MXPA00012271A - Vaccine - Google Patents

Abstract

It is an object of the present invention to provide groups of newly identified antigenic P5-like fimbrin subunit peptides (LBl (f) peptides) of P5-like fimbrin proteins from various ntHi strains. It is a further object to provide chimeric polypeptides that carry these peptides and which induce an immunogenic response in animals to ntHi, and polynucleotides encoding such peptides and polypeptides. The invention also relates to a method of isolating the peptides or chimeric polypeptides, to a method of detecting the presence of the peptides in biological samples, and to a vaccine composition for use in the treatment of Haemophilus influenzae infection.

Description

VACCINE FIELD OF THE INVENTION This invention relates to newly identified peptides and to polynucleotides encoding those peptides; and to chimeric proteins that carry those peptides. The invention is! also relates to a method for isolating the chimeric peptides or proto-proteins, and to a vaccine composition for use in the treatment of Haemophilus influenzae infection.

BACKGROUND OF THE INVENTION Haemophilus influenzae (Hi) is a gram-negative coccobacillus and a strict human commensal. The strains of Hi are encapsulated in a polysaccharide capsule or are not encapsulated and, consequently, are classified into typed (encapsulated) and non-typed (non-encapsulated) strains. The pathogenic Hi strains, encapsulated, cause mainly, although not exclusively, invasive diseases in children under six years of age. Haemophilus influenzae type b (Hib), for example, is a major cause of meningitis and other invasive infections in children. There are effective vaccines against Hib infections, and are based on the production of antibodies to the polysaccharide capsule and, therefore, are safe against non-typeable Haemophilus influenzae (ntHi). Non-typeable Haemophilus influenzae (ntHi) represents most of the colonizing strains and, although they are rarely invasive, they are responsible for a significant proportion of mucous disease, including otitis media, sinusitis, chronic conjunctivitis and chronic infections or exacerbation of infections of the lower respiratory tract. Currently, about 30%, and up to 62% of ntHi are resistant to penicillins. It is estimated that it is carried in approximately 44% of children and approximately 5% of adults, and may persist for months. None of the pathogenic mechanisms nor the immunological response of the host has been completely defined for the otitis media caused by ntHi. 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 a sign of acute local or syndromic malaise. Acute signs include: earache, drainage in the ear, hearing loss, while systemic signs include: fever, lethargy, irritability, anorexia, vomiting, or diarrhea. Streptococcus pneumoniae and non-typeable Haemophilus influenzae (ntHi) are the most predominant bacteria that cause this condition, which reaches 25-50% and 15-30% of the culíivadas species, respectively. In addition, ntHi is responsible for 53% of recurrent otitis media. Approximately 60% and 80% of children have at least one episode of the disease between 1 and 3 years of age, respectively (the peak being around 10 months). There is evidence that there is protective immunity for ntHi; however, antigenic shift in the naturally implicated epitopes (outer membrane proteins P2, P4, P6) plays a major role in the ability of ntHi to evade host immune defense. Therefore, there is a need for additional effective vaccines against Haemophilus influenzae and in particular for vaccines against non-typeable Haemophilus influenzae, which is not affected by currently available Hi-polysaccharide vaccines. The fimbpos, which are superficial appendages found in ntHi, are produced in 100% of the bacteria recovered from the middle ears and the nasopharyngeal region of children with chronic otitis media. A vaccine consisting of fibrin, a filamentous protein derived from the ntHi proteins, has been reported (WO 94/26304). Fimbrin is homologous to the outer membrane protein P5 of ntHl, which has been the subject of another patent application (EP 680765). Fimbrin, a protein similar to P5, is capable of releasing antibodies that react with the surface of the bacterium and which are bactericidal (WO 94/26304). The protein has been purified and shown to induce an immune response against different strains of ntHi. There are methodologies to isolate the fimbrine protein from the outer membrane of the bacterium; but they are tedious and time consuming. One strategy used with other bacterial species has been to produce relatively short, linear peptides of the natural protein. However, this approach has had limited value, since antibodies to said alternative immunogens frequently can not recognize the natural pathogen. LB 1 (f) is a peptide of 19 amino acids (SEQ IDJ No 5) derived from the fimbrine protein, similar to P5, of strain ntHi 1128 (which occupies the region Arg 117 to Gly 135). This peptide was initially defined as a potential B-cell epitope, by analysis of the primary sequence of the P5-like fimbrine protein. Immunization of animals with chimeric fimbrine peptides (termed LB1 peptides), comprising the LB 1 peptide (f), a linker peptide and a T cell epitope, induces an immunological response to the pim-like protein fimbpna, and reduces colonization of ntHi in animals subsequently exposed to ntHi (see US 5,843,464). The peptide LB1 is immunogenic in vivo and the antisera generated against it were immunoreactive [against denatured and natural fimbriae. In this way, the peptide was able to act as an effective immunogen, since it was able to generate antibodies that recognized the epitope in its natural structure, and bound to it. This is partly due to the synptic peptide LB1 (f), which mimics the secondary helical structure of the peptide, within the fimbrin protein. The problem with the use of protein antigens from only one strain of H. influenzae in a vaccine is that the protection conferred tends to be highly restricted to the homologous challenge [Bakaletz and coauthors (1997) Vaccine 15: 955-961; Haase and coauthors (1991), Infecí. Immun., 59: 1278-1284; Sirakova and coauthors (1994) infect. Immun., 62: 2002-2020]. The antigenic diversity of the outer membrane proteins of ntHi means that the development of a widely effective vaccine against a group of such heterogeneous organisms will require a new strategy. As will be seen, this invention refers to the most effective use of the peptide. LB 1 (f) as a vaccine against a broad spectrum of heterologous strains of Haemophilus influenzae, which express the P5-like fimbrin protein (or protein variants that occur in nature).

BRIEF DESCRIPTION OF THE INVENTION It is an object of the present invention to provide antigenic P5-like fimbrine subunit peptide groups (LB1 (f) peptides) of p5-like fimbrin proteins ,? from different strains of ntHi. It is a further objective to provide chimeric polypeptides that carry these peptides and that induce an immunogenic response in animals to ntHi, and polynucleotides encoding said peptides and polypeptides. The invention also relates to a method for isolating the chimeric peptides or polypeptides, to a method for detecting the presence of the peptides in biological samples and to a vaccine composition for use in the treatment of Haemophilus influenzae infection. Peptide groups LB1 (f) contain peptides of about 13 to about 22 amino acids long. The peptides fall into three major groups (one of which contains two subgroups) The chimeric polypeptide comprised one or more of the peptide units LB 1 (f), covalently linked to a carrier protein that additionally acts as a T cell epitope. It is preferred that the carrier protein is derived from Haemophilus influenzae, so that it can also induce an immunogenic response in animals for Haemophilus influenzae (including Haemophilus influenzae non-typeable). The invention will be more fully understood by reference to the following drawings and the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the plasmid Pmglmcs. The DNA sequence of the multiple cloning site is given. Figure 2 is the plasmid P rit 14588. Figure 3 is the plasmid LPD-LB1-A Figure 4 is the plasmid LPD-LB1-II. The DNA and amino acid sequences of the LB1 (f) peptides of group 1 (LB1-GR1) and group 2 (LB1-GR2) are shown with arrows. The arrows include LB 1 (f) within the sequence of its natural context, within the fimbrine protein, similar to p5. Figure 5 is the plasmid LPD-LB 1 -I II. The DNA and amino acid sequences of the peptides LB1 (f) of group 1 (LB1-GR1), of group 2 (LB1-GR2) and of group 3 (LB1-GR3) are indicated by arrows. The arrows include the peptides LB1 (f) within the sequence of their natural context within the fimbpna protein, similar to p5. The LB1 (f) polypeptide (designated LPD-LB1 (f) 2 1 3) extends from Meth to the C-terminal His residue, before the stop codon. Figure 6 is an acrylamide gel stained with Coomassie, which shows the expression products of the following plasmids: Bands: 1. MW markers. 2. pMGMCS 3. pRIT14588 4. LPD-LB1-A 5. LPD-LB1-II. 6. LPD-LB1-II I 7. LPD-LB1 -ill (LPD-LB1 (f) 2, 3 after the purification process) 8. MW markers. Figure 7 is a Western blot (using rabbit anti-LB1 antiserum) of an acrylamide gel showing the expression of products of the following plasmids: Bands: 1. MW markers. 2. pMGMCS. 3 Prit 4588 4. LPD-LB1-A 5. LPD-LB1-II 6. LPD-LB1 -lll 7. LPD-LB1-III (LPD-LB1 (f) 2 1 3 after the purification process). 8. MW markers. Figure 8 is a Western blot (using an anti-LPD monoclonal antibody) of an acrylamide gel, which shows the expression products of the following plasmids: Bands: 1. MW markers. 2. pMGMCS 3. pRIT14588 4. LPD-LB1-A 5 LPD-LB1-M 6. LPD-LB1-III 7. LPD-LB 1 -111 (LPD-LB1 (f) 2 1, 3 after the purification process) 8. MW markers.

Figure 9 is a Western blot (using an antibody against the six-Histidine purification tag) of an acrylamide gel, which shows the expression products of the following plasmids: Bands: 1. MW markers. 2. pMGMCS. 3. pRIT14588 4. LPD-LB1-A 5. LPD-LB1 -II 6. LPD-LB1 -lll 7. LPD-LB1-III (LPD-LB1 (f) 2,?, 3 after the purification process). 8. MW markers. Figure 10 is a passive challenge / transfer experiment. Markings or annotations of inflammation of the middle tympanic membrane during the observation period of 35 days for 5 groups of passively immunized chinchillas. The horizontal line interrupted by an annotation of mean tympanic membrane inflammation of 1.5 indicates the level of inflammation attributable to the adenovirus alone. Values above this line were considered an indication of inflammation induced by ntHi. V = simulation; O = LB1; u = LPD; * = PD; ? = LPD-LB1 (f) 2, 3. Figure 11 is a bar graph showing the percentage of total average ears that are known or suspected to contain an effusion, based on otoscopy and tympanometry in five groups of chinchillas involved with adenovirus, throughout the duration of the experiment. The tier scale was measured with respect to the intranasal challenge of ntHi on day 0. Each animal received a 1: 5 dilution of a specific antiserum by passive transfer, before intranasal challenge with ntHi # 86-028NP. The groups received antisera directed against: be Figure 12 is a Western blot of serum used for passive transfer. Spot A = overflow of anti-LB1 serum. Stain B = overflow of serum anti-LPD-LB1 (f) 2 •, 3; The bands contain: (1) molecular mass standards. (2) LPD; (3) LPD-LB1 (f) 2! 3; (4) LB1; (5) NTHi 86-028NP, preparation of outer membrane protein (OMP) whole; (6) NTHi 1885 MEE, whole OMP; (7) NTHi 1728MEE, whole OMP. Figure 13 is study A. Passive transfer / challenge experiment. Markings of inflammation of the middle tympanic membrane during the observation period of 35 days, for the five groups of passively immunized chinchillas The challenge was with strains 86-028NP or 1885MEE of ntHl. Figure 14 is study B. Passive transfer / challenge experiment. Inflammation marks of mean tympanic membrane, during the observation period of 35 days, for the five groups of passively immunized chinchillas The challenge was with strains 86-028NP or 1728MEE of ntHi. Figure 15 is study A. Graph showing the percentage of total average ears, which are known or suspected to contain an effusion based on otoscopy and tympanometry, in six groups of chinchillas compromised with adenovirus, throughout the duration of the experiment The time scale was measured with respect to the intranasal challenge of nt HI on day 0. Each animal received a 1.5 dilution of a specific antiserum by passive transfer, before intranasal challenge with ntHi # 86-028NP or 1885MEE. Figure 16 is study B. Graph showing the percentage of total average ears that are known or suspected to contain an effusion, based on otoscopy and tympanometry, in six groups of chinchillas committed to adenovirus, throughout the duration of the study. experiment The time scale was measured with respect to the intranasal challenge of ntHi on day 0 Each animal received a 1: 5 dilution of the specified antiserum, by passive transfer before intranasal challenge with ntHi # 86-028NP or 1728MEE DETAILED DESCRIPTION OF THE PREFERRED MODALITY THE PEPTIDES OF THE INVENTION The peptides of the present invention refer to groups of newly identified LB 1 (f) peptides, of p5-like fimbrin proteins, of various strains of ntHi from Europe and the United States. The DNA sequence of the fimbrin protein similar to P5 was determined from 83 strains of ntHi and the peptide sequence of peptide LB1 (f) was noted. The peptides of the present invention are B cell epitopes that occur in approximately the same region (and within the same context) of each priotein, approximately in the region comprising positions 110 and 140 of the amino acid sequence of the protein. In the ntHi10567RM strain, for example, the peptide exists between Arg117 and Gly 135 i (SEQ ID NO: 1). After aligning, the peptide sequences of both the American and European ntHi strains were within the same three groups, with some variation within those groups. The peptides of group 1 [or LB1 (f)?] Represented 71% of the peptides, contained approximately 19 amino acids and had no less than 75% identity with the peptide provided in SEQ ID.; NO: 1. Peptides from group 2 [or LB1 (f) 2] accounted for 19% of the peptides, contained 19 to 22 amino acids and had n? less than 75% identity with the peptide provided in SEQ ID NO: 2. The group could be further divided into two subgroups, group 2a [or LB1 (f) 2a] exemplified by SEQ ID NO: 2, and group 2b [or LB1 (f) 2], exemplified by SEQ ID NO: 4. Peptides of group 3 [or LB1 (f) 3] accounted for 10% of the peptides and contained 13 amino acids (provided in SEQ ID NO: 3) . The sequence identity for the peptides (and polypeptides and polynucleotides) can be calculated, for example, using the UWGCG package, which provides the BESTFIT program for. calculate the homology (identity), preferably in its default settings [Deveraux and coauthors, Nucí. Acids Res., 12: 387-395 (1984)].

From 83 nt H i strains analyzed, the LB1 (f) peptides of the 62 American strains and the 21 European strains were within the groups 1-3 Table 1 shows all the ntHi strains that were analyzed and to which group their respective peptides belong LB1 (f). Tables 2, 3, and 4 have lists of the accumulated sequences of the LB1 (f) peptides of group 1, 2 and 3, respectively. Table 5 has lists of a representative example of a peptide LB 1 (f) of group 1, 2a, 2b and 3. The peptide LB1 (f), previously known (SEQ ID NO: 5) is within group 1. If It is well known that this peptide is an effective immunogen, it confers protection against otitis media caused by ntHi, and until now it has not been known that this peptide was useful in these three antigenically distinct forms, which could potentially be combined to provide protective immunogens against all strains of Haemophilus influenzae that express the fimbrine protein, similar to P5. The peptides of this invention refer to peptides representative of groups 1, 2a, 2b and 3 (SEQ ID NO: 1, 2, 4 and 3, respectively) and variants of these peptides, antigenically related. "Antigenically related variants" may be natural variants (as exemplified by the peptides listed in Tables 2, 3 and 4) or artificially modified variants, which immunologically mimic the antigenic determinant site LB1 (f) of the fimbrin-like protein. P5 Such artificially modified variants may be made by synthetic chemistry or by recombinant DNA mutagenesis techniques, which are well known to those skilled in the art (see, for example, chapter 15 of Sambrook and coauthors Molecular Cloning - A Laboratory Manual ( 1989), Cold Spring Harbor Laboratory Press). Antigenically related variants of the peptides must have an amino acid sequence identity of at least 75% with one of the peptides provided in SEQ ID NO: 1-4 (and, more preferably, at least 85%, and most preferably, at least 95% identity); while the corresponding antigenic determinant site of the P5-like fimbrin protein of non-typeable Haemophilus influenzae is still capable of immunologically mimicking. For this invention, "immunologically mimicking the corresponding antigenic determinant site of the p5-like fimbrin protein of ntHi" is defined as a peptide (variant) that is capable of inducing antibodies that specifically recognize one of the LB 1 (f) sequences of wild type (which appear in the lists in Tables 2, 3 and 4) in the context of the protein fimbrine páarecida to P5, whole, and / or is defined as a peptide (variant) that is able to be recognized by the same immunospecific antibody which recognizes one of the wild-type LB1 (f) sequences (from the lists in Tables 2, 3 and 4) in the context of the whole P5-like fimbrin protein. In the first definition, the variant peptide must be able to induce said antibodies either by itself or together with a carrier molecule. In the second definition, the variant peptide must be capable of being recognized either on its own or together with a carrier molecule. The antigenically related variant peptide does not include those peptides provided in SEQ ID NO: 5 (the previously determined LB 1 (f) peptide of the p5-like fimbrin protein, from the ntH? -1128 strain) and SEQ ID NO. : 6 (the peptide resembling LB1 (f), previously determined, of the P5 protein, derived from ntHi). It may have been added to the related variants, amino acids, or they may have been inserted, substituted or omitted. Preferred variants are those that differ from those referenced by conservative (preferably individual) amino acid substitutions. The peptides of the invention also refer to combinations of peptides LB 1 (f) described above, covalently linked, with optional spacer amino acids between them, to form a single peptide. For such combinations the peptides of SEQ ID NO: 5 and 6 can be used. The method for chemically synthesizing or recombinantly expressing said peptides is well known to those skilled in the art [see, for example, Sambrook and co-authors (1989) ] Preferred optional spacer amino acids should not be more than 18 amino acids on either side of the peptide and should preferably be composed of amino acids from the natural context of peptide LB 1 (f) in the p5-like fimbrin protein (eg, if to bind two peptides LB1 (f), the first N-terminal LB1 (f) peptide would have 9 amino acids from the natural C-terminal context, linked to 9 amino acids from the natural N-terminal context of the second peptide LB1 (f) C-terminal) . One or more peptides LB1 (f) can be linked in this manner. It is preferred to bind from 1 to 10 peptides LB1 (f), more preferably from 1 to 5 and, still more preferable, from 1 to 3. It is preferred more than the examples of at least one peptide LB1 (f) of each group LB1 (f) are linked in that way. It is even more preferred that the bound LB 1 (f) peptides are those provided in SEQ ID NO: 2, 3 and 5. When the three antigenically distinct peptides are combined, a more broadly protective immunogen is thus formed.

THE POLYPEPTIDES OF THE INVENTION The polypeptides of the present invention refer to peptides described above, which are covalently linked to a carrier polypeptide containing at least one T cell epitope (e.g., tetanus toxin, diphtheria toxin, CRM197). , Borrelia burgdorferi, sensu lato OspA, keyhole limpet hemocyanin, P6 protein from H. influenzae, protein fimbrine pa εida to P5 from H. influenzae, OMP26 from H. influenzae, protein D from H. influenzae, or lipoprotein D from H. influenzae), to form a chimeric LB1 (f) polypeptide. This chimeric polypeptide comprises at least one of the peptides LB1 (f) of the invention. It is preferred that the chimeric polypeptide comprises from 1 to 10 peptides, LB 1 (f), more preferably, from 1 to 5 and, even more preferably, from 1 to 3. These peptides may be N-terminally, C-terminally or both N and C-terminally, to the carrier polypeptide. It is preferred that the carrier polypeptide be from Haemophilus influenzae, so that it acts as a good immunogenic carrier, while having some protective efficacy in itself and / or at the same time providing a source of homologous T-cell epitopes, derivatives of H. influenzae. Optionally, the chimeric polypeptide may also comprise a peptide sequence, such as a purification tag (such as a histidine tag or a glutathione-S-transferase tag) in order to aid in the subsequent purification of the polypeptide. Optional short peptide separator sequences can be introduced between elements of the chimeric polypeptide (as defined above in the peptides of the invention). It is preferred that the carrier polypeptide used be OMP26 from H. influenzae (WO 97/01638) or the P6 protein from H. influenzae (Nelson, M. B. and co-authors (1988), Infection and Immunity 56, 128-134). It is highly preferable that the carrier polypeptide used be the protein D (PD) of non-typeable Haemophilus influenzae, or the lipoprotein D (LPD, a lipidated form of PD). PD is an outer surface protein, which binds to human IgD, of 42 kDa, which has been shown to be highly conserved among all strains of Haemophilus influenzae investigated so far (WO 91/18926). Both PD and LPD have been expressed in E. coli.

It has been found that LPD is a virulence factor in H. influenzae and causes bactericidal activity against ntHi in rat antisera. LPD of H. influenzae and the recombinantly expressed equivalent of LPD, in this way, can act as good immunogenic carriers, while they have some protective efficacy in themselves. The non-lipidated form (PD) is more conveniently used for process reasons, and is also a potential carrier polypeptide of this invention. LPD is highly immunogenic due to its adjuvant properties, ie, its ability to induce interleukins in the macrophage and its ability to stimulate the proliferation of B cells (WO 96/32963). PD has no adjuvant properties contained and, thus, these conjugates preferably have added adjuvants, for example (but not limited to), aluminum hydroxide or aluminum phosphate. Antibody responses to LPD can protect against typed and non-typed Hi type strains. In this way, it represents an important carrier molecule to fix other Hi antigens (such as peptides LB 1 (f) in order to obtain more effective vaccines against the organism.) In addition to improving the immunological response to the peptide antigen LB1 (f) , LPD can serve as a protective antigen against both non-encapsulated and encapsulated Hi strains It is preferable that three LB1 (f) peptides are attached to the carrier polypeptide (one from each LB1 (f) group.) Preferably the LB1 (f) peptides used are those provided in SEQ ID NO: 2, 3 and 5, and 1 are preferably C-terminally linked to the carrier polypeptide in the order of SEQ ID NO: 2 (peptide of group 2), SEQ ID NO: 5 (peptide of group 1), SEQ ID NO: 3 (peptide of group 3). Said LPD-linked polypeptide is known as LPD-LB1 (f) 2,?, 3. When the three antigenically distinct peptides are combined, a more broadly protective immunogen is thus formed. While the chimeric polypeptide does not need to have a purification tag, when required, a histidine tag sequence is preferred, and is preferably located at the C-terminus of the polypeptide. The sequence of a preferred chimeric polypeptide LPD-L B 1 (f) 2.1 3 is given in Figure 5. Residues 1-19 are the signal sequence of protein D. This signal peptide can be removed in order to produce the PD version of the chimeric polypeptide. The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include the recombinantly produced polypeptides, the synthetically produced polypeptides or the polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art; however, examples of the method are presented in the examples section.

POLINUCLEOTIDES OF THE INVENTION The polynucleotides of the present invention refer to the wild-type polynucleotide sequences of the LB1 (f) peptides provided in Tables 6-8. They also refer to the wild-type DNA sequence of the polypeptides of the present invention; that is, the construction of the chimeric polypeptide gene such that the wild type genei sequence of the carrier polypeptide and the wild-type polynucleotide sequences of the LB 1 (f) peptides are used. Said polynucleotide is given in Figure 5. The DNA sequence of the optional spacer amino acids is not essential for the invention; however, when the spacer amino acids are from the natural context of the peptide LB 1 (f), it is preferred (although not necessary) to use the natural DNA sequence of those separators. The polynucleotides of the invention also refer to DNA sequences that can be derived from the amino acid sequences of the peptides and polypeptides of the invention, which take into account the degeneracy of codon usage. This is well known in the art, as well as the knowledge of codon usage in different expression hosts, which is useful to optimally carry out the recombinant expression of the peptides and polypeptides of the invention. The invention also provides polynucleotides that are complementary to all of the polynucleotides described above: When the polynucleotides of the invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide can include the coding sequence for the mature polypeptide, itself; or the coding sequence] for the mature polypeptide in the reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a preprotein, proprotein or preproprotein sequence, or other portions of fusion peptide (eg. example, amino acid residues 1 to 19 of Figure 5, the natural signal sequence of LPD). For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexahistidine peptide, such as that provided in the pQE vector (Qiagen, Inc.) and described in Gentz and coauthors, Proc. Natl. Acad. Sci USA (1989) 86: 821-824, a is an HA tag, or is glutathione-s-transferase. The molten LPD is also preferred to its natural signal sequence (amino acid residues 1 to 19 of Figure 5). The polynucleotide may also contain 5 'and 3' non-coding sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosomal binding sites and sequences that stabilize the mRNA.

VECTORS, HOSTS CELLS. EXPRESSION The present invention also relates to vectors comprising a polynucleotide or several of the present invention, and host cells are genetically engineered with vectors of the invention, and to the production of peptides or polypeptides of the invention by recombinant techniques. .

Cell-free translation systems can also be employed to produce said proteins, using the RNAs derived from the DNA constructs of the present invention. For recombinant production, the host cells can be genetically engineered to incorporate expression systems or portions thereof, for the polynucleotides of the present invention. The introduction of polynucleotides1 into the host cells can be effected by methods described in many commonly used laboratory manuals, such as Davis and co-authors, Basic Methods in Molecular Biology (1986) and in Sambrook and co-authors, Molecular cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1989), eg, as calcium phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation transduction, embedding load, ballistic introduction or infection Representative examples of appropriate hosts include bacterial cells, such as meningococci, streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as cells Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 cells and Bowes melanoma cells; and plant cells.

A wide variety of expression systems may be used. Such systems include, but are not limited to, chromosomal, episomal and viral derivative systems; for example, vectors derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from elements of infection, from yeast chromosomal elements, from viruses, such as baculoviruses, papovaviruses, such as SV40, vaccinia virus, adenovirus , poultry pox virus, virus and pseudo-rabies retrovirus, and vectors derived from their combinations, such as plasmid derivatives and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate and also that engender the expression. In general, any suitable system or vector can be used to maintain, propagate or express the polynucleotides to produce a polypeptide in a host. The appropriate nucleotide sequence can be inserted into an expression system, by a variety of well-known and routine techniques, such as, for example, those indicated in Sambrook and co-authors, Molecular Cloning, to Laboratory Manual (supra). For the secretion of the protein transferred to the lumen of the endoplasmic reticulum, in the periplasmic space or in the extracellular environment, appropriate secretion signals can be incorporated into the desired polypeptide. These signals can be endogenous to the polypeptide (residues 1 to 19 in Figure 5) or they can be heterologous signals.

PEPTI DOS / POLY PEPTI TWO EXPRESSED RECOMBINANT WAYS The peptides and polypeptides of the invention can be recovered and purified from recombinant cell cultures, by well-known methods, including precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulase chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It is highly preferable to employ high performance liquid chromatography for purification. Well-known techniques can be employed to refold the proteins, to regenerate the active conformation, when the polypeptide is denatured during isolation or purification. While the gene sequence of the chimeric polypeptide LB 1 (f) can be marked on the vector, with a histidine tag sequence, which aids in the purification of the polypeptide, it is not an essential element for the invention, since Polypeptides without the histidine tag can still be purified by one of the techniques mentioned above. Example 3 describes a purification method for purifying the chimeric polypeptide LPD-LB1 (f) (group 2 / group1 / group 3) (or LPD-LB1 (f) 2.?, 3). It is easier to purify a chimeric LPD-LB1 polypeptide (f) with three or more peptides LB1 (f) at the C-terminus of the polypeptide, than one with only one peptide LB1 (f) at the C-terminus. This is due to degradation observed partial of the polypeptide from the C-terminus, when it contains only one peptide LB 1 (f); what is not observed when there are three peptides LB1 (f) in the exagem C When some degradation has occurred, the full length polypeptide can be separated from the degraded form, incorporating a careful step of changing anions in the purification process.

THE ANTIBODIES The peptides and polypeptides of the invention, or the cells that express them, can also be used as immunogens to produce antibodies immunospecific for the wild type LB 1 (f) peptides. The term "immunospecific" means that the antibodies have substantially greater affinity for the peptides or polypeptides of the invention than their affinity for other related polypeptides in the prior art. Antibodies generated against peptides or polypeptides can be obtained by administering them to an animal, preferably a non-human, using routine protocols in the immunization of an animal with an antigen, the collection of the blood, the isolation of the serum and the use of the antibodies that react with the peptide. The serum or the IgG fraction containing the antibodies can be used in the analysis of the protein. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G and Milstein, C, Nature (1975) 256: 495-4.97), the trioma technique, the Romanian B-cell hybridoma technique (Kozbor and co-authors, Immunology Today ( 1983) 4:72) and the EBV hybridoma technique (Cole and co-authors, Monoclonal Antibodies and Cancer Therapy, pages 77-96, Alan R. Liss, Inc., 1985). Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to peptides or polypeptides of this invention. Transgenic mice or other organisms, including other mammals, can also be used to express the humanized antibodies. The antibodies described above can be used to isolate or identify clones expressing the peptide or to parity the peptides or polypeptides of the invention, by affinity chromatography. The peptides and polypeptides of the present invention are also useful for producing polyclonal antibodies for use in passive immunotherapy against H. influenzae. Human immunoglobulin is preferred because the heterologous immunoglobulin can elicit a damaging immune response to its foreign immunogenic components. Polyclonal antisera are obtained from individuals immunized with the peptides or polypeptides, in any of the ways described. Then the immunoglobulin fraction is enriched. For example, immunoglobulins specific for protein epitopes are enriched by immunoaffinity techniques, employing the peptides or polypeptides of the invention. The antibody is specifically absorbed from antisera in an immunoadsorbent containing epitopes of the polypeptide, and then eluted from the immunoadsorbent, as an enriched immunoglobulin fraction.VACCINES Previous work on peptide LB1 (f) from strain ntHi-1128, indicated that this peptide could be used as an immunogen for the development of a subunit vaccine against Haemophilus influenzae disease, particularly to prevent or reduce susceptibility to otitis media and other diseases caused by non-typed strains. This invention extends that work by discovering three major groups of peptides LB1 (f). The differences between the three groups are such that it is unlikely that efficient cross-protection could be achieved between the strains belonging to different groups. Therefore, the present invention is based on the use of examples of each of these groups of peptides to provide more efficient and complete vaccines against strains of Haemophilus influenzae (preferably ntHi), which express the fimbrin protein, similar to P5 . Accordingly, another aspect of the invention is a vaccine composition comprising an immunogenic amount of at least one peptide or polypeptide of the invention. It is preferable that the composition should also comprise a pharmaceutically acceptable excipient. The vaccine preparation is generally described in Vaccine Design (The subunit and adjuvant approach (editors Powell M. F. and Newman M. J.). 1 (1995) Plenum Press, New York, E. U. A.). Additionally, the peptides and polypeptides of the present invention preferably have adjuvants for the vaccine formulation of the present invention. The adjuvants include an aluminum salt, such as aluminum hydroxide gel (alum) or aluminum phosphate, but there may also be a choke, iron or zinc salt, or it may be an insoluble suspension of acylated tyrosine or acylated sugars , cationic or anionically derived polysaccharides, or polyphosphazenes. Other coinocidal adjuvants include oligonucleotides containing CpG. The oligonucleotides are characterized in that the CpG dinucleotide is not methylated. Such oligonucleotides are well known and are described, for example, in WO 96/02555. Other preferred adjuvants are those that induce a immunological response, preferably of the TH1 type. Elevated levels of Th1-type cytokines tend to favor the induction of cell-mediated immune responses to the given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen. Suitable adjuvant systems include, for example, monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid (3D-MPL) or a combination of 3D-MPL together with an aluminum salt. CpG oligonucleotides also preferentially induce a TH1 response. An increased system comprises the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D.MPL, as described in WO 94/00153, or a less reactogenic composition, where QS21 is activated with cholesterol, as described in WO 96/33739. A particularly potent adjuvant formulation comprising QS21 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210 and is a preferred formulation. Another aspect of the invention relates to a method for inducing an immune response in a mammal, which comprises inoculating the mammal with a peptide or polypeptide of the invention, suitable for producing the antibody and / or the T cell immune response, to protect the animal against the disease due to H influenzae, among others. Yet another aspect of the invention relates to a method for inducing an immune response in a mammal, comprising administering a peptide or polypeptide of the invention, through a vector that directs the expression of a polynucleotide of the invention in vivo, in order to of inducing said immune response, to produce the antibody to protect the animal against the disease. Another additional aspect of the invention relates to an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immunological response in that mammal to a peptide or polypeptide 'LB1 (f), wherein the composition comprises an LB 1 peptide or polypeptide gene (f), or the peptide or polypeptide itself LB1 (f). The vaccine formulation may additionally comprise a suitable carrier. The vaccine composition of LB 1 (f) is preferably administered orally, intranasally or parenterally (including subcutaneous, intramuscular, intravenous, intradermal, transdermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions, which may contain antioxidants, regulators, bacteriostats and solutes that make the formulation isotonic with the recipient's blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in single-dose or multi-dose containers, for example, sealed ampoules and flasks, and may be stored in a dried condition per freezing, which only requires the addition of the sterile liquid carrier, immediately before use. The vaccine formulation may also include the adjuvants, as described above. The dose will depend on the specific activity of the vaccine and can be easily determined by routine experimentation. Another aspect relates to an immunological / vaccine formulation, which comprises the polynucleotide of the invention. Said techniques are known in this field; see, for example, Wolff and coauthors, Science, (1990) 247: 1465-8. The peptides or polypeptides of this invention can be administered as multivalent subunit vaccines, in combination with antigens of other H. influénzae proteins or to obtain an increased bactericidal activity. They can also be administered in combination with polysaccharide antigens, for example, the PRP capsular polysaccharide (preferably conjugated to a protein) of H. influenzae b. For administration in combination with epitopes of other proteins, the peptide or polypeptide LB1 (f) is administered separately, as a mixture, or as a fusion, conjugated or genetic polypeptide. The conjugate is formed by common and current techniques for coupling proteinaceous materials. The peptides or polypeptides of the invention can be used in conjunction with antigens or other organisms (eg, bacteria, viruses, fungi and pajrasites, encapsulated or unencapsulated). For example, the peptides or polypeptides of the invention are useful in conjunction with the antigens of other microorganisms involved in otitis media or other diseases. These include: Streptococcus pneumoniae, Streptococcus pyrogenes, group A, Staphylococcus aureus, the respiratory syncytial virus and Branhemella catarrhalis. Since the polypeptides of the present invention comprise the P5-like fimbrin protein itself, another preferred aspect of the invention is the combination of two or more P5-like fimbp proteins, of different LB 1 (f) groups, in a vaccine formulation. The evaluation of the peptides or polypeptides of the invention is made as potential vaccines against otitis media caused by ntHi, in an animal model with chinchillas, developed by Dr. L. Bakaletz of the Ohio State University. This model mimics the development of otitis media in children, and is based on successive intranasal administrations of adenovirus and ntHi, with a distance of one week. Under these conditions the bacterium is able, after colonizing the nasopharynx, to invade the middle ear through the eustachian tube. Once there, ntHi will proliferate and induce an inflammatory process similar to that seen in children. For evaluation of the vaccine, in the time that the chinchillas have been actively immunized, they are too much travel for the time of challenge, to be inoculated by intranasal route with ntHi; Even with preinfection with adenovirus, none of them will develop otitis media. As an alternative route of challenge, a direct inoculation of the bacteria in the middle ear (bullae) through the skull is used. Passive transfer / challenge protocols can also be used to avoid the necessary transbular challenge. With all these types of challenges, the severity of the disease can be marked by otoscopic observation (through the external ear) or tympanometry, which evaluates the level of inflammation in the middle ear or pressure changes in the middle ear and the presence of fluid in the middle ear, respectively.

The efficacy of a vaccine is determined by reducing the severity and / or duration of inflammation and reducing colonization in the ear and nasopharynx. In previous experiments, the protective efficacy of both LB1 of the ntHi strain was evaluated. 1128 as LPD, after active immunization and intrabular challenge. Repeatedly immunization with LB1 protected the chinchillas against otitis media, as indicated by a reduced duration of otitis, reduced severity and reduced colonization in the ears and nasopharynx. Immunization with LPD only protected the chinchillas against otitis media, but not as well as LB1, and not reproducibly. The vaccines of the invention can be further evaluated by examining silos peptides or polypeptides of the invention inhibit the adhesion of ntHi to chinchilla throat epithelial cells, and whether they can prevent nasopharyngeal colonization by ntHi in vivo. The peptide LB1 of ntHi-1128 has a dose-dependent effect on the inhibition of ntHi adhesion to epithelial cells of chinchilla throat (probably as it acts as a direct steric inhibitor of ntHi binding) and decreases the ntHi in the nasopharyngeal lavage fluid. Nasopharyngeal colonization is an initial step necessary for the development of otitis media, therefore, this inhibition of colonization will also help to inhibit the development of otitis media.

This invention also relates to the use of the peptides or polypeptides of the invention, and the antibodies against those peptides or polypeptides, as diagnostic reagents. of a peptide LB1 (f) will provide a diagnostic tool that can be added to, or define a diagnosis of the disease by Haemophilus influenzae, among others. Biological samples can be obtained for diagnosis of the cells of a subject, such as serum, blood, urine, saliva, sputum tissue biopsy, wash fluids. Polynucleotides of the invention which are identical or sufficiently identical to one of the nucleotide sequences contained in tables 6-8 can be used., as hybridization probes for cDNA and genomic DNA, or as sensitizers for a nucleic acid amplification reaction (PCR) to isolate the full-length cDNAs and genomic clones encoding the P5-like fimbrin protein. Such hybridization techniques are known to those skilled in the art. Typically, these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to those of the referent. The probes will generally comprise at least 15 nucleotides. It is preferred that said probes have at least 30 nucleotides and can have at least 50 nucleotides. Particularly preferred probes will vary between 30 and 50 nucleotides. In this way Haemophilus influenzae can be detected in a biological sample, and under particularly stringent hybridization conditions, the strain or specific strains of Haemophilus influenzae present in a sample could be determined, using the wild-type polynucleotide sequences provided in the tables. 6-8. Thus, in another aspect, the present invention relates to a diagnostic kit for a particular disease, in special disease by Haemophilus influenzae, comprising: (a) a polynucleotide of the invention, preferably a nucleotide sequence provided in the tables 6-8; (b) a nucleotide sequence complementary to that of (a) ,; (c) an LB1 (f) peptide of the invention, preferably the peptides of SEQ ID NO: 1-4, or (d) an antibody to an LB1 (f) peptide of the invention, preferably to the SEQ peptides ID NO: 1-4 It will be appreciated that in any of said equipment (a), (b), (c) or (d) a substantial component may be included. The cited documents are incorporated herein by reference. The invention is further illustrated in the following examples: EXAMPLE The following examples are carried out using common and current techniques, which are well known, and are routine for those skilled in the art, except when set forth in detail in another manner. The examples illustrate, but do not limit the invention.

EXAMPLE 1 DETERMINATION OF VARIABILITY IN THE SEQUENCE OF PEPTIDE AMINO ACIDS LBKf) IN DIFFERENT STRAINS ntHi. 1a) Culture of ntHi isolates - The preparation of samples for PCR analysis. We obtained 53 isolates of ntHi from Dr. L. Bakaletz from Ohio State University and 30 isolates from ntHi from Dr. A. Forsgren from Málmo, Sweden. 0.1 ml of a liquid culture of each ntHi isolate was spread on Gelose chocolate agar (GCA). The purity of the samples was monitored in solidified media (TSA-tri-shoot agar and soup in Petri dishes). The plates were incubated at 35 ° C for 24 hours. The plate colonies were resuspended in 5 ml of filtered TSB (tripose-soy broth + 3 μg / μl of Hemine + 1% horse serum). 50 ml of TSB, liquid medium, was inoculated with 2.5 ml of the culture and incubated at 35 ° C. When the culture concentration grew to 108 cells / ml, 10 ml of culture was centrifuged at 10,000 r.p.m., 4 ° C for 15 minutes. The supernatant was removed and the cells were washed in physiological regulator. The cells were centrifuged at 10,000 r.p.m. for 15 minutes, 4 ° C. The cells were resuspended to a final concentration of 109 cells / ml. The cells were left to boil, at 95-100 ° C for 10-15 minutes, and then placed directly on ice. The samples were frozen at -70 ° C. The samples were then ready for DNA amplification by PCR. 1b) .- Amplification of the DNA fragment of the P5-like fimbrine gene by PCR. The PCR amplification of the fibrin gene fragment was carried out in the ntHi preparations of example 1a). 200 μl of an ntHi preparation was centrifuged at 14,200 r.p.m. for 3 minutes at room temperature. All the supernatant was removed. The cells were resuspended in 25 μl of ADI, then left to boil at 95 ° C for 10 minutes and centrifuged for 3 minutes at 14,200 r.p.m .. 5 μl of supernatant was used for a PCR reaction. DNA amplification was carried out with specific sensitizers: NTHi-01: 5'-ACT-GCA-ATC-GCA-TTA-GTA-GTT-GC-3 'NTHi-02: 5'-CCA-AAT-GCG-AAA- GTT-ACA-TCA-G-3 'The TCP reaction mixture was composed of the following: cell extract supernatant: 5.0 μl; sensitizer NTHi-01 (1/10), 1.0 μl; NTHi-02 sensitizer (1/10), 1.0 μl; DMSO, 2. 0 μl; dNTP mixture, 4.0 μl; 10x regulator, 5.0 μl; ADI 31.5 μl; Taq-polymerase 0.5 μl.

The conditions of the CPR cycle were as follows (94 ° C for one minute 50 ° C for one minute, 72 ° C for 3 minutes) for 25 cycles, and finished with 72 ° C for 10 minutes. The reaction was monitored by electrophoresis on a 3% agarose gel in TBE regulator. The sensitizers used for the identification of which group belonged an ntHi peptide of LB1 (f) of fimbrine similar to P5, are the (used similarly to the previous reaction): Group 1: NTH? -01: 5'-ACT-GCA-ATC-GCA-TTA-GTA-GTT-GC-3 'NTHÍ-GR1: 5'-GTG -GTC-ACG-AGT-ACC-G-3 'Group 2: NTHi-01: 5'-ACT-GCA-ATC-GCA-TTA-GTA-GTT-GC-3' NTHi-GR2bis: 5'-TCT- GTG-ATG-TTC-GCC-TAG-3 'Group 3: NTHi-01: 5'-ACT-GCA-ATC-GCA-TTA-GTA-GTT-GC-3' NTHi-GR-3: 5'-CTA -TCG-ATG-CGT-TTA-TTA-TC-3 ' 1c) .- Purification of DNA The PCR cleaning equipment was used for the purification of DNA fragments from PCR reactions (Coheringer Mannheim). At the end of the procedure, the purified PCR product was eluted twice in 25 μl volumes of redistilled water from the silica resin.

The purified products were analyzed by electrophoresis in a 3% agarose gel, stained with ethidium bromide. Then the DNA was ready for sequencing. 1d) .- DNA sequencing This was carried out in an ABl automatic sequencer, the ABI-PRISM DNA sequencing equipment (using the RCP Terminator cycle sequencer) and Amplitaq DNA Polymerase FS (by Perkin) Elmer). The PCR reaction mixture used was as follows: Mixture (of the equipment), 8 0 μl; DNA (approximately 1 μg), 3.0 μl; sensitizer (see below) 1/5 or 1/10, 1.0 μL; ADI 8.0 μl. The sequencing sensitizers used were the following: NTHi-0.3: 5'-AGG-TTA-CGA-CGA-TTT-CGG-3 \ or NTHi-04 '5'-CGC-GAG-TTA-GCC-ATT-GG- 3 \ or NTHi-05. 5'-AAA-GCA-GGT-GCT-TTA-G-3 \ or NTHi-06: 5'-TAC-TGC-GTA-TTC-TGC-ACC-3 'or NTHi-03: 5'-AGG-TTA -CGA-CGA-TTT-CGG-3 'NTHi-04: 5'-CGC-GAG-TTA-GCC-ATT-GG-3' NTHi-05: 5'-AAA-GCA-GGT-GTT-GCT-TTA -G-3 'NTH -06: 5'-TAC-TGC-GTA-TTC-TTA-TGC-ACC-3' NTH- 14: 5'-GGT-GTA-TTT-GGT-GGT-TAC-C- 3 'NTHi-15: 5'-GTT-ACG-ACG-ATT-ACGGTC-G-3'.

^^^^^ The sequencing conditions of the PCR cycle were the following: (96 ° C for 30 seconds, 50 ° C for 15 seconds, 60 ° C for 4 minutes) for 25 cycles and ending with 72 ° C for 10 minutes. minutes The PCR product was prepared and analyzed: adding 80 μl of ADI to the PCR sequence reaction to obtain a final volume of 100 μ !; adding an equal volume of phenol / chloroform to the DNA solution. The sample was then centrifuged at 14,500 r.p.m. at 4 ° C for 3 minutes and the upper aqueous layer was separated. The phenol / chloroform step and the centrifugation step were repeated once. 10 μl of 3M NaAc pH 4.8 and 220 μl of 100% ethanol (at room temperature) was then added and mixed. The sample was placed at -20 ° C for 5 minutes and then centrifuged at 14,000 r.p.m. 20 minutes, at 4 ° C. The supernatant of ethanol was removed and the pellet was rinsed with | 1 ml of ethanol at 70 ° C (room temperature). This was centrifuged at 14,000 r.p.m. for 10 minutes at 4 ° C and the supernatant was removed, as before. The pellet was air dried and frozen during the night. The pellet was dissolved in the following solution: formamide at 100 μg / deionized water 5 volumes; 0.5M EDTA, pH 8.00, 1 volume. It was stirred until it formed a vortex for a few seconds and was loaded in a sequencing gel. 1e) .- Accumulated results and conclusions Table 1 shows a list of the various ntHi isolates that was analyzed in terms of the sequence of their peptides LB1 (f) of the fimbrine protein similar to P5. The group classification was determined by aligning the peptide LB1 (f) against SEQ ID NO: 5, 2 or 3 (which are representative of the peptides LB1 (f) of group 1, 2 or 3, respectively) The peptides LB1 (f) must have at least 75% identity with the representative peptide of a group in order to assign the group classification to the test peptide. Tables 2, 3 and 4 show the aligned sequences of the peptide sequences LB1 (f) of groups 1, 2 and 3, respectively. Table 5 shows the peptides LB1 (f) representative of groups 1, 2a, 2b and 3, aligned with each other. Tables 6-9 show the DNA sequences of the peptides LB1 (f) of Tables 2-5, respectively.

C U A D RO 1 Serotype No order Strains Group 1 NTHi 1848L H. nfluenzae 1 2 NTHi 1848NP H. influenzae 1 3 NTH i 1885R H. influepzae 1 4 NTH? 1885 EE H. influenzae 2 5 NTHi 10547RMEE H. influenzae 3 6 NTHi 10548LMEE H. nfluepzae 3 7 NTHi 10567RMEE H. influenzae 8 NTHi 10568LMEE H. influenzae 9 NTHi 10567 &8NP H. influenzae 3 NTHi 1371 MEE H. influenzae 11 NTHi 214NP H. influenzae 12 NTHi 1370MEE H. influenzae 13 NTHi 1380 EE H. influenzae 14 NTHi 217NP H. influenzae 15 NTHi 266NP H. influenzae 2 16 NTHi 167NP H. influenzae 17 NTHi 1657 EE H Nfluenzae 18 NTHi 284NP H. influenzae 19 NTHi 1666MEE H. influenzae 20 NTHi 287NP H. nfluenzae '21 NTHi 1236MEE H. influenzae 2 22 NTHi 183NP H. influenzae 2 23 NTHi 165NP H. influenzae 2 24 NTHi 1182 EE H. Nfluenzae 25 NTHi 166NP H. influenzae 26 NTHi 1199 EE H. influenzae 27 NTHi 172NP H. influenzae 28 NTHi 1230MEE H. influenzae 29 NTHi 180NP H. nfluenzae 30 NTHi 1234MEE H. nfluenzae 1 31 NTHi 182NP H. influenzae 32 NTH : 152NP H. pfluenzae 33 NTHi 226NP H. nfluenzae 34 NTHi 1714MEE H. nfluenzae 2 35 NTHi 297NP H. influenzae 2 36 NTHi 1 15MEE H. mfluenzae 2 37 NTHi 1729MEE H. influenzae 3 38 NTHi 1728MEE H. nfluenzae! 3 39 NTHi 250NP H. mfluenzae 1 40 NTHi 1563MEE H. influenzae 1 41 NTHi 1562MEE H. influenzae 1 42 NTHi 10559RMEE H. influepzae 1 43 NTHi 1712IV.EE H. nfluenzae 1 44 NTHi 1521 H. influenzae 1 45 NTHi 1060RMEE H. influenzae 1 46 NTHi 86-027MEE H. i? Fluenzae 2 47 NTHi 86-027NP H. influenzae 1 48 NTHi 86-028NP H. influenzae 1 49 NTHi 86-028LMEE H. nfluenzae 1 50 NTHi 90-100 H. nfluenzae 1 51 NTHi 90-121RMEE H. influenzae 1 52 NTHi 1128 H. nfiuenzae 1 53 NTHi 90-100RME? H. influenzae 1 54 NTHi * 476 H. influenzae 1 55 NTHi "480 H. influenzae 1 56 NTHi" 481 H. influenzae 1 57 NTHi * 482 H. influenzae 1 58 NTHi "484 H. influenzae 1 59 NTHi" 486 H. influenzae 1 60 NTHi "490 H. ntluenzae 1 61 NTHi" 492 H. nfluenzae 2 62 NTHi "494 H. influenzae 1 63 NTHi" 495 H. nfluenzae 2 64 NTHi "498 H. influenzae 1 65 NTHi" 499 H influenzae 1 66 THG 500 H. nfluenzae 2 67 NTHi * 501 H. influenzae 1 68 NTHi * 502 H. influenzae 2 69 NTHi "503 H. nfluenzae 1 70 NTHi * 504 H. influenzae 3 71 NTHi * 506 H. pfluenzae 2 72 NTHi * 507 H. ipfiuenzae 1 73 NTHi * 546 H. influenzae 2 74 NTHi * 567 H. influenzae 1 75 NTHi 544 H. influenzae 3 76 NTHi 565 H. influenzae 1 77 NTHi 600 H. influenzae 3 78 NTHi 601 H. influenzae 1 79 NTHi 603 H. infl ueza 1 80 NTHi 604 H. influenzae 2 51 NTH? 605 H. influenzae 1 82 NTHi 606 H. influenzae 1 83 NTHi 608 H. mfluenzae i Cumulative list of ntHi strains investigated and the classification of the sequence of their respective peptides LB1 (f), of fimbrine protein similar to P5 (strains 1-53 of L. Bakaletz, strains 54-83 of A. Forsgren). The asterisk (*) denotes a European strain of ntHi; all the others were isolated Americans. Strains 1885 and 1128 are available from the American Type Culture Collection (ATCC # 55431 and 55430, respectively) C U A D RO 2 S EC U E N N G L I S E D E P E P E R G U P E 1 NI128 RSDYKFYEDAÍGTRDHKKG N1380ME? RSDYKFYEDANGTRDHKKG N1885R RSDYKFYEDANGTRDHKKG N1562MEE RSDYKFYEDANGTRDHKKG N1563? E RSDYKFYEDANGTRDHKKG Ni8ONP RSDYKFYEDANGTRDHKKG N217NP RSDYKFYEDANGTRDHKKG N284NP RSDYKFYEDANGTRDHKKG N1665ME? N1230MEE RSDYKFYEDANGTRDHKKG NTHI-501 RSDYKFYEDANGTRDHKKG RSDYKFYEDANGTKDHKKG NTHI-507 RSDYKFYEDANGTRDHKKG NTHI-565 RSDYKFYEDANGTRDHKKG NTHI THI-603 RSDYKFYEDANGTRDHKKG N287NP RSDYKFYEDANGTRDHKKG-60S N86028 RSDYKFYEDANGTRDHKKG N152NP RSDYKFYEDANGTRDHKKG N86028NP M RSDYKFYEDANGTRDHKKG N1234MEE RSDYKFYEDADGTRDHKKG RSDYKFYDDANGTRDHKKG N182NP RSDYKFYDDANGTRDHKKG N90IOOR >; RSDYKFYEDEKGTRDHKKG N90100 RSDYKFYEDEKGTRDHKKG N10567RM RSDYKFYEAANGTRDHKKG N1060ME? RSDYKFYEAANGTRDHKKG N172NP RSDYKFY? AANGTRDHKKG N1199MEE RSDYKFYEAANGTRDHKKG N10568LM RSDYKFYEAANGTRDHKKG N90121RM RSDYKFYEAANGTRDHKKG N86027NP RSDYKFYEVANGTRDHKKG NTHI-486 RSDYKFYEVANGTRDHKKG N1712 RSDYKFYEVANGTRDHKKG NTHI-503 EE RSDYKFYEAANGTRDKKKG NTHI-476 RSDYKFYEEANGTRDHKKG N166NP RSDYKFYNDANGTRDHKKS N1I82ME? RSDYKFYNDANGTRDHKKS N1848NP RSDYKFYEVANGTRDHKKS N1371MEE RSDYKFYEVANGTRDHKKS NTHI-498 RSDYKFYEVANGTRDHKKS NTHI-606 RSDYKFYEVANGTRDHKKS N1848L RSDYKFYEVANGTRDHKKS THI-567 RSDYKFYEDANGTRDRKTG NTHI-484 RSDYKFYEDANGTRKHKEG N10559RM RSDYKLYEVANGTRDHKKS THI-601 RSDYKFYEVANGTRDHKQS NTHI-481 RSDYKFYEVANGTRDHKQS NTHI-482 RSDYKFYEVANGTRDHKQS N1370MEE RSDYKFYEVANGTRDHKQS N226NP RSDYKFYEEANGTRDHKRS NTHI-480 RSDYKFYEDANGTRERKRG N1657MEE RSDYKFYEVANGTRERKKG N267NP RSDYKFYEVANGTRERKKG NTHI -490 RSDYKFYEVANGTRERKKG NTHI-494 RSDYKFYEVANGTRERKKG N214NP RSDYKFYEVPNGTRDHKQS N250NP RSDYKRYEEANGTRNHDKG N1521 RSDYKRYEEANGTRNHDKG NTHI-605 RSDYKRYEEANGTRNHDKG NTHI-499 RSDY? FYEAPNSTRDHKKG TABLE 3 ACCUMULATED PEPTIDE SEQUENCES OF GROUP 2 N1715MEE RSDYKLY KNSSSNSTLKNLGE N1714 EE RSDYKLYNKNSSSNSTLKNLGE N86027RM RSDYKLYNKNSSSNSTLKNLGE N297NP RSDYKLYNKNSSSNSTLKNLGE N266NP RSDYKLYNKNSSSNSTLKNLGE N1885MEE RSDYKLYNKNSSSNSTLKNLGE NTHI-546 RSDYKLYNKNSSSNSTLKNLGE NTHI-604 RSDYKL NKNSSSNSTLKNLGE NTHI-492 RSDYK YNKNSS-NSTLKNLGE NTHI-502 RSDYKLYDKNSSSN-TLKKLGE NTHI-506 PSDYKLYN NSS-NSTLKNLGE N1236MEE RSDYKLYNKNSS TLKDLGE NTHI-500 RSDYKLYNKNSS TLKDLGE NTHI-183 RSDYKLYNKNSS TLKDLGE N165NP RSDYKLYNKNSSN-TLKDLGE NTHI-495 RSDYKLYNKNSSD-ALKKLGE TABLE 4 CUMULATIVE PEPTIDE SOLUTIONS OF GROUP 3 N1729ME? RSDYKFYDNKRID NTriI-504 RSDYKFYDNKRID NTHI-544 PSOYKFYDNKRID NTHI-60C RSDYKFYDNKRID N1728MES RSDYKFYDNKRID N10548LX RSDYKFYDNKRID N10547RM RSDYKFYDNKRID N105678R RSDYKFYDNKRID TABLE 5 ACCUMULATED PEPTIDE SEQUENCES OF GROUPS 1, 2a. 2b and 3 N112S RSDYKFYEDANGTRDHKKG N1715MEE RSDYKLYNKNSSSNSTLKNLGE NTHI-183 RSDYKLYNKNSS TLKDLGE N1 29MEE RSDYKFYDN KRID TABLE 6 ACCUMULATED SEQUENCES OF GENE OF GROUP 1 NI128 CGTTCTGATTA AAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NI38OMEE CGTTCTGATTA AAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N1885R CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N1562 EE CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N1563MEE CGTTCTGATTATAAATTT ATGAACATGCAAACGGTAC CGTGACCACAAGAAAGGT NI 8 ONP CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N217NP CGTTCTGATT5TAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N284NP CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NI 666MEE CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NI23 OMEE CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-501 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-507 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-565 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-603 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-608 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N287NP CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N86028L CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT N86028NP CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCACAAGAAAGGT NI52NP CGTTCTGATTATAAATTTTATGAAGATGCAGACGGTACTCGTGACCACAAGAAAGGT NI234MEE CGTTCTGATTATAAATTTTATGATGATGCAAACGGTACTCGTGACCACAAGAAAGGT 182NP CGTTCTGATTATAAATTTTATGATGATGCAAACGGTACTCGTGACCACAAGAAAGGT N9010ORM CGTTCTGATTATAAATTTTATGAAGATGAAAACGGTACTCGTGACCACAAGAAAGGT N9010O CGTTCTGATTATAAATTTTATGAAGA GAAAACGGTACTCGTGACCACAAGA-AAGGT NIO567RM CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT NIO6OMEE CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT NI72NP CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT NI199MEE CGTTCTGATTATAAATTTTATGAAGCTGCAAATGGTACTCGTGACCACAAGAAAGGT NIO568LM CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT N90121RM CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT N86027NP CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI- 86 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAGGT N1712MEE CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-5O3 CGTTCTGATTATAAATTTTATGAAGCTGCAAACGGTACTCGTGACCACAAGAAAGGT NTHI-476 CGTTCTGATTATAAATTTTATGAAGAAGCAAACGGTACTCGTGACCACAAGAAAGGT NI66NP CGTTCTGATTATAAATTTTATAATGATGCAAACGGTACTCGTGACCACAAGAAAAGT N1182MEE CGTTCTGATTATAAATTTTATAATGATGCAAACGGTACTCGTGACCACAAGA-AAAGT NI848NP CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAAGT NI37IMEE CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAAGT NTHI-498 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAAGT NTH1-606 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAAGT NI848L CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGAAAAGT NTHI-567 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGACCGCAAGACAGGT THI-484 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTAAGCACA-AGGAAGGT NIo559R CGTTCTGATTATAAACTTTATGAAGTTGCAAACGGTACTCGTGACCA.CAAGAAAAGT NTHI-6oi CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGCAAAGT NTHI-481 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGCAAAGT NTHI- 82 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGCAAAGT NI37O EE CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGACCACAAGCAAAGT N226NP CGTTCTGATTATAAATTTTATGAAGAAGCAAACGGTACTCGTGACCACAAGAGAAGT NTHI-480 CGTTCTGATTATAAATTTTATGAAGATGCAAACGGTACTCGTGAGCGCAAGAGAGbT NI657MEE CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGAGCGCAAGAAAGGT N267NP CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGAGCGCAAGAAAGGT NTHI-4 0 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGAGCGCAAGAAAGGT NTHI- 9 CGTTCTGATTATAAATTTTATGAAGTTGCAAACGGTACTCGTGAGCGCAAGAAAGGT N214NP CGTTCTGATTATAAATTTTATGAAGTTCCAAACGGTACTCGTGACCACAAGCAAAGT N25ONP CGTTCTGATTATAAACGTTATGAAGAAGCAAACGGTACTCGTAACCACGACAAAGGT NI521 CGTTCTGATTATAAACGTTATGAAGAAGCAAACGGTACTCGTAACCACGACAAAGGT NTHI-6O5 CGTTCTGATTATAAACGTTATGAAGAAGCAAACGGTACTCGTAACCACGACAAAGGT NTHI-499 CGTTCTGATTATGAATTTTATGAAGCTCCAAACAGTACTCGTGACCACAAGAAAGGT TABLE 7 ACCUMULATED GENE SEQUENCES OF GROUP 2 NI 715MEE CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTACTCTTAAAAACCTAGGCGAA NI 714MEE CGTTCTGACTATAAATTGTACA.c \ TAAAAATAGTAGTAGTAATAGTA.CTCTTAAAAACCTAGGCGAA N86 OR 27RM CGTTCTGA.CTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTACTCTTAAAAACCTAGGCGAA N297NP CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTACTC TAAAAACCTAGGCGAA N266NP CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTA.CTCTTAAAAACCTAGGCGAA NI 885MEE CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTACTCTTAAAAACCTAGGCGAA NTE1-546 CGTTCTGACTATAAATTGTACAAT? AAAATAGTAGTAGTAATAGTACTCTTAAAAACCTAGGCGAA NTII1-604 CGTTCTGACTATAAATTGTACAATAAAAAT? GTAGTAGTAATAGTACTCTTAAAAACCtk.GGCGAA NTH1-4 2 CGTTCTGACTATAAATTGTACAATAAAAATAGTAG AATAGTACTCTTAAAAACCTAGGCGAA NTH1-50? CGTTCTGACTATAAATTGTACGATAAAAA? GTAGTAGTAA.T ACTCTTAAAAAACTAGGCGA? NTH1-506 CGTTCTGACTATAAATTGTACAATA? AAA AGTAGT A-ATAGTACTCTTAAAAACCTAGGCGAA NX236 EE CGTTCTGACTATAAATTGTACAATAAAAATAGTAGT ACTCTTAAAGACCTAGGCGAA NTHI-500 CGTTCTGACTATAAATTGTACAATAAAAATAGTAGT ACTCTTAAAGACCTAGGCGAA NTHI-183 CGTTCTGACTATAAATTGTACAATAAAAATAG AGT ACTCTTAAAGACCTAGGCGAA NI65NP CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAAT ACTCTTAAAGACCTAGGCGAA NTHI-495 CGTTCTGACTATAAATTATACAATAAAAATAGTAGTGAT GCTCTTAAAAAACTAGGCGAA TABLE 8 ACCUMULATED SEQUENCES OF GROUP 3 GENE NI729MEE CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT NTHI- NTHI-544 504 CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT CGTTCTGACTATAAATTCTACGA AATAAACGCATCGAT NTHI- 600 CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT NI728MEE CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT N105 8LM CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT NI0547RM CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT NI05678R CGTTCTGACTATAAATTCTACGATAATAAACGCATCGAT TABLE 9 ACCUMULATED GENE SEQUENCES OF GROUPS 1, 2a. 2b and 3 N1128 CGTTCTGATTATAAATTTTATGAAGAT3CAAAC3GTACTCGTGACCACAAGAAAGGT NI715MEE CGTTCTGACTATAAATTGTACAATAAAAATAGTAGTAGTAATAGTACTCTTAAAAACCTAGGCGAA NTH1-18 CGTTCTGACTATAAATTGTACAATAAAAATAGTAGT ACTCTTAAAGACCTAGGCGAA N1729MEE CGTTCTGACTATAAATTCTACGATAAT AAACGCATCGAT The study shows that peptides LB1 (f) of the P5-like fimbrin protein of all 83 ntHi isolates tested can be classified into three groups; and that both the American and European isolates of ntHi remain within that classification.

EXAMPLE 2 EXPRESSION OF FUSION POLYPEPTIDES OF PEPTIDE LPD-LB1 (f) IN E. coli Source material. 1) The expression vector pMG1 The expression vector pMG1 is a derivative of pBR322, in which control elements derived from bacteriophage lambda were introduced for transcription and translation of inserted foreign genes (Young and co-authors (1983) PNAS USA 80, 6105 -6109). In addition, the ampicillin resistance gene was changed to kanamycin resistance gene. The vector contains the lambda PL promoter, the operator O and two sites of use (Nut and NutR) to highlight the polarity effects of transcription. The vectors containing the PL promoter are introduced into a lysogenic host of E. coli to stabilize the plasmid DNA. The lysogenic host strains contain lambda phage DNA with a defect of replication, integrated into the genome. The chromosomal lambda phage DNA directs the synthesis of the repressor protein cl that binds to the repressor 0L of the vector and prevents the binding of RNA polymerase to the promoter P1_ and, thus, the transcription of the inserted gene. The gene of expression strain AR58 contains a temperature-sensitive mutant so that PL-directed transcription can be regulated by the temperature shift, i.e., an increase in culture temperature inactivates the repressor and starts the synthesis of the foreign protein. This expression system allows the controlled synthesis of foreign proteins, especially those that can be toxic to the cell. 2) .- The expression vector pMGMCS The nucleotide sequence was replaced between the restriction sites BamHl and Xbal in pMG1, by a DNA fragment of multiple cloning site (MCS) to generate the expression vector pMGMCS (figure 1) . A poly-His sequence has been added at the 3 'end of the MCS sequence to allow expression of a molten protein product to a 6-histidine tag. The sequence coding for the first three amino acids of NS1 (Met-Asp-Pro) is present in the vector, before the BamH1 restriction site. 3) .- Construction of vector pRIT14588 The cloning strategy for the generation of the expression vector pRIT14588 from the pMGMCS vector is indicated in figure 2. The lipoprotein D gene was amplified by PCR from the vector pHIC348 (Janson and co-authors , (1991) Jnfect, Immun., 59, 119-125) with PCR sensitizers containing BamHl and Ncol restriction sites at the 5 'and 3' ends, respectively. Then the BamHI / Ncol fragment was introduced into pMGMCS between BamHI and Ncol. The lipoprotein D gene product contains its natural signal sequence, except for the first three amino acids that have been replaced by Met-Asp-Pro from NS1. PRIT14588 was used to introduce LB 1 (f) peptides at the 3 'end of the lipoprotein D gene. The LB1 (f) peptides used were the following: Group 1: ntHi-1128 (SEQ ID NO: 5); group 2: ntHI-1715 MEE (SEQ ID NO: 2); group 3: ntHi-1729 MEE (SEQ ID NO: 3). 4) .- The AR58 strain of E. coli. The histagenic strain of E. coli AR58 used for the production of the carrier protein, protein D, is a derivative of the NIH strain N99 of E. coli K12, common and current (F "su" galK2, lacZ "thr") . It contains a defective lysogenic lambda phage (galE "TN10, lambda Kil" cl857 DH1). The KiL phenotype prevents the arrest of the macromolecular synthesis of the host. The mutation of cl857 confers a temperature sensitive lesion to the cl repressor. The omission of DH1 removes the right operon from lambda phage and the bio, uvr3 and ch1A sites of the host. Strain AR58 (Mott and co-authors (1985) PNAS USA 82, 88-92) was generated by transduction of N99 with a P1 phage material previously developed in a SAS500 derivative (galE :: TN10, lambda KM "cl857 DH1). introduction of the defective lysogen in N99 was selected with tetracycline (a TN10 transposon encoding tetracycline resistance is present in the adjacent galE gene) EXAMPLE 2a PRODUCTION OF A LIPOPROTEIN FUSION D-LB1 (f) GROUP 1 The purpose of this construct was to clone the peptide of 19 residues of LB 1 (f) 3 'with respect to the Ncol site of the multiple cloning site of pRIT14588. Immediately 3 'to the Ncol site two glycine residues were introduced to place the peptide gene LB 1 (f) in frame with the LPD gene. After the two Gly residues, the DNA encoding the eight natural residues, N-terminal with respect to the peptide LB 1 (f) (of the fimbpna protein similar to P5), was introduced, followed by the DNA sequence LB 1 (f) , followed by DNA coding for the five C-terminal natural residues with respect to the LB1 peptide (f) The plasmid (named LPD-LB1-A) is shown in Figure 3 and was done as follows: pRIT 14588 was divided with Ncol and Spel and the large linear fragment was dephosphorylated. The peptide gene LB1 (f) was amplified from the p5-like fimbrin gene of ntH-1128, with the following sensitizers 'Sensitizer LB-Baka-01 (containing 5' an Ncol site) 5'-CTA-GCC -ATG-GAT-GGT-GGC-AAA-GCA-GGT-G-3 -í-H-íli-Hl-i-i-i- ^ Sensitizer LB-Baka-05 (containing 3 'a Spel site) '-CAC-TAG-TAC-GTG-CGT-TGT-GAC-GAC-3 'The produced DNA was divided by amplification by RCP, with Ncol and Spel. The DNA fragment of LB1 (f) was purified and ligated into the Ncol and Spel sites of divided pRIT14588. The ligation mixture was transformed into E. coli AR58 and the transformation product was splashed on the liquid medium (BP) LBT-Kanamycin (50 μg / ml). The plates were incubated at 30 ° C overnight. The transformants were checked by PCR and positive candidates were developed in liquid culture at 30 ° C. In order to initiate the expression of the chimeric LPD-LB'1 (f) polypeptide, the culture was subjected to a temperature change of 30 ° C to 39 ° C for 4 hours. The expression on an acrylamide gel was monitored 12. 5% (seen with Coomassie stain and / or Western stain). The molecular size of the chimeric polypeptide was approximately 44 kDa.

EXAMPLE 2b) PRODUCTION OF A GROUP FUSION LPD-LB1 (f) GROUP 2 + LBKf) GROUP 1 The plasmid (named LPD-LB1-II) is shown in Figure 4, and was done as follows: The LPD- plasmid was divided LB1-A with Ncol and the linear DNA was dephosphorylated. The LB1 (f) group 2 peptide gene was amplified, from the p5-like fimbrin gene ntHi-1715MEE, with the following sensitizers 'NT1715-11NCO Sensitizer (containing 5' an Ncol site) 5'-CAT-GCC- ATG-GAT-GGC-GGC-GGT-AAA-GCA-GGT-GTT-GCT-3 'Sensitizer NT1715-12NCO (containing 3' Ncol site) 5'-CAT-GCC-ATG-GCA-CGT-GCT- CTG-TGA-TG-3 '. The DNA produced by PCR amplification was divided with Ncol. The DNA fragment of LB1 (f) was purified and ligated into the open Ncol site of plasmid LPD-L 1-A (5 'to the gene for peptide LB 1 (f) of group 1). The ligation mixture was transformed to the AR58 of E. coli and the transformation product was spread on solid medium (BP) LBT-Kanamycillin (50 μg / ml). The plates were incubated at 30 ° C overnight. The transformants were co-tested by PCR and positive candidates were developed in liquid culture at 30 ° C. In order to initiate the expression of the chimeric polypeptide LPD.LB1 (f) 2 -, the culture was subjected to a change in temperature from 30 ° C to 39 ° C for four hours. The expression was monitored in a 12.5% acrylamide gel (seen with dyeing Coomassie and / or Western blot). The molecular size of the chimeric polypeptide was approximately 50 kDa.

EXAMPLE 2c PRODUCTION OF A LIPOPROTEIN FUSION D-LB1 (f) GROUP 2 + LBKf) GROUP 1 + LB1 (f) GROUP 3 In figure 5 the plasmid (named LPD-LB1-III) is shown and the following was made way: Plasmid LPD-LB1-II was split with Spel and the linear DNA was dephosphoresed. The 3-LB1 (f) peptide gene of type 3 was made from ntHi-1929MEE by hybridization of the following sensitizers: Sensitizer NT1729-18 SPE (5 ', containing a Spel cleavage site at the 5' end). 5-CTA-GTC-GTT-CTG-ACT-ATA-AAT-TCT-ACG-ATA-ATA-AAC-GCA-TCG-ATA-GTA-3 'Sensitizer NT1729-19 SPE (3' - containing a Spel site at the 3 'end) 5'-CTA-GTA-CTA-TCG-ATG-CGT-TTA-TCG-TAG-AAT-TTA-TAG-GCA-GAA-CGA-3' Hybridized DNA contained the gene for the peptide of group 3 LB1 (f) and a Spel divided at each end. Se1 ligated the LB1 (f) DNA fragment into the open Spel site of the plasmid LPD-LB1-II divided (3 'to the gene for the group 1 peptide LB1 (f)). The ligation mixture was transformed into AR58 from E. coli and the transformation product was spread on solid medium (BP) LGT-Kanamycin (50 μg / ml). The plates were incubated at 30 ° C overnight. The transformants were checked by PCR and positive candidates were developed in liquid culture at 30 ° C. In order to initiate the expression of the LPD-LB1 chimeric polypeptide (f ^ 2 1 3), the culture was subjected to a temperature change of 30 ° C to 39 ° C for 4 hours, and the expression was monitored on a gel of acpiamide. 12. 5% (seen with Coomassie staining and / or with Western stain). The molecular size of the chimeric polypeptide was approximately 53 kDa.

EXAMPLE 2d) CHARACTERIZATION OF THE EXPRESSION OF CHEMICAL POLYPEPTIDES The expression of the above chimeric polypeptides was monitored in a 12.5% acrylamide gel which was observed as: a) a Coomassie stained gel (Figure 6) b) a Western blot: 1 ) using rabbit anti-LB1 antibodies (Figure 7) 2) using a monoclonal anti-LPD antibody (Figure 8) 3) using an antibody against the six histidine purification tag (Figure 9). As can be seen, each chimeric polypeptide can be expressed efficiently in E. coli.

EXAMPLE 3 PURIFICATION OF CHEMICAL POLYPEPTIDES Purification of LPE-LB1 (f) 2 1 3 (expressed using the construction shown in Figure 5) was accomplished as follows: E. coli was washed and resuspended in regulator phosphate (50 mM, pH 7.0). The cells were lysed by shaking gently overnight at 4 ° C in the presence of 3% Eiipigen. The solution was then centrifuged for 30 minutes at 8,000 r p.m. in a Beckman JA10 rotor. The supernatant was diluted four times in 50 mM phosphate buffer, 500 mM NaCl, pH 7.0. The first purification step was obtained on a Qiagen NTA N i ++ column, due to the presence of the six histidine tag at the C-terminus of the polypeptide. The column was equilibrated with 10 mM sodium phosphate buffer, 500 mM NaCl, 0.5% Empigen, pH 7.5, and the polypeptide was eluted from the column with an imidazole gradient (0-100 mM) in 20 mM. of sodium phosphate regulator, 0.5% of Empigen, pH 7.0. After the elution, the fractions on SPS-PAGE gels were run. The next step in the purification was in a column Bio-Rad Macro-Prep 50S. The polypeptide was bound to the column equilibrated in 20 mM phosphate buffer, 0.5% Empigen, pH 7.0, and eluted from the column using a 0 to 500 mM gradient of NaCl in the same buffer. The elution was followed by running fractions on SDS-PAGE gels. The last step (polishing) of the process was carried out using a size exclusion column Sephacryl S200 HR. First the polypeptide solution from the previous step was concentrated with a Omega 10 kDa Filter concentrator device. The resulting solution was loaded and operated on the column equilibrated with PBS buffer with 0.5% of Empigen. Elution of the polypeptide was followed by operation of the fractions on SDS-PAGE gels. The collected fractions were filtered through a 0.22 μm filter. The resulting protein is run as a pure band on SDS-PAGE gel stained with Coomassie, and the equivalent Western blot, using an anti-LB1 antibody. The tests showed that the protein remained intact even after seven days at 37 ° C. About 200 mg of the polypeptide per liter of the cell culture can be purified by this method.

EXAMPLE 4 PRECLINICAL EXPERIMENTATION ON THE EFFECTIVENESS OF THE VACCINE OF CHEMICAL PEPTIDES Example 4a): Generation of the antisera Antisera was generated against four types of antigen: LPD, PD, LPD-LB1 (f) 2, - ?, 3 (recombinantly made using the plasmid LPD-LB1 -lll); LB1 (a LB1 (f) peptide fused to a promiscuous T-cell epitope, derived from the mumps virus fusion protein, the sequence of the peptide being: RSDYKFYEDANGTRDHKKGPSLKLLSLIKGVIVHRLEGVE). Four groups comprising 5 chinchillas were immunized, each group with one of the immunogens identified above. the dose was 10 μg of antigen / 200 μl of AIPO4 / 20 μg of MPL (3-O-deacylated monophosphoryl lipid A) for the first three antigens, and 10 μg of antigen delivered in complete or incomplete Freund's adjuvant (CFA or IFA) for LB1. A total of three doses were injected at n month intervals.

Fifteen days after the final immunization, the animals were bled by means of cardiac puncture and torectomy to collect the serum. The group serum was collected and stored at -70 ° C. Titers obtained were 10-50 K for anti-PD serum, 50K for anti-LPD, 50-100K for anti-LB1 and 50-100K for LPD-LB 1 (f> 2.1.3- In addition to peptide LB1, anti-LB1 recognized LPD-LB1 (f) 2,?, 3 in a Western blot.Anti-LPD and anti-PD also recognized LPD-LB1 (f) 2 13. The immuno-gold labeling experiments (using protein A conjugated to gold) showed that polyclonal antisera anti-LB1 and anti-LPD-LB1 (f) 21.3 both recognized the epitopes accessible on the surface, in ntHi 86-028NP cells, similar to those recognized by a monoclonal antibody against p5-like fimbrin protein Additionally, Figure 12 shows a Western blot indicating that the anti-LPD-LB1 (f) 2] 3 3 serum recognizes the P5-like fimbrin protein from the three ntHi strains that represent the three major groups LB1 (f) The recognition of these strains by anti-LPD, LB1 (f) 2, 1.3 is much stronger than by anti-LB1.

EXAMPLE 4b) PASSIVE TRANSFER AND CHALLENGE This study is aimed at conducting an in vivo challenge study of passively immunized chinchillas to determine the relative efficiency among the four immunogen formulations (or simulations) to facilitate the release of ntHi from the nasopharynx. Five groups of eleven chinchillas each (Chinchilla lanigera) free of middle ear disease were inoculated intranasally, on day -7 with 6 x 106 adenovirus TCID50 type 1 On day -1 each group of chinchilla was passively immunized with a 1: 5 dilution of one of the four serum samples described in example 4a, by cardiac puncture. The fifth group (the simulation group) received sterile, pyrogen-free saline solution by cardiac puncture. Approximately 5 ml of serum / kg of animal was administered. On day 0 the groups were challenge intranasally with ntHi: approximately 108 colony forming units (ufe) of ntHÍ # 86-028NP (group 1) per animal. The statistical evaluation of the passive transfer study was carried out before undoing the blind aspect of the study. This sequential inoculation with two pathogens closely mimics both the natural path of acquisition of these agents and their synergistic interaction in the human host.

The severity of the disease was marked by otoscopic observation. This is classified on a 0T4 scale. Signs of inflammation of the tympanic membrane (TM) were observed to obtain a mark: the presence of effusion, small dilatation of vessels, air-fluid interface, opacity, etc. An analysis of repeated measurements of the variation was used to compare the pattern of responses during the time (days) and the ear (left or right) for the five groups. Due to the large number of repeated observations in each animal, the analysis was divided into 5 sections as follows: days 1-7; days 8-14, days 19-21, days 22-28 and days 29-33. There was little variation in the responses on days -7 to 0, 34 and 35 and, therefore, no such analysis was performed at that time. When possible (when there was variability that was not zero in the mean response), tests were carried out to compare the mean responses between the groups at three time points. The Tukey HSD test was used for all post-hoc multiple comparisons. The meaning was determined using an alpha level of 0.05. The results are shown in Figure 10. Inflammation increases over time for all groups, significantly, during the period from day 1 to day 7. During days 29-33 the inflammation decreased with time in a significant way for all groups. As can be seen from the data, the serum containing agents against recombinant LPD-LB1 (f) 2? 1,3 helped reduce the MT inflammation throughout the experiment. An effective vaccine measurer should maintain the MT inflammation at or below 1 5 throughout the time of the study period. The antiserum LPD-LB1 (f) 2 1 3 only allowed the average inflammation mark to rise above 1.5 for two days, as well as inducing a consistent downward trend later. In addition to otoscopy, tympanometry (EarScan, South, Daytona, FL, E.U.A.), which measures changes in middle ear pressure, was also employed. These two measurements can be used together to give a reliable indication of effusion in the middle ear. Tympanometry results indicated an abnormal ear if: a type B tympanogram was obtained, or the middle ear pressure was less than -100 daPa. Figure 11 shows the study of this analysis. Clearly recombinant LPD-LB1 (f) 2? 1? 3 performed well in this study when considering the measures to be taken to prevent both MT inflammation and the development of effusion. LPD-LB1 (f) 2 1 3 is generally classified second, only below the positive control, in peptide LB1. However, peptide LB1 was aided by CFA (a very strong adjuvant) and, therefore, can not be compared directly with the result of LPD-LB1 (f) 2 1 3. Table 10 presents a statistical evaluation of the data presented in figure 11. The evaluation compared the reduction in the percentage of effusion in each immunized group with that observed in animals immunized with simulation during the peak incidence of the disease [the four days of observation in which less 50% of the simulation ears contained an effusion (had otitis media)] The positive control (anti-LB 1 (CFA) was significant at p <0.001 in the four days (days 11-14). -LB1 (f) 2 ... inhibited the development of otitis media at a p-value less than or equal to 0001 on days 11, 12, 13 and 14 also Anti-PD was significant on days 13 and 14 only, while anti-PD -LPD was able to prevent the development of otitis media, with respect to the ani simulation ills, day 14 only (p-value close to 0.02). Therefore, the recombinant polypeptide LPD-LB1 (f) 2 -, 3 significantly inhibited the development of otitis media in chinchillas, which were transferred passively with this pool of sera.

TABLE 10 COMPARISON OF 5 EYES CONTAINING EFUSION IN THE GROUPS LB1. PD, LPD-LB1 (f) 213 AND LPD, WITH% OF EARS THAT CONTAIN EFUSION IN THE SIMULATION GROUP. DAYS 11 TO 14 EXAMPLE 4c! DATA OF INHIBITION OF ADHERENCE An established, single-cell adhesion analysis was performed using human oropharyngeal cells. The average percentage of adhesion inhibition (± sem) of the ntlHi strains for these cells by the immune chinchilla sera produced in example 4a. The results using antisera against LPD-LB1 (f) 2! 3 and LPD can be observed in table 11. It is seen that the antisera against LPD-LB1 (f) 2, 3 are effective for i n h i b i r the adherence of the strains of group 1 and group 2 of ntHi. They are also more effective against all strains than anti-LPD serum TABLE 11 THE AVERAGE PERCENTAGE OF ADHERENCE INHIBITION (± SEM) OF NtHi CEPAS TO HUMAN OROPHARINOGEN CELLS. FOR IMMUNE CHINCHILLS BAGS EXAMPLE 4d) PASSIVE TRANSFER AND CHANGE WITH HETEROGENEOUS STRAINS A study similar to that described in example 4b above was carried out, using ntHi strains of different classifications of the LB 1 (f) group, to challenge the adenovirus coinfection model in chinchillas. total of 132 juvenile chinchillas (approximately 300 g) (Chinchilla lanígera) without evidence of infection in the middle ear, by otoscopy or tympanometry, during two challenge studies using antisera anti-LB1 and anti-LPD-LB1 (f) 2 ? 1? 3 The average weight of the chinchillas for the two challenge studies detailed below were: 296 ± 38 g, for 298 ± 42 g, respectively. The animals were allowed to rest for ten days after arrival, and were nominally bled by cardiac puncture for collection of pre-immunological serum, which was stored at -70 ° C until used. The animals were allowed to rest a minimum of 7 days after the collection of the pre-immunological serum, until they receive the adenovirus. The ntHi used in these studies consists of limited-passage clinical isolates, cultured from the middle or nasopharyngeal ears of children who suffered tympanostomy and tube insertion for chronic otitis media, with effusion at the Children's Hospital of Columbus (86-028NP (group 1), 1885 MEE (group 2) and 1728 MEE (group 3)). All isolates of skim milk, plus 20% glycerol (volume / volume) were kept frozen until they were scratched on chocolate agar and incubated at 37 ° C for 18 hours in a humidified atmosphere containing 5% CO2 Adenovirus serotype 1 of a pediatric patient was also recovered at the Children's Hospital of Columbus. 66 juvenile chinchillas were used to establish six groups of eleven chinchillas each. Natural chinchilla sera from these animals were collected and individually selected by Western blotting for the presence of any previously existing antibody titer, before enrolling them in the study. Experiments were carried out as for Example 4b) above. Two groups received the joint antiserum LB1, two groups received the joint antiserum of LPD-LB1 (f) 2 3, and two groups received sterile, pyrogen-free saline. The observers did not know if each group consisted of animals that received antiserum or nothing . The chinchillas were challenged intranasally by passive inhalation of approximately 108 CFU of: ntHi 86-028NP or 1885 MEE per animal (study A) or ntHi 86-028NP or 1728 iIEE per animal (study B). Each of these three strains was selected to represent a heterogeneous ntHi group of different sequence with respect to peptide LB 1 (f): group 1: strain NTHi 86-028NP; group 2: strain 1885MEE of NTHi; and group 3, strain 1728 MEE of NTHi. As in example 4b) the animals were blindly evaluated by otoscopy and tympanometry daily or for two days from the time of inoculation with adenovirus until 35 days after the NTHi challenge. The signs of tympanic membrane inflammation were classified on an ordinal scale of? 0 to 4 + and tympanometry charts were used to monitor changes in both the middle ear pressure and the tympanic amplitude and the flexibility of the tympanic membrane. Tympanometry results indicated an abnormal ear if: a type B tympanogram was obtained; the flexibility was < _ 0.5 mi or > 1.2 ml; the middle ear pressure was less than -100 daPa; or if the amplitude Tympanic was greater than 150 daPa The Tukey HSD test was used to compare the average inflammation annotations of the timpanid membrane between groups challenged with the same strain of NTHi from day 1 to 35 after the bacterial challenge. Each group of immunized animals had significantly lower mean otoscopy scores (p <0.05) than the simulated group challenged with the same NTHi strain for a minimum of seven days (maximum, 22 days). The results of the otoscopic classification are shown in Figure 13 (Study A) and Figure 14 (Study B). The days in which the average otoscopic annotations were significantly lower for LPD-LB1 (f) 2 1 3 than in the simulated experiments were: days 13-35 (study A, 86-028NP); days 1-8, 12-21 (study A, 1885 MEE) on days 8-14, 23 (study B 86-028NP), days 8-14 (study B, 1728 MEE). An analysis of the percentage of normal ears for studies A and B is shown in Figures 15 and 16, respectively. The passive transfer capacity of specific antisera to protect against the development of otitis media was determined by a Z-test. In both studies, animals receiving anti-LB1 serum did not show signs of developing otitis media with effusion after the NTH challenge 86 -028NP .. The days during which the supply of anti-LPD-LB1 (f) 2,?, 3 serum significantly prevented the development of otitis media compared to simulation animals (measured on days when more than 50% of the simulation animals had effusions) were: days 13-21 (study A, 86-028NP; days 13-18 (study A, 1885 MEE), days 13-14 (study B, 86-028NP), days 9-12 (study B, 1728 MEE) In summary, the challenge of the chinchillas with any of the three ntHi isolates used here resulted in the initial colonization of the nasopharynx. tympanometry indicated that s groups that received antiserum directed against LPD-LB1 (f) 2 -, 3 had significantly lower annotations in otoscopy and a significant reduction in the incidence of otitis mea, compared with the simulation groups, challenged with the same strain of NTHi during many days of observation. Thus, LPD-LB1 (f) 2? 1,3 provided significant protection against the development of otitis media induced by heterologous strains of NTHi in chinchillas compromised with adenovirus. LB1 also gave protection; however, this may have been due in part to the strong adjuvant (CFA) used in conjunction with it. Although certain embodiments of the invention have been shown and described, various adaptations and modifications may be made without departing from the scope of the invention, as described in the appended claims. For example, peptides or polypeptides having substantially the same amino acid sequence as described herein are within the scope of the invention.

SEQ ID NO 1 RSDYKFYEAANGTRDHKKG [from strain ntHi-10567RM (type group 1)] SEQ ID NO 2 RSDYKLYNKNSSSNSTLKNLGE [from strain ntHi-1715MEE (type group 2a)] SEQ ID NO: 3 RSDYKFYDNKRID [from strain ntHi-1729MEE (type group 3)] SEQ ID NO '4 RSDYKLYNKNSSTLKDLGE [of strain ntH? -183NO (type group 2b)] SEQ ID NO: 5 RSDYKFYEDANGTRDHKKG [of strain nt H i-1128 (type group 1)] SEQ ID NO: 6 RSDYKFYEAPNSTRDXKKG [of protein P5 from residue ntHi 119-137 (type group 1)]

Claims (2)

1. CLAIMS 1 - A peptide characterized in that it comprises one or more amino acid sequences, selected from the group consisting of 'SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO. 3 and SEQ ID NO: 4 or any antigenically related variants of said sequences, which have an identity of at least 75% and are capable of immunologically imitating the corresponding antigenic determinant site of the fimbpna protein similar to P5 of non-typeable Haemophilus influenzae, provided that the antigenically related variants do not include the peptides provided in SEQ ID NO: 5 or SEQ ID NO: 6.
2. 2. The peptide according to claim 1, further characterized in that it comprises the amino acid sequence provided in SEQ ID. NO: 1. The peptide according to claim 1, further characterized in that it comprises the amino acid sequence provided in SEQ ID NO: 2. 4. The peptide according to claim 1, further characterized by comprising the sequence of amino acids provided in SEQ ID NO: 3. 5. The peptide according to claim 1, further characterized in that it comprises the amino acid sequence provided in SEQ ID NO: 4. 6. A chimeric polypeptide, characterized in that it comprises one or more peptides of claims 1 to 5, linked covalently to a carrier polypeptide, which conjugates at least one T cell epitope. The chimeric polypeptide according to claim 6, further characterized in that it also comprises a peptide sequence of a purification tag. 8. The polypeptide according to claim 7, further characterized in that the purification tag peptide sequence is a histidine tag sequence. 9. The chimeric polypeptide according to claim 6, further characterized in that the carrier polypeptide is lipoprotein D. 10. The chimeric polypeptide according to claim 6, further characterized in that the amino acid sequences of the peptides used are selected. of the group consisting of SEQ ID NO: 1, 2 and 3. 11. A chimeric polypeptide characterized in that it comprises three subunits LB 1 (f) and lipoprotein D; wherein the amino acid sequences of the subunits LB 1 (f) are provided in SEQ ID NO: 2, 3 and 5. 12. The chimeric polypeptide according to claim 11, further characterized in that it also comprises a purification tag sequence. of histidine. 13 -. 13 - Chimeric polypeptide in accordance! with claim 11, further characterized because the order! of the N-terminal peptide components of the polypeptide is: lipoprotein D, subunit LB1 (f) (SEQ ID NO: 2), subunit LB1 (f) SEQ ID NO: 5) and subunit LB1 (f) (SEQ ID NO: 3). 14. The chimeric polypeptide according to claim 13, further characterized in that the amino acid sequence of the polypeptide is provided in figure 5. 15. A vaccine composition characterized in that it comprises an immunogenic amount of at least one peptide or polypeptide of the rei indications 1 to 14 in a pharmaceutically acceptable excipient, and an optional adjuvant. 16. The use of an immunogenic amount of at least one peptide or polypeptide of claims 1 to 14, in a pharmaceutically acceptable excipient and an optional adjuvant, for preventing or treating a disease by Haemophilus influenzae. 17. The use according to claim 16, wherein the Haemophilus influenzae disease is otitis media, sinusitis, conjunctivitis or lower respiratory tract infection. 18. A method for inducing an immune response in a mammal susceptible to infection by Haemophilus influenzae, characterized in that it comprises administering to the mammal an effective amount of the vaccine according to claim 15. 19.- A method to prevent infection by Haemophilus influenzae, characterized in that it comprises administering to a mammal an effective amount of a vaccine according to claim 15. 20. A DNA or RNA molecule, characterized in that it encodes one of the peptides or polypeptides LB 1 (f) provided in claims 1 to 14 21. The DNA or RNA molecule according to claim 20, further characterized in that the DNA sequence of the LB1 polypeptide (f ) is given in Figure 5. 22. The DNA or RNA molecule according to claim 20 or 21, further characterized in that it is contained within an expression vector, where the expression vector is capable of producing the peptide or polypeptide LB1 (f) when, or is present in, a compatible host cell. 23. A host cell characterized in that it comprises the expression vector of claim 22.- A process for producing a peptide or polypeptide LB1 (f), characterized in that it comprises culturing the host cell of claim 23, under conditions sufficient for the production of the polypeptide, and recovering the peptide or polypeptide LB1 (f). 25.- A process to produce a peptide or polypeptide LB1 (f), according to claim 24, further characterized in that the process comprises the steps of lysing the amphitic cells and purifying the soluble extract using an immobilized nickel column passage, a column step of the cation exchange and one step in column by size exclusion 26.- A process for producing a host cell that produces an LB1 (f) peptide or its polypeptide, characterized in that it comprises transforming or transfecting a host cell with the expression vector of claim 22, so that the host cell, under the appropriate culture conditions, expires that a peptide or polypeptide LB1 (f). 27. A purified antibody, characterized in that it is immunospecific for a peptide provided in one of claims 1 to 5. 28.- A purified antibody, characterized in that it is immunospecific for a chimeric polypeptide provided in claims 6 to 14. 29.- A method to detect the presence of Haemophilus influenzae in a sample, characterized in that said sample is contacted with the antibody of claim 27, in the presence of an indicator. 30.- A method for detecting the presence of Haemophilus influenzae in a sample, characterized in that it comprises contacting the sample with a probe or DNA sensitizer, constructed to correspond to the wild-type nucleic acid sequence, which encodes a peptide LB1 (f) of the fimbrine protein similar to P5 of Haemophilus influenzae; characterized in that the probe is selected from the group consisting of gene sequences as provided in Tables 6-8. 31.- A kit for diagnosing infection with Haemophilus influenzae in a mammal, characterized in that it comprises the DNA probes of claim 30 or an LB 1 (f) peptide of claims 1 to 5 or an antibody of claim 27.