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. The vaccine compositions may be used to prevent, treat, or ameliorate 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 / 699,544 filed September 26, 2024; the entire contents of Patent Application No. 63 / 699,544 are hereby incorporated by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “96573166 Sequence Listing. xml” (77,709 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, treat, 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 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 infection in a human, the method comprising administering a vaccine formulation to a human in need thereof, the vaccine formulation comprising (i) a modulated Transferrin Binding Protein B (TbpB) polypeptide or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, wherein the vaccine formulation is administered to a 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 corresponding reference TbpB polypeptide can be selected from a TbpB polypeptide expressed by a nucleic acid sequence selected from:(a) SEQ.ID NO: 1 , SEQ.ID NO: 3, SEQ. ID NO: 5, SEQ.ID NO: 7, SEQ.ID NO: 9, or SEQ. ID NO: 11 ;(b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a);(c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code;(d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a);(e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, or SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or an immunogenic portion thereof;(f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12 or an immunogenic portion thereof; and(g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f), provided however that, the corresponding reference TbpB polypeptide comprises at least one of(i) a phenylalanine amino acid 55 residue or a phenylalanine amino acid residue located at an equivalent position;(ii) a phenylalanine amino acid 127 residue or a phenylalanine amino acid residue located at an equivalent position;(iii) an arginine amino acid 128 residue or an arginine amino acid residue located at an equivalent position;(iv) a tyrosine amino acid 137 residue or a tyrosine amino acid residue located at an equivalent position;(v) an arginine amino acid 175 residue or an arginine amino acid residue located at an equivalent position;(vi) an arginine amino acid 180 residue, or an arginine amino acid residue located at an equivalent position;(vii) a tyrosine amino acid 186 residue, or a tyrosine amino acid residue located at an equivalent position;(viii) an arginine amino acid 188 residue, or an arginine amino acid residue located at an equivalent position;(ix) an arginine amino acid 189 residue, or an arginine amino acid residue located at an equivalent position;(x) a glutamate amino acid 194 residue, or a glutamate amino acid residue located at an equivalent position;(xi) an aspartate amino acid 197 residue, or an aspartate amino acid residue located at an equivalent position; and(xii) a glutamate amino acid 204 residue, or a glutamate amino acid residue located at an equivalent position.

[0017] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to twenty five amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0018] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to ten amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0019] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to six amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0020] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one or two amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide.

[0021] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, a single amino acid residue can be modulated relative to the corresponding reference TbpB polypeptide.

[0022] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine,proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 55 can be removed.

[0023] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0024] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 127 can be removed.

[0025] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0026] In at least one embodiment, in an aspect, the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 128 can be removed.

[0027] In at least one embodiment, in an aspect, the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the correspondingreference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0028] In at least one embodiment, in an aspect, the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 137 can be removed.

[0029] In at least one embodiment, in an aspect, the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0030] In at least one embodiment, in an aspect, the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 175 can be removed.

[0031] In at least one embodiment, in an aspect, the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0032] In at least one embodiment, in an aspect, the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 180 can be removed.

[0033] In at least one embodiment, in an aspect, the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0034] In at least one embodiment, in an aspect, the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 186 can be removed.

[0035] In at least one embodiment, in an aspect, the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0036] In at least one embodiment, in an aspect, the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 188 can be removed.

[0037] In at least one embodiment, in an aspect, the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0038] In at least one embodiment, in an aspect, the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 189 can be removed.

[0039] In at least one embodiment, in an aspect, the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0040] In at least one embodiment, in an aspect, the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 194 can be removed.

[0041] In at least one embodiment, in an aspect, the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0042] In at least one embodiment, in an aspect, the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the aspartate amino acid residue 197 can be removed.

[0043] In at least one embodiment, in an aspect, the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0044] In at least one embodiment, in an aspect, the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 204 can be removed.

[0045] In at least one embodiment, in an aspect, the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0046] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0047] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be a typable or a non-typable H. influenzae strain.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In at least one embodiment, in an aspect, the clinical signs dan be selected from clinical signs associated with the group of diseases 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.

[0052] In at least one embodiment, in an aspect, the modulated TbpB polypeptide, or immunogenic portion thereof, can be a recombinantly produced polypeptide.

[0053] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the reference TbpB polypeptide corresponding with the modulated TbpB polypeptide is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent, treat, or ameliorate an infection caused by a second H. influenzae strain.

[0054] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable adjuvant.

[0055] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable excipient, carrier, or diluent.

[0056] 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 modulated TbpB protein, or the immunogenic portion thereof, and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

[0057] 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 (i) an effective amount of a modulated TbpB protein, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(I) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0058] In at least one embodiment, in an aspect, the corresponding reference TbpB polypeptide can be selected from a TbpB polypeptide expressed by a nucleic acid sequence selected from:.(a) SEQ.ID NO: 1 , SEQ.ID NO: 3, SEQ. ID NO: 5, SEQ.ID NO: 7, SEQ.ID NO: 9, or SEQ. ID NO: 11 ;(b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a);(c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code;(d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a);(e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ. ID NO: 12, or an immunogenic portion thereof;(f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ. ID NO: 12, or an immunogenic portion thereof; and(g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f), provided however that, the corresponding reference TbpB polypeptide comprises at least one of(i) a phenylalanine amino acid 55 residue or a phenylalanine amino acid residue located at an equivalent position;(ii) a phenylalanine amino acid 127 residue or a phenylalanine amino acid residue located at an equivalent position;(iii) an arginine amino acid 128 residue or an arginine amino acid residue located at an equivalent position;(iv) a tyrosine amino acid 137 residue or a tyrosine amino acid residue located at an equivalent position;(v) an arginine amino acid 175 residue or an arginine amino acid residue located at an equivalent position;(vi) an arginine amino acid 180 residue, or an arginine amino acid residue located at an equivalent position;(vii) a tyrosine amino acid 186 residue, or a tyrosine amino acid residue located at an equivalent position;(viii) an arginine amino acid 188 residue, or an arginine amino acid residue located at an equivalent position;(ix) an arginine amino acid 189 residue, or an arginine amino acid residue located at an equivalent position;(x) a glutamate amino acid 194 residue, or a glutamate amino acid residue located at an equivalent position;(xi) an aspartate amino acid 197 residue, or an aspartate amino acid residue located at an equivalent position; and(xii) a glutamate amino acid 204 residue, or a glutamate amino acid residue located at an equivalent position.

[0059] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to twenty five amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0060] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to ten amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0061] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one to six amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

[0062] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, one or two amino acid residues can be modulated relative to the corresponding reference TbpB polypeptide.

[0063] In at least one embodiment, in an aspect, in the modulated TbpB polypeptide, or immunogenic portion thereof, a single amino acid residue can be modulated relative to the corresponding reference TbpB polypeptide.

[0064] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 55 can be removed.

[0065] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0066] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 127 can be removed.

[0067] In at least one embodiment, in an aspect, the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0068] In at least one embodiment, in an aspect, the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 128 can be removed.

[0069] In at least one embodiment, in an aspect, the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0070] In at least one embodiment, in an aspect, the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 137 can be removed.

[0071] In at least one embodiment, in an aspect, the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0072] In at least one embodiment, in an aspect, the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 175 can be removed.

[0073] In at least one embodiment, in an aspect, the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0074] In at least one embodiment, in an aspect, the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 180 can be removed.

[0075] In at least one embodiment, in an aspect, the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0076] In at least one embodiment, in an aspect, the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 186 can be removed.

[0077] In at least one embodiment, in an aspect, the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0078] In at least one embodiment, in an aspect, the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 188 can be removed.

[0079] In at least one embodiment, in an aspect, the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0080] In at least one embodiment, in an aspect, the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 189 can be removed.

[0081] In at least one embodiment, in an aspect, the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0082] In at least one embodiment, in an aspect, the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 194 can be removed.

[0083] In at least one embodiment, in an aspect, the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0084] In at least one embodiment, in an aspect, the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the correspondingreference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the aspartate amino acid residue 197 can be removed.

[0085] In at least one embodiment, in an aspect, the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0086] In at least one embodiment, in an aspect, the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 204 can be removed.

[0087] In at least one embodiment, in an aspect, the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0088] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0089] In at least one embodiment, in an aspect, the infecting H. influenzae strain can be a typable or non-typable H. influenzae strain.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In at least one embodiment, in an aspect, the clinical signs can be selected from clinical signs associated with the group of diseases 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.

[0094] In at least one embodiment, in an aspect, the modulated TbpB polypeptide, or immunogenic portion thereof, can be a recombinantly produced polypeptide.

[0095] In at least one embodiment, in an aspect, the vaccine formulation can be cross-protective, wherein the reference TbpB polypeptide corresponding with the modulated TbpB polypeptide is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent, treat, or ameliorate an infection caused by a second H. influenzae strain.

[0096] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable adjuvant.

[0097] In at least one embodiment, in an aspect, the vaccine formulation can further comprise a pharmaceutically acceptable excipient, carrier, or diluent.

[0098] 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 modulated TbpB protein, or the immunogenic portion thereof, and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

[0099] 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 infection in a human in need thereof, the vaccine formulation comprising an effective amount of (i) a modulated Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding amodulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, wherein the vaccine formulation is administered to a human in an effective amount to prevent, treat, or ameliorate the infection by the H. influenzae.

[0100] 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.

[0101] In at least one embodiment, in an aspect, the clinical signs can be selected from clinical signs associated with the group of diseases 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.

[0102] 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 (i) a modulated TbpB a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0103] In another aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, 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 modulated TbpB a Transferrin Binding Protein B (TbpB) polypeptide, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0104] 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 modulated TbpB protein, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the modulated TbpB protein, or immunogenic portion thereof; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the modulated 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 (i) the modulated TbpB protein, or immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(D) recovering (i) the modulated TbpB protein, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(E) formulating (i) the recovered modulated TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of (i) the modulated protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, to prevent, treat, or ameliorate an H. influenzae infection in a human.

[0105] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0106] In another aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, an expression vector comprising:(a) a nucleic acid sequence encoding (i) a modulated TbpB polypeptide or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof,; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the modulated TbpB polypeptide, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding aTbpB polypeptide, or an immunogenic portion thereof, in a host cell,wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0107] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0108] 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, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof,; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the modulated TbpB polypeptide, or immunogenic portion thereof or (ii) the mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0109] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0110] Other features and advantages will become apparent from the following detailed description. It should be understood, however, that the detaileddescription, 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

[0111] 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.

[0112] FIG. 1 depicts an amino acid sequence alignment of various example reference H. influenzae TbpB polypeptides, notably TbpB polypeptides obtained from H. influenzae strain Hi036, denoted as “HiO36TbpB”, strain Hi201 , denoted as “Hi201TbpB”, strain Hi038, denoted as “HiO38TbpB”, strain Hi-40, denoted as “Hi40TbpB”, strain Hi210, denoted as “Hi210TbpB”, and strain Hi216, denoted as “Hi216TbpB”. Highlighted are: in reference TbpB polypeptide Hi210TbpB, phenylalanine amino acid residue 55 (denoted as “E”); in reference TbpB polypeptide Hi216TbpB, phenylalanine amino acid residue 127 (denoted as “E”); in reference TbpB polypeptide Hi210TbpB, arginine amino acid residue 128 (denoted as “E”), or an arginine residue located in an equivalent position (denoted as “R”); in reference TbpB polypeptide HiO38TbpB, tyrosine amino acid residue 137 (denoted as “E”), or a tyrosine residue located in an equivalent position (denoted as “Y”); in reference TbpB polypeptide Hi201TbpB, arginine amino acid residue 175 (denoted as “E”), or an arginine residue located in an equivalent position (denoted as “E”); in reference TbpB polypeptide HiO36TbpB, arginine amino acid residue 180 (denoted as “ E”); in reference TbpB polypeptide Hi210TbpB, tyrosine amino acid residue 186 (denoted as “E”), or a tyrosine residue located in an equivalent position (denoted as “Y”); in reference TbpB polypeptide Hi201TbpB, arginine amino acid residue 188 (denoted as “E”), or an arginine residue located in an equivalent position (denoted as “R”); in reference TbpB polypeptide Hi201TbpB, arginine amino acid residue 189 (denoted as “E”),or an arginine residue located in an equivalent position (denoted as “R” or “E”); in reference TbpB polypeptide Hi201TbpB glutamate amino acid residue 194 (denoted as “E ”), or a glutamate residue located in an equivalent position (denoted as “E” or “EEJ”); in reference TbpB polypeptide Hi201TbpB is aspartate amino acid residue 197 (denoted as “E”), or a aspartate residue located in an equivalent position (denoted as “D” or “E’); and in reference TbpB polypeptide Hi216TbpB, glutamate amino acid residue 204 (denoted as “E”), or a glutamate residue located in an equivalent position (denoted as “E”). Boxed residues are residues referenced in the Example section. To illustrate sequence identity, amino residues denoted as “*” are identical in each of the six TbpB polypeptide sequences; amino acid residues denoted as are chemically comparable in each of the six polypeptide sequences.

[0113] FIGS. 2A - 2L depict multiple nitrocellulose membranes representing certain experimental results obtained in the evaluation of example modulated TbpB polypeptides. Notably, the nitrocellulose membranes were obtained in the performance of nitrocellulose solid-phase assays to evaluate the binding between transferrin and multiple example modulated TbpB polypeptides and a corresponding reference TbpB polypeptide. In each of FIGS. 2A - 2L a nitrocellulose membrane is shown, wherein visual detection of a signal on a nitrocellulose membrane signifies binding between a TbpB polypeptide and transferrin, and wherein, conversely, the absence of a signal signifies a lack of binding between a TbpB polypeptide and transferrin. Compared are transferrin binding to a corresponding reference TbpB (labeled “WT”) and transferrin binding to example modulated TbpB polypeptides, notably: a Y55A TbpB polypeptide (labeled “Y55A”) (FIG. 2A); a F127A TbpB polypeptide (labeled “F127A”) (FIG. 2B); a R128E TbpB polypeptide (labeled “R128E”) (FIG. 2C); a Y137A TbpB polypeptide (labeled “Y137A”) (FIG. 2D); a R175DA TbpB polypeptide (labeled “R175D”) (FIG. 2E); a R180E TbpB polypeptide (labeled “R180E”) (FIG. 2F); a Y186A TbpB polypeptide (labeled “Y186A”) (FIG. 2G); a R188E TbpB polypeptide (labeled “R188E”) (FIG. 2H); a R189E TbpB polypeptide (labeled “R189E”) (FIG. 2I); a E194R TbpB polypeptide (labeled “E194R”) (FIG. 2J); a D197R TbpBpolypeptide (labeled “D197R”) (FIG. 2K); and a E204R TbpB polypeptide (labeled “E204R”) (FIG. 2L).

[0114] FIG. 3 is a graph obtained in the performance of certain experiments, notably a bar graph showing the endpoint titre of anti-TbpB antibodies present in mice serum following immunization with a formulation comprising reference TbpB from H. influenzae strain H036 and adjuvant (labeled “H036 WT TbpB"), a formulation comprising a modulated R180E TbpB from H. influenzae strain H036 and adjuvant (labeled “H036 R180E TbpB"), and a formulation comprising adjuvant alone (labeled "AddaVax only”).

[0115] 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

[0116] 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.

[0117] 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”.

[0118] 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.

[0119] 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.

[0120] 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

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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: 1. 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 hybridization conditions 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” includes nucleic acid sequences encoding reference TbpB polypeptides and nucleic acid sequences encoding modulated TbpB polypeptides.

[0125] The terms “nucleic acid sequence encoding a reference TbpB”, and “nucleic acid sequence encoding a reference TbpB polypeptide”, and “nucleic acid sequence encoding a reference TbpB protein”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a reference TbpB polypeptide, including, for example, SEQ.ID NO: 1. Nucleic acid sequences encoding a reference TbpB polypeptide may be naturally occurring in H. influenzae, and isolated from H. influenzae. Nucleic acid sequences encoding a reference TbpB polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the reference TbpB polypeptide sequences set forth herein; or (ii) hybridize to any reference TbpB nucleic acid sequences set forth herein 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.

[0126] The terms “nucleic acid sequence encoding a modulated TbpB”, and “nucleic acid sequence encoding a modulated TbpB polypeptide”, and “nucleic acid sequence encoding a modulated TbpB protein”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a modulated TbpB polypeptide set forth herein, including, for example, SEQ.ID NO: 14. Nucleic acid sequences encoding a modulated TbpB polypeptide furtherinclude any nucleic acid sequences encoding a modulated TbpB polypeptide wherein one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, the modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0127] 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: 2, 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. Furthermore, the term “TbpB polypeptides” includes reference TbpB polypeptides and modulated TbpB polypeptides.

[0128] The terms “reference TbpB polypeptide ” or “reference TbpB protein”, or “reference TbpB”, as may be used herein interchangeably, refer to any and all proteins comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any reference TbpB polypeptide set forth herein, including, for example, SEQ.ID NO: 2, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any reference TbpB polypeptide set forth herein, but for the use of synonymous codons.

[0129] The terms “modulated TbpB polypeptide” or “modulated TbpB protein”, as interchangeably used herein, refer to a TbpB polypeptide wherein, relative to a reference TbpB polypeptide, at least one amino acid has been modified, including all modulated TbpB polypeptide sequences set forth herein, for example, SEQ.ID NO: 14. In order to determine whether a polypeptide is a modulated TbpB polypeptide, the amino acid sequence of the modulated TbpB polypeptide may be aligned with an amino acid sequence of a reference TbpB polypeptide, and the aligned sequences may be evaluated for potential differences in amino acid residues between the aligned sequences. Modulated TbpB polypeptides further include TbpB polypeptides wherein, one or more amino acidresidues are modulated relative to a corresponding reference TbpB polypeptide, the one or more modulated amino acid residues being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 10, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 12, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 10, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 6, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 2, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 10, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 4, or an arginine amino acidresidue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 2, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 4, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, including SEQ.ID NO: 12, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0130] 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.

[0131] 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 skilled in 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 (Log10 [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.

[0132] The term “functional variant”, as used herein in reference to polynucleotides or polypeptides, refers to polynucleotides or polypeptides capable of performing the same function as a noted reference polynucleotide or polypeptide. Thus, for example, a functional variant of the polypeptide set forth in SEQ.ID NO: 2, refers to a polypeptide capable of performing the same function as the polypeptide set forth in SEQ.ID NO: 2. Functional variants include modified a polypeptide wherein, relative to a noted reference polypeptide, the modification includes a substitution, deletion, or addition of one or more amino acids. In some embodiments, substitutions are those that result in a replacement of one amino acid with an amino acid having similar characteristics. Such substitutions include, without limitation substitutions of (i) glutamate and aspartate amino acid residues; (ii) tyrosine, and phenylalanine alanine amino acid residues.

[0133] 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.

[0134] The terms “Haemophilus influenzae” or “H. influenzae”, as interchangeably used herein, refer to any bacteria belonging the bacterial species 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 denotesa H. influenzae strain serotypable as serotype b. Non-serotypable H. influenzae strains may be referred to as NTHi strains.

[0135] 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.

[0136] The terms “immunologically equivalent” or “immunogenic portion”, as used herein, refer to a molecule 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, wherein the binding specificity to the native polyclonal antibodies is comparable to the specificity of native polyclonal antibodies produced when a reference molecule is administered to the subject human. For example, immunologically equivalent portions or immunogenic portions of a full length modulated TbpB polypeptide include immunogenic portions of a modulated TbpB polypeptide which when administered to a subject human elicit a humoral immune response in the form of the production of native antibodies with a specificity to the immunogenic portion of the modulated TbpB polypeptide which is comparable to the binding specificity for a modulated TbpB polypeptide of native antibodies obtained when the corresponding full length modulated TbpB is administered to the human. To compare binding specificity between an immunologically equivalent molecule and a corresponding molecule a radioimmune assay (RIA) may be used, and the extent of binding may be measured. The dissociation constant of an immunologically equivalent molecule is preferably at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dissociation constant of the corresponding molecule.

[0137] 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 a modulated TbpB protein, for example, 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.

[0138] 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).

[0139] 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.

[0140] The terms “treating” and “treatment”, and the like, as used herein, are intended to mean obtaining a desirable physiological, pharmacological, or biological effect. The effect may result in the prevention ( / .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 physiologicaleffect may include, for example, an improved respiratory capacity or lung function, reduced hemorrhaging, reduced mucoid nasal or oral discharge.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] The terms “substantially pure” and “isolated”, as may be used interchangeably herein, describe a compound, e.g., a polypeptide, which has been separated 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 byany appropriate method, e.g., in the case of polypeptides, by chromatography, gel electrophoresis or HPLC analysis.General implementation

[0150] 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.

[0151] The vaccine formulations of the present disclosure are surprisingly potent and can, for example, block binding of human transferrin to TbpB during the immunization process e.g., in a manner more effective than the wildtype reference TbpB polypeptide.

[0152] The binding characteristics of wildtype TbpB polypeptide or modulated TbpB for human transferrin, may be determined, for example, by quantitatively evaluating the dissociation constant (Kd) between TbpB polypeptides and human transferrin, for example, by isothermal calorimetric techniques, surface plasmon resonance, or biolayer interference or other suitable techniques (Rich, R.L. and Myszka, D.G., 2007, Analytical Biochem. 316: 1- 6.; Abdiche, Y. et al., 2008, Anal. Biochem. 377: 209-127; and Velazquez-Campoy A., et al., 2004, Methods Mol. Biol., 261 : 35-54). For example, the Kd between a modulated TbpB polypeptide and a native human transferrin may exceed the Kd between the native non-modulated TbpB polypeptide, and native human transferrin. The binding characteristics of wildtype TbpB polypeptide or modulated TbpB for human transferrin may also be determined qualitatively, for example, by solid phase transferrin binding assays, as described for example, in Example 1.

[0153] Furthermore, the vaccine formulations can provide surprisingly high antibody titres.

[0154] 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 of live attenuated microbial species, and thus avoid infection risks associated with the use of live vaccines.

[0155] 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.

[0156] 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.

[0157] 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, treat, or ameliorate infections caused by H. influenzae in humans. In what follows example embodiments of the compositions and methods of the present disclosure are described.

[0158] Thus, the present disclosure provides, in at least one aspect, in at least one embodiment, a method for treatment of an H. influenzae in a human, 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 polypeptide, or an immunogenic portion thereof, wherein the vaccine formulation is administered in an effective amount to prevent, treat, or ameliorate the infection caused by the H. influenzae.

[0159] The present disclosure further provides, in at least one aspect, in at least one embodiment, a vaccine formulation for the prevention, treatment, or amelioration of an H. influenzae infection in a human, the vaccine formulation comprising (i) an effective amount of a modulated TbpB polypeptide or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof.

[0160] 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 polypeptide, or animmunogenic portion thereof, can be prepared or obtained. These formulations can, 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 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 and (ii) mRNA polynucleotides encoding a modulated TbpB 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 polypeptide, or an immunogenic portion thereof, and suitable example methods of preparing vaccine formulations and administering the same to a human in need thereof will be described.

[0161] In general, modulated TbpB polypeptides in accordance with the present disclosure include TbpB polypeptides wherein in the amino acid sequence constituting the modulated TbpB polypeptide one or more amino acid residues are modulated relative to the amino acid sequence of a corresponding reference TbpB polypeptide. 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 reference TbpB polypeptide. Modulated TbpB polypeptides in accordance herewith may then be prepared or obtained by modification of the amino acid sequence relative the reference TbpB polypeptide. Thus, next suitable reference TbpB polypeptides and modulated TbpB polypeptides will be discussed.

[0162] Initially it is noted that, the TbpB polypeptides which may be used to prepare vaccine formulations, in accordance with the present disclosure, are modulated TbpB polypeptides. In such modulated TbpB polypeptides specific amino acid residues in the amino acid sequence of the TbpB polypeptide are modulated relative to the amino acid sequence of a selected corresponding reference TbpB polypeptide. In accordance with an aspect of the present disclosure, these modulated amino acid residues include the following amino acid residues:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(I) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide .

[0163] It is noted that in the foregoing itemized amino acid residues (a) - (I) amino acid residues are referred to by a specific number (e.g., phenylalanine amino acid residue 55, phenylalanine amino acid residue 127, tyrosine amino acid residue 137, and so on). The numbering in conjunction with an amino acid residue in this respect corresponds with the sequential location of the amino acid residue in the amino acid sequence constituting a reference TbpB polypeptide. In this respect, pursuant to amino acid numbering practices conventionally used in the art, the N-terminal amino residue of the amino acid sequence constituting a polypeptide is referred to as amino acid number 1 , and the following amino acid residues are further sequentially numbered into C-terminal direction. Thus, for example, phenylalanine amino acid residue 55 (or F55) refers to the 55thamino acid residue from the N-terminal end of the amino acid sequence being a phenylalanine amino acid residue, tyrosine amino acid residue 137 (or Y137), refers the 137thamino acid residue from the N-terminal end of the amino acid sequence being a tyrosine, and so on. The numbering in conjunction with specific amino acid residues further refers to specific example reference TbpB polypeptides, notably the reference TbpB polypeptides set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, and SEQ.ID NO: 12. Thus, referring to the example reference TbpB polypeptide having amino acid sequence SEQ.ID NO: 8 (labeled “Hi210TbpB” in FIG. 1), for example, amino acid residue 55 corresponds with a phenylalanine residue, amino acid residue 128 corresponds with an arginine residue; and referring, for example, to reference TbpB polypeptide having amino acid sequence SEQ.ID NO: 4 (labeled “Hi201TbpB” in FIG. 1), for example, amino acid residues 175, 188, and 189 correspond with arginine residues, and amino acid residue 197 corresponds with an aspartate residue.

[0164] In addition to the example reference TbpB polypeptides having amino sequence SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, and SEQ.ID NO: 12 other reference TbpB polypeptides may be selected, as hereinafter further explained. In this respect, it is noted that in the foregoing itemized modulated amino acid residues (a) - (I), reference is made toamino acid residues located at an equivalent position in a reference TbpB polypeptide. By the phrase “located at an equivalent position in the reference TbpB polypeptide”, it is meant that in another selected reference TbpB polypeptide, when such selected reference polypeptide is aligned with a first selected reference TbpB polypeptide, an identical amino acid residue can be identified, however, the amino acid residue may be numbered differently. As will readily be understood by those of skill in the art, different sequential amino acid numbering can arise due to the fact that when a second reference TbpB polypeptide in addition to the first selected reference TbpB polypeptide is selected, the length of such second reference TbpB polypeptide may deviate from the length of the first reference TbpB polypeptide. Examples of such deviations in amino acid numbering when comparing different TbpB polypeptide sequences may be gleaned by referring to FIG. 1 , and the various TbpB sequences shown therein, and as hereinafter further explained. 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 a / ., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et a / ., 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 whereinat least 50% of the total length of one of the two sequences is involved in the alignment.

[0165] Thus, by way of example, FIG. 1 shows six selectable example reference TbpB polypeptides, including the example reference TbpB polypeptides having SEQ.ID NO: 2 (denoted as “HiO36TbpB”), SEQ.ID NO: 4 (denoted as “Hi201TbpB ”), SEQ.ID NO: 6 (denoted as “HiO38TbpB ”), SEQ.ID NO: 8 (denoted as “Hi040TbpB”), SEQ.ID NO: 10 (denoted as “Hi210TbpB ”), and SEQ.ID NO: 12 (denoted as “Hi216TbpB ”) that have been aligned using the method described by Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019 Jul;47(W1) W636-W641. doi:10.1093 / nar / gkz268. PMID: 30976793; PMCID: PMC6602479. In at least one of the of the aligned reference TbpB polypeptides, at least one of the following amino acid residues is indicated: phenylalanine amino acid residue 55, phenylalanine amino acid residue 127, arginine amino acid residue 128, tyrosine amino acid residue 137, arginine amino acid residue 175, arginine amino acid residue 180, tyrosine amino acid residue 186, arginine amino acid residue 188, arginine amino acid residue 189, glutamate amino acid residue 194, aspartate amino acid residue 197, and glutamate amino acid residue 204. In addition, in some of the aligned TbpB polypeptides arginine, tyrosine, arginine, glutamate, or aspartate amino acid residues, located at an equivalent position in another reference TbpB polypeptide are indicated. However, as noted, differences in amino acid numbering may result in different numbering of amino acid residues located in an equivalent position. Thus, referring to FIG. 1 , by way of example, it is noted that in the shown reference H. influenzae TbpB polypeptide denoted as “HiO38TbpB” (SEQ.ID NO: 6), amino acid residue Y137 corresponds with amino acid residues Y137, in the three reference TbpB polypeptides denoted as “HiO36TbpB” (SEQ.ID NO: 2), “Hi201TbpB” (SEQ.ID NO: 4) and “Hi210TbpB” (SEQ.ID NO: 10), and with amino acid residue Y139 in the reference TbpB polypeptide denoted as “Hi040TbpB” (SEQ.ID NO: 8). Furthermore, the TbpB polypeptide denoted as “Hi216TbpB” does not possess a tyrosine amino acid residue in an equivalent position. Therefore, it is to be understood that phenylalanine residues Y137 in “HiO38TbpB”, “HiO36TbpB”, “Hi201TbpB” and “Hi210TbpB” are located at “an equivalent position” as tyrosine residue Y139 in“Hi040TbpB”. By way of a further example, in the shown reference H. influenzae TbpB polypeptide denoted as “Hi201TbpB” (SEQ.ID NO: 4), amino acid residue R189 corresponds with amino acid residue R192 in the TbpB polypeptide denoted as “HiO36TbpB” (SEQ.ID NO: 2) and, with amino acid residue R189 in the TbpB polypeptide denoted as “HiO38TbpB” (SEQ.ID NO: 6), and with amino acid residue R193 in the TbpB polypeptide denoted as “Hi040TbpB” (SEQ.ID NO: 8) and with amino acid residue R190 in the TbpB polypeptide denoted as “Hi210TbpB” (SEQ.ID NO: 10), and with amino acid residue R193 in the TbpB polypeptide denoted as “Hi216TbpB” (SEQ.ID NO: 12). Therefore, it is to be understood that arginine residue R189 in “Hi201TbpB”, is located at “an equivalent position” as arginine residue R192 in “HiO36TbpB”, arginine residue R189 in “HiO38TbpB”, arginine residue R193 in “Hi040TbpB”, arginine residue R190 in “Hi210TbpB”, and arginine residue R193 in “Hi216TbpB”.

[0166] Furthermore, it is noted that reference TbpB polypeptides that may be selected and used in accordance herewith include any and all naturally occurring H. influenzae TbpB, as well as TbpB polypeptides that may have been obtained by modification of naturally occurring TbpB polypeptides.

[0167] Thus, specific suitable example reference TbpB polypeptides that may be selected and used in accordance herewith include any reference TbpB polypeptides set forth herein, notably reference TbpB polypeptides having SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12.

[0168] Yet further specific suitable example reference TbpB polypeptides that may be selected and used in accordance herewith include any TbpB polypeptide expressed by a nucleic acid sequence selected from:.(a) SEQ.ID NO: 1 , SEQ.ID NO: 3, SEQ.ID NO: 5, SEQ.ID NO: 7, SEQ.ID NO: 9, or SEQ.ID NO: 11 ;(b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a);(c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code;(d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a);(e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or an immunogenic portion thereof;(f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ.ID NO: 12, or an immunogenic portion thereof; and(g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f), provided however that, the reference TbpB polypeptide comprises at least one of(i) a phenylalanine amino acid 55 residue or a phenylalanine amino acid residue located at an equivalent position;(ii) a phenylalanine amino acid 127 residue or a phenylalanine amino acid residue located at an equivalent position;(iii) an arginine amino acid 128 residue or an arginine amino acid residue located at an equivalent position;(iv) a tyrosine amino acid 137 residue or a tyrosine amino acid residue located at an equivalent position;(v) an arginine amino acid 175 residue or an arginine amino acid residue located at an equivalent position;(vi) an arginine amino acid 180 residue, or an arginine amino acid residue located at an equivalent position;(vii) a tyrosine amino acid 186 residue, or a tyrosine amino acid residue located at an equivalent position;(viii) an arginine amino acid 188 residue, or an arginine amino acid residue located at an equivalent position;(ix) an arginine amino acid 189 residue, or an arginine amino acid residue located at an equivalent position;(x) a glutamate amino acid 194 residue, or a glutamate amino acid residue located at an equivalent position;(xi) an aspartate amino acid 197 residue, or an aspartate amino acid residue located at an equivalent position; and(xii) a glutamate amino acid 204 residue, or a glutamate amino acid residue located at an equivalent position.

[0169] Discussing further now suitable modulated TbpB polypeptides, it is noted that a modulated TbpB is referred to herein relative to a selected corresponding reference TbpB polypeptide. In particular, in accordance with an aspect hereof, in the modulated TbpB polypeptide, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, wherein at least one modulated amino acid residue is selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0170] In some embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acid residues, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acid residues, may be modulated relative to a corresponding selected reference TbpB polypeptide. Such modulations include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or all 12 amino acid residues selected from the amino acid residues set forth in itemized modulated amino acid residues (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (I) above. Modulations furthermore include at least one amino acid selected from the amino acid residues set forth in itemized modulated amino acid residues (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (I) above, and other amino acids selected from other amino acid residues within the amino acid sequence of the corresponding reference TbpB polypeptide.

[0171] Thus, it will now be understood that upon having selected a reference TbpB, amino acid residues therein may be selected for modulation, and that upon modulation of one or more amino acid residues a modulated TbpB polypeptide corresponding with the selected reference TbpB polypeptide may be obtained. In general, an amino acid residue selected for modulation may be substituted by another amino acid residue, or an amino acid selected for modulation may be removed.

[0172] In an aspect, in an example embodiment, phenylalanine amino acid residue 55 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 10, or a phenylalanine amino acid residue located at anequivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0173] In an aspect, in an example embodiment, phenylalanine amino acid residue 55 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 10, or a phenylalanine amino acid residue located at an equivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0174] In a further aspect, in an embodiment, phenylalanine amino acid residue 127, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or a phenylalanine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0175] In a further aspect, in an embodiment, phenylalanine amino acid residue 127, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or a phenylalanine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0176] In a further aspect, in an embodiment, arginine amino acid residue 128, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 10, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0177] In a further aspect, in an embodiment, arginine amino acid residue 128, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 10, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0178] In an aspect, in an example embodiment, tyrosine amino acid residue 137 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 6, or a tyrosine amino acid residue located at an equivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or removed.

[0179] In an aspect, in an example embodiment, tyrosine amino acid residue 137 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 6, or a tyrosine amino acid residue located at an equivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0180] In a further aspect, in an embodiment, arginine amino acid residue 175, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0181] In a further aspect, in an embodiment, arginine amino acid residue 175, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0182] In a further aspect, in an embodiment, arginine amino acid residue 180, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 2, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0183] In a further aspect, in an embodiment, arginine amino acid residue 180, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 2, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0184] In an aspect, in an example embodiment, tyrosine amino acid residue 186 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or a tyrosine amino acid residue located at an equivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or removed.

[0185] In an aspect, in an example embodiment, tyrosine amino acid residue 186 in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or a tyrosine amino acid residue located at an equivalent position in a reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

[0186] In a further aspect, in an embodiment, arginine amino acid residue 188, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0187] In a further aspect, in an embodiment, arginine amino acid residue188, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0188] In a further aspect, in an embodiment, arginine amino acid residue189, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0189] In a further aspect, in an embodiment, arginine amino acid residue 189, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, or an arginine amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

[0190] In a further aspect, in an embodiment, glutamate amino acid residue 194, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 2, or a glutamate amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0191] In a further aspect, in an embodiment, glutamate amino acid residue 194, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 2, or a glutamate amino acid residue located at an equivalentposition in a corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0192] In a further aspect, in an embodiment, aspartate amino acid residue 197, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an aspartate amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine asparagine, cysteine, glutamate glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0193] In a further aspect, in an embodiment, aspartate amino acid residue 197, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 4, or an aspartate amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0194] In a further aspect, in an embodiment, glutamate amino acid residue 204, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, ora glutamate amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, is selected and substituted by an amino acid residue selected from alanine, arginine asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or removed.

[0195] In a further aspect, in an embodiment, glutamate amino acid residue 204, in a reference TbpB polypeptide, notably, a reference TbpB polypeptide having SEQ.ID NO: 12, ora glutamate amino acid residue located at an equivalent position in a corresponding reference TbpB polypeptide, can be substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

[0196] It is further noted that reference may be made herein to substitutions of specific amino acid residues by other amino acid residues. Such substitutions may be denoted herein by referencing initially the amino acid residue in the reference TbpB polypeptide, then the position of the amino acid residue in the polypeptide chain, and then the substituting amino acid residue. Thus, by way of example “F55A”, signifies that a phenylalanine residue at position 55 in a referenceTbpB polypeptide is substituted by an alanine residue in a modulated TbpB polypeptide; and by way of another example, “Y137A”, signifies that a tyrosine residue at position 137 in a reference TbpB polypeptide is substituted by an alanine residue in a modulated TbpB polypeptide.

[0197] In a further specific aspect, in an example embodiment, the modulated TbpB polypeptide can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0198] Modulated TbpB polypeptides, in accordance herewith, may be prepared using any suitable technique or methodology to produce desired modulated TbpB polypeptides. Thus, for example, nucleic acid sequences expressing modulated TbpB polypeptides may be prepared. Such nucleic acid sequences expressing modulated TbpB polypeptides 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 may be obtained, for example, 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 may be modified to prepare a nucleic acid sequence encoding a modulated TbpB polypeptide. Thus, for example, a nucleic acid sequence encoding a reference TbpB polypeptide may be modified using site directed mutagenesis techniques by introducing altered nucleic acid residues in the nucleic acid sequence encoding the reference TbpB polypeptide, to thereby modify the corresponding expressed TbpB polypeptide sequence, and obtain a modulated nucleic acid sequence capable of expressing a modulated TbpB polypeptide. Suitable methods and techniques for site directed mutagenesis include for example, the methods and techniques described by Liu, H. et al., BMC Biotechnol., 2008, 8 (91), or Zheng, L. et al., Nucleic Acids Research, 2004, 32 (14), 1 - 5.

[0199] Other methods and techniques to obtain a modulated TbpB polypeptide include de novo artificial gene synthesis techniques of a nucleic acid sequence encoding a modulated TbpB polypeptide (see, for example, Kosuri, S etal., 2014, Nature Methods 11 (5) 499-507), or CRISPR / Cas9 based techniques (Jinek, M. et a!., 2012, Science, 337 (6096), 816-821).

[0200] According to an aspect hereof, suitable nucleic acid sequences further include an immunogenic portion of a modulated TbpB polypeptide, notably, in particular, modulated TbpB polypeptide portions which are at least immunologically equivalent to full length modulated TbpB polypeptides, including the polypeptides set forth in SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36. Thus, further example nucleic acid sequences that may be used in selected example embodiments include nucleic acid sequences encoding an amino acid sequence which is at least immunologically equivalent to a modulated TbpB polypeptide, the amino acid sequence corresponding with at least 10 consecutive amino acids and up to 200 amino acids, including 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids of a full length modulated TbpB polypeptide, including of the modulated TbpB polypeptides set forth in SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0201] Furthermore, it is noted that the modulations that may be made to TbpB polypeptides include modulations which diminish binding of the modulated TbpB protein to a native host protein, such as a native transferrin protein. Such diminished binding can generally be assessed by evaluating the affinity of a native non-modulated TbpB protein for a native host protein. The affinity may be quantitatively evaluated by experimentally determining the dissociation constant (Kd) between the TbpB protein and the native host protein. Thus, for example, in this respect, the Kd between a native TbpB protein and a native host protein, for example between an H. influenzae TbpB and an H. influenzae transferrin may be compared with the Kd between a modulated TbpB polypeptide and the same native host protein, i.e., transferrin. In general, the higher the Kd value the weaker the affinity of the TbpB protein for the native host protein. In some embodiments, the Kd between a modulated TbpB polypeptide and a native host protein may exceed the Kd between the native non-modulated TbpB polypeptide and the native host protein by a factor of at least 2x, at least 5x, at least 10x, at least 25x, at least50x, or at least 100x. Techniques to determine the Kd between two proteins are well known to those of skill in the art and include, for example isothermal calorimetry, surface plasmon resonance and bilayer inferometry (see further, for example, Rich R. et al., 2007, Anal. Biochem 361 : 1-6; Abdiche, Y. et al., 2008, Anal. Biochem. 377: 209-217; and Velazquez-Campoy, A. et al., 2004, Methods Mol. Biol. 261 : 35-54).

[0202] Thus, to briefly recap, in accordance with an aspect of the present disclosure a modulated TbpB polypeptide, or immunogenic portion thereof, may be obtained. Within the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0203] 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.

[0204] 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 bacterialhost 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.

[0205] 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 nucleic acid 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.

[0206] 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.

[0207] Thus, in an aspect, the present disclosure provides, in an example embodiment, an expression vector comprising:(a) a nucleic acid sequence encoding a modulated TbpB polypeptide or an immunogenic portion thereof; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the modulated TbpB polypeptide, or immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0208] 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.

[0209] 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 et aL, 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.

[0210] 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.

[0211] 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.

[0212] 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., Mol Biotechnol. (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 promotersof 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.

[0213] 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 art and 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).

[0214] 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.

[0215] 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; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the modulated TbpB polypeptide, or immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0216] 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.

[0217] 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.

[0218] 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. Typical trace 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.

[0219] Upon production by the host cells of a modulated TbpB polypeptide or an immunogenic portion thereof, the modulated TbpB polypeptide or the 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, orimmunogenic 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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 producedin 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.

[0225] In some embodiments, the template can include a 5’-untranslated region (5’-UTR) (i.e., the region that is located immediately upstream (5’) of the start codon, i.e., the first codon of an mRNA translated by a ribosome) and / or a 3’- untranslated region (3’-UTR) (i.e., the region that is located immediately downstream (3’) of the stop codon, i.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.

[0226] 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 (i.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.

[0227] 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).

[0228] Thus, in another aspect, the present disclosure further provides in one embodiment expression vectors and host cells expressing mRNAs encoding modulated TbpB proteins.

[0229] The present disclosure provides, in one embodiment, an expression vector comprising:(a) a nucleic acid sequence encoding an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof,; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(I) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0230] In the modulated TbpB polypeptide can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0231] In another embodiment, the present disclosure provides a host cell comprising a chimeric nucleic acid comprising:(a) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding the mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0232] In embodiment, the modulated TbpB polypeptide can be selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

[0233] 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 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 polynucleotideencoding a modulated TbpB 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.

[0234] It is noted that any modulated TbpB polypeptides, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36 or an immunogenic portion of any of the foregoing. Furthermore, when selecting a modulated TbpB polypeptide consideration or an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, 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, treat, or prevent meningitis caused by a H. influenzae serotype b strain infection, a modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, 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, treat, or prevent otitis media caused by a non-typable H. influenzae strain infection, a modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, 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 al., M. et al., 2003, J. Clin. Microbiol, 41 (1) 393-396).

[0235] It is noted, however, that the vaccine formulations of the present disclosure can be used to prevent, ameliorate, or treat, infections by strains, other than the strain from which a reference TbpB polypeptide, on which thecorresponding modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, included in a vaccine formulation is based. Thus, the vaccine formulations of the present disclosure, surprisingly, do not necessarily need to include modulated TbpB polypeptides or mRNA polynucleotides encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, 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 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, or a single mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, can be said to be cross-protective.

[0236] Notwithstanding the foregoing, in some embodiments, vaccine formulations may comprise two or more TbpB proteins, or two or more mRNA polynucleotides encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, 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 mRNA polynucleotides encoding two or more modulated TbpB polypeptides, obtained from two or more H. influenzae strains, each belonging to a different serotype. In such embodiments, the vaccine can prevent, treat, or ameliorate infection by strains of two or more H. influenzae strains, and such vaccines can be said to be cross-protective.

[0237] In further example embodiments, vaccine formulations may comprise at 2 or least 2, or 3 or at least 3, of the modulated TbpB polypeptides or 2 or at least 2, or 3 or at least 3 mRNA polynucleotides encoding modulated TbpB polypeptides, having any one of the amino acid sequences setforth in SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36, or immunogenic portions thereof.

[0238] 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 an 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, vaccine formulations 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, so that the ratio of modulated TbpB polypeptide or mRNA polynucleotide encoding a modulated TbpB polypeptide 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, than adults. A suitable effective amount can be readily determined by one of skill in the art. Thus, a therapeutically effective amount ofthe 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 prevention, treatment, or amelioration or of disease or condition symptoms, and will fall in a relatively broad range that can be determined through routine trials.

[0239] Vaccine formulations comprising the modulated TbpB polypeptide, or immunogenic portion thereof, or the mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, of the present disclosure preferably further are prepared by combining the modulated TbpB polypeptide, or immunogenic portion thereof, or the 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.

[0240] 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.

[0241] 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, 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 calcium phosphate; 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.

[0242] 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).

[0243] T echniques for producing capsular polysaccharides and conjugating polysaccharides to proteins are well known to those of skill in the art, and guidancecan 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.

[0244] 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 a modulated TbpB protein, or an immunogenic portion thereof, or an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide . the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the modulated TbpB protein, or immunogenic portion thereof; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the modulated TbpB protein, or immunogenic portion thereof in a host cell;(B) introducing the chimeric nucleic acid sequence into a host cell;(C) growing the host cell to produce (i) the modulated TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(D) recovering (i) the modulated TbpB protein, or the immunogenic portion thereof, or the (ii) mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(E) formulating (i) the recovered modulated TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding the modulated TbpB polypeptide, or immunogenic portion thereof,together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of (i) the modulated protein, the immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, to treat a human susceptible to infection by a Gram-negative bacterial species.

[0245] 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: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36, or an immunogenic portion thereof.

[0246] The vaccine formulations of the present disclosure may be used to prevent, treat, or ameliorate 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.

[0247] 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 the case 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.

[0248] 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 .

[0249] 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.

[0250] 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.

[0251] 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 to prepare a vaccine formulation comprising the modulated TbpB polypeptide, or immunogenic portion thereof, or mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, together with a pharmaceutically acceptable adjuvant, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide .

[0252] In light of the foregoing, it will now further 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 mRNA polynucleotide encoding a modulated TbpB polypeptide, or animmunogenic portion thereof, together with a pharmaceutically acceptable adjuvant to ameliorate, treat, or prevent an infection caused by an H. influenzae in a human susceptible to infection by the H. influenzae strain, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

[0253] 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, treat, or prevent infection caused by H. influenzae.

[0254] 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

[0255] SEQ.ID NO: 1 and SEQ.ID NO: 2 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi036 (serotype b).

[0256] SEQ.ID NO: 3 and SEQ.ID NO: 4 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi201 (serotype b).

[0257] SEQ.ID NO: 5 and SEQ.ID NO: 6 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi038 (serotype b).

[0258] SEQ.ID NO: 7 and SEQ.ID NO: 8 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi040 (non-typeable).

[0259] SEQ.ID NO: 9 and SEQ.ID NO: 10 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi210 (non-typeable).

[0260] SEQ.ID NO: 11 and SEQ.ID NO: 12 set forth the polynucleotide sequence and deduced amino acid, respectively, of a corresponding reference TbpB polypeptide of H. influenzae strain Hi216 (serotype b).

[0261] SEQ.ID NO: 13 and SEQ.ID NO: 14 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (F55A) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 10.

[0262] SEQ.ID NO: 15 and SEQ.ID NO: 16 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (F127A) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 12.

[0263] SEQ.ID NO: 17 and SEQ.ID NO: 18 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (R128E) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 10.

[0264] SEQ.ID NO: 19 and SEQ.ID NO: 20 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (Y137A) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 6.

[0265] SEQ.ID NO: 21 and SEQ.ID NO: 22 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (R175D) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 4.

[0266] SEQ.ID NO: 23 and SEQ.ID NO: 24 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (R180E) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 2.

[0267] SEQ.ID NO: 25 and SEQ.ID NO: 26 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (Y186A) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 10.

[0268] SEQ.ID NO: 27 and SEQ.ID NO: 28 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (R188E) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 4.

[0269] SEQ.ID NO: 29 and SEQ.ID NO: 30 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (R189E) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 4.

[0270] SEQ.ID NO: 31 and SEQ.ID NO: 32 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (E194R) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 4.

[0271] SEQ.ID NO: 33 and SEQ.ID NO: 34 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (D197R) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 4.

[0272] SEQ.ID NO: 35 and SEQ.ID NO: 36 set forth the polynucleotide sequence and deduced amino acid, respectively, of a modulated TbpB polypeptide (E204R) based on the corresponding H. influenzae reference TbpB polypeptide of SEQ.ID NO: 12.

[0273] 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 TbpB polypeptide (F55A).Protein expression

[0274] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h210 wherein an alanine residue is substituting phenylalanine 55 (SEQ.IDNO: 13, encoding a polypeptide having SEQ.ID NO: 14) (the “h210 F55A TbpB mutant") was cloned into a custom T7 expression vector (Addgene #: pE5770) that encoded an N-terminal maltose binding protein (MBP) fusion partner to maximize the stability and solubility of the recombinant protein. The vector also encoded a polyhistidine tag upstream of the MBP fusion partner to allow for purification of the protein by affinity chromatography. The TbpB-encoding expression vector was transformed into chemically competent Escherichia coli ER2566 (New England BioLabs) cells. Following a 1-h post-heat shock recovery period at 37°C (with shaking), these cells were then used to inoculate 10 mL of ZYP-5052 medium containing 50 pg / mL of ampicillin, which was then incubated at 37°C for 18 h with shaking. Next, the cells were harvested by centrifugation at 3220 x g for 10 min, the resulting supernatant decanted, and the pellet re-suspended in Resuspension Buffer (50 mM Tris pH 8.0, 300 mM NaCI, 10 mM imidazole). This mixture was then mixed with glass beads (0.1 mm diameter) in 2-mL tubes and the cells mechanically lysed using a tabletop cell disrupter (Scientific Industries). The resulting lysate was then centrifuged at 21 300 x g for 20 min at 4°C, after which the supernatant was mixed with Ni-NTA resin and incubated on a shaker for 1 h at room temperature. Next, the Ni-NTA mixture was centrifuged at 21 300 x g for 1 min, the supernatant decanted, and the Ni-NTA resin washed twice with Wash Buffer (50 mM Tris pH 8.0, 1 M NaCI, 20 mM imidazole). Following the wash step, the recombinant protein captured by the Ni-NTA resin was eluted using Elution Buffer (50 mM Tris pH 8.0, 300 mM NaCI, 300 mM imidazole). Finally, expression of the recombinant protein was verified using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h210 F55A TbpB mutant

[0275] The h210 F55A TbpB mutant was evaluated using a solid-phase human transferrin (hTf) binding assay, which, since binding-defective TbpB mutants are predicted to confer superior protection against infection compared to the intact protein, is used as preliminary screen for suitable TbpB-derived vaccine antigens. Briefly, the recombinant h210 F55A TbpB was spotted onto a nitrocellulose membrane (Pall). After allowing it to dry completely, the membrane was then blocked using 1 % skim milk in Tris-buffered saline (TBS; 50 mM Tris,150 mM NaCI, pH 7.6) and subsequently incubated in a solution containing horseradish peroxidase (HRP)-conjugated hTf and 1 % skim milk (1 pg / mL of HRP- conjugated hTf and 1 % skim milk in TBS) overnight at 4°C with shaking. The following day, the membrane was washed three times with TBS and developed using a solution containing 3 mg / mL of HRP development solution (BioRad) and 0.04% hydrogen peroxide in TBS. The binding properties of the h210 F55A TbpB mutant were compared directly to those of the reference wild-type h210 TbpB (SEQ.ID NO: 10), and the results are shown in FIG. 2A.Example 2 - Preparation and immunological characterization of a second modulated TbpB polypeptide (F127A).Protein expression.

[0276] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h216 wherein an alanine residue is substituting phenylalanine 127 (SEQ.ID NO: 15, encoding a polypeptide having SEQ.ID NO: 16) (the “h216 F127A TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h216 F127A TbpB mutant.

[0277] The binding properties of the h216 F127A TbpB mutant were compared directly to those of the reference wild-type h216 TbpB (SEQ.ID NO: 12), using the methodology described in Example 1 , and the results are shown in FIG.2BExample 3 - Preparation and immunological characterization of a third modulated TbpB polypeptide (R128E).Protein expression.

[0278] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h210 wherein a glutamate residue is substituting arginine 128 (SEQ.ID NO: 17, encoding a polypeptide having SEQ.ID NO: 18) (the “h210 R128E TbpBmutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h210 R128A TbpB mutant.

[0279] The binding properties of the h210 R128E TbpB mutant were compared directly to those of the reference wild-type h210 TbpB (SEQ.ID NO: 10), using the methodology described in Example 1 , and the results are shown in FIG.2C characterization of a fourthProtein expression.

[0280] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h038 wherein an alanine residue is substituting tyrosine 137 (SEQ.ID NO: 19, encoding a polypeptide having SEQ.ID NO: 20) (the “h038 Y137A TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h038 Y137A TbpB mutant.

[0281] The binding properties of the h038 Y137A TbpB mutant were compared directly to those of the reference wild-type h038 TbpB (SEQ.ID NO: 6), using the methodology described in Example 1 , and the results are shown in FIG. 2D characterization of a fifthProtein expression.

[0282] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h201 wherein an aspartate residue is substituting arginine 175 (SEQ.ID NO: 21 , encoding a polypeptide having SEQ.ID NO: 22) (the “h201 R175D TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h201 R175D TbpB mutant

[0283] The binding properties of the h201 R175D TbpB mutant were compared directly to those of the reference wild-type h201 TbpB (SEQ.ID NO: 4), using the methodology described in Example 1 , and the results are shown in FIG. 2EExample 6 - Preparation and immunological characterization of a sixth modulated TbpB polypeptide (R180E).Protein expression.

[0284] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h036 wherein a glutamate residue is substituting arginine 180 (SEQ.ID NO: 23, encoding a polypeptide having SEQ.ID NO: 24) (the “h036 R180E TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h036 R180E TbpB mutant

[0285] The binding properties of the h036 R180E TbpB mutant were compared directly to those of the reference wild-type h036 TbpB (SEQ.ID NO: 2), using the methodology described in Example 1 , and the results are shown in FIG. 2FLarge-scale protein production and purification for mouse immunization.

[0286] The pertinent TbpB-encoding expression vector was transformed into chemically competent E. coli strain ER2566 cells and the transformants used to inoculate 6 L of autoinduction media. The resulting culture 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.Mouse immunizations.

[0287] Immediately priorto immunization, the purified TbpB 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) 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 final blood collection conducted using an intracardiac bleed. Antiserum was then obtained from whole blood by centrifugation.Detection ofanti-TbpB IgG titres by enzyme-linked immunosorbent assay (ELISA).

[0288] TbpB lysates containing the wild-type h036 TbpB 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), after which 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 withthe 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).

[0289] 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. An hTf-HRP control was also used in the ELISA to ensure consistent coating of ELISA wells with the h036 wild-type TbpB. The anti-TbpB titres elicited by the h036 R180E TbpB mutant were compared to those elicited by the wild-type protein (“h036 WT TbpB”) and those detected in mice immunized with the adjuvant alone (“AddaVax only”). The ELISA results are shown in FIG. 3. It is noted that in mice, the modulated h036 R180E TbpB protein elicited an immune response similar to h036 WT TbpB. However, mouse transferrin does not bind to human TbpB proteins. When used for immunization of a human, h036 WT TbpB is expected to bind transferrin. By contrast, modulated h036 R180E TbpB is expected to exhibit substantially reduced binding to human transferrin. Due to transferrin binding, certain epitopes in h036WT TbpB are expected to be blocked, thus restricting an immune response in humans immunized with h036 WT TbpB. The availability of additional epitopes in the modulated h036 R180E TbpB protein can result in the elicitation of a more potent humoral immune response in humans immunized with the modulated h036 R180E TbpB than in humans immunized with h036 WT TbpB. Furthermore, antibodies binding to epitopes in a modulated h036 R180E TbpB that are associated with human transferrin binding in h036 WT TbpB, and that are blocked from antibody access in h036 WT TbpB, may modulate the immune response and / or the course of a pathogenic infection by preventing infecting bacteria from acquiring sufficient exogenous iron for bacterial growth and survival from transferrin, due to transferrin’s restricted capacity to bind to the modulated h036 R180E TbpB protein.Example 7 - Preparation and immunological characterization of a seventh modulated TbpB polypeptide (Y186A).Protein expression.

[0290] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h210 wherein an alanine residue is substituting tyrosine 186 (SEQ.ID NO: 25, encoding a polypeptide having SEQ.ID NO: 26) (the “h210 Y186A TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h210 Y186A TbpB mutant.

[0291] The binding properties of the h210 Y186A TbpB mutant were compared directly to those of the reference wild-type h210 TbpB (SEQ.ID NO: 10), using the methodology described in Example 1 , and the results are shown in FIG. 2GExample 8 - Preparation and immunological characterization of an eighth modulated TbpB (R188E).Protein expression.

[0292] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h201 wherein a glutamate residue is substituting arginine 188 (SEQ.ID NO: 27, encoding a polypeptide having SEQ.ID NO: 28) (the “h201 R188E TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h201 R188E TbpB mutant.

[0293] The binding properties of the h201 R188E TbpB mutant were compared directly to those of the reference wild-type h201 TbpB (SEQ.ID NO: 4), using the methodology described in Example 1 , and the results are shown in FIG. 2HExample 9 - Preparation and immunological characterization of a nineth modulated TbpB (R189E).Protein expression.

[0294] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h201 wherein a glutamate residue is substituting arginine 189 (SEQ.ID NO: 29, encoding a polypeptide having SEQ.ID NO: 30) (the “h201 R189E TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h201 R189E TbpB mutant.

[0295] The binding properties of the h201 R189E TbpB mutant were compared directly to those of the reference wild-type h201 TbpB (SEQ.ID NO: 4), using the methodology described in Example 1 , and the results are shown in FIG. 2IExample 10 - Preparation and immunological characterization of a tenth modulated TbpB (E194R).Protein expression.

[0296] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h201 wherein an arginine residue is substituting glutamate 194 (SEQ.ID NO: 31 , encoding a polypeptide having SEQ.ID NO: 32) (the “h201 E194R TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h201 E194R TbpB mutant.

[0297] The binding properties of the h201 E194R TbpB mutant were compared directly to those of the reference wild-type h201 TbpB (SEQ.ID NO: 4), using the methodology described in Example 1 , and the results are shown in FIG.2 JExample 11 - Preparation and immunological characterization of an eleventh modulated TbpB polypeptide (D197R).Protein expression.

[0298] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h201 wherein an arginine residue is substituting aspartate 197 (SEQ.ID NO: 33, encoding a polypeptide having SEQ.ID NO: 34) (the “h201 D197R TbpB mutant") was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h201 D197R TbpB mutant.

[0299] The binding properties of the h201 D197R TbpB mutant were compared directly to those of the reference wild-type h201 TbpB (SEQ.ID NO: 4),using the methodology described in Example 1 , and the results are shown in FIG. 2KExample 12 - Preparation and immunological characterization of a twelfth modulated TbpB polypeptide (E204R).Protein expression.

[0300] A gene encoding a modulated TbpB polypeptide of H. influenzae strain h216 wherein an arginine residue is substituting glutamate 204 (SEQ.ID NO: 35, encoding a polypeptide having SEQ.ID NO: 36) (the “h216 E204R TbpB mutant”) was cloned and expressed in E. coli as described in Example 1 , and expression was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Evaluation of the transferrin (Tf) binding properties of the h216 E204R TbpB mutant.

[0301] The binding properties of the h216 E204R TbpB mutant were compared directly to those of the reference wild-type h216 TbpB (SEQ.ID NO: 12), using the methodology described in Example 1 , and the results are shown in FIG. 2L

Claims

CLAIMS1. A vaccine formulation comprising (i) an effective amount of a modulated Transferrin Binding Protein B (TbpB) protein, or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

2. A vaccine formulation according to claim 1 , wherein the corresponding reference TbpB polypeptide is selected from a TbpB polypeptide expressed by a nucleic acid sequence selected from:(a) SEQ.ID NO: 1 , SEQ.ID NO: 3, SEQ. ID NO: 5, SEQ.ID NO: 7, SEQ.ID NO: 9, or SEQ. ID NO: 11 ;(b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a);(c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code;(d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a);(e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ. ID NO: 12, or an immunogenic portion thereof;(f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 2, SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: 8, SEQ.ID NO: 10, or SEQ. ID NO: 12, or an immunogenic portion thereof; and(g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f),provided however that, the corresponding reference TbpB polypeptide comprises at least one of(i) a phenylalanine amino acid 55 residue or a phenylalanine amino acid residue located at an equivalent position;(ii) a phenylalanine amino acid 127 residue or a phenylalanine amino acid residue located at an equivalent position;(iii) an arginine amino acid 128 residue or an arginine amino acid residue located at an equivalent position;(iv) a tyrosine amino acid 137 residue or a tyrosine amino acid residue located at an equivalent position;(v) an arginine amino acid 175 residue or an arginine amino acid residue located at an equivalent position;(vi) an arginine amino acid 180 residue, or an arginine amino acid residue located at an equivalent position;(vii) a tyrosine amino acid 186 residue, or a tyrosine amino acid residue located at an equivalent position;(viii) an arginine amino acid 188 residue, or an arginine amino acid residue located at an equivalent position;(ix) an arginine amino acid 189 residue, or an arginine amino acid residue located at an equivalent position;(x) a glutamate amino acid 194 residue, or a glutamate amino acid residue located at an equivalent position;(xi) an aspartate amino acid 197 residue, or an aspartate amino acid residue located at an equivalent position; and(xii) a glutamate amino acid 204 residue, or a glutamate amino acid residue located at an equivalent position.

3. A vaccine formulation according to claim 1 , wherein in the modulated TbpB, polypeptide, or immunogenic portion thereof, one to twenty five amino acid residues is modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

4. A vaccine formulation according to claim 1 , wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one to ten amino acid residues ismodulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

5. A vaccine formulation according to claim 1 , wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one to six amino acid residues is modulated relative to the corresponding reference TbpB polypeptide, or immunogenic portion thereof.

6. A vaccine formulation according to claim 1 , wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or two amino acid residues is modulated relative to the corresponding reference TbpB polypeptide.

7. A vaccine formulation according to claim 1 , wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, a single amino acid residue is modulated relative to the corresponding reference TbpB polypeptide.

8. A vaccine formulation according to claim 1 , wherein the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 55 is removed.

9. A vaccine formulation according to claim 1 , wherein the phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

10. A vaccine formulation according to claim 1 , wherein the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine,glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the phenylalanine amino acid residue 127 is removed.

11. A vaccine formulation according to claim 1 , wherein the phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or the phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

12. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 128 is removed.

13. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

14. A vaccine formulation according to claim 1 , wherein the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 137 is removed.

15. A vaccine formulation according to claim 1 , wherein the tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

16. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 175 is removed.

17. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

18. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 180 is removed.

19. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

20. A vaccine formulation according to claim 1 , wherein the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine, or the tyrosine amino acid residue 186 is removed.

21. A vaccine formulation according to claim 1 , wherein the tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or the tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an aliphatic amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, and proline.

22. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 188 is removed.

23. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

24. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the arginine amino acid residue 189 is removed.

25. A vaccine formulation according to claim 1 , wherein the arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or the arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a negatively charged amino acid residue selected from glutamate or aspartate.

26. A vaccine formulation according to claim 1 , wherein the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 194 is removed.

27. A vaccine formulation according to claim 1 , wherein the glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

28. A vaccine formulation according to claim 1 , wherein the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the aspartate amino acid residue 197 is removed.

29. A vaccine formulation according to claim 1 , wherein the aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or the aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

30. A vaccine formulation according to claim 1 , wherein the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by an amino acid residue selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, or the glutamate amino acid residue 204 is removed.

31. A vaccine formulation according to claim 1 , wherein the glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or the glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide, is substituted by a positively charged amino acid residue selected from histidine, lysine, and arginine.

32. A vaccine formulation according to claim 1 , wherein the modulated TbpB polypeptide is selected from a polypeptide having an amino acid sequence selected from SEQ.ID NO: 14, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, SEQ.ID NO: 22, SEQ.ID NO: 24, SEQ.ID NO: 26, SEQ.ID NO: 28, SEQ.ID NO: 30, SEQ.ID NO: 32, SEQ.ID NO: 34, and SEQ.ID NO: 36.

33. A vaccine formulation according to any one of claims 1 to 32, wherein the infecting H. influenzae strain is a typable or non-typable H. influenzae strain.

34. A vaccine formulation according to any one of claims 1 to 32, 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.

35. A vaccine formulation according to any one of claims 1 to 32, wherein the infecting H. influenzae strain is capable of causing pneumonia, meningitis,bronchitis, otitis media, epiglottitis, cellulitis, infectious arthritis, or septicemia in a human.

36. A vaccine formulation according to any one of claims 1 to 32, wherein the modulated TbpB polypeptide, or immunogenic portion thereof, is a recombinantly produced polypeptide.

37. A vaccine formulation according to any one of claims 1 to 32, wherein the vaccine formulation is cross-protective, wherein the reference TbpB polypeptide corresponding with the modulated TbpB polypeptide is from a first H. influenzae strain, and wherein the vaccine formulation is administered to the human to prevent, treat, or ameliorate an infection caused by a second H. influenzae strain.

38. A vaccine formulation according to any one of claims 1 to 32, wherein the vaccine formulation further comprises a pharmaceutically acceptable adjuvant.

39. A vaccine formulation according to any one of claims 1 to 32, wherein the vaccine formulation further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

40. A vaccine formulation according to any one of claims 1 to 32, wherein the vaccine formulation comprises from about 0.001 % to about 20% by weight per volume of the modulated TbpB protein, or the immunogenic portion thereof, and a pharmaceutically acceptable adjuvant constituting from about 0.1 % to about 50% by weight or volume of the vaccine formulation.

41. A use a vaccine formulation according to any one of claims 1 to 40, for the prevention, treatment, or amelioration of H. influenzae infection in a human in need thereof.

42. A use of a vaccine formulation according to claim 41 , 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.

43. A vaccine formulation according to claim 42, wherein the clinical signs are selected from clinical signs associated with the group of diseases 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.

44. A method for preparing a vaccine formulation according to any one of claims 1 to 40, the method comprising:(A) providing a chimeric nucleic acid sequence comprising as operably linked components:(i) a nucleic acid sequence encoding the modulated TbpB protein, or immunogenic portion thereof; and(ii) one or more nucleic acid sequences capable of controlling expression of the nucleic acid sequence encoding the modulated 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 (i) the modulated TbpB protein, or immunogenic portion thereof, or (ii) a mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(D) recovering (i) the modulated TbpB protein, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof; and(E) formulating (i) the recovered modulated TbpB protein, or immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, together with a pharmaceutically acceptable adjuvant to form a vaccine formulation comprising an effective amount of (i) the modulated protein, or the immunogenic portion thereof, or (ii) the recovered mRNA polynucleotide encoding a modulated TbpB polypeptide, or immunogenic portion thereof, to prevent, treat, or ameliorate an H. influenzae infection in a human.

45. An expression vector comprising:(a) a nucleic acid sequence encoding (i) a modulated TbpB polypeptide or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof,; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the modulated TbpB polypeptide, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

46. A host cell comprising a chimeric nucleic acid comprising:(a) a nucleic acid sequence encoding (i) a TbpB polypeptide or an immunogenic portion thereof, or (ii) an mRNA polynucleotide encoding a modulated TbpB polypeptide, or an immunogenic portion thereof,; and(b) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding (i) the modulated TbpB polypeptide, or immunogenic portion thereof, or (ii) the mRNA polynucleotide encoding a TbpB polypeptide, or an immunogenic portion thereof, in a host cell, wherein in the modulated TbpB polypeptide, or immunogenic portion thereof, one or more amino acid residues are modulated relative to a corresponding reference TbpB polypeptide, at least one modulated amino acid residue being selected from the group consisting of:(a) phenylalanine amino acid residue 55 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(b) phenylalanine amino acid residue 127 in the corresponding reference TbpB polypeptide, or a phenylalanine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(c) arginine amino acid residue 128 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(d) tyrosine amino acid residue 137 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(e) arginine amino acid residue 175 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(f) arginine amino acid residue 180 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(g) tyrosine amino acid residue 186 in the corresponding reference TbpB polypeptide, or a tyrosine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(h) arginine amino acid residue 188 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(i) arginine amino acid residue 189 in the corresponding reference TbpB polypeptide, or an arginine amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(j) glutamate amino acid residue 194 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide;(k) aspartate amino acid residue 197 in the corresponding reference TbpB polypeptide, or an aspartate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide; and(l) glutamate amino acid residue 204 in the corresponding reference TbpB polypeptide, or a glutamate amino acid residue located at an equivalent position in the corresponding reference TbpB polypeptide.

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