WO1993011791A1 - Antigenic preparations that stimulate production of antibodies which bind to the pili of type iv piliated bacteria - Google Patents

Antigenic preparations that stimulate production of antibodies which bind to the pili of type iv piliated bacteria Download PDF

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WO1993011791A1
WO1993011791A1 PCT/US1992/011085 US9211085W WO9311791A1 WO 1993011791 A1 WO1993011791 A1 WO 1993011791A1 US 9211085 W US9211085 W US 9211085W WO 9311791 A1 WO9311791 A1 WO 9311791A1
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Alvin W. Smith
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The State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of Oregon State University
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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
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • 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
    • 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)
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • 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 to an antigenic preparation, capable of generating in vertebrates antibodies which bind to the whole pili of species of Type IV piliated bacteria.
  • a specific embodiment of this invention relates to antigenic preparations active against Bacteroides nodosus .
  • the antigenic preparations use submolecular units of B . nodosus pilin to elicit antibodies capable of blocking the pili function of B . nodosus .
  • This pathogen is the essential causative agent of footrot infection in sheep and other ruminates.
  • Pili are virulence factors for a wide range of bacteria pathogenic to both animal and humans. These pili have multiple functions that include epithelial cell adherence, microcolonization, adherence to other bacteria, twitching motility, and possibly other yet unexplored functions such as proteolytic enzyme or toxin delivery to target tissues.
  • the pili of several genera of these including Bacteroides ( Porphyromonas ) , Moraxella, Pseudomonas , Vibrio, pathogenic E. Coli , and Neisseria are unipolar and have an a ino terminus methionine ⁇ Vibrio and some pathogenic E.
  • Type IV pili phenylalanme which is methylated (NMePhe) or lacking this are otherwise called Type IV pili. All Type IV pili share much sequence homology not only between strains within each bacterial species but between the different genera particularly in the first one third of the molecule (amino end) . This segment (the first 1/3 of the a ino terminal end) is predominantly hydrophobic and seemingly less active biologically than the more antigenically variable remainder of the molecule.
  • B . nodosus is the primary pathogen of sheep footrot. This agent can colonize the feet of sheep, produce proteases which progressively lyse layers of hoof and expose the underlying soft tissues to soil borne secondary infection.
  • B. nodosus to be pathogenic two virulence factors must be present.
  • the organism must have pili and must produce proteases. Included in the proteases of virulent B . nodosus are enzymes that can hydrolyze elastin, collagen type 111, keratin, and other proteins.
  • the pili or fimbria of pathogenic organisms in general are understood to function as organelles of adherence which bind the agent to appropriate host tissue or other organisms. Sometimes they exhibit a secondary functional characteristic of causing gliding or twitching motility. This later phenomena might simply represent release of mechanical forces that build up as the pili extrude from the cell, thus causing the cell to suddenly or gradually move a short distance. Although this motility may not contribute significantly to virulence, pili are thought to be a major, or perhaps the only, mechanism capable of effectively attaching the bacteria to sheep's feet and colonizing host tissue.
  • the pili antigens have been shown to be the protective antigens since antibodies against such pili can prevent sheep footrot (Stewart, D.J. (1978) Res. Vet. Sci.
  • the current commercial vaccines for B. nodosus are made up of whole bacterial cells including their pili each grown as a discrete serotype, (8 serotypes including 2 additional pilin protein variants of one of these type) which are then combined into a single vaccine.
  • the efficacy of these polyvalent vaccines ranges from zero to 80% depending on how well the vaccine strains duplicate those strains which are actually infecting the sheep.
  • the current commercial vaccines use harsh adjuvants to drive up the antibody levels. These adjuvants cause severe tissue reactions sometimes resulting in abcess formation at inoculation sites.
  • the polyvalent vaccines currently being marketed stimulate production of a wide array of poorly targeted antibodies and many of these are of little or no use in conferring immunity. In other words, the sheep's immune reserves are squandered generating inappropriate or useless antibodies.
  • a need continues to exist for a vaccine that elicits the production of antibodies that bind to the whole pili of strains within bacterial species, such as the various serotypes of B. nodosus, or between bacterial species of the Type IV pili class.
  • Such a vaccine would perturb those pili functions conferring virulence and thereby, provide resistance to pathogens of the Type IV pili class.
  • the present invention provides antigenic preparations to produce just such a vaccine using highly conserved antigenic segments of the Type IV pili class.
  • the present invention provides an antigenic preparation active against a species of Type IV piliated bacteria.
  • the antigenic preparation comprises a submolecular unit of pilin protein corresponding to at least one epitope common to structural pilin proteins of the species of Type IV piliated bacteria.
  • the submolecular unit of pilin protein is capable of eliciting antibodies capable of binding to the whole pili of the species of Type IV piliated bacteria. This ability to produce such antibodies provides the basis for effective vaccines against species of Type IV piliated bacteria.
  • Antigenic preparations of the present invention can be prepared against Type IV piliated bacteria species such as Bacteroides nodosus, Neis ⁇ eria gonorrhea, Neisseria meningitis , Moraxella bovis, Vibrio cholera, Escherichia coli, and P ⁇ eudomonas aeroginosa .
  • Type IV piliated bacteria species such as Bacteroides nodosus, Neis ⁇ eria gonorrhea, Neisseria meningitis , Moraxella bovis, Vibrio cholera, Escherichia coli, and P ⁇ eudomonas aeroginosa .
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Bacteroides nodosus is selected from the group of polypeptides consisting of:
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Neisseria gonorrhea has the following sequence:
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Neisseria meningitis has the following sequence:
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Moraxella bovi ⁇ has the following sequence:
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Vibrio cholera has the following sequence:
  • the submolecular unit of pilin protein that is capable of eliciting antibodies against Pseudomonas aeroginosa has the following sequence:
  • the invention further comprises an antigenic preparation of repeating sequences of polypeptides common to structural pilin proteins of the species of Type IV piliated bacteria.
  • the invention further comprises an antigenic preparation of at least one epitope of a polypeptide common to structural pilin proteins of the species of Type IV piliated bacteria.
  • the invention further comprises an antigenic preparation in which the submolecular unit of any part of the submolecular unit of pilin protein suspended in a suitable pharmaceutical carrier is used as a vaccine.
  • FIG. 1 shows the immunoblot results of different B. nodosus serotypes versus a submolecular unit of pilin protein antibody
  • FIG. 2 shows immunoelectron microscopy results for B. nodosus Type XV pili, one of the four known D-set pilin types, versus a submolecular unit of pilin protein antibody;
  • FIG. 3 shows immunoelectron microscopy results for B. nodosus A 198 pili, one of the 17 known A-set pilin Types, versus a submolecular unit of pilin protein antibody;
  • FIG. 4 shows a gene construct coding for a polypeptide of B. nodosu ⁇
  • FIG. 5 shows a gene construct coding for a polypeptide of B. nodo ⁇ u ⁇
  • FIG. 6 shows a gene construct coding for a polypeptide of B. nodosus
  • FIG. 7 shows a gene construct coding for a polypeptide of B . nodo ⁇ u ⁇ j
  • FIG. 8 shows a gene construct coding for a polypeptide of B. nodo ⁇ u ⁇ j
  • FIG. 9 shows a gene construct coding for a polypeptide of N . gonorrhe ;
  • FIG. 10 shows a gene construct coding for a polypeptide of N. meningitis ⁇
  • FIG. 11 shows a gene construct coding for a polypeptide of M. bovis ;
  • FIG. 12 shows a gene construct coding for a polypeptide of V. cholera .
  • FIG. 13 shows a gene construct coding for a polypeptide of P. aeroginosa .
  • the present invention relates to antigenic preparations that produce antibodies that indirectly block or sterically interfere with pili function of pathogens having Type IV pili.
  • Vaccines incorporating these antigenic preparations can provide protection against diseases caused by these pathogens.
  • the approach of the present invention is based on finding a highly conserved antigenic segment, a submolecular unit of the Type IV pilin molecule, which will elicit the production of such antibodies. These antibodies bind to the whole pili of strains within bacterial species or between bacterial species. The result is that the antibodies perturb those pili functions conferring virulence and thereby provide resistance to pathogens of the Type IV pili class.
  • the Type IV pili are made up exclusively or almost exclusively of a structural protein which is a polymerized repeat of a single molecular species.
  • the amino acid sequence and tertiary configuration of this molecule is one basis for the antigenic serotyping of pathogens having Type IV pili.
  • the antigens of the structural protein above are present in far greater numbers (perhaps 1000:1 up to 10,000:1) than any specific adhesion antigen associated with pili.
  • Specific adhesion antigens are amino acid sequences presumably located on the tips or at intervals along the pili.
  • the first step of selecting an antigenic site was accomplished according to the following three procedures. First, computerized predictions of the antigenic profile for known B. nodo ⁇ u ⁇ base sequences were generated. Second, pilin proteins were digested and then tested against a battery of monoclonal antibodies. Third, sequence homology was compared based on published sequences.
  • mice were boosted with 20 ⁇ lg of pili intravenously.
  • Spleen cells from each mouse were harvested, washed with serum-free media, and fused with SP2/0 myeloma cells in 50% polyethylene glycol.
  • Fused cells were seeded into Linbro 96 well plates at 106 cells per well.
  • Cells were fed with RPMI 1640 (Flow Laboratories) containing 15% HyClone defined fetal bovine serum and 1 mg/100 ml gentamicin and HAT.
  • Hybridoma supernatants were screened for antibody production using ELISA. These procedures resulted in production of a family of monoclonals.
  • the pilin protein of eight serotypes of B. nodo ⁇ u ⁇ have been sequenced and compared for homology. Using the methods of Chou and Fasman (Ann Rev Bioche . 47:251-276, 1978) , the secondary structures represented by probable beta-turns were predicted. Also using computer generated models, three of these were compared for regions of hydrophobicity/hydrophilicity of the pilin. Using this rationale two peptides were synthesized where homology occurs between the pilin protein of various B. nodosu ⁇ Australian strains. These were bound to carrier molecules (KLH) and used in rabbits to produce antibodies against the peptides. Although these antibodies did bind to the synthetic peptides, they bound poorly to whole pili and did not block pili adherence. Thus, these two regions were shown not to be of major interest as antigenic sites and focused attention on more highly conserved regions.
  • KLH carrier molecules
  • oligonucleotides up to 50 bases in length. These oligonucleotides correspond to the entire primary structural gene that codes for the pilin of B. nodosu ⁇ A198 incorporating phosphoramidites and standard methods. Also synthesized were complementary sequences to be used as bridges for reconstructing any portion of the genomic code for A198 pilin. Gaps in the second strand can be completed and sealed as desired using DNA polymerase I and DNA ligase. Using this technology, a specified oligonucleotide of 153 bases was assembled. This gene can be amplified using a cloning vector.
  • PCR was used to amplify the desired genomic segments out of native B. nodo ⁇ us cultures. This was accomplished by synthesizing two primers. The first primer was 27 bases with a Bam HI restriction site in the overhang as shown on the gene construct of Fig. 4. The second primer was 30 bases with a stop codon and Hind III site in the overhang as shown in the gene construct of Fig. 4. Such primer construction gave in-frame and directional efficiency for cloning. The primers were purified by acrylamide gel electrophoresis to give 2.5 mg/ml and 40 mg/ml, respectively. PCR amplification was accomplished with 25 cycles at 50°C annealing temperature. The resultant very tight band of B.
  • nodosus DNA was purified by cTAB precipitation in high salt and 3 ammonium acetate precipitations with ethanol, giving a final DNA concentration of 500 ng/ul.
  • the DNA fragment included the partial gene for the pilin protein molecule, and 21 additional bases including a stop codon.
  • This PCR fragment insert was cloned into the over expression vector pTTQ8 (Amersham Cat. No. RPN 1259) and three of these clones were sequenced as follows.
  • Inserts were primed with the ml3/pUC forward sequencing primer using a sequence USB.X
  • This primer matches the pTTQ8 vector at 5 bases downstream from the Hind III site on the 3' side of the pTTQ ⁇ polylinker and allowed direct sequencing of the Bam HI through Hind III insert in the pTTQ ⁇ plasmid. All three clones sequenced were the same 160 base fragment, all have an open reading frame from Bam HI to Hind III, and were of the intended base sequence and number. To insure sufficient antigenicity for the small molecular weight peptide ( ⁇ 10,000 daltons) , the small peptide was expressed as a TrpE fusion protein. This was accomplished by subcloning into the pATH3 vector.
  • the pATH3 system expressed a TrpE fusion protein of approximately 40,000 daltons comprising about 10% of total protein production. This system was scaled up giving approximately 50 mg of pilin-TrpE fusion protein that was purified over a preparative SDS-PAGE gel.
  • the third step of testing antigenic characteristics of peptides was accomplished according to the following procedures. Antibodies were produced against the peptides. These antibodies were tested for binding specificity to B. nodosus pili.
  • Antibodies were generated by administering the fusion protein subcutaneously and intramuscularly into rabbits. This was done using 1 mg amounts contained in polyacrylamide gel and complete Freund's adjuvant after the methods of Rothbard et al., J. Exp. Med. 110:208-221, 1984.
  • the immunoblot procedure used a nitrocellulose membrane to which whole B. nodosu ⁇ pili are fixed.
  • the nitrocellulose binding sites unoccupied by transferred protein were saturated by incubation with 3% gelatin TBS for 1 hour.
  • the treated nitrocellulose was incubated with antiserum dilution of 1:500 in TBS + 1% with gelatin, then washed 4 times 2 x 10 minutes with TBS and 0.05% Tween 20 and 2 x 10 minutes with Tween-free TBS pH 7.5.
  • Antibody bound protein was then visualized by incubating for 1 hour in secondary antibody solution (goat antirabbit) conjugated with horseradish peroxidase diluted 1:2000 with antibody buffer. Then it was washed 4 times as above and developed with horseradish peroxidase color development.
  • polyvalent rabbit antiserum which was made against highly purified whole pilin, also bound the pilin protein.
  • FIG. 1 shows the immunoblot results of different B. nodo ⁇ u ⁇ serotypes versus a submolecular unit of pilin protein antibody.
  • Lane l showed the antiserum of rabbit #684 at pre-injection.
  • Lane 2 showed antipilus antibody being produced in rabbit #684 against B. nodo ⁇ u ⁇ serotypes of A-set and D-set pili 98 days after receiving the 6,270 dalton fusion protein.
  • Lane 3 showed the antiserum of rabbit #685 at pre-injection.
  • Lane 4 showed antipilus antibody being produced in rabbit #685 against B. nodosu ⁇ serotypes of A-set and D-set pili 98 days after receiving the 6,270 dalton fusion protein.
  • the differences in reaction between the samples of B. nodo ⁇ u ⁇ pili serotypes shown in FIG. 1 reflect the differences in pili concentration.
  • the A 198 (A-set pilin) shown in FIG. 1 represent different sample passage
  • grid was blotted and washed five times with TBS/Tween, rinsed three times with distilled water, and stained with 1.3% phosphotungstic acid @pH 7.0; then examined with a transmission electron microscope.
  • FIG. 2 shows immunoelectron microscopy results for B. nodo ⁇ u ⁇ Type II pili, one of the four D-set pilin Types, versus a submolecular unit of pilin protein antibody generated in rabbits using a 10 nm colloidal gold label.
  • the pili without antibody are 5-6 nm in diameter.
  • Those pili coated with antibody are 10-15 nm in diameter and show configurational disruption because of antibody cross binding.
  • FIG. 3 shows immunoelectron microscopy results for B. nodo ⁇ u ⁇ A 198 pili, one of the 17 known"A-set pilin Types, versus a submolecular unit of pilin protein antibody generated in rabbits using a 10 nm colloidal gold label.
  • the pili without antibody are 5-6 nm in diameter and the pili coated antibody which are 10- 15 nm in diameter show configurational disruption.
  • the colloidal gold label is less than the amount of bound antibody because the labeling reaction was not run to completion.
  • Antibodies against the submolecular units of pilin ' proteins bind pili of antigenic groups which represent all currently known B. nodo ⁇ u ⁇ serotypes causing them to clump.
  • Clumping which can be shown to be caused by antibody binding to the structural pilin protein molecule, has the effect of reducing the availability of adhesion proteins for attaching B. nodosus to host tissue.
  • an antibody directed to common epitopes on structural pilin proteins of B. nodosus can mechanically interfere with its adherence to host tissue. This same stearic interference can similarly perturb all pili functions.
  • nodo ⁇ u ⁇ polypeptides of approximately 6000, 7500, 8150, 8500, and 9150 molecular weight, were determined. See the sequences in FIGS. 4, 5, 6, 7, and 8. Also five additional sequences, representing Nei ⁇ seria gonorrhea, N. meningiti ⁇ , Moraxella bovi ⁇ , Vibrio cholera , and P ⁇ eudomona ⁇ aerogino ⁇ a , were determined. All ten of these are constructs which may or may not have the first amino acid (phenylalanine, usually methylated) included. Each construct then continues with specific sequences, cut sites and stop codons such that they can be moved between vector systems. Examples of vectors include, but are not limited to, E. coli, P ⁇ eudomona ⁇ , yeasts, poxviruses. herpesvirus, and irridivirus. In this way either live virus vaccines or purified protein vaccines could be assembled depending upon efficacy, cost, feasibility and need.
  • the first of these constructs is 150 bases with Bam HI and Hind III restriction sites added at the 5' and 3' ends, respectively. Also a stop codon is added at the 3' end.
  • the second construct is identical to the construct in FIG. 4 except that 33 bases are inserted in front of both the stop codon and Hind III restriction site at the 3' end.
  • the construct in FIG. 6 is identical to the one in FIG. 5 except for the 15 bases added.
  • the construct in FIG. 7 is not a modification of those in FIGS. 5 and 6, it is similar. It is made up of 207 bases with Bam HI and Hind III sites added on the 5' and 3' ends , respectively. Also a stop codon is placed on the 3' end.
  • the construct in FIG. 8 differs from the one in FIG. 7 with the addition of 15 bases. All of these constructs are designed to express products with an appropriate and predicted alpha helix for histocompatibility processing. Thus, they may act as stand alone antigens (singlets) or as repeating units of antigens (doublets, triplets) . They are also designed to be expressed with fusion proteins such as Trp E for increasing the size of the molecule carrying the desired epitopes. Furthermore, synthetic peptides representing all or any antigenic portion of these constructs could be combined with a molecular carrier and used as antigens to generate antipili antibodies.
  • the five constructs as shown in FIGS. 9, 10, 11, 12 , and 13 include approximately 150-159 bases, the aforementioned restriction sites, and stop codons. These constructs represent the DNA sequences for N. gonorrhea, N. meningiti ⁇ , M. bovi ⁇ , V. cholera, and P. aerogino ⁇ a. See FIGS. 9, 10, 11, 12 , and 13, respectively. These constructs are designed so they can function in the same manner as the B. nodo ⁇ u ⁇ prototype construct.

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Abstract

Antigenic preparations active against Type IV piliated bacteria comprise submolecular units of pilin protein. The submolecular units correspond to at least one epitope common to structural pilin proteins of Type IV piliated bacteria. The ability of such submolecular units to produce antibodies capable of binding to the whole pili can provide the basis for vaccines.

Description

ANTIGENIC PREPARATIONS THAT STIMULATE PRODUCTION OF ANTIBODIES WHICH BIND TO THE PILI OF TYPE IV PILIATED BACTERIA
FIELD OF THE INVENTION
The present invention relates to an antigenic preparation, capable of generating in vertebrates antibodies which bind to the whole pili of species of Type IV piliated bacteria. A specific embodiment of this invention relates to antigenic preparations active against Bacteroides nodosus . The antigenic preparations use submolecular units of B . nodosus pilin to elicit antibodies capable of blocking the pili function of B . nodosus . This pathogen is the essential causative agent of footrot infection in sheep and other ruminates.
BACKGROUND OF THE INVENTION
Pili are virulence factors for a wide range of bacteria pathogenic to both animal and humans. These pili have multiple functions that include epithelial cell adherence, microcolonization, adherence to other bacteria, twitching motility, and possibly other yet unexplored functions such as proteolytic enzyme or toxin delivery to target tissues. The pili of several genera of these including Bacteroides ( Porphyromonas ) , Moraxella, Pseudomonas , Vibrio, pathogenic E. Coli , and Neisseria are unipolar and have an a ino terminus methionine {Vibrio and some pathogenic E. Coli ) or phenylalanme which is methylated (NMePhe) or lacking this are otherwise called Type IV pili. All Type IV pili share much sequence homology not only between strains within each bacterial species but between the different genera particularly in the first one third of the molecule (amino end) . This segment (the first 1/3 of the a ino terminal end) is predominantly hydrophobic and seemingly less active biologically than the more antigenically variable remainder of the molecule.
One species of Type IV pilin bacteria that has undergone extensive study is B . nodosus. B . nodosus is the primary pathogen of sheep footrot. This agent can colonize the feet of sheep, produce proteases which progressively lyse layers of hoof and expose the underlying soft tissues to soil borne secondary infection. For B. nodosus to be pathogenic two virulence factors must be present. The organism must have pili and must produce proteases. Included in the proteases of virulent B . nodosus are enzymes that can hydrolyze elastin, collagen type 111, keratin, and other proteins. The pili or fimbria of pathogenic organisms in general are understood to function as organelles of adherence which bind the agent to appropriate host tissue or other organisms. Sometimes they exhibit a secondary functional characteristic of causing gliding or twitching motility. This later phenomena might simply represent release of mechanical forces that build up as the pili extrude from the cell, thus causing the cell to suddenly or gradually move a short distance. Although this motility may not contribute significantly to virulence, pili are thought to be a major, or perhaps the only, mechanism capable of effectively attaching the bacteria to sheep's feet and colonizing host tissue.
The pili antigens have been shown to be the protective antigens since antibodies against such pili can prevent sheep footrot (Stewart, D.J. (1978) Res. Vet. Sci.
24:14-19; Emery, D.L. et al. (1984) A st. Vet. J.
61:237-238; Every, D. and Sherman, T.M. (1982) New Z.
Vet. J. 30:156-158). This is also the case for E. coli,
Neisseria, and other piliated pathogenic organisms where the pili are important as organelles of attachment
(Schoolnik, G.K et al. (1983) Prog. Aller. 33:314-331; Haggard, D.L. et al. (1982) Vet. Med. Small Anim. Clin.
77:1391-1394; Beachey, E.H. (1981) J. Infect. Dis.
143:325-345; Issacson, R.E. et al. (1978) Infect. Immun.
21:392-397; Salit, J.E. and Morgan, G. (1981) Infect Immun. 31:430-435) . The serotype specificity of B . nodosus is shown to be dependent upon the antigenic determinants found on the pili (Every, D. (1979) J. Gen.
Microbiol. 115:309-316; Egerton, J.R. (1973) J. Comp.
Path. 83:151-159; Stewart, D.J. (1978) Res. Vet. Sci. 24:293-299). B . nodosus has been shown to carry some cross-reactive minor antigenic determinants on the pili
(Stewart, D.J. et al. (1985) Aust. Vet. J. 62:153-159).
This is the basis for the minor cross protection observed in some vaccine trials using piliated B. nodosus bacterins. Furthermore, a reco binant Pseudomonaε aeroginosa has been constructed which expresses pili for single serotypes of B. nodoεuε (Stewart, D.J. et al.,
(1985) Aust. Vet. J. 62:153-159; Elleman, T.C. et al.
(1986) J. Bacteriol. 168:574-580), but each single serotype affords only minor cross protection. This is because there are many different serotypes (peptide configurations) of pili and antibodies against one does not reliably or very often confer solid protection against the others.
The current commercial vaccines for B. nodosus are made up of whole bacterial cells including their pili each grown as a discrete serotype, (8 serotypes including 2 additional pilin protein variants of one of these type) which are then combined into a single vaccine. However, the efficacy of these polyvalent vaccines ranges from zero to 80% depending on how well the vaccine strains duplicate those strains which are actually infecting the sheep. In addition to such marginal efficacy, the current commercial vaccines use harsh adjuvants to drive up the antibody levels. These adjuvants cause severe tissue reactions sometimes resulting in abcess formation at inoculation sites. The polyvalent vaccines currently being marketed stimulate production of a wide array of poorly targeted antibodies and many of these are of little or no use in conferring immunity. In other words, the sheep's immune reserves are squandered generating inappropriate or useless antibodies.
Therefore, a need continues to exist for a vaccine that elicits the production of antibodies that bind to the whole pili of strains within bacterial species, such as the various serotypes of B. nodosus, or between bacterial species of the Type IV pili class. Such a vaccine would perturb those pili functions conferring virulence and thereby, provide resistance to pathogens of the Type IV pili class. The present invention provides antigenic preparations to produce just such a vaccine using highly conserved antigenic segments of the Type IV pili class.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an antigenic preparation active against a species of Type IV piliated bacteria. The antigenic preparation comprises a submolecular unit of pilin protein corresponding to at least one epitope common to structural pilin proteins of the species of Type IV piliated bacteria. The submolecular unit of pilin protein is capable of eliciting antibodies capable of binding to the whole pili of the species of Type IV piliated bacteria. This ability to produce such antibodies provides the basis for effective vaccines against species of Type IV piliated bacteria. Antigenic preparations of the present invention can be prepared against Type IV piliated bacteria species such as Bacteroides nodosus, Neisεeria gonorrhea, Neisseria meningitis , Moraxella bovis, Vibrio cholera, Escherichia coli, and Pεeudomonas aeroginosa .
The submolecular unit of pilin protein that is capable of eliciting antibodies against Bacteroides nodosus is selected from the group of polypeptides consisting of:
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu; Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly
He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu Gly Asn Asp Asp Lys Gly Lys Tyr Ala Leu Ala; Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu Gly Asn Asp Asp Lys Gly Lys Tyr Ala Leu Ala Thr He Asp Gly Asp; Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr He Ala Arg Ser Gin Val Ser Arg Val Met Ser Glu Thr Gly Gin Met Arg Thr Ala He Glu Thr Cys Leu Leu Asp Gly Lys Glu Gly Lys Asp Cys Phe He Gly Trp Thr Thr Ser Asn Leu; and Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr He Ala Arg Ser Gin Val Ser Arg Val Met Ser Glu Thr Gly Gin Met Arg Thr Ala He Glu Thr Cys Leu Leu Asp Gly Lys Glu Gly Lys Asp Cys Phe He Gly Trp Thr Thr Ser Asn Leu Leu Cys Ser Thr Asp Val Asp Glu Lys Phe Lys Pro Thr.
The submolecular unit of pilin protein that is capable of eliciting antibodies against Neisseria gonorrhea has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He Val Gly He Leu Ala Ala Val Ala Leu Pro Ala Tyr Gin Asp Tyr Thr Ala Arg Ala Gin Val Ser Glu Ala He Leu Leu Ala Glu Gly Gin Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn.
The submolecular unit of pilin protein that is capable of eliciting antibodies against Neisseria meningitis has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He Val Gly He Leu Ala Ala Val Ala Leu Pro Ala Tyr Gin Asp Tyr Thr Ala Arg Ala Gin Val Ser Glu Ala He Leu Leu Ala Glu Gly Gin Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn.
The submolecular unit of pilin protein that is capable of eliciting antibodies against Moraxella boviε has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He He Gly He Leu Ala Ala He Ala Leu Pro Ala Tyr Gin Asp Tyr He Ser Lys Ser Gin Thr Thr Arg Val Val Gly Glu Leu Ala Ala Gly Lys Thr Ala Val Asp Ala Ala Leu Phe Glu Gly Lys Thr Pro. The submolecular unit of pilin protein that is capable of eliciting antibodies against Vibrio cholera has the following sequence:
Met Thr Leu Leu Glu Val He He Val Leu Gly He Met Gly Val Val Ser Ala Gly Val Val Thr Leu Ala Gin Arg Ala He
Asp Ser Gin Asn Met Thr Lys Ala Ala Gin Ser Leu Asn Ser He Gin Val Ala Leu Thr Gin Thr.
The submolecular unit of pilin protein that is capable of eliciting antibodies against Pseudomonas aeroginosa has the following sequence:
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr Val Ala Arg Ser Glu Gly Ala Ser Ala Leu Ser Val Asn Pro Leu Lys Thr Thr Val Glu Glu Ala Leu Ser Arg Gly.
The invention further comprises an antigenic preparation of repeating sequences of polypeptides common to structural pilin proteins of the species of Type IV piliated bacteria.
The invention further comprises an antigenic preparation of at least one epitope of a polypeptide common to structural pilin proteins of the species of Type IV piliated bacteria.
The invention further comprises an antigenic preparation in which the submolecular unit of any part of the submolecular unit of pilin protein suspended in a suitable pharmaceutical carrier is used as a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the immunoblot results of different B. nodosus serotypes versus a submolecular unit of pilin protein antibody;
FIG. 2 shows immunoelectron microscopy results for B. nodosus Type XV pili, one of the four known D-set pilin types, versus a submolecular unit of pilin protein antibody;
FIG. 3 shows immunoelectron microscopy results for B. nodosus A 198 pili, one of the 17 known A-set pilin Types, versus a submolecular unit of pilin protein antibody;
FIG. 4 shows a gene construct coding for a polypeptide of B. nodosuε;
FIG. 5 shows a gene construct coding for a polypeptide of B. nodoεuε;
FIG. 6 shows a gene construct coding for a polypeptide of B. nodosus;
FIG. 7 shows a gene construct coding for a polypeptide of B . nodoεuεj
FIG. 8 shows a gene construct coding for a polypeptide of B. nodoεuε j
FIG. 9 shows a gene construct coding for a polypeptide of N . gonorrhe ;
FIG. 10 shows a gene construct coding for a polypeptide of N. meningitis} FIG. 11 shows a gene construct coding for a polypeptide of M. bovis ;
FIG. 12 shows a gene construct coding for a polypeptide of V. cholera ; and
FIG. 13 shows a gene construct coding for a polypeptide of P. aeroginosa .
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to antigenic preparations that produce antibodies that indirectly block or sterically interfere with pili function of pathogens having Type IV pili. Vaccines incorporating these antigenic preparations can provide protection against diseases caused by these pathogens. The approach of the present invention is based on finding a highly conserved antigenic segment, a submolecular unit of the Type IV pilin molecule, which will elicit the production of such antibodies. These antibodies bind to the whole pili of strains within bacterial species or between bacterial species. The result is that the antibodies perturb those pili functions conferring virulence and thereby provide resistance to pathogens of the Type IV pili class.
Finding such highly conserved antigenic segments is greatly aided by the following. First, the Type IV pili are made up exclusively or almost exclusively of a structural protein which is a polymerized repeat of a single molecular species. Second, the amino acid sequence and tertiary configuration of this molecule is one basis for the antigenic serotyping of pathogens having Type IV pili. Third, using B. nodosus as a modeling system, many serotypes (17 A-set pilin types of 21 B. nodoεuε described to date) bind to a single monoclonal antibody. Also the remaining four serotypes (D-set pilin types) bind with one other monoclonal antibody. Fourth, the antigens of the structural protein above are present in far greater numbers (perhaps 1000:1 up to 10,000:1) than any specific adhesion antigen associated with pili. Specific adhesion antigens are amino acid sequences presumably located on the tips or at intervals along the pili.
Using B. Nodosus as a model, highly conserved antigenic domains on the pilin protein were identified, isolated. and then amplified as immunogens according to the following three steps. First, the conserved antigenic domains on the pilin protein molecule were determined. Second, the polypeptide sequence of these as subunits of the intact pilin protein were reproduced. Third, these subunits were tested as antigenic determinants for stimulating cross reactive antipilus antibodies.
The following are detailed procedures for carrying out the above three steps to determine submolecular units of pilin proteins capable of eliciting protective antibodies against Type IV pilin bacteria. The first step of selecting an antigenic site was accomplished according to the following three procedures. First, computerized predictions of the antigenic profile for known B. nodoεuε base sequences were generated. Second, pilin proteins were digested and then tested against a battery of monoclonal antibodies. Third, sequence homology was compared based on published sequences.
ANTIGENIC PROFILE PREDICTIONS
For antigenic profile predictions, computer generated tertiary configurations of pilin molecules were used. This computer program is based on the composite value of the five parameters of hydrophilicity, alpha helix, beta sheet, random coil, and beta turns and their potential as available antigen sites on any selected region of the pilin polypeptide.
PILIN PROTEIN DIGESTION
A number of enzymatic procedures were used to cleave the 151 AA sequences of B. nodoεuε pilin into specific fragments for testing as conserved epitopes (Smyth, Methods in Enzymoloqy. Vol. XI: ed. by C.H. . Hirs, Academic Press, N.Y., pp. 214-230, 1967; Jacobson et al., J. Biol. Chem. 248:6583-6591, 1973). The cited methods were modified and trypsin digestion was completed after succinylation of lysine residues so that the pilin protein was cleaved on the carboxyl side of arginine residues to produce a peptide of approximately 5000 MW. This digest fragment contains one common epitope shared between 17 serotypes and is bound by the same monoclonal antibody which blocks adherence. A monoclonal antibody was used for demonstrating common antigens following the techniques described below. Adult BALB/c mice (Simonsen Laboratories, Gilroy, California) were injected intraperitoneally with purified pili (100 μg) that have undergone 4 cycles of MgCl2 precipitation and an SDS-PAGE analysis. Three days before fusion (2-7 weeks after the initial injection) , the mice were boosted with 20 μlg of pili intravenously. Spleen cells from each mouse were harvested, washed with serum-free media, and fused with SP2/0 myeloma cells in 50% polyethylene glycol. Fused cells were seeded into Linbro 96 well plates at 106 cells per well. Cells were fed with RPMI 1640 (Flow Laboratories) containing 15% HyClone defined fetal bovine serum and 1 mg/100 ml gentamicin and HAT. Hybridoma supernatants were screened for antibody production using ELISA. These procedures resulted in production of a family of monoclonals. One of these reacts with whole pili of 17 serotypes of B. nodoεuε, with purified pilin protein of these same serotypes, with a 5,000 MW fragment of pilin protein digest, and blocks attachment of B. nodoεuε to epithelial cells.
PEPTIDE SEQUENCING COMPARISONS
Published amino acid sequence data for 8 serotypes of B. nodoεuε are available (Elleman (1988) Microbiol. Rev. 52:233-247). Comparisons of these revealed areas of homology between all 8 serotypes. These areas were further examined for their antigenicity. The second step of reproducing selected sequences was accomplished according to the following procedures. Selected peptides were synthesized. Then portions of B. nodoεuε pilin genome were amplified.
SYNTHETIC PEPTIDE ANALYSIS METHODS
The pilin protein of eight serotypes of B. nodoεuε have been sequenced and compared for homology. Using the methods of Chou and Fasman (Ann Rev Bioche . 47:251-276, 1978) , the secondary structures represented by probable beta-turns were predicted. Also using computer generated models, three of these were compared for regions of hydrophobicity/hydrophilicity of the pilin. Using this rationale two peptides were synthesized where homology occurs between the pilin protein of various B. nodosuε Australian strains. These were bound to carrier molecules (KLH) and used in rabbits to produce antibodies against the peptides. Although these antibodies did bind to the synthetic peptides, they bound poorly to whole pili and did not block pili adherence. Thus, these two regions were shown not to be of major interest as antigenic sites and focused attention on more highly conserved regions.
B. NODOSUS PILIN GENOME AMPLIFICATION
An Applied Biosystems Model 380A Synthesizer was used to synthesize oligonucleotides up to 50 bases in length. These oligonucleotides correspond to the entire primary structural gene that codes for the pilin of B. nodosuε A198 incorporating phosphoramidites and standard methods. Also synthesized were complementary sequences to be used as bridges for reconstructing any portion of the genomic code for A198 pilin. Gaps in the second strand can be completed and sealed as desired using DNA polymerase I and DNA ligase. Using this technology, a specified oligonucleotide of 153 bases was assembled. This gene can be amplified using a cloning vector.
In an alternate and more reliable approach PCR was used to amplify the desired genomic segments out of native B. nodoεus cultures. This was accomplished by synthesizing two primers. The first primer was 27 bases with a Bam HI restriction site in the overhang as shown on the gene construct of Fig. 4. The second primer was 30 bases with a stop codon and Hind III site in the overhang as shown in the gene construct of Fig. 4. Such primer construction gave in-frame and directional efficiency for cloning. The primers were purified by acrylamide gel electrophoresis to give 2.5 mg/ml and 40 mg/ml, respectively. PCR amplification was accomplished with 25 cycles at 50°C annealing temperature. The resultant very tight band of B. nodosus DNA was purified by cTAB precipitation in high salt and 3 ammonium acetate precipitations with ethanol, giving a final DNA concentration of 500 ng/ul. The DNA fragment included the partial gene for the pilin protein molecule, and 21 additional bases including a stop codon. This PCR fragment insert was cloned into the over expression vector pTTQ8 (Amersham Cat. No. RPN 1259) and three of these clones were sequenced as follows. Inserts were primed with the ml3/pUC forward sequencing primer using a sequence USB.X This primer matches the pTTQ8 vector at 5 bases downstream from the Hind III site on the 3' side of the pTTQδ polylinker and allowed direct sequencing of the Bam HI through Hind III insert in the pTTQδ plasmid. All three clones sequenced were the same 160 base fragment, all have an open reading frame from Bam HI to Hind III, and were of the intended base sequence and number. To insure sufficient antigenicity for the small molecular weight peptide ( < 10,000 daltons) , the small peptide was expressed as a TrpE fusion protein. This was accomplished by subcloning into the pATH3 vector. The pATH3 system expressed a TrpE fusion protein of approximately 40,000 daltons comprising about 10% of total protein production. This system was scaled up giving approximately 50 mg of pilin-TrpE fusion protein that was purified over a preparative SDS-PAGE gel.
The third step of testing antigenic characteristics of peptides was accomplished according to the following procedures. Antibodies were produced against the peptides. These antibodies were tested for binding specificity to B. nodosus pili.
ANTIBODY PRODUCTION
Antibodies were generated by administering the fusion protein subcutaneously and intramuscularly into rabbits. This was done using 1 mg amounts contained in polyacrylamide gel and complete Freund's adjuvant after the methods of Rothbard et al., J. Exp. Med. 110:208-221, 1984.
ANTIBODY BINDING SPECIFICITY TO B. NODOSUS PILI
The immunoblot procedure used a nitrocellulose membrane to which whole B. nodosuε pili are fixed. The nitrocellulose binding sites unoccupied by transferred protein were saturated by incubation with 3% gelatin TBS for 1 hour. The treated nitrocellulose was incubated with antiserum dilution of 1:500 in TBS + 1% with gelatin, then washed 4 times 2 x 10 minutes with TBS and 0.05% Tween 20 and 2 x 10 minutes with Tween-free TBS pH 7.5. Antibody bound protein was then visualized by incubating for 1 hour in secondary antibody solution (goat antirabbit) conjugated with horseradish peroxidase diluted 1:2000 with antibody buffer. Then it was washed 4 times as above and developed with horseradish peroxidase color development. Using these immunoblot procedures, polyvalent rabbit antiserum, which was made against highly purified whole pilin, also bound the pilin protein.
Rabbits were inoculated with the 6,270 dalton fusion protein subunit of the pilin molecule contained in polyacrylamide gel and Freund's adjuvant. A 1:250 dilution of serum from the rabbits was used against 3 serotypes of B. nodoεuε, a whole bacteria and purified pilin preparation. The antipilus antibody produced in the rabbits, receiving the fusion protein, was detected by immunoblot techniques. See Table 1. Table 1 IMMUNOBLOT: Three Serotypes of B. nodosuε Purified Pili vs. Antibody to a
6270 Molecular Weight
Replicate of Partial Pilin Expressed as a Fusion Protein with TrpE.
ANTISERUM
* Rabbits were given booster injections
+ = Positive
+ = Very weak positive
= No detectable reaction
FIG. 1 shows the immunoblot results of different B. nodoεuε serotypes versus a submolecular unit of pilin protein antibody. Lane l showed the antiserum of rabbit #684 at pre-injection. Lane 2 showed antipilus antibody being produced in rabbit #684 against B. nodoεuε serotypes of A-set and D-set pili 98 days after receiving the 6,270 dalton fusion protein. Lane 3 showed the antiserum of rabbit #685 at pre-injection. Lane 4 showed antipilus antibody being produced in rabbit #685 against B. nodosuε serotypes of A-set and D-set pili 98 days after receiving the 6,270 dalton fusion protein. The differences in reaction between the samples of B. nodoεuε pili serotypes shown in FIG. 1 reflect the differences in pili concentration. The A 198 (A-set pilin) shown in FIG. 1 represent different sample passages.
The immunoelectron microscopy procedures carried out were a modification of those described by Lindberg et al. (1987) Nature 328:84-87. Whole B. nodoεuε purified pili were allowed to sediment onto a formvar-coated copper grid for 10 minutes. Then they were reacted three minutes against a drop of 1/10 dilution of antibody preparation in RLA-buffer followed by gentle (five minute) washing using P-buffer. The grids treated with the final antibody were washed for five minutes with P-buffer. Next the grids were negatively stained with 1% sodium silicotungstate and examined under the transmission electronmicroscope to detect the structural relationships of the pili.
Slide agglutination tests were run using lyophilized cultures of Eugon agar grown B. nodosus in aqueous suspension to provide approximately 107 bacteria/ml. Drops of this preparation were mixed with test sera on a slide and observed by light microscopy for agglutination or aggregation of the whole bacteria.
The use of colloidal gold label to detect antibody binding to pili was carried out through the following steps:
1. B. nodosus culture for agar plate resuspended in double distilled water, vortexes briefly, and clarified by centrifugation § 1,000 x g for 10 minutes;
2. lOul drop of supernatant place on a Formvar - coated grid for 20 minutes in a moist chamber @ 37°C; 3. grid was blotted and washed two times with tris buffered saline containing 0.3% Tween-20;
4. 10 ul drop of a 1:200 dilution of serum (in TBS/0.3% Tween) was placed on the grid and incubated for 90 minutes in a moist chamber @ 37°C;
5. grid was blotted and washed three times with TBS/Tween;
6. 10 ul drop of a 1:100 dilution of anti-rabbit IgG gold conjugate (lOnm) was placed on the grid and incubated for 120 minutes in a moist chamber @ 37°C; and
7. grid was blotted and washed five times with TBS/Tween, rinsed three times with distilled water, and stained with 1.3% phosphotungstic acid @pH 7.0; then examined with a transmission electron microscope.
An alternate method to show aggregation of serum-treated pili, rather than just attachment of gold to pili uses the following steps:
1. B. nodoεuε culture for agar plate resuspended in double distilled water, vortexes briefly, and clarified by centrifugation @ 1,000 x g for 10 minutes;
2. 10 ul drop of supernatant and lOul drop of 1:100 serum (diluted in TBS/0.3% Tween) mixed together in microcentrifuge tube, and incubated for 90 minutes @ 37°C;
3. 10 ul drop of mixture from Step #2 was placed on a Formvar-coated grid for 20 minutes in a moist chamber @ 37°C;
4. grid was blotted and washed three times with TBS/Tween;
5. 10 ul drop of a 1:100 dilution of anti-rabbit IgG gold conjugate (10 nm) was placed on the grid and incubated for 120 minutes in a moist chamber @ 37°C; and 6. grid was blotted and washed five times with TBS/Tween, rinsed three times with distilled water, and stained with 1.3% phosphotungstic acid §pH 7.0; then examined with a transmission electron microscope.
FIG. 2 shows immunoelectron microscopy results for B. nodoεuε Type II pili, one of the four D-set pilin Types, versus a submolecular unit of pilin protein antibody generated in rabbits using a 10 nm colloidal gold label. In FIG. 2 the pili without antibody are 5-6 nm in diameter. Those pili coated with antibody are 10-15 nm in diameter and show configurational disruption because of antibody cross binding.
FIG. 3 shows immunoelectron microscopy results for B. nodoεuε A 198 pili, one of the 17 known"A-set pilin Types, versus a submolecular unit of pilin protein antibody generated in rabbits using a 10 nm colloidal gold label. In FIG. 3 the pili without antibody are 5-6 nm in diameter and the pili coated antibody which are 10- 15 nm in diameter show configurational disruption. In both FIGS. 2 and 3 the colloidal gold label is less than the amount of bound antibody because the labeling reaction was not run to completion.
Antibodies against the submolecular units of pilin ' proteins bind pili of antigenic groups which represent all currently known B. nodoεuε serotypes causing them to clump. Clumping, which can be shown to be caused by antibody binding to the structural pilin protein molecule, has the effect of reducing the availability of adhesion proteins for attaching B. nodosus to host tissue. Thus, an antibody directed to common epitopes on structural pilin proteins of B. nodosus can mechanically interfere with its adherence to host tissue. This same stearic interference can similarly perturb all pili functions. Five separate and distinct gene configurations, coding for B. nodoεuε polypeptides of approximately 6000, 7500, 8150, 8500, and 9150 molecular weight, were determined. See the sequences in FIGS. 4, 5, 6, 7, and 8. Also five additional sequences, representing Neiεseria gonorrhea, N. meningitiε, Moraxella boviε, Vibrio cholera , and Pεeudomonaε aeroginoεa , were determined. All ten of these are constructs which may or may not have the first amino acid (phenylalanine, usually methylated) included. Each construct then continues with specific sequences, cut sites and stop codons such that they can be moved between vector systems. Examples of vectors include, but are not limited to, E. coli, Pεeudomonaε , yeasts, poxviruses. herpesvirus, and irridivirus. In this way either live virus vaccines or purified protein vaccines could be assembled depending upon efficacy, cost, feasibility and need.
As shown in FIG. 4, the first of these constructs is 150 bases with Bam HI and Hind III restriction sites added at the 5' and 3' ends, respectively. Also a stop codon is added at the 3' end. The second construct, as shown in FIG. 5, is identical to the construct in FIG. 4 except that 33 bases are inserted in front of both the stop codon and Hind III restriction site at the 3' end. In FIG. 6 the construct is identical to the one in FIG. 5 except for the 15 bases added. Although the construct in FIG. 7 is not a modification of those in FIGS. 5 and 6, it is similar. It is made up of 207 bases with Bam HI and Hind III sites added on the 5' and 3' ends , respectively. Also a stop codon is placed on the 3' end.
The construct in FIG. 8 differs from the one in FIG. 7 with the addition of 15 bases. All of these constructs are designed to express products with an appropriate and predicted alpha helix for histocompatibility processing. Thus, they may act as stand alone antigens (singlets) or as repeating units of antigens (doublets, triplets) . They are also designed to be expressed with fusion proteins such as Trp E for increasing the size of the molecule carrying the desired epitopes. Furthermore, synthetic peptides representing all or any antigenic portion of these constructs could be combined with a molecular carrier and used as antigens to generate antipili antibodies.
The five constructs as shown in FIGS. 9, 10, 11, 12 , and 13 include approximately 150-159 bases, the aforementioned restriction sites, and stop codons. These constructs represent the DNA sequences for N. gonorrhea, N. meningitiε, M. boviε, V. cholera, and P. aeroginoεa. See FIGS. 9, 10, 11, 12 , and 13, respectively. These constructs are designed so they can function in the same manner as the B. nodoεuε prototype construct.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made without departing from the spirit or scope of the invention.

Claims

1. An antigenic preparation active against a species of Type IV piliated bacteria comprising a submolecular unit of pilin protein corresponding to at least one epitope common to structural pilin proteins of the species of Type IV piliated bacteria, which submolecular unit is capable of eliciting antibodies capable of binding to the whole pili of the species of Type IV piliated bacteria.
2. An antigenic preparation according to claim 1 in which the submolecular unit of pilin protein is derived from a species selected from the group consisting of:
Bacteroideε nodosuε, Neiεεeria gonorrhea, Neiεεeria meningitiε , Moraxella boviε, Vibrio cholera, Eεcherichia coli, and Pεeudomonaε aeroginoεa .
3. An antigenic preparation according to claim 2 against Bacteroides nodosus, wherein the submolecular unit is selected from the group of polypeptides consisting of:
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly
He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu;
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu Gly Asn Asp Asp Lys Gly Lys Tyr Ala Leu Ala;
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala Phe Ala He Pro Ala Tyr Asn Asp Tyr He Ala Arg Ser Gin Ala Ala Glu Gly Leu Thr Leu Ala Asp Gly Leu Lys Val Arg He Ser Asp His Leu Gly Asn Asp Asp Lys Gly Lys Tyr Ala Leu Ala Thr He Asp Gly Asp; Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr He Ala Arg Ser Gin Val Ser Arg Val Met Ser Glu Thr Gly Gin Met Arg Thr Ala He Glu Thr Cys Leu Leu Asp Gly Lys Glu Gly Lys Asp Cys Phe He Gly Trp Thr Thr Ser Asn Leu; and Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr He Ala Arg Ser Gin Val Ser Arg Val Met Ser Glu Thr Gly Gin Met Arg Thr Ala He Glu Thr Cys Leu Leu Asp Gly Lys Glu Gly Lys Asp Cys Phe He Gly Trp Thr Thr Ser Asn Leu Leu Cys Ser Thr Asp Val Asp Glu Lys Phe Lys Pro Thr.
4. An antigenic preparation according to claim 3, further comprising repeating sequences of any of the polypeptides.
5. An antigenic preparation according to claim 3, further comprising at least one epitope of any of the polypeptides.
6. An antigenic preparation according to claim 2 against Neisseria gonorrhea , wherein the submolecular unit has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He Val Gly He Leu Ala Ala Val Ala Leu Pro Ala Tyr Gin Asp Tyr Thr Ala Arg Ala Gin Val Ser Glu Ala He Leu Leu Ala Glu Gly Gin Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn.
7. An antigenic preparation according to claim 6, further comprising repeating sequences of the polypeptide.
8. An antigenic preparation according to claim 6, further comprising at least one epitope of the polypeptide.
9. An antigenic preparation according to claim 2 against Neisεeria meningitiε , wherein the submolecular unit has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He Val Gly
He Leu Ala Ala Val Ala Leu Pro Ala Tyr Gin Asp Tyr Thr
Ala Arg Ala Gin Val Ser Glu Ala He Leu Leu Ala Glu Gly Gin Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn.
10. An antigenic preparation according to claim 9, further comprising repeating sequences of the polypeptide.
11. An antigenic preparation according to claim 9, further comprising at least one epitope of the polypeptide.
12. An antigenic preparation according to claim 2 against Moraxella boviε , wherein the submolecular unit has the following sequence:
Phe Thr Leu He Glu Leu Met He Val He Ala He He Gly He Leu Ala Ala He Ala Leu Pro Ala Tyr Gin Asp Tyr He Ser Lys Ser Gin Thr Thr Arg Val Val Gly Glu Leu Ala Ala Gly Lys Thr Ala Val Asp Ala Ala Leu Phe Glu Gly Lys Thr Pro.
13. An antigenic preparation according to claim 12, further comprising repeating sequences of the polypeptide.
14. An antigenic preparation according to claim 12, further comprising at least one epitope of the polypeptide.
15. An antigenic preparation according to claim 2 against Vibrio cholera, wherein the submolecular unit has the following structure:
Met Thr Leu Leu Glu Val He He Val Leu Gly He Met Gly Val Val Ser Ala Gly Val Val Thr Leu Ala Gin Arg Ala He Asp Ser Gin Asn Met Thr Lys Ala Ala Gin Ser Leu Asn Ser He Gin Val Ala Leu Thr Gin Thr.
16. An antigenic preparation according to claim 15, further comprising repeating sequences of the polypeptide.
17. An antigenic preparation according to claim 15, further comprising at least one epitope of the polypeptid .
18. An antigenic preparation according to claim 2 against Pεeudomonaε aeroginoεa , wherein the submolecular unit has the following structure:
Phe Thr Leu He Glu Leu Met He Val Val Ala He He Gly He Leu Ala Ala He Ala He Pro Gin Tyr Gin Asn Tyr Val Ala Arg Ser Glu Gly Ala Ser Ala Leu Ser Val Asn Pro Leu Lys Thr Thr Val Glu Glu Ala Leu Ser Arg Gly.
19. An antigenic preparation according to claim 18, further comprising repeating sequences of the polypeptide.
20. ' An antigenic preparation according to claim 18, further comprising at least one epitope of the polypeptide.
21. An antigenic preparation as claimed in any one of claims 1 to 20 in which the submolecular unit or any part of the submolecular unit of pilin protein is suspended in a suitable pharmaceutical carrier and used as a vaccine.
22. An antigenic preparation as claimed in claim 21 which includes an adjuvant.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4622223A (en) * 1984-06-21 1986-11-11 The Board Of Trustees For The Leland Stanford University Broad spectrum vaccine against gonorrhea
US4737363A (en) * 1984-12-24 1988-04-12 Commonwealth Scientific And Industrial Research Organization Bacteroides nodosus vaccine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4622223A (en) * 1984-06-21 1986-11-11 The Board Of Trustees For The Leland Stanford University Broad spectrum vaccine against gonorrhea
US4737363A (en) * 1984-12-24 1988-04-12 Commonwealth Scientific And Industrial Research Organization Bacteroides nodosus vaccine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GENE, Volume 85, No. 1, issued 1989, R. FAAST et al., "Nucleotide Sequence of the Structural Gene, tcp A, for a Major Pilin Subunit of Vibrio Cholerae", pages 227-231. *
J.R. EGERTON et al., "Footrot and Foot Abscess of Ruminants", Published 1989, by CRC PRESS Inc. (BOCA RATON, FL), pages 220-224. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 261, No. 33, issued 25 November 1986, K. JOHNSON et al., "Nucleotide Sequence and Transcriptional Site of Two Pseudomonas Aeruginosa Pilin Genes", pages 15703-15708. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 168, issued September 1988, W.W. REUHL et al., "Purification, Characterization, and Pathogenicity of Moraxella Bovis Pili", pages 983-1002. *
MOLECULAR MICROBIOLOGY, Volume 2, No. 5, issued September 1988, W.J. POTTS et al., "Nucleotide Sequence of the Structural Gene for Class I Pilin from Niesseria Meningitidis: Homologies with the pilE Locus of Neisseria Gonorrhoeae", pages 647-653. *
SCIENCE, Volume 228, issued 24 May 1985, J.F. YOUNG et al., "Expression of Plasmodium Falciparum Circumsporozoite Proteins in Escherichia Coli for Potential Use in a Human Malaria Vaccine", pages 958-962. *

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* Cited by examiner, † Cited by third party
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WO1999055875A3 (en) * 1998-04-29 2000-04-13 American Cyanamid Co VACCINES CONTAINING RECOMBINANT PILIN AGAINST NEISSERIA GONORRHOEAE OR $i(NEISSERIA MENINGITIDIS)
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