ES2356077T3 - CHEMICAL VACCINE FOR A DISEASE INDICATED BY HAEMOPHILUS INFLUENZAE. - Google Patents

Abstract

Chimeric protein comprising a part of the untypifiable H. influenzae PilA protein (NTHi) (SEQ ID NO: 2) and a part of the LB1 peptide (SEQ ID NO: 53), the chimeric protein can cause an immune response to NTHi bacteria

Description

FIELD OF THE INVENTION

The invention described herein refers to a chimeric protein comprising the NTHi contractile pilus (PilA) main subunit protein that has a portion of the NTHi P5 OMP protein. The invention provides vaccine compositions comprising the chimeric protein and methods for eliciting an immune response using the chimeric proteins of the invention.

BACKGROUND

The clinical term for middle ear infections is otitis media (OM). According to Klein, Vaccine, 19 (Suppl. 1): S2-S8, 2000, OM is the most common reason for a sick child to receive medical attention and for a child in the United States to receive antibiotics or undergo anesthesia. general. Statistics indicate that 24.5 million of 10 visits to the doctor's office were made by OM in 1990, which represents an increase of more than 200% compared to those reported in the 1980s. Although rarely associated with mortality, morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often affecting the behavior, education and development of the child's language skills (Baldwin, Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. 15 Dis., 162: 685-694, 1990). The socio-economic impact of OM is also large, with direct and indirect costs of diagnosis and treatment of OM exceeding $ 5 billion annually in the US alone (Kaplan et al., Pediatr. Infect. Dis. J., 16: S9-11, 1997).

It is believed that OM is the result of infectious, environmental and genetic factors of the host. Bacteria such as Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are the most common infectious organisms in OM. Acute OM is a disease characterized by rapid onset and short duration of signs and symptoms of inflammation in the middle ear, while chronic OM refers to a state that is defined by the relatively asymptomatic presence of fluid (or effusion) In the middle ear. However, in chronic OM, despite the absence of certain signs of acute infection (i.e. earache or fever), these anomalous fluids of the middle ear may persist for periods exceeding three months. Treatment of acute OM by antibiotic therapy is common, but multiple antibiotic-resistant bacteria have emerged in the three genera of bacteria responsible for OM. Surgical treatment of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane of the ear while the child is under general anesthesia. Although this procedure is common (prevalence rates are ~ 1 million tubes inserted per year in US Bright et al., Am. J. Public Health, 83 (7): 1026-8, 1993) and is highly effective in As for relieving painful symptoms by draining the 30 accumulated fluids in the middle ear, it is invasive and carries significant risks (Berman et al., Pediatrics, 93 (3): 353-63, 1994; Bright et al., cited above; Cimons , ASM News, 60: 527-528; Paap, Ann. Pharmacother., 30 (11): 1291-7, 1996). Therefore, other approaches to treatment and, preferably, prevention of OM are necessary.

The development of a vaccine against OM is more advanced for S. pneumoniae, the main causative agent of acute OM (AOM), as evidenced by the recent approval and authorization of a heptavalent capsular conjugate vaccine, PREVNAR® (Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). Although PREVNAR® has been extremely effective for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al. , Pedriatr. Infect. Dis J, 19: 187-195, 2000; Eskola et al., Pedriatr. Infect. Dis J., 19: S72-78, 2000; Eskola et al., N. Engl. J. Med. , 344: 403-409, 2001; Snow et al., Otol. Neurotol., 23: 1-2, 2002). 40

H. influenzae is a gram-negative bacterium that, as indicated above, plays a role in OM. The clinical isolates of H. infuenzae are classified either as serotypes "a" to "f" or as non-typifiable depending on the presence or absence, respectively, of specific polysaccharide capsules of the type in bacteria. A vaccine against H. influenzae type b has been developed. Like PREVNAR®, vaccines against H. influenzae type b are directed to the polysaccharide capsule of this organism and therefore the vaccine is composed of a capsular polysaccharide that has been conjugated with a protein carrier. Neither PREVNAR® nor H. influenzae type b vaccine has any efficacy for NTHI-induced respiratory diseases, including OM. Less progress has been made for a vaccine against non-typable H. influenzae (NTHi) that causes approximately 20% of acute OM in children and predominates in chronic OM with effusion (Coleman et al., Inf and Immunity, 59 (5 ), 1716-1722, 1991; Klein, Pedriatr. Infect. Dis J., 16, S5-8, 1997; Spinola et al., J. Infect. Dis., 154, 100-50 109, 1986). NTHi can also cause pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, puerperal and neonatal infections, puerperal and neonatal sepsis, acute and chronic salpingitis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, infection of intra-abdominal infections urinary tract, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpuric fever, occult bacteraemia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchiectasis and cystic fibrosis. An isolate of NTHi prototype 55 is the isolate with number of passes under 86-028NP that was recovered from a child with chronic OM. This strain has been well characterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as in models of Chinchilla OM (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Immun., 62: 1710-8, 1994; DeMaria et al., Infect. Immun., 64: 5187 -92, 1996). NTHi strain 86-026N was deposited in the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110, on October 16, 2001 and was issued 60

assigned registration number PTA-4764. A set of contiguous sequences of the genome of strain 86-028NP can be found on the website of the Columbus Children's Research Institute Center for Microbial Pathogenesis.

Adhesion and colonization are recognized as the first stages in the pathogenesis of diseases induced by H. influenzae. As such, H. influenzae expresses multiple adhesins that include non-fimbrial adhesives, fimbriae and hemagglutinating pili (Gilsdorf et al., Pediatr Res 39, 343-348, 1996; Gilsdorf., Infect. Immun., 65, 2997-3002, 5 1997; and St. Geme III, Cell. Microbiol., 4, 191-200, 2002). In particular, none of the adhesins described have previously been associated with a motility function. In addition, H. influenzae does not express flagella that are also associated with motility. Contractile motility is an independent form of bacterial translocation flagella on wet surfaces and occurs through extension, tying and then retraction of polar structures known as type IV pili (Bardy, Microbiology, 149, 295-304, 2003; Tonjum and Koomey, Gene, 192, 155-163, 1997; 10 Wolfgang et al., EMBO J., 19; 6408-6418; Mattick, Annu. Rev. Microbiol., 56, 289-314, 2002). Type IV pili normally have 5-7 nm in diameter, several micrometers in length and are composed of a single protein subunit assembled in a helical conformation with ~ 5 subunits per turn (Bardy et al., Microbiology, 149, 295- 304, 2003; Wall and Kaiser, Mol. Microbiol., 32, 1-10, 1999). Type IV pilin subunits are usually 145-160 amino acids in length and can be glycosylated or phosphorylated. There are two classes of pilin subunits, type IVa and type IVb, which are distinguished from each other by the average length of the leader peptide and the mature subunit, whose N-methylated amino acid occupies the N-terminal position of the mature protein, and the average length of region D (for disulfide region). Most respiratory pathogens express class IVa pilins, while enteropathogens more commonly express class IVb pilins. Type IVa pili are distinguished by the presence of a highly conserved hydrophobic N-terminal methylated phenylalanine. twenty

Type IV pili act as means of rapid translocation on, and colonization of, new surfaces. Therefore, the expression of type IV pilus is important both for adhesion and for the formation of biofilms by many bacteria (Mattick, Annu. Rev. Microbiol., 56, 289-314 2002; O'Toole and Kolter, Mol Microbiol., 30, 295-304, 1998; Klausen et al., Mol. Microbiol., 50, 61-68, 2003; Jesaitis et al., J. Immunol., 171, 4329-4339, 2003), as well such as the virulence of Neisseria, Moraxella bovis, Vibrio cholerae, enteropathogenic Escherichia coli and 25 Pseudomonas aeruginosa species, among others (OToole and Kolter, cited above; Klausen et al., cited above; Klausen et al., Mol. Microbiol., 48, 1511-1524, 2003; Strom and Lory, Annu. Rev. Microbiol., 47, 565-596, 1993). A biofilm is a complex organization of bacteria that anchor to a surface through a matrix extruded by bacteria, composed of exopolysaccharide or other substances. The matrix involves bacteria and protects them against the human immune system. Ehrlich et al., JAMA, 287 (13), 1710-1715 (2002) describe the formation of biofilms by 30 H. influenzae. It has been postulated that blocking the interaction between type IV pili and the human body can prevent or stop bacterial infection (Meyer et al., US Patent No. 6,268,171 published July 31, 2001).

The expression of type IV pilus is a complex and highly regulated bacterial function. In P. aeruginosa, the biogenesis and function of type IV pili are controlled by more than forty genes (Strom and Lory, cited above). To date, only a subset of the large number of related type IV pilus genes (Tonjum and Koomey, cited above; Darzins and Russell, Gene, 192, 109-115, 1997) have been found in several family members PAH (Haemophilus, Actinobacillus and Pasteurella) (Stevenson et al., Vet. Microbiol., 92, 121-134, 2003; Doughty et al., Vet. Microbiol., 72, 79-90, 2000; Dougherty and Smith, Microbiology , 145, 401-409 1999), but until now neither the expression of type IV pili nor the motility by contraction has been described for any isolate of H. influenzae. In fact, H. influenzae is traditionally described as a bacterium that does not express these structures 40 (Friedrich et al. Appl. Environ. Microbiol., 69, 3695-3700, 2003; Fussenegger et al., Gene, 192, 125-134 , 1997), despite the presence of a cluster of cryptic genes within the genome of strain Rd (Fleischmann et al., Science, 269, 496-512, 1995). The strain Rd is a non-encapsulated derivative of a serotype organism of H. influenzae (Zwahlen et al., Infect. Immun., 42, 708-715, 1983; Bendler and Goodgal, J. Microbiol., 70, 411- 422, 1972; Risberg et al., Eur. J. Biochem., 261, 171-180, 1999). Although strain Rd has some virulence properties, it is generally considered that strains of serotype d are commensal; they do not frequently cause disease (Daines et al., J. Med. Microbiol., 52, 277-282, 2003). Therefore it is important to distinguish between H. influenzae strains that cause disease and strain Rd.

Fimbriae, which are superficial appendages found in nontypable Haemophilus influenzae, are produced by 100% of bacteria recovered from the middle ears and nasopharyngeal region of children with chronic middle otitis 50. A vaccine composed of fimbrine, a filamentous protein derived from the non-typable Haemophilus influenzae fimbriae, was previously developed and is useful for studying, preventing or reducing the severity of otitis media. However, existing methodologies for isolating fimbrine protein from the bacterial outer membrane are tedious and time consuming. Similarly, purification of fimbrine expressed by the fimbrine gene in another host vector is also tedious due to the homology between the fimbrine protein and the outer membrane proteins of the host vector.

The synthetic chimeric vaccine candidate, designated as LB1 and described in US Patent No. 5,843,464, has shown enormous efficacy in multiple preclinical vaccine trials in two rodent hosts. This synthetic peptide comprises a P5 fimbrine B cell epitope synthesized collinearly with promiscuous T cell epitope derived from a measles vaccine fusion protein. Although the LB1 peptide has proven effective in preclinical trials, there is concern about the ability to test and commercialize a vaccine containing a promiscuous T-cell epitope for intended use in very young children. By

therefore, there is a need to develop a vaccine candidate that elicits a specific and controlled immune response against H. influenzae.

SUMMARY OF THE INVENTION

The present invention relates to chimeric proteins comprising a part of the main type IV pilus subunit protein (PilA) of non-typable H. influenzae (NTHi) and a part of the NTHi 5 P5 OMP protein (also called fimbrine P5, fimbrine or adhesive homologous to OMP P5). In particular, the invention provides chimeric proteins comprising modified PilA to present the B cell epitope of the LB1 peptide. Therefore, according to one aspect of the present invention a chimeric protein is provided which comprises a part of non-typable H. influenzae PilA protein (NTHi) (SEQ ID NO: 2) and a part of the LB1 peptides (SEQ ID NO: 53). The invention also provides compositions comprising one or more chimeric proteins of the invention and their use to elicit an immune response.

Peptide LB1 is a peptide derived from synthetic chimeric fimbrine P5 of 40 amino acids (SEQ ID NO: 53) that induces an immunogenic response against NTHi and is advantageous because it does not require tedious purification techniques. The LB1 peptide comprises a N-terminal 19 amino acid peptide that is a B cell epitope (SEQ ID NO: 4). The B-cell epitope was derived from the exposed surface exposed loop 3 of an NTHi outer membrane protein (fimbrine) designated as OMP P5 (also called fimbrine P5 or adhesin homologous to OMP P5). The LB1 peptide further comprises a short 5-mere linker peptide and a promiscuous T cell epitope of 16 residues. The T cell epitope was derived from a measles virus fusion protein. The promiscuous T-cell epitope induces a very intense T-cell response in individuals exposed to this epitope.

The present invention comprises inserting a part or fragment of the LB1 peptide into a safer and more selective carrier protein 20 that does not reduce the effectiveness of inducing a B cell response. Preferably, the part of the LB1 peptide is inserted into a carrier that by itself It also provides protection against diseases induced by NTHi. A carrier of this type that can induce protection against NTHi-induced diseases is the protein comprising the NTHi type IV pilus (contractile pilus) protein, also known as PilA protein (SEQ ID NO: 2). The PilA protein is encoded by the pilA gene (SEQ ID NO: 1). 25

The present invention provides chimeric proteins that comprise a portion of the LB1 peptide in order to present the peptide to induce an immunogenic response. The invention comprises presenting a part of the LB1 peptide having 12 to 35 amino acids, more preferably presenting a part of the LB1 peptide having 15 to 30 amino acids, and still more preferably presenting a part of the LB1 peptide having 18 to 19 amino acids. and be a subunit of the fimbrine protein. A preferred part of the LB1 peptide is the N-terminal amino acid sequence 30 RSDYKFYEDANGTRDHKKG (SEQ ID NO: 4).

In another embodiment, the invention provides a chimeric protein in which the PilA protein is modified to present a 24 amino acid peptide. The 24 amino acid peptide may comprise the B-cell epitope of the modified LB1 peptide as set forth in the amino acid sequence of SEQ ID NO: 5 (LVRSDYKFYEDANGTRDHKKGRHT) in which a leucine and a valine are added to the N-terminal end of the epitope LB1 B-cells and an arginine, a histidine and a threonine are at the C-terminal end of the LB1 B-cell epitope. These modifications to the B cell epitope are contemplated to aid in the folding of the protein and / or the presentation of antigens. The invention further comprises any modification with respect to the LB1 B cell epitope that aids in the folding of the protein and / or the presentation of antigens.

The amino acid sequence of the NTHi OMP P5 surface exposed loop 3 may vary between NTHi strains 40. The invention comprises chimeric proteins comprising a portion of the modified PilA protein to present the B-cell epitope of any variant amino acid sequence of loop 3 of the NTHi P5 OMP. In particular, the invention provides chimeric proteins in which the PilA protein is modified to have one of the following OMP P5 amino acid sequences of NTHi variants: RSDYKLYNKNSSSNSTLKNLGE (SEQ ID NO: 6), RSDYKLYNKNSSTLKDLGE (SEQ ID NO: 7) and RSDYKFYDNKRID (SEQ ID NO: 8). Variant peptides may also be presented with a leucine and a valine added to the N-terminal end and an arginine, a histidine and a threonine added to the C-terminal end or any other modification to aid in the folding of the protein and / or the antigen presentation.

The chimeric proteins of the invention comprise the modified PilA amino acids in which the native PilA amino acids have been replaced by a part of the LB1 peptide. In addition, the chimeric proteins of the invention comprise a sequence of modified PilA amino acids into which a part of the LB1 peptide is inserted and in addition to the native PilA amino acids. The chimeric proteins of the invention have the ability to induce the formation of antibodies directed against two proteins and are therefore more effective and more specific vaccine candidates.

In one embodiment, the chimeric proteins comprise the mature amino acid sequence 55 (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 2) in which a part of the LB1 peptide is inserted between the cysteine residues at positions 62 and 72 of SEQ ID NO: 2 and may substitute for native amino acids, such as the chimeric protein having the amino acid sequence of SEQ ID NO: 54. This chimeric protein comprises the

residues 40-149 of SEQ ID NO: 2 and presents the B cell epitope of LB1 (SEQ ID NO: 5) inserted between residues 62 and 72 of SEQ ID NO: 2. In another embodiment, the part of the peptide LB1 is inserted between cysteine residues at positions 131 and 144 of SEQ ID NO: 2 and can substitute for native amino acids such as the protein having the amino acid sequence of SEQ ID NO: 55. This chimeric protein comprises residues. 40-149 of SEQ ID NO: 2 and presents the B cell epitope of LB1 (SEQ ID NO: 5) inserted between residues 131 and 144 of SEQ ID 5: 2.

In another embodiment, the chimeric proteins comprise the mature amino acid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 2) in which the part of the LB1 peptide is inserted at the C-terminal end of the PilA protein. For example, the chimeric protein of SEQ ID NO: 56 comprises residues 40-149 of SEQ ID NO: 2 and the B-cell epitope of LB1 (SEQ ID NO: 5) is inserted after residue 149 of SEQ ID NO: 2. 10

In another embodiment, the chimeric proteins comprise the mature amino acid sequence (residues 13-149) of the NTHi PilA protein (SEQ ID NO: 2) in which the part of the LB1 peptide is inserted at the N-terminal end of the PilA protein. For example, the chimeric protein of SEQ ID NO: 57 comprises residues 40-149 of SEQ ID NO: 2 and the B-cell epitope of LB1 (SEQ ID NO: 5) is inserted before residue 40 of SEQ ID NO: 2.

In another embodiment, the invention provides chimeric proteins comprising a part of the NTHi PilA protein and one or more of the LB1 peptides described herein. The chimeric proteins of the invention include those that have the same LB1 peptide more than once within a part of the NTHi PilA protein and those that have two or more different LB1 peptides within a part of the NTHi PilA protein.

NTHi pilus polypeptides and polynucleotides of type IV (PilA) of the invention

The chimeric proteins of the invention may comprise the full length or a portion of the main subunit 20 of the NTHi type IV pilus that is encoded by the pilA gene. The PilA protein of the NTHi 86-028NP isolate is encoded by the nucleic acid sequence set forth as SEQ ID NO: 2, which is described in US Patent Publication No. US 2005-018335. Polynucleotides encoding PilA polypeptides are also provided from the clinical isolates of NTHi 1728MEE, 1729MEE, 3224A, 10548MMEE, 1060MEE, 1885MEE, 1714MEE, 1236MEE, 1128MEE and 214NP. The amino acid sequences of these PilA polypeptides are set forth in SEQ ID NOS: 34, 36, 38, 25 40, 42, 44, 46, 48, 50 and 52 respectively. The possibility of an alternative codon use is specifically contemplated in polynucleotides encoding the polypeptides. In one embodiment, the polypeptides are encoded respectively by the nucleotide sequences set forth in SEQ ID NOS: 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51.

The invention provides polynucleotides that hybridize under restrictive conditions with (a) the complement of the nucleotide sequences set forth in SEQ ID NO: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 ; (b) a polynucleotide that is an allelic variant of any polynucleotide mentioned above; (c) a polynucleotide encoding a homologous species of any of the proteins mentioned above; or (d) a polynucleotide encoding a polypeptide comprising a specific domain or truncation of the polypeptides of the present invention. Specifically included are PilA polynucleotides from other strains of non-typable H. influenzae 35 and strains of H. influenzae a, b, c, e and f. These polynucleotides can be identified and isolated by conventional techniques in the art such as hybridization and polymerase chain reaction using part of, or all, the polynucleotides of SEQ ID NO: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 as probes or primers, respectively.

The polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the aforementioned polynucleotides. The polynucleotides according to the invention may, for example, have at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93% or 94% and even more normally at least 95%, 96%, 97%, 98% or 99% sequence identity with the NTHi polynucleotides mentioned above. Four. Five

Within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under restrictive conditions with the NTHi nucleotide sequences of SEQ ID NO: 1, 33, 35, 37, 39, 41 , 43, 45, 47, 49 and 51, or complements thereof, fragment that is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and still more preferably greater than 17 nucleotides. Fragments of, for example, 15, 50, 17 or 20 nucleotides or more are contemplated which are selective for (ie, specifically hybridize with any one of the PilA polynucleotides of the invention). These nucleic acid sequence fragments that can specifically hybridize with an NTHi PilA polynucleotide of the invention can be used as probes to detect NTHi PilA polynucleotides of the invention and / or can differentiate the NTHi PilA polynucleotides of the invention from other bacterial genes. , and are preferably based on unique nucleotide sequences. 55

The term "restrictive" is used herein to refer to conditions that are commonly understood in the art as restrictive. Hybridization restriction is determined primarily by temperature, ionic strength and concentration of denaturing agents such as formamide. The examples of

Restrictive conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68 ° C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42 ° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More restrictive conditions may also be used (such as a higher temperature, a lower ionic strength, a higher concentration of formamide, or other denaturing agent), however, the hybridization rate 5 will be affected. In cases where hybridization of deoxyoligonucleotides is concerned, additional example restrictive hybridization conditions include washing in 6X SSC 0.05% sodium pyrophosphate at 37 ° C (for 14 base oligonucleotides), 48 ° C (for 17 base oligonucleotides), 55 ° C (for 20 base oligonucleotides) and 60 ° C (for 23 base oligonucleotides).

Other agents may be included in the hybridization and wash buffers in order to reduce specific and / or background non-hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), Ficoll, Denhardt's solution, DNA of sonic salmon sperm (or other non-complementary DNA) and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the restriction of hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, under typical ionic strength conditions, the hybridization rate is practically independent of pH. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). One skilled in the art can adjust the hybridization conditions in order to accommodate these variables and allow DNA of different sequence relationships to form hybrids.

As indicated above, the polynucleotides included in the present invention are not limited to the 20 specific PilA polynucleotides of SEQ ID NO: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, but which also include, for example, species and allelic variations thereof. Species and allelic variations can be determined routinely by comparing the sequence provided in SEQ ID NO: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, preferably open reading frames in the same, a representative fragment thereof, or a nucleotide sequence at least identical to 90%, preferably identical to 95%, with the reading frames 25 open within SEQ ID NO: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 with a sequence of another isolated from the same species or another species. Preferred computer program procedures for determining the identity and similarity between two sequences include, but are not limited to, the GCG software package, which includes GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX program is publicly available in the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, MD 20894; Altschul et al., Cited above). The well-known Smith-Waterman algorithm can also be used to determine identity.

The polynucleotides of the invention can be isolated from natural sources or can be synthesized by conventional chemical techniques, for example, the phosphotriester process described in Matteucci et al., J Am Chem 35 Soc., 103: 3185 (1981).

The invention provides chimeric proteins that comprise a portion of the NTHi PilA protein. In one embodiment the polypeptides comprise the 86-028NP amino acid sequences of NTHi set forth respectively in SEQ ID NO: 2. The polypeptides of the invention also include the PilA polypeptides set forth in SEQ ID NO: 34, 36, 38 , 40, 42, 44, 46, 48, 50 and 52. In other embodiments, the PilA polypeptides of the invention are those of other strains of non-typable H. influenzae and strains of H. influenzae a, b , c, e and f.

Polypeptides of the invention specifically include peptide fragments (ie, peptides) or fragments of the PilA polypeptide that retain one or more biological or immunogenic properties of a full length polypeptide of the invention. In one embodiment, the PilA peptide fragments provided by the invention are designated TfpQ2, TfpQ3, TfpQ4 and OLP3 and respectively comprise amino acids 35 to 68 of SEQ ID No. 45: 2, amino acids 69 to 102 of SEQ ID NO: 2, amino acids 103 to 137 of SEQ ID NO: 2, and amino acids 21 to 35 of SEQ ID NO: 2. Another PilA peptide fragment provided by the invention comprises amino acids 40 to 149 of SEQ ID NO: 2.

The invention also provides chimeric proteins that comprise a portion of a PilA polypeptide with one or more conservative amino acid substitutions that do not affect the biological and / or immunogenic activity of the PilA polypeptide. Alternatively, it is contemplated that the PilA polypeptides of the invention have conservative amino acid substitutions that may or may not alter biological activity. The term "conservative amino acid substitution" refers to a substitution of a native amino acid residue with a non-native residue, including naturally occurring and naturally occurring amino acids, so that there is little or no effect on the polarity or load of amino acid residues in that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with another non-polar residue. In addition, any native residue in the polypeptide can also be substituted by alanine, according to the "alanine scavenging mutagenesis" procedures. The naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acids: glutamic acid, aspartic acid; polar unloaded: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, 60

proline, methionine, leucine, norleucine, isoleucine. The following are general rules for amino acid substitutions in Table 1.

 Table 1

 Amino acid substitutions

 Original waste

 Exemplary Substitutions Preferred Substitutions

 To

 Val, Leu, Ile Val

 Arg

 Lys, Gln, Asn Lys

 Asn

 Gln Gln

 Asp

 Glu Glu

 Cys

 Be, Wing Be

 Gln

 Asn asn

 Glu

 Asp Asn

 Gly

 Pro, Wing Wing

 His

 Asn, Gln, Lys, Arg Arg

 Ile

 Leu, Val, Met, Ala, Phe, Leu

 Leu

 Norleucine, Ile, Val, Met,

 Leu

 Lys

 Arg, 1,4-diaminobutyric acid Arg

 Met

 Leu, Phe, Ile Leu

 Phe

 Leu, Val, Ile, Ala, Tyr Arg

 Pro

 Gly wing

 Be

 Thr, Ala, Cys Thr

 Thr

 To be

 Trp

 Tyr, Phe Tyr

 Tyr

 Trp, Phe, Thr, Ser Phe

 Val

 Ile, Met, Leu, Phe, Ala, Leu

The invention also provides chimeric proteins comprising a part of a variant of the NTHi PilA polypeptides of the present invention (for example, a polypeptide having at least about 65%, at least about 70%, at least about 75%, at least 5 about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, usually at least about 95%, 96%, 97%, more usually at least about 98%, or most usually at least about 99% of amino acid identity with respect to a polypeptide of SEQ ID NO: 2, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52) that retain biological and / or immunogenic activity. 10

The invention contemplates that the PilA polynucleotides of the invention can be inserted into a vector for amplification or expression. For their expression, the polynucleotides are functionally connected to appropriate expression control sequences such as the polyadenylation and promoter signal sequences. In addition, host cells comprising polynucleotides of the invention are provided. Exemplary prokaryotic host cells include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia. Polypeptide production methods of the invention are specifically contemplated by growing the host cells and isolating polypeptides from the host cells or growth medium. Alternatively, the polypeptides of the invention can be prepared by chemical synthesis using conventional means. Solid phase techniques are particularly convenient (see, for example, Erikson et al., The Proteins (1976) v. 2, Academic Press, New York, p. 255). Automatic solid phase synthesizers are commercially available. In addition, 20 modifications are easily made to the sequence by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added to the carboxy-terminal end to provide a sulfhydryl group for binding.

convenient to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the binding amino acid at the C-terminal end and the rest of the peptide.

The term "isolated" refers to a substance separated from, and essentially free of, the other components of the environment in which it exists naturally. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from another DNA that flanks it in a genome in which it occurs naturally. 5

A recombinant PilA protein (rPilA) can be generated to act as a more easily renewable product. To do this, the published protocol of Keizer et al. (J. Biol. Chem., 276: 24186-14193, 2001), who studied a pilin that also had four Cys residues since it will be critical that rPilA be folded appropriately in a similar manner to possess functional qualities of the pilin subunit native In summary, a truncated pilin is designed by genetic engineering in which the first 28 residues are removed from the N-10 terminal end to prevent aggregation, and this truncated pilin will be further modified by genetic engineering to be transported to the periplasm by incorporating a OmpA leader sequence in the construct. Using this strategy Keizer et al. generated a recombinant soluble monomeric P. aeruginosa pilin protein that could bind to its receptor (asialo GM1) in in vitro assays and decrease morbidity and mortality in mice when the peptide was administered 15 minutes before heterologous exposure. This truncated, monomeric, soluble form of NTHi PilA will be useful in the studies described herein.

The invention also provides synthetic chimeric proteins. Chimeric proteins can be synthesized, purified and sequenced using conventional techniques. For example, chimeric proteins can be assembled semi-annually by solid phase synthesis with Fmoc-tert-butyl in steps and purified by HPLC. The amino acid composition and sequence of recombinant and synthetic chimeric proteins 20 can be confirmed by amino acid analysis and / or mass spectrometry analysis.

Antibodies

Antibodies that bind antigenic epitopes of the chimeric proteins of the invention are described. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments that retain their ability to bind to their single epitope (eg, Fv, Fab and F (ab) 2 fragments), single chain antibodies and human or humanized antibodies. Antibodies can be generated by conventional techniques in the art using chimeric protein (s) of the invention or host cells expressing chimeric protein (s) of the invention as antigens.

Specific antibodies to the chimeric proteins of the present invention and fragments thereof are described, which show the ability to both destroy H. influenzae bacteria and to protect humans against infection. Antibodies specific for the chimeric proteins of the invention that reduce virulence, inhibit adhesion, inhibit biofilm formation, inhibit contractile motility, inhibit cell division and / or inhibit penetration into the epithelium of H. influenzae bacteria are also described. and / or enhance phagocytosis of H. influenzae bacteria.

In vitro complement-mediated bactericidal assay systems (Musher et al., Infect. 35 Immun. 39: 297-304,1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) can be used for measure the bactericidal activity of chimeric antiprotein antibodies.

It is also possible to confer short-term protection to a host by passive immunotherapy by administering previously formed antibody against a chimeric protein of the invention. Therefore, antibodies described herein can be used in passive immunotherapy. Human immunoglobulin 40 is preferred in human medicine because a heterologous immunoglobulin can elicit an immune response against its foreign immunogenic components. Such passive immunization could be used as an emergency for the immediate protection of non-immunized individuals subject to special risks.

Antibodies described herein can be used in the production of anti-idiotypic antibodies, which in turn can be used as an antigen to stimulate an immune response against epitopes of chimeric proteins or epitopes of H. influenzae.

Procedures for eliciting an immune response and compositions thereof

The invention contemplates the use of the chimeric proteins of the invention to provoke in an individual an immune response against H. influenzae in an individual. These uses cause one or more immune responses, including but not limited to immune responses that inhibit bacterial replication, 50 immune responses that block the adherence of H. influenzae to cells, immune responses that prevent the contraction of H. influenzae, immune responses that destroy H. influenzae bacteria and immune responses that prevent the formation of biofilms. Methods comprising a step consisting of administering an immunogenic dose of a composition comprising one or more chimeric proteins of the invention are described. Methods comprising administering an immunogenic dose of a composition comprising a cell expressing one or more chimeric proteins of the invention are also described. In addition, methods are described comprising administering an immunogenic dose of a composition comprising

one or more polynucleotides encoding one or more chimeric proteins of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or it may be in a vector such as a plasmid or viral vector (eg, adeno-associated virus vector or adenovirus vector). The compositions can be used in combination in a single individual. The compositions can be used before or after H. influenzae infection of an individual. The compositions of the invention can be used to treat or prevent any pathological condition involving H. influenzae (typifiable and non-typifiable strains) such as OM, pneumonia, sinusitis, septicemia; endocarditis, epiglottitis, septic arthritis, meningitis, puerperal and neonatal infections, puerperal and neonatal sepsis, acute and chronic salpingitis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpuric fever, occult bacteremia, chronic obstructive pulmonary disease and exacerbation of underlying lung diseases 10 such as chronic bronchitis, bronchiectasis and cystic fibrosis.

In one embodiment of the invention, a composition of the invention is administered as a sensitization dose followed by one or more booster doses. Co-administration of proteins or polypeptides that beneficially enhance the immune response such as cytokines (eg, IL-2, IL-12, GM-CSF), cytokine-inducing molecules (eg Leaf) or costimulatory molecules is also contemplated. fifteen

An "immunogenic dose" of a composition of the invention is one that generates, upon administration, a detectable humoral (/) and / or cellular (T-cell) immune response compared to the detectable immune response before administration or in comparison. with a conventional immune response before administration. The invention contemplates that the immune response resulting from the procedures can be protective and / or therapeutic. In a preferred embodiment, the immune response of antibodies and / or T 20 cells protects the individual against infection with H. influenzae, particularly infection of the middle ear and / or nasopharynx or lower respiratory tract. In this use, the precise dose depends on the state of health and the weight of the patient, the mode of administration, the nature of the formulation, etc., but generally between about 1.0 μg and approximately 5000 μg per 70 kilograms of patient. , more commonly between about 10 and about 500 μg per 70 kg of body weight. 25

The humoral immune response can be measured by many well-known procedures, such as simple radial immunodiffusion assay (SRID), enzyme immunoassay (EIA) and hemagglutination inhibition assay (HAI). In particular, SRID uses a layer of a gel, such as agarose, that contains the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. The diffusion of the antibody to the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (enzyme-linked immunosorbent assay), is used to determine the total antibodies in the sample. The immunogen is adsorbed on the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme-linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any conventional means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogens present in the test sample. HAI uses the ability of an immunogen such as viral proteins to bind chicken red blood cells (or the like). The assay detects neutralizing antibodies, that is, antibodies that can inhibit hemagglutination. Dilutions of the test serum are incubated with a standard concentration of immunogen, followed by the addition of red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of red blood cells by the immunogen. Tests to measure cellular immune response include the determination of delayed type hypersensitivity or the measurement of lymphocyte proliferative response against the target immunogen.

The invention correspondingly provides compositions suitable for eliciting an immune response against chimeric proteins of the invention. As indicated above, the compositions comprise one or more chimeric proteins, cells expressing one or more chimeric proteins, or one or more polynucleotides encoding one or more chimeric proteins. The compositions may also comprise other components such as vehicles and adjuvants.

In compositions of the invention, a chimeric protein can be fused with another protein when produced by recombinant methods. In one embodiment, the other protein cannot, by itself, produce antibodies, but stabilizes the first protein and forms a fusion protein that retains immunogenic activity. In another embodiment, the fusion protein comprises another protein that is immunogenic, such as glutathione-S-transferase (GST) or beta-galactosidase, relatively large coproteins that solubilize the fusion protein and facilitate its production and purification. . The other protein can act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The other protein can be fused with either the amino-terminal or carboxy-terminal end of the chimeric proteins of the invention. 55

In other compositions of the invention, the chimeric proteins can be otherwise bound to carrier substances. Any procedure known in the art can be used to create such joints. Unions can be formed by hetero-bifunctional agents that generate a disulfide bridge at one end of the functional group and a peptide bond at the other, such as a disulfide amide forming agent, for example, N-succidimidyl-3- (2- pyridyldithio) propionate (SPDP) (see, for example, Jansen et al., Immun. Rev. 62: 185, 1982) and bifunctional coupling agents 60 that form a thioether instead of a disulfide bridge such as acid reactive esters 6 -

maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid and the like, and coupling agent that activates carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4 acid -sulfonic acid, for a sodium salt such as succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC).

Chimeric proteins can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric. Mandelic The salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine and procaine . 10

The compositions of the invention may further comprise adjuvants. Known adjuvants include, for example, emulsions such as Freund's adjuvants and other oil emulsions, Bordetella pertussis, MF59, purified saponin from Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of 15 natural and bacterial products whose use as adjuvants is known include monophosphoryl lipid A (MPL), RC-529 (acylated monosaccharide similar to synthetic MPL), OM-174 which is a lipid A derived from E. coli, holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and thermolabile toxin (LT) of E. coli or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by several factors, such as carrier effect, deposit formation, altered lymphocyte recirculation, stimulation of 20 T lymphocytes, direct stimulation of B lymphocytes and macrophage stimulation.

The compositions of the invention are normally formulated as injectable products, either as suspensions or as liquid solutions; Solid forms suitable for dissolution in, or suspension in, liquid can also be prepared before injection. The preparation can also be emulsified. The active immunogenic principle is frequently mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the efficacy of the vaccine. Vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. 30

Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional carriers and binders may include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 to 2%. Oral formulations include normally used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% active ingredient, preferably 25 to 70%.

The compositions can also be administered by transdermal routes using jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using sprays, aerosols and nasal nebulizers. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly-L-lactate (PLA), D-poly-DL-lactic-co-glycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes, polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine peptides or amphipathic peptides are useful for transdermal delivery systems. In addition, due to its amphipathic nature, liposomes are contemplated for systems of transdermal, transmucosal and intranasal vaccination. Common lipids used for vaccine administration include N- (1) 2,3- (dioleyl-dihydroxypropyl) -N, N, N-trimethylammonium-methyl (DOTAP) sulfate, diootyloxy-propyl-trimethylammonium chloride DOTMA, dimistyloxypropyl -3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N (N ', N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of auxiliary lipids and liposomes 50 will enhance the uptake of liposomes through the skin. These auxiliary lipids include dioleoyl phosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimiristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from Chilean quillay (Quillaja saponaria) and chitosan (deacetylated chitano) have been contemplated as useful adjuvants for intranasal and transmucosal vaccination administration. 55

The formulations may be presented in unit dose or multi-dose containers, for example, sealed ampoules and vials and perhaps stored in a lyophilized state that only requires the addition of the sterile liquid vehicle immediately before use.

H. influenzae inhibition procedures

Alternatively, the invention includes methods of inhibiting the function of type IV pili of H. influenzae in an individual. The methods comprise administering to the individual, for example, one or more antibodies of the invention and / or one or more chimeric proteins of the invention; in an amount that inhibits the function of pili. In vitro assays can be used to demonstrate the ability to inhibit the function of pili. Embodiments of these 5 procedures include, for example, procedures that utilize type IV pili-mediated adhesion inhibitors, inhibitors that alter existing biofilms mediated by type IV pili, and contractility inhibitors.

Inhibition is contemplated for any disease state involving H. influenzae, for example, OM, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, puerperal and neonatal infections, puerperal and neonatal sepsis, acute and chronic salpingitis, pericarditis , cellulitis, osteomyelitis, endocarditis, 10 cholecystitis, intra-abdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpuric fever, occult bacteraemia, chronic obstructive pulmonary disease and exacerbation of underlying lung diseases such as chronic bronchitis, bronchiectasis and cystic fibrosis.

Compositions comprising inhibitors of the function of type IV pili of H. influenzae are provided. The compositions may consist of only one of the above active ingredients, may comprise combinations of the above active ingredients or may comprise additional active ingredients used to treat bacterial infections. As discussed above, the compositions may comprise one or more additional components such as pharmaceutically effective carriers. Also as discussed above, the dosage and frequency of administration of the compositions are determined by conventional techniques and depend, for example, on the weight and age of the individual, the route of administration and the severity of the symptoms. Administration of the pharmaceutical compositions can be carried out by conventional routes in the art, for example, parenteral, intravenous, oral, oral, nasal, pulmonary, rectal, intranasal or vaginal.

Animal model

The methods of the invention can be demonstrated in a widely accepted model of chinchilla as an experimental model for OM. In particular, an OM chinchilla model induced by 25 NTHi has been well characterized (Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol. , 4: 223-225, 1997; Suzuki and Bakaletz, Infect. Immun., 62: 1710-1718, 1994; Mason et al., Infect. Immun., 71: 3454-3462, 2003), and has been used to determine the protection efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, components of chimeric synthetic peptide vaccines and adjuvant formulations against OM (Bakaletz et al., Vaccine, 15: 955-961, 1997 ; Bakaletz et al., Infect. Immun., 67: 2746-2762, 30 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000; Kyd et al., Infect. Immun., 66: 2272-2278, 2003; Novotny and Bakaletz, J. Immunol., 171, 1978-1983, 2003).

In the model, an adenovirus predisposes chinchillas to the average OM induced by H. influenzae, which allowed to establish culture systems of cells, tissues and organs relevant for the biological evaluation of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infection alone has been used to assess the transudation of serum-induced antibodies in the eardrum (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4 (2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protection efficacy of various active and passive immunization regimens that select as a target various proteins from the NTHi outer membrane, combinations of OMP, chimeric synthetic peptide vaccine components and adjuvant formulations as vaccines against vaccines. otitis media (Bakaletz et al., Infect Immunity, 40 67 (6): 2746-62, 1999; Kennedy et al., Infect. Immun., 68 (5): 2756-65, 2000; Novotny et al., Infect Immunity 68 (4): 2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 provides the timeline of the immunization regimen, viral inoculation, exposure to bacteria and the OM disease evaluation period for the efficacy experiments described in Example 45 5.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention, in which example 1 describes the recombinant production of chimeric proteins of the invention, example 2 describes tests to test the immunogenicity of the chimeric proteins of the invention, example 3 describes tests to evaluate passive immunization, Example 4 50 describes assays for evaluating active immunization and Example 5 describes the evaluation of a chimeric protein of the invention.

EXAMPLE 1

Synthesis of chimeric proteins

The chimeric proteins of the invention were produced using recombinant methods.

conventional. Initially, a gene synthesis company (Blue Heron Biotechnology Inc.) was hired to prepare the initial plasmid based on the amino acid sequences of the chimeric protein described herein that were optimized for the use of preferred E. coli codons. . In summary, the native NTHi pilin protein sequence was modified by truncating the N-terminal end (residues 1-39 of SEQ ID NO: 2) and adding an HIS tail sequence and a thrombin cleavage site as it was set forth in SEQ ID NO: 3. The 5 HIS tail was preceded by a sequence (MGSS) to aid expression. The thrombin cleavage site allowed the release of the HIS tail. These plasmids were then cloned into the E. coli expression vector, vector pET-15b (Novagen). The plasmid was then transformed into the E. coli strain "Origami (DE3)" (available from Novagen) as a host for the expression of soluble His tail chimeric proteins. Another E. coli host cell expression strain that can be used is Origami B (DE3) (Novagen). 10

His tail variants of the chimeric proteins will be recovered by nickel column chromatography, then used for initial studies to determine if they are reactive with antisera directed against any of the following: P5-fimbrine from native OMP, LB1 (peptide 40 full-length amino acids), LB1 (1) (a synthetic peptide that only represents the 19-amino acid B-cell epitope of LB1), recombinant PilA protein or native PilA protein. Once the His tail has been removed by cleavage at the thrombin site, the recombinant chimeric proteins will be used as immunogens to determine their immunogenicity and protective capacity.

The exemplary chimeric proteins of the invention have the sequences set forth in Table 2 below. The chimeric proteins that have the amino acid sequences of SEQ ID NO: 10, 12 and 14 have been expressed by E. coli as described above. twenty

 Table 2

 SEQ ID NO:

 Amino acid sequence of the chimeric protein

 9

 10

 eleven

 Table 2 (continued)

 SEQ ID NO:

 Amino acid sequence of the chimeric protein

 12

 13

 14

 fifteen

 16

 17

 18

 19

 twenty

 twenty-one

 22

 2. 3

 24

 Table 2 (continued)

 SEQ ID NO:

 Amino acid sequence of the chimeric protein

 25

 26

 27

 28

 29

 30

 31

 32

Additional exemplary chimeric proteins of the invention have the amino acid sequences set forth in Table 3 below. These chimeric proteins have been expressed by E. coli and purified using an HIS tail, as described above. The chimeric proteins set forth in Table 3 present the His tail sequence removed for use as an immunogen. The chimeric protein presenting the amino acid sequence of SEQ ID NO: 56 was used in the studies described in Example 5. 5

 Table 3

 SEQ ID NO:

 Amino acid sequence of the chimeric protein

 54

 55

 Table 3 (continued)

 SEQ ID NO:

 Amino acid sequence of the chimeric protein

 56

 57

EXAMPLE 2

Immunogenicity of chimeric proteins

Rabbits or chinchillas are immunized with the chimeric proteins. Rabbits receive an initial immunization dose of 500 μg of a chimeric protein in Freund's complete adjuvant. Rabbits receive a second dose of 400 μg of the chimeric protein 21 days later. Rabbits receive a third dose of chimeric protein 5 in Freund's complete adjuvant 42 days after the initial immunization dose with 400 μg of the same peptide either in IFA or in PBS (one rabbit per diluent). Serums are obtained 3 weeks after each dose. Chinchillas receive an initial immunization dose of 10 μg of the chimeric protein in the monophosphoryl lipid A (MPL) adjuvant. One month (~ 30 days) later, chinchillas receive a second identical dose. The third and last dose is given ~ 30 days after the second dose. Serums are obtained ~ 10-14 days after each dose. The sera of all 10 animals are evaluated to determine the titer and specificity against peptide LB1 (40-mer), LB1 (1), PilA protein and chimeric proteins, by ELISA, Western blotting and biosensor. Antisera against complete bacteria are also tested by flow cytometric analysis (FACS).

EXAMPLE 3

Passive immunization evaluation 15

The protection conferred by the immune response of an animal directed against the chimeric proteins of the invention is determined in an experimental otitis media chinchilla model. Chinchillas are passively immunized with 5 ml / kg of chinchilla or human hyperimmune serum directed against a chimeric protein of the invention. Control chinchillas receive normal chinchilla serum or normal human serum. Next, the chinchillas receive a first viral co-pathogen intranasally, then, a week later, an intranasal exposure to the 20 NTHi bacteria. Chinchillas are examined and classified daily or every 2 days for up to 35 days after bacterial exposure. Immunized chinchillas that receive chinchilla or human immune serum show a reduced tympanic membrane pathology and a reduction or absence of signs of infection of the middle ear space as determined both by videootoscopy and tympanometry. In this test, the presence of middle ear fluids in chinchillas that receive chinchilla serum or human chimeric antiprotein is reduced compared to controls.

EXAMPLE 4

Evaluation of active immunization

Cohorts of 5-10 chinchillas each are actively immunized with saline control preparation, a preparation only with adjuvant or one of the chimeric proteins of the invention that has been mixed with an appropriate adjuvant. Immunogens are evaluated to determine the endotoxin content before use as an immunogen by a chromogenic amebocyte lysate assay that is commercially available from Whittaker Bioproducts under the designation QCL-1000. The chinchillas are then injected subcutaneously with 10 μg of immunogen in the MPL adjuvant (or other appropriate adjuvant). Then, 30 days later they receive 10 μg of the same immunogen in MPL. Thirty days after the second immunization, these animals receive the final immunization dose. Approximately 10-14 days after administering the final immunization dose, the chinchillas are exposed both transbularly and intranasally to a strain of NTHi. Chinchillas are evaluated over a period of 35 days to determine: pathology of the tympanic membrane by video-examination and tympanometry; semiquantification of NTHi recovered by epitympanic puncture of the inferior bula and passive lavage of the nasopharynx; and examination using an optical microscope of the fixed middle ear mucous epithelium and tympanic membrane 40 to determine histopathology. For example, chinchillas immunized with the chimeric proteins of the invention will have a reduced pathology of the tympanic membrane, will be free of effusions of the middle ear or will contain effusions that are negative for the culture, will present reduction or absence of biofilm present in the eardrum and there will be minimal thickening of the middle ear mucosa, minimal osteoneogenesis and reduced presence of both red blood cells and inflammatory cells in the subepithelial space. Four. Five

EXAMPLE 5

Chimeric protein evaluation

The protection efficacy of the chimeric protein having the amino acid sequence of SEQ ID NO: 56 (termed "chim-V3" herein) was evaluated using the OM superinfection model, passive transfer of chinchillas. This chimeric peptide comprised the B-cell epitope of peptide LB1 (SEQ ID NO: 5) 50 expressed after the recombinant PilA C-terminal glutamine residue (residues 40-149 of SEQ ID NO: 2). To generate polyclonal antiserum for use in passive transfer efficacy studies, the chim-V3 protein was administered to a cohort of adult chinchillas with the adjuvant, monophosphoryl lipid A (MPL) plus trehalose dimicolate (Corixa). Figure 1 shows a timeline that represents the immunization regimen. To generate combinations of immune sera, chinchillas prone to a subcutaneous alert state were immunized 3 55 times with 30 μg of chim-V3 plus 10 μg of MPL or 10 μg MPL only every 21 days. On day 56, an extraction was collected

of terminal blood from inoculated animals and serum was combined for transfer to previously untreated young animals. To study efficacy, a separate cohort of young chinchillas was first exposed to adenovirus on day -7. Six days later (day -1), the combined antichim-V3 immune serum was passively transferred to those animals compromised by adenovirus. The next day (day 0), animals that received serum antichim-V3 were exposed by passive transfer to the bacterium, Haemmophilus influenzae nontypifiable. 5 These animals were then monitored to determine the incidence and severity of the disease over a period of 26 days (with respect to bacterial exposure) by daily video-typing and tympanometry as well as imaging Xenogen in vivo every two days.

The antichim-V3 antibody titer in the immune serum collected from the inoculated animals was measured using ELISA. This analysis showed that the collected antiserum contained antibodies specific for the chim-V3 protein. The presence of antichim-V3 antibodies in the collected antiserum was also confirmed using Western blot analysis.

FACS analysis was used to measure the ability of serum immunoglobulins of immunized animals to recognize native surface exposed structures expressed by 86-028NP of NTHi. NTHi bacteria were incubated with antiserum against chim-V3, washed, then incubated with previously untreated or immune-treated FITC-protein A, washed and analyzed by FACS analysis. Inoculation with the chim-V3 protein induced a significant increase in antibodies that could recognize the surface proteins of NTHi or chim-V3 protein. The data obtained depended both on the antibody titre and avidity as well as on the relative expression of both the type IV pili and the homologue adhesion of OMP P5 by NTHi when grown in vitro.

The luminous indicator pKMLN-1 of 86-028NP of NTHi was used to detect NTHi infection in the 20 animals in which chim-V3 protein was injected using real-time imaging of Xenogen in vivo. Growth curves of the luminescent strain pKMLN-1 of 86-028NP of NTHi and the original strain 86-028 of NTHi showed that the growth of the luminescent NTHi strain was comparable to the original strain. Obtaining luminescent images of NTHi that were in the nasopharynx of the inoculated animals was easily achieved however, due to the microaerophilic nature of the diseased middle ear, the luminescence of NTHi 25 present in the middle ear could not be monitored along the entire course of the disease because the luminescence depends on the availability of oxygen. The animals were monitored every two days to determine the presence of luminescent bacteria, and if bacteria were detected, it was recorded as a luminescent event. Luminescent infection was detected at least six days after exposure in inoculated animals. The total number of luminescent events in animals in which chim-V3 was inoculated was less than the total number of 30 luminescent events in control animals (in which only MPL was inoculated).

Throughout the course of the study, daily video photoscopy and tympanometry were used to determine the percentage of middle ears of chinchillas with OM. The inoculation of chim-V3 caused a 53% reduction in the number of animals in which the middle ears had OM compared to the control animals (in which only MPL was inoculated). 35

All these studies demonstrate that the chim-V3 protein was immunogenic and the antichim-V3 antibodies were protective in the passive superinfection-transfer model of OM chinchilla.

SEQUENCE LIST

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<211> 148

<212> PRT

<213> Artificial sequence 5

<220>

<223> Chimeric protein

<400> 13

<210> 14 10

<211> 153

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 5

<400> 14

<210> 15 10

<211> 142

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 15

<400> 15

<210> 16

<211> 147

<212> PRT 5

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 16

<210> 17

<211> 139

<212> PRT

<213> Artificial sequence 5

<220>

<223> Chimeric protein

<400> 17

<210> 18 10

<211> 144

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 18

 5

<210> 19

<211> 151

<212> PRT

<213> Artificial sequence

<220> 10

<223> Chimeric protein

<400> 19

<210> 20

<211> 156

<212> PRT 5

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 20

<210> 21 10

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 5

<400> 21

<210> 22

<211> 145 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 22 15

<210> 23

<211> 136

<212> PRT

<213> Artificial sequence 5

<220>

<223> Chimeric protein

<400> 23

<210> 24 10

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 5

<400> 24

<210> 25

<211> 148 10

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 25 15

<210> 26

<211> 153 5

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 26 10

<210> 27

<211> 133

<212> PRT

<213> Artificial sequence 5

<220>

<223> Chimeric protein

<400> 27

<210> 28 10

<211> 138

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 28

<210> 29 5

<211> 130

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 10

<400> 29

<210> 30

<211> 135

<212> PRT 5

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 30

<210> 31 10

<211> 142

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 31 5

<210> 32

<211> 147

<212> PRT 10

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 32

<210> 33

<211> 447

<212> DNA 5

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447)

<400> 33 10

<210> 34

<211> 149

<212> PRT

<213> Haemophilus influenzae

<400> 34 5

<210> 35

<211> 447

<212> DNA 10

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447)

<400> 35 15

<210> 36

<211> 149

<212> PRT 5

<213> Haemophilus influenzae

<400> 36

<210> 37

<211> 447

<212> DNA

<213> Haemophilus influenzae 5

<220>

<221> CDS

<222> (1) .. (447)

<400> 37

<210> 38 10

<211> 149

<212> PRT

<213> Haemophilus influenzae

<400> 38

<210> 39 5

<211> 447

<212> DNA

<213> Haemophilus influenzae

<220>

<221> CDS 10

<222> (1) .. (447)

<400> 39

<210> 40

<211> 149 5

<212> PRT

<213> Haemophilus influenzae

<400> 40

<210> 41

<211> 447

<212> DNA

<213> Haemophilus influenzae 5

<220>

<221> CDS

<222> (1) .. (447)

<400> 41

<210> 42

<211> 149

<212> PRT

<213> Haemophilus influenzae

<400> 42 5

<210> 43

<211> 447

<212> DNA 10

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447)

<400> 43 15

<210> 44

<211> 149

<212> PRT 5

<213> Haemophilus influenzae

<400> 44

<210> 45

<211> 447

<212> DNA 5

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447)

<400> 45 10

<210> 46

<211> 149

<212> PRT

<213> Haemophilus influenzae

<400> 46 5

<210> 47

<211> 447 10

<212> DNA

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447) 15

<400> 47

<210> 48

<211> 149 5

<212> PRT

<213> Haemophilus influenzae

<400> 48

<210> 49

<211> 447

<212> DNA 5

<213> Haemophilus influenzae

<220>

<221> CDS

<222> (1) .. (447)

<400> 49 10

<210> 50

<211> 149

<212> PRT

<213> Haemophilus influenzae

<400> 50

<210> 51 5

<211> 447

<212> DNA

<213> Haemophilus influenzae

<220>

<221> CDS 10

<222> (1) .. (447)

<400> 51

<210> 52

<211> 149

<212> PRT 5

<213> Haemophilus influenzae

<400> 52

<210> 53

<211> 40

<212> PRT

<213> Artificial sequence 5

<220>

<223> Synthetic Peptide

<400> 53

<210> 54 10

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<223> Chimeric protein 15

<400> 54

<210> 55

<211> 126

<212> PRT 5

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 55

 10

<210> 56

<211> 138

<212> PRT

<213> Artificial sequence

<220> 15

<223> Chimeric protein

<400> 56

<210> 57

<211> 144

<212> PRT 5

<213> Artificial sequence

<220>

<223> Chimeric protein

<400> 57

Claims (21)

1. Chimeric protein comprising a part of the untypifiable H. influenzae PilA protein (NTHi) (SEQ ID NO: 2) and a part of the LB1 peptide (SEQ ID NO: 53), the chimeric protein being able to elicit an immune response to NTHi bacteria. 2. Chimeric protein according to claim 1, wherein the part of the LB1 peptide comprises the 5-cell epitope of the LB1 peptide. 3. Chimeric protein according to claim 2, wherein the part of the LB1 peptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO. : 8. 4. Chimeric protein according to claim 1, comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 10 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 , SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEC ID no .: 31, SEQ ID no: 32, SEQ ID no: 54, SEQ ID no: 55, SEQ ID no: 56 or SEQ ID no: 57. 5. Chimeric protein according to claim 4, comprising the amino acid sequence of SEQ ID NO: 57. 15 6. Chimeric protein according to claim 1, wherein the part of the NTHi PilA protein comprises residues 40-149 of SEQ ID NO: 2. 7. Chimeric protein according to claim 6, wherein the LB1 B cell epitope is inserted before residue 40 of SEQ ID NO: 2. 8. Chimeric protein according to any one of claims 1, 2, 6 or 7, wherein the BB cell epitope of LB1 comprises SEQ ID NO: 5. 9. Polynucleotide encoding the chimeric protein according to any one of claims 1 to 8. 10. Vector comprising a polynucleotide according to claim 9. 11. Composition comprising a chimeric protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 25 12. Composition according to claim 11, wherein the composition is formulated for a route of administration selected from the group consisting of parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, intranasal, transdermal, subcutaneous, intramuscular or vaginal. 13. Use of one or more chimeric proteins according to any of claims 1 to 8, for the preparation of a medicament in a patient at risk of suffering an infection by NTHi bacteria to elicit an immune response to NTHi bacteria. 14. Use according to claim 13, wherein the NTHi infection is in the middle ear, nasopharynx or lower respiratory tract. 15. Use according to any of claims 13 or 14, wherein the medicament is formulated for a route of administration selected from the group consisting of parenteral, intravenous, oral, oral, nasal, pulmonary, rectal, intranasal, transdermal administration, subcutaneous, intramuscular or vaginal. 16. Use according to claim 13 or 14, wherein the infection by NTHi bacteria implies a pathological state selected from the group consisting of otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, sepsis, salpingitis , pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpuric fever, occult bacteraemia, chronic obstructive pulmonary disease, chronic bronchitis, bronchiectasis and cystic fibrosis. 17. Chimeric protein according to any one of claims 1 to 8, for use in eliciting an immune response to NTHi bacteria in a patient at risk of suffering an infection with NTHi bacteria. 18. Chimeric protein according to claim 17, wherein the NTHi infection is found in the middle ear, nasopharynx or lower respiratory tract. 19. Chimeric protein according to claim 17, wherein the infection by NTHi bacteria implies a pathological state selected from the group consisting of otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, sepsis, salpingitis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intra-abdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis, fever 50 Brazilian purpuric, hidden bacteraemia, chronic obstructive pulmonary disease, chronic bronchitis, bronchiectasis and cystic fibrosis. 20. Chimeric protein according to any of claims 1 to 8, for use in the treatment or prevention of infection by NTHi bacteria in a patient in need. 21. Chimeric protein according to claim 20, wherein the NTHi infection is found in the middle ear, nasopharynx or lower respiratory tract.