EP0920513A2 - DEFINITION D'EPITOPES DE LA PROTEINE CopB DE MEMBRANE EXTERNE DE MORAXELLA CATARRHALIS - Google Patents

DEFINITION D'EPITOPES DE LA PROTEINE CopB DE MEMBRANE EXTERNE DE MORAXELLA CATARRHALIS

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Publication number
EP0920513A2
EP0920513A2 EP97939404A EP97939404A EP0920513A2 EP 0920513 A2 EP0920513 A2 EP 0920513A2 EP 97939404 A EP97939404 A EP 97939404A EP 97939404 A EP97939404 A EP 97939404A EP 0920513 A2 EP0920513 A2 EP 0920513A2
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European Patent Office
Prior art keywords
peptide
seq
copb
catarrhalis
amino acid
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EP97939404A
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German (de)
English (en)
Inventor
Eric J. Hansen
Leslie D. Cope
Christoph Aebi
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University of Texas System
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University of Texas System
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    • 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
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • 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 present invention relates generally to the fields of microbiology and clinical bacteriology.
  • sequences of the CopB outer membrane protein from various strains of Moraxella catarrhalis provide useful targets in immunodiagnosis and immunoprophylaxis.
  • M. catarrhalis previously known as Branhamella catarrhalis or Neisseria catarrhalis was a harmless saprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990).
  • this organism is an important human pathogen. Indeed, it has been established that this Gram-negative diplococcus is the cause of a number of human infections (Murphy, 1989).
  • M. catarrhalis is now known to be the third most common cause of both acute and chronic otitis media in children and accounts for up to 20% of cases ( Aspin et al. , 1994; Del Beccaro et al, 1992; Gan et al, 1991; Catlin, 1990; Faden et al, 1990;1991; Marchant, 1990), the most common disease for which infants and children receive health care according to the 1989 Consensus Report.
  • This organism also causes acute maxillary sinusitis, generalized infections of the lower respiratory tract (Murphy and Loeb, 1989), and is an important cause of bronchopulmonary infections in patients with underlying chronic lung disease and, less frequently, of systemic infections in immunocompromised patients (Melendez and Johnson, 1990; Sarubbi et al, 1990; Schonheyder and Ejlertsen, 1989; Wright and Wallace, 1989).
  • M. catarrhalis DNA could be detected by polymerase chain reaction (PCRTM) in middle ear fluid from 46% of patients with chronic otitis media with effusion (Post et al, 1995).
  • PCRTM polymerase chain reaction
  • M. catarrhalis is a frequent cause of acute exacerbations of chronic obstructive pulmonary disease (Davies and Maesen, 1988; Hager et al, 1987; McLeod et al, 1986; Nicotra et al, 1986).
  • Invasive infections with this organism such as bacteremia, meningitis, skeletal infections and endocarditis, are rare and occur mainly in immunocompromised individuals (Doern et al, 1981 ; Malkamaki e/ ⁇ /., 1983).
  • catarrhalis is inversely related to the prevalence of nasopharyngeal colonization and incidence of otitis media involving M. catarrhalis (Goldblatt et al, 1990b; Vaneechoutte et al, 1990). (iii) Passive immunization with M. catarrhalis- ⁇ i ⁇ ected antibodies as well as active immunization with M. catarrhalis outer membrane proteins enhanced pulmonary clearance of M. catarrhalis in an animal model (Helminen et al, 1993a; 1994; Maciver ⁇ ?/ ⁇ /., 1993).
  • M. catarrhalis antigens that would serve as potentially important targets of the human immune response to infection (Murphy, 1989; Goldblatt et al, 1990; Murphy et al, 1990).
  • OMPs outer membrane proteins
  • LOS lipooligosaccharide
  • fimbriae fimbriae.
  • M. catarrhalis appears to be somewhat distinct from other Gram-negative bacteria in that attempts to isolate the outer membrane of this organism using detergent fractionation of cell envelopes has generally proven to be unsuccessful in that the procedures did not yield consistent results (Murphy, 1989; Murphy and Loeb, 1989).
  • preparations were found to be contaminated with cytoplasmic membranes, suggesting an unusual characteristic of the M. catarrhalis cell envelope.
  • OMPs have been grouped into classes A-H, beginning with bands of molecular weight around 98 kD (OMP-A) and proceeding to bands with molecular weights of about 21 kD (OMP-H) (Murphy and Loeb, 1989; Murphy, 1989).
  • CopB OMP designated CopB
  • M catarrhalis CopB outer membrane protein appears to be fairly well conserved among strains of M. catarrhalis, with the CopB-specific monoclonal antibody (MAb) 10F3 binding to the majority (70%) of 23 M. catarrhalis strains tested, suggesting that a strain-common epitope is present in many, but not all, strains of this pathogen.
  • Southern blot analysis results also suggest some degree of conservation of the copB gene at the nucleotide sequence level among different strains.
  • polyclonal antisera raised against outer membrane vesicles from two MAb 10F3- unreactive strains, TTA24 and B21 , reacted with the recombinant CopB protein encoded by the copB gene which was derived from the M. catarrhalis 10F3-reactive strain O35E, indicating some conservation of the protein structure of the CopB protein among the strains.
  • M. catarrhalis The lack of a polysaccharide capsule surrounding M. catarrhalis indicates that surface-exposed outer membrane antigens are the likely targets for a protective immune response.
  • Different M. catarrhalis strains share remarkably similar outer membrane protein profiles (Bartos and Murphy, 1988; Murphy and Bartos, 1989), and at least three surface-exposed proteins of this organism appear to be well-conserved antigenically (Helminen et al , 1993a; 1994; Hsiao et al, 1995; Murphy et al, 1993).
  • OMP B2 80 kDa CopB protein
  • CopB expression is iron-regulated (Aebi et al, 1996; Campagnari et al, 1994) and that CopB is involved at some level in the ability of M. catarrhalis to acquire iron from human transferrin and lactoferrin (Aebi et al, 1996).
  • Expression of CopB is apparently essential for virulence of M. catarrhalis, at least in an animal model, because an isogenic copB mutant was less able than its wild-type parent strain to resist clearance from the lungs of mice (Helminen et al, 1993b).
  • CopB of M. catarrhalis It is also a goal to provide methods for the diagnosis and prevention of M. catarrhalis infections using these polypeptide and nucleic acids, and derivatives thereof. It is a further goal of the present invention to screen for agents that may be used both in diagnosis and immunoprophylaxis of M. catarrhalis infections.
  • an isolated peptide of about 5 to about 60 amino acids comprising the consecutive amino acid sequence KYAGK (SEQ ID NO:35) which is reactive with the antibody 10F3.
  • the consecutive residues of the isolated peptide may further comprise the amino acid residues KYAGKG (SEQ ID NO:36), KYAGKGY (SEQ ID NO:37), NKYAGK (SEQ ID NO:35).
  • NKYAGKG SEQ ID NO:39
  • NKYAGKGY SEQ ID NO:40
  • the isolated peptide may comprise at least about 20 consecutive residues of the amino acid sequence LDIEKDKKKRTDEQLQAELDNKYAGKGY (SEQ ID NO:41) or LDIKKDDKTLTETELQAELDNKYAGKGY (SEQ ID NO:42).
  • the isolated peptide may be about 5, 7, 10, 15, 20, 30, 40, 50 or 60 amino acids in length.
  • an isolated peptide of about 20 to about
  • amino acids comprising at least about 20 consecutive residues of the amino acid sequence
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO:43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45).
  • the isolated peptide may be about 20, 30, 40, 50 or 60 amino acids in length.
  • an antigenic composition comprising (a) an isolated peptide of about 5 to about 60 amino acids comprising the amino acid sequence KYAGK (SEQ ID NO:35) and (b) a pharmaceutically acceptable buffer or diluent.
  • the an antigenic composition comprises (a) an isolated peptide of about 20 to about 60 amino acids comprising the amino acid sequence
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO:43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45).
  • the antigenic composition may further comprise a carrier conjugated to said peptide.
  • the carrier may be KLH.
  • the antigenic composition may further comprise an adjuvant.
  • the antigenic composition may comprise a peptide is covalently linked to a second antigen.
  • the second antigen may be a peptide antigen or a non-peptide antigen.
  • a vaccine composition comprising (a) an isolated peptide of about 5 to about 60 amino acids comprising the amino acid sequence KYAGK (SEQ ID NO:35) and (b) a pharmaceutically acceptable buffer or diluent.
  • the an antigenic composition comprises an isolated peptide of about 20 to about 60 amino acids comprising the amino acid sequence
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO:43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45)
  • a method for inducing an immune response in a mammal comprising the step of providing to said mammal an antigenic composition comprising (a) an isolated peptide of about 5 to about 60 amino acids comprising the amino acid sequence KYAGK (SEQ ID NO:35) and (b) a pharmaceutically acceptable buffer or diluent.
  • an antigenic composition comprises (a) an isolated peptide of about 20 to about 60 amino acids comprising the amino acid sequence
  • an isolated and purified M. catarrhalis CopB antigen comprising the amino acid sequence KYAGK (SEQ ID NO:35), or in still a further embodiment
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO:43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45)
  • CopB antigen of the M. catarrhalis isolates TTA24, 012E and 046E are also provided. Also provided are copB DNA sequences of the M. catarrhalis isolates TTA24 (SEQ ID NO: 34), 012E (SEQ ID NO:28) and 046E (SEQ ID NO:32).
  • a method for generating a strain-specific antibody to M. catarrhalis CopB antigen comprising the step of providing to a mammal an antigenic composition comprising (a) an isolated peptide comprising an amino acid sequence corresponding to residues of variable regions of said CopB antigen and (b) a pharmaceutically acceptable buffer or diluent.
  • M. catarrhalis infection comprising the step of determining the presence, in a sample, of an M. catarrhalis, amino acid sequence corresponding to residues of variable regions of said CopB antigen.
  • the step of determining may comprise PCR or immunologic reactivity of an antibody with an M. catarrhalis antigen.
  • a method for generating a strain-common antibody to M. catarrhalis CopB antigen comprising the step of providing to a mammal an antigenic composition comprising (a) an isolated peptide comprising an amino acid sequence corresponding to residues of common regions of said
  • CopB antigen and (b) a pharmaceutically acceptable buffer or diluent.
  • M. catarrhalis infection comprising the step of determining the presence, in a sample, of an M. catarrhalis amino acid sequence corresponding to residues of common regions of said CopB antigen.
  • the step of determining may comprise PCR or comprises immunologic reactivity of an antibody with an M. catarrhalis antigen.
  • a method for treating an individual having an M. catarrhalis infection comprising providing to said individual an isolated peptide of about 20 to about 60 amino acids comprising at least the consecutive residues KYAGK (SEQ ID NO.35) or about 20 consecutive residues of the amino acid sequence
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO.43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45).
  • a method for preventing or limiting an M. catarrhalis infection comprising providing to a subject an antibody that reacts immunologically with an epitope formed by the amino acid sequence
  • LDIEKDKKKRTDEQLQAELDDKYAGKGY (SEQ ID NO:43), LDIEKNKKKRTEAELQAELDDKYAGKGY (SEQ ID NO:44) or IDIEKKGKIRTEAELLAELNKDYPGQGY (SEQ ID NO:45).
  • a method for screening a peptide for reactivity with a CopB antibody comprising the steps of (a) providing said peptide; (b) contacting said peptide with an antibody that binds immunogically to CopB; and (c) determining the binding of said antibody to said peptide.
  • the antibody may be 10F3, and the determining may comprise an immunoassay selected from the group consisting of a Western blot, an ELISA, an RIA and immunoaffinity separation.
  • a method for screening a peptide for the ability to induce a protective immune response against M. catarrhalis comprising the steps of (a) providing said peptide; (b) administering a peptide in a suitable form to an experimental animal; (c) challenging said animal with M. catarrhalis; and (d) assaying the infection of said animal with M. catarrhalis.
  • the animal may be a mouse, the challenge is a pulmonary challenge, and the assaying comprises assessing the degree of pulmonary clearance by said mouse.
  • the peptide is a CopB peptide, and more preferably, the CopB peptide encompasses residues 296-300 of M. catarrhalis strain 035E.
  • the preferred embodiment may employ a CopB peptide is a peptide having at least 6 consecutive amino acids from M. catarrhalis strain 035E, including residue 295.
  • an isolated peptide having at least about 5 consecutive amino acids from the CopB protein of M. catarrhalis and including residues 296-300, or the analogous position thereof when compared to strain 035E or having about 6 consecutive amino acids from the CopB protein of M. catarrhalis, wherein said peptide includes residue 295, or the analogous position thereof when compared to strain 035E.
  • the isolated peptide may be between 5 and 60 amino acids in length.
  • the peptide may comprise non-CopB sequences and even non- catarrhalis sequences.
  • an antigen composition comprising (i) an isolated peptide having at least about 6 consecutive amino acids from the CopB protein of M. catarrhalis, wherein said peptide includes residue 295, or the analogous position thereof when compared to strain 035E and (ii) a pharmaceutically acceptable buffer or diluent.
  • FIG. 1A and FIG. IB Western blot of oligopeptide (region 1) antisera against
  • CopB antigen from each of strains 035E, 01 2E, 046E and TTA24 Outer membrane vesicles of the M. catarrhalis strains 035E (lane 1), 012E (lane 2), TTA24 (lane 3) and 046E (lane 4) probed with MAb 10F3 (FIG. 1A) or mouse antiserum raised against peptide from region 1 conjugated with KLH (FIG. IB).
  • FIG. 2 Amino acid sequence and reactivity with antibodies of both region 1 from CopB of four different M. catarrhalis strains and synthetic peptides derived from these sequences.
  • the numbers (i.e., 275-300) above the region 1 sequences indicate the amino acid positions in the intact CopB protein.
  • the numbers (i.e., 1-26) above the RI synthetic peptide indicate residue positions in the RI peptide and its derivatives. Boxes indicates residues differing from those in 035E (SEQ ID NO:l).
  • FIG. 3 Fine mapping of the MAb 10F3-reactive epitope using a dot blot assay involving immobilized synthetic decapeptides. Binding of MAb 10F3 to individual peptides was detected by incubating the membrane with radioiodinated goat anti-mouse immunoglobulin. The autoradiograph of the dot blot (peptides 1-8) is shown on the left, the corresponding amino acid sequence of each of the overlapping decapeptides with their respective positions in the CopB protein is listed in Table V. The numbers written vertically denote the amino acid positions in the intact CopB protein; the RI region is underlined.
  • FIG. 4 Hydropathy plot and secondary structure analysis of region 1 from the
  • CopB protein of M. catarrhalis strain 035E The hydropathy plot was generated by the method of Kyte and Doolittle (1982) using a window size of seven residues and secondary structure predictions were made using the method of Chou and Fasman (1978) as found in MacVector® sequence analysis software (Version 6.0). The numbers on the horizontal scale denote the amino acid positions in the intact CopB protein; the RI region is underlined.
  • FIG. 5A and FIG. 5B Western blot analysis of the reactivity of MAbs and polyclonal serum antibodies with outer membrane proteins of M. catarrhalis strains, the GST-26 fusion protein, and GST alone. Proteins present in outer membrane vesicles from strains 035E (lane 1), 012E (lane 2), TTA24 (lane 3), and 046E (lane 4), purified
  • GST-26 (lane 5) and GST (lane 6) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with MAb 10F3 (FIG. 5 A) or with mouse GST-26 antiserum (dilution 1 :1000) (FIG. 5B).
  • the CopB protein from strain 035E gave rise to a doublet reactive with MAb 10F3 in this study. Molecular weight position markers are shown on the left sides of the blots.
  • FIGS. 6A-6D Bactericidal activity of MAb 10F3 against M. catarrhalis strains. Suspensions of the MAb 10F3-reactive strains 035E (FIG. 6A) and 012E (FIG. 6B) and the MAb 10F3-unreactive strains TTA24 (FIG. 6C) and 046E (FIG. 6D) were incubated with: normal human serum (closed triangles), normal human serum and MAb 10F3 (open squares), heat-inactivated normal human serum and MAb 10F3 (closed squares), normal human serum and GST-26 antiserum (open circles) or normal human serum and GST antiserum (closed circles). Portions of the reaction mixture were removed over time and spread on BHI agar plates to determine the number of viable bacteria. Note that the open circles and open squares in FIG. 6B are superimposed.
  • the present invention relates to the identification of CopB outer membrane protein sequences from multiple strains of M. catarrhalis, and the nucleic acid sequences corresponding thereto.
  • the present invention provides insights into the antigenic structure of CopB, based on an analysis of the amino acid sequences. This information is useful in developing both strain-specific and strain-common reagents for the diagnosis of M. catarrhalis infection.
  • an understanding of which regions of the molecule are conserved permits the development of agents that will be useful in protecting against M. catarrhalis infections, e.g., in the preparation of vaccine reagents.
  • Particular embodiments relate to peptides, polypeptides and nucleic acids corresponding to the CopB molecules, peptides and antigenic compositions derived therefrom, and methods for the diagnosis and treatment of M. catarrhalis disease.
  • CopB represents an important antigenic determinant, as the monoclonal antibody (MAb) termed 10F3 has been shown to protect experimental animals, as measured in a pulmonary clearance model, when provided in passive immunizations.
  • MAb monoclonal antibody
  • the present invention by virtue of new information regarding the structure of CopB provides such improved compositions and methods.
  • the present invention provides for the identification of conserved and non-conserved portions of the CopB polypeptide.
  • a homology comparison illustrates at least ten discrete regions of low homology. These regions are designated region 1 (about residues 275-302), region 2 (about residues 322-329), region 3 (about residue 348), region 4 (about residues 400-416), region 5 (about residues 437-
  • region 6 (about residues 485-486), region 7 (about residues 526-541), region 8 (about residues 583-596), region 9 (about residues 637-640) and region 10 (about residues 683-695).
  • the numbering is based on the amino acid sequence of 035E strain (SEQ ID NO:29) which is encoding by SEQ ID NO:30 beginning at position 14.
  • the remaining regions of the molecule may be considered "conserved.” Of particular note in this regard are the regions from about residue 10 to about residue 270, from about residue 350 to about residue 395, from about residue 455 to about residue 480, from about residue 490 to about residue 520, from about residue 545 to about residue 580, from about residue 600 to about residue 635 and from about residue 645 to about residue 680. Again, the numbering is based on the 035E strain (SEQ ID NO:29).
  • the present invention has identified the specific epitope to which 10F3 binds.
  • this same region from the CopB protein from M. catarrhalis strain 012E (SEQ ID NO:2) is bound by 10F3, although there are differences in the identified region between these two strains.
  • Two other strains, 046E and TTA24 are not bound by 10F3.
  • sequences from the identified region of these strains contain differences in common to each other but not in common with 035E or 012E. It is clear that this region plays an important role in the biology of the pathogen and, from this information, one can deduce amino acids residues that are critical to and not critical to 10F3 antibody binding.
  • the specific epitope to which 10F3 binds is even further defined.
  • Peptides LDNKYAGKGY SEQ ID NO:25; peptide 7; residues 293-302 of the CopB protein of strain O35E
  • KYAGKGYKLG SEQ ID NO:26; peptide 8; residues 296-305 of the CopB protein of strain O35E
  • peptides 1 - 6 FIG. 3; Table V
  • the small peptide KYAGK (SEQ ID NO:35) is preferred for binding to 10F3
  • a more preferred peptide is
  • KYAGKG SEQ ID NO:36
  • an even more preferred peptide is KYAGKGY (SEQ ID NO:37).
  • the peptide sequence, LDNKYAGKGY allows maximal binding of MAb 10F3 compared to other peptides examined herein.
  • the asparagine (N) residue (position 3 of SEQ ID NO:25) appears to improve binding.
  • N asparagine residue
  • this position influences the binding kinetics of the epitope, because when aspartic acid (D) is substituted for N at this position, reactivity with 10F3 is abolished.
  • NKYAGK SEQ ID NO:38
  • a more preferred peptide is NKYAGKG (SEQ ID NO:39)
  • an even more preferred peptide is NKYAGKGY (SEQ ID NO:40).
  • MAb 10F3 with a surface-exposed epitope Helminen et al, 1993a
  • bactericidal activity of MAb 10F3 against strains that react with this MAb FGS. 6A-6D
  • the likely function of the CopB protein in a TonB-dependent physiologic process Aebi et al, 1996.
  • FrpB an iron-regulated outer membrane protein of Neisseria meningitidis that is 49% identical to the CopB protein of strain 035E, has also been shown to be a target for bactericidal antibody (Pettersson et al, 1990; 1995).
  • FIG. 3 10F3-reactive epitope using overlapping decapeptides (FIG. 3) which demonstrated optimal binding of MAb 10F3 to a decapeptide (peptide 7; SEQ ID NO:25) that corresponded to the residues 19-28 of region 1 (i.e. , residues 293-302 of the CopB protein).
  • This epitope comprises the C-terminal portion of a highly hydrophilic domain when analyzed by means of the hydropathy algorithm of Kyte and Doolittle (1982) (FIG.
  • Haemophilus influenzae for which it has been demonstrated that some surface-exposed domains exhibit strain-specific antigenicity (Duim et al, 1994; 1996).
  • CopB protein of the equally MAb 10F3-reactive strain 012E (FIG. 5B).
  • This result suggested that these polyclonal antibodies did not possess the same antigenic specificity as MAb 10F3.
  • the strong reactivity of the GST-26 antiserum with the MAb 10F3-unreactive peptides RIB (SEQ ID NO:3; FIG. 2) and R1-TTA24 (SEQ ID NO:4; FIG. 2) suggested that most of these polyclonal antibodies are directed against epitopes of peptide RI (SEQ ID NO:l ) other than that which binds MAb 10F3.
  • CopB protein among strains of this pathogen was crucial for localization of the protective epitope on the CopB protein of M. catarrhalis strain 035E that is recognized by the bactericidal MAb 10F3.
  • Immunization with a fusion protein reactive with MAb 10F3 induced the synthesis of CopB-specific antibodies bactericidal for at least one MAb 1 OF 3 -reactive strain of M. catarrhalis. It is important to note that screening methods for diagnosis and prophylaxis are readily available, as set forth below.
  • test peptides, mutant peptides and antibodies for their reactivity with each other and test peptides and antibodies for the ability to prevent infections in vivo provide powerful tools to develop clinically important reagents.
  • the present invention in one embodiment, encompasses the three new amino acid sequences presented in SEQ ID NO:27, SEQ ID NO:31 and SEQ ID NO:33.
  • hybrid molecules containing portions from the CopB of one strain fused with portions of the CopB from another strain For example, one may fuse residues 1-350 of strain TTA24 CopB with residues 351-759 of strain 035E CopB.
  • a fusion could be generated with sequences from three or even four strains represented in a single CopB molecule, thus combining epitopes from each of the various strains.
  • CopB molecules also encompassed are fragments of the disclosed CopB molecules, as well as insertion, deletion or replacement mutants in which non-CopB sequences are introduced, CopB sequences are removed, or CopB sequences are replaced by or fused with non-CopB sequences, respectively.
  • non-CopB sequences are introduced, CopB sequences are removed, or CopB sequences are replaced by or fused with non-CopB sequences, respectively.
  • CopB may be advantageously cleaved into fragments for use in further structural or functional analysis, or in the generation of reagents such as CopB-related polypeptides and CopB-specific antibodies. This can be accomplished by treating purified or unpurified CopB with a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN). Treatment with CNBr is another method by which CopB fragments may be produced from natural CopB. Recombinant techniques also can be used to produce specific fragments of CopB.
  • a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN).
  • Treatment with CNBr is another method by which CopB fragments may be produced from natural CopB. Recombinant techniques also can be used to produce specific fragments of CopB.
  • amino acids may be substituted for other amino acids in a protein structure in order to modify or improve its antigenic or immunogenic activity (see, e.g., Kyte & Doolittle, 1982; Hopp, U.S. Patent 4,554,101, incorporated herein by reference).
  • substitution of alternative amino acids small conformational changes may be conferred upon a polypeptide which result in increased activity or stability.
  • amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide-molecule conjugates which retain enough antigenicity of the starting peptide to be useful for other purposes.
  • a selected CopB peptide that may be covalently bound to a solid support may be constructed which would have particular advantages in diagnostic embodiments.
  • amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with substrate molecules.
  • substitutions which result in an antigenically equivalent CopB peptide or CopB protein will generally involve amino acids having index scores within ⁇ 2 units of one another, and more preferably within ⁇ 1 unit, and even more preferably, within ⁇ 0.5 units.
  • isoleucine which has a hydropathic index of ⁇ 4.5
  • an amino acid such as valine (+4.2) or leucine (+3.8).
  • lysine (-3.9) will preferably be substituted for arginine (-4.5), and so on.
  • one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent CopB peptide or protein.
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like.
  • preferred substitutions which take various of the foregoing characteristics into consideration will be known to those of skill in the art and include, for example, the following combinations: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • peptides derived from these polypeptides are contemplated.
  • such peptides may comprise about 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 consecutive residues.
  • a peptide that comprises 6 consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3 to 8 and so on of the CopB protein.
  • Such peptides may be represented by the formula
  • x to (x + n) amino to carboxy residues of the first and last consecutive residues
  • Syntheses of CopB peptides are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of 7.0 to 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • the peptide(s) are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled, deionized) or buffer prior to use.
  • An epitope is a region of a molecule that stimulates a response from a T-cell or B-cell, and hence, elicits an immune response from these cells.
  • An epitopic core sequence is a relatively short stretch of amino acids that is structurally and biochemically “complementary” to, and therefore will bind to, binding sites on antibodies or T-cell receptors.
  • Antibody binding sites also may be termed "linear" epitopes, as the residues that are involved in the actual physical interaction of antigen and antibody are linear, or at least in a discrete region of the molecule.
  • Other epitopes may be non-linear in that widely separated regions of the molecule participate in the interaction. Operationally, these two types of epitopes may be distinguished on the basis of binding in denaturing Western blot analysis - positive binding indicates linear, negative binding indicates nonlinear.
  • the identification of epitopic core sequences is known to those of skill in the art. For example U.S. Patent 4,554,101 teaches identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity, and by Chou-Fasman analyses. Numerous computer programs are available for use in predicting antigenic portions of proteins, examples of which include those programs based upon Jameson-Wolf analyses
  • Epitopic core sequences of the present invention also may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the corresponding CopB antigen to the corresponding CopB- directed antisera.
  • immunoassays may be employed to screen for reactivity of these molecules with previously developed immunoreagents.
  • new antibodies and antisera, based on these variants may be generated in order test their reactivity with existing "native" or "wild-type” molecules.
  • availability of an art-recognized model for M. catarrhalis provides a ready means for testing variant in vivo for their ability to protect subjects from M. catarrhalis infections.
  • variable region 1 it is clear that it should be possible to substitute specific amino acids of the 10F3 epitope of region 1 with other amino acids and still retain, and possibly even improve upon, the reactivity of the epitope with 10F3 or other antibodies that recognize the CopB protein of Moraxella.
  • Which amino acids may be interchanged is dependent upon several factors, including but not limited to, the hydropathicity, hydrophilicity, polarity and tertiary structure of the targeted amino acid as well as other amino acids proximal to the targeted residue which may influence its characteristics.
  • the analysis of variable region 1 is described below.
  • the fourth position of region 1 may be at least either glutamate or lysine and not adversely affect reactivity with 10F3. Both of these amino acids are charged residues, but glutamate is negative and lysine is positive; therefore, it is reasonable to speculate that any charged residue may be present in this position and still result in a protein or peptide that binds to the 10F3 antibody. This data suggests that the residue at position 4 is unlikely to be critical for strain-specific antibody binding. But as indicated above, other factors may affect the ability of one amino acid to be substituted for another without causing an undesired effect. Mutated peptides, polypeptides and proteins resulting from such substitutions could be easily screened for reactivity using the immunoassays described in the instant specification.
  • position 6 is occupied by aspartate, a negatively charged amino acid.
  • strains TTA24 and 046E which do not bind 10F3 the homologous position is occupied by either a positively charged lysine or a weakly basic asparagine. If one were to only consider the charge of the amino acids with regard to antibody binding then one would deduce that position 6 may be critical to reactivity with 10F3; however, data disclosed herein show that position 6 alone does not determine reactivity with 10F3. Other factors appear to be more important for 10F3 binding. But amino acids at position 6 could have a synergistic effect upon reactivity with 10F3 or other antibodies. Such an assertion could be tested as indicated.
  • Position 7 may be occupied by either lysine or aspartate and still result in a peptide that binds to 10F3. Given the analysis of positively and negatively charged residues described previously, it is reasonable to deduce that any one of lysine, arginine, histidine, aspartate or glutamate could also be present at the seventh position of a 10F3- binding peptide. Further residues may also occupy this position and still result in a peptide that retains 10F3 antibody binding.
  • Position 9 is occupied by either a positively charged lysine or a neutral amino acid in both strains that react or do not react with 10F3. It is possible that other positively charged or even neutral amino acids may occupy position 9. At position 10 of each of the currently known 10F3-binding species there is a positively charged amino acid. However, as 035E contains arginine at this position and 012E contains lysine, it seems reasonable to propose that histidine, another positively charged amino acid, could also be present at this position in a 10F3-binding peptide.
  • Position 20 is occupied by either the negatively charged aspartate or by the weakly basic asparagine in strains that bind 10F3, but as this position is also occupied by aspartate in the strains that do not bind 10F3 little can be deduced about its importance in the reactivity of the region. But as indicated earlier, its role could be easily determined by preparing mutant peptides, polypeptides or proteins and screening them for reactivity with 10F3 or another antibody as described in the instant specification.
  • Position 21 is occupied by only the weakly basic asparagine in the 035E and 012E strains. In TTA24 and 046E it is occupied by the negatively charged aspartate and the positively charged lysine, respectively. Although there are significant differences in charge between the residues, there is no pattern other than charge. Nonetheless, data described herein indicate that position 21 does indeed contain the key residue for determining maximal binding to 10F3.
  • Positions 22 and 26 of the region are both occupied by either the positively charged lysine or by one of two negatively charged residues. As there is no apparent relationship to 10F3 binding associated with either positively or negatively charged residues at this position. It is. reasonable to assume that these residue do not play a important roles in strain-specific reactivity given their current, surrounding residues. But if proximal residues are altered, then residues at either of these positions may exert an effect on antibody binding.
  • the present invention also encompasses nucleic acids encoding the CopB antigens of SEQ ID NO:27, SEQ ID NO:31 and SEQ ID NO:33 which are provided as SEQ ID NO:28, SEQ ID NO:32 and SEQ ID NO:34, respectfully.
  • many other nucleic acids also may encode a given CopB, however.
  • four different three-base codons encode the amino acids alanine, glycine, proline, threonine and valine, while six different codons encode arginine, leucine and serine. Only methionine and tryptophan are encoded by a single codon.
  • Table I provides a list of amino acids and their corresponding codons for use in such embodiments.
  • codon table provided herein.
  • substitution of the natural codon with any codon encoding the same amino acid will result in a distinct nucleic acid that encodes CopB.
  • this can be accomplished by site-directed mutagenesis of an existing copB gene or de novo chemical synthesis of one or more nucleic acids.
  • the nucleic acids of the present invention provide for a simple way to generate fragments (e.g., trunications) of CopB, CopB-CopB fusion molecules (discussed above) and CopB fusions with other molecules.
  • fragments e.g., trunications
  • utilization of restriction enzymes and nuclease in the copB gene permits one to manipulate the structure of these genes, and the resulting gene products.
  • Vectors and cloning strategies are well known to those of skill in the art.
  • nucleic acid sequence information provided by the present disclosure also allows for the preparation of relatively short DNA (or RNA) sequences that have the ability to specifically hybridize to gene sequences of the selected copB gene.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of the coding sequence of the copB gene, or flanking regions near the cop B gene, such as regions downstream and upstream in the M. catarrhalis chromosome.
  • the ability of such nucleic acid probes to specifically hybridize to copB gene sequences lends them particular utility in a variety of embodiments.
  • the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • these oligonucleotides can be inserted, in frame, into expression constructs for the purpose of screening the corresponding peptides for reactivity with existing antibodies or for the ability to generate diagnostic or prophylactic reagents.
  • they may be utilized to generate mutants by site- directed mutagenesis.
  • the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 20, or so, nucleotide stretch of the sequence, although sequences of 30 to 60 or so nucleotides are also envisioned to be useful. Such molecules may be term oligonucleotides or probes. A size of at least 9 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of the specific hybrid molecules obtained.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
  • oligos or probes that would be useful may be derived from any portion of the sequences of strains TTA24 (SEQ ID NO:34), 012E (SEQ ID NO:28) or 046E (SEQ ID NO:34),
  • oligos or probes are specifically contemplated that comprise nucleotides 1 to 9, or 2 to 10, or 3 to 1 1 and so forth up to a probe comprising the last 9 nucleotides of the nucleotide sequence of TTA24 (SEQ ID NO:34), 012E (SEQ ID NO:34),
  • each probe would comprise at least about 9 linear nucleotides of the nucleotide sequence of TTA24 (SEQ ID NO:34), 012E (SEQ ID NO:32).
  • n is an integer from 1 to the number of nucleotides in the sequence, less the length of the probe -
  • the probes of the present invention will find particular utility as the basis for diagnostic hybridization assays for detecting copB genes in clinical samples.
  • Exemplary clinical samples that can be used in the diagnosis of infections are thus any samples which could possibly include Moraxella nucleic acid, including middle ear fluid, sputum, mucus, bronchoalveolar fluid, amniotic fluid or the like.
  • a variety of hybridization techniques and systems are known which can be used in connection with the hybridization aspects of the invention, including diagnostic assays such as those described in Falkow et al, U.S. Patent 4,358,535.
  • relatively stringent conditions for example, one will select relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
  • mutant clone colonies growing on solid media which contain variants of the copB nucleic acid sequence could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to obtain hybridization only between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies.
  • small hybridization probes containing short variant sequences of the copB gene may be utilized to identify those clones growing on solid media which contain sequence variants of the entire copB gene. These clones can then be grown to obtain desired quantities of the variant copB nucleic acid sequences or the corresponding CopB antigen.
  • nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples. Biotin/avidin- based reagents also may be employed, where one of these binding partners is attached to the probe.
  • the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase.
  • the test DNA (or RNA) from suspected clinical samples such as exudates, body fluids (e.g., amniotic fluid, middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • specific hybridization is detected, or even quantified, by means of the label.
  • the nucleic acid sequences which encode for the CopB antigens, or their variants may be useful in conjunction with polymerase chain reaction (PCR) methodology to detect M. catarrhalis.
  • PCR polymerase chain reaction
  • Patent 4,60,102 one may utilize various portions of the copB gene sequence as oligonucleotide primers for the PCR amplification of a defined portion of a copB nucleic acid in a sample.
  • the amplified portion of the copB sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of M. catarrhalis nucleic acid may detected in a sample utilizing CopB sequences.
  • nucleic acids of the present invention may be used advantageously to produce recombinant CopB protein.
  • M. catarrhalis cells may be grown on agar plates using brain heart infusion broth as a medium. Plates are incubated at 37°C in candle extinction jars. Outer membrane fragments may be subsequently prepared from these cells by the EDTA-based extraction procedure of Murphy et al.
  • recombinant clones bearing DNA segments encoding CopB were deposited with the American Type Culture Collection (ATCC) on August 4, 1992, under the provisions of the Budapest Treaty.
  • ATCC American Type Culture Collection
  • plasmid pMEH 120 (ATCC accession number 75285) bearing a nucleic segment encoding the CopB antigen of strain 035E was deposited in the form of purified plasmid DNA. Further the nucleotide sequences of the copB genes from M.
  • catarrhalis strains TTA24, 012E and 046E have been submitted to GenBank and assigned the accession numbers U69980 (SEQ ID NO:34), U69981 (SEQ ID NO:28), and U69982 (SEQ ID NO:32), respectively.
  • the gene copB encoding the CopB protein of strain 035E, was originally cloned in a pBR322-based recombinant plasmid, designated pMEHlOO. Subsequently, this gene was subcloned in pBluescript for sequencing analysis.
  • This new plasmid, designated pMEH120 is what was deposited with the ATCC.
  • Recombinant plasmid pMEH120 is a pBluescript II SK+ vector containing an insert of M. catarrhalis chromosomal NA approximately 4.5 kb in size, and encodes a protein of about 80 kD that is reactive with monoclonal antibody 10F3.
  • CopB protein may be isolated from cultures of M. catarrhalis, it may prove more efficient to generate the protein in other host cell systems.
  • the synthesis of mutant polypeptides, the nucleic acids for which must be generated outside the M. catarrhalis genome, also will likely be produced most effectively in a recombinant system.
  • the expression cassette contains a CopB-encoding nucleic acid trader transcriptional control of a promoter.
  • a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrase "under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • Those promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems
  • the appropriate expression cassette can be inserted into a commercially available expression vector by standard subcloning techniques.
  • the E. coli vectors pUC or pBluescript may be used according to the present invention to produce recombinant CopB polypeptide in vitro.
  • the manipulation of these vectors is well known in the art.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example,
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al, 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as a transforming vector in connection with these hosts.
  • the phage lambda GEMTM- 11 may be utilized in making recombinant phage vector which can be used to transform host cells, such as E. coli LE392.
  • the protein is expressed as a fusion protein with ⁇ -gal, allowing rapid affinity purification of the protein.
  • fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA).
  • both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the protein to its native conformation.
  • Other fusion systems produce proteins where it is desirable to excise the fusion partner from the desired protein.
  • the fusion partner is linked to the recombinant protein by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA).
  • E coli is a preferred prokaryotic host.
  • E coli strain RR1 is particularly useful.
  • Other microbial strains which may be used include E coli strains such as E coli LE392, E. coli B, and E coli X 1776 ,(ATCC No. 31537).
  • the aforementioned strains, as well as E coli W31 10 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used. These examples are, of course, intended to be illustrative rather than limiting.
  • Recombinant bacterial cells for example E coli
  • E coli are grown in any of a number of suitable media, for example LB, and the expression of the recombinant polypeptide induced by adding IPTG to the media or switching incubation to a higher temperature.
  • the cells are collected by centrifugation and washed to remove residual media.
  • the bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed trader conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed.
  • the recombinant protein is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as ⁇ -mercaptoethanol or dithiothreitol (DTT).
  • urea e.g. 8M
  • chaotropic agents such as guanidine hydrochloride
  • DTT dithiothreitol
  • polypeptide may be advantageous to incubate the polypeptide for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein.
  • conditions generally include low protein concentrations less than 500 ⁇ g/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulphide bonds within the protein molecule.
  • the refolding process can be monitored, for example, by SDS-PAGE or with antibodies which are specific for the native molecule.
  • the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
  • the expression system may be eukaryotic in origin.
  • the expression system used is the insect baculovirus system, and the expression cassette comprises the baculovirus polyhedron promoter.
  • the gene encoding the protein can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector.
  • a preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA).
  • the vector carrying the copB gene is transfected into Spodoptera afrugiperda (e.g., Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant protein.
  • the expression construct may comprise a virus or engineered construct derived from a viral genome.
  • viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • the first viruses used as vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoassociated viruses. Retroviruses also are attractive gene transfer vehicles (Nicolas and Rubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) adeno- associated virus (Ridgeway, 1988) and HSV (Glorioso et al, 1995). Such vectors may be used to (i) transform cell lines in vitro for the purpose of expressing proteins of interest or (ii) to transform cells in vitro or in vivo to provide protective polypeptides.
  • papovaviruses simian virus 40, bovine papilloma virus, and polyoma
  • Retroviruses also
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • Preferred promoters include those derived from HSV, including ⁇ 4 promoter.
  • Another preferred embodiment is the tetracycline controlled promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • Tables II and III list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of a transgene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof.
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic Promoter Data Base EPDB any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a transgene.
  • Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Host cells include eukaryotic microbes, such as yeast cultures may also be used.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al, 1979; Kingsman et al, 1979; Tschemper et al, 1980). This plasmid already contains the t ⁇ l gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1
  • Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, 1980) or other glycolytic enzymes (Hess et al,
  • enolase such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and
  • HeLa cells Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in from of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • Antibodies that react immunologically with CopB polypeptides or peptides derived therefrom may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. , purified or partially purified protein, synthetic protein or fragments thereof, as discussed in the section on vaccines.
  • Animals to be immunized are mammals such as cats, dogs and horses, although there is no limitation other than that the subject be capable of mounting an immune response of some kind.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells) are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed.
  • Spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x IO 7 to 2 x IO 8 lymphocytes.
  • the antibody-producing B cells from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas.”
  • any one of a number of myeloma cells may be used and these are known to those of skill in the art.
  • the immunized animal is a mouse
  • NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
  • P3-NS-l-Ag4-l NS-1 myeloma cell line
  • Mutant Cell Repository by requesting cell line repository number GM3573.
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20: 1 to about 1 :1 , respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate.
  • Fusion procedures usually produce viable hybrids at low frequencies, about
  • the selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single- clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art.
  • Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, i.e., hybrids of two or more antibody molecules.
  • monoclonal antibodies according to the invention are chimeric monoclonal antibodies.
  • the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene.
  • the antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.
  • monoclonal antibodies according to the present invention is a "humanized” monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counte ⁇ arts.
  • CDRs mouse complementary determining regions
  • Humanized monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and prophylactic methods for treating Moraxella infections.
  • the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art.
  • Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogen
  • Monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo.
  • Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
  • fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.
  • the monoclonal conjugates of the present invention are prepared by methods known in the art, e.g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as H, I, I P, 3 S, C, Cr, CI, Co, Co, Fe, 3 Se, Eu, and "mTc, are other useful labels which can be conjugated to antibodies.
  • Radioactively labeled monoclonal antibodies of the present invention are produced according to well- known methods in the art.
  • monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labeled with technetium- m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • a reducing agent such as SNC1 2
  • a buffer solution such as sodium-potassium phthalate solution
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and western blot methods, as well as other procedures which may utilize antibodies specific to CopB epitopes. While ELISAs are preferred forms of immunoassays, it will be readily appreciated that assays also include RIAs and other non-enzyme linked antibody binding assays or procedures. Additionally, it is proposed that monoclonal antibodies specific to the particular CopB epitope may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant CopB species or variants thereof.
  • CopB peptides of the invention will find use as antigens for raising antibodies and in immunoassays for the detection of anti-CopB antigen-reactive antibodies and CopB antigens (competitive assays).
  • immunoassays may be exploited to determine the antigen relationship between certain CopB mutant peptides.
  • screening assays may involve (i) the generation of antisera or antibodies against mutant peptides, the reactivity of which can then be checked against other peptides, or (ii) the testing of mutant peptide reactivity with a battery of immunoreagents developed against heterologous antigens, such as antibody 10F3. In this way, a mutational analysis of various epitopes may be performed.
  • Diagnostic immunoassays include direct culturing of bodily fluids, either in liquid culture or on a solid support such as nutrient agar.
  • a typical assay involves collecting a sample of bodily fluid from a patient and, optionally, placing the sample in conditions optimum for growth of the pathogen. A determination can then be made as to whether a particular microbe exists in the sample. Further analysis can be carried out to determine the properties of the microbe, e.g., whether or not it is hemolyzing.
  • Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays
  • ELISAs ELISAs
  • RIAs radioimmunoassays
  • the anti-CopB antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection also may be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the CopB antigen are immobilized onto the well surface and then contacted with the anti-CopB antibodies. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.
  • test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells.
  • Peptide antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized peptide or antibody.
  • the presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well, and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adso ⁇ tion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C. Following incubation, the anti sera-contacted surface is washed so as to remove non- immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first or for a distinct epitope of the bound antigen.
  • the second antibody will preferably be an antibody having specificity in general for human IgG.
  • the detecting antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS-Tween).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. Alternatively, the label may be a chemi luminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.
  • active and passive immunoprophylaxis there are provided methods for active and passive immunoprophylaxis.
  • Active immunoprophylaxis will be discussed first, followed by a discussion of passive immunoprophylaxis. It should be noted that the discussion of formulating vaccine compositions in the context of active immunoprophylaxis is relevant to raising antibodies in experimental animals for passive immunoprophylaxis and for the generation of diagnostic methods.
  • CopB polypeptides or CopB-derived peptides may be used as vaccine formulations to generate protective anti- . catarrhalis antibody responses in vivo.
  • protective it is meant only that the immune system of a treated individual is capable of generating a response that reduces, to any extent, the clinical impact of the bacterial infection. This may range from a minimal decrease is bacterial burden to outright prevention of infection. Ideally, the treated subject will not exhibit the more serious clinical manifestations of M. catarrhalis infection.
  • immunoprophylaxis involves the administration, to a subject at risk, a vaccine composition.
  • the vaccine composition will contain a CopB polypeptide or immunogenic derivative thereof in a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • those of skill in the art are able, through a variety of mechanisms, to identify appropriate antigenic characteristics of CopB and, in so doing, develop vaccines that will achieve certain goals. These goals include (i) generation of immune responses against specific strains of M. catarrhalis, (ii) generation of immune responses against certain groups of M catarrhalis strains and (iii) generation of immune responses against all M. catarrhalis strains.
  • CopB antigens may vary and, therefore, it may be desirable to couple the antigen to a carrier molecule.
  • exemplary carriers are keyhole limpet hemocyanin (KLH) and human and bovine serum albumin, myoglobin, ⁇ - galactosidase, penicillinase and bacterial toxoids.
  • KLH keyhole limpet hemocyanin
  • myoglobin myoglobin
  • ⁇ - galactosidase penicillinase and bacterial toxoids.
  • Synthetic carriers such as multi-poly-DL-alanyl-poly-L-lysine and poly-L-lysine also are contemplated. Coupling generally is accomplished through amino-or carboxy-terminal residues of the antigen, thereby affording the peptide or polypeptide the greatest chance of assuming a relatively "native" conformation following coupling.
  • adjuvants which are known to stimulate the appropriate portion of the immune system of the vaccinated animal.
  • Suitable adjuvants for the vaccination of subjects include, but are not limited to oil emulsions such as Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol 52:Montanide 888 (Marcol is a Trademark of Esso,
  • Montanide is a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), mineral gels such as aluminum hydroxide, aluminum phosphate, calcium phosphate and alum, surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N,N'- bis(2-hydroxyethyl)- propanediamine, methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate.
  • Adjuvant 65
  • Agents include synthetic polymers of sugars (Carbopol), emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) also may be employed.
  • Carbopol emulsion in physiologically acceptable oil vehicles
  • mannide mono-oleate mannide mono-oleate
  • Fluosol-DA perfluorocarbon
  • vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 , 4,601,903, 4,599,231, 4,599,230, 4,596,792 and 4.578,770, all of which are inco ⁇ orated herein by reference.
  • Such vaccines are prepared as injectables. Liquid solutions are preferred; solid forms suitable for solution or suspension in liquid prior to injection also may be prepared. The preparation further may be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • the vaccine preparations of the present invention also can be inco ⁇ orated into non-toxic carriers such as liposomes or other microcarrier substances, or by conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "iscoms" (immunostimulating complexes). These complexes can serve to reduce the toxicity of the antigen, delay its clearance from the host and improve the immune response by acting as an adjuvant.
  • suitable adjuvants for use in this embodiment of the present invention include INF, IL-2, IL-4, IL-8, IL-12 and other immunostimulatory compounds.
  • conjugates comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT may prove advantageous.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the peptides may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient to one milligram or so per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size of the host. In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
  • the vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
  • the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
  • the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932, 4,174,384 and 3,949,064, as illustrative of these types of assays.
  • Passive immunity is defined, for the pu ⁇ oses of this application, as the transfer to one organism of an immune response effector that was generated in another organism.
  • the classic example of establishing passive immunity is to transfer antibodies produced in one organism into a second, immunologically compatible animal.
  • immunologically compatible it is meant that the antibody can perform at least some of its immune functions in the new host animal.
  • other effectors such as certain kinds of lymphocytes, including cytotoxic and helper T cells, NK cells and other immune effector cells.
  • the present invention contemplates both of these approaches.
  • Antibodies, antisera and immune effector cells are raised using standard vaccination regimes in appropriate animals, as discussed above.
  • the primary animal is vaccinated with at least CopB polypeptide or peptide product according to the present invention, with or without an adjuvant, to generate an immune response.
  • the immune response may be monitored, for example, by measurement of the levels of antibodies produced, using standard ELISA methods.
  • immune effector cells can be collected on a regular basis, usually from blood draws.
  • the antibody fraction can be purified from the blood by standard means, e.g., by protein A or protein G chromatography.
  • monoclonal antibody - producing hybridomas are prepared by standard means (Coligan et al, 1991).
  • Monoclonal antibodies are then prepared from the hybridoma cells by standard means. If the primary host's monoclonal antibodies are not compatible with the animal to be treated, it is possible that genetic engineering of the cells can be employed to modify the antibody to be tolerated by the animal to be treated. In the human context, murine antibodies, for example, may be "humanized" in this fashion.
  • Antibodies, antisera or immune effector cells are injected into hosts to provide passive immunity against microbial infection.
  • an antibody composition is prepared by mixing, preferably homogeneously mixing, at least one antibody with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or excipient using standard methods of pharmaceutical or veterinary preparation.
  • the amount of antibody required to produce a single dosage form will vary depending upon the microbial species being vaccinated against, the individual to be treated and the particular mode of administration.
  • the specific dose level for any particular individual will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the individual, time of administration, route of administration, rate of excretion, drug combination and the severity of the microbial infectation.
  • the antibody composition may be administered intravenously, subcutaneously, intranasally, orally, intramuscularly, vaginally, rectally, topically or via any other desired route. Repeated dosings may be necessary and will vary, for example, depending on the clinical setting, the particular microbe, the condition of the patient and the use of other treatments.
  • Murine short-term pulmonary clearance models have now been developed which permit an evaluation of the interaction of bacteria with the lower respiratory tract as well as assessment of pathologic changes in the lungs.
  • This model reproducibly delivers an inoculum of bacteria to a localized peripheral segment of the murine lung. Bacteria multiply within the lung, but are eventually cleared as a result of (i) resident defense mechanisms, (ii) the development of an inflammatory response, and/or (iii) the development of a specific immune response.
  • serum IgG antibody can enter the alveolar spaces in the absence of an inflammatory response and enhance pulmonary clearance of non-typable H. influenzae (McGehee et al. , 1989), a pathogen with a host range and disease spectrum nearly identical to those of M. catarrhalis.
  • transformants were transferred to nitrocellulose pads, cross-linked using the UV Stratalinker 1800TM (Stratagene Inc., La Jolla, CA) and hybridized with a P-labeled 1.3 kb PvuII/ Cbal copB gene fragment from M. catarrhalis O35E.
  • This gene probe was shown to hybridize with a 7.8 kb Pstl fragment of M. catarrhalis TTA24 chromosomal DNA in a Southern blot analysis.
  • Reactive clones were tested for expression of the CopB protein by western blot analysis of whole cell lysates with polyclonal antiserum raised against M. catarrhalis TTA24 outer membrane vesicles.
  • a recombinant plasmid containing a 7.8 kb insert that encoded the CopB protein was designated pTFAlOO. This plasmid was purified using an alkaline lysis protocol and digested with Hinfl.
  • the O12E and O46E chromosomal DNAs were extracted from whole cell isolates as follows. One to two loopfuls of cells of each respective isolate was resuspended into 1 ml of lysis buffer (150 mM NaCl , 50mM Tris, pH 8.0, 10 mM EDTA, pH 8.0). Sodium dodecyl sulfate (SDS) and RNase were added to yield final concentrations of 1% SDS and 50 ⁇ g/ml RNase. Resuspended cells were incubated for 1 to 10 minutes at 55-65°C until cell lysis. DNAs were extracted from cell lysates with an equal volume of a mixture of phenol, chloroform and isoamyl alcohol.
  • the upper phase of the extract was separated and DNAs were precipitated with two volumes of 100% ethanol.
  • the isolated DNAs were resuspended in 300-500 ⁇ l of Tris EDTA (TE) buffer. SDS and NaCl were added to each resuspension for final concentrations of 0.1 % and 50 mM, respectively.
  • TE Tris EDTA
  • PCR with the 5' primer 5'-CAAGCCTCATAATCGGAG-3' (SEQ ID NO:9) and the 3' primer 5'-CCTCCAGTGAAATCGAATC-3' (SEQ ID NO: 10) in order to obtain DNA fragments containing the copB gene.
  • the PCR reactions were cycled as follows: 1 minute 30 seconds at 94°C; 1 minute 30 seconds at 51 °C; 1 minute 30 seconds at 72°C. These steps were repeated 29 times. The reactions were held for 10 minutes at 72°C after the final cycle.
  • the PCR reactions were then electrophoresed in a 0.7% agarose gel and the respective 2.5 kb bands were cut out and stored at -20°C.
  • the respective DNAs in the frozen gel slices were extracted by spinning in a CoStar Spin-X column, followed by an isopropanol precipitation of the column flow through liquid.
  • the DNAs were resuspended in distilled water and quantitated on a UV spectrophotometer.
  • the 2.5 kb PCR products of O12E and O46E were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit With AmpliTaq DNA Polymerase, FS, in a ABI 373A automated sequencer following the manufacturer's protocol. Sequencing primers were designed from the O35E copB DNA sequence or were derived from the individual DNA sequences of O12E or O46E. The chromosomal DNA isolated from TTA24 was sequenced using standard methods by Lark Sequencing Technologies Inc., Houston, TX.
  • copB DNA gene sequences cloned from TTA24, O12E and O46E are SEQ ID NO:34, SEQ ID NO:28 and SEQ ID NO:32. respectively.
  • the deduced CopB amino acid sequences from TTA24, O12E and O46E are SEQ ID NO:33, SEQ ID NO:27 and SEQ ID NO:31 , respectively.
  • Region 3 comprised about 14 amino acids corresponding to about positions 343-356 of O35E.
  • Region 4 comprised about 19 amino acids corresponding to about positions 392-410 of O35E.
  • Region 5 comprised about 1 1 amino acids corresponding to about positions 445455 of O35E.
  • the inventors synthesized oligopeptides from the CopB protein of O35E that encompassed the five regions of variability identified above using standard methods.
  • the inventors tested the ability of the synthesized oligopeptides to bind to MAb 10F3. Of the five, only the oligopeptide corresponding to variable region 1 (SEQ ID NO:l), specifically bound to MAb 10F3.
  • Direct ELISAs were conducted as follows. Flat-bottomed microtiter ELISA plates (Coming Glass Ware, Coming, NY) were coated with outer membrane vesicles of M. catarrhalis strain O35E (5 mg) or synthetic peptides (0.3-300 mg) in coating buffer
  • the oligopeptide from variable region 1 (SEQ ID NO: l) which was shown to lack cross-reactivity with MAb 10F3 in the preceding example was covalently coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde.
  • mice were either immunized intraperitoneally with 50 ⁇ g of oligopeptide-KLH conjugate in Freund's complete adjuvant and boosted with the same dose of oligopeptide-KLH conjugate in Freund's incomplete adjuvant on day 28 of the test or they were immunized with a peptide-KLH conjugate containing an unrelated and non-cross-reacting peptide from Borrelia burgdorferi. Blood was drawn from the mice on day 42 post-primary immunization and antisera was isolated from it.
  • MAb 10F3 was shown to be bactericidal against MAb 10F3- reactive strains. Further comparison of the deduced amino acid sequences of the CopB proteins from four strains of M. catarrhalis revealed a high degree of identity among these proteins which in turn facilitated mapping of the MAb 10F3-reactive epitope.
  • M. catarrhalis strain 035E has been described in detail (Helminen et al, 1993a; 1993b). M. catarrhalis strains were routinely cultured at 37°C on Brain-Heart Infusion (BHI) agar plates (Difco Laboratories, Detroit, MI) in an atmosphere of 95% air-5% CO 2 or in BHI broth.
  • BHI Brain-Heart Infusion
  • the Escherichia coli cloning strains RR1 , HB101, DH5 ⁇ and recombinant strains were grown on Lubria- Bertani (LB) medium (Sambrook et al, 1989) supplemented, when necessary, with an appropriate antimicrobial compound.
  • Lubria- Bertani (LB) medium Standardbrook et al, 1989
  • M. catarrhalis strain TTA24 chromosomal DNA containing the copB gene pTTA150 pBluescript II SK+ with a 2.6 kb Hinfl This study fragment from pTTAlOO containing the copB gene pGEX-4T-2 Cloning vector Pharmacia pEP10F3 pGEX-4T-2 with a 78-bp insert encoding This study amino acids 275-300 of the CopB protein of M. catarrhalis strain 035E
  • Outer membrane protein preparations and western blot analysis Outer membrane vesicles were prepared from BHI broth-grown M. catarrhalis strains as described (Mu ⁇ hy and Loeb, 1989). Proteins present in these outer membrane vesicles were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by western blot analysis as described (Helminen et al, 1993a). Monoclonal antibodies (MAbsl and polyclonal antisera.
  • MAb 10F3 is a murine IgG 2a antibody reactive with a surface-exposed epitope of the CopB outer membrane protein of the M.
  • catarrhalis strains 035E and 012E Helminen et al, 1993a.
  • This MAb does not bind to the CopB protein expressed by M. catarrhalis strains TTA24 and 046E (Helminen et al, 1993a).
  • This MAb was used in the form of hybridoma culture supernatant fluid for ELISA assays, epitope mapping, western blot analysis and in the indirect antibody-accessibility assay.
  • MAb 10F3 was purified using protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ) as described (Ey et al, 1978).
  • MAb 17C7 is a murine IgG antibody directed against the UspA surface antigen of M. catarrhalis (Helminen et al, 1994).
  • MAb 3F12 is a murine IgG antibody directed against the UspA surface antigen of M. catarrhalis (Helminen et al, 1994).
  • MAb 3F12 is a murine IgG antibody specific for the major outer membrane protein (MOMP) of H. ducreyi (Gulig et al, 1982). These latter two MAbs were used as negative controls in the ELISA and, indirect antibody-accessibility assays, respectively. Polyclonal rabbit antiserum raised against M. catarrhalis strain TTA24 outer membrane vesicles was described previously (Helminen et al. , 1993a).
  • catarrhalis strains 012E and 046E was extracted by standard methods and used as template DNA in a PCRTM system together with primers derived from the nucleotide sequence of the copB gene from M. catarrhalis strain 035E (Helminen et al, 1993a). Using a PTC 100
  • sequencing reactions were performed with the Applied Biosystems PRISM Ready Reaction DiDeoxy Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase according to the manufacturer's directions (Applied Biosystems, Inc., Foster City, CA). Sequencing primers were designed from the nucleotide sequence of the copB gene from M. catarrhalis strain 035E (Helminen et al, 1993a) or from the individual copB sequences.
  • nucleotide sequence accession numbers The nucleotide sequences of the copB genes from M. catarrhalis strains TTA24, 012E and 046E have been submitted to
  • GenBank and assigned the accession numbers U69980 (SEQ ID NO:34), U69981 (SEQ ID NO:28), and U69982 (SEQ ID NO:32), respectively.
  • Oligopeptide synthesis Oligopeptides were synthesized on a Symphony Peptide Synthesizer (Rainin Instrument Co., Inc., Wobum, MA). Molecular weight and purity of the peptides were determined by high performance liquid chromatography and mass spectrometry.
  • PBS-Tween PBS containing 0.05% [v/v] Tween-20 and 0.025% [w/v] sodium azide
  • PBS-Tween containing 1% (w/v) bovine serum albumin (BSA) for 1 hr
  • lOO ⁇ l portions of MAb 10F3 or MAb 17C7 (negative control) or antiserum in dilutions of 1 :10 to 1 : 1000 in blocking buffer were added and the plates were incubated at 37°C for 1 hr.
  • the ligation product was transformed into competent E. coli DH5 ⁇ and recombinant clones were screened for reactivity with MAb 10F3 using a colony blot assay described elsewhere (Gulig et al, 1982).
  • the plasmid construct present in one of the MAb 10F3-reactive clones was designated pEP10F3.
  • M. catarrhalis strain 035E was diluted in PBS buffer containing 10% (v/v) fetal bovine serum and 0.025% (w/v) sodium azide to a density of 110 Klett units (ca. IO 9 colony forming units [cfu]/ml) as measured with a Klett-Summerson colorimeter (Klett Manufacturing Co., New York, N.Y.). Identical portions (100 ⁇ l) of this suspension were added to 1 ml of MAb 10F3 or MAb 3F12 hybridoma culture supernatant or to 1 ml of the PBS buffer described above containing antiserum at a dilution of 1 :500.
  • the bacterial cells were washed once and then resuspended in 1 ml of the buffer solution. Radioiodinated goat anti-mouse immunoglobulin was added and the mixture was incubated for 1 hr at 4°C with gentle agitation. The cells were then washed four times with 1 ml of the buffer solution, resuspended in 500 ⁇ l of triple detergent (Gulig et al, 1982) and transferred to a 12 x 75 mm glass tube. The radioactivity present in each sample was determined by using a gamma counter.
  • Portions (100 ⁇ l) of this cell suspension were added to 100 ⁇ l of native or heat-inactivated normal human serum containing either 15 ⁇ g of purified MAb 10F3 or heat-inactivated antiserum in final dilutions of 1 :20 to 1 :2000. This mixture was then incubated at 37°C. At time 0 and at 60 and 120 min after the start of the assay, 10 ⁇ l aliquots were removed, suspended in 75 ⁇ l of BHI broth and spread onto prewarmed BHI agar plates which were then incubated overnight to determine the number of cfu in each sample.
  • the recombinant plasmid was designated pTTAlOO and a 2.6 kb Hinfl fragment from this plasmid that was predicted to contain the entire copB gene was subcloned into pBluescript to obtain the recombinant plasmid pTTA150. Nucleotide sequence analysis of both strands of this 2.6 kb DNA fragment confirmed that it contained the complete copB gene from strain TTA24.
  • copB genes from additional M. catarrhalis strains were amplified from the chromosome of each strain by PCRTM.
  • the oligonucleotide primers (5'-CAAGCCTCATAATCGGAG-3' (SEQ ID NO:9) and 5'-
  • CCTCCAGTGAAATCGAATC-3' (SEQ ID NO: 10) were derived from the sequence of the copB gene of strain 035E (Helminen et al, 1993a) and were located just outside the open reading frame. Both strands of these two PCRTM products were sequenced in their entirety.
  • CopB proteins of strains 035E and TTA24 shared the highest degree of identity but differed in their ability to bind MAb 10F3 provided the necessary information to begin localizing the MAb 10F3-reactive epitope. Sequence deviations between these two CopB proteins were confined to five distinct regions (designated 1-5) located between amino acid residues 280 and 450. Because MAb 10F3 was known to bind to both native and denatured CopB (Helminen et al, 1993a) and thus in all likelihood to a linear epitope, only one of these five regions was expected to be involved in the binding of this MAb.
  • CopB protein of strain 035E were designated RI through R5 and had the following amino acid sequences:
  • the negative control MAb 17C7 did not bind any of these peptides.
  • R1A did not affect binding of this MAb.
  • the presence of N in position 21 of peptide RI (SEQ ID NO:l) (corresponding to residue 295 in the CopB protein) thus appeared critical for binding of MAb 10F3.
  • a peptide (R1-TTA24; SEQ ID NO:4) containing the amino acid sequence from the same region of the CopB protein from M. catarrhalis strain TTA24 did not bind this MAb (FIG. 2).
  • PEPTIDE 1 LDIEKDKKKR SEQ ID NO: 19 PEPTIDE 2 EKDKKKRTDE SEQ ID NO:20 PEPTIDE 3 KKKRTDEQLQ SEQ ID NO.21 PEPTIDE 4 RTDEQLQAEL SEQ ID NO:22 PEPTIDE 5 EQLQAELDNK SEQ ID NO.23 PEPTIDE 6 QAELDNKYAG SEQ ID NO:24 PEPTIDE 7 LDNKYAGKGY SEQ ID NO:25 PEPTIDE 8 KYAGKGYKLG SEQ ID NO:26
  • region 1 within the context of the CopB protein of strain 035E revealed that this region is part of a highly hydrophilic domain (FIG. 4).
  • CopB-specific antibody in mouse antiserum raised against GST- 26 was assessed by ELISA using the synthetic peptides RI (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO: l), R1A (SEQ ID NO:
  • MAb 10F3 also bound to an 82 kDa antigen from strain 012E (FIG. 5A, lane 2); the CopB protein of strain 012E is known to migrate slightly more slowly in SDS-PAGE than the CopB protein of strain 035E.
  • the GST-26 antiserum did not react with any antigens in the 80-82 kDa range from strains 012E, TTA24, and 046E (FIG. 5B, lanes 2-4, respectively).
  • catarrhalis strains 035E, TTA24, 012E and 046E as antigen demonstrated that, as expected, MAb 10F3 readily bound to the surface of strains 035E and 012E, but not to strains TTA24 and 046E (Table VI).
  • MAb 10F3 readily bound to the surface of strains 035E and 012E, but not to strains TTA24 and 046E (Table VI).
  • antibodies in the GST-26 antiserum exhibited specific binding to whole cells of strain 035E, while antibodies present in the control antiserum raised against GST did not (Table VI).
  • MAb 10F3 Bactericidal activity of MAb 10F3 and of GST-26 antiserum.
  • MAb 10F3 was previously shown to enhance pulmonary clearance of M. catarrhalis from the lungs of mice passively immunized with this MAb (Helminen et al, 1993a). However, the functional basis for this MAb-accelerated elimination of bacteria from the lower respiratory tract was not determined. Therefore, the ability of MAb 10F3 to kill M. catarrhalis in the presence of human complement was evaluated by performing bactericidal assays with the MAb 10F3-reactive strains 035E and 012E and with the MAb 10F3-unreactive strains TTA24 and 046E. Similarly, the antisera raised against GST-26 and GST (negative control) were tested for their bactericidal activity because
  • GST-26 contained antibodies directed at the surface of strains 035E, 012E and TTA24.
  • the GST-26 antiserum did not exert detectable bactericidal activity against strains 035E, TTA24 and 046E (FIG. 6A, FIG. 6C and FIG. 6D, open circles). However, this same GST-26 antiserum did kill strain 012E (FIG. 6B, open circles). The possibility that a prozone effect had prevented killing of strain 035E by the GST-26 antiserum was addressed by incubating the M. catarrhalis strain 035E cells with dilutions of the GST- 26 antiserum ranging from 1 :20 to 1 :2000, none of which showed bactericidal activity.
  • Faden et al Ann. Otol Rhinol Laryngol, 100:612-615, 1991. Faden, Hong, Mu ⁇ hy, "Immune response to outer membrane antigens of Moraxella catarrhalis in children with otitis media," Infect. Immun., 60:3824-3829, 1992.
  • Nicolas and Rubenstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 494-513, 1988. Nicotra, Rivera, Liman, Wallace, "Branhamella catarrhalis as a lower respiratory tract pathogen in patients with chronic lung disease," Arch. Intern. Med, 146:890-893, 1986. Pettersson, Haas, van Wassenaar, van der Ley, Tommassen, "Molecular cloning of F ⁇ B, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis," Infect. Immun., 63:4181-4184, 1995.

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Abstract

Cette invention a trait à une analyse de la protéine CopB de membrane externe de M. Catarrhalis. Quatre antigènes de CopB ont maintenant été séquencés et leurs séquences d'acides aminés prévues, comparées. Dans cet antigène, des régions de conservation et de non-conservation ont été identifiées, y compris une région non conservée représentant une région de liaison provenant de la souche 035E. Un seul changement d'acide aminé (N pour D) dans cet épitope, sur le résidu 295, abolit la réactivité de l'anticorps 10F3 vis-à-vis de CopB. Des peptides ne contenant que des résidus de cette région possédant une extrémité carboxylique pour le résidu 295 retiennent bien la réactivité mais leur réactivité est inférieure à la réactivité maximale obtenue en présence d'une asparagine en position 295. L'invention porte également sur des compositions ainsi que sur des méthodes tant diagnostiques qu'immunoprophylactiques.
EP97939404A 1996-08-12 1997-08-12 DEFINITION D'EPITOPES DE LA PROTEINE CopB DE MEMBRANE EXTERNE DE MORAXELLA CATARRHALIS Withdrawn EP0920513A2 (fr)

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PCT/US1997/014221 WO1998006851A2 (fr) 1996-08-12 1997-08-12 DEFINITION D'EPITOPES DE LA PROTEINE CopB DE MEMBRANE EXTERNE de $i(MORAXELLA CATARRHALIS)

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WO1999003884A2 (fr) * 1997-07-21 1999-01-28 North American Vaccine, Inc. Compositions de pneumolysine immunogene modifiee utiles en tant que vaccins
AU4260099A (en) * 1998-05-12 1999-11-29 Smithkline Beecham Biologicals (Sa) Compounds from moraxella catarrhalis
GB9810285D0 (en) * 1998-05-13 1998-07-15 Smithkline Beecham Biolog Novel compounds
DE60039108D1 (de) * 1999-05-24 2008-07-17 Glaxosmithkline Biolog Sa Neue verbindungen aus moraxella catarrhalis
GB9915031D0 (en) * 1999-06-25 1999-08-25 Smithkline Beecham Biolog Novel compounds
ES2290056T3 (es) * 1999-09-14 2008-02-16 Glaxosmithkline Biologicals S.A. Polipeptidos de moraxella (branhamella) catarrhalis.
WO2004069163A2 (fr) * 2003-01-30 2004-08-19 Wyeth Holdings Corporation Epitopes a protection croisee de moraxella catarrhalis et methodes d'utilisation correspondantes
CN111675753A (zh) * 2020-05-19 2020-09-18 深圳市疾病预防控制中心 禽流感病毒抗原表位筛选方法和应用

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