Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the invention relates to Haemophilus adhesion and penetration proteins, nucleic acids, and vaccines
• Haemophilus influenzae is a common commensal organism of the human respuatory tiact (Kuklmska and Ki an, 1984, Eur J Clin Microbiol 3 249-252) It is a human-specific oigamsm that normally resides in the human nasopharynx and must colonize this site m order to avoid extinction
• This microbe has a numbei of surface structuies capable of promoting attachment to host cells (Gue ⁇ na et al , 1982, J Infect Dis 146 564, Pichichero et al , 1982, Lancet u 960-962, St Geme et al , 1993, Proc Natl Acad Sci U S A 90 2875-2879)
• H influenzae has acquired the capacity to enter and survive within these cells (Forsgren et al , 1994, Infect Immun 62 673-679, St Geme and Falkow, 1990, Infect Immun 58 4036-4044, St Geme and Falkow, 1991
• the initial step in the pathogenesis of disease due to H influenzae involves colonization of the uppei respiratory mucosa (Murphy et al , 1987, J Infect Dis 5 723-731) Colonization with a paiticulai strain may persist for weeks to months, and most individuals remain asymptomatic throughout this period (Spmosa et al , 1986, 1 Infect Dis 154 100-109) However, m certain circumstances colonization will be followed by contiguous spread within the respiratory tract, resulting in local disease in the middle ear, the sinuses, the conjunctiva, or the lungs Alternatively, on occasion bacteria will penetrate the nasopharyngeal epithelial barrier and enter the bloodstream In vitro observations and animal studies suggest that bacterial surface appendages called pili (or fimbriae) play an important role in H.
• nonpiliated organisms retained a capacity for colonization, though at reduced densities; moreover, among monkeys originally infected with the piliated strain, virtually all organisms recovered from the nasopharynx were nonpiliated. All of these observations are consistent with the finding that nasopharyngeal isolates from children colonized with H. influenzae are frequently nonpiliated (Mason et al, 1985, Infect. Immun. 49:98-103; Brinton et al, 1989, Pediatr. Infect. Dis. J. 8:554-561).
• H. influenzae are capable of entering (invading) cultured human epithelial cells via a pili-independent mechanism (St. Geme and Falkow, 1990, supra; St. Geme and Falkow, 1991, supra). Although//, influenzae is not generally considered an intracellular parasite, a recent report suggests that these in vitro findings may have an in vivo correlate (Forsgren et al, 1994, supra). Forsgren and coworkers examined adenoids from 10 children who had their adenoids removed because of longstanding secretory otitis media or adenoidal hypertrophy. In all 10 cases there were viable intracellular H. influenzae.
• Electron microscopy demonstrated that these organisms were concentrated in the reticular crypt epithelium and in macrophage-like cells in the subepithelial layer of tissue.
• One possibility is that bacterial entry into host cells provides a mechanism for evasion of the local immune response, thereby allowing persistence in the respiratory tract.
• HAP Haemophilus Adherence and Penetration
• An additional object of the invention is to provide monoclonal antibodies for the diagnosis of Haemophilus infection.
• a further object of the invention is to provide methods for producing the HAP proteins, and a vaccine comprising the HAP proteins of the present invention. Methods for the therapeutic and prophylactic treatment of Haemophilus infection are also provided.
• the present invention provides recombinant HAP proteins, and isolated or recombinant nucleic acids which encode the HAP proteins of the present invention. Also provided are expression vectors which comprise DNA encoding a HAP protein operably linked to transcriptional and translational regulatory DNA, and host cells which contain the expression vectors.
• the invention also provides methods for producing HAP proteins which comprises culturing a host cell transformed with an expression vector and causing expression of the nucleic acid encoding the HAP protein to produce a recombinant HAP protein.
• the invention also includes vaccines for Haemophilus influenzae infection comprising an HAP protein for prophylactic or therapeutic use in generating an immune response in a patient.
• Methods of treating or preventing Haemophilus influenzae infection comprise administering a vaccine.
• Figures 1 A and IB depict light micrographs of/ , influenzae strains DB117(pGJB103) and DB117(pN187) incubated with Chang epithelial cells. Bacteria were incubated with an epithelial monolayer for 30 minutes before rinsing and straining with Giemsa stain.
• Figure 1A H. influenzae strain DB117 carrying cloning vector alone (pGJB103);
• Figure IB H. influenzae strain DB117 harboring recombinant plasmid pN187. Bar represents 3.5 ⁇ m.
• Figures 2A, 2B, 2C and 2D depict thin section transmission electron micrographs demonstrating interaction between H. influenzae strains Nl 87 and DB117(pN187) with Chang epithelial cells. Bacteria were incubated with epithelial monolayers for four hours before rinsing and processing for examination by transmission electron microscopy.
• Figure 2A strain Nl 87 associated with the epithelial cell surface and present in an intracellular location
• Figure 2B H. influenzae DBl ll (pN187) in intimate contact with the epithelial cell surface
• Figure 2C strain DB117(pN187) in the process of entering an epithelial cell
• Figure 2D strain DB117(pN187) present in an intracellular location. Bar represents 1 ⁇ m.
• Figure 3 depicts outer membrane protein profiles of various strains. Outer membrane proteins were isolated on the basis of sarcosyl insolubility and resolved on a 10% SDS-polyacrylamide gel. Proteins were visualized by staining with Coomassie blue. Lane 1, //. influenzae strain DB117(pGJB103); lane 2, strain DB117(pN187); lane 3, strain DB117(pJS106); lane 4, E. coli HB101(pGJB103); lane 5, HB101(pN187). Note novel proteins at ⁇ 160 kD and 45 kD marked by asterisks in lanes 2 and 3.
• Figure 4 depicts a restriction map of pN187 and derivatives and locations of mini-Tn/0 kan insertions.
• pN187 is a derivative of pGJB103 that contains an 8.5-kb Sau3Al fragment of chromosomal DNA from H. influenzae strain Nl 87.
• Vector sequences are represented by hatched boxes. Letters above top horizontal line indicate restriction enzyme sites: Bg, Bglll; C, CM; E, EcoBl; P, Vstl.
• Numbers and lollipops above top horizontal line show positions of mini-Tn/ 0 kan insertions; open lollipops represent insertions that have no effect on adherence and invasion, while closed lollipops indicate insertions that eliminate the capacity of pN187 to promote association with epithelial monolayers.
• Heavy horizontal line with arrow represents location of hap locus within pN187 and direction of transcription. (+): recombinant plasmids that promote adherence and invasion; (-): recombinant plasmids that fail to promote adherence and invasion.
• Figure 5 depicts the identification of plasmid-encoded proteins using the bacteriophage T7 expression system.
• Bacteria were radiolabeled with [ 35 S] methionine, and whole cell lysates were resolved on a 10% SDS-polyacrylamide gel. Proteins were visualized by autoradiography.
• Lane 1 E. coli XL-1 Blue(pT7-7) uninduced; lane 2, XL-1 Blue(pT7-7) induced with IPTG; lane 3, XL-1 Blue(pJS103) uninduced; lane 4, XL-1 Blue(pJS103) induced with IPTG; lane 5, XL-1 Blue(pJS104) uninduced; lane 6, XL-1 Blue(pJS104) induced with IPTG.
• the plasmids pJS103 and pJS104 are derivatives of pT7-7 that contain the 6J-kb Vstl fragment frompN187 in opposite orientations. Asterisk indicates overexpressed protein in XL-1 Blue(pJS104).
• Figures 6A, 6B, and 6C depict the nucleotide sequence (SEQ ID NO: 1) and predicted amino acid sequence (SEQ ID NO: 2) of hap gene. Putative -10 and -35 sequences 5' to the hap coding sequence are underlined; a putative 7'Ao-independent terminator 3 ' to the hap stop codon is indicated with inverted arrows. The first 25 amino acids of the protein, which are boxed, represent the signal sequence.
• Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H depict a sequence comparison of the hap product (SEQ ID NO: 2) and the cloned /, influenzae IgAl proteases (SEQ ID NO: 3-6) .
• Amino acid homologies between the deduced hap gene product and the iga gene products from//, influenzae HK368 (SEQ ID NO: 3), HK61 (SEQ ID NO: 6), HK393 (SEQ ID NO: 4), and HK715 (SEQ ID NO: 5) are shown. Dashes indicate gaps introduced in the sequences in order to obtain maximal homology.
• a consensus sequence for the five proteins is shown on the lower line.
• the conserved serine-type protease catalytic domain is underlined, and the common active site serine is denoted by an asterisk.
• the conserved cysteines are also indicated by asterisks.
• Figure 8 depicts the IgAl protease activity assay. Culture supernatants were assayed for the ability to cleave IgAl. Reaction mixtures were resolved on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was probed with antibody against human IgAl heavy chain. Lane 1, H. influenzae strain N187; lane 2, strain DBl 17(pGJB103); lane 3, strain DB117(pN187). The cleavage product patterns suggest that strain Nl 87 contains a type 2 IgAl protease while strains DBl 17(pGJB103) and DBl 17(pN187) contain a type 1 enzyme. The upper band of ⁇ 70-kD seen with the DBl 17 derivatives represents intact IgAl heavy chain.
• Figures 9A and 9B depict southern analysis of chromosomal DNA from strain H. influenzae Nl 87, probing with hap versus iga. DNA fragments were separated on a 0.7% agarose gel and transferred bidirectionally to nitrocellulose membranes prior to probing with either hap or iga.
• Lane 1 N187 chromosomal DNA digested with scoRI; lane 2, Nl 87 chromosomal DNA digested with BgHl; lane 3, Nl 87 chromosomal DNA digested with BamBl; lane 4, the 4.8-kb Clal-Pstl fragment from pN 187 that contains the intact hap gene.
• Figure 9A Hybridization with the 4.8-kb Clal-Pstl fragment containing the hap gene
• Figure 9B hybridization with the iga gene from H. influenzae strain Rd, carried as a 4.8- kb Clal-Ec ⁇ Sl fragment in pVDl 16.
• Figure 10 depicts a SDS-polyacrylamide gel of secreted proteins. Bacteria were grown to late log phase, and culture supernatants were precipitated with trichloroacetic acid and then resolved on a 10% SDS-polyacrylamide gel. Proteins were visualized by staining with Coomassie blue. Lane l, / .
• Asterisk indicates 110-kD secreted protein encoded by hap.
• Figure 11 depicts an alignment of the deduced amino acid sequence of HAP proteins obtained from various H. influenzae strains.
• the strains include N187 (SEQ ID NO: 7), TN106 (SEQ ID NO: 11) and 860295 (SEQ ID NO: 13) .
• Figure 12 depicts Western blot of Hap proteins.
• Panel A shows outer membrane proteins
• panel B shows culture supernatants after precipitation with trichloroacetic acid.
• lane 1 contains DB 117/pGJB 103 (vector)
• lane 2 contains DB 117/pJS 106 (encoding HapN 187)
• lane 3 contains
• lane 4 contains DBl 17/pHapTN106.
• Immunoblotting was performed with guinea pig antiserum GP74, which was raised against purified Hap s from strain N187 and recognizes full-length Hap in outer membranes and Hap s in culture supernatants. Without being bound by theory, it is thought that the lower band in lane 3 of panel B presumably reflects autoproteolysis of multiple sites in Hap from strain P860925 (Fink et al; 2001).
• Figure 13 Adherence to Chang epithelial cells by H. influenzae strain DBl 17 expressing HapN187, HapTN106, or HapP860295 and the inhibitory effect of preincubation with anti-Haps antiserum. Adherence was determined in 30 minute assays and was calculated by dividing the number of adherent bacteria by the number of inoculated bacteria. For all strains, inocula were approximately 2xl0 7 CFU/ml. Bars represent the mean + standard error of the mean.
• Figure 14 depicts SDS-PAGE of purified HAPs proteins from both strain P860295 and strain N187. Amino terminal amino acid sequencing confirmed that purified protein was Haps.
• Figure 15 depicts clearance of NTHi TN106.P2 in Balb/c mice vaccinated with Hap s P860295 with and without CT-E29H.
• Six week old female Balb/c mice were vaccinated intranasally with Hap s from P860295, HAP+CT-E29H, or Formalin Fixed TN106.P2 in a 40 ⁇ l volume at weeks 0, 1, 3, & 5.
• Five animals from each group were IN challenged with ⁇ 1 x 10 6 CFU/;mouse in 10 ⁇ l, 2 and 3 weeks post vaccination. Nasal tissue were harvested 3 days after challenge.
• Figure 16 depicts the nucleotide sequence for NTHi strain 11 hap gene (SEQ ID NO: 8) (start codon to stop codon).
• Figure 17 depicts the amino acid sequence for NTHi strain 11 Hap protein (SEQ ID NO: 9) (first amino acid to last amino acid).
• Figure 18 depicts the nucleotide sequence for NTHi strain TN106 hap gene (SEQ ID NO: 10) (start codon begins at position 422, stop codon begins at position 4595).
• Figure 19 depicts the amino acid sequence for NTHi strain TN106 Hap protein (SEQ ID NO: 11) (first amino acid to last amino acid).
• Figure 20 depicts the nucleotide sequence for NTHi strain 860295 hap gene (SEQ ID NO: 12) (start codon begins at position 430, stop codon begins at position 4738).
• Figure 21 depicts the amino acid sequence for NTHi strain 860295 Hap protem (SEQ ID NO: 13) (first amino acid to last amino acid).
• Figure 22 depicts the nucleotide sequence for NTHi strain 3219B hap gene (SEQ ID NO: 14) (start codon begins at position 388, stop codon begins at position 4561).
• Figure 23 depicts the amino acid sequence for NTHi strain 3219B Hap protein (SEQ ID NO: 15) (first amino acid to last amino acid).
• Figure 24 depicts the nucleotide sequence for NTHi strain 1396B hap gene (SEQ ID NO: 16)(start codon begins at position 313, stop codon begins at position 4546).
• Figure 25 depicts the amino acid sequence for NTHi strain 1396B Hap protein (SEQ ID NO: 17) (first amino acid to last amino acid).
• HAP proteins are from Haemophilus strains, and in the preferred embodiment, from Haemophilus influenzae.
• HAP proteins from other Haemophilus influenzae strains including but not limited to NTHI TN 106, TN106.P2, N187, P860295, 11, 3219B, 1396B or from other bacterial species such as Neisseria spp. or Bordetella spp. may also be obtained.
• a HAP protein may be identified in several ways.
• a HAP nucleic acid or HAP protein is initially identified by substantial nucleic acid and/or amino acid sequence homology to the sequences shown in Figure 6. Such homology can be based upon the overall nucleic acid or amino acid sequence.
• a HAP protein is identifiable by substantial amino acid sequence homology or identity to the sequences shown in Figures 11 (SEQ ID NOS 7, 11, 13), 17 (SEQ ID NO 9), 19 (SEQ ID NO 11), 21 (SEQ ID NO 13), 23 (SEQ ID NO 15) or 25 (SEQ ID NO 17).
• HAP nucleic acid is identified by substantial nucleic acid sequence indentity to the sequences set forth in Figures 16 (SEQ ID NO 8), 18 (SEQ ID NO 10), 20 (SEQ ID NO 12), 22 (SEQ ID NO 14), or 24 (SEQ ID NO 16).
• the HAP proteins of the present invention have limited homology to Haemophilus influenzae and N. gonorrhoeae serine-type IgAl proteases. This homology, shown in Figure 7, is approximately 30-35% at the amino acid level, with several stretches showing 55-60% identity, including amino acids 457- 549, 399-466, 572-622, and 233-261. However, the homology between the HAP protein and the IgAl protease is considerably lower than the similarity among the IgAl proteases themselves.
• the full length HAP protein has homology to Tsh, a hemagglutinin expressed by an avian E. coli strain (Maritime and Curtiss 1994, Infect. Immun. 62: 1369-1380). The homology is greatest in the N-terminal half of the proteins, and the overall homology is 30.5% homologous.
• the full length HAP protein also has homology with pertactin, a 69 kD outer membrane protein expressed by B. pertussis, with the middle portion of the proteins showing 39% homology.
• HAP also has 34 - 52% homology with six regions of HpmA, a calcium-independent hemolysin expressed by Proteus mirabilis (Uphoff and Welch, 1990, J. Bacteriol. 172:1206-1216).
• a protein is a "HAP protein" if the overall homology of the protein sequence to one of the amino acid sequences shown in Figure 6, 11, 17, 19, 21, 23, or 25, is preferably greater than about 40 - 50%, more preferably greater than about 60% and most preferably greater than 80%. In some embodiments the homology will be as high as about 90 to 95 or 98%. This homology will be determined using standard techniques known in the art, such as the Best Fit sequence program described by Devereux et al, Nucl. Acid Res. 12:387-395 (1984). The alignment may include the introduction of gaps in the sequences to be aligned.
• HAP proteins of the present invention may be shorter than the amino acid sequence shown in Figure 6 or 11. As shown in the Examples, the HAP protein may undergo post-translational processing similar to that seen for the serine-type IgAl proteases expressed by Haemophilus influenzae and N. gonorrhoeae.
• proteases are synthesized as preproteins with three functional domains: the N- terminal signal peptide, the protease, and a C-terminal helper domain. Following movement of these proteins into the periplasmic space, the carboxy terminal ⁇ -domain of the proenzyme is inserted into the outer membrane, possibly forming a pore (Poulsen et al, 1989, Infect. Immun. 57:3097-3105; Pohlner et al, 1987, Nature (London). 325:458-462; Klauser et al, 1992, EMBO J. 11:2327-2335; Klauser et al, 1993, J. Mol. Biol. 234:579-593).
• the amino end of the protein is exported through the outer membrane, and autoproteolytic cleavage occurs to result in secretion of the mature 100 to 106-kD protease.
• the 45 to 56-kD C-terminal ⁇ -domain remains associated with the outer membrane following the cleavage event.
• the HAP nucleic acid is associated with expression of a 155 kD outer membrane protein.
• the secreted gene product is an approximately 110 kD protein, with the simultaneous appearance of a 45 kD outer membrane protein.
• the 45 kD protein corresponds to amino acids from about 1037 to about 1395 of Figure 6. Any one of these proteins is considered a HAP protein for the purposes of this invention.
• HAP protein fragments included within the defintion of HAP proteins are portions or fragments of the sequence shown in Figure 6 or 11.
• the fragments may be fragments of the entire sequence, the 110 kD sequence, or the 45 kD sequence.
• the HAP protein fragments may range in size from about 10 amino acids to about 1900 amino acids, with from about 50 to about 1000 amino acids being preferred, and from about 100 to about 500 amino acids also preferred.
• Particularly preferred fragments are sequences unique to HAP; these sequences have particular use in cloning HAP proteins from other organisms or to generate antibodies specific to HAP proteins. Unique sequences are easily identified by those skilled in the art after examination of the HAP protein sequence and comparison to other proteins; for example, by examination of the sequence alignment shown in Figure 7.
• unique sequences include, but are not limited to, amino acids 11-14, 16-22, 108-120, 155-164, 257-265, 281-288, 318-336, 345-353, 398-416, 684-693, 712-718, 753-761, 871-913, 935-953, 985-1008, 1023-1034, 1067-1076, 1440-1048, 1585-1592, 1631- 1639, 1637-1648, 1735-1743, 1863-1871, 1882-1891, 1929-1941, and 1958-1966 (using the numbering of Figure 7).
• HAP protein fragments which are included within the definition of a HAP protein include N- or C-terminal truncations and deletions which still allow the protein to be biologically active; for example, which still exhibit proteolytic activity in the case of the 110 kD putative protease sequence.
• the HAP protein when the HAP protein is to be used to generate antibodies, for example as a vaccine, the HAP protein must share at least one epitope or determinant with either the full length protein, the 110 kD protein or the 45 kD protein, shown in Figure 6.
• the epitope is unique to the HAP protein; that is, antibodies generated to a unique epitope exhibit little or no cross-reactivity with other proteins.
• epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller HAP protein will be able to bind to the full length protein.
• the HAP protein contains sequences conserved among HAP proteins from different species or strains. As shown in Figure 11, alignment of the amino acid sequences of Hap TN106, HapP860295, and HapN187 revealed absolute identity through the first 47 amino acids, divergence over the next 350 amino acids, and then marked similarity over the final 1000-1050 amino acids. Accordingly, in a preferred embodiment the HAP protein of the invention includes amino acids 1-47 of the proteins shown in Figure 11.
• the HAP protein of the invention includes amino acids that are invariant among the sequences set forth in Figure 11 and contiguous for at least about 3, preferably at least about 5 and most preferably at least about 8 amino acids in length.
• Preferred peptides are included in the following Table 1.
• the fragment of the HAP protein used to generate antibodies are small; thus, they may be used as haptens and coupled to protein carriers to generate antibodies, as is known in the art.
• the fragment of the HAP protein is a fragment of one of the peptides listed above. In this embodiment the fragment need only comprise a single epitope.
• the invention provides a composition comprising at least one of the peptides as shown in Table I.
• the invention provides a polypeptide comprising at least one of the peptides as shown in Table I.
• the antibodies are generated to a portion of the HAP protein which remains attached to the Haemophilus influenzae organism.
• the HAP protein can be used to vaccinate a patient to produce antibodies which upon exposure to the Haemophilus influenzae organism (e.g. during a subsequent infection) bind to the organism and allow an immune response.
• the antibodies are generated to the roughly 45 kD fragment of the full length HAP protein
• the antibodies are generated to the portion of the 45 kD fragment which is exposed at the outer membrane
• the antibodies bmd to the mature secreted 110 kD fragment may be administered therapeutically to generate neutralizing antibodies to the 110 kD putative protease, to decrease the undesirable effects and/or bmdmg activity of the 100 kD fragment
• the nucleic acid sequence homology may be either lower or higher than that of the protem sequence
• the homology of the nucleic acid sequence as compared to the nucleic acid sequence of Figure 6, 16, 18, 20, 22 or 24, is preferably greater than 40%, more preferably greater than about 60% and most preferably greater than 80% In some embodiments the homology will be as high as about 90 to 95 or 98%
• nucleic acid homology is determined through hybridization studies
• nucleic acids which hybridize under high stringency to all or part of the nucleic acid sequence shown in Figure 6 are considered HAP piotem genes
• High stringency conditions include washes with 0 1XSSC at 65°C for 2 hours
• nucleic acid of the invention are preferably greater than 40%, more preferably greater than about 60% and most preferably greater than 80% identical to the nucleic acids as set forth in Figure 6, 16, 18, 20, 22 or 24 In some embodiments the identity will be as high as about 90 to 95 or 98% up to 100%
• nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ⁇ bonucleotides
• nucleic acids mclude genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids Specifically included withm the definition of nucleic acid are anti-sense nucleic acids
• An anti-sense nucleic acid will hybridize to the corresponding non-codmg strand of the nucleic acid sequence shown in Figure 6, but may contam ⁇ bonucleotides as well as deoxyribonucleotides
• anti-sense nucleic acids function to prevent expression of mRNA, such that a HAP protem is not made, or made at reduced levels
• the nucleic acid may be double stranded, smgle stranded, or contam portions of both double stranded or smgle strand
• nucleic acids once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
• a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
• a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated away from some or all of the proteins and compounds with which it is normally associated in its wild type host, or found in the absence of the host cells themselves. Thus, the protein may be partially or substantially purified.
• the definition includes the production of a HAP protein from one organism in a different organism or host cell.
• the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
• the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions.
• HAP protein from other organisms, including, but not limited to various H. influenza strains, which are cloned and expressed as outlined below.
• an anti-sense nucleic acid is defined as one which will hybridize to all or part of the corresponding non-coding sequence of the sequence shown in Figure 6.
• the hybridization conditions used for the determination of anti-sense hybridization will be high stringency conditions, such as 0.1XSSC at 65°C.
• the HAP protein nucleic acid Once the HAP protein nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire HAP protein nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant HAP protein nucleic acid can be f rther used as a probe to identify and isolate other HAP protein nucleic acids. It can also be used as a "precursor" nucleic acid to make modified or variant HAP protein nucleic acids and proteins.
• the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
• these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the HAP protein.
• "Operably linked" in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence of the HAP protein in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5 ' to the HAP protein coding region.
• the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the HAP protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus will be used to express the HAP protem in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
• the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
• the regulatory sequences include a promoter and transcriptional start and stop sequences.
• Promoter sequences encode either constitutive or inducible promoters.
• the promoters may be either naturally occurring promoters or hybrid promoters.
• Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
• the expression vector may comprise additional elements.
• the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
• the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
• the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
• the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
• Selection genes are well known in the art and will vary with the host cell used.
• the HAP proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a HAP protein, under the appropriate conditions to induce or cause expression of the HAP protein.
• the conditions appropriate for HAP protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
• the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
• the timing of the harvest is important.
• the baculo viral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
• Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli. Bacillus subtilis. SF9 cells, C129 cells, 293 cells, Neurospora,
• HAP proteins are expressed in bacterial systems.
• Bacterial expression systems are well known in the art.
• a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of HAP protein into mRNA.
• a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples mclude promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan.
• Promoters from bacteriophage may also be used and are known in the art.
• synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences.
• a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
• the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon.
• SD Shine-Delgarno
• the expression vector may also include a signal peptide sequence that provides for secretion of the HAP protein in bacteria.
• the signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art.
• the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
• the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
• Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
• Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
• Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
• the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
• HAP proteins are produced in insect cells.
• Expression vectors for the transformation of insect cells and in particular, baculo virus-based expression vectors, are well known in the art. Briefly, baculovirus is a very large DNA virus which produces its coat protein at very high levels. Due to the size of the baculoviral genome, exogenous genes must be placed in the viral genome by recombination.
• the components of the expression system include: a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the HAP protein; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene into the baculovirus genome); and appropriate insect host cells and growth media.
• a transfer vector usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the HAP protein
• a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector this allows for the homologous recombination of the heterologous gene into the baculovirus genome
• appropriate insect host cells and growth media are appropriate insect host cells and growth media.
• a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for HAP protein into mRNA.
• a promoter will have a transcription initiating region, which is usually place proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct R ⁇ A polymerase II to begin R ⁇ A synthesis at the correct site.
• a mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 base pairs upstream of the TATA box.
• An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
• mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, and herpes simplex virus promoter.
• transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
• the 3' terminus of the mature mR ⁇ A is formed by site-specific post-translational cleavage and polyadenylation.
• transcription terminator and polyadenlytion signals include those derived form SV40.
• HAP protein is produced in yeast cells.
• Yeast expression systems are well known in the art, and include expression vectors for Saccharomvces cerevisiae, Candida albicans and C. maltosa, Hansenula polvmorpha, Kluweromvces fragilis and K. lactis. Pichia guillerimondii and P. pastoris, Schizosaccharomvces pombe, and Yarrowia lipolytica.
• Preferred promoter sequences for expression in yeast include the inducible GAL1J0 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase, hexokinase, phosphofhictokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene.
• Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the G418 resistance gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.
• a recombinant HAP protein may be expressed intracellularly or secreted.
• the HAP protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, if the desired epitope is small, the HAP protein may be fused to a carrier protein to form an immunogen. Alternatively, the HAP protein may be made as a fusion protein to increase expression.
• HAP proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the HAP protein, using cassette mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant HAP protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques.
• Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the HAP protein amino acid sequence.
• the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed HAP protein variants screened for the optimal combination of desired activity.
• Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, PCR primer mutagenesis. Screening of the mutants is done using assays of HAP protein activities; for example, mutated HAP genes are placed in HAP deletion strains and tested for HAP activity, as disclosed herein. The creation of deletion strains, given a gene sequence, is known in the art.
• nucleic acid encoding the variants may be expressed in a Haemophilus influenzae strain deficient in the HAP protein, and the adhesion and infectivity of the variant Haemophilus influenzae evaluated.
• the variant HAP protein may be expressed and its biological characteristics evaluated, for example its proteolytic activity.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to 30 residues, although in some cases deletions may be much larger, as for example when one of the domains of the HAP protein is deleted. Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.
• substitutions that are less conservative than those shown in Chart I.
• substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
• the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
• leucyl isoleucyl, phenylalanyl, valyl or alanyl
• a cysteine or proline is substituted for (or by) any other residue
• a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
• an electronegative residue e.g. glutamyl or aspartyl
• a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
• the variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the polypeptide as needed.
• the variant may be designed such that the biological activity of the HAP protein is altered.
• the proteolytic activity of the larger 110 kD domain of the HAP protein may be altered, through the substitution of the amino acids of the active site.
• the putative catalytic domain of this protein was considered to be GDSGSPMF (SEQ ID NO: 53).
• the residues of the active site may be individually or simultaneously altered to decrease or eliminate proteolytic activity. This may be done to decrease the toxicity or side effects of the vaccine.
• the cleavage site between the 45 kD domain and the 100 kD domain may be altered, for example to eliminate proteolytic processing to form the two domains.
• the catalytic triad has been defined as His98, Asp 140 and Ser 243. Each of these amino acids has been mutated; the mutations eliminated proteolytic activity.
• four sites have been identified at which autoproteolytic cleavage occurs (Hendrixson et al., 1997; Hendrixson and St. Geme, 1998; Fink et al., 2001).
• the HAP protein is purified or isolated after expression.
• HAP proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
• the HAP protein may be purified using a standard anti-HAP antibody column. Ultrafiltration and diafilfration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the HAP protein. In some instances no purification will be necessary.
• HAP proteins are useful in a number of applications.
• the HAP proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify antibodies from samples obtained from animals or patients exposed to the Haemophilus influenzae organism. The purified antibodies may then be used as outlined below.
• the HAP proteins are useful to make antibodies to HAP proteins. These antibodies find use in a number of applications.
• the antibodies are used to diagnose the presence of an Haemophilus influenzae infection in a sample or patient. This will be done using techniques well known in the art; for example, samples such as blood or tissue samples may be obtained from a patient and tested for reactivity with the antibodies, for example using standard techniques such as ELISA.
• monoclonal antibodies are generated to the HAP protein, using techniques well known in the art. As outlined above, the antibodies may be generated to the full length HAP protein, or a portion of the HAP protein.
• Antibodies generated to HAP proteins may also be used in passive immunization treatments, as is known in the art. Antibodies generated to unique sequences of HAP proteins may also be used to screen expression libraries from other organisms to find, and subsequently clone, HAP nucleic acids from other organisms.
• the antibodies may be directly or indirectly labelled.
• labelled herein is meant a compound that has at least one element, isotope or chemical compound attached to enable the detection of the compound.
• labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
• the labels may be incorporated into the compound at any position.
• the HAP protein antibody may be labelled for detection, or a secondary antibody to the HAP protein antibody may be created and labelled.
• the antibodies generated to the HAP proteins of the present invention are used to purify or separate HAP proteins or the Haemophilus influenzae organism from a sample.
• antibodies generated to HAP proteins which will bind to the Haemophilus influenzae organism may be coupled, using standard technology, to affinity chromatography columns. These columns can be used to pull out the Haemophilus organism from environmental or tissue samples.
• antibodies generated to the soluble 110 kD portion of the full-length portion of the protein shown in Figure 7 may be used to purify the 110 kD protem from samples.
• the HAP proteins of the present invention are used as vaccines for the prophylactic or therapeutic treatment of a Haemophilus influenzae infection in a patient.
• vaccine herein is meant an antigen or compound which elicits an immune response in an animal or patient.
• the vaccine may be administered prophylactically, for example to a patient never previously exposed to the antigen, such that subsequent infection by the Haemophilus influenzae organism is prevented.
• the vaccine may be administered therapeutically to a patient previously exposed or infected by the Haemophilus influenzae organism. While infection cannot be prevented, in this case an immune response is generated which allows the patient's immune system to more effectively combat the infection. Thus, for example, there may be a decrease or lessening of the symptoms associated with infection.
• the HAP proteins of the invention protect against infection by //. influenza. That is, administration of at least one of the HAP proteins of the invention to a patient results in protection against H. influenza infection. In another embodiment administration of at least one HAP protein of the invention results in reduced colonization by H. influenza. In a particularly preferred embodiment administration of at least one of the HAP proteins of the invention results in protection against infection or colonization by a heterologous strain of Haemophilus influenzae.
• heterologous is meant a strain that is not the same strain from which the HAP protein is obtained. Accordingly, the present invention provides a method of vaccinating against infection by a heterologous strain of//, influenza.
• a "patient” for the purposes of the present invention includes both humans and other animals and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
• the administration of the HAP protein as a vaccine is done in a variety of ways, including but not limited to intramuscular or subcutaneous injection, intranasal delivery, oral delivery, intravenous delivery and intradermal delivery as is known in the art.
• the HAP proteins can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby therapeutically effective amounts of the HAP protein are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are well known in the art. Such compositions will contain an effective amount of the HAP protein together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions for effective administration to the host.
• the composition may include salts, buffers, earner proteins such as serum albumin, targeting molecules to localize the HAP protein at the appropriate site or tissue within the organism, and other molecules.
• the composition may include adjuvants as well.
• the HAP protein is administered combined with an adjuvant as is known in the art, such as aluminum hydroxide.
• the adjuvant is a modified cholera toxin adjuvant.
• CT-E29H is a mutant form of CT that contains a histidine in place of a glutamic acid at residue 29 in the enzymatic A subunit. This mutant lacks enzymatic activity and has ⁇ 1% of the cellular toxicity of native cholera toxin but remains fully active as an adjuvant, suggesting considerable utility in humans (Tebbey et al, 2000, Vaccine 18:2723-34).
• the invention provides a composition comprising a HAP protein of the invention and cholera toxin CT-E29H.
• the invention provides a method of improving immunization by administering an immunogenic protein of the invention and an adjuvant.
• the adjuvant is CT-E29H.
• the vaccine is administered as a single dose; that is, one dose is adequate to induce a sufficient immune response to prophylactically or therapeutically treat a Haemophilus influenzae infection.
• the vaccine is administered as several doses over a period of time, as a primary vaccination and "booster" vaccinations.
• therapeutically effective amounts herein is meant an amount of the HAP protein which is sufficient to induce an immune response. This amount may be different depending on whether prophylactic or therapeutic treatment is desired.
• H influenzae strain Nl 87 is a clinical isolate that was originally cultivated from the middle ear fluid of a child with acute otitis media. This strain was classified as nontypable based on the absence of agglutination with typing antisera for H. influenzae types a-f (Burroughs Wellcome) and the failure to hybridize with pU038, a plasmid that contains the entire cap b locus (Kroll and Moxon, 1988, J. Bacteriol. 170:859-864).
• H. influenzae strain DBl 17 is a rec-I mutant of Rd, a capsule-deficient serotype d strain that has been in the laboratory for over 40 years (Alexander and Leidy, 1951, J. Exp. Med. 83:345-359); DBl 17 was obtained from G. Barcak (University of Maryland, Baltimore, MD) (Sellow et al, 1968). DBl 17 is deficient for in vitro adherence and invasion, as assayed below.
• H. influenzae strain 12 is the nontypable strain from which the genes encoding the HMW1 and
• HMW2 proteins were cloned (Barenkamp and Leininger, 1992, Infect. Immun. 60:1302-1313); HMW1 and HMW2 are the prototypic members of a family of nontypable Haemophilus antigenically-related high-molecular- weight adhesive proteins (St. Geme et al, 1993).
• E. coli HB101 which is nonadherent and noninvasive, has been previously described (Sambrook et al, 1989, Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold
• E. coli DH5c. was obtained from Bethesda Research Laboratories.
• E. coli MC1061 was obtained from H. Kimsey (Tufts University, Boston, MA).
• E. coli XL-1 Blue and the plasmid pBluescript KS- were obtained from Stratagene. Plasmid pT7-7 and phage mGPl-2 were provided by S. Tabor (Harvard Medical School, Boston, MA) (Tabor and Richardson, 1985, Proc. Natl. Acad. Sci. USA. 82:1074-1078).
• the E. coli-Haemophilus shuttle vector pGJB103 (Tomb et al, 1989, Rd. J.
• Plasmid pVDl 16 harbors the IgAl protease gene from/ , influenzae strain Rd (Koomey and Falkow, 1984, Infect. Immun. 43:101- 107) and was obtained from M. Koomey (University of Michigan, Ann Arbor, MI).
• H. influenzae strains were grown as described (Anderson et al, 1972, J. Clin.
• Invest. 51 :31-38 They were stored at -80°C in brain heart infusion broth with 25% glycerol.
• E. coli strains were grown on LB agar or in LB broth. They were stored at -80°C in LB broth with 50% glycerol.
• H. influenzae tetracycline was used in a concentration of 5 ⁇ g/ml and kanamycin was used in a concentration of 25 ⁇ g/ml.
• antibiotics were used in the following concentrations: tetracycline, 12.5 ⁇ g/ml; kanamycin, 50 ⁇ g/ml; ampicillin, 100 ⁇ g/ml.
• Plasmids were introduced into E. coli strains by either chemical transformation or electroporation, as described (Sambrook et al, 1989, supra; Dower et al, 1988, Nucleic Acids Res. 16:6127-6145).
• H. influenzae transformation was performed using the MIV method of Herriott et al. (1970, J. Bacteriol. 101:517-524), and electroporation was carried out using the protocol developed for E. coli (Dower et al, 1988, supra).
• Transposon mutagenesis Mutagenesis of plasmid DNA was performed using the mini-Tn/0 kan element described by Way et al. (1984, supra). Initially, the appropriate plasmid was introduced mto E. c ⁇ /z ' MC1061. The resulting strain was infected with ⁇ 1105, which carries the kan transposon. Transductants were grown overnight in the presence of kanamycin and an antibiotic to select for the plasmid, and plasmid DNA was isolated using the alkaline lysis method. In order to recover plasmids containing a transposon insertion, plasmid DNA was electroporated into E. coli DH5 ⁇ , plating on media containing kanamycin and the appropriate second antibiotic.
• Plasmid pT7-7 contains the T7 phage ⁇ lO promoter and ribosomal binding site upstream of a multiple cloning site (Tabor and Richardson, 1985, supra).
• the T7 promoter was induced by infection with the recombinant Ml 3 phage mGPl-2 and addition of isopropyl-/3-D-thiogalactopyranoside (final concentration, 1 mM).
• Phage mGPl-2 contains the gene encoding T7 RNA polymerase, which activates the ⁇ lO promoter in pT7-7 (Tabor and Richardson, 1985, supra).
• strain DBl 17 carrying pJS 106 expressed new outer membrane proteins 160-kD and 45-kD in size ( Figure 3, lane 3).
• this fragment of DNA was ligated into the bacteriophage T7 expression vector pT7-7.
• the resulting plasmid containing the insert in the same orientation as in pNl 87 was designated pJS104, and the plasmid with the insert in the opposite orientation was designated pJS103.
• Both pJS104, and pJS103 were introduced into E. coli XL-1 Blue, producing XL-1 Blue(pJS104) and XL-1 Blue(pJS103), respectively.
• pT7-7 was also transformed into XL-1
• T7 promoter was induced in these three strains by infection with the recombinant M13 phage mGPl-2 and addition of isopropyl- 3-D-thiogalactopyranoside (final concentration, 1 mM), and induced proteins were detected using [ 35 5] methionine.
• induction of XL-1 Blue(pJS104) resulted in expression of a 160-kD protein and several smaller proteins which presumably represent degradation products.
• Adherence and invasion assays were performed with Chang epithelial cells [Wong-Kilbourne derivative, clone l-5c-4 (human conjunctiva)], which were seeded into wells of 24- well tissue culture plates as previously described (St. Geme and Falkow, 1990). Adherence was measured after incubating bacteria with epithelial monolayers for 30 minutes as described (St. Geme et al, 1993). Invasion assays were carried out according to our original protocol and involved incubating bacteria with epithelial cells for four hours followed by treatment with gentamicin for two hours (100 ⁇ g/ml) (St. Geme and Falkow, 1990).
• This gene encodes a 1394 amino acid polypeptide, which we have called Hap, with a calculated molecular mass of 155.4-kD, in good agreement with the molecular mass of the larger of the two novel outer membrane proteins expressed by DBl 17(pN187) and the protein expressed after induction of XL-1 Blue/pJS104.
• the hap gene has a G+C content of 39.1%, similar to the published estimate of 38.7% for the whole genome (Kilian, 1976, J. Gen. Microbiol. 93:9-62). Putative -10 and -35 promoter sequences are present upstream of the initiation codon. A consensus ribosomal binding site is lacking.
• a sequence similar to a 7"/zo-independent transcription terminator is present beginning 39 nucleotides beyond the stop codon and contains interrupted inverted repeats with the potential for forming a hairpin structure containing a loop of three bases and a stem of eight bases. Similar to the situation with typical E. coli terminators, this structure is followed by a stretch rich in T residues. Analysis of the predicted amino acid sequence suggested the presence of a 25 amino acid signal peptide at the amino terminus. This region has characteristics typical of procaryotic signal peptides, with three positive H-terminal charges, a central hydrophobic region, and alanine residues at positions 23 and 25 (-3 and -1 relative to the putative cleavage site) (von Heijne, 1984, J. Mol. Biol.
• the hap product also contains two cysteines corresponding to the cysteines proposed to be important in forming the catalytic domain of the IgA proteases (Pohlner et al, 1987, supra). Overall there is 30-35%> identity and 51-55% similarity between the hap gene product and the H. influenzae and N. gonorrhoeae IgA proteases.
• the deduced amino acid sequence encoded by hap was also found to contain significant homology to Tsh, a hemagglutinin expressed by an avian-5. coli strain ( Portugal and Curtiss, 1994, supra).
• Tsh is also synthesized as a preprotein and is secreted as a smaller form; like the IgAl proteases and perhaps Hap, a carboxy terminal peptide remains associated with the outer membrane (D. Portugal, personal communication). While this protein is presumed to have proteolytic activity, its substrate has not yet been determined. Interestingly, Tsh was first identified on the basis of its capacity to promote agglutination of erythrocytes. Thus Hap and Tsh are possibly the first members of a novel class of adhesive proteins that are processed analogously to the IgAl proteases.
• pertactin a 69-kD outer membrane protein expressed by B. pertussis (Charles et al, 1989, Proc. Natl. Acad. Sci. USA. 86:3554-3558). The middle portions of these two molecules are 39% identical and nearly 60% similar.
• This protein contains the amino acid triplet arginine-glycine-aspartic acid (RGD) and has been shown to promote attachment to cultured mammalian cells via this sequence (Leininger et al, 1991, Proc. Natl. Acad. Sci. USA. 88:345-349).
• Bordetella species are not generally considered intracellular parasites, work by Ewanowich and coworkers indicates that these respiratory pathogens are capable of in vitro entry into human epithelial cells (Ewanowich et al, 1989, Infect. Immun. 57:2698-2704; Ewanowich et al, 1989, Infect. Immun. 57: 1240-1247).
• Recently Leininger et al. reported that preincubation of epithelial monolayers with an RGD-containing peptide derived from the pertactin sequence specifically inhibited B. pertussis entry (Leininger et al, 1992, Infect. Immun. 60:2380-2385).
• HpmA calcium-independent hemolysin expressed by Proteus mirabilis
• the hap locus is distinct from the H. influenzae IgAl protease gene. Given the degree of similarity between the hap gene product and H. influenzae IgAl protease, we knew whether we had isolated the IgAl protease gene of strain Nl 87. To examine this possibility, we performed IgAl protease activity assays. Among H. influenzae strains, two enzymatically distinct types of IgAl protease have been found (Mulks et al, 1982, J. Infect. Dis. 146:266-274).
• Type 1 enzymes cleave the Pro-Ser peptide bond between residues 231 and 232 in the hinge region of human IgAl heavy chain and generate fragments of roughly 28-kD and 31-kD; type 2 enzymes cleave the Pro-Thr bond between residues 235 and 236 in the hinge region and generate 26.5-kD and 32.5-kD fragments.
• the recombinant plasmid associated with adherence and invasion encodes a secreted protein.
• the IgAl proteases are synthesized as preproteins with three functional domains: the N-terminal signal peptide, the protease, and a C-terminal helper domain, which is postulated to form a pore in the outer membrane for secretion of the protease (Poulsen et al, 1989, supra; Pohlner et al, 1987, supra).
• the C-terminal peptide remains associated with the outer membrane following an autoproteolytic cleavage event that results in release of the mature enzyme.
• DB117(pN187) produced a secreted protein approximately 110-kD in size that was absent from DBl 17(pGJB103) ( Figure 10).
• This protein was also produced by DBl 17(pJS106), but not by DBl 17(pJ5102) or DBl 17(pJS105).
• the two mutants with transposon insertions within the hap coding region were deficient in this protein.
• this protein was transferred to a PVDF membrane and N-terminal amino acid sequencing was performed. Excessive background on the first cycle precluded identification of the first amino acid residue of the free amino terminus.
• the sequence of the subsequent seven residues was found to be HTYFGID (SEQ ID NO: 55), which corresponds to amino acids 27 through 33 of the hap product.
• H. influenzae promoter or ribosomal binding site was poorly recognized in E. coli. Indeed the putative -35 sequence upstream of the hap initiation codon is fairly divergent from the ⁇ 70 consensus sequence, and the ribosomal binding site is unrecognizable.
• an accessory gene may be required for proper export of the Hap protein, although the striking homology with the IgA proteases, which are normally expressed and secreted in E. coli, argues against this hypothesis.
• Filamentous hemagglutinin is a 220-kD protein expressed by B. pertussis that mediates in vitro adherence and facilitates natural colonization (Relman et al, 1989, Proc. Natl. Acad. Sci. U.S.A.
• Outer membrane protems were isolated on the basis of sarcosyl insolubility according to the method of Carlone et al. (1986, J. Clin. Microbiol. 24:330-332). Secreted proteins were isolated by centrifuging bacterial cultures at 16,000 g for 10 minutes, recovering the supernatant, and precipitating with trichloroacetic acid in a final concentration of 10%. SDS-polyacrylamide gel electrophoresis was performed as previously described (Laemmli, 1970, Nature (London). 227:680-685).
• DB117(pN187) and DB117(pGJB103) were compared.
• DBl 17(pN187) expressed two new outer membrane protems: a high-molecular-weight protein approximately 160-kD in size and a 45-kD protein.
• E. coli HB101 harboring pN187 failed to express these proteins, suggesting an explanation for the observation that HB101(pN187) is incapable of adherence or invasion.
• PVDF polyvinylidene difluoride
• IgAl protease activity In order to assess IgAl protease activity, bacteria were inoculated into broth and grown aerobically overnight. Samples were then centrifuged in a microphage for two minutes, and supernatants were collected. A 10 ⁇ l volume of supernatant was mixed with 16 ⁇ l of 0.5 ⁇ g/ml human IgAl (Calbiochem), and chloramphenicol was added to a final concentration of 2 ⁇ g/ml.
• reaction mixtures were electrophoresed on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with goat anti-human IgAl heavy chain conjugated to alkaline phosphatase (Kirkegaard & Perry).
• the membrane was developed by immersion in phosphatase substrate solution (5-bromo-4- chloro-3-indolylphosphate toluidinium-nitro blue tetrazolium substrate system; Kirkegaard .& Perry).
• Immunoblot analysis Immunoblot analysis of bacterial whole cell lysates was carried out as described (St. Geme et al, 1991).
• Microscopy i. Light microscopy. Samples of epithelial cells with associated bacteria were stained with Giemsa stain and examined by light microscopy as described (St. Geme and Falkow, 1990).
• NTHi strains Nl 87 and P860295 were isolated from middle ear fluid of children with acute otitis media, while NTHi strain TNI 06 was isolated from a patient with pneumonia.
• Strain N187 is the strain from which the hap gene was originally cloned (Sanders et al, 993, Infect. Immun. 61:3966-3975; St. Geme et al, 1994, Mol. Microbiol. 14:217-233).
• Strain P860295 was obtained from Dr. Charles Brinton (University of Pittsburgh), and strain TN106 was obtained from Dr. Eric Hansen (University of Texas, Southwestern School of Medicine).
• Sfrain TN106.P2 is a derivative of TN106 that was recovered after plating on medium containing 100 ⁇ g/ml of streptomycin, then inoculating into the nasopharynx of a Balb/c mouse. Strains TN106.P2 and TN106 are indistinguishable in terms of morphology and growth characteristics.
• H. influenzae strain DBl 17 is a reel mutant of Rd, a capsule-deficient serotype d strain (Setlow et al, 1968, J. Bacteriol. 95:546-558).
• DBl 17 contains a nonfunctional hap gene because of a spontaneous nonsense mutation at codon 710 and is nonadherent in assays with cultured epithelial cells (Fleischmann et al, 1995. Rd. Science 269:496-512.).
• H. influenzae strains were grown on chocolate agar supplemented with 1% IsoVitaleX, on brain heart infusion agar supplemented with hemin and NAD (BHI-XV agar), or in brain-heart infusion broth supplemented with hemin and NAD (BHIs), as described previously (St. Geme and Falkow, 1990, supra). These strains were stored at-80°C in BHI broth with 20% glycerol. E. coli strains were grown on Luria-Bertani (LB) agar or in LB broth and were stored at -80°C in LB broth with 50% glycerol.
• LB Luria-Bertani
• Antibiotic concentrations used to select for plasmids included 5mg/ml tetracycline in H. influenzae and 100 mg/ml ampicillin and 12.5 mg/ml tetracycline in E. coli. DNA ligations, restriction endonuclease digestions and gel electrophoresis were performed according to standard techniques (Sambrook et al, 1989, supra). Plasmids were introduced into E. coli by electroporation (Dower et al, 1988, supra). In H. influenzae, transformation was performed using the MIV method of Herriott et al. (Herriott et al, 1970, supra).
• PCR primers corresponding to sequence flanking hap in strain Nl 87. Reactions were performed with Expand polymerase (Roche Molecular Biochemicals) to enhance long range amplification and to minimize PCR-related errors.
• the 5' primer was based on sequence beginning approximately 500 base pairs upstream of hap (5'-TGCAGGATCCCCGCAGACTGGATTGTTG-3') (SEQ ID NO: 56), and the 3' primer corresponded to sequence beginning roughly 50 base pairs downstream of hap (5'-TGCAGGATCCGATCTGCCCCACCTTGTT-3')(SEQ ID NO: 57). To facilitate initial cloning, both the 5' and the 3' primers included a BamHI site. The amplified genes were cloned into BamHI-digested pUC19 and Bglll-digested pGJB103.
• Nucleotide sequencing was performed using an Applied Biosystems automated sequencer and the Big Dye Terminator Premix-20 kit (Applied Biosystems/Perkins Elmer). Double-stranded plasmid DNA was used as template, and sequencing was carried out along both strands. With strain TN106, clones from two separate PCR assays were sequenced, and the two sequences were identical. With strain P860295, a single clone was sequenced.
• the A pTNlO ⁇ gene encodes a protein with 1392 amino acids
• the A ⁇ p>P860295 gene encodes a slightly larger protein with a total of 1436 amino acids.
• HapTN106 is 80%o similar and 77%> identical to Ha ⁇ N187
• HapP860295 is 85% similar and 83% identical to HapN187.
• the predicted amino acid sequences of Hap TN106 and Hap P860295 are 82% similar and 79% identical to each other.
• Adherence assays were performed with Chang conjunctival epithelial cells (Wong-Kilbourne derivative, clone l-5c-4 [human conjunctiva], as previously described (St. Geme, et al, 1993, supra). Percent adherence was calculated by dividing the number of adherent colony- forming units per monolayer by the number of inoculated colony-forming units.
• DB 117/pHap(TN106) and DBl 17/pHap(P860295) were compared with DBl 17/pJS106 in adherence assays with Chang conjunctival epithelial cells.
• both Hap TN106 and HapP860295 promoted appreciable levels of adherence to these cells, similar to levels associated with HapNl 87.
• the resulting precipitate was dissolved in 50 mM sodium phosphate buffer, pH 5.8, 1 mM EDTA, 50 mM NaCl and was dialyzed at 4DC against the same buffer (Buffer 1), then centrifuged at 100,000 x g for 1 hour at 4DC to remove insoluble material.
• Buffer 1 buffer 1
• 70 ml of the above soluble material was loaded onto the column at a flow rate of 5 ml/min.
• the column was washed with Buffer 1 until the OD280 reached baseline, and the flow through material was discarded.
• Hap s from NTHi strain N187 was purified as previously described (Hendrixson, et al, 1997, Mol. Microbiol. 26:505-518; Hendrixson and St. Geme, 1998, Mol. Cell 2:841-850).
• Strain P860295 was purified as previously described (Hendrixson, et al, 1997, Mol. Microbiol. 26:505-518; Hendrixson and St. Geme, 1998, Mol. Cell 2:841-850).
• Hap s was purified from the native strain, while strain N187 Hap s was purified from DBl 17 harboring pJS106 (encoding HapN187). Using this purification scheme, highly pure protem from both strain P860295 and strain Nl 87 ( Figure 14) was recovered. Amino terminal amino acid sequencing (described below) confirmed that purified protein was Hap s .
• N-terminal amino acid sequence To confirm the identity of purified Hap s , protein was resolved by SDS-PAGE, then electrotransferred to a polyvinylidene membrane. After staining with Coomassie brilliant blue R-250, protein was excised from the membrane and submitted to Midwest Analytical, Inc. (St. Louis, MO). Amino-terminal sequence determination was performed by automated Edman degradation using a Perkin-Elmer Biosystems model 477A sequencing system.
• mice Intranasal immunization of mice. Groups often, 6-week old, female Balb/c mice were immunized intranasally with Hap s purified from either strain P860295 or strain Nl 87. Hap s was diluted in Dulbecco's PBS (D-PBS) to a final concentration of 5 or 15 ⁇ g/40 ⁇ l, with or without 0.1 ⁇ g CT-
• D-PBS Dulbecco's PBS
• E29H (a mutant cholera toxin used as an adjuvant) (Tebbey, et al, 2000, Vaccine 18:2723-34). Control mice received D-PBS alone or D-PBS with 0.1 ⁇ g CT-E29H, again in 40 ⁇ l volumes.
• mice Prior to intranasal immunization, mice were anesthetized with an injectable mixture of ketamine (0.008 mis x body weight)/xylazene (0.007 mis x body weight), a mixture that maintains a state of anesthesia for 15-20 minutes. Immunizing preparations were delivered by pipette in a volume of 20 ⁇ l/nostril. The pipette was positioned so that the tip touched the opening of the nostril and liquid was drawn into the nasopharynx during breathing. Immediately following immunization, mice were placed in a supine position for a 3 to 5 minutes. Mice were immunized at weeks 0, 1, 3, and 5.
• mice Intranasal challenge of mice. Either two or three weeks after the final immunization, animals were challenged intranasally with approximately 1 x 10 6 CFU of strain TN106.P2.
• the TN106.P2 challenge strain was prepared for challenge by first inoculating three BBL Chocolate II agar plates from frozen stocks. Plates were incubated overnight at 37°C in 5% C0 2 . Five ml of D-PBS was added to each plate and bacteria were resuspended with a curved glass rod. Bacteria from all three plates were combined with an additional 10 ml of D-PBS and the suspension was poured over a D- PBS pre-wetted nylon wool column to remove clumps of bacteria and debris.
• This suspension was used for challenge. Mice were anesthetized as described for immunization, and 5 ⁇ l of bacteria were administered in each nostril. Twenty minutes after the challenge began, an aliquot of the bacterial suspension was diluted in D-PBS and plated onto BHI-XV agar to determine the actual inoculum. Three days after challenge, nasal tissue was harvested, weighed, homogenized and plated on BHI-XV plates containing 100 ⁇ g/ml streptomycin. Following incubation of plates overnight, colonies were counted, and CFU/g of nasal tissue were determined. Statistical differences among groups were analyzed using the Student t-test (JMP Software v3.2.1)
• Serum antibody responses Animals immunized IN will primarily produce a secretory immune response. Addition of CT-E29H increases the secretory immune response and also helps induce a serum antibody response. The volumes of the immunogens used in this experiment (40 ⁇ l) probably resulted in some of the material being aspirated into the mice's lungs, further increasing the immune response in the serum.
• the anti-Hap s ELISA titers of the sera obtained from immunized mice are shown in Table 2. The titers are somewhat lower than those usually seen with parenteral immunization since animals were immunized via the IN route. Significant increases in anti-Hap s titers were seen in the sera.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Communicable Diseases (AREA)
• Biochemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Wood Science & Technology (AREA)
• Biophysics (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Pharmacology & Pharmacy (AREA)
• Microbiology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Immunology (AREA)
• Biotechnology (AREA)
• Pulmonology (AREA)
• Epidemiology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Mycology (AREA)
• Oncology (AREA)
• Gastroenterology & Hepatology (AREA)
• General Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=33129591

Family Applications (1)

Country Status (5)

Families Citing this family (1)

Family Cites Families (1)

• 2003
  • 2003-02-19 WO PCT/US2003/005226 patent/WO2003072800A2/en not_active Ceased
  • 2003-02-19 AU AU2003219832A patent/AU2003219832A1/en not_active Abandoned
  • 2003-02-19 CA CA002476666A patent/CA2476666A1/en not_active Abandoned
  • 2003-02-19 EP EP03716109A patent/EP1480997A4/de not_active Ceased
  • 2003-02-19 BR BR0303226-4A patent/BR0303226A/pt not_active IP Right Cessation

Also Published As

Similar Documents

Legal Events