Immunogenic proteins from bordetella pertussis

Abstract

The latest evidence accumulated in the past ten years shows that the production of CD4 + T cells is crucial for sustained immunization against Bordetella pertussis. The Bordetella pertussis contains hundreds of antigens which are processed and presented on MHC class II, and are recognized by CD4 'T cells. The present disclosure relates to a vaccine comprising a Bordetella pertussis antigen peptide to prevent Bordetella pertussis bacterial infection.

Description

[0001] Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Grant No. AI153829 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] Cross - Reference to Related Applications

[0004] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 408,244, filed on September 20, 2022, entitled "IMMUNOGENIC PROTEINS FROM BORDETELLA PERTUSSIS", which is incorporated herein by reference in its entirety.

[0005] Reference to Sequence Listing

[0006] A sequence listing was submitted in XML file format on September 15, 2023, named "103361 - 361WO1.xml", created on September 15, 2023, with a file size of 6,384 bytes, and is hereby incorporated by reference in accordance with 37 C.F.R.§1.52(e)(5). Technical Field

[0007] The present disclosure relates to vaccines comprising Bordetella pertussis antigenic peptides for preventing Bordetella pertussis bacterial infections. Background Art

[0008] The latest evidence accumulated over the past decade indicates that the generation of CD4+ T cells is crucial for sustained immunity against Bordetella pertussis (B. pertussis, the bacterium that causes whooping cough disease). Bordetella pertussis contains hundreds of antigens that are processed and presented on major histocompatibility complex class II (HLA - DR) and recognized by CD4+ T cells. However, there is a need to develop the next generation of whooping cough vaccines that elicit strong long - term systemic and mucosal immunity against Bordetella pertussis. Current acellular pertussis vaccines (aPV) contain 3 - 5 proteins adjuvanted with alum. These proteins were selected because of their role in the pathogenesis of Bordetella pertussis, with the aim of generating neutralizing antibodies that clear the bacterium from the host. However, the efficacy of these vaccines is not long - lasting.

[0009] Given the above limitations, there is a need to identify new Bordetella pertussis proteins to incorporate into vaccines, leading to the development of new vaccine therapies that generate better and more long - lasting immune responses.

[0010] The compositions and methods disclosed herein address these needs. Summary of the Invention

[0011] The present disclosure provides a vaccine composition for preventing Bordetella pertussis infection and eliciting an immune response against Bordetella pertussis infection and methods of using the same.

[0012] In one aspect, the present disclosure provides a pertussis vaccine comprising a BP0205 antigen or variant thereof and at least one peptide from a Bordetella pertussis bacterium, wherein the BP0205 peptide and the at least one peptide are to be presented to MHC class II cells and recognized by CD4+ T cells.

[0013] In some embodiments, the BP0205 antigen comprises at least 70% sequence identity with SEQ ID NO:1. In some embodiments, the BP0205 antigen comprises at least 80% sequence identity with SEQ ID NO:1. In some embodiments, the BP0205 antigen comprises at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the BP0205 antigen comprises SEQ ID NO:1.

[0014] In some embodiments, the vaccine comprises an epitope that comprises SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the vaccine comprises an epitope that comprises SEQ ID NO:2 and SEQ ID NO:3.

[0015] In some embodiments, the at least one peptide is derived from a protein involved in at least one biological pathway within the Bordetella pertussis bacterium. In some embodiments, the at least one peptide is derived from a protein of Table 2 that includes the Bordetella pertussis protein of interest.

[0016] In some embodiments, the at least one pathway is selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation, or stress response.

[0017] In some embodiments, proteins selected from the cell adhesion and motility pathway include an adherence and elimination protein, a fimbrial protein, a flagellar biosynthesis protein (FlhF), or a combination thereof.

[0018] In some embodiments, the proteins selected from the cell cycle pathway include proteins containing a β-lactamase domain, cell division coordination protein (CpoB), cell division protein (ZapD), peptidyl-prolyl cis-trans isomerase, peptidyl-prolyl cis-trans isomerase (Cbf2), phospho-N-acetylmuramoyl-pentapeptide-transferase, tol-Pal system protein (TolB), tol-Pal system protein (TolQ), or combinations thereof.

[0019] In some embodiments, the proteins selected from the cell signaling pathway include dermonecrotic toxin, cyclic di-GMP phosphodiesterase response regulator (RpfG), enterobactin outer membrane receptor, or combinations thereof.

[0020] In some embodiments, the proteins selected from the cell structure pathway include metal-binding protein, BrkB, D-alanine-D-alanine ligase, inner membrane protein (ybhN), inner membrane protein (YiaH), lipoprotein, lipoteichoic acid synthase 2, outer membrane protein assembly factor (BamA), P.93, penicillin-binding protein 1A, pertussis toxin release protein F, membrane protein, DMT superfamily permease, spermidine synthase with an N-terminal membrane domain, lipoic acid-protein ligase A, HflK, assembly protein, inner membrane protein, HI_0719, soluble lytic murein transglycosylase, virulence-associated outer membrane protein Vir-90, or combinations thereof.

[0021] In some embodiments, the proteins selected from the cellular transport pathways include Xaa-Pro aminopeptidase, 2-aminoethylphosphonate ABC transporter substrate-binding protein, aerobactin synthase (IucC), antimicrobial resistance protein, arabinose efflux permease, BFD-like [2Fe-2S] binding domain, bicarbonate transport ATP-binding protein (CmpD), bicyclomycin / multidrug efflux system, biotin transporter, BrkA autotransporter, ComEC family competence protein, D-methionine-binding lipoprotein (metQ), drug efflux system protein (MdtG), efflux pump membrane transporter, extracytoplasmic solute receptor protein, extracytoplasmic solute receptor protein (yiaO), ferrichrome receptor (FcuA), filamentous hemagglutinin, hemin-inhibitable hemoglobin-binding protein, hemin import ATP-binding protein (HmuV), inner membrane metabolite transporter (yhjE), inner membrane protein (yhjX), inner membrane transporter (ydhP), iron-sulfur cluster carrier protein, leucine ABC transporter subunit substrate-binding protein (LivK), leucine-, isoleucine-, valine-, threonine-, and alanine-binding protein, LIV-I protein F, LIV-I protein H, major facilitator superfamily protein, manganese transport system membrane protein (mntB), methyl viologen resistance protein (SmvA), multidrug export ATP-binding / permease protein (SAV1866), multidrug resistance protein B, multiple antibiotic resistance and pH homeostasis protein A, multiple antibiotic resistance and pH homeostasis protein D, multiple antibiotic resistance and pH homeostasis protein E, Neu5Ac permease, nickel / cobalt efflux system, BP0840, preprotein translocase subunit (SecD), ABC transporter ATP-binding protein (HI_0664), amino acid metabolite efflux pump, chorismate pyruvate-lyase, membrane transporter, protein translocase subunit (SecD), purine efflux pump (PbuE), putrescine transport system permease protein (PotH), RTX-I toxin determinant B, sec-independent protein translocase protein (TatC), short-chain fatty acid transporter, sn-glycerol-3-phosphate transport system permease protein (ugpA), spermidine / putrescine transport system permease protein (PotB), TonB family C-terminal domain, TpsB transporter, transport permease protein, TRAP-C4 type-dicarboxylate transport system, small permease component, TRAP type uncharacterized transport system, fusion permease component, tricarboxylate transport protein (TctA) or a combination thereof.

[0022] In some embodiments, the proteins selected from the gene regulatory pathways include type IV secretion system protein virB11, 30S ribosomal protein S10, 50S ribosomal protein L10, 50S ribosomal protein L14, 50S ribosomal protein L25, 50S ribosomal protein L31 B type, ATP-dependent DNA helicase Rep, ATP-dependent RNA helicase (HrpA), bacterial DNA-binding protein, BpH3, cyn operon transcriptional activator, DNA polymerase III subunit γ / τ, DNA protection protein 2 during starvation, DNA repair protein (RecN), galactose-binding protein regulator, Gcv operon activator, HTH-type transcriptional regulator (gabR), leucine-responsive regulator, LigA, polynucleotide nucleotide transferase, protein (ApaG), ribonuclease (TTHA0252), ribosomal RNA small subunit methyltransferase B, ribosome-binding factor A, ribosome recycling factor, single-stranded DNA-binding protein, transcriptional regulator, y4mF family, transcription-repair coupling factor, or a combination thereof.

[0023] In some embodiments, the proteins selected from the metabolic pathways include ACR, COG1565, 1,4-α-glucan branching enzyme (GlgB), 1,4-dihydroxy-2-naphthoyl-CoA synthase, 2-(hydroxymethyl)glutarate dehydrogenase, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylic acid N-acetyltransferase, 3-(3-hydroxyphenyl)propionate / 3-hydroxycinnamate hydroxylase, 3-hydroxyacyl-CoA dehydrogenase, 3-oxoadipate enol-lactonase 2, 3-oxosteroid 1-dehydrogenase, 4-aminobutyrate aminotransferase PuuE, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin), 5-formyltetrahydrofolate cyclo-ligase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, 6-aminocaproate-cyclic dimer hydrolase aldehyde dehydrogenase 2, acetolactate synthase isozyme 3 large subunit, acetone carboxylase α subunit, acetone carboxylase β subunit, acetyl-CoA acetyltransferase, acetyltransferase component of pyruvate dehydrogenase complex, acyl carrier protein, acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase, acylating enzyme (ACY 1), acyl-CoA dehydrogenase (AidB), short-chain specific acyl-CoA dehydrogenase, S-adenosylhomocysteine hydrolase, aldehyde dehydrogenase, thermostable aliphatic sulfonate transport permease protein (ssuC), amidophosphoribosyltransferase, aminotransferase, arginine transport ATP-binding protein (ArtM), argininosuccinate lyase, aspartate 1-decarboxylase, aspartate acylase, aspartate kinase, ATP synthase subunit β, ATP-dependent desulfobiotin synthase (BioD), β-ketoacyl-acyl-carrier-protein synthase I, biotin carboxyl carrier protein of acetyl-CoA carboxylase, biotin carboxylase, carbamoyl-phosphate synthase large chain, carbamoyl-phosphate synthase small chain, cytochrome c, cytochrome c oxidase subunit 2, D-amino acid dehydrogenase, dihydrolipoamide dehydrogenase, dihydrolipoamide lysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, dihydropteroate synthase, D-malate degradation protein R, D-malate dehydrogenase (decarboxylating), export protein, ferredoxin-dependent glutamate synthase 1, Flp pilus assembly protein (TadD), formate-dependent phosphoribosylglycinamide formyltransferase, formyl-CoA transferase, glutamate dehydrogenase, glutamine-fructose-6-phosphate aminotransferase, glyceraldehyde-3-phosphate dehydrogenase, glycine dehydrogenase, guanidinobutyrase, imidazoleglycerol-phosphate dehydrase, iron-sulfur cluster assembly scaffold protein (IscU), L-aspartate dehydrogenase, long-chain fatty acid-CoA ligase, malate dehydrogenase, maltose α-D-glucosyltransferase, MmgE / PrpD family, N5-carboxyaminoimidazole ribonucleotide synthase, 6-phosphogluconate dehydrogenase NAD +A binding domain, nicotinamidase / pyrazinamidase, nucleoside recognition, oxoglutarate dehydrogenase, phosphoserine aminotransferase, an α / β-hydrolase fold hydrolase, a heme IX biogenesis protein, soluble aldehyde dehydrogenase (yliI), sporulation inhibitor (kipI), stringent starvation protein A, succinate dehydrogenase flavoprotein subunit, succinate-semialdehyde dehydrogenase [NADP(+)] (GabD), a thiamine biosynthesis protein (HI_0357), thiosulfate sulfurtransferase, threonine synthase, thymidylate synthase, UDP-glucose 6-dehydrogenase, Vi polysaccharide biosynthesis protein (TviD), xanthine dehydrogenase accessory protein (XdhC), or a combination thereof.

[0024] In some embodiments, the proteins selected from the oxidation-reduction pathway include 3-α-(or 20-β)-hydroxysteroid dehydrogenase, 3-ketosteroid-9-α-hydroxylase reductase subunit, 3-oxoacyl-[acyl-carrier-protein] reductase (FabG), acetoin:2,6-dichlorophenolindophenol oxidoreductase subunit β, alkyl hydroperoxide reductase C, benzaldehyde dehydrogenase [NAD(+)], benzene 1,2-dioxygenase subunit α, catalase, dTDP-4-dehydrorhamnose reductase, γ-glutamylputrescine oxidoreductase, gluconate 2-dehydrogenase cytochrome c subunit, glutathione hydrolase proenzyme, glutathione import ATP-binding protein (GsiA), glutathione transport system permease protein (gsiD), glutathione-binding protein (gsiB), glyoxylate / hydroxypyruvate reductase A, glyoxylate / hydroxypyruvate reductase B, heme A synthase, L-2-hydroxyglutarate oxidase (LhgO), methylmalonic-semialdehyde dehydrogenase, Mg-chelatase subunit child, muconolactone δ-isomerase, NAD(P)H-hydrate epimerase, NADH-quinone oxidoreductase, NADH-quinone oxidoreductase subunit L, NADH-quinone oxidoreductase subunit M, NADH-quinone oxidoreductase subunit N, aldehyde-ketone reductase, NAD(P)H quinone oxidoreductase, PIG3, quinone oxidoreductase 1, rhodocoxin reductase, sarcosine oxidase subunit β, sulfite reductase, thiol:disulfide exchange protein (DsbD), thioredoxin reductase, pantothenol oxidase subunit 1, or a combination thereof.

[0025] In some embodiments, the proteins selected from the protein regulation pathways include apolipoprotein N-acyltransferase, ATP-dependent Clp protease ATP-binding subunit (ClpX), capsular polysaccharide biosynthesis protein, carboxypeptidase G2, chaperonin GroEL, chorismate synthase, co-chaperonin GroES, dihydroxy-acid dehydratase, FtsH protease regulator (HflK), small-chain glutamate synthase, glutamine synthetase, ion protease, membrane dipeptidase M19, membrane protein insertase (YidC), methionine-tRNA ligase, molybdopterin molybdenum transferase, oligopeptidase A, periplasmic serine endoprotease DegP-like, C-terminal PDZ domain protease, HtpX, protein-L-is aspartate O-methyltransferase, glutamate-cysteine ligase 2, tRNA pseudouridine synthase A, tryptophan-tRNA ligase, tyrosine-tRNA ligase, or a combination thereof.

[0026] In some embodiments, the proteins selected from the stress response pathways include Rv1996 / MT2052, SAV1710, or a combination thereof.

[0027] In some embodiments, the pertussis vaccine further comprises any combination of an adjuvant, a preservative, or a stabilizer. In some embodiments, the vaccine comprises a pharmaceutically acceptable carrier selected from excipients, diluents, salts, buffers, stabilizers, lipids, emulsions, or nanoparticles.

[0028] In one aspect, a method of preventing a subject from being infected with Bordetella pertussis bacteria is disclosed herein, the method comprising administering to the subject a pharmaceutically effective amount of the pertussis vaccine of any of the foregoing aspects.

[0029] In one aspect, a method of improving the immune response of a subject against Bordetella pertussis bacteria is disclosed herein, the method comprising administering to the subject a pharmaceutically effective amount of the pertussis vaccine of any of the foregoing aspects.

[0030] In some embodiments, infection with Bordetella pertussis bacteria causes the subject to develop pertussis. In some embodiments, the immune response comprises presenting an antigen to class II MHC cells, recognition of the antigen by CD4+ T cells, or a combination thereof. In some embodiments, the immune response is against the BP0205 antigen and at least one protein involved in a biological pathway within Bordetella pertussis bacteria. In some embodiments, the at least one pathway is selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation, or stress response.

[0031] In some embodiments, the method further comprises administering a pertussis vaccine by injection via an intranasal, intramuscular, intraperitoneal, or subcutaneous route.

[0032] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings incorporated in and forming a part of this specification illustrate several aspects described below.

[0034] Figure 1 Showing immunoprecipitation of MHC-II epitopes.

[0035] Figure 2A and 2B Showing IFNγ secretion of splenocytes stimulated with target peptides as evaluated by ELISA. Splenocytes from non-immunized ( Figure 2A ) and convalescent mice ( Figure 2B ) were stimulated with peptides or with media alone as unstimulated (NS) negative control. One-way ANOVA with Tukey's multiple comparisons was used to detect differences between all experimental groups. Significance of each group is marked above (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Each point represents an individual well. Data are shown as mean and SEM.

[0036] Figure 3A and 3B Showing cell proliferation assay performed by 3H thymidine incorporation. The target peptides we screened and confirmed were used to stimulate splenocytes from non-immunized mice ( Figure 3A ) or mice immunized with heat-killed bacteria ( Figure 3B ). Media alone was used as unstimulated (NS) negative control. Significance of each group is marked above (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Each point represents an individual well. Results are shown as mean and SEM.

[0037] Figure 4 Showing human AIM gating strategy.

[0038] Figure 5 Showing that Bp0840 and Bp0205 stimulate T cells from DTP-sensitized Tdap-boosted patients to express early activation markers. The promising peptides we screened and confirmed were used to stimulate human PBMC isolated from vaccinated donors. T cell stimulation was evaluated by flow cytometry by comparing the expression of activation-induced markers (AIM). Media alone was used as unstimulated (-) negative control. Each symbol represents an individual participant (7 per group). DETAILED DESCRIPTION

[0039] The following provides a description of the present disclosure that can teach the present disclosure in the best currently known embodiments. To this end, those skilled in the relevant art will recognize and understand that many changes can be made to the various embodiments of the invention described herein while still achieving the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some features of the present disclosure without utilizing other features. Therefore, those skilled in the art will recognize that many modifications and variations to the present disclosure are possible and may even be desirable in certain cases, and are part of the present disclosure. Accordingly, the following description illustrates the principles of the present disclosure and is not intended to limit it.

[0040] Reference will now be made in detail to the embodiments of the present invention, which are illustrated in the accompanying drawings and examples. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0041] The term

[0042] 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. As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof, and are open-ended and non-limiting terms. Although the terms "comprising" and "including" are used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments and are also disclosed.

[0043] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0044] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by use of the antecedent “about”, it should be understood that the particular value forms another embodiment. It will be further understood that each of the endpoints of each range is significant with respect to the other endpoint and independent of the other endpoint. It will also be understood that a plurality of values are disclosed herein, and each value is also disclosed herein as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It will also be understood that when a value is disclosed as “less than or equal to” the value, “greater than or equal to the value” and the possible ranges between the values are also disclosed, as would be appropriately understood by one of ordinary skill in the art. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It will also be understood that throughout the application, the data is provided in a variety of different formats, and such data represents ranges of endpoints and starting points and any combination of data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, then it is to be understood that greater than, greater than or equal to, less than, less than or equal to and equal to 10 and 15 and between 10 and 15 are considered to be disclosed. It will also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

[0045] “Optional” or “optionally” means that the subsequent described event or circumstance may or may not occur, and the description includes both the instance where the event or circumstance occurs and the instance where it does not occur.

[0046] “Increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median or average increase that is a statistically significant amount of a condition, symptom, activity, composition. Thus, an increase can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% increase, so long as the increase is statistically significant.

[0047] "Reduce" can refer to any change that results in a smaller amount of a symptom, disease, component, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product of that substance is smaller relative to the output of the gene product without that substance. Also, for example, a reduction can be a change in the symptoms of a disorder such that the symptoms are fewer than those previously observed. A reduction can be any individual, median, or average reduction in a statistically significant amount of a condition, symptom, activity, or component. Thus, a reduction can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction, so long as the reduction is statistically significant.

[0048] "Enhance" can refer to any change that results in a greater amount of a symptom, disease, component, condition, response, or activity. An increase can be any individual, median, or average increase in a statistically significant amount of a condition, symptom, activity, or component. Thus, an increase can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% improvement, so long as the change is statistically significant.

[0049] "Prevent" or other forms of the word mean to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chance of a particular event or characteristic occurring. Prevention does not require a comparison to a control because it is generally more absolute than, for example, reduction. As used herein, some things can be reduced but not prevented, and some things that are reduced can also be prevented. Similarly, some things can be prevented but not reduced, and some things that are prevented can also be reduced. It is understood that the use of other terms is also expressly disclosed in the context of using reduction or prevention, unless specifically stated otherwise.

[0050] "Lower" or other forms of the word mean to lower an event or characteristic (e.g., a bacterial infection). It is understood that this is generally relative to some standard or expected value, in other words, it is relative, but it does not always require reference to a standard or relative value. For example, "reduce bacterial infection" means to lower the rate of bacterial growth and spread in a subject relative to a standard or control.

[0051] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, such as a mammal. In one aspect, the subject can be a human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, a subject can be a human or veterinary patient.

[0052] The term "pharmaceutically effective amount" refers to an amount of the composition being used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need only be a reduction or alteration and not necessarily an elimination.

[0053] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements but do not preclude other elements. "Consisting essentially of" when used to define compositions and methods will mean including the recited elements but excluding other elements that have any significant meaning for the combination. Thus, a composition consisting essentially of the elements as defined herein will not preclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, etc. "Consisting of" will mean excluding other ingredients in amounts greater than trace elements and substantial method steps for administering the compositions provided and / or claimed in the present disclosure. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0054] The term "administering" refers to oral, topical, intravenous, subcutaneous, transdermal, percutaneous, intramuscular, intra-articular, parenteral, intra-arterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, or via an implanted reservoir. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0055] As used herein, the term "infection" refers to the invasion of a tissue by a pathogen, their multiplication, and the response of the host tissue to the infectious agent and any toxins they release. Infections can be caused by many types of pathogens, most commonly bacteria and viruses.

[0056] "Protein", "polypeptide", or "peptide" each refers to a polymer of amino acids and does not imply a specific length of the polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides having post-expression modifications such as glycosylation (e.g., addition of sugars), acetylation, phosphorylation, etc.

[0057] The term "amino acid" includes, but is not limited to, amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term "amino acid residue" may also include amino acid residues contained in the group consisting of homocysteine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, β-aminopropionic acid, allo-hydroxylysine, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline, pipecolic acid, 6-aminohexanoic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 2-aminoisobutyric acid, N-methylglycine, sarcosine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminoheptanedioic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminoheptanedioic acid, norleucine, 2,3-diaminopropionic acid, ornithine, and N-ethylglycine. Generally, the amide bond of a peptide is formed by the amino group of the backbone of one amino acid and the carboxyl group of the backbone of another amino acid.

[0058] Peptides, polypeptides, proteins, and compositions containing peptides, polypeptides, and proteins are also mentioned herein. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids that is typically ≥100 amino acids in length (Garrett & Grisham, Biochemistry, 2nd ed., 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids that is typically 20 or fewer amino acids in length and more typically 12 or fewer amino acids in length (Garrett & Grisham, Biochemistry, 2nd ed., 1999, Brooks / Cole, 110).

[0059] The peptides, polypeptides, and proteins disclosed herein can be modified to include non-amino acid moieties. Modifications can include, but are not limited to, carboxylation (e.g., N-terminal carboxylation via addition of a dicarboxylic acid having 4-7 straight-chain or branched carbon atoms such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as an alkyl amide or dialkyl amide), polyethylene glycolylation (e.g., N-terminal or C-terminal polyethylene glycolylation via addition of polyethylene glycol), acylation (e.g., O-acylation (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., addition of an acetyl group at the N-terminus of a protein or at a lysine residue), formylation lipoylation (e.g., attachment of lipoic acid, a C8 functional group), myristoylation (e.g., attachment of myristic acid, a C14 saturated acid), palmitoylation (e.g., attachment of palmitic acid, a C16 saturated acid), alkylation (e.g., addition of an alkyl group such as a methyl group at a lysine or arginine residue), isoprenylation / prenylation (e.g., addition of an isoprenoid group such as farnesol or geranylgeraniol), C-terminal amidation, glycosylation (e.g., addition of a sugar moiety to asparagine, hydroxylysine, serine, or threonine to produce a glycoprotein). Different from glycation which is considered to be a non-enzymatic linkage of sugars, polysialylation (e.g., addition of polysialic acid), glycosylphosphatidylinositolization (e.g., glycosylphosphatidylinositol (GPI) anchor formation), hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., generally addition of a phosphate group to serine, tyrosine, threonine, or histidine).

[0060] The phrases “percent identity” and “% identity” as applied to polypeptide sequences refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods for aligning polypeptide sequences are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, more fully explained above, generally preserve the charge and hydrophobicity at the substitution site, thereby preserving the structure (and thus the function) of the polypeptide. The percent identity of amino acid sequences can be determined as understood in the art (see, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410) provides a set of commonly used and freely available sequence alignment algorithms, which can be obtained from several sources, including NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs, including “blastp,” which is used to align a known amino acid sequence with other amino acid sequences from a variety of databases.

[0061] The percent identity can be measured over the length of the entire defined polypeptide sequence or can be measured over a shorter length, such as over the length of a fragment taken from the larger defined polypeptide sequence, such as a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are merely exemplary and it is to be understood that any fragment length can be used to describe the length over which the percent identity can be measured.

[0062] It should also be noted that amino acids and derivatives (except glycine) exist in two isomeric forms: the L-form or the D-form. The L- and D-forms represent amino acids of the same atoms, however these atoms may have different arrangements, which may affect the properties and functions of the amino acids. The similarity between the two forms is that they are both naturally occurring and contain a central carbon atom, at least one hydrogen atom, a carboxyl group, an amine group and a variable group. The difference between the two forms is that they are usually mirror images of each other, with the position of the amine group being different. L-amino acids are used in protein synthesis, while D-amino acids are less common in protein synthesis. L-amino acids rotate counterclockwise or to the left during a process called left-handed rotation. D-amino acids rotate clockwise or to the right during a process called right-handed rotation. L-amino acids are used to synthesize proteins, while D-amino acids are found in the cell walls of bacteria.

[0063] The term "variant" means a polypeptide derived from a parental albumin by one or more (several) alterations (i.e., substitutions, insertions and / or deletions) at one or more (several) positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, preferably 1-3 amino acids, adjacent to the amino acid occupying a certain position. With respect to substitution, "adjacent" can be the N-side ("upstream") or the C-side ("downstream") of the amino acid occupying a certain position ("the named amino acid"). Thus, for an amino acid named / numbered "X", the insertion can be at position "X+1" ("downstream") or position "X-1" ("upstream").

[0064] A "variant" of a specific polypeptide sequence can be defined as a polypeptide sequence that has at least 50% sequence identity over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool available on the National Center for Biotechnology Information website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences—a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). In some embodiments, the variant polypeptide may exhibit, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or higher sequence identity over a certain defined length relative to the reference polypeptide.

[0065] The variant polypeptide may have substantially the same functional activity as the reference polypeptide. For example, the variant polypeptide may exhibit one or more biological activities associated with binding to a ligand.

[0066] Variants encompassing fragments of the reference amino acid sequence are covered herein. A "fragment" is a part of an amino acid sequence that has the same sequence as the reference sequence but is shorter in length than the reference sequence. A fragment may comprise up to the full length of the reference sequence minus at least one amino acid residue. For example, a fragment may comprise 5 to 1000 or contiguous amino acid residues of the reference polypeptide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250 or 500 contiguous amino acid residues of the reference polypeptide, respectively. Fragments may preferably be selected from certain regions of the molecule, such as the N-terminal region and / or the C-terminal region of the polypeptide. The term "at least fragment" encompasses the full-length polypeptide.

[0067] A "vaccine" refers to a biological preparation that provides active acquired immunity against a specific infectious disease caused by a virus, bacterium, parasite or any other microorganism. Vaccines typically contain an agent or several agents, also known as antigens, that are expressed by the disease-causing microorganism and are usually made from a weakened or killed form of the microorganism, its toxin or its surface protein / peptide. Vaccines are also formulated to contain additional components, such as adjuvants, preservatives and / or stabilizers, to enhance the immune response, improve safety and facilitate vaccine storage.

[0068] "Antigen" refers to a molecule, moiety, foreign particulate matter, or allergen that can bind to a specific antibody or T cell receptor. The presence of an antigen in a host can trigger an immune response against the molecule, moiety, foreign particulate matter, or allergen.

[0069] "Adjuvant" refers to a drug, molecule, substance, or combination thereof used to increase the efficacy or potency of certain therapeutic agents (such as vaccines and / or antibodies). An "adjuvant" is typically at least one component used in some vaccines that helps to generate a stronger immune response in a host receiving the vaccine.

[0070] The terms "cell", "cell line", and "cell culture" include progeny. It is also understood that due to intentional or unintentional mutations, the DNA content of all progeny may not be precisely the same. Variant progeny screened for the same function or biological characteristics in the originally transformed cells are included. The "host cell" used in the present invention is typically a prokaryotic or eukaryotic host.

[0071] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significantly undesirable biological effects or interacting in a harmful manner with any other component of the formulation containing it. When referring to administration to a human, the term generally means that the component has met the required standards of toxicological and production testing or is listed in the Inactive Ingredients Guide established by the U.S. Food and Drug Administration.

[0072] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that can be used to prepare a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline solutions, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents.

[0073] As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer or other substance well known in the art for use in pharmaceutical formulations. The choice of carrier for a composition will depend on the intended route of administration of the composition. Pharmaceutically acceptable carriers and the preparation of formulations containing such materials are described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, edited by University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffers and buffers with other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG) and PLURONICS TM (BASF; (Florham Park, NJ). To provide administration of such doses for the desired therapeutic treatment, based on the weight of the total composition including the carrier or diluent, the compositions disclosed herein may advantageously contain in total between about 0.1 wt% and 99 wt% of one or more of the subject compounds.

[0074] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the prior art in the field to which this application pertains. The materials contained in the disclosed references that are discussed in the sentences that rely on those references are also incorporated herein by reference individually and specifically.

[0075] Compounds and Compositions

[0076] A pertussis vaccine is a vaccine used to protect against the types of Bordetella pertussis bacteria known to cause "whooping cough" in humans. There are two types of pertussis vaccines: whole cell vaccines and acellular vaccines. Compared to whole cell pertussis vaccines, acellular vaccine types protect the respiratory tract and have fewer side effects; however, the efficacy of acellular pertussis vaccines declines at a much faster rate, and acellular vaccines do not cause clearance of the upper respiratory tract. Given the current limitations of pertussis vaccines, there is a need to produce improved pertussis vaccines with increased efficacy and long-lasting protection.

[0077] It is known that Bordetella pertussis bacteria infect the upper and lower respiratory tracts, leading to an immune response. Infection with this bacterium induces innate (neutrophil and macrophage immune cells) and adaptive (B and T cell) immune responses. Generally, antigen-presenting cells such as dendritic cells, macrophages, and some endothelial cells can present Bordetella pertussis peptides bound to major histocompatibility complex II (MHCII) in mice and HLA-DR in humans on their cell surfaces. This recognition activates the CD4+ T cell response, which is part of the adaptive immune response. Then the presentation of Bordetella pertussis peptides on the surface of immune cells is recognized by T cells such as CD4+ T cells. An increase in the number of CD4+ T cells leads to the clearance of Bordetella pertussis from the respiratory organs.

[0078] In this article, unbiased biochemical methods were used to identify peptides from Bordetella pertussis presented on MHC class II. These peptides are derived from various Bordetella pertussis proteins that are not part of the current acellular pertussis vaccines. Experimental evidence supports the immunogenicity of these peptides and shows that including these novel proteins in the next generation of pertussis vaccines will generate a better and more long-lasting immune response than current vaccines.

[0079] The present disclosure relates to an improved vaccine against Bordetella pertussis bacterial infection, which comprises novel proteins expressed by Bordetella pertussis and presented as peptides on HLA-DR and MHC class II molecules.

[0080] In one aspect, the present disclosure provides a pertussis vaccine comprising a combination of the BP0205 antigen or a variant thereof and at least one additional peptide from Bordetella pertussis bacteria, wherein the BP0205 peptide and at least one additional peptide are to be presented to MHC class II cells and recognized by CD4+ T cells.

[0081] In some embodiments, the pertussis vaccine comprises an acellular pertussis (aP) vaccine.

[0082] In some embodiments, the at least one peptide is derived from a protein involved in at least one biological pathway within Bordetella pertussis bacteria. In some embodiments, the at least one peptide is derived from the proteins of Table 2 that include the Bordetella pertussis protein of interest.

[0083] For the commercial benefit of vaccine manufacturers working to improve pertussis vaccine efficacy, vaccine targets or antigens are experimentally confirmed and validated. Further, the antigens are intracellular and extracellular components of Bordetella pertussis bacteria. Herein, the antigens include cell surface proteins, lipopolysaccharides, peptidoglycans, and intracellular proteins that are essential for bacterial gene regulation, bacterial protein regulation, bacterial metabolism, bacterial cell cycle, bacterial cell adhesion and motility, and bacterial cell stress response.

[0084] The present disclosure provides a pertussis vaccine comprising the BP0205 peptide or protein as an antigen. BP0205 is a lipoprotein or lipopeptide that includes a signal peptide (exemplified in SEQ ID NO:4) and is found in the inner membrane of Bordetella pertussis bacteria. BP0205 is thought to be involved in the insertion, folding, and complex formation of integral membrane proteins into the cell membrane. The so-called "BP0205 peptide" or "BP0205 protein" means the complete peptide or a fragment thereof that is recognized as an antigen. Those skilled in the art will understand that variants, fragments, or portions of the BP0205 peptide can be used as antigens. The antigenic response of a peptide or its fragment or variant can be measured by determining the immune response to the peptide. SEQ ID NO:1 represents the full length of BP0205. The BP0205 sequence is also found in NP_879093 (WP_010929682.1). Vaccines comprising peptides having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 are also disclosed herein.

[0085] It is expected that BP0205 contains an adjuvant region near the amino terminus of the peptide. It should also be understood that an antigen differs from an adjuvant in that an antigen is used as a target component or constituent of a vaccine to elicit an immune response, specifically by activating the adaptive immune response (e.g., presentation to class II MHC cells and recognition by CD4+ T cells). An adjuvant, on the other hand, is used as a co-administered vaccine component or constituent to increase the magnitude and persistence of the immune response against an antigen (Moyer et al., "Beyond antigens and adjuvants: formulating future vaccines. 2016). Thus, the antigenic region of BP0205 in combination with at least one additional Bordetella pertussis peptide elicits an immune response.

[0086] In some embodiments, the BP0205 peptide comprises at least one peptide fragment. In some embodiments, the BP0205 peptide comprises 1, 2, 3, 4, 5 or more peptide fragments. In some embodiments, the 1, 2, 3, 4, 5 or more peptide fragments are operably linked together by a peptide linker. As used herein, "peptide linker" refers to a short sequence of amino acids that separates two or more peptide fragments within a single peptide or protein. The length of the peptide linker can vary from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids. In some embodiments, the BP0205 peptide comprises SEQ ID NO:2 and / or SEQ ID NO:3. In some embodiments, SEQ ID NO:2 and SEQ ID NO:3 are separated by 1, 2, 3, 4, 5 or more amino acids. In some embodiments, SEQ ID NO:2 and SEQ ID NO:3 are operably linked by a peptide linker.

[0087] As used herein, "operably linked" means the juxtaposition of two or more components (such as peptide fragments), wherein the components are arranged such that the two or more components maintain their normal functions (such as eliciting an immune response).

[0088] As used herein, "antigenic epitope", "epitope" or "antigenic determinant" refers to the part of an antigen, molecular structure or foreign particulate that can bind to a specific antibody or T cell receptor. The presence of an antigen or an epitope of an antigen in a host can elicit an immune response. It should be understood that "antigenic epitope", "epitope" or "antigenic determinant" can be used interchangeably.

[0089] In some embodiments, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 can be used alone as an epitope. In some embodiments, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 can be arranged in any order or combination to obtain the desired immunogenic effect. Thus, in some embodiments, the pertussis vaccine comprises an epitope comprising SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:3 and SEQ ID NO:2; SEQ ID NO:2 and SEQ ID NO:4; SEQ ID NO:4 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:4 and SEQ ID NO:3; SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:3; SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:4; SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:3; or SEQ ID NO:4, SEQ ID NO:3, and SEQ ID NO:2.

[0090] In some embodiments, the BP0205 peptide comprises at least one peptide fragment 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 or more amino acids. In some embodiments, the amino acids are contiguous. In some embodiments, the amino acids are non-contiguous. In some embodiments, the amino acids are near the carboxyl terminus of BP0205. In some embodiments, the amino acids are near the amino terminus of BP0205. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in the peptide fragment are altered. In some embodiments, the amino acids are conservative.In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or more amino acids are deleted from the BP0205 peptide. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or more amino acids are added to the BP0205 peptide.

[0091] In some embodiments, the pertussis vaccine comprises at least 50% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 55% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 60% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 65% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 70% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 75% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 80% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 85% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 96% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 97% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 98% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises at least 99% sequence identity with SEQ ID NO:1. In some embodiments, the pertussis vaccine comprises SEQ ID NO:1.

[0092] In some embodiments, the pertussis vaccine comprises the BP0205 peptide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides from Bordetella pertussis bacteria. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides are derived from those in Table 2 or from at least one biological pathway selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation or stress response, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303 proteins or combinations thereof.

[0093] In some embodiments, the proteins selected from the cell adhesion and motility pathways include attachment and elimination proteins, fimbrial proteins, flagellar biosynthesis protein (FlhF), or combinations thereof.

[0094] In some embodiments, the proteins selected from the cell cycle pathway include the protein containing a β-lactamase domain, cell division coordination protein (CpoB), cell division protein (ZapD), peptidyl-prolyl cis-trans isomerase, peptidyl-prolyl cis-trans isomerase (Cbf2), phospho-N-acetylmuramoyl-pentapeptide-transferase, tol-Pal system protein (TolB), tol-Pal system protein (TolQ), or combinations thereof.

[0095] In some embodiments, the proteins selected from the cell signaling pathway include dermonecrotic toxin, cyclic di-GMP phosphodiesterase response regulator (RpfG), enterobactin outer membrane receptor, or combinations thereof.

[0096] In some embodiments, the proteins selected from the cell structure pathway include metal-binding proteins, BrkB, D-alanine-D-alanine ligase, inner membrane protein (ybhN), inner membrane protein (YiaH), lipoprotein, lipoteichoic acid synthase 2, outer membrane protein assembly factor (BamA), P.93, penicillin-binding protein 1A, pertussis toxin release protein F, membrane protein, DMT superfamily permease, spermidine synthase with an N-terminal membrane domain, lipoic acid-protein ligase A, HflK, assembly protein, inner membrane protein, HI_0719, soluble lytic murein transglycosylase, virulence-associated outer membrane protein Vir-90, or combinations thereof.

[0097] In some embodiments, proteins selected from cell transport pathways include Xaa-Pro aminopeptidase, 2-aminoethylphosphonate ABC transporter substrate-binding protein, aerobactin synthase (IucC), antimicrobial resistance protein, arabinose efflux permease, BFD-like [2Fe-2S] binding domain, bicarbonate transport ATP-binding protein (CmpD), bicyclomycin / multidrug efflux system, biotin transporter, BrkA autotransporter, ComEC family competence protein, D-methionine-binding lipoprotein (metQ), drug efflux system protein (MdtG), efflux pump membrane transporter, extracytoplasmic solute receptor protein, extracytoplasmic solute receptor protein (yiaO), ferrichrome receptor (FcuA), filamentous hemagglutinin, hemin-inhibitable hemoglobin-binding protein, hemin import ATP-binding protein (HmuV), inner membrane metabolite transporter (yhjE), inner membrane protein (yhjX), inner membrane transporter (ydhP), iron-sulfur cluster carrier protein, leucine ABC transporter subunit substrate-binding protein (LivK), leucine-, isoleucine-, valine-, threonine-, and alanine-binding protein, LIV-I protein F, LIV-I protein H, major facilitator superfamily protein, manganese transport system membrane protein (mntB), methyl viologen resistance protein (SmvA), multidrug export ATP-binding / permease protein (SAV1866), multidrug resistance protein B, multiple antibiotic resistance and pH homeostasis protein A, multiple antibiotic resistance and pH homeostasis protein D, multiple antibiotic resistance and pH homeostasis protein E, Neu5Ac permease, nickel / cobalt efflux system, BP0840, preprotein translocase subunit (SecD), ABC transporter ATP-binding protein (HI_0664), amino acid metabolite efflux pump, chorismate pyruvate-lyase, membrane transporter, protein translocase subunit (SecD), purine efflux pump (PbuE), putrescine transport system permease protein (PotH), RTX-I toxin determinant B, sec-independent protein translocase protein (TatC), short-chain fatty acid transporter, sn-glycerol-3-phosphate transport system permease protein (ugpA), spermidine / putrescine transport system permease protein (PotB), TonB family C-terminal domain, TpsB transporter, transport permease protein, TRAP-C4 type-dicarboxylate transport system, small permease component, TRAP-type uncharacterized transport system, fusion permease component, tricarballylate transport protein (TctA), or a combination thereof.

[0098] In some embodiments, the proteins selected from the gene regulatory pathways include type IV secretion system protein virB11, 30S ribosomal protein S10, 50S ribosomal protein L10, 50S ribosomal protein L14, 50S ribosomal protein L25, 50S ribosomal protein L31 B type, ATP-dependent DNA helicase Rep, ATP-dependent RNA helicase (HrpA), bacterial DNA-binding protein, BpH3, cyn operon transcriptional activator, DNA polymerase III subunit γ / τ, DNA protection protein 2 during starvation, DNA repair protein (RecN), galactose-binding protein regulator, Gcv operon activator, HTH-type transcriptional regulatory protein (gabR), leucine-responsive regulatory protein, LigA, polynucleotide nucleotide transferase, protein (ApaG), ribonuclease (TTHA0252), ribosomal RNA small subunit methyltransferase B, ribosome-binding factor A, ribosome recycling factor, single-stranded DNA-binding protein, transcriptional regulatory factor, y4mF family, transcription-repair coupling factor, or a combination thereof.

[0099] In some embodiments, the proteins selected from metabolic pathways include ACR, COG1565, 1,4-α-glucan branching enzyme (GlgB), 1,4-dihydroxy-2-naphthoyl-CoA synthase, 2-(hydroxymethyl)glutaryl dehydrogenase, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylic acid N-acetyltransferase, 3-(3-hydroxyphenyl)propionic acid / 3-hydroxycinnamic acid hydroxylase, 3-hydroxyacyl-CoA dehydrogenase, 3-oxoadipate enol-lactonase 2, 3-oxosteroid 1-dehydrogenase, 4-aminobutyrate aminotransferase PuuE, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin), 5-formyltetrahydrofolate cyclo-ligase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, 6-aminocaproate-cyclic dimer hydrolase aldehyde dehydrogenase 2, acetolactate synthase isozyme 3 large subunit, acetone carboxylase α subunit, acetone carboxylase β subunit, acetyl-CoA acetyltransferase, acetyltransferase component of pyruvate dehydrogenase complex, acyl carrier protein, acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase, acylating enzyme (ACY 1), acyl-CoA dehydrogenase (AidB), short-chain specific acyl-CoA dehydrogenase, S-adenosylhomocysteine hydrolase, aldehyde dehydrogenase, thermostable aliphatic sulfonate transport permease protein (ssuC), amidophosphoribosyltransferase, aminotransferase, arginine transport ATP-binding protein (ArtM), argininosuccinate lyase, aspartate 1-decarboxylase, aspartate acylase, aspartate kinase, ATP synthase subunit β, ATP-dependent desulfobiotin synthetase (BioD), β-ketoacyl-acyl-carrier-protein synthase I, biotin carboxyl carrier protein of acetyl-CoA carboxylase, biotin carboxylase, carbamoyl-phosphate synthase large chain, carbamoyl-phosphate synthase small chain, cytochrome c, cytochrome c oxidase subunit 2, D-amino acid dehydrogenase, dihydrolipoamide dehydrogenase, dihydrolipoamide lysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, dihydropteroate synthase, D-malate degradation protein R, D-malate dehydrogenase (decarboxylating), export protein, ferredoxin-dependent glutamate synthase 1, Flp pilus assembly protein (TadD), formate-dependent phosphoribosylglycinamide formyltransferase, formyl-CoA transferase, glutamate dehydrogenase, glutamine-fructose-6-phosphate aminotransferase, glyceraldehyde-3-phosphate dehydrogenase, glycine dehydrogenase, guanidinobutyrase, imidazoleglycerol-phosphate dehydrase, iron-sulfur cluster assembly scaffold protein (IscU), L-aspartate dehydrogenase, long-chain fatty acid-CoA ligase, malate dehydrogenase, maltose α-D-glucosyltransferase, MmgE / PrpD family, N5-carboxyaminoimidazole ribonucleotide synthase, 6-phosphogluconate dehydrogenase NAD +A binding domain, nicotinamidase / pyrazinamidase, nucleoside recognition, oxoglutarate dehydrogenase, phosphoserine aminotransferase, an α / β - hydrolase fold hydrolase, a heme IX biogenesis protein, soluble aldehyde dehydrogenase (yliI), sporulation inhibitor (kipI), stringent starvation protein A, succinate dehydrogenase flavoprotein subunit, succinate - semialdehyde dehydrogenase [NADP(+)] (GabD), a thiamine biosynthesis protein (HI_0357), thiosulfate sulfurtransferase, threonine synthase, thymidylate synthase, UDP - glucose 6 - dehydrogenase, Vi polysaccharide biosynthesis protein (TviD), xanthine dehydrogenase accessory protein (XdhC), or a combination thereof.

[0100] In some embodiments, the proteins selected from the oxidation - reduction pathway include 3 - α - (or 20 - β) - hydroxysteroid dehydrogenase, 3 - ketosteroid - 9 - α - hydroxylase reductase subunit, 3 - oxoacyl - [acyl - carrier - protein] reductase (FabG), acetoin: 2,6 - dichlorophenolindophenol oxidoreductase subunit β, alkyl hydroperoxide reductase C, benzaldehyde dehydrogenase [NAD(+)], benzene 1,2 - dioxygenase subunit α, catalase, dTDP - 4 - dehydro - rhamnose reductase, γ - glutamylputrescine oxidoreductase, gluconate 2 - dehydrogenase cytochrome c subunit, glutathione hydrolase proenzyme, glutathione import ATP - binding protein (GsiA), glutathione transport system permease protein (gsiD), glutathione - binding protein (gsiB), glyoxylate / hydroxypyruvate reductase A, glyoxylate / hydroxypyruvate reductase B, heme A synthase, L - 2 - hydroxyglutarate oxidase (LhgO), methylmalonate - semialdehyde dehydrogenase, Mg - chelatase subunit child, muconolactone δ - isomerase, NAD(P)H - hydrate epimerase, NADH - quinone oxidoreductase, NADH - quinone oxidoreductase subunit L, NADH - quinone oxidoreductase subunit M, NADH - quinone oxidoreductase subunit N, aldehyde - ketone reductase, NAD(P)H quinone oxidoreductase, PIG3, quinone oxidoreductase 1, rhodocoxin reductase, sarcosine oxidase subunit β, sulfite reductase, thiol: disulfide exchange protein (DsbD), thioredoxin reductase, pantothenol oxidase subunit 1, or a combination thereof.

[0101] In some embodiments, the proteins selected from the protein regulatory pathways include apolipoprotein N-acyltransferase, ATP-dependent Clp protease ATP-binding subunit (ClpX), capsular polysaccharide biosynthesis protein, carboxypeptidase G2, chaperonin GroEL, chorismate synthase, co-chaperonin GroES, dihydroxy-acid dehydratase, FtsH protease regulator (HflK), small chain glutamate synthase, glutamine synthetase, ion protease, membrane dipeptidase M19, membrane protein insertase (YidC), methionine-tRNA ligase, molybdopterin molybdenum transferase, oligopeptidase A, periplasmic serine endoprotease DegP-like, C-terminal PDZ domain protease, HtpX, protein-L-is aspartate O-methyltransferase, glutamate-cysteine ligase 2, tRNA pseudouridine synthase A, tryptophan-tRNA ligase, tyrosine-tRNA ligase, or a combination thereof.

[0102] In some embodiments, the proteins selected from the stress response pathways include Rv1996 / MT2052, SAV1710, or a combination thereof.

[0103] It should be noted that the pertussis vaccine of the present disclosure is also an immunogenic composition comprising the BP0205 antigen or a variant thereof in combination with at least one peptide from Bordetella pertussis bacteria. It should be further understood that the terms vaccine and immunogenic composition can be used interchangeably. Accordingly, the vaccine further comprises a pharmaceutically acceptable carrier and one or more adjuvants, preservatives or stabilizers or any combination thereof. As used herein, the term "pharmaceutically acceptable" means that the carrier or excipient will not cause adverse or harmful effects in the subject to which it is administered at the dosages and concentrations used. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington. The Science and Practice of Pharmacy, Mack Publishing Company 1990; Frokjaer, S. & Hovgaard, L. Pharmaceutical Formulation Development of Peptides and Proteins, 2000; Handbook of Pharmaceutical Excipients, Pharmaceutical Press 2000). The composition is preferably formulated and administered as a sterile solution. The sterile solution is prepared by sterile filtration or by other methods known per se in the art. The solution can then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution is generally in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5. The components of the composition are generally in a solution with a suitable pharmaceutically acceptable buffer, and the solution may also contain salts. In some embodiments, a detergent is present in the vaccine. In some embodiments, the vaccine can be formulated as an injectable preparation. These vaccine preparations contain an effective amount of the antigen and / or peptide component and are in the form of a sterile liquid solution, a liquid suspension or a lyophilized form. The vaccine preparation may also contain a non-limiting amount of adjuvants, stabilizers, preservatives and / or excipients or combinations thereof.

[0104] Further examples of suitable formulations for storage and / or pharmaceutical administration of the pertussis vaccine are known ("Vaccines", 5th edition, S. Plotkin et al.). PBS or saline are examples of suitable diluents. As used herein, the pertussis vaccine may have at least one preservative present, including but not limited to phenoxyethanol, thimerosal or parabens. If a preservative is present, it is preferably present at a low level.

[0105] In some embodiments, the pertussis vaccine comprises at least one adjuvant. Adjuvants are known in the art to further increase the immune response to the applied antigenic determinants (for a review of adjuvants, see, e.g., Montomoli, 2011, Expert Rev. Vaccines 10:1053-1061). Examples of suitable adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or water-in-oil compositions), including squalene water emulsions such as MF59 (see, e.g., WO 90 / 14837); saponin formulations such as QS21 and immunostimulating complexes (ISCOMs) (see, e.g., U.S. Patent No. 5,057,540; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); Toll-like receptor (TLR) agonists such as TLR7 agonists (see, e.g., WO 2012 / 117377, e.g., pages 15-18), e.g., in combination with an aluminum salt such as aluminum hydroxide to which the TLR agonist can adsorb; bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing CpG motifs, ADP-ribosylated bacterial toxins or mutants thereof such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT, etc.

[0106] Methods for preventing infection and / or improving immune response

[0107] In one aspect, methods are disclosed herein for preventing a subject from being infected with Bordetella pertussis bacteria, the method comprising administering to the subject a pharmaceutically effective amount of a pertussis vaccine of any of the foregoing aspects.

[0108] In one aspect, methods are disclosed herein for improving the immune response of a subject against Bordetella pertussis bacteria, the method comprising administering to the subject a pharmaceutically effective amount of a pertussis vaccine of any of the foregoing aspects.

[0109] In one aspect, methods are disclosed herein for preventing a subject from being infected with Bordetella pertussis bacteria, wherein a pharmaceutically effective amount of a pertussis vaccine is administered to the subject to enhance the immune response.

[0110] In some embodiments, infection with Bordetella pertussis bacteria causes whooping cough in a subject. It should be noted that whooping cough, caused by Bordetella pertussis infection, is a highly contagious and highly infectious disease that affects the respiratory system. Individuals affected by this infection exhibit severe dry coughs followed by high-pitched inhalations that sound like "whooping." While this disease was once considered a childhood disease, it is now known that whooping cough can cause severe illness in individuals of all ages, including infants, toddlers, children, adolescents, young adults, adults, the elderly, and pregnant women. It is also recognized in the art that the best way to prevent Bordetella pertussis and thus prevent whooping cough is through vaccine administration ("Whooping Cough Vaccination", August 2022, www.cdc.gov / pertussis / vaccines).

[0111] In some embodiments, the immune response comprises presenting an antigen to MHC class II cells, recognition of the antigen by CD4+ T cells, or a combination thereof. In some embodiments, the immune response comprises a cellular (T cell) and / or humoral (antibody) response. In some embodiments, the immune response is directed against the BP0205 antigen and at least one protein involved in a biological pathway within Bordetella pertussis bacteria. In some embodiments, the at least one pathway is selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation, or stress response.

[0112] In some embodiments, the method further comprises administering a pertussis vaccine by intranasal, intramuscular, intraperitoneal or subcutaneous injection. In some embodiments, the method comprises administering an acellular pertussis vaccine comprising a combination of the BP0205 antigen or a variant thereof and at least one peptide from Bordetella pertussis bacteria, wherein the BP0205 peptide and the at least one peptide are to be presented to class II MHC cells and recognized by CD4+ T cells. In some embodiments, the method comprises administering a pertussis vaccine (e.g., an aP vaccine) that comprises the BP0205 antigen and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides from Bordetella pertussis bacteria. In some embodiments, the method comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides of 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303 proteins or combinations thereof.

[0113] The pertussis vaccine can be administered in such amounts, at such times, and by such routes as are considered necessary to achieve the desired results. The exact amount of the pertussis vaccine will vary from subject to subject, depending on the type, age, and general condition of the subject, the severity of the Bordetella pertussis infection, the particular vaccine formulation, its mode of administration, its mode of action, etc. For ease of administration and uniformity of dosage, the pertussis vaccine is preferably formulated in dosage unit form. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the infection being treated and the severity of the infection; the activity of the pertussis vaccine being used; the particular pertussis vaccine being used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the particular pertussis vaccine being used; the duration of the treatment; drugs used in combination with or concurrently with the particular pertussis vaccine being used; and similar factors well known in the medical arts.

[0114] The pertussis vaccine can be administered by any route. In some embodiments, the pertussis vaccine is administered by multiple routes, including intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by endotracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Generally, the most appropriate route of administration will depend on a variety of factors, including the nature of the pertussis vaccine (e.g., its stability in the subject's body environment), the condition of the subject (e.g., whether the subject can tolerate the administration), etc. In one embodiment, the vaccine is administered by intramuscular injection.

[0115] The exact amount of the pertussis vaccine required to achieve a therapeutically effective amount will vary from subject to subject, depending on the type, age, and general condition of the subject, the severity of side effects, the characteristics of the particular compound, the mode of administration, etc. The amount to be administered to, for example, children or adolescents can be determined by a medical practitioner or a person skilled in the art and can be lower than or equal to the amount administered to adults.

[0116] The pertussis vaccine is also suitable for use as a booster vaccine for groups that have previously been vaccinated with other vaccines, where the other vaccines are whole cell pertussis vaccines (wP) or aP vaccines that are different in composition from the vaccines of the present disclosure, or combination vaccines that contain wP or aP that are different in composition from the vaccines of the present disclosure. Such boosters can be used, for example, to vaccinate adults or the elderly who have not been vaccinated against Bordetella pertussis for more than a decade. It may be useful to repeat the booster vaccination approximately every five, ten, or fifteen years. In certain embodiments, aP is suitable for administration to infants, children, adolescents, adults, the elderly, or pregnant women.

[0117] The dose of the vaccine is the amount administered in a single administration to a subject. The subject can suitably be an animal or a human, and in some embodiments, the subject is a human. In some embodiments, the subject is a mammal. The vaccine disclosed herein is suitably administered to the same individual at least once to obtain the desired effect against infection. The vaccine disclosed herein is suitably administered to the same individual more than once at sufficient time intervals to obtain a booster effect in the individual (e.g., the time interval is at least four weeks to six months to one year to several years and up to twenty years). Non-limiting examples of pertussis vaccine administrations include two or three or more times, at least 4 weeks apart, such as one or two months apart between each administration. One non-limiting example is administration at 6 weeks of age, 10 weeks of age, and 14 weeks of age according to the EPI schedule. Another protocol is at 2 months of age, 4 months of age, and 6 months of age. In some embodiments, a booster vaccination containing a pertussis vaccine is given 10 - 20 years later (e.g., during puberty). In some embodiments, the pertussis vaccine administration includes two or three vaccinations in the first year after birth, a further booster administration in the second year after birth, and a further booster administration at four to five years of age, followed by a puberty booster administration at about twelve years of age.

[0118] Numerous embodiments of the present disclosure have been described. However, it will be understood that various modifications can be made without departing from the essence and scope of the invention. Accordingly, other embodiments are also within the scope of the following claims.

[0119] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0120] Examples

[0121] The following examples are given to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalent and variant embodiments of the invention that are obvious to those skilled in the art.

[0122] Example 1: Unbiased biochemical confirmation of immunogenic epitopes presented by MHC class II from Bordetella pertussis.

[0123] Methods

[0124] Immunoprecipitation of MHC-II epitopes. Bone marrow was isolated from WT C57BL / 6J mice and the cells were resuspended in dendritic cell (DC) differentiation medium consisting of RPMI1640 supplemented with 10% FBS, gentamicin, β-mercaptoethanol, and 40 ng / μl GM-CSF. Then 10x10 6Cells were seeded in a 10 cm non - tissue culture dish and differentiated for 7 days. Then bone marrow - derived dendritic cells (BMDDC) were transferred to a tissue culture - treated plate and allowed to sit for 24 hours; meanwhile, Bordetella pertussis was cultured overnight. The next day, live H762 or heat - killed BP536 was co - cultured with BMDC at an MOI of 100 for 24 hours. Then BMDC were harvested by scraping and frozen until ready for immunoprecipitation (IP). For MHCII IP, the frozen cell pellet was rapidly thawed and lysed at 5×10 7 cells / ml lysis buffer / sample in a cold IP lysis buffer consisting of 150 mM NaCl, 4 mM MgCl2, 0.25 mM CaCl2, 20 mM Tris pH 8, 1% CHAPS, 6 μg / ml DNaseI from bovine pancreas (Sigma Aldrich), protease inhibitor mixture (Sigma Aldrich), and 1 mM PMSF (RPI). The clarified lysate was gently rotated overnight at 4 °C for immunoprecipitation. Immunoprecipitation was performed using protein G agarose beads (GE Healthcare) conjugated to an αMHC - II antibody (BioXcell, clone M5 / 114). Each sample contained approximately 2 mg of bound antibody. Then the beads were washed three times with sterile cold PBS, followed by washing with sterile cold ultrapure water. Finally, the samples were lyophilized and stored at - 80 until peptide isolation and detection. Peptide elution and detection were performed using liquid chromatography - tandem mass spectrometry (LC - MS / MS) according to a protocol that provides an exhaustive list of peptide epitopes presented by MHCII. Figure 1 Shows the experimental workflow.

[0125] Analysis and refinement of the MHCII epitope library. To refine the list of antigens, the list of in - silico - predicted epitopes was checked for their MHC - II binding affinity using the Immune Epitope Database (IEDB). This online database contains an extensive repository of experimentally validated immune epitopes. The entire peptide library was interrogated using IEDB for binding to mouse and human MHCII alleles, and high - affinity binders (defined as having an adjusted percentile rank of ≤10) were detected.

[0126] Spleen cell stimulation and cytokine ELISA assays. The ability of the confirmed peptides to activate T cells from immunized or convalescent mice was tested. Mice challenged with 5x10 5 CFU of bacteria (BP536 or H762) were used > 35 days after challenge. Two doses of 1x10 8Mice immunized intramuscularly with heat-killed bacteria of CFU and analyzed at least 2 weeks after booster immunization were used as wPV-immunized mice. After dissociation and red blood cell lysis using ACK buffer, the single-cell suspension was plated at 2.5×10 6 cells / well in complete T cell medium (RPMI, 10% FBS, 10 μg / ml gentamicin, 5×10 -5 M β-mercaptoethanol) and stimulated with 1 μg / ml of the selected peptide or with medium alone as a negative control. Supernatants were collected on day 3 after stimulation and IFNγ production was quantified by sandwich ELISA (R&D Cat. DY485-05) according to the manufacturer's instructions. Plates were read at A450 on a SpectraMax microplate reader and concentrations were calculated based on the standard curve.

[0127] 3 H-thymidine incorporation. The ability of the confirmed peptides to induce proliferation of T cells isolated from immunized or convalescent mice was tested. After dissociation and red blood cell lysis using ACK buffer, the single-cell suspension was plated at 4×10 5 cells / well in complete T cell medium (RPMI, 10% FBS, 10 μg / ml gentamicin, 5×10 -5 M β-mercaptoethanol) and stimulated with 10 μg / ml of the selected peptide or with medium alone as a negative control for 3 days. The cells were then pulsed with 3 H-thymidine medium (RPMI 1640 containing 1.25% HEPES buffer and 2 μCi / ml 3 H-thymidine) for 18 h. The cells were then harvested onto a Unifilter plate using a FilterMate harvester (PerkinElmer, Shelton, CT) and stored overnight at room temperature to dry. 30 μl of Microscint20 (PerkinElmer) was added to each well and the plate was sealed. 3 H-thymidine incorporation was read using a TopCount NXT machine (PerkinElmer).

[0128] PBMC collection. Peripheral blood obtained from consenting volunteers was mixed 1:1 with 0.9% saline, loaded onto Ficoll-paque (at 25 °C), and centrifuged at 1500 rpm, 25 °C for 30 minutes in a swinging bucket centrifuge (Eppendorf 5810R) without using the brake. PBMCs at the interface were collected and washed twice with RPMI 1640 + 2% HI-FBS. The cells were pelleted at 1500 rpm, 4 °C for 5 minutes, the supernatant was aspirated, and the cells were resuspended in 90% FBS + 10% DMSO, with 1.0 x 10^6 cells aliquoted per cryovial. The harvested PBMCs were frozen and stored at -80 °C for downstream applications.

[0129] Activation-induced marker (AIM) assay. Frozen PBMC samples were resuspended in T cell medium (RPMI 1640 + 10% AB serum + 10 μg / ml gentamicin + 0.2% β-mercaptoethanol). The cells were pelleted at 1500 rpm, 4 °C for 5 minutes and resuspended in T cell medium for counting. 100 μL of PBMCs per well were added to a sterile U-bottom 96-well plate (Falcon Ref. 353077) (2 x 10 6 cells / well). 100 μL of the following prepared stimulants were added (1 μg / ml selected peptide, phytohemagglutinin (PHA) for positive control, and DMSO for negative control), and mixed by pipetting. The samples were incubated overnight - 18 hours at 37 °C + 5% CO2. The next day, the cells were pelleted and washed 2x with cold sterile 1x PBS. The cells were resuspended in Live / Dead stain (Zombie Nir 1:10,000 dilution) and incubated for 30 minutes at 4 °C in the dark. The cells were washed 2x in PBS and the samples were blocked with 10% FBS PBS at 4 °C for 5 minutes. After blocking, antibodies (Table 1) were added and FMO was prepared using pooled cells for gating control. The samples were incubated at 4 °C for 15 minutes. After labeling, the samples were washed as emphasized above, the cells were resuspended in 1% FBS PBS, and transferred to FAC tubes. The cells were analyzed using a Cytek Aurora spectral flow cytometer. Labeling compensation beads (eBioscience Ultra comp beads) were used for compensation. The data was analyzed using FlowJo and graphs were generated using GraphPad Prism.

[0130] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. Considering the specification and practice of the methods disclosed herein, other embodiments of the present disclosure will be apparent to those skilled in the art. The specification and examples are intended to be considered only exemplary, and the true scope and spirit of the invention are indicated by the following claims.

[0131] Table

[0132] Table 1: Human AIM Assay Antibody Panel

[0133]

[0134] Table 2: Bordetella pertussis Proteins of Interest

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] Sequence

[0145] 1. SEQ ID NO:1 – (Full-length BP0205 Sequence)

[0146] MQLTIRKLAYTLAFSTLVLAGCTTASKKTDGQAATPADQASSQQASAASVEFYVAQAKAGDGLMEVKVPDGSLYMQRQPVLTRADLTEAAALVDRQGQNFVGLRFTEAGARKLNDISSKNIGNMLALVIDRELVAAPRIAEPLNRGVLAFGVPSAKAASEIAAKIRGDAGAPAAGVPAAPAPKPAPK

[0147] 2. SEQ ID NO:2 – (Epitope)

[0148] IDRELVAAPRIAEPL

[0149] 3. SEQ ID NO:3 – (Epitope)

[0150] GVLAFGVPSAKAASEI

[0151] 4. SEQ ID NO:4 – (Signal Peptide)

[0152] MQLTIRKLAYTLAFSTLVLAGC

Claims

1. A pertussis vaccine comprising a combination of the BP0205 antigen and at least one additional peptide from Bordetella pertussis bacteria, wherein the BP0205 peptide and the at least one additional peptide are to be presented to MHC class II cells and recognized by CD4+ T cells.

2. The vaccine according to claim 1, wherein the BP0205 antigen comprises at least 70% sequence identity with SEQ ID NO:

1.

3. The vaccine according to claim 1, wherein the BP0205 antigen comprises at least 80% sequence identity with SEQ ID NO:

1.

4. The vaccine according to claim 1, wherein the BP0205 antigen comprises at least 90% sequence identity with SEQ ID NO:

1.

5. The vaccine according to claim 1, wherein the BP0205 antigen comprises SEQ ID NO:

1.

6. The vaccine according to any one of claims 1-5, wherein the vaccine comprises: an epitope comprising SEQ ID NO:2 or SEQ ID NO:

3.

7. The vaccine according to any one of claims 1-5, wherein the vaccine comprises: epitopes comprising SEQ ID NO:2 and SEQ ID NO:

3.

8. The vaccine according to any one of claims 1-7, wherein the at least one peptide is derived from a protein involved in at least one biological pathway within the Bordetella pertussis bacteria.

9. The vaccine according to any one of claims 1-8, wherein the at least one pathway is selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation, or stress response.

10. The vaccine according to claim 9, wherein the protein selected from the cell adhesion and motility pathway comprises an adhesion and elimination protein, a pilin protein, a flagellar biosynthesis protein (FlhF), or a combination thereof.

11. The vaccine according to claim 9, wherein the protein selected from the cell cycle pathway comprises a protein containing a β-lactamase domain, a cell division coordination protein (CpoB), a cell division protein (ZapD), a peptidyl-prolyl cis-trans isomerase, a peptidyl-prolyl cis-trans isomerase (Cbf2), a phospho-N-acetylmuramoyl-pentapeptide-transferase, a tol-Pal system protein (TolB), a tol-Pal system protein (TolQ), or a combination thereof.

12. The vaccine according to claim 9, wherein the protein selected from the cellular signaling pathway comprises dermonecrotizing toxin, cyclic di-GMP phosphodiesterase response regulator (RpfG), enterobactin outer membrane receptor, or a combination thereof.

13. The vaccine according to claim 9, wherein the protein selected from the cellular structure pathway comprises metal-binding protein, BrkB, D-alanine-D-alanine ligase, inner membrane protein (ybhN), inner membrane protein (YiaH), lipoprotein, lipoteichoic acid synthase 2, outer membrane protein assembly factor (BamA), P.93, penicillin-binding protein 1A, pertussis toxin release protein F, membrane protein, DMT superfamily permease, spermidine synthase having an N-terminal membrane domain, lipoic acid-protein ligase A, HflK, assembly protein, inner membrane protein, HI_0719, soluble lytic murein transglycosylase, virulence-associated outer membrane protein Vir-90, or a combination thereof.

14. The vaccine according to claim 9, wherein the protein selected from the cellular transport pathway comprises Xaa-Pro aminopeptidase, 2-aminoethylphosphonate ABC transporter substrate-binding protein, aerobactin synthase (IucC), antimicrobial resistance protein, arabinose efflux permease, BFD-like [2Fe-2S] binding domain, bicarbonate transport ATP-binding protein (CmpD), bicyclomycin / multidrug efflux system, biotin transporter, BrkA autotransporter, ComEC family competence protein, D-methionine-binding lipoprotein (metQ), drug efflux system protein (MdtG), efflux pump membrane transporter, extracytoplasmic solute receptor protein, extracytoplasmic solute receptor protein (yiaO), ferrichrome receptor (FcuA), filamentous hemagglutinin, hemin-inhibitable hemoglobin-binding protein, hemin import ATP-binding protein (HmuV), inner membrane metabolite transporter (yhjE), inner membrane protein (yhjX), inner membrane transporter (ydhP), iron-sulfur cluster carrier protein, leucine ABC transporter subunit substrate-binding protein (LivK), leucine-, isoleucine-, valine-, threonine-, and alanine-binding protein, LIV-I protein F, LIV-I protein H, major facilitator superfamily protein, manganese transport system membrane protein (mntB), methyl viologen resistance protein (SmvA), multidrug export ATP-binding / permease protein (SAV1866), multidrug resistance protein B, multiple resistance and pH homeostasis protein A, multiple resistance and pH homeostasis protein D, multiple resistance and pH homeostasis protein E, Neu5Ac permease, nickel / cobalt efflux system, BP0840, preprotein translocase subunit (SecD), ABC transporter ATP-binding protein (HI_0664), amino acid metabolite efflux pump, chorismate pyruvate-lyase, membrane transporter, protein translocase subunit (SecD), purine efflux pump (PbuE), putrescine transport system permease protein (PotH), RTX-I toxin determinant B, sec-independent protein translocase protein (TatC), short-chain fatty acid transporter, sn-glycerol-3-phosphate transport system permease protein (ugpA), spermidine / putrescine transport system permease protein (PotB), TonB family C-terminal domain, TpsB transporter, transport permease protein, TRAP-C4 type-dicarboxylate transport system, small permease component, TRAP type uncharacterized transport system, fusion permease component, tricarboxylate transport protein (TctA) or a combination thereof.

15. The vaccine according to claim 9, wherein the protein selected from the gene regulatory pathway comprises type IV secretion system protein virB11, 30S ribosomal protein S10, 50S ribosomal protein L10, 50S ribosomal protein L14, 50S ribosomal protein L25, 50S ribosomal protein L31 B type, ATP-dependent DNA helicase Rep, ATP-dependent RNA helicase (HrpA), bacterial DNA-binding protein, BpH3, cyn operon transcriptional activator, DNA polymerase III subunit γ / τ, DNA protection protein 2 during starvation, DNA repair protein (RecN), galactose-binding protein regulator, Gcv operon activator, HTH-type transcriptional regulator (gabR), leucine-responsive regulator, LigA, polynucleotide nucleotide transferase, protein (ApaG), ribonuclease (TTHA0252), ribosomal RNA small subunit methyltransferase B, ribosome-binding factor A, ribosome recycling factor, single-stranded DNA-binding protein, transcriptional regulator, y4mF family, transcription-repair coupling factor or a combination thereof.

16. The vaccine according to claim 9, wherein the proteins selected from the metabolic pathways include ACR, COG1565, 1,4-α-glucan branching enzyme (GlgB), 1,4-dihydroxy-2-naphthoyl-CoA synthase, 2-(hydroxymethyl)glutamate dehydrogenase, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylic acid N-acetyltransferase, 3-(3-hydroxyphenyl)propionate / 3-hydroxycinnamate hydroxylase, 3-hydroxyacyl-CoA dehydrogenase, 3-oxoadipate enol-lactonase 2, 3-oxosteroid 1-dehydrogenase, 4-aminobutyrate aminotransferase PuuE, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin), 5-formyltetrahydrofolate cyclo-ligase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, 6-aminocaproate-cyclic dimer hydrolase aldehyde dehydrogenase 2, acetolactate synthase isozyme 3 large subunit, acetone carboxylase α subunit, acetone carboxylase β subunit, acetyl-CoA acetyltransferase, acetyltransferase component of pyruvate dehydrogenase complex, acyl carrier protein, acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase, acylating enzyme (ACY 1), acyl-CoA dehydrogenase (AidB), short-chain specific acyl-CoA dehydrogenase, S-adenosylhomocysteine hydrolase, aldehyde dehydrogenase, thermostable aliphatic sulfonate transport permease protein (ssuC), amidophosphoribosyltransferase, aminotransferase, arginine transport ATP-binding protein (ArtM), argininosuccinate lyase, aspartate 1-decarboxylase, aspartate acylase, aspartate kinase, ATP synthase subunit β, ATP-dependent desulfobiotin synthetase (BioD), β-ketoacyl-acyl-carrier-protein synthase I, biotin carboxyl carrier protein of acetyl-CoA carboxylase, biotin carboxylase, carbamoyl-phosphate synthase large chain, carbamoyl-phosphate synthase small chain, cytochrome c, cytochrome c oxidase subunit 2, D-amino acid dehydrogenase, dihydrolipoamide dehydrogenase, dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, dihydropteroate synthase, D-malate degradation protein R, D-malate dehydrogenase (decarboxylating), export protein, ferredoxin-dependent glutamate synthase 1, Flp pilus assembly protein (TadD), formate-dependent phosphoribosylglycinamide formyltransferase, formyl-CoA transferase, glutamate dehydrogenase, glutamine-fructose-6-phosphate aminotransferase, glyceraldehyde-3-phosphate dehydrogenase, glycine dehydrogenase, guanidinobutyrase, imidazoleglycerol-phosphate dehydrase, iron-sulfur cluster assembly scaffold protein (IscU), L-aspartate dehydrogenase, long-chain fatty acid-CoA ligase, malate dehydrogenase, maltose α-D-glucosyltransferase, MmgE / PrpD family, N5-carboxyaminoimidazole ribonucleotide synthase, 6-phosphogluconate dehydrogenase NAD +A binding domain, nicotinamidase / pyrazinamidase, nucleoside recognition, oxoglutarate dehydrogenase, phosphoserine aminotransferase, an α / β - hydrolase fold hydrolase, a heme IX biogenesis protein, soluble aldehyde dehydrogenase (yliI), sporulation inhibitor (kipI), stringent starvation protein A, succinate dehydrogenase flavoprotein subunit, succinate - semialdehyde dehydrogenase [NADP(+)] (GabD), thiamine biosynthesis protein (HI_0357), thiosulfate sulfurtransferase, threonine synthase, thymidylate synthase, UDP - glucose 6 - dehydrogenase, Vi polysaccharide biosynthesis protein (TviD), xanthine dehydrogenase accessory protein (XdhC), or a combination thereof.

17. The vaccine according to claim 9, wherein the protein selected from the oxidation - reduction pathway comprises 3 - α - (or 20 - β) - hydroxysteroid dehydrogenase, 3 - ketosteroid - 9 - α - hydroxylase reductase subunit, 3 - oxoacyl - [acyl - carrier - protein] reductase (FabG), acetoin: 2,6 - dichlorophenol indophenol oxidoreductase subunit β, alkyl hydroperoxide reductase C, benzaldehyde dehydrogenase [NAD(+)], benzene 1,2 - dioxygenase subunit α, catalase, dTDP - 4 - dehydro - rhamnose reductase, γ - glutamylputrescine oxidoreductase, gluconate 2 - dehydrogenase cytochrome c subunit, glutathione hydrolase proenzyme, glutathione import ATP - binding protein (GsiA), glutathione transport system permease protein (gsiD), glutathione - binding protein (gsiB), glyoxylate / hydroxypyruvate reductase A, glyoxylate / hydroxypyruvate reductase B, heme A synthase, L - 2 - hydroxyglutarate oxidase (LhgO), methylmalonate - semialdehyde dehydrogenase, Mg - chelatase subunit child, muconolactone δ - isomerase, NAD(P)H - hydrate epimerase, NADH - quinone oxidoreductase, NADH - quinone oxidoreductase subunit L, NADH - quinone oxidoreductase subunit M, NADH - quinone oxidoreductase subunit N, aldehyde - ketone reductase, NAD(P)H quinone oxidoreductase, PIG3, quinone oxidoreductase 1, rhodomycin reductase, sarcosine oxidase subunit β, sulfite reductase, thiol: disulfide exchange protein (DsbD), thioredoxin reductase, pantothenol oxidase subunit 1, or a combination thereof.

18. The vaccine according to claim 9, wherein the proteins selected from the protein regulation pathways include apolipoprotein N-acyltransferase, ATP-dependent Clp protease ATP-binding subunit (ClpX), capsular polysaccharide biosynthesis protein, carboxypeptidase G2, chaperonin GroEL, chorismate synthase, co-chaperonin GroES, dihydroxy-acid dehydratase, FtsH protease regulator (HflK), small chain glutamate synthase, glutamine synthetase, ion protease, membrane dipeptidase M19, membrane protein insertase (YidC), methionine-tRNA ligase, molybdopterin molybdenum transferase, oligopeptidase A, periplasmic serine endoprotease DegP-like, C-terminal PDZ domain protease, HtpX, protein-L-is aspartate O-methyltransferase, glutamate-cysteine ligase 2, tRNA pseudouridine synthase A, tryptophan-tRNA ligase, tyrosine-tRNA ligase, or a combination thereof.

19. The vaccine according to claim 9, wherein the proteins selected from the stress response pathways include Rv1996 / MT2052, SAV1710, or a combination thereof.

20. The vaccine according to any one of claims 1-19, wherein the pertussis vaccine further comprises any combination of an adjuvant, a preservative, or a stabilizer.

21. The vaccine according to any one of claims 1-20, wherein the vaccine comprises a pharmaceutically acceptable carrier selected from excipients, diluents, salts, buffers, stabilizers, lipids, emulsions, or nanoparticles.

22. A method for preventing a subject from being infected with Bordetella pertussis bacteria, the method comprising administering to the subject a pharmaceutically effective amount of the pertussis vaccine according to any one of claims 1-21.

23. A method for improving the immune response of a subject against Bordetella pertussis bacteria, the method comprising administering to the subject a pharmaceutically effective amount of the pertussis vaccine according to any one of claims 1-21.

24. The method according to claim 22 or 23, wherein infection with the Bordetella pertussis bacteria causes the subject to develop whooping cough.

25. The method according to any one of claims 22-24, wherein the immune response comprises presenting the antigen to MHC class II cells, the antigen being recognized by CD4+ T cells, or a combination thereof.

26. The method according to any one of claims 22-25, wherein the immune response is against the BP0205 antigen and at least one protein involved in at least one biological pathway within the Bordetella pertussis bacteria.

27. The method according to claim 26, wherein the at least one pathway is selected from cell adhesion and motility, cell cycle, cell signaling, cell structure, cell transport, gene regulation, metabolism, oxidation-reduction, protein regulation or stress response.

28. The method according to any one of claims 22-27, wherein the method further comprises administering the pertussis vaccine by injection via an intranasal, intramuscular, intraperitoneal or subcutaneous route.

29. The method according to any one of claims 22-28, wherein the subject is a mammal.

30. The method according to any one of claims 22-29, wherein the subject is a human.

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