Haemophilus influenzae vaccine and its use

Abstract

The present disclosure relates to fusion proteins comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins comprises a lipid moiety, as well as vaccines and immunogenic compositions comprising such fusion proteins. Methods of treating or preventing disorders associated with Hi infection in a subject are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 076,461, filed September 10, 2020, which is incorporated by reference herein in its entirety.

[0002] The present disclosure is in the field of Haemophilus influenzae vaccines.

[0003] Incorporation by reference of sequence listing This application contains a sequence listing in computer-readable format (filename: 55894PCT_SeqListing.txt; 83,018 byte-ASCII text file; created on September 8, 2021), which is incorporated by reference herein and forms part of this disclosure. [Background technology]

[0004] Haemophilus influenzae (Hi) is a gram-negative coccobacillus and a strict human commensal. Hi strains are either encapsulated or non-encapsulated and are therefore classified as encapsulated and acapsulated (non-encapsulated) strains.

[0005] Polysaccharide capsular types of Hi include serotypes a, b, c, d, e, and f. Encapsulated Hi type b was the primary cause of invasive Hi infections before the invention of the Hib vaccine. Hib vaccines are not available worldwide due to cost and administration challenges. Invasive Hi capsular types a, c, d, e, and f have emerged in recent years. Non-encapsulated Haemophilus influenzae (Hi) represent the majority of colonizing strains and, while rarely invasive, are responsible for a significant proportion of mucosal diseases, including otitis media, sinusitis, chronic conjunctivitis, and chronic or exacerbated lower respiratory tract infections. Currently, approximately 30% and as many as 62% of Hi strains are resistant to penicillin. Carriage rates are estimated at 44% in children and 5% in adults and can persist for several months. Neither the pathogenic mechanism nor the host immunological response has been fully defined for Hi-induced otitis media.

[0006] Otitis media is a common disease in children under 2 years of age. It is defined by the presence of fluid in the middle ear accompanied by signs of acute local or systemic disease. Acute signs include ear pain, otorrhea, and hearing loss, while systemic signs include fever, lethargy, irritability, loss of appetite, vomiting, or diarrhea. Streptococcus pneumoniae and Hi are the most prevalent bacteria causing this condition, accounting for 25-50% and 15-30% of cultured species, respectively. Additionally, Hi is responsible for 53% of recurrent otitis media cases. Approximately 60% and 80% of children develop at least one episode of the disease by age 1 and 3 years, respectively (peaking at approximately 10 months).

[0007] Acapsular Hi is the most common cause of acute exacerbations of chronic bronchitis (AECB), and the presence of new strains of Hi in the sputum of patients with chronic bronchitis increases the relative risk of exacerbations by twofold (Sethi et al., N. Engl. J. Med., 347:464-471, 2002).

[0008] Bacterial infections (including Hi infections) have also been found to be associated with chronic obstructive pulmonary disease (COPD) (Albertson et al., J. Am. Geriat. Soc., 58:570-579, 2010). The syndrome of COPD consists of chronic bronchitis (CB), bronchiectasis, emphysema, and reversible airway disease, which are uniquely combined in each patient. Elderly patients are at risk for COPD and its components: emphysema, chronic bronchitis (CB), and bronchiectasis. Bacterial and viral infections play a role in acute exacerbations of COPD (AECOPD) and acute exacerbations of CB without COPD features (AECB). Elderly patients are at risk for bacterial infections (including acapsular Hi infections) during episodes of AECOPD and AECB.

[0009] CB is a progressive disease characterized by chronic sputum production and is defined by at least 3 months of cough and sputum in each of 2 consecutive years after the elimination of tuberculosis, lung cancer, and other causes of cough (Balter et al., Can. J. Respir. J. 10, 3B-32B, 2003; Bronton et al., Am. J. Manage Care, 10:689-696, 2004). CB has been reported in the majority of patients with COPD (Balter, supra). AECB causes recurrent attacks in these patients, which are associated with worsening bronchial inflammation.

[0010] Although there is evidence that protective immunity exists for Hi, antigenic drift in epitopes naturally involved in infection (outer membrane proteins P2, P4, and P6) plays a major role in Hi's ability to evade host immune defenses. Summary of the Invention [Means for solving the problem]

[0011] In one aspect, described herein is a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, and the group consisting of PD and PF, wherein at least one of the Hi proteins is lipidated. In some embodiments, the fusion protein comprises Omp26 and P6, wherein the Omp26 protein is lipidated. In some embodiments, the fusion protein comprises Omp26 and P6, wherein the P6 protein is lipidated. In some embodiments, the fusion protein comprises two lipidated Hi proteins.

[0012] In various embodiments, the construct is LOmp26φP6 (SEQ ID NO: 31), LP6φOmp26 (SEQ ID NO: 29), L-P6φNL-PD (SEQ ID NO: 39), L-PDφNL-PF (SEQ ID NO: 40), L-PDφNL-P6 (SEQ ID NO: 41), L-P6φNL-PD (SEQ ID NO: 42), L-Omp26φNL-PD (SEQ ID NO: 43), L-PDφNL-Omp26 (SEQ ID NO: 44), or L-PFφNL-P6 (SEQ ID NO: 45). Exemplary sequences are L-PDφNL-PF, signal sequence in bold, linker underlined.

[0013] [Table 1]

[0014] In various embodiments, the fusion protein can include a signal sequence selected from the group consisting of SS1: the native P6 signal sequence, MNKFVKSLLVAGSVAALAAC (SEQ ID NO: 36); SS2: the E. coli native signal sequence of the Pal protein, MQLNKVLKGLMIALPVMAIAAC (SEQ ID NO: 37), and SSP4: the Haemophilus influenzae signal sequence of the P4 protein, MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38). When a signal sequence is used in the fusion protein sequence, it is contemplated that the signal sequence can be cleaved at the C residue and a lipid moiety attached.

[0015] In some embodiments, at least one Hi protein comprises a lipid moiety comprising one or more of C18, C16, C14, C12, and C10 fatty acids. In some embodiments, at least one Hi protein comprises a lipid moiety selected from diacyl and / or triacyl fatty acids, or combinations thereof. In some embodiments, at least one Hi protein comprises a lipid moiety selected from N-acylated or O-acylated fatty acids. In some embodiments, the Hi protein comprises a lipid moiety at the N-terminus of the protein. In some embodiments, two Hi proteins in the fusion protein are lipidated.

[0016] In some embodiments, the fusion protein comprises lipidated Omp26 and non-lipidated P6, wherein the lipid comprises a C16 fatty acid. In some embodiments, the fusion protein comprises lipidated P6 and non-lipidated Omp26, wherein the lipid comprises a C16 fatty acid.

[0017] In some embodiments, Omp26 comprises all or a portion of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, P6 comprises all or a portion of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the P4 protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the PD protein comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the PF protein comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0018] In various embodiments, the fusion protein optionally comprises a linker. In some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In another embodiment, the linker is GlySerGlyGlyGlyGly (SEQ ID NO: 12).

[0019] In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:29 or 31.

[0020] Vaccines and immunogenic compositions comprising the fusion proteins described herein are also contemplated.

[0021] The present disclosure also provides a method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins comprises a lipid moiety, the method comprising: (i) providing a nucleic acid sequence encoding a lipid moiety signal sequence region; (ii) providing a first nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF; and (iii) providing a second nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF, wherein the second nucleic acid is not a nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF. (iv) optionally providing a third or additional nucleic acid sequence encoding all or part of one or more additional Hi proteins Omp26, P6, P4, PD, or PF, wherein the third or additional nucleic acid encodes a Hi protein different from (ii) and (iii); v) inserting nucleic acid sequences (i)-(iv) into a plasmid vector capable of expressing the nucleic acid; (vi) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule and expressing the fusion protein; and (vii) purifying the recombinant fusion protein expressed in the host cell. In various embodiments, the host cell is grown in a minimal medium.

[0022] In another embodiment, a method for producing a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins is lipidated, comprises: i) inserting a nucleic acid encoding a lipid moiety signal sequence region upstream of a first nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF in a plasmid vector; ii) inserting a second nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF into the plasmid vector; iii) optionally inserting a third or additional nucleic acid sequence encoding one or more additional Hi proteins or fragments thereof selected from the group consisting of Omp26, P6, P4, PD, and PF; iv) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule; and v) purifying the fusion protein expressed by the plasmid. In various embodiments, the host cell is grown in a minimal medium.

[0023] In various embodiments, the lipid moiety signal sequence is selected from the group consisting of MNKFVKSLLVAGSVAALAAC (SEQ ID NO: 36), with or without a terminal C residue, MQLNKVLKGLMIALPVMAIAAC (SEQ ID NO: 37), with or without a terminal C residue, MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38), or MKTTLKMTALAALSAFVLAG (SEQ ID NO: 11). In various embodiments, the lipid moiety signal sequence is the P4 signal sequence MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38) or MKTTLKMTALAALSAFVLAG (SEQ ID NO: 11).

[0024] In another aspect, a method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins is lipidated, comprises: i) inserting a nucleic acid encoding a P4 lipid moiety signal sequence region upstream of a first nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF in a plasmid vector; ii) inserting a second nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF into the plasmid vector; iii) optionally inserting a third or additional nucleic acid sequence encoding one or more additional Hi proteins or fragments thereof selected from the group consisting of Omp26, P6, P4, PD, and PF; iv) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule; and v) purifying the fusion protein expressed by the plasmid.

[0025] In another aspect, a method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins is lipidated, comprising: i) providing a nucleic acid encoding a P4 lipid moiety signal sequence region; ii) providing a first nucleic acid encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF; and iii) providing a second nucleic acid encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF. wherein the second nucleic acid encodes a Hi protein different from i); iv) optionally providing a third or additional nucleic acid sequence encoding one or more additional proteins or fragments thereof selected from the group consisting of Omp26, P6, P4, PD, and PF; v) inserting the nucleic acid sequences i-iv) into a plasmid vector capable of expressing the nucleic acid; vi) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule and expressing the fusion protein; and (vii) purifying the recombinant fusion protein expressed in the host cell.

[0026] In another aspect, described herein are methods for treating or preventing diseases associated with a non-encapsulated Haemophilus influenzae (Hi) infection, comprising administering a vaccine or immunogenic composition comprising a fusion protein described herein. Disorders associated with Hi infection include, but are not limited to, otitis media, bronchitis, pneumonia, sinusitis, sepsis, endocarditis, acute epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpura fever, occult bacteremia, and exacerbations of underlying lung diseases such as chronic acute obstructive pulmonary disease (AECB), bronchiectasis, or cystic fibrosis, chronic obstructive pulmonary disease (COPD), and acute exacerbations of COPD (AECOPD).

[0027] In some embodiments, the disease associated with acapsular Hi infection is otitis media. In some embodiments, the disease associated with acapsular Hi infection is acute exacerbation of chronic bronchitis (AECB). In some embodiments, the disease associated with acapsular Hi infection is acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

[0028] Also provided are compositions comprising the fusion proteins, vaccines, or immunogenic compositions described herein for use in treating or preventing disease associated with non-encapsulated Haemophilus influenzae (Hi) infection. In various embodiments, the present disclosure provides for the use of a composition comprising the fusion proteins, vaccines, or immunogenic compositions described herein in the preparation of a medicament for use in treating or preventing disease associated with non-encapsulated Haemophilus influenzae (Hi) infection.

[0029] In various embodiments, the vaccine or immunogenic composition reduces or prevents colonization of one or more of the sinuses, lungs, and ears.

[0030] In various embodiments, the vaccine or immunogenic composition comprises an LOmp26φP6 fusion protein or an LP6φOmp26 fusion protein.

[0031] In various embodiments, the vaccine or immunogenic composition is administered orally, intravenously, intramuscularly, intranasally, or subcutaneously, hi various embodiments, the vaccine or immunogenic composition is administered intranasally.

[0032] It is understood that each feature or embodiment, or combination, described herein is a non-limiting illustrative example of any of the aspects of the invention and is therefore intended to be combinable with any other feature, embodiment, or combination described herein. For example, when a feature is described with terms such as "one embodiment," "some embodiments," "an embodiment," "a further embodiment," "a particular exemplary embodiment," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without necessarily listing every possible combination. Such features or combinations of features apply to any of the aspects of the invention. When example values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of upper and lower limits are envisioned.

[0033] Headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments, and variations of the invention will become apparent from the detailed description and / or drawings and / or claims. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a graph comparing IgG antibody titers of proteins P6 and Omp26 in their lipidated and non-lipidated forms, as determined by ELISA. [Figure 2] 1 is a bar graph comparing IgG antibody titers of lipidated and non-lipidated fusion proteins as determined by ELISA. [Figure 3A]Bar graphs comparing Hi burden in nasal washes (Figure 3A) and middle ear washes (Figure 3B) of mice vaccinated with three doses (10 μg of each protein) of nonlipidated P6, lipidated P6, and alum as a control. In the P6 study, 4-5 adult mice were used per group. p values ​​were calculated using Student's t-test between groups after logarithmic transformation of CFU counts. [Figure 3B] Bar graphs comparing Hi burden in nasal washes (Figure 3A) and middle ear washes (Figure 3B) of mice vaccinated with three doses (10 μg of each protein) of nonlipidated P6, lipidated P6, and alum as a control. In the P6 study, 4-5 adult mice were used per group. p values ​​were calculated using Student's t-test between groups after logarithmic transformation of CFU counts. [Figure 4] Figure 1 shows the type of lipidation found on different Hi lipidated peptides as measured by HEK-BLUE™ TLR2-TLR1 cell stimulation, expressed as % stimulation. The PAM3CSK4 positive control is 100% stimulation. *P<0.0001 [Figure 5] 1 shows an analysis of signal sequences used to attach lipids to proteins as measured by activation of HEK-BLUE™ cells stimulation. P values ​​are for differences among three signal sequences at two different concentrations using Anova. [Figure 6A] Figure 6A shows the immunogenicity of the lipidated fusion protein L-P6φNL-PD in vivo as measured by antibody responses against the PD protein (Figure 6A) and P6 protein (Figure 6B) (p values ​​were calculated between groups using Student's t test on log2-transformed antibody levels). [Figure 6B] Figure 6A shows the immunogenicity of the lipidated fusion protein L-P6φNL-PD in vivo as measured by antibody responses against the PD protein (Figure 6A) and P6 protein (Figure 6B) (p values ​​were calculated between groups using Student's t test on log2-transformed antibody levels). [Figure 6C]Figure 6 shows the immunogenicity of the lipidated fusion protein L-P6φNL-PD in vivo as measured by antibody responses to the PD protein (Figure 6A) and P6 protein (Figure 6B) (p values ​​were calculated between groups using Student's t-test on log2-transformed antibody levels). The L-OMP26φNL-P6 fusion induces a significant increase in antibodies to OMP26 and P6. p values ​​were calculated using Student's t-test between groups. [Figure 7] 1 shows antibody titers after intranasal immunization with the L-OMP26φNL-P6 fusion in animal model B. [Figure 8A] The effect of diacyl moieties on immunogenicity, as measured by antibody production (FIG. 8A) or induction of cytokines such as CCL5 (FIG. 8B), is shown compared to triacyl lipid moieties. [Figure 8B] The effect of diacyl moieties on immunogenicity, as measured by antibody production (FIG. 8A) or induction of cytokines such as CCL5 (FIG. 8B), is shown compared to triacyl lipid moieties. [Figure 9A] Comparison of immunogenicity for L-OMP26φNL-P6 fusion protein administered intranasally (FIG. 9A) or intraperitoneally (FIG. 9B) in the presence or absence of adjuvant. Animal model B was used. [Figure 9B] Comparison of immunogenicity for L-OMP26φNL-P6 fusion protein administered intranasally (FIG. 9A) or intraperitoneally (FIG. 9B) in the presence or absence of adjuvant. Animal model B was used. [Figure 10A] Lipidated P6 and OMP26 and the fusions L-P6φNL-OMP26 and LOMP26φNL-P6 protect against nasopharyngeal colonization (FIG. 10A) and ear infection (FIG. 10B). p values ​​were calculated using Student's t-test between groups after logarithmic transformation of CFU. [Figure 10B]Lipidated P6 and OMP26 and the fusions L-P6φNL-OMP26 and LOMP26φNL-P6 protect against nasopharyngeal colonization (FIG. 10A) and ear infection (FIG. 10B). p values ​​were calculated using Student's t-test between groups after logarithmic transformation of CFU. [Figure 11A] Figure 11 shows L-PDφNL-PF fusion immunized intranasally (IN) in a modified colonization model in which influenza infection was not administered. Colonization was measured in CFU in the nasopharynx (Figure 11A) and ear (Figure 11B). [Figure 11B] Figure 11 shows L-PDφNL-PF fusion immunized intranasally (IN) in a modified colonization model in which influenza infection was not administered. Colonization was measured in CFU in the nasopharynx (Figure 11A) and ear (Figure 11B). [Figure 12A] The effect of different lipidated proteins after intramuscular (IM) immunization as measured by nasal wash (FIGS. 12B, 12C) and otic bulla (FIGS. 12A, 12D) is shown. [Figure 12B] The effect of different lipidated proteins after intramuscular (IM) immunization as measured by nasal wash (FIGS. 12B, 12C) and otic bulla (FIGS. 12A, 12D) is shown. [Figure 12C] The effects of different lipidated proteins after intramuscular (IM) immunization as measured by nasal washes (Figures 12B, 12C) and otic bulla (Figures 12A, 12D) are shown, from the same experiment, but with LPDφNL-OPM26 compared to control alone. [Figure 12D] The effects of different lipidated proteins after intramuscular (IM) immunization as measured by nasal washes (Figures 12B, 12C) and otic bulla (Figures 12A, 12D) are shown, from the same experiment, but with LPDφNL-OPM26 compared to control alone. DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following reference provides one of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed., 1994).

[0036] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded, double-stranded, or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can comprise a polymer of synthetic subunits, such as phosphoramidates, and thus can be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-8; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-23; Schultz et al. (1996) Nucleic Acids Res. 24:2966-73. Phosphorothioate bonds can be used instead of phosphodiester bonds. Braun et al. (1988) J. Immunol. 141:2084-9; Latimer et al. (1995) Molec. Immunol. 32:1057-1064. Furthermore, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by de novo synthesis of the complementary strand using DNA polymerase with appropriate primers. A reference to a polynucleotide sequence (eg, a reference to a SEQ ID NO) also includes the complementary sequence.

[0037] As used herein, "vaccine" refers to a composition comprising a fusion protein as described herein, which is useful for establishing immunity to Hi in a subject. It is contemplated that the vaccine will include a pharmaceutically acceptable carrier and / or adjuvant. In various embodiments, the vaccine will include a vector comprising the fusion protein. It is contemplated that the vaccine will be prophylactic or therapeutic.

[0038] A "prophylactic" treatment is a treatment administered to a subject who shows no signs of disease or who shows only early signs, with the intent of reducing the risk of developing a condition. The compounds of the present disclosure can be administered as a prophylactic treatment to reduce the likelihood of developing a condition or to minimize its severity if the condition does develop. In some embodiments, a prophylactic vaccine is administered to a subject to reduce the likelihood of the subject developing otitis media or sinusitis.

[0039] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of a pathology with the intent of reducing or eliminating those signs or symptoms, which may be biochemical, cellular, histological, functional, subjective, or objective.

[0040] A "protective" immune response means that the vaccine or immunogenic composition is capable of eliciting a humoral and / or cellular immune response that protects an individual from infection. The protection provided need not be absolutely protective, i.e., infection need not be completely prevented or eradicated, provided that the protection is statistically significantly improved compared to a control population of infected individuals, such as infected subjects not administered the vaccine or immunogenic composition. Protection can be limited to reducing the severity or rapid onset of symptoms of infection. Generally, a "protective immune response" includes the induction of an increase in the amount of antibodies specific to a particular antigen in at least 50% of individuals, including some increase in a measurable functional antibody response against each antigen. In certain instances, a "protective immune response" can include at least a two-fold or four-fold increase in the amount of antibodies specific to a particular antigen, including some measurable functional antibody response against each antigen. In certain embodiments, antibodies are associated with a protective immune response. A protective immune response can be measured by analyzing for the presence of antigen-specific antibodies in serum using assays known in the art. A protective immune response can also be analyzed by measuring the percentage reduction in bacterial counts in a serum bactericidal activity (SBA) assay. Such assays are well known in the art. In some embodiments, bacterial counts are reduced by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more compared to bacterial counts in the absence of the immunogenic composition.

[0041] As used herein, "purified" refers to a fusion protein or immunogenic composition that has been isolated under conditions that reduce or eliminate the presence of extraneous materials (i.e., contaminants, including endogenous materials from which the composition is derived). For example, but not by way of limitation, a purified fusion protein is substantially free of host cell or culture components (including tissue culture or cellular proteins and non-specific pathogens). In various embodiments, purified material that is substantially free of contaminants is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, biological assays, and other methods known in the art.

[0042] As used herein, a "pharmaceutical composition" refers to a composition suitable for administration to a subject animal, including humans and mammals. A pharmaceutical composition comprises a pharmacologically effective amount of a fusion protein, vaccine, or immunogenic composition of the present disclosure, and also comprises a pharmaceutically acceptable carrier. Pharmaceutical compositions encompass compositions comprising the active ingredient(s) and the inactive ingredient(s) that comprise the pharmaceutically acceptable carrier, as well as any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more components. Thus, pharmaceutical compositions of the present disclosure encompass any composition made by mixing a fusion protein, immunogenic composition, or vaccine of the present disclosure with a pharmaceutically acceptable carrier.

[0043] As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance may be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, or when administered using routes well known in the art, such as those described below.

[0044] As used herein, "pharmaceutically acceptable carrier" includes any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and excipients (e.g., phosphate-buffered saline solution, 5% aqueous solutions of dextrose or mannitol, and emulsions (e.g., oil-in-water emulsions or water-in-oil emulsions), and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Co., Easton, 1995). Pharmaceutical carriers useful in the compositions depend on the intended mode of administration of the active agent. Typical modes of administration include, but are not limited to, enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration). "Pharmaceutically acceptable salts" are salts that can be formulated into compounds or conjugates for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0045] Haemophilus influenzae (Hi) Haemophilus influenzae is a small, nonmotile, Gram-negative bacterium. Some Hi strains are polysaccharide-encapsulated, and capsular types are designated a, b, c, d, e, and f. Acapsular (Hi) strains lack the polysaccharide capsule and are sometimes referred to as "non-encapsulated." Non-encapsulated Hi strains are genetically distinct from encapsulated strains and are more heterogeneous than H. influenzae type b isolates. Hi presents a complex array of antigens to the human host. Potential antigens that can elicit protection include outer membrane proteins (OMPs), lipopolysaccharides, lipoproteins, adhesion proteins, and non-capsular proteins. Among these antigens are components shared by encapsulated and non-encapsulated strains, including proteins Omp26, P6, P4, PD, and PF.

[0046] Humans are the sole host of Haemophilus influenzae. Hi strains commonly reside in the upper and lower respiratory tract, including the nasopharynx and posterior oropharynx, and the female genital tract. Hi causes a wide range of illnesses in humans, including, but not limited to, otitis media, bronchitis, pneumonia, sinusitis, sepsis, endocarditis, acute epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infections, conjunctivitis, Brazilian purpura fever, occult bacteremia, and exacerbations of underlying lung diseases such as chronic bronchitis, bronchiectasis, or cystic fibrosis, chronic obstructive pulmonary disease (COPD), and acute exacerbations of COPD (AECOPD).

[0047] Antigenic Hi proteins include Omp26, P6, P4, PD, and PF. In some embodiments, the OMP26 protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the P6 protein comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the P4 protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the PD protein comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the PF protein comprises the amino acid sequence set forth in SEQ ID NO: 10. Cross-sections of any of SEQ ID NOs: 2, 4, 6, 8, or 10 that elicit an immune response are also contemplated.

[0048] fusion proteins The present disclosure provides fusion proteins comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of OMP26, P6, P4, PD, and PF, wherein at least one of the Hi proteins or fragments thereof is lipidated. Contemplated fusion proteins include LOmp26φP6, LP6φOmp26, L-P6φNL-PD, L-PDφNL-PF, L-PDφNL-P6, L-P6φNL-PD, L-Omp26φNL-PD, L-PDφNL-Omp26, and L-PFφNL-P6.

[0049] As used herein, the term "lipidated" refers to a protein that includes a lipid moiety. Fusion proteins are contemplated to be lipidated, for example, by including one or more Hi proteins that include a lipid moiety. As used herein, the term "lipidated Hi protein" refers to a Hi protein or fragment thereof that includes a lipid moiety. A "non-lipidated" Hi protein or fragment thereof lacks a lipid moiety. "L" means lipidated, and "NL" means non-lipidated. Exemplary lipid moieties include the fatty acids provided in Table A below.

[0050] [Table 2]

[0051] In various embodiments, the lipidated fusion protein comprises a mixture of lipidated and non-lipidated Hi protein. As used herein, the term "lipid moiety" refers to a saturated, unsaturated, or branched fatty acid having a chain length of C10 to C18 (e.g., C10, C11, C12, C13, C14, C15, C16, C17, or C18). In some embodiments, the lipid moiety comprises a C18, C16, C14, C12, or C10 fatty acid. In some embodiments, the lipid moiety comprises a diacyl and / or triacyl fatty acid. In some embodiments, the lipid moiety is an N-acylated and / or O-acylated fatty acid. In various embodiments, the lipid moiety comprises a mixture of diacyl and triacyl fatty acids. In various embodiments, the mixture of diacyl and triacyl fatty acids is in a ratio of 1:1, 1.5:1, 2:1, 2.5:1, or 3:1.

[0052] In some embodiments, the fusion protein comprises Omp26 or a fragment thereof and P6 or a fragment thereof, wherein P6 or a fragment thereof is lipidated and Omp26 is non-lipidated. In some embodiments, the fusion protein comprises Omp26 or a fragment thereof and P6 or a fragment thereof, wherein Omp26 or a fragment thereof is lipidated and P6 is non-lipidated.

[0053] In some embodiments, the fusion protein comprises two or more lipidated Hi proteins or fragments thereof.

[0054] In some embodiments, the fusion protein comprises all or a portion of Omp26 and P6, where Omp26 comprises one or more C16 fatty acids. In some embodiments, the fusion protein comprises all or a portion of Omp26 and P6, where P6 comprises one or more C16 fatty acids.

[0055] In some embodiments, the fusion protein comprises lipidated Omp26 comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the fusion protein comprises lipidated P6 comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the fusion protein comprises lipidated PD comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the fusion protein comprises lipidated PF comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 20.

[0056] In some embodiments, two or more Hi proteins or fragments thereof are linked. In some embodiments, two or more Hi proteins or fragments thereof are linked by a peptide linker. As used herein, "linker" refers to an amino acid or peptide sequence located between two polypeptide sequences and connecting the two polypeptides. A linker can be 1 to 80 amino acids in length. In some embodiments, a linker can be 2 to 40, 3 to 40, 3 to 30, or 3 to 20 amino acids in length. In some embodiments, a linker can be a peptide at least about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids in length. In other embodiments, a linker can be 3 to 25, 3 to 18, 5 to 20, 6 to 18, or 10 to 20 amino acids in length. The linker can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Often, the linker lacks a free cysteine ​​residue (i.e., it is not involved in disulfide bonds) and does not contain an N-glycosylation site (i.e., Asn-Xxx-Ser / Thr, where X can be any amino acid except proline). Examples of other suitable linkers include, among others, G2, G3, G3S, G3P, G3q, G5, and G5S. Each capital letter in the above linkers refers to the conventional single-letter code for an amino acid, and each number refers to the number of tandem repeats of that amino acid in the linker. For example, "G3SG2" refers to a linker having the sequence Gly-Gly-Gly-Ser-Gly-Gly. "G4S" refers to a linker having the sequence Gly-Gly-Gly-Gly-Ser. In various embodiments, the linker is a GS linker having a combination of Gly and Ser residues. In another embodiment, the linker comprises GlySerGlyGlyGlyGly (SEQ ID NO: 12).

[0057] In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:29 or 31.

[0058] Immunogenic compositions comprising one or more of the fusion proteins described herein are also contemplated. In some embodiments, the immunogenic composition comprises a mixture of lipidated fusion proteins. For example, in some embodiments, at least 10% of the fusion proteins in the immunogenic composition comprise a diacyl fatty acid. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the fusion proteins in the immunogenic composition comprise a diacyl fatty acid. In some embodiments, at least 10% of the fusion proteins in the immunogenic composition comprise a triacyl fatty acid. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the fusion proteins in the immunogenic composition comprise a triacyl fatty acid.

[0059] Vaccines comprising one or more of the fusion proteins described herein are also contemplated. In some embodiments, the vaccine comprises a mixture of lipidated fusion proteins. For example, in some embodiments, at least 10% of the fusion proteins in the vaccine comprise a diacyl fatty acid. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the fusion proteins in the vaccine comprise a diacyl fatty acid. In some embodiments, at least 10% of the fusion proteins in the vaccine comprise a triacyl fatty acid. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the fusion proteins in the vaccine comprise a triacyl fatty acid.

[0060] Manufacturing method The present disclosure also provides a method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins comprises a lipid moiety, the method comprising: (i) providing a nucleic acid sequence encoding a lipid moiety signal sequence region; (ii) providing a first nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF; and (iii) providing a second nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF, wherein the second nucleic acid is not a nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF. (iv) optionally providing a third or additional nucleic acid sequence encoding all or part of one or more additional Hi proteins Omp26, P6, P4, PD, or PF, wherein the third or additional nucleic acid encodes a Hi protein different from (ii) and (iii); v) inserting nucleic acid sequences (i)-(iv) into a plasmid vector capable of expressing the nucleic acid; (vi) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule and expressing the fusion protein; and (vii) purifying the recombinant fusion protein expressed in the host cell. In various embodiments, the host cell is grown in a minimal medium.

[0061] In another embodiment, a method for producing a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins is lipidated, comprises: i) inserting a nucleic acid encoding a lipid moiety signal sequence region upstream of a first nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF in a plasmid vector; ii) inserting a second nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF into the plasmid vector; iii) optionally inserting a third or additional nucleic acid sequence encoding one or more additional Hi proteins or fragments thereof selected from the group consisting of Omp26, P6, P4, PD, and PF; iv) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule; and v) purifying the fusion protein expressed by the plasmid. In various embodiments, the host cell is grown in a minimal medium.

[0062] In various embodiments, the lipid moiety signal sequence is selected from the group consisting of MNKFVKSLLVAGSVAALAAC (SEQ ID NO: 36), with or without a terminal C residue, MQLNKVLKGLMIALPVMAIAAC (SEQ ID NO: 37), with or without a terminal C residue, MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38), or MKTTLKMTALAALSAFVLAG (SEQ ID NO: 11). In various embodiments, the lipid moiety signal sequence is the P4 signal sequence MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38) or MKTTLKMTALAALSAFVLAG (SEQ ID NO: 11).

[0063] The present disclosure also provides a method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins comprises a lipid moiety, the method comprising: (i) providing a nucleic acid sequence encoding a P4 lipid moiety signal sequence region; ii) providing a first nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF; and iii) providing a second nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF, wherein the second nucleic acid is not a nucleic acid sequence encoding all or a portion of the Hi protein Omp26, P6, P4, PD, or PF. (iv) optionally providing a third or additional nucleic acid sequence encoding all or part of one or more additional Hi proteins Omp26, P6, P4, PD, or PF, wherein the third or additional nucleic acid encodes a Hi protein different from (ii) and (iii); v) inserting nucleic acid sequences (i)-(iv) into a plasmid vector capable of expressing the nucleic acid; (vi) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule and expressing the fusion protein; and (vii) purifying the recombinant fusion protein expressed in the host cell. In various embodiments, the host cell is grown in a minimal medium.

[0064] In another embodiment, a method for producing a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, P4, PD, and PF, wherein at least one of the Hi proteins is lipidated, comprises: (i) inserting a nucleic acid encoding a P4 lipid moiety signal sequence region upstream of a first nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF in a plasmid vector; (ii) inserting a second nucleic acid encoding all or a portion of the Haemophilus influenzae (Hi) protein Omp26, P6, P4, PD, or PF into the plasmid vector; (iii) optionally inserting a third or additional nucleic acid sequence encoding one or more additional Hi proteins or fragments thereof selected from the group consisting of Omp26, P6, P4, PD, and PF; (iv) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule; and (iv) purifying the fusion protein expressed by the plasmid. In various embodiments, the host cell is grown in minimal medium.

[0065] In some embodiments, the nucleic acid sequence encoding the Hi protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 3, 5, 7, or 9. In some embodiments, the nucleic acid sequence encoding Omp26 comprises SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding P6 comprises SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encoding P4 comprises SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding PD comprises SEQ ID NO: 7. In some embodiments, the nucleic acid sequence encoding PF comprises SEQ ID NO: 9. Fragments of any of SEQ ID NOs: 1, 3, 5, 7, or 9 that encode a Hi protein capable of eliciting an immune response are also contemplated.

[0066] In some embodiments, the nucleic acid sequence encoding a lipidated Hi protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, 15, 17, or 19. In some embodiments, the nucleic acid sequence encoding lipidated Omp26 comprises SEQ ID NO: 13. In some embodiments, the nucleic acid sequence encoding lipidated P6 comprises SEQ ID NO: 15. In some embodiments, the nucleic acid sequence encoding lipidated PD comprises SEQ ID NO: 17. In some embodiments, the nucleic acid sequence encoding lipidated P4 comprises SEQ ID NO: 19. Nucleic acids encoding any of the fusion proteins LOmp26φP6 (SEQ ID NO: 31), LP6φOmp26 (SEQ ID NO: 29), L-P6φNL-PD (SEQ ID NO: 39), L-PDφNL-PF (SEQ ID NO: 40), L-PDφNL-P6 (SEQ ID NO: 41), L-P6φNL-PD (SEQ ID NO: 42), L-Omp26φNL-PD (SEQ ID NO: 43), L-PDφNL-Omp26 (SEQ ID NO: 44), or L-PFφNL-P6 (SEQ ID NO: 45) are also contemplated.

[0067] In some embodiments, the nucleic acid encoding the P4 lipid moiety signal sequence is inserted upstream of the first nucleic acid sequence in the plasmid vector, and the second nucleic acid sequence, as well as the optional third additional nucleic acid sequence, are inserted downstream of the first nucleic acid sequence in the plasmid vector. In some embodiments, the P4 lipid moiety signal sequence comprises the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the P4 lipid moiety signal sequence comprises the nucleotide sequence set forth in SEQ ID NO: 38. In some embodiments, the nucleic acid encoding the P4 lipid moiety signal sequence is inserted upstream of the second nucleic acid sequence in the plasmid vector, and the first nucleic acid sequence, as well as the optional third additional nucleic acid sequence, are inserted upstream of the second nucleic acid sequence in the plasmid vector. In some embodiments, the nucleic acid encoding the P4 lipid moiety signal sequence is inserted upstream of the second nucleic acid sequence in the plasmid vector, and the first nucleic acid sequence is inserted upstream of the second nucleic acid sequence in the plasmid vector, and the plasmid vector optionally comprises the third additional nucleic acid sequence.

[0068] In some embodiments, the method further includes inserting a nucleic acid sequence encoding a peptide linker between the first nucleic acid sequence, the second nucleic acid sequence, and the optional third additional nucleic acid sequence. Nucleic acids encoding any of the peptide linkers described herein are contemplated herein. In another embodiment, the linker sequence encodes GlySerGlyGlyGlyGly (SEQ ID NO: 12).

[0069] In some embodiments, the first nucleic acid sequence encodes a P6 protein or a fragment thereof and the second nucleic acid sequence encodes an Omp26 protein or a fragment thereof, hi some embodiments, the first nucleic acid sequence encodes an Omp26 protein or a fragment thereof and the second nucleic acid sequence encodes a P6 protein or a fragment thereof.

[0070] In some embodiments, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO:28 or 30.

[0071] In various embodiments, the first, second, and linker nucleic acid sequences provided herein (and optionally third and subsequent nucleic acid sequences) are assembled into an expression vector, preferably under the control of a promoter suitable for expression of a mature lipoprotein, in a suitable host organism, such as E. coli, thereby causing initial translation of the fusion mRNA and expression of a recombinant mature lipidated fusion protein in the host organism, in accordance with further aspects of the invention.

[0072] The nucleic acids of the present disclosure can be cloned into vectors such as plasmids, cosmids, bacmids, phages, artificial chromosomes (BACs, YACs), or viruses, into which other genetic sequences or elements (either DNA or RNA) can be inserted to effect replication of the linked sequences or elements. In some embodiments, the expression vector contains a constitutively active promoter segment (such as, but not limited to, CMV, SV40, elongation factor, or LTR sequences) or an inducible promoter sequence, such as the steroid-inducible pIND vector (Invitrogen), to regulate nucleic acid expression. The expression vectors of the present invention can further comprise regulatory sequences (e.g., internal ribosome entry sites). The expression vector can be introduced into cells, for example, by transfection.

[0073] A secretory signal peptide sequence can also optionally be encoded by the expression vector operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell for easier isolation of the polypeptide of interest from the cell, if desired.

[0074] Recombinant host cells containing such vectors and expressing the fusion proteins described herein are also provided. Recombinant host cells can be prokaryotic cells, such as Escherichia coli cells, or eukaryotic cells, such as mammalian or yeast cells. Yeast cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia coli cells. Mammalian cells include VERO, HeLa, Chinese hamster ovary (CHO), W138, baby hamster kidney (BHK), COS-7, MDCK, human embryonic kidney line 293, normal dog kidney cell line, normal cat kidney cell line, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH / 3T3 cells, LMTK31 cells, mouse Sertoli cells, human cervical carcinoma cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse breast carcinoma cells, TRI cells, MRC5 cells, and FS4 cells. Recombinant protein-producing cells of the present disclosure also include any known insect expression cell line, such as, for example, fall armyworm cells. In one embodiment, the cell is a mammalian cell. In a specific embodiment, the mammalian cell is a CHO cell.

[0075] It is contemplated that host cells are grown in a medium applicable to the growth of the particular cell type. For example, bacteria can be cultured in LB, TB, SXYT liquid culture medium, or minimal medium. In various embodiments, the host cells are bacteria and grown in minimal medium. In various embodiments, the minimal medium is M9 medium.

[0076] Protein purification methods are known in the art and are utilized herein for the recovery of recombinant proteins from cell culture media. For example, protein purification methods are known in the art and can be used in conjunction with the production of fusion proteins of the present disclosure. In some embodiments, methods for protein purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. The filtration step can include ultrafiltration, and optionally, ultrafiltration and diafiltration. Filtration is preferably performed at least about 5-50 times, more preferably 10-30 times, and most preferably 14-27 times. Affinity column chromatography can be performed, for example, using PROSEP® affinity chromatography (Millipore, Billerica, Massachusetts). In various embodiments, the affinity chromatography step includes PROSEP®-vA column chromatography. The eluate can be washed in a solvent wash. Examples of cation exchange chromatography include SP-Sepharose cation exchange chromatography. Examples of anion exchange chromatography include, but are not limited to, Q-Sepharose Fast Flow Anion Exchange. The anion exchange step is preferably non-binding, thereby allowing for the removal of contaminants including DNA and BSA. The fusion protein is preferably nanofiltered, for example, using a Pall DV 20 Nanofilter. The fusion protein can be concentrated, for example, using ultrafiltration and diafiltration. This method can further include a size exclusion chromatography step.

[0077] Vaccines and immunogenic compositions and routes of administration The present disclosure encompasses compositions comprising the fusion protein(s), immunogenic compositions, or vaccines, and a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and excipients (e.g., phosphate-buffered saline solution, 5% aqueous solutions of dextrose or mannitol, and emulsions (e.g., oil / water emulsions or water / oil emulsions), as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Co., Easton, 1995). The pharmaceutical carriers useful in the compositions depend on the intended mode of administration of the active agent.

[0078] The amount of fusion protein(s) or vector(s) administered will depend on several factors (e.g., the route of administration, the condition of the individual, the aggressiveness of the malignancy, and the particular vector used). Also, the vectors may be used in combination with other therapeutic modalities.

[0079] It is contemplated that an effective amount of the fusion protein(s), immunogenic composition, or vaccine is administered. An "effective amount" is an amount sufficient to achieve a desired biological effect, such as inducing sufficient humoral or cellular immunity. This may depend on the type of vaccine and the age, sex, health, and weight of the recipient. Examples of desired biological effects include, but are not limited to, asymptomatic, reduced symptoms, reduced bacterial titers in tissues or mucosal secretions, complete protection against infection with H. influenzae, and partial protection against infection with H. influenzae.

[0080] A vaccine or immunogenic composition of the present disclosure is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient that enhances at least one primary or secondary humoral and / or cellular immune response to H. influenzae. In one embodiment, a vaccine or immunogenic composition of the present disclosure is provided either before the onset of infection (to prevent or attenuate an anticipated infection) or after the onset of an actual infection, thereby protecting against further bacterial infection.

[0081] Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.One example of such acceptable carrier is a physiologically balanced culture medium containing one or more stabilizers, such as stabilized hydrolyzed protein, lactose, etc.The carrier is preferably sterile.The formulation should be suitable for the mode of administration.

[0082] If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can be in the form of a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, inhalation formulation, or powder.Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0083] Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. When the composition is administered by injection, an ampoule of sterile diluent can be provided so that the ingredients can be mixed prior to administration.

[0084] The precise dosage of the fusion protein(s), immunogenic composition, or vaccine employed in the formulation will also depend on the route of administration and the nature of the patient, and should be determined according to the judgment of the physician and standard clinical techniques in accordance with each patient's circumstances. The precise amount of vector or virus utilized in a given preparation is not critical, so long as it provides the minimum amount of virus necessary to produce immunological activity. Dosage ranges from as little as about 10 mg to amounts of 1 milligram or more are contemplated.

[0085] Effective dosages of the fusion protein(s), immunogenic compositions, or vaccines of the present disclosure can be extrapolated from dose-response curves derived from animal model test systems. In some embodiments, the immunogenic compositions or vaccines described herein comprise an amount of fusion protein in the range of 20 to 200 μg (e.g., about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, or about 200 μg).

[0086] In various embodiments, the immunogenic composition further comprises an adjuvant. Exemplary adjuvants include saponin, nonionic surfactants, vegetable oils, mineral gels such as aluminum hydroxide and aluminum phosphate, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, polysaccharides such as curdlan, chitosan, glucan, mannose, and keyhole limpet hemocyanin, as well as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, BCG (bacilli Calmette-Guerin), Corynebacterium parvum, and the like. Potentially useful human adjuvants include, but are not limited to, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-38, PEG-39, PEG-40, PEG-41,

[0087] ISCOM is an acronym for Immune Stimulating Complex, first described by Morein et al. (Nature 308:457-460, 1984). ISCOMs are a novel vaccine delivery system and are distinct from traditional adjuvants. ISCOMs are formed in two ways. In some embodiments, the antigen is physically incorporated into the structure during its formulation. In other embodiments, the ISCOM matrix (e.g., as supplied by Isconova) does not contain an antigen but is mixed with an antigen selected by the end user prior to immunization. After mixing, the antigen is in solution with the ISCOM matrix but is not physically incorporated into the structure.

[0088] In one embodiment, the adjuvant is an oil-in-water emulsion. Oil-in-water emulsions are well known in the art and have been suggested to be useful as adjuvant compositions (EP 399843; WO 95 / 17210; U.S. 20080014217). In one embodiment, the metabolizable oil is present in an amount of 0.5% to 20% (final concentration) of the total volume of the antigenic composition or isolated virus, in an amount of 1.0% to 10% of the total volume, or in an amount of 2.0% to 6.0% of the total volume.

[0089] In some embodiments, oil-in-water emulsion systems useful as adjuvants have small oil droplet sizes, hi particular embodiments, the droplet sizes range from 120 to 750 nm, or 120 to 600 nm in diameter.

[0090] For any oil-in-water composition to be suitable for human administration, the oil phase of the emulsion system contains a metabolizable oil. The oil can be any vegetable, fish, animal, or synthetic oil that is not toxic to the recipient and can be converted metabolically. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this disclosure and can include commercially available oils such as NEOBEE®. A particularly suitable metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil found in large amounts in shark liver oil and in small amounts in olive oil, wheat germ oil, rice bran oil, and yeast, making it a particularly suitable oil for use in this disclosure. Squalene is a metabolizable oil due to the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th ed., entry 8619). Exemplary oils useful in oil-in-water emulsions include, but are not limited to, sterols, tocols, and alpha-tocopherol.

[0091] In further embodiments, immune system stimulants are added to the immunogenic composition, such as cells, growth factors, chemokines, supernatants from cell cultures of lymphocytes, monocytes, or cells from lymphoid organs, cell preparations and / or extracts from plants, cell preparations and / or extracts from bacteria (e.g., BCG, Mycobacterium, Corynebacterium), parasites, or mitogens, and novel nucleic acids from other viruses or other sources (e.g., double-stranded RNA, CpG), polysaccharides, block copolymers, nanobeads, or other compounds known in the art, used alone or in combination.

[0092] Specific examples of adjuvants and other immunostimulatory agents include, but are not limited to, lysolecithin; glycosides (e.g., saponins and saponin derivatives, such as QuilA (QS7 and QS21) or GPI-0100); cationic surfactants (e.g., DDA); quaternary hydrocarbon ammonium halides; pluronic polyols; polyanions and polyatomic ions; polyacrylic acid, nonionic block polymers (e.g., Pluronic F-127); and 3D-MPL (3de-O-acylated monophosphoryl lipid A). See, e.g., U.S. Patent Publication Nos. 20080187546 and 20080014217.

[0093] In various embodiments, the adjuvant is an immune checkpoint inhibitor. In various embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, or PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody. Antibodies against checkpoint inhibitors include ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, and durvalumab. Antibodies specific for CTLA-4 include tremelimumab and ipilimumab (YERVOY®), which are approved for the treatment of melanoma. Antibodies against PD-1 include pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp) and nivolumab (OPDIVO®, Bristol-Myers Squibb). Antibodies that target PD-L1 include atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®).

[0094] Many methods can be used to administer or introduce the fusion protein(s), immunogenic composition, vaccine to an individual (i.e., including a subject or patient), including, but not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes.

[0095] How to use The present disclosure provides methods for treating or preventing diseases associated with a non-encapsulated Haemophilus influenzae (Hi) infection, comprising administering a vaccine or immunogenic composition comprising a fusion protein described herein. Disorders associated with Hi infection include, but are not limited to, otitis media, bronchitis, pneumonia, sinusitis, sepsis, endocarditis, acute epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infections, conjunctivitis, Brazilian purpura fever, occult bacteremia, and exacerbations of underlying lung diseases such as chronic acute respiratory bronchitis (AECB), bronchiectasis, and cystic fibrosis, chronic obstructive pulmonary disease (COPD), and acute exacerbation of COPD (AECOPD). In some embodiments, the disease associated with acapsular Hi infection is otitis media. In some embodiments, the disease associated with acapsular Hi infection is acute exacerbation of chronic bronchitis (AECB). In some embodiments, the disease associated with acapsular Hi infection is acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

[0096] In some embodiments, the immunogenic composition is administered concomitantly with other vaccines, for example, parenteral vaccines such as DTPw or DtPa vaccines (vaccines against Bordetella pertussis, diphtheria, and tetanus), meningitis from Haemophilus influenzae type b, hepatitis B, or vaccines against measles, mumps, and rubella (MMR), or pneumococcus, to optimize the number of visits to the doctor.

[0097] Also provided are compositions comprising the fusion proteins, vaccines, or immunogenic compositions described herein for use in treating or preventing disease associated with non-encapsulated Haemophilus influenzae (Hi) infection. In various embodiments, the present disclosure provides for the use of a composition comprising the fusion proteins, vaccines, or immunogenic compositions described herein in the preparation of a medicament for use in treating or preventing disease associated with non-encapsulated Haemophilus influenzae (Hi) infection.

[0098] In various embodiments, the vaccine or immunogenic composition reduces or prevents colonization of one or more of the sinuses, lungs, and ears.

[0099] Packaging and dosage form The fusion proteins, immunogenic compositions, or vaccines described herein can be packaged in unit doses or multiple doses (e.g., two doses, four doses, or more than four doses). For multiple dose formats, vials are typically, but not necessarily, superior to prefilled syringes. Suitable multiple dose formats include, but are not limited to, 2-10 doses per container, with 0.1-2 mL per dose. In a specific embodiment, the dose is a 0.5 mL dose.

[0100] The composition may be provided in a vial or other suitable storage container, or in a pre-filled transfer device, such as a single- or multi-component syringe, which may or may not be equipped with a needle. The syringe typically contains a single dose of the preservative-containing immunogenic composition of the present invention, although multi-dose pre-filled syringes are also contemplated. Similarly, a vial can contain a single dose or can contain multiple doses.

[0101] Although effective dose volumes can be routinely established, a typical dose of an injectable composition has a volume of 0.5 mL. In certain embodiments, the dose is formulated for administration to a human subject. In certain embodiments, the dose is formulated for administration to a human subject, whether adult, adolescent, infant, or young child (i.e., 1 year old or younger), and in preferred embodiments, may be administered by injection.

[0102] The liquid immunogenic compositions disclosed herein are also suitable for reconstituting other immunogenic compositions provided in lyophilized form. When the immunogenic composition is intended to be reconstituted for its own use, the present invention provides kits with two or more vials, two or more pre-filled syringes, or one or more vials and alternatively filled syringes, in which the contents of the syringe are used to reconstitute the contents of the vial prior to injection, or vice versa.

[0103] Alternatively, the immunogenic compositions disclosed herein can be lyophilized and reconstituted, e.g., using one of many methods well known in the art for lyophilization, to form dry, regular (e.g., spherical) particles, such as microparticles or microspheres, with particle characteristics, such as average diameter dimensions, that can be selected and controlled by varying the exact method used to prepare the particles. The immunogenic compositions can optionally further include an adjuvant, which can be prepared with or contained within each of the dry, regular (e.g., spherical) particles, such as microparticles or microspheres. In these embodiments, the present disclosure further provides immunogenic composition kits that include a first component comprising a stable dry immunogenic composition, optionally including one or more preservatives, and a second component for reconstituting a sterile aqueous solution of the first component. In certain embodiments, the aqueous solution includes one or more preservatives and, optionally, at least one adjuvant (see, e.g., WO 2009 / 109550, incorporated herein by reference).

[0104] In another embodiment, the container for the multi-dose form is selected from one or more of the group consisting of, but not limited to, common laboratory glassware, flasks, beakers, graduated cylinders, decanters, bioreactors, tubing, piping, bags, bottles, vials, vial closures (e.g., rubber stoppers, screw caps), ampoules, syringes, double- or multi-chamber syringes, syringe stoppers, syringe plungers, rubber fasteners, plastic fasteners, glass fasteners, cartridges, and disposable pens. The container is not limited by the material of manufacture and includes a variety of materials, such as glass, metals (e.g., steel, stainless steel, aluminum, etc.), and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a specific embodiment, the container for this form is a 5 mL Schott Type 1 glass vial with a butyl stopper. One of skill in the art will recognize that the above list is not exhaustive and merely serves as a guide to the skilled artisan regarding the many forms that can be used in the present invention. Other forms contemplated for use in the present invention can be found among laboratory equipment suppliers and manufacturers, such as the VWR Public Catalog of United States Plastic Corp. (Lima, Ohio).

[0105] Further aspects and details of the present disclosure will become apparent from the following examples, which are intended to be illustrative rather than limiting.

[0106] Example Example 1 - Cloning of recombinant HirV lipoproteins All of the H. influenzae recombinant vaccine (HirV) candidates in the natural H. influenzae host contain signal sequences that target the proteins to the membrane, but only P6, PD, and P4 are naturally occurring lipoproteins. The constructs described below have their native signal sequences replaced with the P4 lipid-encoding signal sequence (SEQ ID NO: 11). Initial transformation of the clones was into DH5 E. coli cells. Restriction analysis and DNA sequencing of the recombinant isolated plasmid DNA were performed to confirm cloning.

[0107] Lipidated Omp26 (LOmp26): The mature Omp26 gene containing the P4 lipid encoding N-terminal signal sequence (SEQ ID NO: 13) was first synthesized and cloned into the Kpn1 and Sph1 sites of pBAD18Cm using GenScript (Piscataway, NJ). Primers were designed (see Table 1) and used to PCR the Omp26 gene from pBAD18Cm and clone it into the Nde1 and Xho1 sites of the pET21a vector, adding a poly-His tag to the carboxy terminus of the protein. The full-length LOmp26 amino acid sequence is provided in SEQ ID NO: 14.

[0108] Lipidated P6 (LP6): The mature P6 gene sequence containing the P4 lipid encoding N-terminal signal sequence (SEQ ID NO: 15) was first synthesized and cloned into the Kpn1 and Sph1 sites of pBAD18Cm using GenScript (Piscataway, NJ). Primers were designed (see Table 1) and used to PCR the P6 gene from pBAD18Cm and clone it into the Nde1 and Xho1 sites of the pET21a vector, adding a poly-His tag to the carboxy terminus of the protein. The full-length LP6 amino acid sequence is provided in SEQ ID NO: 16.

[0109] Lipidated PD (LPD): The mature PD gene sequence containing the P4 lipid encoding the N-terminal signal sequence (SEQ ID NO: 17) was first synthesized and cloned into the Kpn1 and Sph1 sites of pBAD18Cm using GenScript (Piscataway, NJ). Primers were designed (see Table 1) and used to PCR the PD gene from pBAD18Cm and clone it into the Nde1 and Xho1 sites of the pET21a vector, adding a poly-His tag to the carboxy terminus of the protein. The full-length LPD amino acid sequence is provided in SEQ ID NO: 18.

[0110] Lipidated P4 (LP4): The mature P4 gene sequence, including the P4 lipid encoding the N-terminal signal sequence (SEQ ID NO: 19), was synthesized and directly cloned into pET-21a(+) at the NdeI and XhoI sites using GenScript (Piscataway, NJ). The P4 gene also contained the N218Q mutation, which reduces its phosphomonoesterase activity, as previously reported. The full-length LP4 amino acid sequence is provided in SEQ ID NO: 20.

[0111] [Table 3]

[0112] Example 2 - Recombinant protein expression and purification Recombinant clones produced as described in Example 1 were transformed into BL21 or BLR-expressing cell lines, which are B / r E. coli and lack the cytoplasmic Ion protease. The lipoprotein clones were then transformed into C41(DE3) or C43(DE3), which overcome the toxicity of overproduced membrane proteins compared to BL21(DE3) (Mirouz et al., J. Mol. Biol., 260:289-298, 1996; Dumon-Seignovert et al., Protein Exp. Purif., 37:203-206, 2004; Chen et al., Vaccine, 27:1400-1409, 2009). Table 2 lists the genotypes of the various E. coli strains.

[0113] [Table 4] (DE3): T7 polymerase (I gene) inserted into the lambda phage integrase gene. Expression of T7 polymerase is controlled by lacUV5 and produced in a λ-lysogen under lacUV5 control (IPTG-inducible).

[0114] Cells were grown in Luria-Bertanni (LB) medium + 100 μg / ml ampicillin in 50 ml culture tubes at 37°C with shaking at 200 rpm. A 1:100 dilution of overnight-grown bacteria into fresh LB medium + 100 μg / ml ampicillin was grown to an OD600 of approximately 0.5 (approximately 2.5-3 hours at 37°C, 200 rpm) before being induced with 0.4 mM IPTG. Cells were grown at 30°C for 4 hours and harvested by centrifugation at 10K rpm. Pellets were stored at -20°C. All recombinant proteins were expressed using the above conditions with the following modifications: LP6:C43(DE3) cells were used. LPD:C41(DE3)pLysS cells were used, and pre-growth, growth, and incubation media also contained 34 μg / mL chloramphenicol. LPF:C41(DE3)pLysS cells were used. Pre-growth, growth, and incubation media also contained 34 μg / mL chloramphenicol. Growth and induction media also contained 1% glucose. Induction was at 37°C for 4 hours. LP4:C43(DE3) cells were used. Growth and induction medium also contained 1% glucose.

[0115] Cell lysis: For each thawed protein pellet produced from a 1 L batch, 30 ml of a buffer solution containing 50 mM Tris, 300 mM NaCl, and 100 μg / ml lysosomal acid, pH 8, was added. The protease inhibitor PMSF was added to a concentration of 1 mM. After suspending the pellet, cells were lysed by sonication (W-225 Sonicator Ultrasonic W-225) on ice for 3-4 cycles of 15 seconds at a 30-dose duty cycle, followed by incubation in a 37°C water bath for 30 minutes and rotation at 10 rpm for 1 hour.

[0116] Next, for each thawed protein pellet produced from a 1 L batch, 30 ml of buffer containing 50 mM Tris / 300 mM NaCl / 100 μg / ml lysosomal pH=8 was added. The protease inhibitor PMSF was added to a concentration of 1 mM. After suspending the pellet, the cells were lysed by sonication (W-225 Sonicator Ultrasonic W-225) for 3-4 cycles of 15 seconds at a 30-dose duty cycle on ice, followed by incubation in a 37°C water bath for 30 minutes and rotation at 10 K rpm for 1 hour. For each thawed protein pellet produced from a 1 L batch, 30 ml of buffer containing 50 mM Tris / 300 mM NaCl / 100 μg / ml lysosomal pH=8 was added. The protease inhibitor PMSF was added to a concentration of 1 mM. After suspending the pellet, cells were lysed by sonication (W-225 Sonicator Ultrasonic W-225) on ice for 3-4 cycles of 15 seconds at a 30-dose duty cycle, followed by incubation in a 37°C water bath for 30 minutes and rotation at 10K rpm for 1 hour.

[0117] Purification of lipidated recombinant protein: For each thawed protein pellet produced from a 1 L batch, 30 ml of buffer containing 50 mM Tris, 300 mM NaCl, and 100 μg / ml lysosomal acid, pH 8, was added. The protease inhibitor PMSF was added to a concentration of 1 mM. After suspending the pellet, cells were lysed by sonication (W-225 Sonicator Ultrasonic W-225) on ice for 3-4 cycles of 15 seconds at a 30-dose duty cycle. The pellet was then incubated for 30 minutes in a 37°C water bath and rotated at 10 rpm for 1 hour.

[0118] For binding and elution of His-tagged lipoproteins, the dissolved protein extract was applied to the equilibrated beads, and the flow-through was collected for further analysis by SDS-PAGE gel. The beads were washed with 5x column volumes of wash buffer. Lipoproteins were then eluted with 1-5 ml of elution buffer (250 mM imidizole + 0.5% Zwittergent), and 1 ml fractions were collected. Protein concentrations of the fractions were determined on a Nanodrop.

[0119] Fractions were analyzed by SDS-PAGE for lipoprotein recovery and purity, and if necessary, size exclusion or ion exchange was used to further purify the lipoproteins.

[0120] Imidazole was removed using Millipore Centricon tube filters (3 or 10 kDa). The lipoprotein fraction was loaded onto the filter and spun at 3 rpm for 20-30 minutes. 10-15 ml of PBS buffer containing 0.05% Zwittergent was added to 1 ml of the concentrate and spun again. This process was repeated twice.

[0121] Protein concentrations were determined using a Bradford assay, diluted with PBD buffer containing 0.05% Zwittergent to a final concentration of >0.5 mg / ml, and aliquots were stored at -80°C. Lipoprotein size and purity were assessed by SDS-PAGE (data not shown). Final lipoprotein yields were 1-4 mg per liter of induced culture.

[0122] Purification of non-lipidated recombinant proteins: All non-lipidated proteins were expressed using the same conditions as lipoproteins. No lysozyme was added during lysis, but 1% Triton was added before sonication. After the first centrifugation, the supernatant containing the protein was purified using the Ni-bead protocol as described above, but without the addition of Zwittergent in the buffer.

[0123] The final yield of non-lipidated protein was 5-15 mg per liter of induced culture.

[0124] Example 3 - Mass Spectrometry Lipidated proteins were prepared for LC-MS analysis by reduction and alkylation, followed by protein precipitation. After precipitation, proteins were digested into peptides using trypsin and chymotrypsin separately. Small sample volumes were run on a nanoLC system using two different gradients to ensure complete elution of highly lipidated peptides. Mass spectra were then generated using an HCD-IT (ion trap) and an HCD-OT (orbitrap). Data processing was performed using Proteome Discoverer 2.2. The combination of trypsin digestion, a high organic solvent gradient, and MS using an HCD-IT (ion trap) showed the best results in identifying lipidated peptides.

[0125] A control triacylated peptide, PAM3CSK4, was run to optimize the detection of triacylated peptides generated from recombinant lipidated proteins. Results showed reliable detection of fully triacylated peptides, as well as O-linked diacylated and N-linked monoacylated products generated during the collision / fragmentation process.

[0126] Based on published literature, the theoretical monoisotopic and average masses for each predicted lipid composition (1, 2, 3, A, A', B, B', C, D, E) were used in the analysis of the generated spectra by Skyline and Proteome Discoverer software. Modifications included neutral mass (M), +1 charge (M+H), and +2 charge (M+2). O-linked diacylation (diacylglycerol) was detected in many PSMs for lipid structures 1, 2, 3, and B. Lipid structure D also yielded high confidence with only one PSM due to its well-defined peak (data not shown). Triacylated rLP6 was detected with moderate confidence with only one PSM for lipid D structure. Table 3 shows a summary MS analysis of two different lots of LP6 and LP4, and one lot of LPF.

[0127] [Table 5]

[0128] Example 4 - Addition of N-acetyltransferase enzyme promotes triacylation. Lipoproteins produced in Gram-negative and certain Gram-positive bacteria carry both O- and N-acylated fatty acids. 1,2 The addition of N-acylated fatty acids occurs after diacylation of glycerol at the N-terminal cysteine ​​residue. 3 In Escherichia coli, the enzyme N-acyltransferase (Lnt, originally named CuTE) 4 transfers the sn-1-acyl chain of phosphatidylethanolamine to the amine group of cysteine ​​to generate triacylated lipoproteins. The crystal structure of Lnt has been determined and it is a monomeric eight-transmembrane protein localized in the inner membrane. 5

[0129] The level of recombinantly expressed triacylated lipoprotein relative to diacylated lipoprotein produced in E. coli may reflect the amount of Lnt available to catalyze the final step in lipoprotein maturation.

[0130] This approach involves cloning the Lnt gene, containing the native Shine-Dalgarno and ribosome binding regions, from E. coli BLR21 cells into a plasmid compatible with our pET21 expression vector. A p15A plasmid origin vector, such as pACYC184, which is a low-copy plasmid and uses a non-inducible promoter, is used when overexpression of Lnt is lethal. The chloramphenicol resistance gene on pACYC184 is replaced with the Lnt gene, and Lnt expression is under constitutive control of the chloramphenicol promoter. The pACYC184_Lnt plasmid is cloned into DH5 competent cells and tested for expression of the plasmid-encoded lnt RNA. After confirming the correct clone and RNA expression, the plasmid is transformed into HirV-recombinant lipoprotein-resistant C41(DE3) or C43(DE3) strains, co-selecting for tetracycline resistance. The level of triacylation of our HirV purified lipidated proteins is determined by mass spectrometry (MS), for example by the method described in Example 2.

[0131] References for Example 4: 1. Nguyen MT, Gotz F. Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence. Microbio lMol Biol Rev2016;80(3):891-903. 2.Nakayama H, Kurokawa K, Lee BL.Lipoproteins in bacteria: structures and biosynthetic pathways.The FEBS journal2012;279(23):4247-68. 3.Okuda S, Tokuda H.Lipoprotein sorting in bacteria.Annu Rev Microbiol2011;65:239-59 4.Gupta SD, Gan K, Schmid MB, Wu HC.Characterization of a temperature-sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-acyltransferase.J Biol Chem 1993;268(22):16551-6. 5.Noland CL, Kattke MD, Diao J, et al.Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase.Proc Natl Acad Sci USA 2017;114(30):E6044-e53. 6. Luo Y, Friese OV, Runnels HA, et al. The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease. The AAPS journal 2016;18(6):1562-75. 7.Martinon-Torres F, Gimenez-Sanchez F, Bernaola-Iturbe E, Diez-Domingo J, Jiang Q, Perez JL. A randomized,phase 1 / 2 trial of the safety, tolerability, and immunogenicity of bivalent rLP2086 meningococcal B vaccine in healthy infants.Vaccine 2014;32(40):5206-11.

[0132] Example 5 - Cloning of recombinant lipidated fusion proteins The following examples describe the process for making LP6φOmp26 and LOmp6φP6 fusion proteins.

[0133] LP6φOmp26 fusion protein: The P6 and Omp26 genes used to generate the fusion were derived from the pET21 clone construct described in Example 1. The fusion linker sequence is provided in SEQ ID NO: 12. To generate the LP6φOmp26 construct, an LP6 DNA fragment was PCR-generated using primers Nde1-LP4Fwd and Lnk-P6Rev (see Table 1 for primer sequences). In a separate reaction, an Omp26 DNA fragment was PCR-generated using primers Lnk-Omp26Fwd and Xho1Omp26Rev. The purified DNA fragments were added together, and after the first PCR reaction, primers Nde1-LP4Fwd and Xho1Omp26Rev were added to the PCR reaction. The purified final fusion PCR product was digested with Nde1 and Xho1 and cloned into the Nde1 and Xho1 sites of pET21. The nucleotide sequence cloned into pET21a is set forth in SEQ ID NO: 28, and the encoded amino acid sequence is set forth in SEQ ID NO: 29.

[0134] LOmp26φP6 fusion protein: The LOmp26 and P6 genes used to generate the fusion were derived from the pET21 clone construct described in Example 1. The fusion linker sequence is provided in SEQ ID NO: 12. To generate the LOmp26φP6 construct, an LOmp26 DNA fragment was PCR-generated using primers Nde1-LP4Fwd and Lnk-Omp26Rev (see Table 1 for primer sequences). In a separate reaction, a P6 DNA fragment was PCR-generated using primers Lnk-P6Fwd and Xho1P6Rev. The purified DNA fragments were added together, and after the first PCR reaction, primers Nde1-LP4Fwd and Xho1P6Rev were added to the PCR reaction. The purified final fusion PCR product was digested with Nde1 and Xho1 and cloned into the Nde1 and Xho1 sites of pET21. The DNA sequence cloned into pET21a is set forth in SEQ ID NO: 30, and the predicted full-length His-tag fusion protein sequence is set forth in SEQ ID NO: 31.

[0135] Example 6 - Cloning of recombinant non-lipidated fusion proteins The following examples describe processes for producing non-lipidated P6φOmp26 and Omp6φP6 fusion proteins.

[0136] P6φOmp26: The P6 and Omp26 genes used to generate the fusion were derived from the pET21 clone construct described in Example 1. The fusion linker sequence is set forth in SEQ ID NO: 12. To generate the P6φOmp26 construct, a P6 DNA fragment was PCR-generated using primers Nde1P6Fwd and Lnk-P6Rev (see Table 1 for primer sequences). In a separate reaction, an Omp26 DNA fragment was PCR-generated using primers Lnk-Omp26Fwd and Xho1Omp26Rev. The purified DNA fragments were added together, and after the first PCR reaction, primers Nde1P6Fwd and Xho1Omp26Rev were added to the PCR reaction. The purified final fusion PCR product was digested with Nde1 and Xho1 and cloned into the Nde1 and Xho1 sites of pET21. The DNA sequence cloned into pET21a is set forth in SEQ ID NO: 32, and the predicted full-length His-tag fusion protein sequence is set forth in SEQ ID NO: 33.

[0137] The Omp26 and P6 genes used to generate the Omp6φP6-fusion were derived from the pET21 clone construct described in this report. The fusion linker sequence is set forth in SEQ ID NO: 12. To generate the Omp26φP6 construct, an Omp26 DNA fragment was PCR-generated using primers Nde1Omp26Fwd and Lnk-Omp26Rev (see Table 1 for primer sequences). In a separate reaction, a P6 DNA fragment was PCR-generated using primers Lnk-P6Fwd and Xho1P6Rev. The purified DNA fragments were added together, and after the first PCR reaction, primers Nde1Omp26Fwd and Xho1P6Rev were added to the PCR reaction. The purified final fusion PCR product was digested with Nde1 and Xho1 and cloned into the Nde1 and Xho1 sites of pET21. The DNA sequence cloned into pET21a is set forth in SEQ ID NO: 34, and the predicted full-length His-tag fusion protein sequence is set forth in SEQ ID NO: 35.

[0138] Example 7 - Immunogenicity studies of lipidated versus non-lipidated forms of P6 and Omp26 Mice (n = 4-5) were vaccinated at the indicated times with a 10 μg dose of each protein, and serum was collected 2 weeks after the third dose. Results showed that significantly higher mouse serum IgG antibodies were induced by the lipidated versions of P6 and OMP26 compared to the nonlipidated versions (Figure 1).

[0139] Example 8 - Immunogenicity studies of lipidated fusion proteins Fusion constructs of the OMP26φP6 protein (LOmp26φP6), in which a signal sequence was linked to OMP26, were generated as described in Example 5. Mice (n = 4-5) were vaccinated with a 10 μg dose of LOmp26φP6 or non-lipidated Omp26φP6 at the indicated times, and serum was collected 2 weeks after the third dose. The results show that LOmp26φP6 increased the IgG response to both OMP26 and P6, even though P6 was not lipidated (Figure 2). IgG OMP26 titers showed that LOMP26 was more immunogenic and showed no difference in immunogenicity when fused to other proteins, indicating that the fusion construct still maintained its native folding and structure. Interestingly, a significant increase in P6 IgG titers was observed when fused to LOMP26 compared to the non-fusion protein, demonstrating the trans-effect and similar functionality of the fusion construct.

[0140] Example 9 - Animal model of otitis media A murine Hi acute otitis media (AOM) model, mimicking the pathogenesis of natural human infection, was generated by introducing NP virus infection 1 week before Hi challenge using the mouse-adapted influenza strain PR8 / 36 (PR8). Data showed that HiNP colonization densities exceeded the pathogenic threshold for establishing AOM, and we validated this model in vaccine protection using protein D.

[0141] Using heat-killed Hi as an immunogen (positive control), results showed a highly significant reduction in NP colonization and complete protection against AOM. In challenge experiments in this mouse model (n = 4-5 mice per group) with lipidated and non-lipidated P6 protein, lipidated P6 resulted in a >1-log reduction in NP colonization by Hi (Figure 3). Hi burden was lowest in mice receiving lipidated P6 in both nasal wash and otic bulla samples.

[0142] Example 10 - Additional in vivo studies Lipidated and non-lipidated proteins of P6, OMP26, OMP26φP6, and P6φOMP26 fusions were expressed in E. coli strains BLR21(DE3) and C43(DE3), respectively, and purified using standard established protocols (Fletcher et al., Infection and Immunity, 74:6383-6845, 2005). For purification of lipidated proteins, 1% Zwittergent was used during cell lysis to extract proteins from membranes, and the final lipidated construct proteins were stored at -80°C in 0.05% Zwittergent buffer to keep the proteins stable. Mass spectrometry analysis was performed on all four lipidated proteins to confirm their triacylated state (fully palmitoylated).

[0143] Four-week-old C57BL / 6 mice (n=10, 5 males and 5 females per group) were intramuscularly immunized on days 0, 7, and 21 with two different concentrations of the vaccine formulation, containing 10 μg and 25 μg of each protein (the molar amount of each protein, including the fusion construct, was taken into account in the calculation) (vertebrate section), along with aluminum phosphate adjuvant. Control mice received AlPO4 alone (negative control) or HK-Hi (positive control). In one group, both P6 and OMP26 proteins were mixed in lipidated and non-lipidated versions (e.g., L-OMP26 + NL-P6, L-P6 + NL-OMP26, and NLP6 + NLOMP26) to test for synergistic effects in challenge compared to the fusion construct. Blood samples were collected on day 35, and serum IgG and IgM were measured by standardized ELISA against the individual non-lipidated P6 and OMP26 proteins. On day 35, mice were infected with PR8 virus using the dose used in the standard HiAOM33 model. One week later, mice were challenged intranasally with 10 μl of Hi strain 575 given to each nostril. (Strain 575 is from a repository characterized for surface expression of both proteins and whole genome analysis.) Three days after infection, ear washes, otic bullae, nasal washes (NW), NP mucosal tissue (embedded in paraffin for preservation), NALT, blood, and spleens were collected. Measurement of Hi bacteria in NW and middle ear samples was performed by plating different dilutions onto chocolate plates to enumerate them.

[0144] Mucosal IgG and IgA antibody levels are then determined from nasal washes (NW) and ear washes. Nasal-associated lymphoid tissue (NALT), the primary mucosal site of respiratory tract infection and a reservoir for local T cells, is isolated. Spleens are homogenized, and isolated cells are stored in liquid nitrogen for T cell analysis.

[0145] The levels of IL-17, IL-6, and IL-22 produced by memory Th17 cells were determined from nasal and ear washes using Luminex (Biorad). In humans, IL-6 is not produced by Th17 cells. To measure Th17 cell responses, CD4+ T cells were isolated from the spleen (using MACS microbead technology). Th17 cells were quantified by intracellular staining (ICS) of IL-17A. Because the number of mice tested in each group served as their own control for variance, duplicate assays were not performed. Antipathogenic Th17 responses in NALT to lipidated and nonlipidated proteins (by pooling NALT from 2–3 mice from each group to obtain sufficient numbers of cells) were also determined by standard ICS staining.

[0146] Without wishing to be bound by any particular theory, it is contemplated that a reduction in Hi is observed in ear and NP samples from mice vaccinated with the lipidated proteins described herein (alone or as fusion constructs) compared to non-lipidated antigens.

[0147] Next, the levels of IL-8 and TNF-α, known neutrophil chemokines, will be determined using Luminex (Biorad) in stored (-20°C) nasal and middle ear washes from vaccinated mice. Neutrophil infiltration will be assessed by histopathology of NP mucosal tissue as previously described (Luet al., PLoS Pathogens, 4:e10001592008; van Rossum et al., Infect. Immun., 73:718-7726, 2005). To confirm that neutrophils play a major role in reducing Hi in NP, immunized neutrophil-depleted mice will be analyzed for reduced protection. Six mice per group will be immunized with lipidated and nonlipidated P6 and OMP26 and treated with monoclonal antibody RB6-8C5 at the time of challenge, as previously described for Spn carriage (Lu, supra), and differential protection against Hi in the NP and middle ear will be observed. Antibody, cytokine levels and neutrophil counts correlate with Hi bacterial load in NP and ME lavage fluids.

[0148] Without wishing to be bound by any particular theory, it is contemplated that higher IL-8 and TNF-α levels and neutrophil infiltration in nasal washes and mucosa are observed after challenge from mice immunized with lipidated vaccine antigens. Depletion of neutrophils upon challenge reduces protection. Similar results are expected compared to lipidated and non-lipidated protein vaccination.

[0149] Example 11 - Passive Immunization The following example provides a protocol for determining whether immune serum alone and / or passive transfer of immune T cells confers protection in naive infant and adult mice. Ten adult mice per group are vaccinated with lipidated and non-lipidated P6 and OMP26 proteins (according to the schedule described in Example 9). Two weeks after the third vaccination, mice are sacrificed, blood and spleens are collected, and processed for serum and splenocytes. Antibody levels are quantified, and serum samples with high titers for each vaccinating protein are pooled. Serum samples containing 10 μg of antibody are adoptively transferred into naive mice via the tail vein. A second group of naive mice receives isolated immune CD4+ T cells alone, and a third group of naive mice receives a combination of immune serum and CD4+ T cells, which are then given to infant (day 14) and adult (week 6) mice via the tail vein. Mice (n = 8 per group) were given PR8 / / 36 7 days before transfer and challenged with Hi575 strain 7 days later (4-6 hours after antibody and T cell transfer). Bacterial loads in ear and nasal washes were determined 3 days after challenge.

[0150] Without wishing to be bound by any particular theory, it is contemplated that protection in adult mice requires only CD4+ T cells. Employing antibodies and CD4+ T cell transfer better passively protects infant mice from colonization and AOM.

[0151] Example 12 - Effect of tri- or diacylation on immune responses Protein production methods can result in varying levels of protein lipidation. To determine whether the type of lipidation has any effect on the immunogenicity of lipidated Hi proteins, the type and predicted level of lipidation on the fusion protein was examined.

[0152] To examine the effect of lipidation, HEK-BLUE™ hTLR2-TLR1 cells (SEAP reporter 293 cells expressing human TLR2 and TLR1 genes (InvivoGen, San Diego, CA)), which respond to high levels of triacylated peptides, were cultured according to the manufacturer's recommended protocol.

[0153] Figure 4 shows the level (%) of cell stimulation by various peptides, along with the positive control PAM3CSK4 (trilipidated peptide), used as 100% stimulation, and the negative control PAM2CSK4 (dilipidated peptide). Tests were performed at three different lipoprotein concentrations (10 μg, 1 μg, and 0.1 μg of protein). Data shown are for 1 μg. By this method, compared to the positive control Pam3CSK4, lipidated P6 (LP6) was estimated to be 86% triacyl lipoprotein, LOMP26 was 21% triacyl, LPD was 26% triacyl, LPF was 62% triacyl, LP6φNLOMP26 was 21% triacyl, and LPDφNLPF was 46% triacyl. LOMP26φNLP6 showed no triacyl stimulation, while all non-lipidated proteins showed no stimulation (negative control).

[0154] The type of signal sequence used to express the fusion proteins was also evaluated to determine whether there were any differences in the type or level of acylation on the protein as a result of the signal sequence. Constructs were made using signal sequences and tested in HEK-BLUE™ hTLR2-TLR1 cells. The different constructs were grown and purified under the same conditions. In all constructs, the signal sequence is cleaved at the C residue and the lipid moiety is attached. The sequences of the different signals used were: native P6 signal sequence SS1: MNKFVKSLLVAGSVAALAAC... (SEQ ID NO: 36); E. coli native signal sequence SS2 of Pal protein: MQLNKVLKGLMIALPVMAIAAC... (SEQ ID NO: 37); Haemophilus influenzae signal sequence SSP4 of P4 protein, MKTTLKMTALAALSAFVLAGC... (SEQ ID NO: 38).

[0155] The data in Figure 5 show that the signal sequence affects the amount of triacyl lipidation: constructs using the P4 signal sequence have approximately the same triacyl lipidation levels as constructs using the P6 native signal sequence.

[0156] Example 13 - Characterization of lipidated proteins To characterize the lipidation of the proteins, mass spectrometry analysis was performed on LOMP26, LP6, and the fusion protein LOMP26P6. Selected tripalm precursor m / zs were subjected to full-scan DDA runs (same as the previous run in Example 3 with a new gradient to further fragment the top 10 MS1 precursors) and parallel reaction monitoring (PRM) analysis. The readout detected triacylation modifications, diacylglycerol modifications on cysteines, and palmitoyl modifications on the N-terminus of cysteines.

[0157] Di- and triacyl-lipidated moieties may be present in different peaks in mass spectrometry, representing variations in lipid tail carbon length and number of bonds (mono or cyclo). The various compositions shown in the table indicate analysis by nomenclature modification T1 or D1, modification TA or DA, etc.

[0158] [Table 6]

[0159] Next, we tested whether the medium used to culture cells producing lipidated proteins affected the type and amount of lipidation. Cells were grown in 2XYT liquid culture medium versus minimal medium (M9) and the level of lipidation was measured. 2XYT liquid culture medium contains twice the amount of yeast extract as regular LB medium. M9 minimal medium contains a minimal salt formulation and nitrogen source (see, e.g., the Thermo Fisher Scientific catalog). While low lipidation yields were observed using 2XYT liquid culture medium, the use of minimal medium (M9) improved the yield of diacyl and triacyl lipoproteins. Lipidated proteins were extracted from membranes using 1% Triton and 1% zwitterionic detergent, purified by Ni column chromatography, and characterized by SDS-PAGE with Western blot for confirmation. Mass spectrometry comparison of L-OMP26 grown in 2XYT and M9 media showed that the height and size of the lipoprotein peaks correlated with the amount of a particular diacyl or triacyl lipoprotein.

[0160] The results demonstrated that when L-OMP26 was grown in minimal medium (M9), significantly more diacyl and / or triacyl products were present compared to 2XYT. Furthermore, L-OMP26 grown in minimal medium exhibited triacylation, whereas L-OMP26 grown in 2XYT did not produce triacylated products. Higher PSMs were observed for diacyl peaks grown in minimal medium compared to 2XYT medium. [PSM is the peptide spectral match, which refers to the number of matches between a theoretical spectrum (generated in silico based on the protein sequence) and the actual spectrum from the sample. More matches indicate a higher abundance of the target peptide.]

[0161] Example 14 - Protection by Hi fusion proteins in vaccinated mice Two models of Hi infection were used to evaluate the effect of lipidated protein vaccines on animal infection and colonization. In the first model (Model A), mice were immunized with different lipidated Hi protein constructs on Days 0, 7, and 21. After three doses of vaccine, on Day 35, mice were infected with PR8 influenza virus as described in the standard model of NTHi acute otitis media (AOM) (Michel et al., J Med Microbiol 2018 67(10):1527-1532). One week later, mice were challenged intranasally with 10 μl of NTHi strain 575 or 86-028NP administered into each nostril (strain 575 has been characterized for surface expression of target proteins and whole genome analysis). Three days after infection, ear washes, otic bullae, nasal washes (NW), and blood were collected. NTHi bacteria in NW and middle ear samples were enumerated by plating on chocolate plates.

[0162] In the second animal model (Model B), mice were immunized with different lipidated Hi protein constructs on Days 0, 7, and 21. On Day 35, after three doses of vaccine, NTHi inoculation was performed to establish colonization, followed by challenge with fluX31 influenza virus (10 ED50). Influenza strain X31 is known to induce a milder infection than previously used strains. The time between NTHi inoculation (10 cfu / mouse) and influenza inoculation was 6 hours. Five days after infection, ear washes, otic bullae, nasal washes (NW), and blood were collected.

[0163] The immunogenicity of lipidated proteins was analyzed (Model B). Alum hydroxide was used as an adjuvant. N=6 mice / group. Vaccines were administered intraperitoneally (IP). Considering the molar amounts during immunization, 10 μg of fusion protein, 7 μg of PD, and 3 μg of P6 protein were administered per mouse for IP injection. The results are shown in Figure 6.

[0164] When non-lipidated protein D fused to lipidated P6 was administered, a trans effect was observed for protein D responses. The trans effect refers to the enhanced immunogenicity of non-lipidated components of a vaccine construct when fused to a lipidated protein in a vaccine. After three doses, a greater than 10-fold increase in antibodies was measured for PD in the alum-free L-P6φNL-PD fusion compared to NL-PD with alum.

[0165] Three doses versus two doses were observed to increase immunogenicity. Furthermore, fusions can reduce the induced lipidated protein antibody response. For example, a two-fold reduction in P6 antibodies was observed with the L-P6φNL-PD fusion compared to administration of the L-P6 protein alone.

[0166] Furthermore, protein D immunogenicity was tested for lipidated fusion constructs in the presence or absence of alum, but no difference in antibody levels was measured. For P6 antibodies, the L-P6φNL-PD fusion and the L-P6 vaccine with and without alum showed no difference after two and three doses.

[0167] In a separate experiment, mice were administered fusions containing LOMP26, as shown in Figure 6C, at a protein dose of 10 μg per mouse. Alum hydroxide was used as the adjuvant (N = 4 mice per group, intraperitoneal (IP) injection). Figure 6C shows that the L-OMP26φNL-P6 fusion resulted in a significant increase in antibodies against OMP26 when the lipidated protein was given without alum compared to when the protein was composed of alum hydroxide. Consistent with previous results, three doses of the vaccine increased antibody levels compared to two doses. Furthermore, alum reduced OMP26 antibody levels but had no adverse effect on P6 antibody levels.

[0168] Mice were immunized with OMP26 or P6 (10 μg protein dose / mouse) with or without curdlan adjuvant (N=3-4 mice / group). The results are shown in Figure 7. Intranasal (IN) immunization with the fusion protein L-OMP26φNL-P6 induced a significant antibody response, with three doses of vaccine increasing antibody levels more than two doses. There is also an improvement for IN immunization with lipidated protein constructs when an adjuvant is used.

[0169] The level of lipidation was analyzed for immunogenicity. Figure 8A shows that diacyl moieties elicit more antibodies than triacyl lipid moieties. L-OMP26, which contains primarily triacyl moieties, showed a lower antibody response compared to diacyl-LOMP26. The diacylated and triacylated states of L-OMP26 are confirmed by mass spectrometry analysis based on the peak ratio, which indicates 10-fold higher tri-lipidation (black bars in the figure).

[0170] Immunogenicity markers were also analyzed. No differences were measured in the inflammatory markers IL-8, IL-6, IL-17, or TNF-α after L-OMP26 immunization with or without one, two, or three doses of alum, comparing the triacyl- and diacyl-predominant compositions, consistent with the very low reactogenicity of both constructs. When the CLL5 cytokine, a surrogate marker of inflammation, was analyzed, no significant differences were observed in CCL5 24 hours after vaccination between the two and three doses (Figure 8B).

[0171] The effect of vaccination on colonization after intranasal immunization was examined. Similar to Model B, mice were inoculated with L-OMP26φNL-P6 with or without curdlan adjuvant. The results are shown in Figure 9A and demonstrate protection from ear infection by intranasal immunization with the fusion protein L-OMP26φNL-P6. The L-OMP26φNL-P6 fusion provided significant protection against NTHi ear infection (Figure 9B). However, alum hydroxide in the composition appeared to reduce protection from ear infection.

[0172] Additional fusion protein constructs, NLOMP26+NLP6, LOMP26NLP6, LP6NLOMP26, and LP6+LOMP26, were used in Model B immunizations. Lipidated P6 combined with lipidated OMP26 without alum and lipidated P6 fused to nonlipidated OMP26 protect against nasopharyngeal colonization compared to nonlipidated P6 combined with nonlipidated OMP26 with alum hydroxide, as evidenced by nasal wash and otic bulla colonization levels. Lipidated LP6 combined with lipidated LOMP26 without alum protects against ear infection compared to nonlipidated P6 combined with nonlipidated OMP26 with alum hydroxide (Figure 10A).

[0173] L-PDφNL-PF fusions are protective in preventing NTHi colonization after IN immunization. (Figures 11A, 11B.) Balb / c mice (n=4 / group) were challenged with the 14-02-575 autologous clinical strain of NTHi using 10 μg of protein per dose, administered three times. In the colonization model, CFUs were detectable in the ear 12 hours after challenge, and a significant difference in colonization levels was observed with the fusion L-PDφPF.

[0174] Intramuscular immunization was also tested. IM immunization (10 μg / dose) of the L-PDφNL-OMP26 fusion protein conferred protection against ear infection and nasopharyngeal colonization (Figures 12A and 12B). Separate administration of L-PD and NL-OMP26 did not confer protection. In a separate experiment, different combinations of Hi proteins were administered three times at 10 μg / dose in model B (n = 5-6 mice / group). Figures 12C and 12D show that IM administration of the L-PDφNL-OMP26 fusion protein (10 μg protein / dose) conferred protection against both nasopharyngeal colonization and ear infection.

[0175] Numerous modifications and variations of the invention as described in the illustrative examples above are expected to occur to those skilled in the art, and therefore only limitations that appear in the appended claims should be placed on the invention.

Claims

1. A fusion protein comprising all or part of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, PD, and PF, wherein at least one of the Hi proteins is lipidated with a saturated, unsaturated, or branched fatty acid.

2. Omp26 and P6, (i) the Omp26 protein or fragment thereof is lipidated with saturated, unsaturated, or branched fatty acids; or (ii) the P6 protein or fragment thereof is lipidated with saturated, unsaturated, or branched fatty acids; The fusion protein of claim 1.

3. (i) the saturated, unsaturated, or branched fatty acid is selected from a C18, C16, C14, C12, or C10 fatty acid; (ii) the saturated, unsaturated, or branched fatty acid is selected from diacyl and / or triacyl fatty acids; (iii) the saturated, unsaturated, or branched fatty acid is selected from N-acylated or O-acylated fatty acids, and / or (iv) the two Hi proteins in the fusion protein are lipidated with saturated, unsaturated, or branched fatty acids; The fusion protein according to claim 1 or 2.

4. lipidated Omp26 and non-lipidated P6, or lipidated P6 and non-lipidated Omp26, wherein the saturated, unsaturated, or branched fatty acid comprises a C16 fatty acid; The fusion protein is selected from the group consisting of LOmp26φP6 (lipidated Omp26 linked to P6, as set forth in SEQ ID NO:31), LP6φOmp26 (lipidated P6 linked to Omp26, as set forth in SEQ ID NO:29), L-P6φNL-PD (lipidated P6 linked to non-lipidated PD, as set forth in SEQ ID NO:39), L-PDφNL-PF (lipidated PD linked to non-lipidated PF, as set forth in SEQ ID NO:40), L-PDφNL-P6 (lipidated P6 linked to non-lipidated P6, as set forth in SEQ ID NO:41), and L-PDφNL-P6 (lipidated P6 linked to non-lipidated P6, as set forth in SEQ ID NO:42). L-P6φNL-PD (lipidated P6 linked to non-lipidated PD, as set forth in SEQ ID NO:42), L-Omp26φNL-PD (lipidated Omp26 linked to non-lipidated PD, as set forth in SEQ ID NO:43), L-PDφNL-Omp26 (lipidated PD linked to non-lipidated Omp26, as set forth in SEQ ID NO:44), or L-PFφNL-P6 (lipidated PF linked to non-lipidated P6, as set forth in SEQ ID NO:45), The fusion protein according to any one of claims 1 to 3.

5. (i) Omp26 comprises all or part of the amino acid sequence set forth in SEQ ID NO: 2; or (ii) P6 comprises all or part of the amino acid sequence set forth in SEQ ID NO: 4; The fusion protein of claim 4.

6. (i) the at least one Hi protein comprises a saturated, unsaturated, or branched fatty acid at the N-terminus of the protein; (ii) the Hi proteins are linked by a peptide linker; (iii) the Hi proteins are linked by a peptide linker, and the peptide linker is GlySerGlyGlyGlyGly (SEQ ID NO: 12); (iv) the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 29 or 31, and / or (v) the fusion protein comprises a pharmaceutically acceptable carrier or excipient; The fusion protein according to any one of claims 1 to 5.

7. A vaccine comprising a fusion protein described in any one of claims 1 to 6. (i) the fusion protein in the vaccine elicits an antibody response against the lipidated Hi protein and at least one non-lipidated Hi protein. (ii) at least 10% of the fusion proteins in the vaccine comprise a diacyl fatty acid; and / or (iii) at least 10% of the fusion proteins in the vaccine comprise a triacyl fatty acid; The vaccine of claim 7. (i) at least 10% of the fusion proteins in the immunogenic composition comprise a diacyl fatty acid; and / or (ii) at least 10% of the fusion proteins in the immunogenic composition comprise a triacyl fatty acid; An immunogenic composition comprising the fusion protein of any one of claims 1 to 6.

10. 10. A medicament for use in the treatment or prevention of a disorder associated with Haemophilus influenzae (Hi) infection in a subject, comprising the vaccine of claim 7 or the immunogenic composition of claim 9, (i) the disorders associated with Hi infection are otitis media, bronchitis, pneumonia, sinusitis, sepsis, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chronic salpingitis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infections, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpura fever, occult bacteremia, and exacerbations of underlying lung diseases such as chronic bronchitis, bronchiectasis or cystic fibrosis, chronic obstructive pulmonary disease (COPD), and acute exacerbations of COPD (AECOPD); (ii) the vaccine or immunogenic composition is administered orally, intravenously, intramuscularly, intranasally, or subcutaneously; (iii) the vaccine or immunogenic composition reduces or prevents colonization of one or more of the sinuses, lungs, and ears; and / or (iv) the vaccine or immunogenic composition comprises LOmp26φP6 (lipidated Omp26 linked to P6) or LP6φOmp26 (lipidated P6 linked to Omp26); The drug.

11. 1. A method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, PD, and PF, wherein at least one of the Hi proteins is lipidated with a saturated, unsaturated, or branched fatty acid; (i) inserting a nucleic acid encoding a lipid moiety signal sequence region upstream of a first nucleic acid encoding all or part of a Haemophilus influenzae (Hi) protein Omp26, P6, PD, or PF in a plasmid vector; (ii) inserting into said plasmid vector a second nucleic acid encoding all or part of Haemophilus influenzae (Hi) protein Omp26, P6, PD, or PF; (iii) transfecting the plasmid vector into a host cell capable of expressing the nucleic acid molecule; (iv) purifying the fusion protein expressed by the plasmid.

12. The method of claim 11, further comprising inserting into the plasmid vector a third or additional nucleic acid sequence encoding one or more additional Hi proteins or fragments thereof selected from the group consisting of Omp26, P6, PD, and PF.

13. 1. A method for making a fusion protein comprising all or a portion of two or more Haemophilus influenzae (Hi) proteins selected from the group consisting of Omp26, P6, PD, and PF, wherein at least one of the Hi proteins is lipidated with a saturated, unsaturated, or branched fatty acid; (i) providing a nucleic acid encoding a lipid moiety signal sequence region; (ii) providing a first nucleic acid encoding all or part of a Hi protein Omp26, P6, PD, or PF; (iii) providing a second nucleic acid encoding all or part of a Hi protein Omp26, P6, PD, or PF, wherein the second nucleic acid encodes a Hi protein different from (ii); (iv) inserting the nucleic acid sequences (i) to (iii) into a plasmid vector capable of expressing the nucleic acid; (v) transfecting the plasmid vector of (iv) into a host cell capable of expressing the nucleic acid molecule and expressing the fusion protein; (vi) purifying the recombinant fusion protein expressed in the host cell.

14. The method of claim 13, further comprising providing a third or additional nucleic acid sequence encoding one or more additional proteins or fragments thereof selected from the group consisting of Omp26, P6, PD, and PF. (i) a nucleic acid encoding the lipid moiety signal sequence region is inserted upstream of the first nucleic acid in the plasmid vector, and the second nucleic acid is inserted downstream of the first nucleic acid in the plasmid vector; or (ii) a nucleic acid encoding the lipid moiety signal sequence region is inserted upstream of the second nucleic acid in the plasmid vector, and the first nucleic acid is inserted upstream of the nucleic acid encoding the lipid moiety signal sequence region; 14. The method of claim 11 or 13. (i) a nucleic acid encoding the lipid moiety signal sequence region is inserted upstream of the first nucleic acid in the plasmid vector, and the second nucleic acid and a third or additional nucleic acid are inserted downstream of the first nucleic acid in the plasmid vector; or (ii) a nucleic acid encoding the lipid moiety signal sequence region is inserted upstream of the second nucleic acid in the plasmid vector, the first nucleic acid is inserted upstream of the nucleic acid encoding the lipid moiety signal sequence region, and a third or additional nucleic acid is inserted downstream of the second nucleic acid in the plasmid vector; 15. The method of claim 12 or 14.

17. (i) the lipid moiety signal sequence is selected from the group consisting of MNKFVKSLLVAGSVAALAAC (SEQ ID NO: 36), with or without a terminal C residue, MQLNKVLKGLMIALPVMAIAAC (SEQ ID NO: 37), with or without the terminal C residue, MKTTLKMTALAALSAFVLAGC (SEQ ID NO: 38), or MKTTLKMTALAALSAFVLAG (SEQ ID NO: 11); (ii) the method further comprises a nucleic acid encoding a peptide linker sequence inserted between the first nucleic acid and the second nucleic acid, and / or between the second nucleic acid and a third or additional nucleic acid, wherein the linker is GlySerGlyGlyGlyGly (SEQ ID NO: 12); (iii) the method further comprises an additional nucleic acid encoding a lipid moiety signal sequence upstream of the nucleic acid encoding a Hi protein or a fragment thereof selected from the group consisting of Omp26, P6, PD, and PF; (iv) the first nucleic acid encodes a P6 protein or a fragment thereof, and the second nucleic acid encodes an Omp26 protein or a fragment thereof; (v) the first nucleic acid encodes an Omp26 protein or a fragment thereof, and the second nucleic acid encodes a P6 protein or a fragment thereof; (vi) the saturated, unsaturated, or branched fatty acid comprises a C18, C16, C14, C12, or C10 fatty acid; (vii) the saturated, unsaturated, or branched fatty acids comprise diacyl and / or triacyl fatty acids; (viii) the saturated, unsaturated, or branched fatty acid is an N-acylated or O-acylated fatty acid; (ix) the nucleic acid encoding Omp26 comprises all or part of the nucleic acid sequence set forth in SEQ ID NO: 1; (x) the nucleic acid encoding P6 comprises all or part of the nucleic acid sequence set forth in SEQ ID NO: 3, and / or (xi) the saturated, unsaturated, or branched fatty acid is at the N-terminus of the lipidated protein; The method according to any one of claims 11 to 16.

18. (i) The fusion protein is selected from the group consisting of LOmp26φP6 (lipidated Omp26 linked to P6, as set forth in SEQ ID NO: 31), LP6φOmp26 (lipidated P6 linked to Omp26, as set forth in SEQ ID NO: 29), L-P6φNL-PD (lipidated P6 linked to non-lipidated PD, as set forth in SEQ ID NO: 39), L-PDφNL-PF (lipidated PD linked to non-lipidated PF, as set forth in SEQ ID NO: 40), L-PDφNL-P6 (lipidated P6 linked to non-lipidated P6, as set forth in SEQ ID NO: 41), L-P6φNL-PD (lipidated P6 linked to non-lipidated PD, as set forth in SEQ ID NO:42), L-Omp26φNL-PD (lipidated Omp26 linked to non-lipidated PD, as set forth in SEQ ID NO:43), L-PDφNL-Omp26 (lipidated PD linked to non-lipidated Omp26, as set forth in SEQ ID NO:44), or L-PFφNL-P6 (lipidated PF linked to non-lipidated P6, as set forth in SEQ ID NO:45); or (ii) the method further comprises the nucleic acid sequence set forth in SEQ ID NO: 13 or 15; 18. The method of claim 17.

19. (i) the host cell is a bacterial cell; (ii) the host cell is Escherichia coli; (iii) the host cells are cultured in shake flasks, and / or (iv) the cells are sonicated prior to purification of the fusion protein; The method according to any one of claims 11 to 18.

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