polypeptide (SEQ.ID NO: 40) encompassing the three C-terminal halves (SEQ.ID NO: 6; SEQ.ID NO: 34; SEQ.ID NO: 22). Correspondingly, in a further embodiment, the present disclosure provides (i) a first polypeptide comprising an N-terminal half-domain or a C-terminal half-domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal half-domain or the C-terminal half-domain comprises multiple β-strands connected by multiple loop domains, and wherein at least one loop domain of the multiple loop domains of the N-terminal half-domain or the C-terminal half-domain has been modified, the first polypeptide being linked to (ii) multiple polypeptides, each polypeptide comprising a HIBP surface receptor protein or a portion thereof obtainable from a Gram-negative bacterial species. In a preferred embodiment, the portion of the HIBP surface receptor protein is an N-terminal half-domain or a C-terminal half-domain. In a further preferred embodiment, the portion of the HIBP surface protein is an N-terminal half-domain or a C-terminal half-domain of a HIBP surface receptor protein obtainable from or obtained from a Gram-negative pathogenic bacterial species, wherein the N-terminal half-domain or the C-terminal half-domain comprises multiple β-strands connected by multiple loop domains, and wherein at least one loop domain of the multiple loop domains of the N-terminal half-domain or the C-terminal half-domain has been modified.

[0159] In other embodiments, after removing one or more amino acids from one or more loop domains of the C-terminal or N-terminal half of the HIBP surface receptor protein, these residues are replaced with one or more replacement amino acid residues. In one embodiment, the replacement amino acid residues comprise a heterologous polypeptide antigen determinant capable of eliciting an immune response in a vertebrate host organism. In other embodiments, the replacement amino acid residues comprise two or more heterologous polypeptide antigen determinants capable of eliciting an immune response in a vertebrate host organism. The heterologous antigen determinants may be immunologically cross-reactive to the same or different pathogenic organisms. Thus, it should be clear that the modified C-terminal or N-terminal half domain of the HIBP surface receptor protein can be used as a scaffold to generate and present one or more antigen determinants. In a preferred embodiment, one or more loop regions of the C-terminal or N-terminal half domain of TbpB are replaced with one or more polypeptide moieties obtainable from or derived from an IOM protein, the IOM proteins including transferrin binding protein (“TbpA”) or lactoferrin binding protein A (“LbpA”). LbpA and TbpA polypeptides that can be used accordingly include those shown in SEQ.ID NO: 162 and SEQ.ID NO: 152. Portions of the LbpA and TbpA polypeptides that can be used accordingly include those shown in SEQ.ID NO: 286 and SEQ.ID NO: 287. These fragments can be used to construct chimeric nucleic acid sequences and polypeptides, including those shown in SEQ.ID.NO: 163; SEQ.ID.NO: 164; SEQ.ID.NO: 165; SEQ.ID.NO: 166; SEQ.ID.NO: 167; and SEQ.ID.NO: 168. This embodiment of the present disclosure is further detailed in Examples 7 and 8 in this regard. In a further preferred embodiment, one or more loop domains of the C-terminal or N-terminal half of TbpB are replaced with a lysine-rich polypeptide sequence, as further described in Example 9.

[0160] In all of the above-described embodiments of HIBP surface receptor polypeptides that include an N-terminal half domain, it is preferred that the N-terminal half domain is modified in such a way that the N-terminal anchoring polypeptide or most of it is removed from the N-terminal half domain. The length of the anchoring polypeptide can vary depending on the HIBP surface receptor polypeptide, but typically ranges from 40 to 75 amino acids in length and is located at the N-terminus of the mature HIBP polypeptide. Thus, referring to Figure 1, wherein the anchoring polypeptide of the depicted mature TbpB polypeptide is 43 amino acids in length. Accordingly, in a preferred embodiment in this regard of a HIBP polypeptide comprising an N-terminal half domain, the length of the anchoring polypeptide is reduced by at least 10 amino acid residues, preferably at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 residues. In embodiments where a portion of the anchoring polypeptide is selected for length reduction, preferably a continuous portion at the N-terminus of the anchoring polypeptide is removed, such as 10 terminal amino acid residues of the anchoring peptide, although other portions of the anchoring polypeptide may also be removed. In a further preferred embodiment, the TbpB polypeptide in which the anchoring peptide has been truncated comprises SEQ.ID NO: 279 to SEQ.ID NO: 283. The inventors of the present disclosure have found that the foregoing embodiments are particularly desirable because removal of the anchoring polypeptide reduces aggregation of the modified HIBP surface receptor polypeptide, thereby making the polypeptide easier to produce, yet does not substantially affect the immunogenic properties of the polypeptide. Thus, such modification of the HIBP polypeptide can be used in conjunction with any of the modified peptides that are substantially unable to bind the host iron-binding protein shown herein, including the HIBP polypeptides comprising an N-terminal half domain or a C-terminal half domain containing a modified loop domain as described herein, and the HIBP polypeptides comprising an N-terminal half domain or a C-terminal half domain in which a single amino acid has been replaced in such a way that the HIBP polypeptide is substantially unable to bind the host iron-binding protein.

[0161] As mentioned above, according to the present disclosure, the HIBP surface receptor protein is modified in such a way that it is substantially unable to bind the host iron-binding protein. The binding of the host iron-binding protein to the modified HIBP surface receptor protein can be evaluated using any chemical or biochemical assay capable of assessing such binding, including, for example, solid-phase binding assays, affinity capture assays or biophysical assays. General methods for performing these assays are known to those skilled in the art and are described, for example, in (12, 23 - 25, 50, 51). By performing these assays, the differences in the binding characteristics between the native HIBP surface receptor protein and the native host iron-binding protein and between the modified HIBP surface receptor protein and the native host iron-binding protein can be readily determined, and a series of modified HIBP surface receptor proteins can be evaluated to determine whether they can bind the host iron-binding protein. Different binding characteristics, including the binding constant (Kd), can be determined. As mentioned above, the Kd characterizing the binding between the host iron-binding protein and the modified HIBP surface receptor protein of the present disclosure is at least 2-fold that of the Kd characterizing the binding between the host iron-binding protein and the native HIBP surface receptor protein. An exemplary assay method for determining the binding constant using host transferrin is further described in Example 11 in this regard.

[0162] The present disclosure further includes a method for identifying a modified HIBP surface receptor protein, the method comprising:

[0163] (i) providing a modified HIBP surface receptor protein and a native HIBP surface receptor protein;

[0164] (ii) determining the binding characteristics between the modified HIBP surface receptor protein and a host iron-binding protein to obtain the binding characteristics of the modified HIBP surface receptor protein;

[0165] (iii) determining the binding characteristics between the native HIBP surface receptor protein and a host iron-binding protein to obtain the native HIBP surface receptor protein binding characteristics;

[0166] (iv) comparing the binding characteristics of the modified HIBP surface receptor protein characteristics with the native HIBP surface receptor protein characteristics; and

[0167] (v) identifying a HIBP surface receptor protein that exhibits binding characteristics that are substantially modulated relative to the binding characteristics of the native HIBP surface receptor protein.

[0168] As used herein, "substantially modulated" means that the binding interaction force between the modified HIBP surface receptor protein and the host iron-binding protein is substantially weaker than the binding interaction force between the native HIBP surface receptor protein and the host iron-binding protein. In a preferred embodiment, the binding characteristic used is the Kd related to the binding interaction between the HIBP surface receptor protein and the host iron-binding protein, wherein the Kd value of the binding interaction between the modified HIBP surface receptor protein and the host iron-binding protein is at least 2 times the Kd value of the binding interaction between the native HIBP surface receptor protein and the host iron-binding protein. Further, it should be noted that the foregoing method can be used to screen multiple different candidate HIBP surface receptor proteins simultaneously or sequentially, and to identify among the screened candidate HIBP surface receptor proteins those that exhibit more or less significant modulation of the binding characteristics relative to the native HIBP surface receptor protein.

[0169] The present disclosure further includes a method for preparing a modified HIBP surface receptor protein for use as a vaccine, the method comprising:

[0170] (i) providing a modified HIBP surface receptor protein and a native HIBP surface receptor protein;

[0171] (ii) determining the binding characteristics between the modified HIBP surface receptor protein and a host iron-binding protein to obtain the binding characteristics of the modified HIBP surface receptor protein;

[0172] (iii) Determine the binding characteristics between the native HIBP surface receptor protein and the host iron-binding protein to obtain the binding characteristics of the native HIBP surface receptor protein;

[0173] (iv) Compare the binding characteristics of the modified HIBP surface receptor protein with the binding characteristics of the native HIBP surface receptor protein;

[0174] (v) Identify HIBP surface receptor proteins that exhibit binding characteristics that are substantially modulated relative to the binding characteristics of the native HIBP surface receptor protein; and

[0175] (vi) Prepare modified HIBP surface receptor proteins having binding characteristics that are substantially modulated relative to the binding characteristics of the native HIBP surface receptor protein for use as vaccines.

[0176] According to the foregoing, the identified HIBP surface receptor proteins that exhibit binding characteristics that are substantially modulated relative to the binding characteristics of the native HIBP surface receptor protein can be used to prepare immunogenic preparations, for example, by recombinantly producing the HIBP surface receptor protein, isolating the HIBP surface protein, and preparing a vaccine preparation comprising the HIBP surface receptor protein.

[0177] In other embodiments, the present disclosure includes methods for assessing the cross-reactivity of antisera against surface receptor protein variants. Accordingly, the present disclosure further includes a method for assessing the cross-reactivity of antisera against surface receptor protein variants, the method comprising:

[0178] (i) Providing a plurality of nucleic acid sequences encoding surface receptor proteins;

[0179] (ii) Determining nucleic acid sequence variations among the plurality of surface receptor proteins;

[0180] (iii) Selecting variant portions of the surface receptor protein;

[0181] (iv) Linking a nucleic acid sequence encoding an N-terminal or C-terminal portion of the variant surface receptor protein to a nucleic acid sequence encoding a peptide that is readily susceptible to enzymatic biotinylation and a nucleic acid sequence capable of controlling expression in a host cell to form a chimeric nucleic acid sequence;

[0182] (v) Introducing the chimeric nucleic acid sequence into a host cell and expressing the chimeric nucleic acid sequence to produce a fusion polypeptide comprising an N-terminal or C-terminal portion of the variant surface receptor protein fused to a peptide that is readily susceptible to biotinylation;

[0183] (vi) Preparing a cell lysate from the host cell;

[0184] (vii) applying the cell extract to a streptavidin-coated immunoassay substrate material; and

[0185] (viii) applying an antiserum to the immunoassay substrate material, washing the immunoassay substrate material and applying a labeled second conjugate to evaluate the cross-reactivity between the antiserum and the variant portion of the surface receptor protein.

[0186] The nucleic acid sequence encoding a peptide that is readily susceptible to biotinylation may additionally comprise a nucleic acid sequence of sufficient length to permit a polypeptide extension of the fusion polypeptide upon expression such that the surface receptor protein is away from the immunoassay substrate material and thus fully accessible to antibody binding. The streptavidin-coated immunoassay substrate material can be any substrate material, including, for example, an ELISA plate.

[0187] In other embodiments, the present disclosure includes methods for evaluating the cross-reactivity or protective properties of an antiserum, a surface receptor protein variant, the method comprising:

[0188] (i) providing a plurality of nucleic acid sequences encoding a surface receptor protein;

[0189] (ii) determining nucleic acid sequence variations in the plurality of surface receptor proteins;

[0190] (iii) providing a host cell comprising a reverse selectable marker capable of replacing the nucleic acid sequence encoding the surface receptor protein;

[0191] (iv) PCR amplifying multiple variant portions of one or more nucleic acid sequences encoding the surface receptor protein to obtain a plurality of PCR products encoding surface receptor variants, wherein the PCR amplification is carried out in a manner that permits integration of the PCR products into a host cell comprising a reverse selectable marker, and wherein the PCR products comprise unique foreign nucleic acid sequences to permit identification of each PCR product;

[0192] (v) introducing and expressing the plurality of PCR products in a host cell comprising a reverse selectable marker to provide a library of antigenic HIBP variants; and

[0193] (vi) using all or a portion of the library in an in vivo or in vitro immunoassay to analyze the cross-reactivity or cross-protective properties of the library or a portion thereof.

[0194] The in vivo or in vitro immunoassay can be any assay, including any ELISA assay, functional immunoassay or animal infection model.

[0195] In other embodiments, the present disclosure includes a method of assessing the efficacy of a vaccine for preventing colonization of Gram-negative strains expressing surface receptor protein variants in the upper respiratory tract of a mammal, the method comprising:

[0196] (i) providing (a) a transgenic mouse line expressing a mammalian CEACAM receptor from the host species of the pathogen against which the vaccine is directed, and (b) a mouse line genetically identical to the transgenic mouse line but not expressing the CEACAM receptor;

[0197] (ii) demonstrating that a Gram-negative strain expressing a variant surface receptor protein is capable of colonizing the upper respiratory tract of the transgenic mouse line and is not capable of colonizing the upper respiratory tract of the mouse line not expressing the CEACAM receptor;

[0198] (iii) determining whether immunization with an antigen derived from the surface receptor protein results in the absence of colonization in the upper respiratory tract of transgenic mice infected with a Gram-negative strain expressing a surface receptor protein variant;

[0199] (iv) determining whether the provision of antiserum from animals immunized with an antigen derived from the surface receptor protein results in the absence of colonization in the upper respiratory tract of non-transgenic immunized mice infected with a Gram-negative strain expressing a surface receptor protein variant

[0200] (v) preparing a library comprising portions of the surface receptor protein from the Gram-negative strain and using the library in an animal upper respiratory tract colonization model to assess colonization of the upper respiratory tract of animals challenged with the surface receptor variant; and

[0201] (vi) optionally, extracting and preparing DNA obtained from the library used to challenge the animals and / or from samples obtained from animals challenged at an appropriate time after exposure, and determining the proportion of strains expressing different receptor variants.

[0202] Generally, those skilled in the art will understand upon reading the present disclosure that, in accordance with the present disclosure, a series of different regulated polypeptides can be prepared and obtained, all of which are modified HIBP surface receptor proteins, wherein the modification is made in such a way that the modified HIBP surface receptor protein cannot substantially bind to host iron-binding proteins. These regulated polypeptides and methods of preparing the regulated polypeptides are all intended to be included within the scope of the compositions and methods provided herein.

[0203] Modified C-terminal or N-terminal domain polypeptides are conveniently prepared by providing a nucleic acid sequence encoding a HIBP surface receptor protein and modulating the native nucleic acid sequence in a manner such that a polypeptide comprising the modified C-terminal or N-terminal domain is expressed in a recombinant host organism (e.g., a microbial cell). Modulation of the nucleic acid sequence can be carried out using a variety of nucleic acid modification techniques, which will generally be known to those skilled in the art, including, for example, site-directed mutagenesis, targeted mutagenesis, random mutagenesis, addition of organic solvents, gene rearrangement, or combinations of these techniques and other techniques known to those skilled in the art, each method being designed to target the loop domain of the C-terminal or N-terminal domain in a manner such that the loop domain is modified. Alternatively, a modulated nucleic acid sequence encoding a size-modified C-terminal or N-terminal domain polypeptide can be prepared de novo using gene synthesis techniques. General techniques for preparing and modifying nucleic acid sequences are readily available to the skilled person, for example in Green and Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, (33).

[0204] In other embodiments of the present disclosure, methods for preparing immunogenic compositions are provided. Accordingly, the present disclosure provides a method for preparing an immunogenic composition, the method comprising:

[0205] (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components:

[0206] (i) a nucleic acid sequence encoding a polypeptide comprising a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide has been modified in a manner such that it cannot substantially bind to a host iron-binding protein; and

[0207] (ii) a nucleic acid sequence capable of controlling expression in a recombinant host cell;

[0208] (b) introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the polypeptide comprising the C-terminal or N-terminal domain;

[0209] (c) recovering the polypeptide comprising the C-terminal or N-terminal domain from the host cell; and

[0210] (d) preparing an immunogenic composition

[0211] In certain embodiments, the C-terminal or N-terminal half-domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified, and wherein the polypeptide has been modified in such a way that it cannot substantially bind to the host iron-binding protein.

[0212] In a further preferred embodiment, the present disclosure provides a method for preparing an immunogenic composition, the method comprising:

[0213] (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components:

[0214] (i) a first nucleic acid sequence encoding a polypeptide comprising a first C-terminal or first N-terminal half-domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species;

[0215] (ii) a second nucleic acid sequence encoding a polypeptide comprising a second C-terminal or second N-terminal half-domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species; and

[0216] (iii) a nucleic acid sequence capable of controlling expression in a recombinant host cell;

[0217] (b) introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce a polypeptide comprising the first and second C-terminal or first and second N-terminal half-domains;

[0218] (c) recovering the polypeptide comprising the first and second C-terminal or first and second N-terminal half-domains from the host cell; and

[0219] (d) preparing an immunogenic composition.

[0220] In a preferred embodiment, the first and second nucleic acids are operably linked in such a way that a heteropolymeric fusion polypeptide comprising the first and second C-terminal or first and second N-terminal half-domains is produced, and wherein the heteropolymeric fusion polypeptide cannot substantially bind to the host iron-binding protein.

[0221] Accordingly, the nucleic acid sequence encoding the HIBP surface receptor protein is linked to a nucleic acid sequence capable of controlling the expression of the HIBP surface receptor protein in a host cell. Correspondingly, the present disclosure also provides a nucleic acid sequence encoding the HIBP surface receptor protein linked to a promoter capable of controlling expression in a host cell. Nucleic acid sequences capable of controlling expression in a host cell useful herein include any transcriptional promoter capable of controlling the expression of a polypeptide in a host cell. Generally, promoters obtained from bacterial cells are used when a bacterial host is selected accordingly, while fungal promoters will be used when a fungal host is selected, plant promoters will be used when plant cells are selected, and so on. Other nucleic acid elements capable of controlling expression in a host cell include transcriptional terminators, enhancers, etc., all of which may be included in the chimeric nucleic acid sequences of the present disclosure.

[0222] According to the present disclosure, a chimeric nucleic acid sequence comprising a nucleic acid sequence encoding a polypeptide comprising a C-terminal half domain or an N-terminal half domain of the HIBP surface receptor protein obtainable or obtained from a Gram-negative bacterial species and linked to a promoter capable of controlling expression in a host cell can be integrated into a recombinant expression vector to ensure good expression in a host cell, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified. Correspondingly, the present disclosure includes a recombinant expression vector, the vector comprising the following as operably linked components:

[0223] (i) a nucleic acid sequence capable of controlling expression in a host cell; and

[0224] (ii) a nucleic acid sequence encoding a polypeptide comprising a C-terminal half domain or an N-terminal half domain of the HIBP surface receptor protein obtainable or obtained from a Gram-negative bacterial species, wherein the polypeptide has been modified in such a way that it cannot substantially bind to the host iron-binding protein.

[0225] Wherein the expression vector is suitable for expression in a host cell. The term "suitable for expression in a host cell" means that the recombinant expression vector contains the chimeric nucleic acid sequence of the present disclosure linked to genetic elements required for expression in the host cell. Genetic elements that may be included in the expression vector in this regard include transcriptional termination regions, one or more nucleic acid sequences encoding marker genes, one or more origins of replication, and the like. Typically, those skilled in the art also know that the genetic elements are operably linked by, for example, connecting a promoter in the 5' to 3' transcriptional direction to the coding sequence. In a preferred embodiment, the expression vector further contains genetic elements required for integrating the vector or a portion thereof into the genome of the host cell. In other embodiments, the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and at least one of the plurality of loop domains has been modified.

[0226] According to the present disclosure, the expression vector may further contain a marker gene. Marker genes that can be used according to the present disclosure include all genes that distinguish transformed cells from non-transformed cells, including all selectable and screenable marker genes. The marker gene may be a resistance marker, such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable marker genes that can be used to identify transformants by visual inspection include β-galactosidase, β-glucuronidase (GUS) (U.S. Patent Nos. 5,268,463 and 5,599,670), and green fluorescent protein (GFP) (52).

[0227] A host cell that can be used particularly conveniently is Escherichia coli. The preparation of an Escherichia coli vector can be achieved using generally known techniques, such as restriction digestion, conjugation, conjugation-independent cloning, gel electrophoresis, DNA sequencing, polymerase chain reaction (PCR), and other methods. A variety of cloning vectors can be used to perform the necessary steps required for the preparation of a recombinant expression vector, including the custom vectors developed by the present inventors. Vectors such as those of the pUC or pET series are among the vectors having a replication system that is functional in Escherichia coli. Typically, these cloning vectors contain markers that allow for the selection of transformed cells. Nucleic acid sequences can be introduced into these vectors, and the vectors can be introduced into Escherichia coli by preparing competent cells, electroporation, or using other methods well known to those skilled in the art. Escherichia coli can be grown in a suitable medium such as Luria-Broth medium and harvested. The recombinant expression vector can be easily recovered from the cells upon collection and lysis of the cells. In addition, general guidance on the preparation of recombinant vectors and the growth of recombinant organisms can be found, for example, in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, 3rd Edition (33).

[0228] Production of the recombinant protein can occur throughout the growth of an E. coli strain, preferably by inducing expression after the growth phase to achieve significant biomass. This will result in the production of polypeptides comprising the C-terminal half domain or the N-terminal half domain or the C-terminal half domain or the N-terminal half domain with a modified loop. The polypeptide can then be recovered, isolated, and separated from other host cell components by a variety of different protein purification techniques, including, for example, metal chelate chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, etc. Other general guidance on protein purification can be found, for example, in Protein Purification: Principles, High Resolution Methods, and Applications (53). As used herein, the term "recovered" means that the polypeptide is obtained in a more or less pure form. In a preferred embodiment, a substantially immunogenic polypeptide comprising the C-terminal half domain of the HIBP surface receptor protein obtainable from a Gram-negative bacterial species can be obtained thereby, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands linked by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified. Thus, the HIBP polypeptide obtained thereby can be prepared in a substantially pure form. "Substantially pure" means that the immunogenic protein is separated from other host cell components. Accordingly, the immunogenic protein is at least 95% pure, and more preferably at least 96%, 97%, 98%, or 99% pure. Alternatively, a relatively crude composition comprising the HIBP polypeptide can be obtained, such as cells containing the polypeptide, cell lysates containing the polypeptide, or cell components containing the polypeptide.

[0229] In other embodiments, the present disclosure provides methods for eliciting an immune response in a vertebrate subject. The immune response can be elicited by delivering the immunogenic protein or by delivering an expression vector comprising a nucleic acid sequence encoding the immunogenic protein. Accordingly, the present disclosure further provides a method for eliciting an immune response in a vertebrate subject, the method comprising administering to the subject:

[0230] (a) an immunogen comprising a polypeptide comprising the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; or

[0231] (b) an expression vector comprising a nucleic acid sequence encoding an immunogen comprising a polypeptide comprising the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; and

[0232] Wherein the immunogen is administered or expressed in an amount sufficient to elicit an immune response in the vertebrate subject.

[0233] The present disclosure also provides the use of the following in eliciting an immune response in a vertebrate subject:

[0234] (a) An immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; or

[0235] (b) An expression vector comprising a nucleic acid sequence encoding an immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein.

[0236] The present disclosure further provides the use of the following in the manufacture of a medicament for eliciting an immune response in a vertebrate subject:

[0237] (a) An immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; or

[0238] (b) An expression vector comprising a nucleic acid sequence encoding an immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein.

[0239] The present disclosure also provides the following for eliciting an immune response in a vertebrate subject:

[0240] (a) An immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein; or

[0241] (b) An expression vector comprising a nucleic acid sequence encoding an immunogen comprising a polypeptide that comprises a C-terminal or N-terminal domain of a HIBP surface receptor protein obtainable from a Gram-negative bacterial species, wherein the polypeptide does not substantially bind to a host iron-binding protein.

[0242] In a preferred embodiment, the polypeptide comprises at least two C-terminal half domains or at least two N-terminal half domains. In a further preferred embodiment, the polypeptide comprises at least three C-terminal half domains or at least three N-terminal half domains.

[0243] In certain embodiments, the C-terminal half domain or N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified in such a way that the C-terminal half domain or N-terminal half domain cannot substantially bind to a host iron-binding protein.

[0244] The present disclosure further includes an immunogen comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor polypeptide for use as a medicament, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified.

[0245] The present disclosure further includes an immunogen comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor polypeptide, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified, the immunogen for preventing an infection or disease caused by an infectious Gram-negative bacterium, the bacterium including a bacterium belonging to the genus Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella or Moraxella.

[0246] The present disclosure further includes an immunogen comprising a C-terminal half domain or an N-terminal half domain of a HIBP surface receptor polypeptide, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified, the immunogen for manufacturing a medicament for preventing an infection or disease caused by an infectious Gram-negative bacterium, the bacterium including a bacterium belonging to the genus Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella or Moraxella.

[0247] Vaccine Preparation

[0248] The present disclosure further provides a vaccine preparation. Accordingly, the present disclosure further provides a vaccine composition comprising an antigen derived from a HIBP surface receptor protein from a Gram-negative pathogenic bacterial species, wherein the protein derived from the HIBP surface receptor protein has been modified in such a way that it cannot substantially bind to a host iron-binding protein. The vaccine composition of the present disclosure preferably comprises a vaccine comprising a polypeptide comprising a HIBP surface receptor protein, a C-terminal half domain or an N-terminal half domain obtainable from a Gram-negative pathogenic bacterial species, wherein the polypeptide has been modified in such a way that it cannot substantially bind to a host iron-binding protein. In a preferred embodiment, the vaccine preparation comprises a mixture of C-terminal half domains belonging to two different bacterial species or two different strains. In a further preferred embodiment, the polypeptide comprises at least two or at least three N-terminal half domains or C-terminal half domains. In a further preferred embodiment, the at least two or at least three N-terminal half domains or C-terminal half domains form a heteropolymer. In other embodiments, the C-terminal half domain or N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one of the plurality of loop domains has been modified.

[0249] The vaccine preparation of the present disclosure comprises an immunogenic HIBP polypeptide in a more or less pure form. Accordingly, a substantially pure HIBP polypeptide can be obtained and used to prepare a vaccine preparation. In other embodiments, a cruder HIBP polypeptide preparation can be obtained and used to prepare a vaccine preparation. Thus, for example, in such embodiments, cells, cell lysates or cell components comprising the HIBP polypeptide can be used to prepare a vaccine preparation.

[0250] To enhance the immune response in a subject, the compositions provided herein further preferably include an adjuvant, such as a pharmacological agent, a cytokine, etc. Suitable adjuvants include any substance that enhances the immune response of the subject to the immunogenic polypeptide of the present disclosure. Non-limiting examples of adjuvants include cytokines, such as IL-1, IL-2, IL-12, IL-6, and further include inorganic salts, such as aluminum hydroxide, aluminum phosphate, and calcium phosphate; oil emulsions, such as mineral oil, MF59, QS-21, Montamide ISA51, and ISA-720; Isocom, such as ISCOMATRIX; microbial derivatives, such as MPLA, macrophage activating protein-2, virus particles, LT / CT, CpG; natural polymers, such as polysaccharides; and synthetic polymers, such as polyanhydrides and polyesters, as reviewed by Wilson-Welder et al. (54). The adjuvant can be administered, for example, simultaneously with the administration of the polypeptide antigen as a protein or other macromolecule, before or after the administration of the peptide antigen.

[0251] The dosage of the immunogenic protein generally ranges from about 0.1 μg to about 20 mg, preferably from 10 μg to about 3 mg, for human subjects. However, the exact amount will vary depending on the age and general condition of the subject to be treated, the severity of the condition being treated, the particular formulation being delivered, the site of administration, and other factors. The appropriate therapeutically effective amount can be readily determined by those skilled in the art. Thus, a "therapeutically effective amount" of the compositions of the invention will be an amount sufficient to effect treatment or prevention of the symptoms of a disease or condition or to prevent colonization by pathogenic bacteria and will fall within a relatively broad range that can be determined by routine experimentation.

[0252] Vaccine formulations comprising the immunogenic compositions of the present disclosure preferably further comprise a vehicle, excipients, and auxiliary substances that may be present in the excipients or vehicle, such as wetting or emulsifying agents, pH buffering substances, and the like. These vehicles, excipients, and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the subject and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included therein, such as inorganic acid salts, e.g., hydrochloride, phosphate, sulfate, etc.; and salts of organic acids, e.g., acetate, propionate, benzoate, etc. Although not required, it is also preferred that the formulation will contain a pharmaceutically acceptable excipient as a stabilizer, particularly for stabilizing the polypeptides of the present disclosure. Examples of suitable carriers that also serve as stabilizers for peptides include, but are not limited to, pharmaceutical grade dextran, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable carriers also include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, glycine, polyethylene glycol (PEG), and combinations thereof.

[0253] The vaccine formulations of the present disclosure can be used to prevent infections or diseases caused by infectious Gram-negative bacteria, including but not limited to bacteria belonging to the genera Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella, or Moraxella, including but not limited to Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Neisseria meningitidis. The vaccine formulations can be used to immunize any vertebrate subject, including any vertebrate subject expressing a host iron-binding protein and including but not limited to any mammalian subject, including any human subject, porcine subject, bovine subject, equine subject, ovine subject, caprine subject, canine subject, feline subject, rabbit subject and further including any ruminant subject and murine subject. Other vertebrate subjects that can be immunized include any avian subject and fish subject. The vaccine formulations of the present disclosure can exhibit enhanced cross-reactivity and / or cross-protective immune responses in a recipient host organism. It should be further noted that the vaccine formulations of the present disclosure can prevent infections and / or colonization by infectious Gram-negative bacteria, including bacterial colonization of the respiratory or genital tract of a vertebrate subject.

[0254] The present disclosure further includes a vaccine comprising the HIPB surface receptor protein, the C-terminal half domain or the N-terminal half domain of the HIBP surface receptor polypeptide, wherein the C-terminal half domain or the N-terminal half domain comprises a plurality of β-strands connected by a plurality of loop domains, and wherein at least one loop domain has been modified, the vaccine being for preventing an infection or disease caused by an infectious Gram-negative bacteria, including bacteria belonging to the genera Actinobacillus, Neisseria, Haemophilus, Mannheimia, Histophilus, Pasteurella, or Moraxella.

[0255] Test Vaccine Preparation

[0256] Accordingly, the efficacy of the vaccine formulations of the present disclosure can be evaluated, for example, by determining the antibody titer present in the serum of a subject immunized with the vaccine formulation, for example, by performing an enzyme-linked immunosorbent assay (ELISA). Accordingly, the present disclosure further encompasses a method for evaluating the efficacy of a vaccine formulation comprising the C-terminal half domain or the N-terminal half domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species, wherein the polypeptide is modified in such a way that it cannot substantially bind to a host iron-binding protein, the method comprising:

[0257] (a) administering to a vertebrate subject a vaccine formulation comprising a HIPB surface receptor protein or the C-terminal half domain or the N-terminal half domain of a HIBP surface receptor protein obtainable from a Gram-negative pathogenic bacterial species, wherein the polypeptide is modified in such a way that it cannot substantially bind to a host iron-binding protein;

[0258] (b) Obtaining serum from the vertebrate subject; and

[0259] (c) Analyzing the presence of antibodies against the HIBP surface receptor polypeptide in the serum.

[0260] The vertebrate serum can be analyzed after administration of a single or multiple (e.g., 2, 3, or 4) doses of the vaccine preparation. The analysis can be performed using isolates of the HIBP surface receptor protein from different strains or species of a single or multiple microorganisms, such as an ELISA assay. The ELISA assay can involve conjugating the HIBP surface receptor polypeptide to a carrier protein, such as maltose-binding protein. When analyzing the reactivity of antibodies against multiple isolates of the HIBP surface receptor protein, it is possible to evaluate the cross-reactivity of the vaccine preparation.

[0261] Vaccination Regimen

[0262] As will be apparent to those skilled in the art in light of the teachings of this specification, vaccination using the above polypeptide or using a nucleic acid sequence encoding the polypeptide (DNA vaccine) can be carried out continuously or intermittently in a treatment course with one dose. Methods for determining the most effective mode and dose of administration are well known to those skilled in the art and will vary with the delivery vehicle, the nature of the composition, the specific prophylaxis or therapy being sought, the target cells, and the subject being treated. Single and multiple administrations can be carried out at dose levels and patterns selected by a suitable medical practitioner.

[0263] Administration of the above pharmaceutical preparation can be carried out continuously or intermittently in a treatment course with one dose. Delivery will most typically be via conventional needles and syringes for liquid compositions and for liquid suspensions of particulate compositions. Additionally, various liquid jet injectors are known in the art and can be used to administer the compositions of the present invention. The vaccine delivery route can vary. Thus, the vaccines of the present disclosure can be delivered intravenously, subcutaneously, intramuscularly, vaginally, intraperitoneally, intranasally, orally, or via other mucosal routes. Methods for determining the most effective mode and dose of administration are well known to those skilled in the art and will vary with the delivery vehicle, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out at dose levels and patterns selected by a attending physician or veterinarian. Examples

[0264] The following are examples for carrying out specific embodiments of the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0265] Example 1 - Immune responses using the C-terminal half domain of TbpB, the N-terminal half domain of TbpB, and mixtures thereof.

[0266] This example provides an illustration of the value of using subdomains of the TbpB receptor protein to obtain a more desirable immune response. By a more desirable immune response, we consider both the magnitude of the antibody response and the cross-reactivity of the antibody with variant TbpB proteins.

[0267] Figure 3A Illustrate the results of the first experiment for this example, in which different host species (mouse, rabbit, and pig) were immunized with whole TbpB from the human pathogen Neisseria meningitidis (strain B16B6 - SEQ.ID NO: 117) or from the porcine pathogen Actinobacillus pleuropneumoniae (strain H49 - SEQ.ID NO: 2). Sera from the immunized animals were tested against the immunizing antigen in our custom ELISA assay (see below). The results illustrate that the magnitude (titer) of the antibody response in pigs using TbpB from the porcine pathogen Actinobacillus pleuropneumoniae (gray bars) is substantially lower than in other host species compared to TbpB from the human pathogen Neisseria meningitidis (black bars), indicating that the binding of host transferrin affects the development of the antibody response against TbpB.

[0268] The second experiment ( Figure 3B ) involved immunizing pigs with whole TbpB from the bovine pathogen Mannheimia haemolytica (strain H196 - SEQ.ID NO: 206) or whole TbpB (SEQ.ID NO: 2), the N-terminal half of TbpB (SEQ.ID NO: 8), or the C-terminal half of TbpB (SEQ.ID NO: 6) from the porcine pathogen Actinobacillus pleuropneumoniae (strain H49). Figure 3B Illustrate the immune response (bars) in individual pigs before immunization (white bars), after the first immunization (light gray bars), after the second immunization (dark gray bars), and after the third immunization (black bars). Note that titers are expressed as binary logarithms to reflect the two-fold dilutions used to assess titers. Most pigs exhibited high antibody titers (between 26,000 and 256,000) after the third immunization, but several pigs immunized with whole TbpB or the N-terminal half of TbpB from Actinobacillus pleuropneumoniae exhibited substantially reduced titers (between 5,300 and 8,000). The observation that two out of four pigs immunized with whole TbpB and three out of three pigs immunized with the N-terminal half of TbpB exhibited substantially reduced titers indicates that the binding of host transferrin affects the development of the antibody response only in a subset of the animals.

[0269] The third experiment illustrated in this example ( Figure 3C) is designed to evaluate the ability of sera against full-length TbpB and its subdomains to react with different representative TbpBs from porcine pathogens in order to assess the cross-reactivity of the antiserum. Pigs were immunized with full-length TbpB (SEQ.ID NO: 2), the C-terminal half-domain of TbpB (SEQ.ID NO: 6), the N-terminal half-domain of TbpB (SEQ.ID NO: 8), or a mixture of the N-terminal half and the C-terminal half of TbpB. The sera were tested against (i) full-length TbpB (SEQ.ID NO: 2) from Actinobacillus pleuropneumoniae strain H49, (ii) full-length TbpB (SEQ.ID NO: 115) from Haemophilus parasuis strain HP5, or (iii) full-length TbpB (SEQ.ID NO: 12) from Actinobacillus pleuropneumoniae strain H87.

[0270] Figure 3C The results in illustrate that the C-terminal half-domain of TbpB from Actinobacillus pleuropneumoniae strain H49 induces a more cross-reactive immune response than full-length TbpB (higher titers against heterologous TbpB from strain H87) or the N-terminal half of TbpB (higher titers against strains H87 and HP5). The TbpBs used in this analysis were designed to represent the overall sequence and structural diversity present in clinical disease isolates of Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis from pigs worldwide (SEQ.ID NO: 2; SEQ.ID NO: 12; SEQ.ID NO: 28; and SEQ.ID NO: 107 to SEQ.ID NO: 115)( Figure 4 ). Thus, in this example, we did not limit our analysis to a single porcine pathogen, but targeted three different porcine pathogens that are problematic in the global swine industry. The fact that these pathogens share a common mechanism for obtaining iron from the host provides a unique opportunity to develop a vaccine targeting the three pathogens from the common antigen TbpB. These results indicate that by using one or more C-terminal half-domains as antigens, it is feasible to generate a broad cross-reactive response against porcine pathogens worldwide and thus potentially consider vaccination to eliminate the global presence of these pathogens.

[0271] Surprisingly, the enhanced cross-reactivity induced by the C-terminal half-domain is retained even when the C-terminal half is mixed with the N-terminal half in the immunization mixture ( Figure 3C , "N + C-terminal half"). These results teach us that antigens with smaller variable loop regions (the "C-terminal half" compared to the "N-terminal half", Figure 1) It is capable of inducing a more cross-reactive antibody response, and this enhanced ability to induce a cross-reactive immune response is retained even when the C-terminal half is combined with other antigens in an immunological mixture. More specifically, these results indicate that the tendency of the N-terminal half to generate a more strain-specific immune response can only effectively inhibit the cross-reactive immune response induced by the C-terminal half when the C-terminal half is physically linked to the N-terminal half. In other words, unexpectedly, the tendency of the N-terminal half to generate a more specific immune response is substantially reduced when the N-terminal half is mixed with the C-terminal half. To our knowledge, this phenomenon has not been previously described.

[0272] The results also showed a reduced response to intact TbpB and the TbpB N-terminal half relative to the C-terminal half ( Figure 3B 、 3C ), which may indicate that the binding of host transferrin (a feature present only in intact TbpB and the TbpB N-terminal half) can modulate the immune response in pigs.

[0273] Importantly, in contrast to previously published studies, we did not use the standard ELISA method for measuring antibody levels because we identified a major flaw in the standard ELISA method. We observed that, in contrast to what is commonly assumed, purified proteins do not necessarily bind randomly to the ELISA plate, potentially providing significant bias or flaws in assessing the binding of antibodies to epitopes on the surface of the protein. In particular, we noted that TbpB and especially the N-terminal half of TbpB bind to the solid surface of the ELISA plate in a substantially single orientation, masking the cap region of the N-terminal half and preventing the detection of transferrin binding ( Figure 5 ). In contrast, recombinant fusion proteins with an N-terminal maltose-binding protein (Mbp) chaperone are commonly used for transferrin binding ( Figure 5 ), and the chaperone is a precursor for the purified TbpB N-terminal half. This phenomenon was observed with the TbpB N-terminal half from human, porcine, and bovine pathogens and to a lesser extent with intact TbpB. Since the sequences of these proteins are extremely different (<30% overall sequence identity), this indicates that this is not a unique property of a specific protein, but rather that the degree of non-random binding can vary and affect solid-phase binding assays.

[0274] To overcome this flaw, we designed a method for binding recombinant proteins to streptavidin-coated ELISA plates by means of biotin residues that are enzymatically added to the N-terminus during protein expression in the cytoplasm. As Figure 5As shown, the addition of the enzymatically biotinylated N-terminal peptide restored the ability of the N-terminal half of TbpB to bind transferrin. This approach more effectively exposes the transferrin-binding region, in contrast to the results using the N-terminal half of Tbp fused to Mbp, as indicated on the left and right sides of this figure. This novel and innovative ELISA assay format is used in all of our ELISA assays for monitoring antibody reactivity because it ensures complete and equal access to all epitopes on the target protein, thus providing a true comparison when assessing the degree of cross-reactivity.

[0275] Recombinant antigens for immunization experiments were produced in the cytoplasm of Escherichia coli using a custom T7 expression vector encoding an N-terminal polyhistidine tag, maltose-binding protein, and a tobacco etch virus (TEV) protease cleavage site. The recombinant fusion protein was separated by Ni-NTA chromatography, and the antigen was released by (TEV) cleavage and purified by a combination of Ni-NTA and Q-agarose chromatography.

[0276] FVB mice (albino MHC haplotype H2q from Charles River), 3-month-old New Zealand white rabbits, and 51-day-old Large White Landrace F1 hybrid pigs were used for Figure 3A the immunization experiments. The purified recombinant protein was mixed with phosphate-buffered saline and 33% ( Figure 3A ) or 20% ( Figure 3B , Figure 3C ) Emulsigen D (MVP Technologies) to a final concentration of 25 μg / 0.1 ml (mice), 50 μg / 0.5 ml (rabbits), and 100 μg / 2 ml (pigs). The three injection solutions were administered subcutaneously to mice and rabbits and subcutaneously ( Figure 3A ) or intramuscularly ( Figure 3B , Figure 3C ) to pigs. The animals were immunized on days 0, 21, and 42 and the final blood was collected on day 56.

[0277] Serum obtained at 8 weeks was tested against representative proteins in our custom solid-phase ELISA assay. Recombinant fusion proteins for ELISA assays were produced in the cytoplasm of Escherichia coli using a custom T7 expression vector encoding an N-terminal optimized biotinylation sequence, polyhistidine tag, maltose-binding protein, and a tobacco etch virus (TEV) protease cleavage site. These proteins were biotinylated in vivo at the N-terminal biotinylation sequence, enabling their application to streptavidin-coated ELISA plates.

[0278] Based on previous optimization experiments, the crude extract from a small-scale overnight protein expression experiment using an expression vector for a biotinylated fusion protein was sufficient to saturate the ELISA plates. Dilutions of the antiserum of interest were prepared in 2.5% skim milk in phosphate-buffered saline (PBST) and applied to the plates for 1 hour at room temperature. After removal and washing, the primary antibody was detected at room temperature for 1 hour by HRP-conjugated goat anti-mouse, anti-rabbit, or anti-pig IgG at a dilution of 1:100,000 (1:25,000 for anti-pig). The titer was expressed as the reciprocal of the last dilution at A450 > 0.3 (greater than the mean plus three standard deviations of the background readings of the wells without added serum).

[0279] Calculation of SEM error was performed via ANOVA, where Tukey's HSD (honestly significant difference) test was performed as a post hoc test. Statistics showed that for all sera, H49 was significantly different from H87 and the N-terminal half was significantly different from the C-terminal half or N + C-terminal half. Figure 3C The asterisks shown indicate specific immune / protein pairs that are significantly different from the C-terminal half or N + C-terminal half tested against H49.

[0280] Example 2 - Generation of a trimer of the C-terminal half of porcine pathogen TbpB

[0281] In this example, we demonstrated that three recombinant engineered C-terminal halves representing the extensive diversity of TbpB present in three porcine pathogens could be joined together and retain antigenicity. The engineered C-terminal halves used herein were those obtained from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis ( Figure 4 ). Thus, the genes encoding the TbpB C-terminal halves from strains Actinobacillus pleuropneumoniae H49 (SEQ.ID NO: 6), Actinobacillus suis H57 (SEQ.ID NO: 35), and Actinobacillus pleuropneumoniae H87 (SEQ.ID NO: 22) were ligated to form a gene encoding Figure 6 a single polypeptide (SEQ.ID NO: 40) in Figure A that encompasses the three C-terminal halves. In this C-terminal half trimer, there are relatively short peptides (indicated by underlining) that link the secondary structure elements of the C-terminal halves. The linker peptides consist of the putative inter-half sequences from the individual C-terminal half domains. Figure 6 Figure B illustrates the generation of the C-terminal half trimer and compares it to preparations of recombinant engineered TbpB N-terminal and C-terminal halves from the human pathogen Neisseria meningitidis strain M982. The results indicate that the C-terminal half trimer was produced in good quality and was stable. However, Figure 6 the preparations illustrated in

[0282] Since there is no major barrier to the production of the stable C-terminal half-trimer, the C-terminal half-trimer is produced and purified for immunization experiments to determine whether it retains immunogenicity. As Figure 7 illustrated therein, the C-terminal half-trimer is capable of inducing an immune response against the three representative TbpBs, indicating that the three individual C-terminal halves linked together do not substantially alter their immunological properties. The results also suggest that a single protein antigen may be capable of inducing an immune response that can react with most, if not all, of the Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis porcine pathogen strains, showing potential promise for the development of a broadly cross-protective porcine vaccine.

[0283] Example 3 - Reduction of the loop region in the N-terminal half of Actinobacillus pleuropneumoniae TbpB

[0284] In this example, the loop regions of the N-terminal half domains of three representative TbpBs from porcine pathogens were modified to determine their effect on the induction of cross-reactive immune responses. The three representative TbpBs are from Actinobacillus suis strain H57 (SEQ.ID NO: 28), Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 12), and Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 2). These particular TbpBs were chosen not only because they represent sequence diversity but also because high-resolution structural data are available; Actinobacillus suis strain H57 (3PQU.pdb), Actinobacillus pleuropneumoniae strain H87 (3PQS.pdb), and Actinobacillus pleuropneumoniae strain H49 (3HOL.pdb). The loop reduction process resulted in an overall loss of 74 amino acids (297 - 224) for the TbpB N-terminal half from Actinobacillus suis strain H57 (SEQ.ID NO: 36, 38), an overall loss of 45 amino acids (248 - 203) for the TbpB N-terminal half from Actinobacillus pleuropneumoniae strain H87 (SEQ.ID NO: 24, 26), and an overall loss of 27 amino acids (274 - 247) for the TbpB N-terminal half from Actinobacillus pleuropneumoniae strain H49 (SEQ.ID NO: 8, 10). The design of the loop reduction for Actinobacillus pleuropneumoniae strain H49 is described in more detail below for illustrative purposes. In each case, the first-listed SEQ.ID refers to the TbpB N-terminal half sequence, and the second SEQ.ID refers to the engineered loop reduction.

[0285] For loop reduction, the structure of TbpB from strain H49 (3HOL.pdb) was overlaid with the structure of TbpB from strain H87 (3PQS.pdb), while examining the multiple sequence alignment of a collection of representative TbpBs from porcine pathogens. By examining the structure and variable regions, several regions were identified for potential loop reduction that were predicted not to disrupt the overall structure of the N-terminal half. These regions include loops 1, 5, 8a, 8c, and 12 according to standard nomenclature ( Figure 2 ), and are illustrated on the structural models of the side view (Figure A) and top view (Figure B) of the TbpB N-terminal half in Figure 8 . Loop reduction was designed to minimize potential disruption to the overall fold and structure of the N-terminal half by, in addition to removing amino acids, selecting appropriate amino acid substitutions when needed. The amino acid sequences of the initial N-terminal half (top sequence) and the modified N-terminal half (bottom sequence) are illustrated in a sequence alignment in Figure 8 Figure C, where the loop regions are highlighted in gray and labeled in gray font. The DNA sequences encoding the amino acid sequences illustrated in Figure C were optimized for expression in an E. coli strain and then synthesized.

[0286] A similar strategy was employed to generate loop reduction for the TbpB N-terminal half from Actinobacillus pleuropneumoniae strain H87 and Actinobacillus suis strain H57, as they represent significant sequence and structural diversity of TbpB from porcine pathogens ( Figure 4 ) and the related structures (3PQS.pdb and 3PQU.pdb) were available (12). The potential to generate engineered antigens that together may induce cross-reactive responses against the N-terminal half regions of most, if not all, clinical isolates would have significant potential for enhancing the effectiveness of vaccines targeting these three porcine pathogens.

[0287] The resulting genes were cloned into a custom expression vector encoding an N-terminal polyhistidine region, maltose binding protein, and a TEV (tobacco etch virus) cleavage site upstream of the coding sequence of the cloned N-terminal half. The expression plasmid for the engineered form of the TbpB N-terminal half from Actinobacillus pleuropneumoniae strain H49 was transformed into the E. coli expression strain ER256 carrying a chromosomal copy of the T7 RNA polymerase gene inserted downstream of the lacZ promoter. Small-scale expression analysis was performed using an autoinduction medium that utilizes glucose repression and lactose induction of the lac promoter (55). Thus, by using a specific ratio of glucose to lactose in the medium, the expression of the T7 RNA polymerase was optimally initiated in mid-logarithmic growth phase. Cells were lysed using a bead mill after overnight growth, the recombinant protein was captured using Ni-NTA resin or porcine Tf-agarose resin, washed, and the bound protein was eluted in SDS-PAGE buffer.

[0288] As Figure 9 described, loop reduction in the N-terminal half of TbpB from Actinobacillus pleuropneumoniae strain H49, Actinobacillus suis strain H57, or Actinobacillus pleuropneumoniae strain H87 results in the production of a stable protein, thus not interfering with the overall folding of the N-terminal half and providing material suitable for immunization. The upper panel shows recombinant proteins produced in small-scale expression experiments captured on Ni-NTA resin, with wild-type full-length TbpB and the N-terminal half of TbpB as controls. The results also show that, unlike the wild-type protein, the mutant protein is no longer able to substantially bind to porcine transferrin (Tf). Thus, in the middle panel, the engineered N-terminal half is not captured by porcine Tf-agarose. In the bottom panel, the material described in the upper panel is used in a solid-phase binding assay using HRP-conjugated porcine transferrin and, unlike the control, the engineered N-terminal half does not exhibit any binding activity. We expect enhanced immunogenicity of these proteins in the natural host pig. Thus, this example shows that the strategy for removing antigenic variable regions in the N-terminal half of the TbpB protein is feasible and that such removal can lead to enhanced immunogenicity.

[0289] Example 4 - Cross-Reactivity of the C-Terminal Half of Neisseria meningitidis TbpB

[0290] This example illustrates the use of engineered derivatives of TbpB from the human pathogen Neisseria meningitidis according to the present disclosure. As a first step, we examined the diversity of TbpB from Neisseria meningitidis and ensured that we would have a representative set of TbpB for our assessment of cross-reactivity. Figure 10 illustrates the overall diversity of TbpB in Neisseria meningitidis strains collected globally over a long period plus additional sequences from the Neisseria Bacterial Isolate Genome Sequence Database (BIGSDB http: / / pubmlst.org / neisseria / ) (56)(41). Regarding those from Figure 10ASequences of a representative set of TbpB of strains indicated by arrows, double arrows or lines therein (SEQ.ID NO: 117; SEQ.ID NO: 123; SEQ.ID NO: 132 to SEQ.ID NO: 147; SEQ.ID NO: 177; and SEQ.ID NO: 178) are included in the present application to define the sequence diversity within each group. There are four main phylogenetic groupings of Neisseria meningitidis TbpB. Group 1 includes strains with isoform I TbpB. Isoform I TbpB is characterized by a smaller TbpB (about 65-70 kDa) compared to isoform II TbpB (80-85 kDa). Comparison of the sequences by multiple sequence alignment revealed that the difference in size is mainly attributed to the larger C-terminal half in isoform II TbpB. Isoform II TbpB clusters into three main phylogenetic groups (groups 2-4, Figure 10A ).

[0291] A phylogenetic tree ([[]] Figure 10B Figure 10B ) illustrating the sequence diversity of the C-terminal half of TbpB supports the conclusion that the C-terminal half sequences are mainly responsible for the identification of the two TbpB isoforms. Figure 10A Members of group 2 in []] Figure 10A Figure 10B do not cluster together in the C-terminal half phylogenetic tree but are distributed throughout the tree in []] Figure 10B

[0292] , indicating that group 2 is mainly defined by the N-terminal half sequences. This indicates that if immunological cross-reactivity is to be determined by reactivity against the C-terminal half, then specific representatives of group 2 will not be required. In contrast, the set of TbpB sequences used to represent the overall TbpB diversity by arrows and lines does not adequately represent the overall C-terminal half diversity, and therefore two additional strains were selected to provide a more comprehensive representation of the C-terminal half diversity. C-terminal half sequences from strains identified by arrows, double arrows or lines are included in the present application to provide a representative sample of the sequence diversity (SEQ ID NO: 87; SEQ.ID NO: 93; and SEQ.ID NO: 147 to SEQ.ID NO: 163).

[0292] To address the question of whether vaccines targeting TbpB from Neisseria meningitidis might induce an immune response against Neisseria gonorrhoeae, we performed an analysis of the sequence diversity of TbpB and the C-terminal half of TbpB from Neisseria gonorrhoeae. Sequences of representative TbpB and the C-terminal half of TbpB from Neisseria meningitidis studies (Figure 10; SEQ.D NO: 119; SEQ.ID NO: 125; SEQ.ID NO: 179 to SEQ.ID NO: 195; SEQ.ID NO: 117; SEQ.ID NO: 123; SEQ.ID NO: 132 to SEQ.ID NO: 147; SEQ.ID NO: 177; and SEQ.ID NO: 178) were included in the analysis. As Figure 26A illustrated, Neisseria gonorrhoeae TbpB is most closely related to type 2 TbpB and forms two subgroups within the Neisseria meningitidis type 2 cluster. This suggests that in the case of a strategy using site-directed mutants of recombinant TbpB as preferred vaccine antigens, broad cross-protection may be achieved mainly by antigens derived from meningococcal TbpB. Figure 26B Illustrating a contrast to the case of using full-length TbpB, the C-terminal half of Neisseria gonorrhoeae TbpB is a different subgroup from the C-terminal half of Neisseria meningitidis. Thus, in the case of a strategy using the C-terminal half to provide broad cross-protection, the C-terminal half from Neisseria gonorrhoeae strains will be required.

[0293] To compare the ability of truncated TbpB and the C-terminal half of TbpB to induce cross-reactive antibody responses, recombinant truncated TbpB and the C-terminal half of TbpB derived from Neisseria meningitidis strain B16B6 (marked by the black arrow in Figure 10) were selected for immunoassays. Recombinant truncated TbpB and the C-terminal half of TbpB from strain B16B6 were used to immunize rabbits and the cross-reactivity of the sera was tested using our novel ELISA assay ( Figure 5 ). Importantly, it is recognized that the multiple sequences of the C-terminal half included in this application start just after the end of the last β-strand of the barrel domain on the N-terminal half and thus include the linker region (L15, Figure 2 ) between the N-terminal half and the C-terminal half. Thus, it will be possible to prepare stable, functional C-terminal halves with N-terminal truncations that remove the L15 region (14 amino acids in the B16B6 C-terminal half) for immunization experiments.

[0294] A representative set of TbpB from strains distributed in the phylogenetic tree (black and grey arrows, Figure 10) was selected to evaluate the cross-reactivity of the sera. Two additional TbpB indicated by two double-headed arrows have been included in the analysis to provide more comprehensive coverage but were not available at the time of analysis.

[0295] The protein was expressed in our custom expression vector with an N-terminal biotinylation sequence and applied to streptavidin-coated ELISA plates. The ability of sera from rabbits immunized with the engineered C-terminal half and truncated TbpB derived from strain B16B6 to recognize a panel of TbpB variants was tested. The antiserum against truncated TbpB had a higher titer against homologous TbpB (B16B6) than the antiserum against the C-terminal half, and a slightly higher or equal titer against TbpB from one of the heterologous strains (H44 / 76). However, the antiserum against the C-terminal half had a higher antibody titer against TbpB from all other heterologous strains than the anti-TbpB antiserum. Thus, the C-terminal half was superior to full-length TbpB in its ability to induce cross-reactive antibodies.

[0296] In this immunization experiment, the recombinant antigen was produced as described above and used to immunize rabbits (New Zealand white rabbits, 3 months old, female) subcutaneously in the hind area with 50 μg of purified antigen in 20% Emulsigen D (VSA) adjuvant. The rabbits were immunized at 0, 3, and 6 weeks.

[0297] Sera obtained at week 8 were tested against representative proteins in our custom solid-phase ELISA assay. The recombinant fusion proteins used in the ELISA assay were produced as described above. The recombinant proteins tested in the custom ELISA assay were truncated forms (lacking the first 19 - 36 amino acids) of full-length TbpB from Neisseria meningitidis strains; (i) B16B6 (SEQ.ID NO: 117), (ii) H44 / 76 (SEQ.ID NO: 133), (iii) S3131 (SEQ.ID NO: 132), (iv) M990 (SEQ.ID NO: 134), (v) M978 (SEQ.ID NO: 135), (vi) M992 (SEQ.ID NO: 138), (vii) P3006 (SEQ.ID NO: 139), (viii) 120M (SEQ.ID NO: 137), (ix) MC58 (SEQ.ID NO: 136), and (x) M982 (SEQ.ID NO: 123).

[0298] Based on previous optimization experiments, the crude extract from a small-scale overnight protein expression experiment using an expression vector for a biotinylated fusion protein was sufficient to saturate the ELISA plates. Dilutions of the antiserum of interest were prepared in 2.5% skim milk in phosphate-buffered saline (PBST) and applied to the plates at room temperature for 1 hour. The primary antibody was detected at room temperature with HRP-conjugated goat anti-rabbit IgG at a dilution of 1:100,000 for 1 hour and the titer was expressed as the reciprocal of the last dilution at A450 > 0.3.

[0299] Example 5 - Generation of a dimer of the C-terminal half of Neisseria meningitidis TbpB

[0300] Figure 11 The results in illustrate that the C-terminal half domain of TbpB from the human pathogen Neisseria meningitidis is capable of inducing enhanced cross-reactive responses relative to full-length TbpB. In this example, we demonstrate the possibility of generating a stable and immunogenic single polypeptide encompassing the two C-terminal halves. The two C-terminal halves are from strains with antigenically divergent TbpB, namely strains B16B6 and M982, which represent two isoforms of TbpB (Figure 10). ​ Figure A of shows a single gene constructed to encode two representative C-terminal halves of TbpB (SEQ.ID NO: 150) for the immunization experiments described in Example 4. ​ Figure B of shows the generation of the C-terminal half dimer compared to preparations of the individual component TbpB C-terminal halves. There is a reasonable level of C-terminal half dimer generation, which appears to be relatively stable but will require additional purification before use in immunization experiments.

[0301] ​ Demonstrates that a dimer composed of the C-terminal halves of TbpB from Neisseria meningitidis strains M982 and B16B6 is capable of inducing an effective immune response against full-length TbpB from both species.

[0302] Example 6 - Reduction of the loop region of the C-terminal half from Neisseria meningitidis.

[0303] In this example, we demonstrate the possibility of substantially reducing the loop region of the C-terminal half from Neisseria meningitidis. The reduction is applicable as it provides derivatives with large loop regions that lack a convenient'scaffold' available for presenting epitopes from other antigens or other pathogens and further may present the desired immunological properties.

[0304] In this embodiment, the loop regions of the C-terminal half domain from Neisseria meningitidis strain M982 were modified to remove a total of 82 amino acids from the large flexible loops. Structural models of the native C-terminal half and the engineered C-terminal half with a significant reduction of the four large flexible loops are illustrated in ​ Figure A of

[0305] The strategy for loop removal was developed by examining the detailed structure (3VE2.pdb) and simultaneously evaluating sequence variations of the sequence alignment of the selected C-terminal half. However, providing the most effective bridging between the loops without potentially disrupting the structure was the main criterion for selecting the bridging amino acids. The sequence alignment of the native C-terminal half (SEQ.ID NO: 125), the engineered derivatives lacking each of the targeted loops, and the 'loopless' C-terminal half (SEQ.ID NO: 129) in which all four loops were removed is illustrated in ​ Figure B of

[0306] Using the overlap extension splicing method (SOEing) (57), genes encoding individual loop deletions and the 'loopless' C-terminal half in which all four loops were removed were prepared. The SOEing method was performed on the expression plasmid used to generate the recombinant C-terminal half from Neisseria meningitidis M982, such that the stability of the resulting engineered protein could be easily evaluated. The vector encodes an N-terminal polyhistidine tag, the gene encoding maltose binding protein, and a TEV (tobacco etch virus) cleavage site before the inserted gene encoding the C-terminal half of TbpB. The expression plasmid was transformed into the Escherichia coli expression strain ER256 carrying a chromosomal copy of the T7 RNA polymerase gene inserted into the lacZ gene and was thus under the control of the lac promoter. Small-scale expression analysis was performed using an autoinduction medium (55). After overnight growth, the cells were collected and lysed, and the supernatant fraction after centrifugation was applied to Ni-NTA resin, washed, and the bound protein was eluted in SDS-PAGE buffer.

[0307] As ​ illustrated, the yields of the recombinant C-terminal halves with a significant reduction of loop 18, loop 21, loop 23, or loop 27 were comparable to that of the native C-terminal half protein (WT), indicating that the removal of individual loops did not adversely affect protein stability. ​It is also shown that removal of 81 amino acids in all four loops does not affect the level of the resulting protein, indicating that removal of all four loops does not interfere with the overall folding of the C-terminal half. Importantly, it is mentioned that the 'loopless' C-terminal half is readily produced and purified in good quality, and crystal structures have been obtained from the purified 'loopless' C-terminal half. This indicates that the engineered antigen can be readily produced in a substantially stable form at production levels suitable for commercial applications. Thus, this example not only shows that the strategy for removing antigenic variable regions is practicable, but also that the resulting protein is suitable as an epitope presentation scaffold since the folding of the core structure is not affected by changes in the size and nature of the loops.

[0308] Finally, as ​ illustrated in, the 'loopless' C-terminal half (SEQ.ID NO: 129, last two) is immunogenic and capable of inducing a strong antibody response against the intact (native) TbpB antigen (SEQ.ID NO: 123). This figure shows that the loopless C-terminal half actually induces a higher antibody titer against TbpB from Neisseria meningitidis strain M982 than the initial TbpB C-terminal half (SEQ ID NO: 125) antigen in mice (compare lane 3 and lane 1 in this figure). Surprisingly, the following observation was made: the 'loopless' C-terminal half induces antibody levels against heterologous TbpB from strain B16B6 similar to those of the native TbpB C-terminal half from B16B6, although in fact its sequence is extremely divergent (Figure 10). ​ Comparison of lane 2 and lane 4 in shows that these two proteins induce similar antibody levels against intact TbpB from strain B16B6.

[0309] In these experiments, FvB female mice were immunized with 25 μg of purified protein antigen with 20% emulisgen D on days 0, 21, and 42 and the endpoint titers of sera obtained on day 56 were evaluated using our custom ELISA assay. The B16B6 TbpB C-terminal half, M982 TbpB C-terminal half, or modified M982 TbpB ('loopless') was used for each of the four mice immunized. Endpoint titers were evaluated against biotinylated intact M982 or B16B6 TbpB proteins. Titers were determined using goat anti-mouse IgG H+L peroxidase-conjugated antibody at 1:100,000. The endpoint titer was determined as the reciprocal of the last dilution at which a positive signal could be reliably detected. Each serum was assayed in triplicate and the results are shown as the mean + / - SEM for all mice receiving the treatment.

[0310] Example 7 - Engineering of partial insertion of TbpA into the engineered C-terminal half of TbpB.

[0311] In this embodiment, DNA encoding segments of extracellular surface loops of the complete outer membrane protein transferrin-binding protein A (TbpA) was spliced into a gene encoding the modified or 'acycled' C-terminal half of TbpB from Neisseria meningitidis strain M982, resulting in genes encoding various hybrid TbpA-TbpB proteins. The reason for splicing the selected regions of TbpA onto the TbpB C-terminal half backbone is that it provides a more efficient production method than the complete TbpA protein and provides the ability to specifically target surface regions of TbpA to induce antibodies.

[0312] The gene encoding the modified C-terminal half of the TbpB polypeptide (SEQ ID NO: 129) prepared in Example 6 was used as a starting point (see further: ​ Figure A (middle model). DNA encoding segments of different surface loops from TbpA ( ​ , Figure A) was spliced into the site ( ​ , Figure B) where the larger loops had been removed from the TbpB C-terminal half polypeptide. Portions of extracellular loops 3, 10, and 11 of the β-barrel of TbpA (58) (the regions that are space-filled and labeled in Figure A) were inserted into modified loop regions 18, 21, and 23 of the engineered TbpB C-terminal half (Figure B). Similarly, a segment from the N-terminal plug region between the C1 and C2 domains of human transferrin inserted into the TbpA-transferrin structure (the plug loop, Figure A) was inserted into modified loop 27 of the modified TbpB C-terminal half.

[0313] Assembly of the TbpA loops onto the TbpB C-terminal half was performed using the splicing by overlap extension method (SOEing) (57). The SOEing method was performed on the expression plasmid used to generate the recombinant C-terminal half from Neisseria meningitidis M982 such that the stability of the resulting engineered protein could be readily evaluated. The vector encodes an N-terminal polyhistidine tag, the gene encoding maltose-binding protein, and a TEV (tobacco etch virus) cleavage site before the inserted gene encoding the TbpB C-terminal half. The expression plasmid was transformed into the Escherichia coli expression strain ER256 carrying a chromosomal copy of the T7 RNA polymerase gene inserted into the lacZ gene and was thus under the control of the lac promoter. Small-scale expression analysis was performed using an autoinduction medium (55). After overnight growth, the cells were harvested and lysed, and the supernatant fraction after centrifugation was applied to Ni-NTA resin, washed, and the bound protein was eluted in SDS-PAGE buffer.

[0314] As ​ illustrated in Figure A, insertion of the foreign TbpA segments into the loops of the modified TbpB C-terminal half resulted in the production of stable recombinant proteins. ​The recombinant protein illustrated in FIG. A contains an N-terminal polyhistidine tag, a maltose binding protein fusion partner, and a TEV (tobacco etch virus) protease cleavage site. ​ FIG. B illustrates the release of wild-type and mutant C-terminal halves from the recombinant protein fusion partner by cleavage with TEV protease. The results demonstrate that the insertion of the foreign protein segment does not substantially affect the stability of the engineered C-terminal half, indicating that the foreign segment does not interfere with the normal folding of the core structural elements of the C-terminal half. These results suggest that the C-terminal half appears to be a stable and versatile protein scaffold for presenting foreign epitopes, which may ultimately be used to present epitopes from a variety of antigens and antigen variants, thus providing an additional strategy for generating engineered antigens capable of eliciting a broadly cross-protective immune response.

[0315] Finally, as ​ illustrated, a modified TbpB C-terminal half containing a TbpA region spliced into a region that reduces or removes a large loop is immunogenic. This figure illustrates that the hybrid TbpA-TbpB C-terminal half protein induces antibody titers equal to or higher than those of the parental modified (‘loopless’) C-terminal half. It should be noted that the proteins presenting the regions of loop 10 and loop 11 from TbpA have the highest titers ( ​ lanes 2 and 3 in

[0316] In this experiment, FvB female mice were immunized on days 0, 21, and 42 with 25 μg of purified protein antigen with 20% emulisgen D and the endpoint titers of sera obtained on day 56 were evaluated using our custom ELISA assay. Three mice were immunized with the 'loopless' C-terminal half (SEQ.ID NO: 97) in which all four loops were removed. Five mice were used to immunize with any one of four other hybrid antigens; (i) the 'loopless' C-terminal half (SEQ.ID NO: 154) with TbpA loop 10 inserted into loop 21 of the TbpB C-terminal half, (ii) the 'loopless' C-terminal half (SEQ.ID NO: 156) with TbpA loop 11 inserted into loop 23 of the TbpB C-terminal half, (iii) the 'loopless' C-terminal half (SEQ.ID NO: 158) with the TbpA loop 3 helix inserted into loop 27 of the TbpB C-terminal half, or (iv) the 'loopless' C-terminal half (SEQ.ID NO: 160) with the TbpA plug loop inserted into loop 18 of the TbpB C-terminal half. Sera were tested against the biotinylated recombinant form (SEQ.ID NO: 131) of the modified M982 C-terminal half presenting all four inserted TbpA loops. Titers were determined with goat anti-mouse IgG H+L peroxidase-conjugated antibody at 1:100,000. The endpoint titer was determined as the reciprocal of the last dilution at which a positive signal could be reliably detected. Each serum was run in triplicate and the results are shown as the mean + / - SEM of all mice using the treatment.

[0317] Example 8 - Portions of LbpA were inserted into modified C-terminal halves of TbpB.

[0318] In this example, a segment of the extracellular surface loop of the full-length outer membrane protein lactoferrin-binding protein A (LbpA) from strain MC58 (SEQ.ID NO: 162) was spliced into the modified TbpB C-terminal half (SEQ.ID NO: 129) from Neisseria meningitidis strain M982 described in Example 6. The reason for splicing the selected region of LbpA onto the TbpB C-terminal half backbone is that it provides a more efficient production method than the full-length LbpA protein and the ability to specifically target surface regions of LbpA to induce antibodies. In combination with Example 7, we were able to demonstrate how the production of hybrid proteins could potentially provide an opportunity to induce an immune response against three different proteins present on the surface of Neisseria meningitidis, thus providing a greater barrier to potential 'vaccine escape' in which antigenic variants of key target proteins can evade the effects of the immune response generated against vaccine antigens.

[0319] Since there is no available structure for LbpA, three web-based protein prediction servers, SWISS-Model, I-TASSER, and PHYRE2, were used to perform structure modeling of LbpA in an attempt to obtain the most appropriate model. An initial BLAST search was performed to identify the most appropriate LbpA for modeling from the known TbpA structure (58), and LbpA from strain MC58 (SEQ.ID NO: 130) was disclosed as the most appropriate. The alignment of MC58 LbpA with K454 TbpA was generated by ClustalW and used as the input for the alignment pattern in SWISS-Model. In PHYRE2, the PDB ID for the K454 TbpA structure and the FASTA sequence of MC58 LbpA were submitted as the template and target, respectively. Only the FASTA sequence of MC58 LbpA was submitted to I-TASSER. The root mean square deviation (RMSD) was used to evaluate the similarity of the models after overlapping different models generated by the template structure in Pymol ( ​ ). The LbpA model generated by PHYRE2 was selected as the most appropriate model and used to select the loop regions for generating hybrid or chimeric proteins ( ​ , Figure A).

[0320] The 'acycle' C-terminal half (SEQ.ID NO: 129) of the TbpB polypeptide prepared in Example 6 was used as the starting point. The DNA encoding the regions from LbpA extracellular loops 3 and 2 was inserted between the DNA encoding the β-strands of loops 18 and 21 flanking the engineered TbpB C-terminal half ( ​ , Figure B). Pymol was used to analyze the distance between the loop regions in the TbpB C-terminal half and the corresponding loops in LbpA for insertion to ensure that the LbpA replacement loops were structured within the distance parameters predicted by the loops. The LbpA loops were assembled onto the TbpB C-terminal half as described in Examples 6 and 7 using SOE PCR, and sequence analysis confirmed the insertion of MC58 LbpA helix 3 and loop 2 into the acycle M982 TbpB C-terminal half. The design of the hybrid protein involved inserting 15 amino acids (protein sequence: 383-YGTDEAEKFRDKSGV) from the LbpA helix 3 region into the loop 18 region (SEQ.ID NO: 166) of the M982 C-terminal half, and inserting 11 amino acids (protein sequence: LNRWVKERIEQL) from the LbpA loop 2 region into the loop 21 region (SEQ.ID NO: 164) of the M982 C-terminal half ( ​ >, Figure B).

[0321] The methods for transforming the recombinant plasmid and performing the preliminary expression experiments were as described in Example 7. Preliminary screening demonstrated a high yield of the recombinant protein (bottom left, Figure B, ​) can be equivalent to or better than the results obtained from the native C-terminal half or the acyclic C-terminal half used as a scaffold (data not shown). Clearly, the results further demonstrate that the insertion of the foreign protein segment does not substantially affect the stability of the engineered C-terminal half, indicating that the foreign segment does not interfere with the normal folding of the core structural elements of the C-terminal half.

[0322] Example 9 - Engineering of the C-terminal half of TbpB from Haemophilus influenzae for use in conjugate capsular vaccine applications

[0323] Most conjugate capsular vaccines developed to date have used a toxin-based vaccine component as a carrier for conjugating polysaccharide capsular materials. There are several drawbacks to the strategy of conjugating capsular polysaccharides to tetanus or diphtheria toxins or toxoids. One is the potential to adversely affect the induction of an effective immune response due to continuous exposure to the carrier protein that is also present in vaccines used for routine immunization; the development of immune tolerance. The second is that the carrier is not related to the native exposure to the pathogen and thus will not adequately elicit the most relevant T cell help when encountering the pathogen.

[0324] Conjugate capsular vaccines have been very successful in preventing infections by bacterial pathogens expressing specific capsular polysaccharides (commonly referred to as serotypes or serogroups), and in fact preventing colonization. However, there is essentially no cross-protection against bacteria expressing other polysaccharide capsular types, which can ultimately lead to diseases caused by strains expressing polysaccharides not covered by the vaccine. The need to extend the range of polysaccharide capsular types covered by the conjugate capsular vaccines has led to the ongoing development of extended-range vaccines and it is thought that the ultimate solution lies in protein-based vaccines that can provide significant cross-protection. However, if a protein-based vaccine capable of inducing broad cross-protection is developed, it will likely not be accepted as a replacement for existing conjugate capsular vaccines. Adding another vaccine to an already approved routine immunization schedule may be seen as a potential barrier to the introduction of protein-based vaccines.

[0325] In this example, we have engineered a conjugate loop into the C-terminal half of TbpB from Haemophilus influenzae containing 42 lysine residues, which substantially exceeds the total number of lysines (lysine residues) in the remainder of the TbpB C-terminal half, which is predicted to minimize the modification of lysines in key epitopes, simply because those lysines that react with activated carbohydrate moieties will be modified and the carbohydrate-to-protein ratio can be controlled during the conjugation process.

[0326] The sequence of the gene encoding the engineered C-terminal half of TbpB from Haemophilus influenzae strain H36 is described in ​In Figure A, a DNA region (SEQ.ID NO: 167) with a conjugated loop indicated by an increased font size (14 vs. 12) is shown. The insertion site and sequence of the conjugated loop were designed using the large negatively charged loop in LbpB from Neisseria meningitidis MC58 as a model (59). Essentially, the sequence of the LbpB loop was used as a template and lysine was used to replace aspartic or glutamic acid residues in the loop. The conjugated loop was engineered onto the handle domain of the C-terminal half of TbpB between β-strands 22 and 23, at the position of loop 23 ( ​ ). The position of the loop is illustrated in ​ Figure B using a structural model of the C-terminal half of Haemophilus influenzae generated by replacing loop 23 with a much smaller loop (containing 11 amino acids). The inserted residues are illustrated as space-filling spheres. The engineered loop actually contains 91 amino acids, which is more than 1 / 4 of the size of the overall C-terminal half (352 amino acid residues), and the loop is shown as highlighted residues throughout the C-terminal half, where the engineered loop is shown in SEQ.ID NO: 205. This shows that the loop domain can accommodate a large number of additional amino acids.

[0327] The conjugation region includes, but is not limited to, insertion into the loop regions of the N-terminal or C-terminal halves, but may be provided, for example, by including a cluster of lysine residues at the N-terminus of the full-length TbpB or TbpB half.

[0328] Example 10 - Generation of Amino Acid Substitutions in the Surface-Binding Loops of TbpB and Evaluation of Their Tf-Binding Properties

[0329] A series of site-directed mutants were constructed in the surface loops of the TbpB protein to investigate their effects on function and immunological properties. To target surface-exposed amino acids for modification, site-directed mutagenesis was performed in TbpB for which we had a structure derived from x-ray crystallography (12, 13). Mutations were introduced into the gene encoding the truncated TbpB protein from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis, porcine pathogens, using the splicing overlap extension polymerase chain reaction (SOE PCR) method. It includes Actinobacillus pleuropneumoniae TbpB from strain H49 20-528 (amino acids 20 - 528) (ApH49 TbpB, SEQ.ID NO: 2), and the F171A mutant of ApH49 TbpB 20-528 (SEQ ID.NO: 4). It also includes Actinobacillus pleuropneumoniae TbpB from strain H87 26-528(Amino acids 26 - 528) (ApH87 TbpB, SEQ.ID NO: 12) and Y95A mutant (SEQ.ID NO: 14), Y121A mutant (SEQ.ID NO: 16), Y174A mutant (SEQ.ID NO: 18) and R179E mutant (SEQ.ID NO: 20). Porcine Actinobacillus pleuropneumoniae TbpB derived from strain H57 was also generated. 27-577 (Amino acids 27 - 577) (AsH57 TbpB, SEQ.ID NO: 28), F63A mutant (SEQ.ID NO: 30) and F152A mutant (SEQ ID NO: 32). Finally, Haemophilus parasuis TbpB derived from Nagasaki strain Hp5 was also prepared. 27-577 (Amino acids 27 - 577) (Hp5TbpB, SEQ.ID NO: 115), Y93A mutant (SEQ.ID NO: 170), Y117AA mutant (SEQ.ID NO: 172), Y167A mutant (SEQ.ID NO: 174) and W176A mutant (SEQ.ID NO: 176).

[0330] The recombinant proteins were initially produced with an N - terminal fusion partner containing a poly - histidine tag, a maltose - binding protein and a TEV protease cleavage site. This enabled us to isolate recombinant proteins suitable for solid - phase binding assays either using nitrocellulose membranes and enzymatically labeled porcine transferrin or by capturing the recombinant fusion proteins from crude extracts with porcine - agarose affinity resin (13). Using these assays, strong binding of native TbpB was readily observed, while binding of multiple site - directed mutants was reduced.

[0331] Although it was possible to derive semi - quantitative binding constants for binding to pigs from these experiments, we chose to use a variety of different biophysical and biochemical methods to obtain more precise and quantitative measurements of binding affinity. As ​ shown, using isothermal titration calorimetry, surface plasmon resonance or biolayer interferometry (23 - 25), the affinity constants (Kd) for native TbpB were generally in the range of 20 to 60 nM, with the exception of TbpB from Actinobacillus pleuropneumoniae, whose binding kinetics were slightly unique and had an estimated Kd of 120 nM (12).

[0332] Several mutations caused a ≥100 - fold increase in the affinity constant (Kd), such as the F171A mutation in TbpB from Actinobacillus pleuropneumoniae strain H49, the Y174A mutation in TbpB from Actinobacillus pleuropneumoniae strain H87 or the Y167A and W176A double - mutation in TbpB from Haemophilus parasuis strain HP5. Interestingly, it was noted that all of these mutants mapped to loop 8.

[0333] Example 11 - Evaluation of the immunological properties of TbpB derivatives with amino acid substitutions in the surface - binding loop

[0334] To test the immunological properties of mutant proteins derived from TbpB that are defective in binding Tf, it is important to test the proteins in the natural host and preferably directly test their ability to protect the host from infection by the targeted pathogen. Thus, experiments were initiated in a well - established infection model for Haemophilus parasuis, where colostrum - deprived piglets were immunized starting at 28 days after birth and challenged with Haemophilus parasuis at day 63 (37, 60). In this infection model, a commercial vaccine (Porcillis Glasser) derived from the challenge strain provided complete protection from death and a recombinant form of the transferrin receptor had previously provided 20 - 30% survival 15 days after challenge.

[0335] Groups of five or six pigs were immunized with recombinant full - length TbpB, site - directed Y167A TbpB protein, Porcillis Glasser vaccine, or adjuvant alone from the Hp5 challenge strain. The pigs were challenged with a standard 10 8 challenge dose of the Haemophilus parasuis Hp5 (Nagasaki) strain and monitored for a 15 - day period. As ​ illustrated, only 1 out of 5 pigs immunized with the control Porcilis Glasser vaccine withstood the challenge, indicating that a more virulent variant of the Hp5 strain was used in this experiment. Follow - up experiments, where isolates from infected pigs had lower survival rates, supported this conclusion. Despite the enhanced virulence of the challenge strain, all six pigs immunized with Y167A TbpB survived for 15 days, and 5 out of the 6 pigs had minimal or no symptoms and minimal or no pathology was observed at necropsy. This degree of protection was in contrast to six pigs immunized with the wild - type protein, of which only 3 pigs survived after the challenge. The 3 surviving pigs had significant clinical symptoms after challenge and showed significant pathology at necropsy. In summary, this experiment demonstrates that the Y167A TbpB mutant protein induces a superior protective immune response compared to the wild - type protein, and since the two proteins are virtually identical except for their transferrin - binding properties ( ​ ), the sub - optimal immune response of the native protein can be attributed to the binding of host transferrin.

[0336] To provide further evidence of the effect of the mutation on the immune response, B - cell and T - cell responses were evaluated. Adaptive immune responses were analyzed in blood samples taken immediately before challenge (after two immunizations) and 96 hours after challenge. The mature B cells (αIgM + CD21+ ) and T helper cells (CD4 + CD8α - ) subsets. ​ The results in demonstrated that prior to challenge, the Y167A mutant TbpB antigen had induced stronger B cell responses (Figure A) and T helper cell responses (Figure B) than the native TbpB antigen or the commercial Porcilis Glasser vaccine. At 96 hours post-challenge, the response to mutant TbpB (51.48% ± 1.18%) was significantly higher than the response to native TbpB (45.65% ± 1.20%) or the PG vaccine (44.83% ± 1.59%) ( ​ , Figure C). A similar trend was observed in the T helper cell responses; however, the percentage differences between the three groups were less obvious ( ​ , Figure D). However, only 3 / 6 and 2 / 5 surviving pigs remained in the groups immunized with native TbpB or the Porcilis Glasser vaccine after 96 hours, and since the low responders were the pigs more prone to earlier death, the differences between the groups observed at the 96 h time point were actually underestimates.

[0337] Example 12 - Immune responses against TbpB derivatives can prevent colonization

[0338] Although infection models do provide opportunities to evaluate the potential efficacy of vaccines, they rarely mimic the natural infection process, in which the spread of the pathogen usually results in colonization of the host upper respiratory tract prior to the establishment of infection. Conjugate capsular vaccines designed to prevent meningitis, pneumonia, and invasive infections have been shown to eliminate the targeted bacteria from the upper respiratory tract (17), thus providing the additional advantage of herd immunity that protects non-immune individuals. The ability to prevent colonization has since become an important feature for making decisions regarding vaccine implementation (18). Therefore, it may be prudent to design vaccines that prevent colonization such that, along with preventing infection, they can eliminate the reservoir of the disease-causing pathogen.

[0339] Using previous studies (61) that characterized the interaction between Neisseria meningitidis and the human CEACAM receptor, we developed a transgenic humanized mouse model (62) that is capable of supporting the colonization of Neisseria meningitidis. This model is based on the specific interaction between the Neisseria meningitidis Opa protein and the human CEACAM1 receptor and may potentially be extended to other pathogens that naturally or artificially utilize this interaction. Immunization of the transgenic mice with a meningococcal group C conjugate capsular vaccine resulted in sterilizing mucosal immunity in the colonization model or, in other words, prevented the colonization of group C Neisseria meningitidis but not strains with other capsular types.

[0340] This model was used to test the ability of TbpB and its derivatives to prevent colonization by Neisseria meningitidis. Since human transferrin is not present in these mice during the immunization phase, it was not necessary to use engineered non-binding TbpB as described in Example 11. As ​ illustrated in A, 8 out of 9 mice immunized with recombinant truncated TbpB did not have detectable levels of Neisseria meningitidis 3 days after intranasal challenge with 1 × 10 7 CFU of Neisseria meningitidis strain M982. In control mice treated with adjuvant alone, 6 out of 8 mice had detectable levels of Neisseria meningitidis present. It is important to mention that this is the first protein antigen shown to be able to prevent colonization and, due to our limited understanding of the mechanisms involved, it cannot be assumed that this property is shared by surface protein antigens.

[0341] In a tracking experiment, we compared TbpB with another surface lipoprotein, namely factor H-binding protein, which is a key component in two vaccines, and we compared TbpB with individual TbpB subdomains. As ​ illustrated in B, the C-terminal half was able to prevent colonization as well as or better than the full-length TbpB or the N-terminal half of TbpB, and was also able to induce sterilizing immunity as effectively or more effectively than factor H-binding protein in this experiment. The ability of the TbpB C-terminal half to induce sterilizing mucosal immunity under systemic immunization is an especially exciting finding since its lack of Tf binding means that it will be equally effective in the natural host, and its enhanced ability to induce cross-reactive immune responses (Figure 3, ​ ) will facilitate the development of a broadly cross-protective vaccine.

[0342] Colonization studies were performed as previously described (62). Groups of 8 or more C57 / B16 (bred in-house) expressing the human CEACAM-1 transgene received 100 μl of the designated immunization subcutaneously on days 0 and 21. Each group received the designated protein (25 μg) or a protein-free control diluted to a volume of 100 μl per injection in sterile phosphate-buffered saline (PBS) (Gibco) and adjuvanted with 20% Emulsigen D (MVP Laboratories).

[0343] On day 35, mice were anesthetized with Isofluran (Baxter) and inoculated via intranasal instillation, and two animals were passaged with Neisseria meningitidis strain M982. To prepare the inoculum, the strain to be infected was grown overnight on GC agar (Beckton Dickinson); the overnight-grown bacterial lawn was collected into 1 ml of PBS (PBS / Mg) containing 1 mM MgCl2 and the OD600 was measured to adjust the bacterial number. The culture was adjusted such that each final 10 μl inoculum contained approximately 1×10 7 colony-forming units. The density of the colonization dose was confirmed by serial dilution plating on GC agar.

[0344] Three days after infection (day 38), the mice were sacrificed by carbon dioxide asphyxiation. The colonization burden was evaluated by tracheal lavage with 250 μl of PBS / Mg followed by direct swabbing of the nostrils with a polyester applicator (Puritan Medical Products) resuspended in 500 μl of PBS / Mg. Samples were counted after overnight growth on GC agar supplemented with VCNT inhibitor (Becton Dickinson) to prevent the growth of nasal colonies. Animal experiments were conducted according to the Animal Ethics Review Committee of the University of Toronto.

[0345] Example 13 - Vaccine formulation comprising a mixture of TbpB or a portion thereof

[0346] Since pathogens with TbpB specifically reside in their specific hosts (humans, pigs, cattle, and / or related ruminants) and since TbpB can prevent colonization ( ​ ), a broadly cross-protective vaccine based on engineered antigens targeting TbpB has the potential to eliminate said pathogens.

[0347] To broaden the efficacy of vaccine formulations against a spectrum of Gram-negative pathogens, TbpB or portions thereof, such as the C-terminal half-domain, obtained from different bacterial species or strains can be combined. In this example, we provide preferred combinations of TbpB polypeptides or their combinations for preparing vaccine formulations.

[0348] An important consideration when identifying effective combinations of TbpB polypeptides is the extent to which different strains, species, and genera can readily exchange tbpB genes and thus act as potential reservoirs of TbpB variants not covered by the vaccine. An important factor influencing the horizontal exchange of the tbpB gene is the nature of the uptake signal sequence (USS) that is inherently present in these naturally transformable species (63, 64). These bacteria preferentially take up DNA containing the specific USS and incorporate it into their genome, thus providing a highly effective mechanism for incorporating antigenic variants of their surface antigens.

[0349] In the case of Neisseria meningitidis, we have a broad collection of strains that appropriately represent the overall sequence diversity ( ​ ). An especially large collection of sequences from around the world is available on public databases for this pathogen, representing an extremely comprehensive understanding of sequence diversity. Since other human pathogens (Haemophilus influenzae, Moraxella catarrhalis) that have TbpB and typically reside in the human upper respiratory tract do not contain the USS specific to Neisseria in their genomic DNA, they do not constitute a ready reservoir for antibody variants. Accordingly, embodiments of the invention include vaccine formulations comprising combinations of engineered TbpB antigens, the combinations comprising at least two TbpB polypeptides or portions thereof (e.g., C-terminal half-domains) obtained from two different phylogenetic clusters of Neisseria meningitidis strains selected from ​ . The vaccine formulations have the potential to induce cross-reactive ( ​ ) and cross-protective antibody responses and may potentially be used to eliminate Neisseria meningitidis from the human population.

[0350] The related pathogen Neisseria gonorrhoeae, which typically resides in the human urogenital tract, shares the same USS and may thus potentially serve as a reservoir for antigenic variation due to the occasional presence of both species on the same mucosal surface. However, analysis of the sequence diversity of gonococcal TbpB relative to that in Neisseria meningitidis ( ​ ) indicates that it is primarily a subset of the sequence diversity present in Neisseria meningitidis, such that it is anticipated that with a slight extension of our method, a collection of engineered antigens may be used in vaccines that have the potential to eliminate colonization by either pathogen. Regarding the engineered C-terminal half ([[]] ​), which will involve specifically targeting the C-terminal half of the Neisseria gonorrhoeae TbpB variant. The presence of TbpB in some commensal Neisseria isolates represents another potential reservoir of antigenic variants, and thus our approach extends to include representative variants from commensal Neisseria which may be necessary to effectively eliminate Neisseria expressing TbpB capable of causing disease. Accordingly, embodiments of the invention include vaccine formulations comprising combinations of engineered TbpB antigens, said combinations comprising Neisseria meningitidis TbpB polypeptide or a portion thereof (e.g., C-terminal half domain) and Neisseria gonorrhoeae TbpB polypeptide or a portion thereof (e.g., C-terminal half domain).

[0351] The porcine pathogens Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis share the same USS and thus, TbpB sequence diversity is distributed among the three species ( ​ ), such that major phylogenetic clusters have representatives from at least two species. Accordingly, it is important to consider the overall TbpB sequence variation in all three species when developing TbpB-based vaccines against these pathogens. This is the basis for our rather unusual approach to developing engineered antigens capable of inducing an immune response against antigens from more than one species ( ​ , ​ ), and since TbpB can prevent colonization, using methods that may be used to eliminate all three pathogens from their porcine host. Accordingly, embodiments of the invention include a vaccine formulation comprising a combination of engineered TbpB antigens, said combination comprising at least two TbpB polypeptides or portions thereof (e.g., C-terminal half domain) obtained from Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis.

[0352] Regarding Haemophilus influenzae, the distinct disease spectra caused by strains with a type b polysaccharide capsule and non-typeable strains lacking a polysaccharide capsule have led to interest in vaccines that specifically target each group. The recent increase in invasive disease caused by strains expressing group A polysaccharide capsule has prompted consideration of developing a vaccine targeting group A strains (65). Assessment of TbpB diversity in Haemophilus influenzae strains indicates the presence of three major phylogenetic clusters ( ​ ), with non-typeable strains distributed among all three groups. Since all Haemophilus influenzae strains share the same USS, it is possible that the distribution of TbpB diversity will not be affected by capsule type, and the development of cross-protective vaccines based on engineered TbpB antigens will effectively target type b strains, non-typeable strains, and strains expressing other capsule types. Accordingly, our approach will facilitate the development of a broadly protective TbpB-based vaccine against Haemophilus influenzae as a standalone vaccine, or as a carrier for conjugate capsular vaccines ( ​)。Thus, embodiments of the present invention include vaccine formulations comprising a combination of engineered TbpB antigens, said combination comprising at least two TbpB polypeptides obtained from two different phylogenetically clustered Haemophilus influenzae strains selected from ​ as shown in

[0353] Unlike Neisseria meningitidis and Haemophilus influenzae, no obvious USS is present in the genome of Moraxella catarrhalis strains, yet it is naturally transformable and has a strong preference for Moraxella catarrhalis DNA. Thus, developing a broadly cross-protective vaccine against Moraxella catarrhalis from engineered antigens targeting TbpB only requires considering the diversity of TbpB from Moraxella catarrhalis ( ​ ). Antigens derived from strains constituting the three main groups should be sufficient to induce a broadly cross-protective vaccine capable of preventing colonization by Moraxella catarrhalis. Thus, embodiments of the present invention include vaccine formulations comprising a combination of engineered TbpB antigens, said combination comprising at least two TbpB polypeptides obtained from two different phylogenetically clustered Moraxella catarrhalis strains selected from ​ as shown in

[0354] The bovine pathogen Mannheimia haemolytica (previously called Pasteurella haemolytica) is a major cause of bovine respiratory disease (shipping fever) in cattle and respiratory infections in sheep. Pasteurella trehalosi, a sheep pathogen that has been reclassified into two species, Mannheimia glucosida and Bibersteinia trehalosi, shares USS with Mannheimia haemolytica. This pathogen can mainly lead to the following finding: these species share a common gene pool (66). In contrast to the bovine pathogen, Histophilus somni (previously called Haemophilus somnus) has a different USS and is thus not a reservoir for antigenic variants of Mannheimia haemolytica. There are three main phylogenetic lineages of TbpB from Mannheimia haemolytica, Mannheimia glucosida, and Bibersteinia trehalosi, which clearly cover pathogens of sheep and cattle (66)( ​ ), where the clustering of variants is mainly limited to cattle or sheep. Thus, it will be possible to consider developing TbpB-derived engineered antigens targeting diseases in cattle, sheep, or both ruminant species. Thus, embodiments of the present invention include vaccine formulations comprising a combination of an engineered TbpB antigen or a portion thereof (e.g., the C-terminal half domain), said combination comprising at least two TbpB polypeptides obtained from two different phylogenetically clustered Mannheimia haemolytica, Mannheimia glucosida, and Bibersteinia trehalosi strains selected from ​ as shown in

[0355] All publications, patents, and patent applications are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0356]

[0357]

[0358]

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[0368] ​

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Claims

1. An immunogenic composition comprising a transferrin-binding protein B (TbpB) polypeptide from a Gram-negative pathogenic bacterial species and an adjuvant, wherein the TbpB polypeptide is modified and the sequence of the modified TbpB polypeptide is SEQ.ID NO:129, SEQ.ID NO:154, SEQ.ID NO:156, SEQ.ID NO:158, SEQ.ID NO:160 or SEQ.ID NO:

174.

2. A vaccine composition comprising the immunogenic composition according to claim 1.

3. A method for preparing an immunogenic composition, the method comprising: (a) Providing a chimeric nucleic acid sequence comprising the following as operably linked components: (i) A nucleic acid sequence encoding a TbpB polypeptide from a Gram-negative pathogenic bacterial species, wherein the TbpB polypeptide is modified and the sequence of the modified TbpB polypeptide is SEQ.ID NO:129, SEQ.ID NO:154, SEQ.ID NO:156, SEQ.ID NO:158, SEQ.ID NO:160 or SEQ.ID NO:174; and (ii) A nucleic acid sequence capable of controlling expression in a recombinant host cell; (b) Introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the modified TbpB polypeptide; (c) Recovering the modified TbpB polypeptide from the host cell; and (d) Preparing an immunogenic composition.

4. Use of the following (a) or (b) in the manufacture of a medicament for preventing infection caused by Neisseria meningitidis or Haemophilus parasuis in a vertebrate subject: (a) An immunogen comprising a TbpB polypeptide from a Gram-negative pathogenic bacterial species, wherein the TbpB polypeptide is modified and the sequence of the modified TbpB polypeptide is SEQ.ID NO:129, SEQ.ID NO:154, SEQ.ID NO:156, SEQ.ID NO:158 or SEQ.ID NO:160, and the corresponding medicament is directed against infection caused by Neisseria meningitidis; or the sequence of the modified TbpB polypeptide is SEQ.ID NO:174, and the corresponding medicament is directed against infection caused by Haemophilus parasuis; or (b) An expression vector comprising a nucleic acid sequence encoding an immunogen comprising a TbpB polypeptide from a Gram-negative pathogenic bacterial species, wherein the TbpB polypeptide is modified and the sequence of the modified TbpB polypeptide is SEQ.ID NO:129, SEQ.ID NO:154, SEQ.ID NO:156, SEQ.ID NO:158 or SEQ.ID NO:160, and the corresponding medicament is directed against infection caused by Neisseria meningitidis; or the sequence of the modified TbpB polypeptide is SEQ.ID NO:174, and the corresponding medicament is directed against infection caused by Haemophilus parasuis; and wherein the immunogen is present in an amount sufficient to elicit an immune response in the vertebrate subject.

5. Use of an immunogen comprising a polypeptide, which is a TbpB protein from a Gram-negative pathogenic bacterial species, in the manufacture of a medicament for preventing infection caused by Neisseria meningitidis or Haemophilus parasuis in a vertebrate subject, wherein the TbpB protein is modified and the sequence of the modified TbpB protein is SEQ.ID NO: 129, SEQ.ID NO: 154, SEQ.ID NO: 156, SEQ.ID NO: 158 or SEQ.ID NO: 160, and the corresponding medicament is directed against infection caused by Neisseria meningitidis; or the sequence of the modified TbpB protein is SEQ.ID NO: 174, and the corresponding medicament is directed against infection caused by Haemophilus parasuis.

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