Vaccines and methods for the treatment of haemophilus influenzae caused diseases

Abstract

Disclosed are novel vaccine compositions comprising modulated Transferrin Binding Protein B (TbpB) polypeptides or immunogenic portions thereof. Notably, the TbpB polypeptides are modulated with respect to domains known as loop domains located in the C-terminal lobe of TbpB polypeptides. The vaccine compositions may be used to ameliorate, treat or prevent pathogenic infections in humans caused by pathogenic H. influenzae strains. Related methods and uses are also disclosed.

Description

TITLE: VACCINES AND METHODS FOR THE TREATMENT OF HAEMOPHILUS INFLUENZAE CAUSED DISEASESRELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Application No. 63 / 699,545 filed September 26, 2024; the entire contents of Patent Application No. 63 / 699,545 are hereby incorporated by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “96573182 Sequence Listing. xml” (51 ,280 bytes), filed herewith by electronic submission and created on September 22, 2025, is herein incorporated by reference.FIELD OF THE DISCLOSURE

[0003] The methods and compositions disclosed herein relate to the treatment of infectious diseases. In particular, the methods and compositions disclosed herein relate to methods and vaccines to prevent, treat, or ameliorate infections in humans caused by pathogenic strains of the microbial species Haemophilus influenzae.BACKGROUND OF THE DISCLOSURE

[0004] The following paragraphs are provided by way of background to the present disclosure. They are not however an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art.

[0005] Bacteria of the species Haemophilus influenzae (H. influenzae) can cause a variety of localized and invasive infections in humans, ranging from mild infections, such as ear infections, to more serious infections, such as blood stream infections. Some H. influenzae infections are considered invasive since the bacteria can invade parts of the body that are normally free from microbial germs. Upon invasion, H. influenzae can cause serious illnesses. For example, H. influenzae can invade the fluid around the spine and brain and cause meningitis, and H. influenzae can invade the bloodstream and cause bacteremia. The most common types of diseases transmitted by H. influenzae, both in adults andchildren, include pneumonia, meningitis, bronchitis, epiglottitis (swelling of the throat), cellulitis, infectious arthritis, and septicemia (bloodstream infection).

[0006] H. influenzae strains are commonly classified depending on the capsule type they possess. The term “capsule” in this respect refers to a protective polysaccharide layer that is located outside the bacterial cellular envelope. Commonly distinguished are capsulated H. influenzae strains (also known as “typable strains”) and non-capsulated H. influenzae strains (also known as “non- typable strains”, or“NTHi strains”). Moreover, typable H. influenzae strains can be further defined depending on the chemically distinct capsular polysaccharide antigens they possess. In this respect, the six generally recognized capsular H. influenzae strain types are known as “serotype a” through “serotype f” strains (also designated “Hia” through “Hif”). Capsulated H. influenzae strains can be identified serologically using anti-capsular serotype specific sera. By contrast, NTHi strains fail to react with antisera against capsular serotypes.

[0007] Among the H. influenzae strains, H. influenzae serotype b (Hib) is most commonly identified as the causative agent in invasive disease. In particular, prior to the introduction of infant Hib vaccination in the 1980s, H. influenzae serotype b was responsible for almost all H. influenzae related morbidity and mortality.

[0008] Treatment options for H. influenzae infections include antibiotics. However, H. influenzae strains resistant to some antibiotics, notably ampicillin, have started to emerge, and treatment failure due to antibiotic resistance is becoming a concern.

[0009] Current vaccines available for infections in humans caused by pathogenic H. influenzae species are exclusively efficacious against Hib. But as the incidence of Hib caused disease has decreased, the prevalence of disease caused by H. influenzae strains of the other capsular types, and non-capsular strains has increased. There currently are no vaccines against disease caused by non-Hib strains and non-typable strains. Furthermore, infection by H. influenzae serotype b continues to be a public health concern in countries that have not implemented an Hib vaccination program.

[0010] In light of the foregoing, there exists a need in the art for improved methods and compositions to, prevent, treat, or ameliorate infectious diseases in humans caused by H. influenzae.SUMMARY OF THE DISCLOSURE

[0011] The following paragraphs are intended to introduce the reader to the more detailed description that follows, not to define or limit the claimed subject matter of the present disclosure.

[0012] In one aspect, the present disclosure relates to vaccine formulations.

[0013] In another aspect, the present disclosure relates to vaccine formulations to prevent or ameliorate diseases in humans that are caused by pathogenic Haemophilus influenzae.

[0014] In another aspect, the present disclosure relates to a class of proteins known as Transferrin Binding Protein B (TbpB) proteins. In particular, the present inventors have discovered that certain modulated forms of naturally occurring TbpB proteins can be used to formulate surprisingly potent vaccine formulations.

[0015] Accordingly, the present disclosure provides, in accordance with the teachings herein, in at least one aspect, in at least one embodiment, a method for prevention, treatment, or amelioration of Haemophilus influenzae (H. influenzae) infection in a human, the method comprising administering to a human in need thereof, a vaccine formulation comprising (i) a TbpB a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, wherein the vaccine formulation is administered to the human in an effective amount to prevent, treat, or ameliorate the infection by the H. influenzae.

[0016] In at least one embodiment, in an aspect, the modulated C-lobe domain can further comprise:(iii) a third joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 26, SEQ.ID NO: 28, or SEQ.ID NO: 30, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 32, SEQ.ID NO: 34, or SEQ.ID NO: 36, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 7 amino acid residues.

[0017] In at least embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0018] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be a typable or a non-typable H. influenzae strain.

[0019] In at least one embodiment, in an aspect, the typable H. influenzae strain can be a serotype a strain, a serotype b strain, a serotype c strain, a serotype d strain, a serotype e strain, or a serotype f strain.

[0020] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be capable of causing pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia in a human.

[0021] In at least one embodiment, in an aspect, the treatment or amelioration of the H. influenzae infection can comprise the reduction of clinical signs of any disease caused by the H. Influenzae infection.

[0022] In at least one embodiment, in an aspect, the clinical signs can be clinical signs associated with a disease selected from the group consisting of pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia relative to a human infected by H. influenzae not having been administered the vaccine formulation.

[0023] In at least one embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be a recombinantly produced polypeptide.

[0024] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by a second H. influenzae strain.

[0025] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a H036 H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by one or more H. influenzae strains selected from the group consisting of a HO H. influenzae strain, a H026 H. influenzae strain, a H040 H. influenzae strain, a H210 H. influenzae strain, a H036 H. influenzae strain, a WP050 H. influenzae strain, a WP11880 H. influenzae strain, a PRI75995 H. influenzae strain, a WP111 H. influenzae strain, a WP11887 H. influenzae strain, a PRI69814 H. influenzae strain, a WP221 H. influenzae strain, and a WP112 H. influenzae strain.

[0026] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable adjuvant.

[0027] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable excipient, carrier, or diluent.

[0028] In at least one embodiment, in an aspect, the vaccine formulation can comprise from about 0.001 % to about 20% by weight per volume of the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain, and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

[0029] In another aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, a vaccine formulation for the prevention, treatment, or amelioration of an H. influenzae infection in a human, the vaccine formulation comprising an effective amount of (i) a TbpB protein, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, orSEQ.ID NO: 24, ora C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptideare contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues.

[0030] In at least one embodiment, in an aspect, the modulated C-lobe domain, can further comprise:(iii) a third joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 26, SEQ.ID NO: 28, or SEQ.ID NO: 30, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 32, SEQ.ID NO: 34, or SEQ.ID NO: 36, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 7 amino acid residues.

[0031] In at least embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0032] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be a typable or non-typable H. influenzae strain.

[0033] In at least one embodiment, in an aspect, the typable H. influenzae strain can be a serotype a strain, a serotype b strain, a serotype c strain, a serotype d strain, a serotype e strain, or a serotype f strain.

[0034] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be capable of causing pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia in a human.

[0035] In at least one embodiment, in an aspect, the administration of the vaccine formulation to a human in need thereof can prevent clinical signs or result in a reduction of clinical signs of any disease caused by the H. influenzae infection.

[0036] In at least one embodiment, in an aspect, the clinical signs can be clinical signs associated with a disease selected from the group consisting ofpneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia, relative to a human infected by H. influenzae not having been administered the vaccine formulation.

[0037] In at least one embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be a recombinantly produced polypeptide.

[0038] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by a second H. influenzae strain.

[0039] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a H036 H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by one or more H. influenzae strains selected from the group consisting of a HO H. influenzae strain, a H026 H. influenzae strain, a H040 H. influenzae strain, a H210 H. influenzae strain, a H036 H. influenzae strain, a WP050 H. influenzae strain, a WP11880 H. influenzae strain, a PRI75995 H. influenzae strain, a WP111 H. influenzae strain, a WP11887 H. influenzae strain, a PRI69814 H. influenzae strain, a WP221 H. influenzae strain, and a WP112 H. influenzae strain.

[0040] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable adjuvant.

[0041] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable excipient, carrier, or diluent.

[0042] In at least one embodiment, in an aspect, the vaccine formulation can comprise from about 0.001 % to about 20% by weight per volume of the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

[0043] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a use of a vaccine formulation for the prevention, treatment, or amelioration of H. influenzae infectionin a human in need thereof, the vaccine formulation comprising an effective amount of (i) a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and wherein the vaccine formulation is administered in an effective amount to prevent, treat, or ameliorate the H. influenzae infection.

[0044] In at least one embodiment, in an aspect, use of the vaccine formulation in a human in need thereof can prevent clinical signs or results in a reduction of clinical signs of any disease caused by the H. influenzae infection.

[0045] In at least one embodiment, in an aspect, the clinical signs can be clinical signs associated with a disease selected from the group consisting of pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia, relative to a human infected by H. influenzae not having been administered the vaccine formulation.

[0046] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a use of a vaccine formulation in the manufacture of a medicament for the prevention, treatment, or amelioration of H. influenzae infection in a human in need thereof, the vaccine formulation comprising an effective amount of (i) a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loopflanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and wherein the vaccine formulation is administered in an effective amount to prevent, treat, or ameliorate the H. influenzae infection.

[0047] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a vaccine formulation for use in the prevention, treatment or amelioration of H. influenzae infection in a human, the vaccine formulation comprising an effective amount of comprising (i) a TbpB a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues.

[0048] In another aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, a method for preparing a vaccine formulation for administration to a human to prevent, treat, or ameliorate an H. influenzae infection, the vaccine formulation comprising (i) a TbpB protein, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, orSEQ.ID NO: 24, ora C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the TbpB protein, or immunogenic portion thereof comprising the modulated C-lobe domain or; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the TbpB protein or immunogenic portion thereof in a host cell;(B) introducing the chimeric nucleic acid sequence into the host cell;(C) growing the host cell to produce the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain, or an mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain; and(D) recovering (i) the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain or (ii) the mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain; and(E) formulating (i) the recovered TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain together or (ii) the recovered mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of (i) the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain, to prevent, treat, or ameliorate an H. influenzae in a human.

[0049] In at least embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0050] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, an expression vector comprising:(a) a nucleic acid sequence encoding (i) a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or animmunogenic portion thereof, comprising a modulated C-lobe domain the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues,(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain, or (ii) the mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

[0051] In at least embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0052] In another aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, a host cell comprising a chimeric nucleic acid comprising:(a) a nucleic acid sequence encoding (i) a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the TbpB polypeptide, or immunogenicportion thereof, comprising the modulated C-lobe domain, or (ii) the mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

[0053] In at least embodiment, in an aspect, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0054] Other features and advantages will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect. The figures are not intended to limit the present disclosure.

[0056] FIGS. 1A, 1B (I) and 1B (ii) are schematic drawings illustrating certain secondary structural features of an example TbpB polypeptide, namely a TbpB polypeptide found in H. influenzae strain H036. Indicated are a N-lobe domain (“N-lobe”); a C-lobe domain (“C-lobe”); a lobe-linking peptide (“Linker peptide”); within the N-lobe and C-lobe domain, an N-lobe and C-lobe p-barrel structure (“N-lobe p-barrel domain”; “C-lobe p-barrel domain”) and an N-lobe and C-lobe p-sheet (“N-lobe “handle” p-sheet domain”; “C-lobe “handle” p-sheet domain”) structure; and within the C-lobe domain, loop domains 20, 23 and 31 (“Loop 20”; “Loop 23”; “Loop 31”) (FIG. 1A). FIGS. 1 B (I) and 1B (ii) show the polypeptide sequence of the same example TbpB polypeptide, / .e., a TbpB polypeptide found in H. influenzae strain H036. Indicated are an N-lobe domain (FIG. 1 B (i)), a C -lobe domain (FIG. 1B (ii)), a lobe-linking peptide (“Loop 15 / 16(Linker peptide)”), and loop domains 1 - 32. It is noted that the sequence shown in FIGS. 1B (i) and 1B (ii) corresponds with SEQ.ID NO: 42 set forth herein.

[0057] FIGS. 2A, 2B, and 2C are schematic drawings illustrating an example polypeptide portion of a non-modified reference TbpB polypeptide (10) comprising an example loop domain (FIG. 2A); separation of the same loop domain from the polypeptide portion (FIG. 2B); and the corresponding polypeptide portion of an example modulated TbpB polypeptide (20), lacking the same loop domain (FIG. 2C).

[0058] FIG. 3 shows a polypeptide sequence alignment of example reference TbpB polypeptides obtained from H. influenzae strain H036 (“HiO36TbpB”, corresponding with SEQ.ID NO: 42), H. influenzae strain H040 (“Hi040TbpB”, corresponding with SEQ.ID NO: 44), and H. influenzae strain H201 (“Hi201TbpB”, corresponding with SEQ.ID NO: 46). Indicated with the polypeptide sequences are loop domains 20, 23, and 31 (“Loop 20”; “Loop 23”; “Loop 31”).

[0059] FIG. 4 depicts a graph representing certain experimental results, notably a bar graph obtained in the performance of experimental assays, notably enzyme-linked immunosorbent (ELISA) assays, to evaluate the in vivo efficacy of a vaccine formulation comprising an example modulated TbpB polypeptide, notably an ELISA assay to quantitatively evaluate anti-TbpB end-point titres (vertical axis) elicited by vaccine formulations comprising (i) H036 wildtype TbpB (“h036 WT TbpB”); (ii) a trivalent vaccine formulation comprising h036, h040, and h201 wild-type TbpBs (“Trivalent”); (iii) an example modulated TbpB polypeptide (h036 LCL; SEQ.ID NO: 40) (“H036 LCL”); or (iv) an adjuvant only control (“Adjuvant only”).

[0060] FIGS. 5A, 5B, and 5C depict various graphs representing certain experimental results, notably bar graphs obtained in the performance of experimental assays, notably enzyme-linked immunosorbent (ELISA) assays to evaluate the in vivo efficacy of a vaccine formulation comprising an example modulated TbpB polypeptide, notably ELISA assays to quantitatively evaluate cross-reactivity by normalized OD450 (vertical axis) between antisera elicited by a vaccine formulation comprising an example modulated TbpB polypeptide (h036 LCL; SEQ.ID NO: 40) (“h036 LCL”) (FIG. 5A) and a trivalent vaccine formulation comprising h036, h040, and h201 wild-type TbpBs (“Trivalent Vaccine”) (FIG. 5B) and a panel of 15 immobilized wild-type H. influenzae TbpB polypeptides(designated as “h014”; “h026”; “h036”; “h040”; “h201”; “h216”; “WP_050”; “WP-11880”; “PRI757995”; “WP_111”; “WP_11887”; “PRI6984”; “WP_221”; and “WP_112”), and a mock lysate control (“Mock lysate”), each identified on the horizontal axis of the bar graphs. In the graph of FIG. 5C, the results shown in FIGS. 5A and 5B are combined to facilitate a more direct comparison. In each of FIGS. 5A, 5B, and 5C, the TbpB polypeptides used to coat ELISA plates that match those included in the vaccine formulation are highlighted by arrows.

[0061] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice.DETAILED DESCRIPTION

[0062] Various compositions, methods, or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or methods that differ from those described below. The claimed subject matter is not limited to compositions, processes or methods having all of the features of any one composition, method or process described below or to features common to multiple or all of the compositions, methods or processes described below. It is possible that a composition, method, or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, method or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) orowner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[0063] As used herein and in the claims, the singular forms, such “a”, “an” and “the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or moreother non-stated integers or groups of integers. The term “or” is inclusive unless modified, for example, by “either”.

[0064] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1 % and 15% of the stated number or numerical range, as will be readily recognized by context. Furthermore, any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed (e.g., a range of 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). Similarly, otherterms of degree such as "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.

[0065] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0066] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.Terms and definitions

[0067] The terms “nucleic acid”, or “nucleic acid sequence”, as used herein, refer to a sequence of nucleoside or nucleotide monomers, consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acids of the present disclosure may be deoxyribonucleic nucleic acids (DNA) or ribonucleic acids (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The nucleic acids may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. A sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, nucleic acid sequence, a nucleotide sequence, or a nucleoside sequence.

[0068] The terms “polypeptide” and “protein”, as may be used interchangeably herein, in conjunction with a reference SEQ.ID NO, refer to any and all polypeptides and proteins comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequence constituting the polypeptide having such reference SEQ.ID NO, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the polypeptide having such reference SEQ.ID NO, but for the use of synonymous codons. A sequence of amino acid residues may be referred to as an amino acid sequence, or polypeptide sequence.

[0069] The terms “nucleic acid sequence encoding a polypeptide” and “nucleic acid sequence encoding a protein”, as used herein in conjunction with a reference SEQ.ID NO, refer to any and all nucleic acid sequences encoding a polypeptide or protein having such reference SEQ.ID NO. Nucleic acid sequences encoding a polypeptide, in conjunction with a reference SEQ.ID NO, further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the polypeptide having such reference SEQ.ID NO; or (ii) hybridize to any nucleic acid sequences encoding polypeptides having such reference SEQ.ID NO under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[0070] The terms “TbpB polypeptide” or “TbpB protein”, or “TbpB”, as may be used herein, interchangeably refer to any and all protein comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any TbpB polypeptide set forth herein, including, for example, SEQ.ID NO: 42, SEQ.ID NO: 44, and SEQ.ID NO: 46, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any TbpB polypeptide set forth herein, but for the use of synonymous codons. TbpB polypeptides generally contain an N-terminal portion referred to as the N-lobe domain and a C-terminal portion referred to as the C-lobe domain, separated and connected by a lobelinking peptide. Furthermore, the term “TbpB polypeptides” can include TbpB polypeptides and modulated TbpB polypeptides, including TbpB polypeptides comprising a modulated C-lobe domain.

[0071] The terms “modulated TbpB polypeptide” or “modulated TbpB protein”, or “modulated TbpB”, as may be used herein, generally refer to a TbpB polypeptide of which the polypeptide sequence has been altered relative to a reference or native TbpB polypeptide. In this respect, reference TbpB polypeptides include any naturally occurring TbpB polypeptides, and further include TbpB polypeptides comprising SEQ.ID NO: 42, SEQ.ID NO: 44, and SEQ.ID NO: 46, or any TbpB polypeptides substantially identical thereto. Modulated TbpB polypeptides include TbpB polypeptides comprising a modulated C-lobe domain, and modulated TbpB polypeptides include polypeptides having SEQ.ID NO: 38 and SEQ.ID NO: 40.

[0072] The terms “nucleic acid sequence encoding a TbpB”, and “nucleic acid sequence encoding a TbpB polypeptide”, and “nucleic acid sequence encoding a TbpB protein”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a TbpB polypeptide, including, for example, SEQ.ID NO: 41 , SEQ.ID NO: 43, and SEQ.ID NO: 45. Nucleic acid sequences encoding a TbpB polypeptide may be naturally occurring in H. influenzae bacterial strains, and isolated from H. influenzae. Nucleic acid sequences encoding a TbpB polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the TbpB polypeptide sequences set forth herein; or (ii) hybridize to any TbpB nucleic acid sequences set forth herein under at least moderately stringent hybridizationconditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons. Furthermore, the term “nucleic acid sequences encoding a TbpB polypeptide” can include nucleic acid sequences encoding modulated TbpB polypeptides including nucleic acid sequences encoding TbpB polypeptides comprising a modulated C-lobe domain.

[0073] The term “C-lobe domain”, as used herein, refers generally to the C- terminal portion of a TbpB polypeptide, comprising a plurality of p-strands connected by a plurality of loop domains, wherein some of the p-strands can be configured to together form a p-barrel and an adjacent p-sheet structure termed a handle domain. For example, referring to FIG. 1A and FIGS. 1B (I) and 1B (ii) , the C-lobe domain is the contiguous polypeptide domain located C-terminal of the lobe-linking peptide separating the N-lobe domain and C-lobe domain. It is noted that the term C-lobe domain, as used herein, is intended to include, the C-lobe p- barrel domain, as well as the C-lobe handle domain forming a p-sheet structure, comprised typically of approximately 90 or more amino acid residues, and located N-terminally relative to the C-lobe p-barrel structure.

[0074] The term “modulated C-lobe domain”, as used herein refers to a C- lobe domain which has been modified relative to a reference or native C-lobe domain, and includes C-lobe domains in which one or more loop domains have been altered so that an N-terminal loop flanking polypeptide, typically including at least some p-strand structure, is contiguously joined to a C-terminal loop flanking polypeptide, typically including at least some p-strand structure, or joined by a short linker peptide, e.g., 10 amino acids or less in length. Modulated C-lobe domains further include, for example, C-lobe domains comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loopflanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues.

[0075] The terms “loop domain” and “loop”, as used herein, refer to the TbpB polypeptide sequences within a TbpB protein that connect two p-strands. Loop polypeptide sequences may vary considerably in length from several amino acid residues to 150 or more amino acid residues. To distinguish loop domains from one another, loop domains are consecutively numbered from the N-terminal end of a TbpB polypeptide to the C-terminal end of a TbpB polypeptide. Thus, for example “loop 1” refers to the loop domain located closest to the N-terminal end of the TbpB polypeptide, “loop 2” refers to the loop domain located 2ndclosest to the N-terminal end of the TbpB polypeptide, and so forth. Thus, for example, indicated in FIGS. 1B (i) and 1B (ii) are loop domains 1 - 31 of an example H. influenzae TbpB polypeptide, namely a TbpB polypeptide found in H. influenzae strain h036 (SEQ.ID NO: 42). TbpB polypeptides may be aligned with, for example, SEQ.ID NO: 42 to identify corresponding loop domains therein, or, alternatively or additionally, a protein secondary structure-prediction program, such as PyMOL Molecular Graphics System (www.pymol.org), for example, may be used to identify corresponding loop domains in TbpB polypeptides.

[0076] The term “N-lobe domain”, as used herein, refers generally to the N- terminal portion of a TbpB polypeptide, comprising a plurality of p-strands connected by a plurality of loop domains, wherein some of the p-strands can be configured to together form a p-barrel and an adjacent p-sheet structure termed a handle domain. For example, referring to FIG. 1A and FIGS. 1B (I) and 1B (ii), theN-lobe domain is the contiguous polypeptide domain N-terminal of the lobe-linking peptide separating the N-lobe domain and C-lobe domain. It is noted that the term N-lobe domain, as used herein, is intended to include, the N-lobe p-barrel domain, as well as the N-lobe handle domain forming a p-sheet structure.

[0077] By the term “substantially identical” it is meant that two amino acid sequences preferably are at least 70% identical, and more preferably are at least 85% or 90% identical, and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. In order to determine the percentage of identity between two amino acid sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981 , 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1 , so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

[0078] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilledin the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (LoglO [Na+])+0.41 (% (G+C)-600 / l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 ° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5xDenhardt's solution / 1.0% SDS at Tm (based on the above equation) -5° C, followed by a wash of 0.2xSSC / 0.1 % SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3xSSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts, and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

[0079] The term “chimeric”, as used herein in the context of nucleic acids, refers to at least two linked nucleic acids which are not naturally linked. Chimeric nucleic acids include linked nucleic acids of different natural origins. For example, a nucleic acid constituting a microbial promoter linked to a nucleic acid encoding a plant polypeptide is considered chimeric. Chimeric nucleic acids also may comprise nucleic acids of the same natural origin, provided they are not naturally linked. For example, a nucleic acid constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid encoding a polypeptide obtained from that same cell-type, but not normally linked to the nucleic acid constituting the promoter. Chimeric nucleic acids also include nucleic acids comprising any naturally occurring nucleic acids linked to any non-naturally occurring nucleic acids.

[0080] The terms “Haemophilus influenzae” or “H. influenzae”, as interchangeably used herein, refer to any bacteria belonging to the bacterial species taxonomically classified as such and include any subspecies thereof, and further include any H. influenzae strains, including any typable and non-typable strains, variants, serogroups, serovars, serotypes (including serotype a through serotype f), or genotypes. It is noted that H. influenzae strains may be referred to by serotype. Thus, for example, a strain referred to as H. influenzae serotype b or Hib denotes a H. influenzae strain serotypable as serotype b. Non-serotypable H. influenzae strains may be referred to as NTHi strains.

[0081] The term “effective amount”, as used herein, refers to an amount of an active agent or pharmaceutical formulation, including a human vaccine formulation, sufficient to induce a desired biological or therapeutic effect, including a prophylactic effect. Such effect can include an effect with respect to the signs, symptoms or causes of a disorder, or disease or any other desired alteration of a biological system. The effective amount can vary depending, for example, on the health condition, injury stage, disorder stage, or disease stage, of the human being treated, timing of the administration, manner of the administration, age of the human, size of the human, and the like, all of which can be determined by those of skill in the art.

[0082] The terms “immunological portion” or “immunogenic portion”, as used herein, refers to a portion of molecule, notably a portion of a protein, that is capable of eliciting a humoral immune response in the form of the production of native polyclonal antibodies in a subject human when administered thereto. The binding specificity to the native polyclonal antibodies may be comparable to the specificity of native polyclonal antibodies produced when the entire molecule, such as a full length protein, is administered to the subject human. For example, immunologically equivalent portions or immunogenic portions of a TbpB polypeptide include a portion of a TbpB polypeptide lacking the N-lobe domain or a portion thereof, and comprising or consisting substantially of the C-lobe domain of a TbpB polypeptide. To compare binding specificity between an entire molecule, e.g., a full length protein, and a portion thereof, e.g., a portion of a protein, a radioimmune assay (RIA) may be used, and the extent of binding may be measured. The dissociation constant of the portion of the molecule is preferably atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dissociation constant of the entire molecule.

[0083] The term “cross-protective”, as used herein, refers to a vaccine formulation capable of providing protection against infection by multiple strains (e.g., 2, 3, 4, 5, or more strains) of a pathogenic microbial organism, for example, strains belonging to multiple serotypes, or belonging to a serotypable strain, and a non-serotypable strain. A cross-protective vaccine formulation may comprise multiple antigenic substances, or a single antigenic substance, for example, multiple immunogenic polypeptides or a single immunogenic polypeptide. To evaluate cross-protection, a vaccine formulation comprising an immunogenic substance obtained from a selected strain of a microbial organism, a TbpB protein or modulated TbpB protein, for example, a TbpB protein comprising a modulated C-lobe domain, may be used to immunize a subject human (or test animal). The subject human (or test animal) then may be exposed to another pathogenic strain of a pathogenic microbial organism, and the immunological response and the development of disease symptoms of the human (or test animal) may be evaluated. In the event the infection results in the development of an improved immune response, or less severe or no disease symptoms in the infected human (or test animal than in an unvaccinated human (or test animal) exposed to the same infection, the vaccine formulation can be said to be cross-protective.

[0084] The terms “vaccine”, “vaccine formulation”, “human vaccine”, and “human vaccine formulation”, as used herein, refer to a pharmaceutically acceptable preparation that may be administered to a human (or test animal) to induce a humoral immune response (including eliciting a soluble antibody response) and / or cell-mediated immune response (including eliciting a cytotoxic T lymphocyte (CTL) response).

[0085] The term “pharmaceutically acceptable”, as used herein, refers to materials, including carriers, diluents, or auxiliary agent that are compatible with other materials in a pharmaceutical formulation, including a vaccine formulation, and within the scope of reasonable medical judgement suitable for use in contact with humans without excessive toxicity, allergic response, irritation, or other adverse response commensurate with a reasonable risk / benefit ratio.

[0086] The terms “treating” and “treatment”, and the like, as used herein, are intended to mean obtaining a desirable physiological, pharmacological, orbiological effect. The effect may result in the prevention (i.e., prophylactic treatment), inhibition, attenuation, reversal of a sign, symptom or cause of a disorder, or disease, attributable to the disorder, or disease. Clinical evidence of the treatment may vary with the disorder, or disease, the age of the human, and the selected treatment. In the context of the treatment of indication a physiological effect may include, for example, an improved respiratory capacity or lung function, improved brain and / or spinal cord functioning, reduced hemorrhaging, reduced mucoid nasal or oral discharge.

[0087] The term “bronchitis”, as used herein, refers to the accepted medical definition of bronchitis, and includes, in general, a respiratory disease state caused by H. influenzae.

[0088] The term “cellulitis”, as used herein, refers to the accepted medical definition cellulitis, and includes, in general, a skin disease state caused by H. influenzae.

[0089] The term “epiglottitis”, as used herein, refers to the accepted medical definition of epiglottitis, and includes, in general, a swollen throat disease state caused by H. influenzae.

[0090] The term “infectious arthritis”, as used herein, refers to the accepted medical definition infectious arthritis, and includes, in general, a joint and joint fluid disease state caused by H. influenzae.

[0091] The term “meningitis”, as used herein, refers to the accepted medical definition of meningitis, and includes, in general, a brain and / or spinal cord disease state caused by H. influenzae.

[0092] The term “otitis media”, as used herein, refers to the accepted medical definition otitis media, and includes, in general, an ear infection disease state caused by H. influenzae.

[0093] The term “pneumonia”, as used herein, refers to the accepted medical definition of bronchitis, and includes, in general, a respiratory disease state caused by H. influenzae.

[0094] The term “septicemia”, as used herein, refers to the accepted medical definition of septicemia, and includes, in general, a blood disease state caused by H. influenzae.

[0095] The terms “substantially pure” and “isolated”, as may be used interchangeably herein, describe a compound, e.g., a polypeptide, which has beenseparated from components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides, by chromatography, gel electrophoresis or HPLC analysis.General implementation

[0096] As hereinbefore mentioned, the present disclosure relates to vaccine formulations, notably human vaccine formulations. In general, the herein provided methods and compositions can be used to prevent, treat, or ameliorate infections caused by pathogenic H. influenzae in humans. In this respect, the methods and compositions of the present disclosure, in particular, may be used to treat multiple diseases caused by H. influenzae, including pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, and septicemia. The vaccine formulations of the present disclosure can provide long-term, effective protection against infections caused by pathogenic H. influenzae strains, including typable and non-typable H. influenzae strains.

[0097] The vaccine formulations of the present disclosure comprise modulated TbpB polypeptides, notably TbpB polypeptides comprising a modulated C-lobe domain, and are surprisingly potent.

[0098] The inventors have discovered that the vaccine formulations of the present disclosure can be cross-protective, and can provide protection against infection by multiple H. influenzae strains. Surprisingly, the vaccine formulations of the present disclosure can be cross-protective using a single immunogenic active agent. Moreover, vaccines comprising one modulated TbpB polypeptide of the present invention can be cross-protective across a wider range of H. influenzae strains, than vaccines comprising multiple native TbpB polypeptides combined.

[0099] Furthermore, the vaccine formulations can provide surprisingly high antibody titres.

[0100] Furthermore, the vaccine formulations of the present disclosure involve the use of polypeptide-based immunogenic active agents, and as such, the compositions and methods of the present disclosure do not involve the use oflive attenuated microbial species, and thus avoid infection risks associated with the use of live vaccines.

[0101] Furthermore, the vaccine formulations of the present disclosure can limit the administration of antibiotics to humans, and thus limit the development of antibiotic resistant microbial strains.

[0102] Furthermore, the polypeptide-based immunogenic active agents included in the vaccine formulations of the present disclosure may be prepared using a convenient recombinant production system, and the immunogenic active agents may be stably stored.

[0103] The present inventors have discovered that Transferrin Binding Protein B (TbpB) polypeptides may be modulated, and that the modulated TbpB polypeptides may be used as immunogenic active agents in the formulation of vaccines to prevent or ameliorate infections caused by H. influenzae in humans, and in this respect are surprisingly potent. In what follows example embodiments of the compositions and methods of the present disclosure are described.

[0104] Thus, the present disclosure provides, in at least one aspect, in at least one embodiment, a method fortreatmentof a human susceptible to infections caused by H. influenzae, the method comprising administering to the human a vaccine formulation comprising (i) a modulated TbpB protein, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof, wherein the vaccine formulation is administered in an effective amount to prevent or ameliorate the infection caused by the H. influenzae.

[0105] The present disclosure further provides, in at least one aspect, in at least one embodiment, a vaccine formulation for the prevention or amelioration of infection caused by H. influenzae in a human susceptible to infection by H. influenzae, the vaccine formulation comprising an effective amount of (i) a modulated TbpB polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a TbpB a modulated polypeptide, or an immunogenic portion thereof.

[0106] In general, according to an aspect, vaccine formulations comprising (i) a modulated TbpB polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof can be prepared or obtained. These formulationscan, in an aspect hereof, be administered in effective amounts to a human in need thereof. Thus, in what follows next suitable example preparations comprising (i) a modulated TbpB polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof, will be described, as well as suitable example methods of making (i) modulated TbpB polypeptides, or an immunogenic portion thereof or (ii) mRNA polynucleotides encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof. Thereafter, suitable example vaccine formulations comprising (i) a modulated TbpB polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof, and suitable example methods of preparing vaccine formulations and administering the same to a human in need thereof, in particular, to prevent, treat, or ameliorate an H. influenzae infection, will be described.

[0107] In general terms, modulated TbpB polypeptides in accordance with the present disclosure include TbpB polypeptides wherein in the amino acid sequence constituting the modulated TbpB polypeptide multiple amino acid residues are modulated relative to the amino acid sequence of a corresponding TbpB polypeptide. Notably, the modulated TbpB polypeptides of the present disclosure include TbpB polypeptides in which a C-lobe domain has been modified with respect to its loop domains. In this respect, in accordance with the present disclosure, in one aspect, modulated TbpB polypeptides can be obtained or prepared by initially referring to the amino acid sequence of a selected suitable native reference TbpB polypeptide, and identifying loop domains therein. Modulated TbpB polypeptides in accordance herewith may then be prepared or obtained by modification of the loop domains of the selected reference TbpB polypeptide. Accordingly, next loop domains of TbpB polypeptides will be discussed, as well as modulation of loop domains.

[0108] Initially it is noted that in accordance herewith multiple amino acid residues in the amino acid sequence forming loop domains of TbpB polypeptides are modulated. In particular, in accordance with the present disclosure, loop domains within C-lobe domains of TbpB polypeptides are modulated. In this respect, it is understood that C-lobe domains of TbpB polypeptides generally comprise multiple loop domains. This is illustrated further, for example, in FIG. 1A and FIGS. 1B (I) and 1B (ii), which show an example native reference TbpBpolypeptide, notably a TbpB polypeptide of H. influenzae strain h036, and consecutively numbered loop domains 1 to 31 . The present disclosure provides, in particular, modulated TbpB polypeptides lacking loop domains in the C-lobe domains, relative to the C-lobe domains of the corresponding reference TbpB polypeptides.

[0109] Thus, to illustrate this further, referring next to FIGS. 2A, 2B, and 2C, by way of example, shown therein in FIG. 2A is a schematic representation of a polypeptide portion 18 of a C-lobe domain of a native TbpB polypeptide 10, including an example loop domain 12. It is to be understood that polypeptide portion 18 and loop domain 12 are intended to be an example polypeptide portion and an example loop domain of a C-lobe domain of a TbpB polypeptide, respectively, and polypeptide portion 18 and loop domain 12 do not refer to a specific polypeptide portion of a specific loop domain, but can be any polypeptide portion of a C-lobe domain of a TbpB polypeptide comprising a loop domain. Loop domain 12 is flanked at its N-terminal end by an N-terminal loop flanking polypeptide 14, and at its C-terminal end, by a C-terminal loop flanking polypeptide 16. In modulations of a TbpB polypeptide involving the removal of a loop domain (illustrated in FIG. 2B), N-terminal loop flanking polypeptide 14 of a loop domain 12 is joined to C-terminal loop flanking polypeptide 16 of the same loop domain 12 to obtain a polypeptide portion 18 of a C-lobe domain of a modulated TbpB polypeptide 20, lacking loop domain 12 (illustrated in FIG. 2C). It is noted that the joint N-terminal loop flanking polypeptide 14 and C-terminal loop flanking polypeptide 16 together may be referred to as a “joining polypeptide” 22.

[0110] Next, referring to an initial selected example reference TbpB polypeptide, notably a TbpB polypeptide found in H. influenzae strain H036 (SEQ.ID NO: 42) (see: FIGS. 1B (I) and 1B (ii)), modulation of specific loop domains in accordance herewith will be discussed. Thereafter, modulation of other selected example reference TbpB polypeptides in accordance with the present disclosure will be discussed.

[0111] Thus, referring again to FIGS. 1 B (I) and 1B (ii), shown therein is a selected example TbpB polypeptide found in H. influenzae strain H036 (SEQ.ID NO: 42). In accordance herewith, in particular, included herein are example modulated TbpB polypeptides that can be prepared from the selected TbpB polypeptide having SEQ.ID NO: 42 (encoded by nucleic acid sequence SEQ.IDNO: 41 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), wherein, in an example embodiment, the modulated TbpB polypeptide comprises a C-lobe domain lacking loop domains 23 and 31 (the modulated TbpB polypeptide being set forth in SEQ.ID NO: 38 (encoded by nucleic acid sequence SEQ.ID NO: 37, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), or, wherein, in another example embodiment, the modulated TbpB polypeptide further optionally lacks, loop domain 20 (the modulated TbpB polypeptide being set forth in SEQ.ID NO: 40 (encoded by nucleic acid sequence SEQ.ID NO: 39, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions).

[0112] It is noted that the N-terminal loop flanking polypeptide of loop 23 within SEQ.ID NO: 38 and SEQ.ID NO: 40 comprises SEQ.ID NO: 2 (encoded by nucleic acid sequence SEQ.ID NO: 1 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and that the C-terminal loop flanking polypeptide of loop 23 within SEQ.ID NO: 38 and SEQ.ID NO: 40 comprises SEQ.ID NO: 8 (encoded by nucleic acid sequence SEQ.ID NO: 7, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). Thus, removal of loop 23 results in the formation of a joining polypeptide comprising SEQ.ID NO: 2 and SEQ.ID NO: 8, wherein SEQ.ID NO: 2 and SEQ.ID NO: 8 are contiguously joined, or, in other embodiments, joined by a short linker peptide, generally 10 amino acids or less in length (as hereinafter further described).

[0113] Similarly, It is noted that the N-terminal loop flanking polypeptide of loop 31 within SEQ.ID NO: 38 and SEQ.ID NO: 40 comprises SEQ.ID NO: 14 (encoded by nucleic acid sequence SEQ.ID NO: 13, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and that the C-terminal loop flanking polypeptide of loop 31 within SEQ.ID NO: 38 and SEQ.ID NO: 40 comprises SEQ.ID NO: 20 (encoded by nucleic acid sequence SEQ.ID NO: 19, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). Thus, removal of loop31 results in the formation of a joining polypeptide comprising SEQ.ID NO: 14 and SEQ.ID NO: 20, wherein SEQ.ID NO: 14 and SEQ.ID NO: 20 are contiguously joined, or, in other embodiments, joined by a short linker peptide, generally 10 amino acids or less in length (as hereinafter further described).

[0114] Similarly, It is noted that the N-terminal loop flanking polypeptide of loop 20 within SEQ.ID NO: 40 comprises SEQ.ID NO: 26 (encoded by nucleic acid sequence SEQ.ID NO: 25, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and that the C-terminal loop flanking polypeptide of loop 20 within SEQ.ID NO: 40 comprises SEQ.ID NO: 32 (encoded by nucleic acid sequence SEQ.ID NO: 31 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). Thus, removal of loop 20 results in the formation of a joining polypeptide comprising SEQ.ID NO: 26 and SEQ.ID NO: 32, wherein SEQ.ID NO: 26 and SEQ.ID NO: 32 are contiguously joined, or, in other embodiments, joined by a short linker peptide, generally 10 amino acids or less in length (as hereinafter further described).

[0115] Thus, it will be clear that, in accordance with the foregoing, the present disclosure includes, in one example embodiment, a TbpB polypeptide comprising or consisting of or consisting substantially of a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 4, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, or a polypeptide sequence that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, or a polypeptide sequence that is at least 90% thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide arecontiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues.

[0116] In one example embodiment, the TbpB polypeptide comprises, or consists of or consists substantially of a modulated C-lobe domain, wherein the C-lobe domain lacks loops 23 and 31 , can comprise or consist of SEQ.ID NO: 38, or a polypeptide sequence that is substantially identical thereto, and can be encoded by nucleic acid sequence SEQ.ID NO: 37, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions.

[0117] It will further be clear that, in at least one example embodiment, the modulated C-lobe domain can further additionally comprise:(iii) a third joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 26, or a polypeptide sequence that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 32, or a polypeptide sequence that is at least 90% thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 7 amino acid residues.

[0118] In one example embodiment, the TbpB polypeptide comprising a modulated C-lobe domain, wherein the C-lobe domain lacks loops 20, 23 and 31 , can comprise or consist of SEQ.ID NO: 40, or a polypeptide sequence that is substantially identical thereto and be encoded by nucleic acid sequence SEQ.ID NO: 39, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions.

[0119] Turning now to other example reference TbpB polypeptides that may be selected and used in accordance herewith. In general, any and all TbpB polypeptides naturally found in any H. influenzae strain may be selected and used in accordance herewith, as well as any TbpB sequences that may be modulated, including for example, immunogenic portions of TbpB polypeptides, notably, for example immunogenic portions of TbpB polypeptides comprising a C-lobe domain, and lacking all or part of the N-lobe domain.

[0120] Thus, for example, a reference TbpB polypeptide from H. influenzae strain 036 (SEQ.ID NO: 42) (encoded by a nucleic acid sequence having SEQ.ID NO: 41 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), or an immunogenic portion thereof, a reference TbpB polypeptide from H. influenzae strain 40 (SEQ.ID NO: 44) (encoded by a nucleic acid sequence having SEQ.ID NO: 43, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), or an immunogenic portion thereof, and a reference TbpB polypeptide from H. influenzae 201 (SEQ.ID NO: 46) (encoded by a nucleic acid sequence having SEQ.ID NO: 45, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), or an immunogenic portion thereof, may each be selected and used. When immunogenic portions of reference TbpB polypeptides are selected they generally preferably contain at least a C-lobe domain of a TbpB polypeptide, and more preferably, at least a portion of a C-lobe domain comprising loop 20, loop 23 and loop 31 , or preferably, in particular, at least loop 23 and 31 . Furthermore, any other TbpB polypeptide of H. influenzae that is substantially identical, e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ.ID NO: 42, SEQ.ID NO: 44, or SEQ.ID NO: 46, may be selected and used.

[0121] Referring next to FIG. 3, shown therein are aligned selected example TbpB polypeptide sequences of H. influenzae strain H036, H040 and H201. Indicated within the polypeptide sequences are loop domains 20, 23, and 31. Similar to the TbpB polypeptide sequence of H036 (SEQ.ID NO: 42) as described above, the TbpB polypeptide sequences of H. influenzae strain H040 (SEQ.ID NO: 44) and H201 (SEQ.ID NO: 46) may be used to modulate loop domains 23 and 31 , and optionally loop domain 20. Thus, for example, the N-terminal loop flanking polypeptide of loop 23 of strain H040 comprises SEQ.ID NO: 4 (encoded by nucleic acid sequence SEQ.ID NO: 3, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C-terminal loop flanking polypeptide comprises SEQ.ID NO: 10 (encoded by nucleic acid sequence SEQ.ID NO: 9, or a nucleic acid sequence substantially identical thereto or capable of hybridizingthereto under at least moderately stringent hybridization conditions). The N- terminal loop flanking polypeptide of loop 31 of strain H040 comprises SEQ.ID NO: 16 (encoded by nucleic acid sequence SEQ.ID NO: 15, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C-terminal loop flanking polypeptide comprises SEQ.ID NO: 22 (encoded by nucleic acid sequence SEQ.ID NO: 21 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). The N-terminal loop flanking polypeptide of loop 20 of strain H040 comprises SEQ.ID NO: 28 (encoded by nucleic acid sequence SEQ.ID NO: 27, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C- terminal loop flanking polypeptide comprises SEQ.ID NO: 34 (encoded by nucleic acid sequence SEQ.ID NO: 33, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). Similarly, for example, the N-terminal loop flanking polypeptide of loop 23 of strain H201 comprises SEQ.ID NO: 6 (encoded by nucleic acid sequence SEQ.ID NO: 5, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C-terminal loop flanking polypeptide comprises SEQ.ID NO: 12 (encoded by nucleic acid sequence SEQ.ID NO: 11 , or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). The N- terminal loop flanking polypeptide of loop 31 of strain H201 comprises SEQ.ID NO: 18 (encoded by nucleic acid sequence SEQ.ID NO: 17, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C-terminal loop flanking polypeptide comprises SEQ.ID NO: 24 (encoded by nucleic acid sequence SEQ.ID NO: 23, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions). The N-terminal loop flanking polypeptide of loop 20 of strain H201 comprises SEQ.ID NO: 30 (encoded by nucleic acid sequence SEQ.ID NO: 29, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions), and the C-terminal loop flanking polypeptide comprises SEQ.ID NO: 36 (encoded by nucleic acid sequence SEQ.ID NO: 35, or a nucleic acid sequence substantially identical thereto or capable of hybridizing thereto under at least moderately stringent hybridization conditions).

[0122] As noted, in general any H. influenzae TbpB sequences may be selected and used as a reference TbpB polypeptide, and using alignment tools and methods a selected TbpB polypeptide may be aligned with other reference TbpB sequences, such as SEQ.ID NO: 42, SEQ.ID NO: 44, and SEQ. ID NO: 46, to identify loops 20, 23, and 31 in a reference TbpB polypeptide. Methods for aligning polypeptide sequences are generally art recognized and include, for example, the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981 , 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Furthermore, the highest order identity may be calculated and expressed as a percentage identity. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1 , so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

[0123] In order to further facilitate the identification of loop domains in reference TbpB sequences, secondary structure-prediction programs, such asPyMOL Molecular Graphics System (www.pymol.org), for example, may be used and generate TbpB protein representations, such as shown In FIG. 1A.

[0124] Furthermore, as noted H. influenzae TbpB reference polypeptide sequences comprising substantial sequence identity to any TbpB sequences set forth herein, including SEQ.ID NO: 42, SEQ.ID NO: 44, and SEQ.NO: 46, may be selected and used to identify loops 23, 31 and 20, and the N-terminal and C- terminal loop flanking polypeptide sequence, and to modulate the C-lobe domain, as described herein. Thus, the TbpB polypeptides comprising a modulated C-lobe domain, in addition to comprising a joining peptide comprising an N-terminal loop flanking polypeptide of loop 23 (SEQ.ID NO: 2, SEQ.ID NO: 4, and SEQ.ID NO: 6) contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 23 (SEQ.ID NO: 8, SEQ.ID NO: 10, and SEQ.ID NO: 12) may comprise an N-terminal loop flanking polypeptide of loop 23 that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ.ID NO: 2, SEQ.ID NO: 4, and SEQ.ID NO: 6 contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 23 that is at least 80%, at least 85%, at least 90%, at least, 95%, or at least 99% identical to SEQ.ID NO: 8, SEQ.ID NO: 10, and SEQ.ID NO: 12. Thus, for example, SEQ.ID NO: 2 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 8; SEQ.ID NO: 4 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 10; and SEQ.ID NO: 6 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 12).

[0125] Similarly, the TbpB polypeptides comprising a modulated C-lobe domain, in addition to comprising a joining peptide comprising an N-terminal loop flanking polypeptide of loop 31 (SEQ.ID NO: 14, SEQ.ID NO: 16, and SEQ.ID NO: 18) contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 31 ((SEQ.ID NO: 20, SEQ.ID NO: 22, and SEQ.ID NO: 24), may comprise an N-terminal loop flanking polypeptide of loop 31 that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ.ID NO: 14, SEQ.ID NO: 16, and SEQ.ID NO: 18 contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 31 that is at least 80%, at least 85%, at least 90%, at least, 95%, or at least 99% identical to SEQ.ID NO: 20, SEQ.ID NO: 22, and SEQ.ID NO: 24. Thus, for example, SEQ.ID NO: 14 may be contiguously linked (or linked through a shortpeptide linker) to SEQ.ID NO: 16; SEQ.ID NO: 18 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 20; and SEQ.ID NO: 22 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 24).

[0126] Similarly, the TbpB polypeptides comprising a modulated C-lobe domain, in addition to comprising a joining peptide comprising an N-terminal loop flanking polypeptide of loop 20 (SEQ.ID NO: 26, SEQ.ID NO: 28, and SEQ.ID NO: 30) contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 20 (SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36), may comprise an N-terminal loop flanking polypeptide of loop 20 that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ.ID NO: 26, SEQ.ID NO: 28, and SEQ.ID NO: 30 contiguously linked (or linked through a short peptide linker) to a C-terminal loop flanking polypeptide of loop 20 that is at least 80%, at least 85%, at least 90%, at least, 95%, or at least 99% identical to SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36. Thus, for example, SEQ.ID NO: 26 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 28; SEQ.ID NO: 30 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 32; and SEQ.ID NO: 34 may be contiguously linked (or linked through a short peptide linker) to SEQ.ID NO: 36).

[0127] Regarding the short peptide linker optionally used to couple an N- terminal loop flanking polypeptide to a C-terminal loop flanking polypeptide, it is generally 10 amino acids or less in length. Thus, the peptide linker can be e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. Furthermore, the peptide linker may contain any combination of amino acid residues, including one or more aliphatic amino acid residues (alanine, isoleucine, leucine, proline, and valine).

[0128] Thus, it will now be understood that upon having selected a reference TbpB polypeptide of H. influenzae, amino acid residues corresponding with loops 23 and 31 , and additionally, optionally, loop 20, therein may be selected for modulation, and that upon modulation a modulated TbpB polypeptide lacking loops 23 and 31 and, optionally loop 20, can be obtained. In such modulated TbpB polypeptides, the N-terminal loop flanking domain and C-terminal loop flanking domain of these loops are contiguously joined, or can be optionally joined by a short peptide linker.

[0129] In a further specific aspect, in example embodiments, the modulatedTbpB polypeptide can be selected from a polypeptide having an amino acidsequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0130] Modulated TbpB polypeptides, in accordance herewith, may be prepared using any suitable technique or methodology to produce desired modulated TbpB polypeptides, or immunogenic portions thereof, notably immunogenic portions comprising a modulated C-lobe domain. Thus, nucleic acid sequences expressing modulated TbpB polypeptides, or immunogenic portions may be obtained. Such nucleic acid sequences expressing modulated TbpB polypeptides, or immunogenic portions thereof, may subsequently be introduced in a host cell for expression in the host cell and to thereby produce the modulated TbpB polypeptides, as hereinafter further described. In order to prepare a modulated TbpB polypeptide, initially a nucleic acid sequence encoding a reference TbpB polypeptide including a nucleic acid sequences comprising SEQ.ID NO: 41 , SEQ.ID NO: 43, orSEQ.ID NO: 45, may be obtained, forexample, by isolation (cloning) of such a nucleic acid sequence from a source microbial organism, notably H. influenzae. Subsequently, the nucleic acid sequence encoding the reference TbpB, or immunogenic portion thereof, may be used to prepare a nucleic acid sequence encoding a modulated TbpB polypeptide, or immunogenic portion thereof. Thus, for example, a nucleic acid sequence encoding a reference TbpB polypeptide, or an immunogenic portion thereof, may be used to obtain a nucleic acid sequence encoding a modulated TbpB polypeptide using de novo artificial gene synthesis techniques of a nucleic acid sequence encoding a modulated TbpB polypeptide (see, for example, Kosuri, S et al., 2014, Nature Methods 11 (5) 499-507), or CRISPR / Cas9 based techniques (Jinek, M. et a!., 2012, Science, 337 (6096), 816-821).

[0131] As will be understood from the foregoing, nucleic acid sequences encoding modulated TbpB polypeptides that may be prepared include nucleic acid sequences having SEQ.ID NO: 37 (encoding a modulated TbpB polypeptide lacking loop 23 and 31 , corresponding with SEQ.ID NO: 38), and SEQ.ID NO: 39 (encoding a modulated TbpB polypeptide lacking loop 20, 23 and 31 , corresponding with SEQ.ID NO: 40).

[0132] Thus, to briefly recap, in accordance with an aspect of the present disclosure a nucleic acid sequence encoding a modulated TbpB polypeptide, or immunogenic portion thereof, may be obtained. Within the modulated TbpBpolypeptide, or immunogenic portion thereof, loop domains 23 and 31 , and optionally loop domain 20 may be modulated relative to a corresponding reference TbpB polypeptide.

[0133] Turning now to methods of making the modulated TbpB polypeptides, once a suitable nucleic acid sequence encoding a modulated TbpB has been obtained, preparations containing a modulated TbpB polypeptide, or an immunogenic portion thereof can be made biosynthetically using a host cell system. In this respect, an isolated nucleic acid encoding an amino acid sequence corresponding with a modulated TbpB polypeptide, or an immunogenic portion thereof, can be introduced in host cells and expressed therein.

[0134] As is known to those of skill in the art, expression of nucleic acids in a host cell, to thereby biosynthetically produce a protein, can be achieved by providing one or more nucleic acids capable of controlling expression in a host cell, and operably linking the one or more nucleic acids capable of controlling expression in a host cell to the nucleic acid one wishes to express. Such operable linking of a nucleic acid controlling expression generally involves linking in the 5’ to 3’ direction of expression the nucleic acid capable of controlling expression in a host cell to the nucleic acid one wishes to express, i.e., within the context of the instant disclosure, a modulated TbpB polypeptide. Nucleic acid sequences capable of controlling expression in host cells that may be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells. Generally, promoters obtained from bacterial cells are used when a bacterial host cell is selected, while a yeast promoter will be used when a yeast host cell is selected, a plant promoter will be used when a plant cell is selected, and so on. The obtained nucleic acid comprising a promoter and the nucleic acid expressing a modulated TbpB polypeptide is generally a chimeric nucleic acid. Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers, and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.

[0135] In accordance with the present disclosure, the chimeric nucleic acid sequences can be integrated into a recombinant expression vector which ensures good expression in the host cell, wherein the recombinant expression vector is suitable for expression in a host cell. The term “suitable for expression in a host cell” means that the recombinant expression vector comprises the chimeric nucleicacid linked to genetic elements required to achieve expression in a cell. As noted, such genetic elements can include transcriptional promoters, terminators, and enhancers, and the like. Further genetic elements that may be included in the expression vector are one or more nucleic acid sequences encoding marker genes, and one or more origins of replication. In some embodiments, the expression vector can freely replicate in the host cell. In other embodiments, the chimeric nucleic acid can be integrated into the host cell’s genomic DNA. In some embodiments, the expression vector further can comprise genetic elements required for the integration of the vector or a portion thereof in the host cell's genome, for example, if a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome can be included in the vector.

[0136] Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from nontransformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin, chloramphenicol, methotrexate, or ampicillin. In other instances, a marker gene may be a gene which allows a cell to produce an essential nutrient, for example amino acids.

[0137] Thus, in an aspect, the present disclosure provides, in an example embodiment, an expression vector comprising:(a) a nucleic acid sequence encoding a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminalloop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain in a host cell.

[0138] In example embodiments, the expression vector can comprise a chimeric nucleic acid comprising a nucleic acid sequence encoding a promoter linked to a nucleic acid sequence encoding a modulated TbpB polypeptide, or an immunogenic portion thereof.

[0139] Turning now to the host cell, it is noted, initially, that any host cell which upon cultivation expresses the chimeric nucleic acid can be selected and used in accordance with the present disclosure. Suitable host cells in this respect include, for example, microbial cells, such as bacterial cells, yeast cells, for example, and algal cells or plant cells. A variety of techniques and methodologies to manipulate host cells to introduce nucleic acid sequences in cells and attain expression exists and are well known to the skilled artisan. These methods include, for example, cation based methods, for example, lithium ion or calcium ion based methods, electroporation, biolistics, and glass beads based methods. As will be known to those of skill in the art, depending on the host cell selected, the methodology to introduce nucleic acid material in the host cell may vary, and, furthermore, methodologies may be optimized for uptake of nucleic acid material by the host cell, for example, by comparing uptake of nucleic acid material using different conditions. Detailed guidance can be found, for example, in Sambrook etal., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. It is noted that the chimeric nucleic acid is a non-naturally occurring chimeric nucleic acid sequence and can be said to be heterologous to the host cell.

[0140] One example host cell that conveniently may be used is Escherichia coli. The preparation of the E. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, the polymerase chain reaction (PCR) and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc. Suitable promoter sequences for use in E. coli include, for example, the T7 promoter, the T5 promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan / lactose (tac) promoter, lipoprotein (Ipp) promoter, and A phage PL promoter. Typically, cloning vectors contain a marker, for example, an antibiotic resistance marker, such as ampicillin or kanamycin resistance marker, allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E. coli by preparing competent cells, electroporation or using other well-known methodologies to a person of skill in the art. E. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells.

[0141] Another example host cell that may be conveniently used is a yeast cell. Example yeast host cells that can be used are yeast cells belonging to the genus Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, and Yarrowia. In specific example embodiments, the yeast cell can be a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, or Pichia pastoris cell.

[0142] A number of vectors exist for the expression of recombinant proteins in yeast host cells. Examples of vectors that may be used in yeast host cells include, for example, Yip type vectors, YEp type vectors, YRp type vectors, YCp type vectors, pGPD-2, pAO815, pGAPZ, pGAPZa, pHIL-D2, pHIL-S1 , pPIC3.5K, pPIC9K, pPICZ, pPICZa, pPIC3K, pHWO10, pPUZZLE and 2 pm plasmids. Such vectors are known to the art and are, for example, described in Cregg et al., MolBiotechnol. (2000) 16(1): 23-52. Suitable promoter sequences for use in yeast host cells are also known and described, for example, in Mattanovich et al., Methods Mol. Biol., 2012, 824:329-58, and in Romanos et al., 1992, Yeast 8: 423- 488. Examples of suitable promoters for use in yeast host cells include promoters of glycolytic enzymes, like triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), P. pastoris glucose-6- phosphate isomerase promoter (PPGI), the 3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF), S. cerevisiae enolase (ENO-1 ), S. cerevisiae galactokinase (GAL1), S. cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2 / GAP), S. cerevisiae triose phosphate isomerase (TPI), S. cerevisiae metallothionein (CUP1), and S. cerevisiae 3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL). Marker genes suitable for use in yeast host cells are also known to the art. Thus, antibiotic resistance markers, such as ampicillin resistance markers, can be used in yeast, as well as marker genes providing genetic functions for essential nutrients, for example, leucine (LEU2), tryptophan (TRP1 and TRP2), uracil (URA3, URA5, URA6), histidine (HIS3), and the like. Methods for introducing vectors into yeast host cells can, for example, be found in S. Kawai et al., 2010, Bioeng. Bugs 1 (6): 395-403.

[0143] Yet other example host cells that may be used in accordance herewith are plant cells. Methods for introducing nucleic acids in plant cells are known to those of skill in the art. Agrobacterium mediated plant cell transformation methods are described, for example, by Gelvin S. in Microbiol. Mol. Biol. Rev., 2003, 67(1): 16-37, and physical transformation based methods for plant cells are described by Rivera A.L. et al., 2012, Phys. Life Rev. 9(3): 308-345. Plant selectable marker genes are known to those of skill in the art and include antibiotic resistance genes, for example kanamycin resistance genes, and herbicide resistance genes, such as the bar and pat genes (Wohlleben et al., 1988, Gene 70:25-37). Screenable markers that may be employed to identify plant transformants through visual inspection include p-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14: 403). Plant promoters are also known to those in the artand include, for example, constitutive promoters, such as the 35S cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987, Gene 53: 153-161), the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171 ; U.S. Pat. No. 6,429,357), a ubiquitin promoter, such as the corn ubiquitin promoter (U.S. Pat. Nos 5,879,903 and 5,273,894), and the parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol. 21 :673-684), and organ specific promoters, such as seed specific promoters, for example, a phaseolin promoter (Sengupta-Gopalan et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3320-3324), or an oleosin promoter (U.S. Pat. No. 5,792,922).

[0144] Further, guidance with respect to the preparation of expression vectors and introduction thereof into host cells, including in E. coli cells, yeast cells, and other host cells, may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0145] Thus, in another aspect, the present disclosure provides, in one example embodiment, a host cell comprising a chimeric nucleic acid comprising: (a) a nucleic acid sequence encoding a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptidethat is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain in a host cell.

[0146] In example embodiments, the expression vector can comprise a chimeric nucleic acid comprising a nucleic acid sequence encoding a promoter linked to a nucleic acid sequence encoding a modulated TbpB polypeptide, or an immunogenic portion thereof.

[0147] Thus, to briefly recap, a host cell comprising a chimeric nucleic acid comprising (i) a nucleic acid sequence encoding a modulated TbpB polypeptide, or an immunogenic portion thereof; and (ii) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding a modulated TbpB polypeptide, or an immunogenic portion thereof in a host cell can be prepared in accordance with the present disclosure.

[0148] In accordance herewith, host cells are grown to multiply and to express a chimeric nucleic acid. Expression of the chimeric nucleic acid results in the biosynthetic production in the host cell of a modulated TbpB polypeptide, or an immunogenic portion thereof. Growth media and growth conditions can vary depending on the host cell that is selected, as will be readily appreciated to those of ordinary skill in the art. Growth media typically contain a carbon source, one or several nitrogen sources, essential salts including salts of potassium, sodium, magnesium, phosphate and sulphate, trace metals, water soluble vitamins, and process aids including but not limited to antifoam agents, protease inhibitors, stabilizers, ligands, and inducers. Typical carbon sources are e.g., mono- or disaccharides. Typical nitrogen sources are, e.g., ammonia, urea, amino acids, yeast extract, corn steep liquor and fully or partially hydrolyzed proteins. Typicaltrace metals are e.g., Fe, Zn, Mn, Cu, Mo and H3BO3. Typical water soluble vitamins are e.g., biotin, pantothenate, niacin, thiamine, p- aminobenzoic acid, choline, pyridoxine, folic acid, riboflavin, and ascorbic acid. Further, specific example media include liquid culture media for the growth of yeast cells and bacterial cells including, Luria-Bertani (LB) broth for bacterial cell cultivation, and yeast extract peptone dextrose (YEPD or YPD), for yeast cell cultivation. Further media and growth conditions can be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0149] Upon production by the host cells of a modulated TbpB polypeptide, or an immunogenic portion thereof, the modulated TbpB polypeptide, or immunogenic portion thereof may be recovered from the host cells, and separated from other constituents, such as cellular debris, or media constituents, for example. Separation techniques will be known to those of skill in the art and include a variety of different protein purification techniques including, e.g., ionexchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, etc. Further general guidance with respect to protein purification may for example be found in: Cutler, P. Protein Purification Protocols, Humana Press, 2004, Second Ed. Thus, substantially pure preparations of modulated TbpB polypeptides or immunogenic portions thereof may be obtained. The recovered modulated TbpB polypeptides may be obtained in a more or less pure form, for example, a preparation of a modulated TbpB polypeptide, or immunogenic portion, thereof having a purity of at least about 60% (w / v), about 70% (w / v), about 80% (w / v), about 90% (w / v), about 95% (w / v), or about 99% (w / v) may be obtained.

[0150] Furthermore, it is noted that the recombinant production of the modulated TbpB polypeptides or immunogenic portions thereof in host cell system permits the production thereof in a manner in which the modulated TbpB polypeptide, or immunogenic portions, thereof are substantially free from other constituent materials of pathogenic H. influenzae bacterial species capable of causing infectious diseases in humans, such as, for example, H. influenzae proteins, membrane materials, lipopolysaccharides, and the like, naturally associated with native H. influenzae TbpB polypeptides, respectively.

[0151] It is noted that the cells, in some embodiments, may secrete a certain portion of the total quantity of the produced modulated TbpB polypeptide, or an or immunogenic portion thereof, in the cell growth medium. Thus, a certain portion the produced modulated TbpB polypeptides, or immunogenic portion thereof, may be recovered from the cells, and a further portion of the modulated TbpB polypeptides, or immunogenic portion thereof, may be recovered from the growth medium.

[0152] It is further noted that the vaccine formulations of the present disclosure may comprise modulated TbpB polypeptides or an immunogenic portion thereof in more or less pure form. Thus, in accordance herewith, a substantially pure modulated TbpB polypeptides or immunogenic portion thereof may be obtained and used to prepare vaccine formulations. In other embodiments, more crude preparations comprising modulated TbpB polypeptides or an immunogenic portion thereof may be obtained and used to prepare vaccine formulations. Thus, for example, in such embodiments, host cells, host cell lysates or host cell fractions comprising the modulated TbpB polypeptides or an immunogenic portion thereof may be used to prepare the vaccine formulations.

[0153] In another embodiment, an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, may be prepared and included in a vaccine formulation.

[0154] It is noted that in embodiments wherein an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, is produced, the mRNA encoding the modulated TbpB polypeptide may be produced in vivo, or in vitro. In vitro methods for synthesizing mRNA may include, for example, an in vitro transcription (IVT) system, using a template polynucleotide, for example, a template cDNA or linearized plasmid DNA, encoding a modulated TbpB polypeptide, or an immunogenic portion thereof. IVT systems typically include a transcription buffer, nucleotide triphosphates (NTPs), and RNAse inhibitor, and an RNA-polymerase. An example IVT system that may be used in this respect is described in PCT / US2014 / 026835. The mRNAs may be non- chemically modified and contain the conventional naturally occurring ribonucleotides, i.e., adenosine, guanosine, cytosine, and uridine. In other embodiments, the mRNAs may comprise chemically modified ribonucleotides, e.g., 1-methyl-pseudouridine (m1 i ), 1-ethyl-pseudouridine (e1 qj), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine ( 1), 5- methoxymethyl uridine, 5-methylthio uridine, 1 -methoxymethyl pseudouridine, 5- methyl cytidine, and / or 5-methoxy cytidine. When using an IVT system, nonmodified and chemically modified NTPs may be included in the IVT system.

[0155] In some embodiments, the template can include a 5’-untranslated region (5’-UTR) ( / .e., the region that is located immediately upstream (5’) of the start codon, / .e., the first codon of an mRNA translated by a ribosome) and / or a 3’- untranslated region (3’-UTR) ( / .e., the region that is located immediately downstream (3’) of the stop codon, / .e., the last codon of an mRNA translated by a ribosome). The 5’-UTR and 3’-UTR do not encode protein, however they may contain stabilizing elements, such as a 3’-polyA tail, or a 5’-terminal cap.

[0156] The 5’-UTR may contain a promoter sequence, for example, a T7- promoter sequence, or any other suitable promoter sequence. The 5’-UTR may further contain a 5’ -terminal cap, for example, a 3'-0-Me-m7G(5')ppp(5') G (ARCA cap; New England BioLabs, Ipswich, Mass., USA), which may be included concomitantly with in vitro transcription. The 3’-UTR may contain a polyA tail, including, for example, 10 - 300 monophosphates, or it may contain AU rich elements (AREs). 5’-UTRs and 3’-UTRs may be heterologous to the mRNA sequence being expressed or synthetic ( / .e., not occurring in nature), including natural sequences that have been mutated. Example 5'-UTRs include Xenopus or human derived a-globin or p-globin (see: U.S. Pat. No. 8,278,036; 9,012,219), human cytochrome b-245a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (see: U.S. Pat. Nos. 8,278,036, 9,012,219). Example 3’-UTRs include including Xenopus p-globin UTRs and human p -globin UTRs (see: U.S. Pat. Nos. 8,278,036; 9,012,219; US20110086907). A further modified p-globin construct which provides enhanced stability in some cell types by cloning two sequential human p-globin 3'-UTRs head to tail that may be used is well known in the art (see: Holtkamp, S. et al. , 2006, Blood, 109 (13), 4009 - 4017; WO2017 / 060314). Those of ordinary skill in the art will understand that 5'-UTRs that are heterologous or synthetic may be used with any desired 3'-UTR sequence. For example, a heterologous 5-'UTR may be used with a synthetic 3'-UTR with or with heterologous 3'-UTR.

[0157] Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Cleanup may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass., USA), poly- T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or F1 PLC based purification methods such as, but not limited to, strong anion exchange F1 PLC, weak anion exchange F1 PLC, reverse phase F1 PLC (RP- F1 PLC), and hydrophobic interaction F1 PLC (F1 IC-F1 PLC).

[0158] Thus, in another aspect, the present disclosure further provides in one embodiment expression vectors and host cells expressing mRNAs encoding modulated TbpB proteins.

[0159] The present disclosure provides, in one embodiment, an expression vector comprising:(a) a nucleic acid sequence encoding an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flankingpolypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues,(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

[0160] In one embodiment, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0161] In another embodiment one embodiment, in accordance with the teachings herein, a host cell comprising a chimeric nucleic acid comprising:(a) a nucleic acid sequence encoding an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO:24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

[0162] In one embodiment, the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

[0163] T urning next to the preparation of vaccine formulations, in an aspect hereof, in order to prepare a vaccine formulation, a preparation comprising (i) a modulated TbpB polypeptide, or immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof, may be combined with at least one other pharmaceutically acceptable in ingredient, including, but not limited to, a diluent, an excipient, a carrier, an adjuvant, or mixtures thereof, whereby (i) the modulated TbpB polypeptides or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a modulated TbpB a polypeptide, or an immunogenic portion thereof, and at least one other ingredient are mixed together or blended or homogenized or otherwise prepared until the vaccine formulation is formed.

[0164] It is noted that any modulated TbpB polypeptides or immunogenic portion thereof, or any mRNA polynucleotide encoding a modulated TbpB a polypeptide, or immunogenic portion thereof, may be used to formulate the vaccine formulations of the present disclosure, including modulated TbpB polypeptides having any one of the amino acid sequences set forth in SEQ.ID NO; 38, orSEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto, or an immunogenic portion of any of the foregoing. Furthermore, when selecting a modulated TbpB polypeptide, or an mRNA polynucleotideencoding a modulated TbpB a polypeptide, consideration may be given to the H. influenzae strain causing an infection in a human, as well as the disease of the human one wishes to address. Thus, for example, when a vaccine formulation is prepared to ameliorate or prevent meningitis caused by a H. influenzae serotype b strain infection, a modulated TbpB polypeptide or an mRNA polynucleotide encoding a modulated TbpB a polypeptide, may be prepared by referring to a reference TbpB polypeptide that corresponds with the infecting H. influenzae serotype b strain; or, for example, when a vaccine formulation is prepared to ameliorate or prevent otitis media caused by a non-typable H. influenzae strain infection, a modulated TbpB polypeptide or an mRNA polynucleotide encoding a modulated TbpB a polypeptide, may be prepared by referring to a reference TbpB polypeptide that corresponds with the infecting non-typable H. influenzae strain. It is noted that specific H. influenzae strains can be obtained (e.g., from a collection of microbial species, such as the American Type Culture Collection (ATCC)) or isolated, for example, from infected humans, and the serotype of a strain, can be determined using methods known to those of skill in the art (see: for example, LaClaire L. et ai., M. et al., 2003, J. Clin. Microbiol, 41 (1 ) 393-396).

[0165] It is noted, however, that the vaccine formulations of the present disclosure can be used to ameliorate or prevent infections by strains, other than the strain from which a reference TbpB polypeptide, on which the corresponding modulated TbpB polypeptide included in a vaccine formulation is based, is obtained. Thus, the vaccine formulations of the present disclosure, surprisingly, do not necessarily need to include modulated TbpB polypeptides or mRNA polynucleotides encoding modulated TbpB a polypeptides, from a plurality of pathogenic H. influenzae strains in order to be used for the treatment of a human, even if the human can be or has been exposed to, or can be or has been infected by a plurality of H. influenzae strains. Thus, for example, a vaccine formulation comprising a modulated TbpB polypeptide or mRNA polynucleotide encoding a TbpB a modulated polypeptide, based on a corresponding reference TbpB polypeptide from a H. influenzae strain of a first serotype, may be used to treat a human for an infection caused by a H. influenzae strain of another serotype. In this respect, the vaccine formulations, even if they include a single modulated TbpB polypeptide, or immunogenic portion thereof, can be said to be cross-protective. Thus, for example, the TbpB polypeptide, or immunogenic portion thereof,comprising the modulated C-lobe domain can be from a H036 H. influenzae strain, and the vaccine formulation can be administered to a human to prevent or ameliorate an infection caused by one or more H. influenzae strains selected from the group consisting of a HO H. influenzae strain, a H026 H. influenzae strain, a H040 H. influenzae strain, a H210 H. influenzae strain, a H036 H. influenzae strain, a WP050 H. influenzae strain, a WP11880 H. influenzae strain, a PRI75995 H. influenzae strain, a WP111 H. influenzae strain, a WP11887 H. influenzae strain, a PRI69814 H. influenzae strain, a WP221 H. influenzae strain, and a WP112 H. influenzae strain.

[0166] Notwithstanding the foregoing, in some embodiments, vaccine formulations may comprise two or more TbpB proteins or two or more mRNAs encoding TbpB proteins, including at least one modulated TbpB polypeptide of the present disclosure. Thus, in example embodiments, vaccine formulations may comprise two or more modulated TbpB proteins or two or more mRNAs encoding modulated TbpB proteins obtained from two or more H. influenzae strains, each belonging to a different serotype. In such embodiments, the vaccine can prevent or ameliorate infection by strains of two or more H. influenzae strains, and such vaccines can be said to be cross-protective.

[0167] In further example embodiments, vaccine formulations may comprise at the modulated TbpB polypeptides protein having any one of the amino acid sequences set forth in SEQ.ID NO: 38, and SEQ.ID NO: 40, or polypeptides having an amino acid sequence that is at least 90% identical thereto, or immunogenic portions thereof.

[0168] The amount of modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof in the vaccine formulation may vary. In general, consideration is given to the dose to be administered to a human. Doses for the modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, may be formulated to include modulated TbpB polypeptide, or an immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, in quantities ranging from about 1 pg / kg of body weight to about 0.25 mg / kg of body weight, preferably about 1 pg / kg of body weight to about 100 pg / kg of body weight. Furthermore, vaccineformulations are preferably formulated so that a dose comprises at least about 0.001 % by weight or volume, at least 0.025% or about 0.025%, at least 0.05% or about 0.05%, at least 0.1 % or about 0.1 %, at least 0.5 or about 0.5%, at least 1 % or about 1 %, at least 5% or about 5%, at least 10% or about 10%, at least 15% or about 15%, at least 20% or about 20%, or at least 25% or about 25%, by weight of the modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, so that the ratio of modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof to other vaccine constituents (e.g., adjuvants, diluents, carriers, excipients) of the vaccine formulation by weight or volume is at least 0.001 :99.999, 0.025:99.975, 0.05: 99,95, 0.01 :99.99, 0.5:99.5, 1 :99, 5:95, 15:85, 20:80, or 25:75, respectively, by weight. The exact amount necessary, however, will vary depending on the species, age, and general condition of the recipient human to be treated, the severity of the condition being treated, the particular preparation delivered, the site of administration, as well as other factors. In this respect, vaccine formulations may, in particular, vary with respect to the quantity of modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, included in a dose, depending on the age of the human to which the vaccine formulation is administered, wherein, as will be understood those of skill in the art, neonates, babies, infants, toddlers, and children, may receive a lower quantities of the modulated TbpB polypeptide, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof than adults. A suitable effective amount can be readily determined by one of skill in the art. Thus, a therapeutically effective amount of the modulated TbpB polypeptide, or immunogenic portion, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion, to be included in the vaccine formulations of the present disclosure will be an amount sufficient to bring about amelioration or prevention of disease or condition symptoms, and will fall in a relatively broad range that can be determined through routine trials.

[0169] Vaccine formulations comprising the modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, of the present disclosurepreferably further are prepared by combining the modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, with e.g., carriers, excipients, diluents, and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like. These carriers, excipients, diluents, and auxiliary substances are pharmaceutically acceptable ingredients. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts can also be included in the formulation, for example, mineral acid salts such as hydrochlorides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, benzoates, and the like. It is also preferred, although not required, that the vaccine formulation will contain a pharmaceutically acceptable carrier that serves as a stabilizer, particularly in order to stabilize the polypeptides of the present disclosure. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, glycine, polyethylene glycols (PEGs), and combinations thereof. Carriers may constitute, for example, from about 10% to about 95% by weight of the vaccine formulation.

[0170] Further, auxiliary agents such as freeze drying stabilizers, wetting, or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives may also be included in the vaccine formulations of the present disclosure. Vaccine formulations generally comprise less than about 5% by weight of such auxiliary agents.

[0171] In order to augment an immune response in a human, the vaccine formulations provided herein further preferably include one or more adjuvants, such as pharmacological agents, cytokines, or the like. Suitable adjuvants include any substance that enhances the immune response of the recipient human to the immunogenic modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, of the disclosure. Non-limiting examples of adjuvants include cytokines, e.g., IL-1 , IL-2, IL-12, IL-6, and further include inorganic salts, e.g., aluminum hydroxide, aluminum phosphate, and calciumphosphate; oil emulsions, e.g., mineral oil, MF59, QS-21 , MontanideTmISA51 , MontanideTmISA61 , MontanideTmGel 02, MontanideTmISA-720, or Emulsigen D®; Isocoms, e.g., ISCOMATRIX; microbial derivatives, e.g., monophosphorul lipid A (MPLA), macrophage-activating protein-2, virosomes, LT / CT, CpG; natural polymers, e.g., polysaccharides; and synthetic polymers, e.g., polyanhydrides and polyesters, or nucleic acid analogs, such as Poly l:C. Adjuvants may be administered, for example, as proteins or other macromolecules at the same time (e.g., by inclusion in the vaccine formulation), prior to, or subsequent to, administration of the polypeptide antigens. When included in a vaccine formulation, adjuvants may constitute, for example, from 0.1 % or about 0.1 % to 50% or about 50%, from 0.1 % or about 0.1 % to 20% or about 20%, or from 1 % or about 1 % to 10% or about 10% by weight or volume of a vaccine formulation.

[0172] Furthermore, capsular polysaccharides, present on the surface of pathogenic bacteria may be conjugated to the TbpB proteins, or immunogenic portions thereof of the present disclosure, and polysaccharide-conjugated TbpB proteins, or immunogenic portions thereof, may be included in vaccine formulations in accordance with the present disclosure. The conjugation of capsular polysaccharides to TbpB proteins, or immunogenic portions thereof, may enhance the immunogenic response. Polysaccharides that may be conjugated to the TbpB proteins, or immunogenic portions thereof, include polysaccharides obtainable from H. influenzae strains, including, for example, capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1 -> 1 )-p-D-ribose-3- phosphate) (see: Mawa, F. et al., 2007, Biologicals, 35, 235 - 245).

[0173] T echniques for producing capsular polysaccharides and conjugating polysaccharides to proteins are well known to those of skill in the art, and guidance can be found, for example, in: Frasch, C., 2009, Vaccine, 27, 6468-6470; PCT Patent Application having Publication No WO 2018 / 144439; and Pawlowski A. et al., 2000, Vaccine 18, 1873 - 1885.

[0174] In light of the foregoing, it will now be understood that, in another aspect, the present disclosure provides, in an example embodiment, a method for preparing a vaccine formulation for administration to a human, the vaccine formulation comprising (i) a TbpB protein, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encodinga modulated TbpB polypeptide, or an immunogenic portion thereof, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, orSEQ.ID NO: 24, ora C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the TbpB protein, or immunogenic portion thereof comprising the modulated C-lobe domain, or an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, comprising the modulated C-lobe domain; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the TbpB protein or immunogenic portion thereof, comprising the modulated C-lobe domain, or the mRNA polynucleotide encoding a modulated TbpBpolypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain, in a host cell;(B) introducing the chimeric nucleic acid sequence into the host cell;(C) growing the host cell to produce the TbpB protein, or immunogenic portion thereof, the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C- lobe domain; and(D) recovering (i) the TbpB protein, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof comprising the modulated C-lobe domain; and(E) formulating (i) the recovered TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding the modulated TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain, or the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain, to treat a human susceptible to infection by H. influenzae.

[0175] In at least embodiment, in an aspect, the modulated TbpB polypeptide can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or an amino acid sequence that is at least 90% identical thereto or an immunogenic portion thereof.

[0176] The vaccine formulations of the present disclosure may be used to prevent infection or disease caused by pathogenic H. influenzae strain in humans. The vaccine formulations further may be used to immunize any human, including a neonate, infant, toddler, child, adult, or elderly human.

[0177] The vaccine formulations of the present may be administered to a human using any convenient administration means. Thus, for example, the vaccine formulations may be injected, for example, intramuscularly or subcutaneously, or the vaccine formulations may be orally administered to the human, for example, as a food supplement. It will be understood that, in this respect, the administration means and techniques, such as, for example, in thecase of injections, the gauge of the injection needle, may vary depending on the human, for example, the age of the animal. The dosage of the vaccine formulation will be dependent upon the disease, the route of administration, the age, body weight, and other standard factors. In this respect, a person of ordinary skill in the art can readily titrate the appropriate dosage for an effective amount as well as select a suitable method of administration.

[0178] It is further noted that the vaccine formulations of the present disclosure may be administered prophylactically, i.e., in order to prevent infection caused by H. influenzae, or in order to ameliorate symptoms associated with an infection caused by H. influenzae following the occurrence of an infection in a human .

[0179] The administration of the vaccine formulations of the present disclosure generally elicits an immune response in the subject human. It will be understood that vaccine formulations comprising mRNAs encoding modulated TbpB proteins may upon administration to a subject human elicit an in vivo production of the modulated TbpB proteins in the subject human. Moreover, antibodies against the modulated TbpB polypeptide included in the vaccine formulation, or modulated TbpB proteins produced in vivo, may be formed by the human. In some embodiments, anti-TbpB antibodies can be detected in the blood serum of the human at least 13 weeks, at least 26 weeks, or at least 52 weeks following administration of the vaccine formulation.

[0180] Furthermore, the treatment or amelioration of the H. influenzae infection can result into a reduction of clinical signs of any disease caused by an H. influenzae infection. Thus, for example, the clinical signs can be clinical signs associated with pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia, and the reduction can be a reduction of clinical signs relative to a human infected by H. influenzae not having been administered the vaccine formulation.

[0181] In light of the foregoing, it will now be understood that, in another aspect, the present disclosure provides a use of a modulated TbpB polypeptide, or an immunogenic portion thereof, or an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, the modulated TbpB polypeptide comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, to prepare a vaccine formulation comprising the TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, comprising the modulated C-lobe domain, together with a pharmaceutically acceptable adjuvant.

[0182] In light of the foregoing, it will now further also be understood that, in another aspect, the present disclosure provides a use of a vaccine formulation comprising a modulated TbpB polypeptide, or an immunogenic portion thereof, or an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO:6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, together with a pharmaceutically acceptable adjuvant to ameliorate or prevent an infection caused by H. influenzae in a human susceptible to infection by H. influenzae.

[0183] As can now be understood, vaccine formulations comprising a modulated TbpB protein or an immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, may be prepared. The vaccine formulations can be administered to a human to ameliorate or prevent infection caused by H. influenzae.

[0184] Of course, the above described example embodiments of the present disclosure are intended to be illustrative and in no way limiting. The embodiments are susceptible to many modifications or composition, details, and order of operation. The invention and this disclosure is intended to encompass all such modifications within its scope, as defined by the claims, which should be given a broad interpretation consistent with the description as a whole.SUMMARY OF SEQUENCES

[0185] SEQ.ID NO: 1 and SEQ.ID NO: 2 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0186] SEQ.ID NO: 3 and SEQ.ID NO: 4 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0187] SEQ.ID NO: 5 and SEQ.ID NO: 6 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0188] SEQ.ID NO: 7 and SEQ.ID NO: 8 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0189] SEQ.ID NO: 9 and SEQ.ID NO: 10 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0190] SEQ.ID NO: 11 and SEQ.ID NO: 12 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 23 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0191] SEQ.ID NO: 13 and SEQ.ID NO: 14 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0192] SEQ.ID NO: 15 and SEQ.ID NO: 16 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0193] SEQ.ID NO: 17 and SEQ.ID NO: 18 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0194] SEQ.ID NO: 19 and SEQ.ID NO: 20 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0195] SEQ.ID NO: 21 and SEQ.ID NO: 22 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0196] SEQ.ID NO: 23 and SEQ.ID NO: 24 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 31 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0197] SEQ.ID NO: 25 and SEQ.ID NO: 26 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0198] SEQ.ID NO: 27 and SEQ.ID NO: 28 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0199] SEQ.ID NO: 29 and SEQ.ID NO: 30 set forth the polynucleotide sequence and deduced amino acid, respectively, of a N-terminal loop-flanking polypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0200] SEQ.ID NO: 31 and SEQ.ID NO: 32 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0201] SEQ.ID NO: 33 and SEQ.ID NO: 34 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flankingpolypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H040 (non- typeable).

[0202] SEQ.ID NO: 35 and SEQ.ID NO: 36 set forth the polynucleotide sequence and deduced amino acid, respectively, of a C-terminal loop-flanking polypeptide of loop 20 of a TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0203] SEQ.ID NO: 37 and SEQ.ID NO: 38 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide of H. influenzae strain H036 (serotype b) comprising a modulated loop 23 and loop 31.

[0204] SEQ.ID NO: 39 and SEQ.ID NO: 40 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide of H. influenzae strain H036 (serotype b) comprising a modulated loop 20, loop 23 and loop 31 .

[0205] SEQ.ID NO: 41 and SEQ.ID NO: 42 set forth the polynucleotide sequence and deduced amino acid, respectively, of a reference native TbpB polypeptide of H. influenzae strain H036 (serotype b).

[0206] SEQ.ID NO: 43 and SEQ.ID NO: 44 set forth the polynucleotide sequence and deduced amino acid, respectively, of a reference native TbpB polypeptide of H. influenzae strain H040 (non-typable).

[0207] SEQ.ID NO: 45 and SEQ.ID NO: 46 set forth the polynucleotide sequence and deduced amino acid, respectively, of a reference native TbpB polypeptide of H. influenzae strain H201 (serotype b).

[0208] Hereinafter are provided examples of specific implementations for performing the methods of the present disclosure, as well as implementations representing the compositions of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.EXAMPLESExample 1 - Preparation and immunological characterization of a first modulated H. influenzae TbpB polypeptideTbpB protein preparation:

[0209] A polynucleotide encoding an H. influenzae strain H036 TbpB polypeptide having SEQ.ID NO: 40, and lacking loops 20, 23 and 31 (the polypeptide also being referred to herein as “h036 LCL”), was cloned into a custom T7 expression vector (Addgene #: pE5770) that encodes an N-terminal maltose binding protein (MBP) fusion partner to maximize the stability and solubility of the recombinant protein. The vector also encodes a polyhistidine tag upstream of the MBP fusion partner to allow for purification of the protein by affinity chromatography. The LCL-encoding expression vector was then transformed into chemically competent E. coli strain ER2566 cells and the transformants used to inoculate 6 L of autoinduction medium. The inoculated medium was then incubated at 37°C for 18 h, followed by an incubation at 20°C for 24 h. Following this, the cells were lysed using a cell homogenizer (Avestin) and the recombinant MBP- TbpBs purified from the resulting crude lysate using a HisTrap Ni-NTA column (Cytiva). TEV protease was then used to separate the TbpB antigen from its MBP fusion partner and the cleaved TbpB subsequently isolated from MBP using anion- exchange chromatography (HiTrap column; Cytiva). The purified TbpB was stored in 100-pL aliquots at -80°C until needed to prepare a vaccine formulation.Mouse immunizations:

[0210] Immediately prior to immunization, the purified LCL was thawed and diluted to a concentration of 0.5 mg / mL in Dulbecco’s phosphate-buffered saline (D-PBS, with calcium and magnesium; Multicell). The diluted protein was then mixed with the squalene-based oil-in water emulsion adjuvant AddaVax (InvivoGen) to prepare a vaccine formulation, such that each vaccine dose contained 50% v / v AddaVax. Eight male C57BL / 6 mice (Charles River, 6 weeks old) were each immunized with 100 pL of the vaccine formulation, which amounted to a dose of 25 pg of protein. Vaccine doses were administered on days 0, 21 , and 42 via the intraperitoneal route. Following the third immunization, the mice were euthanized on day 56 and a final blood collection conducted using an intracardiac bleed. Antiserum was then obtained from whole blood by centrifugation.Detection of anti-TbpB IgG titres by enzyme-linked immunosorbent assay (ELISA):

[0211] Lysates containing wild-type H. influenzae TbpB variants were prepared for coating ELISA plates essentially as described above except for instead of subjecting the lysates to further purification by affinity chromatography via an Ni-NTA column, the crude lysates were diluted by a factor of 5 using PBS with 0.05% Tween-20 (PBST; Sigma-Aldrich), afterwhich 100 pL of diluted lysate was added to each well of a 96-well streptavidin-coated ELISA plate (Greiner BioOne). The lysate-coated plates were then incubated overnight at 4°C. Next, the plates were washed three times with 250 pL PBST (all wash steps were conducted thrice with the same volume of PBST) and subsequently blocked using 200 pL / well 5% skim milk (all skim milk solutions mentioned herein were diluted in PBST) for 2 h at room temperature. Following another wash step, the wells were subsequently coated with 100 pL of antiserum diluted in 2.5% skim milk and the plates incubated overnight at 4°C. The next day, the plates were washed and then treated with 100 pL / well of HRP-labelled goat anti-mouse IgG secondary antibody solution (Sigma-Aldrich) diluted by a factor of 10,000 in 2.5% skim milk and the plates incubated for 1 h at room temperature. A final wash step was conducted, and the plates were then developed via the addition of 50 pL of 3, 3’, 5,5’- tetramethylbenzidine (TMB; Sigma-Aldrich) substrate solution to each well and subsequent incubation for 20 min in a dark compartment at room temperature. The reaction was then quenched by adding 25 pL of 4 N HCI to each well and the resulting OD450 absorbance values determined using an ELISA plate reader (BioTek Synergy HTX Multimode Reader; Agilent).

[0212] To ascertain the approximate number of anti-TbpB IgG molecules in each serum sample - and thereby gain important insights as to the immunogenicity of the protein antigen - the endpoint titre was determined for each sample. To this end, twofold serial dilutions were performed with a starting dilution of 1 / 200. The endpoint titre was defined as the reciprocal of the last dilution in the series that produced an absorbance value that was greater than double the absorbance value of the negative control, which consisted of wells that had been treated with every step / reagent in the ELISA protocol except for the addition of serum ( / .e., a “no serum” control). If the endpoint titre was not reached in the initial dilution series,the ELISA was repeated with a higher starting dilution. A positive control well consisting of lysate-coated wells treated with human transferrin conjugated to horseradish peroxidase (hTf-HRP) instead of the secondary antibody was also used in the ELISA to ensure consistent coating of ELISA wells with wild-type TbpBs. The anti-TbpB titres elicited by the h036 LCL were compared to those elicited by the wild-type protein - both in the context of a monovalent vaccine (“h036 WT TbpB” (SEQ.ID NO: 42)) and a trivalent vaccine formulation consisting of the h036, h040, and h201 wild-type TbpBs (“Trivalent”) (SEQ.ID NO: 42, SEQ.ID NO: 44, and SEQ.ID NO: 46, respectively) - and those detected in mice immunized with the adjuvant alone (“Adjuvant only”).

[0213] Referring to FIG. 4, shown therein is a graph showing anti-h036 TbpB IgG titres elicited by a monovalent h036 TbpB vaccine formulation, a trivalent vaccine formulation containing the h036, h040, and h201 wild-type TbpBs, and a vaccine formulation containing the modulated TbpB h036 LCL. Endpoint titres are defined as the reciprocal of the final dilution in the series that produces an absorbance value that is greater than double the absorbance value produced by the negative (no serum) control. Significance was determined using the Brown- Forsythe and Welch ANOVA tests, followed by a Dunnett’s T3 multiple comparisons test comparing each group to the “adjuvant only” group (*p<0.05, **p<0.01 ).

[0214] The cross-reactivity of the antiserum elicited by the modulated TbpB h036 LCL when tested against a panel of 15 wild-type H. influenzae TbpBs was also compared with the cross-reactivity of the antiserum elicited by the trivalent vaccine (FIGS. 5A - 5C). Given the large number of variants used in the crossreactivity ELISA, the results of this assay are expressed as fractions of the absorbance values detected in the hTf-HRP control wells instead of as endpoint titres. Furthermore, referring to FIGS. 5A - 5C, shown, in a quantitative manner, is the cross-reactivity between antisera elicited by a vaccine formulation comprising the modulated polypeptide h036 LCL (FIG. 5A) and a trivalent vaccine formulation containing the h036, h040, and h201 wild-type TbpBs (FIG. 5B), and the panel of immobilized 15 wild-type H. influenzae TbpB variants. In FIG. 5C, the results shown in FIGS. 5A and 5B are more directly compared. The TbpB polypeptides used to coat ELISA plates in this experiment that matched those included in the vaccine formulation are highlighted by arrows. A serum dilutionof 1 / 1 ,000 was used for all samples assessed here. Absorbance readings at 450 nm (OD450) were normalized to account for slight differences in coating of each TbpB variant by expressing each value as a fraction of the absorbance value produced by the binding of HRP-labelled hTf to each variant. Significance was determined using the Brown-Forsythe and Welch ANOVA tests, followed by a Dunnett’s T3 multiple comparisons test comparing each mean to the “mock lysate” mean (*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001).

[0215] As can be seen in FIGS. 5A - 5C, surprisingly, the vaccine formulation comprising the single modulated h036 LCL polypeptide exhibits substantial cross-reactivity against all of the 15 wild-type H. influenzae TbpB variants, notably a HO H. influenzae strain, a H026 H. influenzae strain, a H040 H. influenzae strain, a H210 H. influenzae strain, a H036 H. influenzae strain, a WP050 H. influenzae strain, a WP11880 H. influenzae strain, a PRI75995 H. influenzae strain, a WP111 H. influenzae strain, a WP11887 H. influenzae strain, a PRI69814 H. influenzae strain, a WP221 H. influenzae strain, and a WP112 H. influenzae strain, while, in contrast, the vaccine formulation comprising three wildtype TbpB polypeptides exhibited limited cross-reactivity to a substantial number of the 15 wild-type H. influenzae TbpB variants.Example 2 - Preparation of a second modulated H. influenzae TbpB polypeptide

[0216] A polynucleotide encoding an H. influenzae strain H036 TbpB polypeptide having SEQ.ID NO: 38, the TbpB polypeptide lacking loops 23 and 31 , can be cloned into a custom T7 expression vector (Addgene #: pE5770) that encodes an N-terminal maltose binding protein (MBP) fusion partner to maximize the stability and solubility of the recombinant protein. The vector also encodes a polyhistidine tag upstream of the MBP fusion partner to allow for purification of the protein by affinity chromatography. The LCL-encoding expression vector can then be transformed into chemically competent E. coli strain ER2566 cells and the transformants can be used to inoculate 6 L of autoinduction medium. The inoculated medium can then be incubated at 37°C for 18 h, followed by an incubation at 20°C for 24 h. Following this, the cells can be lysed using a cell homogenizer (Avestin) and the recombinant MBP-TbpBs purified from theresulting crude lysate using a HisTrap Ni-NTA column (Cytiva). TEV protease can then be used to separate the TbpB antigen from its MBP fusion partner and the cleaved TbpB polypeptide can subsequently be isolated from MBP using anion- exchange chromatography (HiTrap column; Cytiva). The purified TbpB polypeptide can be used to prepare a vaccine formulation, or alternatively can be stored in 1OO-pL aliquots at -80°C, as necessary.

Claims

CLAIMS1. A vaccine formulation comprising an effective amount of (i) a Transferrin Binding Protein B (TbpB) protein, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, orSEQ.ID NO: 24, ora C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues.

2. A vaccine formulation according to claim 1 , wherein the modulated C-lobe domain, further comprises:(iii) a third joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 26, SEQ.ID NO: 28, or SEQ.ID NO: 30, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptidehaving SEQ.ID NO: 32, SEQ.ID NO: 34, or SEQ.ID NO: 36, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 7 amino acid residues.

3. A vaccine formulation according to claims 1 or 2, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 38 and SEQ.ID NO: 40, or a polypeptide comprising an amino acid sequence that is at least 90% identical thereto.

4. A vaccine formulation according to claims 1 or 2, wherein the infecting H. influenzae strain is a typable or non-typable H. influenzae strain.

5. A vaccine formulation according to claims 1 or 2, wherein the typable H. influenzae strain is a serotype a strain, a serotype b strain, a serotype c strain, a serotype d strain, a serotype e strain, or a serotype f strain.

6. A vaccine formulation according to claims 1 or 2, wherein the infecting H. influenzae strain is capable of causing pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia in a human.

7. A vaccine formulation according to claims 1 or 2, wherein the administration of the vaccine formulation to a human in need thereof prevents clinical signs or result in a reduction of clinical signs of any disease caused by the H. influenzae infection.

8. A vaccine formulation according to claim 7, wherein the clinical signs are clinical signs associated with a disease selected from the group consisting of pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia, relative to a human infected by H. influenzae not having been administered the vaccine formulation.

9. A vaccine formulation according to claims 1 or 2, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is a recombinantly produced polypeptide.

10. A vaccine formulation according to claims 1 or 2, wherein the vaccine formulation is cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by a second H. influenzae strain.

11. A vaccine formulation according to claims 1 or 2, wherein the vaccine formulation is cross-protective, wherein the TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain is from a H036 H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent or ameliorate an infection caused by a H. influenzae strain selected from the group consisting of a HO H. influenzae strain, a H026 H. influenzae strain, a H040 H. influenzae strain, a H210 H. influenzae strain, a H036 H. influenzae strain, a WP050 H. influenzae strain, a WP11880 H. influenzae strain, a PRI75995 H. influenzae strain, a WP111 H. influenzae strain, a WP11887 H. influenzae strain, a PRI69814 H. influenzae strain, a WP221 H. influenzae strain, and a WP112 H. influenzae strain.

12. A vaccine formulation according to claims 1 or 2, wherein the vaccine formulation further comprises a pharmaceutically acceptable adjuvant.

13. A vaccine formulation according to claims 1 or 2, wherein the vaccine formulation further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

14. A vaccine formulation according to claims 1 or 2, wherein the vaccine formulation omprises from about 0.001 % to about 20% by weight per volume of the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

15. A use a vaccine formulation according to any one of claims 1 to 14, for the prevention, treatment, or amelioration of H. influenzae infection in a human in need thereof.

16. A use according to claim 15, wherein the use of the vaccine formulation in a human in need thereof prevents clinical signs or results in a reduction of clinical signs of any disease caused by the H. influenzae infection.

17. A use according to claim 16, wherein the clinical signs are clinical signs associated with a disease selected from the group consisting of pneumonia, meningitis, bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia, relative to a human infected by H. influenzae not having been administered the vaccine formulation.

18. A method for preparing a vaccine formulation for administration to a human, the vaccine formulation comprising (i) a TbpB protein, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identicalthereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, orSEQ.ID NO: 24, ora C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the TbpB protein or immunogenic portion thereof, comprising the modulated C-lobe domain, in a host cell;(B) introducing the chimeric nucleic acid sequence into the host cell;(C) growing the host cell to produce the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain or an mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain; and(D) recovering (i) the TbpB protein, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof comprising the modulated C-lobe domain; and(E) formulating (i) the recovered TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding the modulated TbpB polypeptide, or immunogenic portion thereof, comprising the modulated C-lobe domain together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of (i) the TbpB protein, or immunogenic portion thereof, comprising the modulated C-lobe domain, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, comprisingthe modulated C-lobe domain, to treat a human susceptible to infection by H. influenzae.

19. An expression vector comprising:(a) a nucleic acid sequence encoding (i) a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N- terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues,(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the TbpB polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) the mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

20. A host cell comprising a chimeric nucleic acid comprising:(a) a nucleic acid sequence encoding (i) a TbpB polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) an mRNA polynucleotide encoding a TbpB a polypeptide, or an immunogenic portion thereof, comprising a modulated C-lobe domain, the modulated C-lobe domain comprising:(i) a first joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or a C- terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C- terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues; and(ii) a second joining polypeptide comprising an N-terminal loop flanking polypeptide having SEQ.ID NO: 14, SEQ.ID NO: 16, or SEQ.ID NO: 18, or an N-terminal loop flanking polypeptide that is at least 90% identical thereto, joined to a C-terminal loop flanking polypeptide having SEQ.ID NO: 20, SEQ.ID NO: 22, or SEQ.ID NO: 24, or a C-terminal loop flanking polypeptide that is at least 90% identical thereto, wherein the N-terminal loop flanking polypeptide and the C-terminal loop flanking polypeptide are contiguously joined, or joined by a peptide linker separating the N-terminal and C-terminal loop flanking polypeptides, the peptide linker containing 1 to 10 amino acid residues, and(b) a nucleic acid sequence capable of controlling expression of (i) the TbpB polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, or (ii) the nucleic acid sequence encoding the mRNA polynucleotide encoding a TbpB a polypeptide, or immunogenic portion thereof, comprising a modulated C-lobe domain, in a host cell.

Images