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WO2001078650A2 - Vaccins destines a proteger contre borrelia burgdorferi - Google Patents

Vaccins destines a proteger contre borrelia burgdorferi Download PDF

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Publication number
WO2001078650A2
WO2001078650A2 PCT/US2001/011243 US0111243W WO0178650A2 WO 2001078650 A2 WO2001078650 A2 WO 2001078650A2 US 0111243 W US0111243 W US 0111243W WO 0178650 A2 WO0178650 A2 WO 0178650A2
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oms66
polypeptide
vaccine
protein
immunogen
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PCT/US2001/011243
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English (en)
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WO2001078650A3 (fr
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Maurice M. Exner
Michael A. Lovett
David R. Blanco
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The Regents Of The University Of California
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Priority to AU2001259042A priority Critical patent/AU2001259042A1/en
Publication of WO2001078650A2 publication Critical patent/WO2001078650A2/fr
Publication of WO2001078650A3 publication Critical patent/WO2001078650A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to Borrelia burgdorferi polypeptides and vaccines for the prevention of Borrelia burgdorferi infection.
  • the disease is difficult to diagnose, because the symptoms mimic other diseases.
  • a characteristic red rash usually occurs at the site of the bite; however, the bite may go unnoticed. A few months after the bite, muscle paralysis, joint inflammation, neurological symptoms and sometimes heart symptoms may occur.
  • OspA The abundant Borrelia burgdorferi lipoprotein, OspA, is currently being used in a recombinant form as a human and animal vaccine against Lyme disease (Sigal, et al. (1998) New England Journal of Medicine 339:1638-1639; Steere, et al. (1998) New
  • OspA is now recognized to be downregulated when organisms are within a mammalian host (de Silva, et al. (1996) JExp Med. 183:271-275; Margolis, et al. (1993)
  • the OspA vaccine reduces the risk of acquiring Lyme disease by only 49-68% after two injections, and 76-92% after 3 injections (Steere et al. and Sigal et al., supra).
  • protection against heterologous strains may be limited (Lovrich, et al. (1995) Infect Immun. 63:2113-9; Masuzawa, et al. (1997) Microbiology and Immunology 41:733-6). Because of these concerns about the OspA vaccine, there has been a search for additional protective immimogens.
  • a surface exposed outer membrane protein of B. burgdorferi designated p66 (Bunikis, et al. (1995) Ferns Microbiol Lett. 131:139-145; Bunikis, et al. (1996) Infectlmmun. 64:5111-5116, Probert, et al. (1995) Infect Immun. 63:1933-1939) is expressed during human infection, as judged by the presence of specific antibodies in patients with Lyme disease (Bunikis, et al. 1996. Infect Immun. 64:5111-5116). This protein is conserved within Borrelia species, although there is some sequence variability between--?, burgdorferi sensu lato strains (Bunikis, et al. (1998) JBacteriol.
  • the invention provides vaccines for inducing immunoprotection in a mammal against infection by Borrelia burgdorferi, comprising a pharmaceutically acceptable excipient and an Oms66 polypeptide in a unit dosage form, wherein the polypeptide is capable of specifically binding a polyclonal antibody generated against a native conformation of the polypeptide displayed at positions 21 to 618 of SEQ ID NO:l, where the antibody has been cross-adsorbed with a denatured Oms66 polypeptide as displayed in SEQ ID NO:l, and where native confirmation is determined in a channel assay.
  • the Oms66 polypeptide is capable of forming a channel in a channel assay.
  • the Oms66 polypeptide is a recombinant polypeptide.
  • the vaccine comprises an adjuvant.
  • the adjuvant can be selected from the following: incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide and alum.
  • the vaccine further comprises a second immunogen from Borrelia burgdorferi.
  • the second immunogen can be selected from OspA, OspB, OspC, p35, p37 or the decorin binding protein DbpA.
  • the second immunogen can be a whole cell vaccine.
  • the invention also provides methods of preventing infection by Borrelia burgdorferi in a mammal, comprising administering to the mammal a vaccine comprising a pharmaceutically acceptable excipient and an Oms66 polypeptide in a unit dosage form, wherein the polypeptide is capable of specifcally binding a polyclonal antibody generated against a native conformation of the polypeptide displayed at positions 21 to 618 of SEQ ID NO:l, where the antibody has been cross-adsorbed with a denatured Oms66 polypeptide as displayed in SEQ ID NO:l, and where native confirmation is determined in a channel assay.
  • the Oms66 polypeptide is capable of forming a channel in a channel assay. In one aspect of the methods of the invention, the Oms66 polypeptide is a recombinant polypeptide.
  • the vaccine comprises an adjuvant.
  • the adjuvant can be selected from the following: incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide and alum.
  • the vaccine further comprises a second immunogen from Borrelia burgdorferi.
  • the second immunogen can be selected from OspA, OspB, OspC, p35, p37 or the decorin binding protein DbpA.
  • the second immunogen can be a whole cell vaccine.
  • Immunoprotection refers to the generation of an immunologic memory response wherein a subsequent exposure of an animal to an epitope generates an increased immune response, thereby preventing infection by a pathogen such as B. burgdorferi.
  • a "channel assay” is an assay for determining whether a polypeptide is capable of forming a functional channel in a lipid bilayer. One such assay is detailed below.
  • a “Oms66 polypeptide” refers to a sequence of about 550 to about 650 amino acids, comprising an amino acid sequence substantially identical to SEQ ID NO:l.
  • the precursor of the Oms66 protein is 618 amino acids and is processed into a mature protein of 597 amino acids (displayed at positions 21 to 618 of SEQ ID NO.T).
  • a full length Oms66 polypeptide, or f agments thereof, capable of forming a channel in a lipid bilayer, as described below, are particularly preferred.
  • Oms66 polypeptides of the invention are capable of being bound by antisera which was 1) generated against a polypeptide comprised of positions 21 to 618 of SEQ ID NO:l which is capable of forming a channel in a lipid bilayer, and 2) which has been cross-absorbed with a denatured polypeptide of SEQ ID NO: 1.
  • a Oms66 polypeptide is "denatured” if it is not capable of forming a channel in a lipid bilayer as described below.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to native conformation Oms66 can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with Oms66 and not with other proteins, except for polymorphic variants, orthologs, and alleles of Oms66. This selection may be achieved by subtracting out antibodies that cross-react with molecules such as denatured Oms66 polypeptides.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • substantially identity of polypeptide sequences means that a polypeptide comprises a sequence that has at least 70% sequence identity, at least 80% sequence identity, preferably at least 85%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. Accordingly, Oms66 sequences of the invention include polypeptide sequences that have substantial identity to SEQ ID NO: 1.
  • SEQ ID NO: 1 One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
  • Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • BLAST algorithm Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul (1990) J. Mol. Biol. 215: 403-410.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ ; see also Zhang (1997) Genome Res. 7:649-656 (1997) for the "PowerBLAST" variation.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra.).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as -defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad. Sci.
  • BLAST refers to the BLAST algorithm which performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • This invention provides vaccines comprising an Oms66 polypeptide or fragments thereof.
  • the present invention provides the surprising discovery that Oms66 in its native, porin, confirmation can act as a vaccine to raise an immunoprotective response against B. burgdorferi, the causal agent of Lyme disease.
  • the Oms66 polypeptide comprises at least one epitope that the native conformation of Oms66 has but the linear, denatured conformation lacks.
  • the Oms66 polypeptide of the vaccine is capable of forming a channel in a lipid bilayer.
  • the invention also provides methods of preventing infection by B. burgdorferi in a mammal by administering a vaccine comprising an Oms66 polypeptide.
  • the mammal is a human. In other preferred embodiments, the mammal is a dog.
  • Oms66 polypeptides are polypeptides, or fragments thereof, which are at least 70% identical to the polypeptide displayed in SEQ ID NO:l. In a preferred embodiment, the polypeptide will be in the native conformation.
  • Native conformation is determined either by the ability of a polypeptide to form a channel of between about 7 to about 11 nS in a lipid bilayer or the ability to bind to antisera which was 1) generated against a polypeptide comprised of positions 21 to 618 of SEQ ID NO:l which is capable of forming a channel in a lipid bilayer, and 2) which has been cross- absorbed with a denatured polypeptide of SEQ ID NO: 1. bind antisera generated capable of forming a channel in a channel assay (see below).
  • Oms66 polypeptides of the invention are not limited to the embodiments expressly described herein, but also includes modified polypeptides. So long as they meet the criteria discussed above, Oms66 polypeptides of the invention can include polypeptide fragments, peptides, peptide analogs or polypeptides with synthetic amino acids or other chemical modifications known to those of skill in the art. Without intending to be bound by any theory, the native conformation of the Oms66 polypeptide presents epitopes useful for generating an immunoprotective response in vaccinated animals. Therefore, the Oms66 polypeptide, or portions thereof which comprise one or more epitopes of the native conformation are preferred.
  • polypeptides provided herein, or portions thereof, under in vivo conditions may require their chemical modification since the polypeptides themselves may not have a sufficiently long serum and/or tissue half-life. It may also be advantageous to modify the polypeptides in order to impose a conformational restraint upon them. This might be useful, for example, to mimic a naturally-occurring conformation of the polypeptide in the context of the native protein in order to optimize the effector immune responses that are elicited. Modified polypeptides are referred to herein as "analog" polypeptides.
  • analog extends to any functional and/or structural equivalent of a peptide characterized by its increased stability and/or efficacy in- vivo or in- vitro in respect of the practice of the invention.
  • analog also is used herein to extend to any amino acid derivative of the polypeptides as described herein.
  • Analogs of the polypeptides or peptides contemplated herein include, but are not limited to, modifications to side chains, and incorporation of unnatural amino acids and/or their derivatives, non-amino acid monomers and cross-linkers. Other methods which impose conformational constraint on the polypeptides or their analogs are also contemplated.
  • polypeptides of the invention can be modified in a variety of different ways without significantly affecting the functionally important immunogenic behavior thereof. Possible modifications to the polypeptide sequence may include the following:
  • One or more individual amino acids can be substituted by amino acids having comparable or similar properties, thus: V may be substituted by I;
  • One or more of the amino acids of polypeptides of the invention can be replaced by a "retro-inverso" amino acid, i.e., a bifunctional amine having a functional group corresponding to an amino acid, as discussed in WO 91/13909.
  • One or more amino acids can be deleted.
  • Structural analogs mimicking the 3 -dimensional structure of the polypeptide can be used in place of the polypeptide itself.
  • side chain modifications contemplated by the present invention include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylatiori of amino groups with 2, 4, 6, trinitrobenzene sulfphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 '-phosphate followed by reduction with NaBH4.
  • the guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • Sulfhydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; and carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- " bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3 -nitro tyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butyglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and/or D-isomers of amino acids.
  • polypeptides could be conformationally constrained by, for example, incorporation of alpha-methylamino acids, introduction of double bonds between adjacent C atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between N and C termini, between two side chains or between a side chain and the N or C terminus.
  • polypeptides of the invention or their analogs may occur as single length or as multiple tandem or non-tandem repeats.
  • a single type of polypeptide or analog may form the repeats or the repeats may be composed of different molecules including suitable carrier molecules.
  • the immunogenicity of the polypeptides of the present invention may also be modulated by coupling to fatty acid moieties to produce lipidated polypeptides.
  • Convenient fatty acid moieties include glycolipid analogs, N-palmityl-S-(2RS)-2,3-bis- (palmitoyloxy)propyl-cysteinyl-serine (PAM3 Cys-Ser), N-palmityl-S-[2,3 bis (palmitoyloxy)-(2RS)-propyl-[R]-cysteine (TPC) or a dip almityl-ly sine moiety.
  • PAM3 Cys-Ser N-palmityl-S-[2,3 bis (palmitoyloxy)-(2RS)-propyl-[R]-cysteine
  • polypeptides may also be conjugated to a lipidated amino acid, such as an octadecyl ester of an aromatic acid, such as tyrosine, including octadecyl-tyrosine (OTH).
  • a lipidated amino acid such as an octadecyl ester of an aromatic acid, such as tyrosine, including octadecyl-tyrosine (OTH).
  • peptide vaccines there are a number of strategies for amplifying an immunogen's effectiveness, particularly as related to the art of vaccines. For example, cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency. See U.S. Patent No. 5,001,049 which is incorporated by reference herein.
  • a small peptide antigen can be conjugated to a suitable carrier, usually a protein molecule.
  • a suitable carrier usually a protein molecule.
  • This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule.
  • the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.
  • Suitable carriers include, e.g., keyhole limpet hemocyanin (KLH), serum albumin, purified protein derivative of tuberculin (PPD), ovalbumin, non-protein carriers and many others.
  • KLH keyhole limpet hemocyanin
  • PPD purified protein derivative of tuberculin
  • ovalbumin non-protein carriers and many others.
  • the conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, "A
  • nucleic acid sequences encoding individual chimera partners or domains are obtained from cDNA and genomic DNA libraries or isolated using amplification techniques with oligonucleotide primers.
  • a source that is rich in the desired target mRNA.
  • the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra).
  • the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb.
  • the fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro. Recombinant phage are analyzed by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977).
  • An alternative method of isolating nucleic acids encoding the Oms66 polypeptide combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences encoding Oms66 directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • Oligonucleotides can be designed to amplify nucleic acids encoding known sequences.
  • the Oms66 polypeptides of the invention are synthesized using recombinant nucleic acid techniques. After the gene encoding Oms66 is isolated, it is ligated into an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein. Techniques sufficient to guide one of skill through such procedures are found in, e.g., Sambrook, Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Ausubel et al. Finally, synthetic oligonucleotides can be used to construct recombinant genes for expression of polypeptides of this invention.
  • Oligonucleotides can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res.
  • oligonucleotides Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • this method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and nonsense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific subsequence of the gene of interest. The specific subsequence is then ligated into an expression vector. The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al, Gene 16:21-26 (1981).
  • the desired gene is cloned, it is expressed to obtain the Oms66 polypeptide.
  • the gene of interest e.g., an Oms66 gene
  • an expression vector that contains a strong promoter to direct transcription, a transcription translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation.
  • Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
  • Bacterial expression systems for expressing the protein are available in, e.g., E.
  • a preferred system for expressing the Oms66 polypeptides of the invention is the E. coli strain B21. Kits for such expression systems are commercially available. ⁇ ukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the chimera partner-encoding nucleic acid in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding the Oms66 polypeptide and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell may be included in the construct. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
  • Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra).
  • the transfected cells are cultured under conditions favoring expression of the fusion or fusion partner, which is recovered from the culture using standard techniques identified below.
  • Oms66 polypeptide may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).
  • Oms66 polypeptides can be expressed as a fusion protein. The Oms66 protein can then be released from the fusion by enzymatic cleavage prior to or after purification of the protein.
  • Oms66 polypeptides are purified.
  • proteins having established molecular adhesion properties can be reversibly fused to the polypeptide.
  • the polypeptide With the appropriate ligand, the polypeptide can be selectively adsorbed to a purification (affinity) column and then freed from the column in a relatively pure form. The ligand is then removed by enzymatic activity.
  • the Oms66 polypeptide could be purified using immunoaffinity columns.
  • the Oms66 polypeptides of the invention can also be purified using an anion exchange column.
  • An anion exchange column for instance, can be utilized with a column buffer consisting of 50 mM Tris, pH 8.0. A salt gradient of 0-1 M NaCl can be used for elution.
  • the polypeptides can be purified using Fast Protein Liquid Chromatography (FPLC).
  • FPLC Fast Protein Liquid Chromatography
  • rOms66 Purified recombinant Oms66 (rOms66) can be renatured using dialysis. Those of skill in the art will recognize that dialysis can be repeated to better remove salts from the polypeptide and to renature the protein.
  • Various dialysis buffers may be used in the dialysis. For example, a buffer containing 100 mM Tris, pH 8.0, 200 mM NaCl, 10 mM EDTA, and 0.05% Zwittergent 3-10 can be used.
  • the protein sample can be further purified using gel filtration chromatography. For instance, a Sephacryl S-300 column may be used.
  • Renaturation of proteins of the invention can be confirmed using the channel assay test as described below.
  • Preferred polypeptides of the invention are capable of forming a channel (or pore) in a channel assay.
  • the method used to test a polypeptide for channel activity is essentially as described by Skare, J. T., et al. JClin Invest. 96:2380-2392 (1995).
  • Lipid bilayers are formed using a lipid solution consisting of 20 mg/ml of azolectin in n- heptane. The lipid solution is painted across a 0.5 mm diameter aperture separating two sides of a chamber that contain a buffer solution.
  • Exemplary buffer solutions include, e.g., 1.0 M KC1 and 10 mM Tris pH 8.0. Bilayer formation is monitored by viewing the membrane through a telescope.
  • agar-agar silver chloride/silver electrode is inserted into the solution on each side of the membrane.
  • One electrode is connected to a voltage source, and the other to a current amplifier and chart recorder (with the output being monitored on a storage oscilloscope).
  • a voltage of 50 mV is then applied across the membrane.
  • a purified protein sample 25 ng is diluted.
  • the protein can be diluted in a solution of 1.0 M KC1, 10 mM Tris pH 8.0, and 0.01% Triton X-100.
  • the protein is added to the solution bathing a lipid bilayer, and conductance is measured. Conductance increases (indicating the insertion of a pore-forming molecule into the lipid bilayer) can then be recorded.
  • Channel conductance of approximately 7-11 nS indicates that the protein has formed a channel of the appropriate channel conductance and thus that it is an Oms66 protein of the proper conformation.
  • a preferred channel conductance of about 9.62 nS, i.e. the same channel conductance as the native protein see Exner et al, May 2000 Infect, and Immun., and Skare et al, Infect. Immun. 65: 3654-3661, 1997), is particularly preferred.
  • Oms66 polypeptide can be helpful to establish that a protein of interest is an Oms66 protein of the invention.
  • the antisera is preferably cross-absorbed with denatured Oms66 protein, thereby insuring that the antisera specifically binds Oms66 in the native confirmation as described herein.
  • antisera can be generated against Oms66 capable of forming a functional channel in a lipid bilayer and then can be cross absorbed with denatured Oms66 protein.
  • the cross-absorbed antisera can then be used as described below in a variety of immuno logical binding assays to determine if a protein of interest binds to the antisera and thus is an Oms66 protein of the invention.
  • polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against sample proteins, using a competitive binding immunoassay to determine if the sample protein has an equivalent conformation to that of native Oms66.
  • Specific polyclonal antisera and monoclonal antibodies will usually bind with a K d of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • Antibodies specific only for a particular Oms66 ortholog can also be made by subtracting out cross-reacting orthologs from a species such as non-E. burgdorferi bacteria.
  • the Oms66 can be detected and/or quantified using any of a number of .well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168 See, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • Methods in Cell Biology Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).
  • Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case native conformation Oms66 or an antigenic subsequence thereof).
  • the antibody e.g., anti-Oms66
  • the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
  • Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
  • the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
  • the labeling agent may be a labeled Oms66 polypeptide or a labeled anti-Oms66 antibody.
  • the labeling agent may be a third moiety, such a secondary antibody, which specifically binds to the antibody/Oms66 complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
  • the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
  • detectable moieties are well known to those skilled in the art. Throughout the assays, incubation and/or washing steps may be required after each combination of reagents.
  • Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. .However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. Non-competitive assay formats
  • Immunoassays for detecting the native conformation Oms66 in samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
  • antibodies generated against native conformation Oms66 proteins can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture Oms66 present in the test sample. The Oms66 proteins are thus immobilized and then bound by a labeling agent.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
  • the amount of the native conformation Oms66 present in the sample is measured indirectly by measuring the amount of known, added (exogenous) native conformation Oms66 displaced (competed away) from an antibody generated against native conformation Oms66 by the unknown sample Oms66 present in a sample.
  • a known amount of the native conformation Oms66 is added to a sample and the sample is then contacted with an antibody that specifically binds to the native conformation Oms66.
  • the amount of exogenous Oms66 bound to the antibody is inversely proportional to the concentration of the native conformation Oms66 present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of native conformation Oms66 bound to the antibody may be determined either by measuring the amount of Oms66 present in a Oms66/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
  • the amount of native confirmation Oms66 may be detected by providing a labeled Oms66 molecule.
  • a hapten inhibition assay is another preferred competitive assay. In this assay the known native conformation Oms66 is immobilized on a solid substrate. A known amount of antibody generated against native conformation Oms66 is added to the sample, and the sample is then contacted with the immobilized Oms66.
  • the amount of anti-Oms66 antibody bound to the known immobilized Oms66 is inversely proportional to the amount of native conformation Oms66 present in the sample.
  • the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
  • Immunoassays in the competitive binding format can also be used for crossreactivity determinations for native conformation Oms66.
  • a native conformation Oms66 protein can be immobilized to a solid support.
  • the ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the native conformation OMS 66 or immunogenic portion thereof to compete with itself.
  • the percent cross-reactivity for the above proteins is calculated, using standard calculations.
  • Those antisera with less than 10% cross- reactivity with each of the added proteins listed above are selected and pooled.
  • the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., denatured Oms66.
  • Antibodies that specifically bind to the native conformation of Oms66, as described herein, can also be made using this methodology.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immimoassay as described above to compare a second protein, thought to be perhaps an allele, ortholog, or polymorphic variant of Oms66, to the immunogen protein.
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the native conformation Oms66 protein that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to the respective Oms66 immunogen.
  • the immunogen may be combined or mixed with various solutions and other compounds as are known in the art.
  • Vaccines may be prepared as injectables, as liquid solutions or emulsions.
  • the polypeptides of the invention may be mixed with pharmaceutically-acceptable excipients which are compatible with the polypeptides. Excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the vaccine may further contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness of the vaccines.
  • adjuvants or agents examples include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • aluminum hydroxide aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide
  • Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N. J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
  • Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Only aluminum is approved for human use.
  • the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al 2 O 3 basis).
  • the amount of the immunogen can range from about 5 ⁇ g to about 100 ⁇ g protein per patient.
  • a preferable range is from about 20 ⁇ g to about 40 ⁇ g per dose.
  • a suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 20 ⁇ g of immunogen in admixture with 0.5% aluminum hydroxide.
  • the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
  • the vaccine may be incorporated into a sterile container that is then sealed and stored at a low temperature, for example 4 °C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
  • the vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (e.g., subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
  • the immunogen of the invention can be combined with appropriate doses of compounds including other epitopes of the target bacteria.
  • the immunogen could be a component of a recombinant vaccine that could be adaptable for oral administration.
  • Suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction.
  • Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.
  • Oral formulations may include normally employed excipients such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of the polypeptides of the invention.
  • the peptides of this invention can be formulated for administration via the nasal passages.
  • Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the active ingredient.
  • aqueous or oily solutions of the active ingredient include aqueous or oily solutions of the active ingredient.
  • the pharmaceutical formulation for nasal administration may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • the unit dosage for nasal administration can be from 7 to 3000 mg, preferably 70 to mg, and most preferably, 1 to 10 mg of active ingredient per unit dosage form.
  • Vaccines of the invention may be combined with other vaccines for the same or other diseases to produce multivalent vaccines.
  • a pharmaceutically effective amount of the immunogen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans.
  • Examples of other B. burgdorferi vaccines that can be combined with the Oms66 polypeptides of the invention include OspA (Sigal, et al. (1998) New England Journal of Medicine 339:1638-1639; Steere, et al. (1998) New England Journal of Medicine 339:209-15; Van Hoecke, et al. (1996) Vaccine. 14:1620-6), OspB, OspC and decorin binding protein A (DbpA)(Fikrig, et al. (1992) Infect. Immun. 60:657-661; Hanson, et al. (1998) Infect Immun. 66:2143-53; Mbow, et al. (1999) Infect Immun. 67:5470-5472).
  • Other preferred polypeptides that can be combined with Oms66 include . B. burgdorferi p35 and/or p37 proteins (Fikrig et al, Immunity 6(5):531-539 (1997).
  • the epitopes are typically segments of amino acids that are a small portion of the whole protein.
  • Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions within the natural amino acid sequence for the select target protein.
  • certain amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein.
  • Example 1 Isolation of the Oms66 gene
  • coli strain DH5 ⁇ was used for all cloning experiments.
  • the oms66 gene was cloned into pCR-TOPO for DNA sequencing (Invitrogen, Carlsbad, CA), and into pGEX 4T-1 (Pharmacia, Alameda, CA) for recombinant expression.
  • the polymerase chain reaction was used to amplify the oms66 gene from B. burgdorferi strain B313, using B313 chromosomal DNA as a template.
  • the primers used which included a BamHI restriction site at the 5' end of the gene, and an Xhol site at the 3' end of the gene, were as follows:
  • CGGCTCGAGTTAGCTTCCGCTGTAGGCTAT Amplification was carried out using 0.25 ⁇ g of genomic DNA template at 94°C for 30 seconds, 52 °C for 30 seconds, and 72°C for 2 min for 28 cycles in a Perkin Elmer DNA Thermal Cycler (Perkin-Elmer Corp., Norwalk, CT). Cloning of the resulting product into the BamHI and Xhol sites in pGEX 4T-1 generated a plasmid designated as pOms66. This construct was engineered such that expression of a GST-Oms66 fusion protein, followed by thrombin cleavage, would release a full length Oms66 protein with 2 additional amino acids at the N- terminus.
  • the amplified oms66 gene was cloned into the T-tailed pCR-TOPO vector as described in the Invitrogen cloning manual. This construct was used for DNA sequence analysis, which was performed at the University of Montana Molecular Biology Sequencing Facility using the dideoxy chain termination method (Sanger, F., et al. Proc. Natl. Acad. Sci. USA. 74:5463-5467 (1977)). Sequencing data confirmed that the cloned polynucleotide sequence was the Oms66 gene.
  • Example 2 Purification of Oms66 protein A. Purification of native conformation Oms66 protein This section demonstrates that the purification of Oms66 protein in its native conformation from E. burgdorferi. B.
  • burgdorferi 313 was first inoculated into 2.5 L of media and incubated at 34°C for 5 days.
  • Cells (approximately 2.5 x 10 11 ) were then pelleted by centrifugation at 10,000 x g for 20 min and washed 3 times in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Final cell pellets were then subjected to a sequential detergent solubilization. This was done by first resuspending the pellets in 40 ml of 1% sodium lauryl sarcosinate. The suspension was then frozen at -20°C, thawed, and sonicated for 15 seconds.
  • insoluble material which included most of the Oms66, was pelleted at 100,000 x g for 1 h at 4°C. The supernatant was removed, and the pellet was then resuspended in 20 ml of 0.5% octyl-polyoxyethylene (OPOE), sonicated, and centrifuged as described above. The supernatant was removed, and the pellet, which again included most of the Oms66, was resuspended in 20 ml of 1% hydrogenated Triton X-100 (hTX-100) and again sonicated and centrifuged.
  • OPOE octyl-polyoxyethylene
  • the supernatant from the hTX-100 solubilization was subjected to anion exchange and chromato focusing chromatography using a Fast Protein Liquid Chromatography (FPLC) system (Pharmacia, Alameda, CA).
  • FPLC Fast Protein Liquid Chromatography
  • the buffer used was 50 mM Tris pH 8.0, containing either 0.02% hTXIOO or 0.75%o octyl glucoside, and elution was performed with a salt gradient of 0 to 1 M NaCl.
  • the starting buffer was 25 mM Bis-Tris pH 6.7, containing either 0.1% hTXIOO or 0.75% octyl glucoside, and the eluent was 10% Polybuffer 74 (Pharmacia,
  • Oms66 was isolated from B. burgdorferi strain B313. This strain is deficient in the expression of a number of abundant lipoproteins (Sadziene, A., et al. Infect Immun. 63:1573-1580 (1995)), which facilitated the purification of nOms66.
  • a sequential detergent solubilization procedure on insoluble extracts of the B313 strain was used in order to enrich for the Oms66 protein, and to eliminate contaminating proteins.
  • the first two detergents used in the extraction procedure (sodium lauryl sarcosinate and octyl-polyoxyethylene, respectively) were found to release many contaminating proteins without releasing Oms66 from the insoluble extract.
  • the supernatant from the final hTX-100 detergent solubilization was markedly enriched with the Oms66 protein.
  • the Oms66 protein was shown, by gel analysis, to be pure and free from other B. burgdorferi proteins..
  • Western blot analyses were carried out using the Amersham ECL system (Amersham, Piscataway, NJ).
  • preparations of the Oms66 protein were spotted onto nitrocellulose membranes with a pore size of 0.45 ⁇ m (Schleicher &Schuell, Keene, NH).
  • 2 ⁇ l of protein solution, containing 0.5 ⁇ g of protein was spotted on the membrane, dried, and then subjected to ECL detection.
  • DH5 ⁇ harboring the pOms66 plasmid was grown in 6L of Luria broth containing lOO ⁇ g/ml of ampicillin. After growing the cells to mid-log phase, expression of the Oms66-GST fusion protein was induced by the addition of 1.0 mM (final concentration) isopropyl- ⁇ -D-thiogalactoside (IPTG) (Pharmacia, Alameda, CA). Expression was allowed to proceed for 3 h, at which time the cells were pelleted and resuspended in 100 ml of PBS. Cells were then lysed by sonication, followed by the addition of Triton X-100 to a final concentration of 1%.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the suspension was incubated for 1 hour at room temperature, and insoluble material was pelleted by centrifugation at 10,000 x g for 15 min.
  • the insoluble pellet was resuspended in 50 ml a solution containing 50 mM Tris, pH 8.0, and 8.0 M urea.
  • Insoluble material was pelleted at 10,000 x g for 15 min.
  • the supernatant was then dialyzed 3 times in 1 L of 50 mM Tris, pH 8.0. After dialysis, 100 U of thrombin (Pharmacia, Alameda, CA) was added to the sample, followed by incubation for 16 hours at 22°C.
  • Oms66 recombinant Oms66 protein
  • the GST moiety could not be used for affinity purification of the fusion protein, because denaturation by urea abrogated binding to glutathione-conjugated sepharose beads. Therefore, the cleaved Oms66 protein was purified to homogeneity by gel purification or Fast Protein Liquid Chromatography (FPLC).
  • FPLC Fast Protein Liquid Chromatography
  • the 66 kDa band corresponding to the cleaved Oms66 moiety of the fusion protein was excised from the membrane.
  • the protein was eluted from the membrane by mcubatmg membrane strips for 30 min at 22°C in 50 mM Tris, pH 8.0, with 2% SDS and 1% Triton X-100. After the incubation, the strips were centrifuged at 10,000 x g for 15 minutes, and the supernatant was saved.
  • the Oms66 protein was purified using Fast Protein Liquid Chromatography (FPLC) as follows.
  • FPLC Fast Protein Liquid Chromatography
  • An anion exchange column was utilized with a column buffer consisting of 50 mM Tris, pH 8.0.
  • a salt gradient of 0-1 M NaCl was used for elution.
  • rOms66 Purified recombinant Oms66 (rOms66) was then renatured using the following procedure: One mg of rOms66 was solubilized in a solution of 100 mM Tris, pH 8.0, 200 mM NaCl, 10 mM ethylene diamine tetraacetic acid (EDTA), 8 M urea, and 2% Zwittergent 3-10. The soluble protein was then dialyzed twice in 800 ml of a buffer containing 100 mM Tris, pH 8.0, 200 mM NaCl, 10 mM EDTA, and 0.05% Zwittergent 3-10. The dialysis proceeded with no stirring or mixing, such that a rapid removal of urea would not occur.
  • the protein sample was subjected to gel filtration chromatography on a column containing Sephacryl S-300, with a column buffer of 100 mM Tris, pH 8.0, 200 mM NaCl, 10 mM EDTA, and 0.05% Zwittergent 3-10.
  • agar-agar silver chloride/silver electrode was inserted into the solution on each side of the membrane.
  • One electrode was connected to a voltage source, and the other to a current amplifier and chart recorder (with the output being monitored on a storage oscilloscope).
  • a voltage of 50 mV was applied across the membrane.
  • Purified Oms66 protein 25 ng was diluted in a solution of 1.0 M KC1, 10 mM Tris pH 8.0, and 0.01% Triton X-100. The protein was added to the solution bathing a lipid bilayer, and conductance increases (indicating the insertion of a pore forming molecule into the lipid bilayer) were recorded.
  • Recombinant, renatured Oms66 protein was judged to be in its native conformation when it formed pores in this lipid bilayer system, and when the channel conductance was approximately 9 nS, i.e. the same channel conductance that the native protein formed (see Exner et al, May 2000 Infect, and Immun., and Skare et al, Infect. Immun. 65: 3654-3661, 1997).
  • Example 3 Conformation of the Oms66 plays a role in its function as an immunogen
  • This example demonstrates that the native conformation of Oms66 is important for the induction of a complement-dependent immune response that recognizes and kills B. burgdorferi.
  • Antisera generated against native confirmation Oms66 was capable of inducing complement-dependent bactericidal activity against a strain of B. burgdorferi.
  • the antisera was still capable of inducing the complement-dependent bacterial killing, indicating that the native conformation of Oms66 was important for inducing the immune response to B. burgdorferi.
  • nOms66 protein-binding protein
  • rabbits and mice were immunized with small amounts of protein (as low as 1 ⁇ g and 500 ng, respectively) via the popliteal lymph node and spleen, respectively.
  • sera were tested for complement-dependent killing activity as follows. In a microtiter plate, serial dilutions of test sera were made in normal rabbit serum (NRS) which was heat inactivated (56°C for 30 min). Ten ⁇ l of each test serum dilution was combined with 55 ⁇ l PBS, 25 ⁇ l of guinea pig serum (GPS) (Sigma, St. Louis, MO) as a source of active complement, and 10 ⁇ l of a E. burgdorferi suspension.
  • NBS normal rabbit serum
  • GPS guinea pig serum
  • the organisms used were harvested during the logarithmic phase of growth by centrifugation at 2000 X g for 10 min followed by washing in a 1 : 1 mixture of PBS and NRS. The concentration of organisms was adjusted to 2 x 10 organisms/ml, such that 2 x 10 6 organisms were added to each well. Assays were performed using either B. burgdorferi strain B313, or virulent, low passage B. burgdorferi strain B31. B31 was obtained from culture of skin biopsies of infected rabbits and was passaged only once prior to use. In order to determine whether complement mediated killing was responsible for bactericidal activity, assays were also performed using heat inactivated guinea pig serum.
  • a control was included using 25 ⁇ l GPS, 55 ⁇ l PBS, lO ⁇ l NRS, and lO ⁇ l organisms without the addition of immune serum. All test mixtures were made in duplicate and were incubated at 34°C for 6 hours at which time 5 ⁇ l of each sample was transferred to 200 ⁇ l of BSK II medium. The mixture was incubated an additional 12 hours, and another 5 ⁇ l was then transferred to 200 ⁇ l of medium.
  • both the rabbit serum and mouse sera possessed equally high titers (1:320 dilution) of complement-dependent bactericidal activity against strain B313. This activity was 8 times greater than that observed using serum from infection-derived immune rabbits (1 :40 dilution). No bactericidal activity was observed in the presence of heat-inactivated guinea pig serum as a complement source, indicating the complement-dependent nature of this activity. In comparison, these same sera showed no bactericidal activity against strain B31, even though this strain expresses Oms66. Recently, Bunikis et al. (Bunikis, J., et al. Infect. Immun.
  • Oms66-l to 6 represent results for the 6 different mice that were immunized with nOms66 protein.
  • rOms66 was conjugated to Sepharose 4-B beads (Pharmacia, Alameda, CA) as follows: 500 ⁇ g of rOms66, in 1 ml of 0.1 M NaHCO3, pH 8.0, 0.5 M NaCl, was added to 500 ⁇ l of CNBr activated Sepharose 4B that had been washed in 1 mM HC1.
  • the solution was mixed for 2 h at 22°C and then washed with the NaHCO3/NaCl buffer. Remaining active groups on the beads were blocked by incubating for 2 h in a solution of 0.1 M Tris, pH 8.0. The final product was then washed 3 times in alternating cycles of low pH buffer (0.1 M NaCH3COO, pH 4.0) and high pH buffer (0.1 M Tris, pH 8.0. 0.5 M NaCl). After the coupling process, 500 ⁇ l of anti-nOms66 rabbit serum was added directly to 150 ⁇ l the rOms66-conjugated beads, and to Sepharose 4B beads that were not coupled to a ligand. The suspensions were incubated for 2 h at 22°C, and the beads were then pelleted at 500 x g and removed. The cross-absorption was repeated three times.
  • IEM was performed to compare the abilities of different rabbit antisera to bind whole B. burgdorferi. IEM was performed as follows: 1 x 10 motile B. burgdorferi B31 and B313 were pelleted at 2000 x g for 10 min. The pellets were resuspended in 20 ⁇ l of BSK II medium, followed by the addition of test sera, diluted in BSK II medium. Final dilutions of 1 :40 were used for anti-nOms66 serum, anti-hOms66 serum, infection immune serum, and basal serum. The cross-absorbed serum was diluted 1:10, thus giving a final effective concentration of 1 :40 (since the cross-absorbed serum was diluted 1 :4 in the adsorption procedure).
  • Organisms were incubated with the sera for 4 hours, after which time 1.5 ml of a solution containing 0.15 M NaCl, 10 mM CaC12, and 10 mM MgC12 (SCM) was added. Organisms were pelleted at 2000 x g for 10 min, and were washed again in 1.5 ml of SCM. The organisms were then resuspended in 20 ⁇ l of SCM, and were incubated on a Parlodion 300 mesh copper grid for 10 min at 22°C. Grids were then washed 4 times in SCM and blocked for 30 min using 50% normal goat serum (NGS) and 50% SCM at room temperature in a humidified chamber.
  • NGS normal goat serum
  • Grids were then washed 8 times in SCM, followed by incubation for 1 h at 22°C in a humidified chamber with a 1 :20 dilution (in 10% NGS/90% SCM) of goat anti-rabbit immunoglobulin which was conjugated to 10 nm colloidal gold particles (Sigma, St. Louis, MO). Grids were washed 8 times in SCM, and 8 times in distilled water, and were then stained for 30 seconds using 2% uranyl acetate in water. Grids were then washed 6 times in distilled water, and were examined in a JEOL electron microscope using an accelerating voltage of 80 kV. For the enumeration of gold particle binding, particles were counted on the surface of 10 organisms.
  • the sera tested were antiserum against nOms66 (anti-nOms66), anti- - ⁇ nOms66 that was cross-absorbed with recombinant Oms66 (Ads-anti-nOms66), antiserum against heated and denatured nOms66 (anti-hOms66), serum from rabbits infected with B. burgdorferi and shown to be immune to challenge reinfection (IRS), and normal rabbit serum (NRS). All organisms used in the enumeration of particle binding were structurally intact, as judged by the absence of extracellular flagella and by the maintenance of cell structure.
  • anti-nOms66 antibody showed significant binding to the surface of strain B313 (17.1 gold particles/ ⁇ m2) whereas only 5.1 gold particles/ ⁇ m2 bound to strain B31.
  • This result correlated with the findings of bactericidal activity described above and confirmed that the accessibility of antibody binding to Oms66 on strain B31 is limited as Bunikis and coworkers have reported (Bunikis, J., et al. Infect. Immun. 67:2874-2883 (1999)).
  • the surface binding of anti- nOms66 on strain B313 was 2.28 times greater than that of IRS binding on these same organisms, which is also consistent with the greater level of bactericidal activity of anti- nOms66 as compared to IRS.
  • Ads-anti-nOms66 also bound to the surface of strain B313, although the amount of binding was reduced almost 2-fold (9.8 particles/ ⁇ m2) relative to the non-cross-absorbed serum. Interestingly, this level of binding by Ads-anti- nOms66 was comparable to that of IRS (7.5 particles/ ⁇ m2), yet Ads-anti-nOms66 showed 4-fold greater killing activity against strain B313 than IRS.
  • Oms66 protein was used in either its native conformation as described above, or in a denatured form.
  • Native Oms66 (nOms66) was in a solution containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.02% Triton X-100.
  • the denatured form (hOms66) was prepared by boiling native Oms66 in a 2% SDS solution, followed by acetone precipitation, and resuspension in the original Tris/NaCl/Triton X-100 buffer.
  • Example 5 Induction of an immunoprotective response to B. burgdorferi in mice
  • Ketamine and injected with l ⁇ g of protein via the popliteal lymph node as follows. The popliteal region was shaved and swabbed with ethanol. The lymph node was then located and palpitated until it was visible beneath the skin, and the antigen was injected through " the skin, directly into the lymph node. Boost immunizations were repeated at 2 and 3 months by popliteal lymph node injection. Serum was obtained prior to immunization (basal serum) and also prior to and 2 weeks after each boost. Infection derived immune rabbit serum was produced by infecting rabbits intradermally with 6 x 10 7 virulent B. burgdorferi B31. Serum was obtained two weeks after infection at which time the animals were shown to have cleared the infection and were resistant to challenge reinfection. Immune serum was stored at -76°C until ready for use.
  • soluble, nOms66 in 50 mM Tris, 0.02% hTXIOO was used to immunize 6 eight week old C3H HeJ mice.
  • the initial injection and the first 3 boost immunizations were administered subcutaneously with a dose of 1 ⁇ g for the primary injection, and 500 ng for each boost.
  • the fourth boost immunization (l ⁇ g) was given intrasplenically as follows. Mice were anesthetized using 200 mg/kg of ketamine and 10 mg/kg xylazine.
  • mice were challenged with infected tissue from a donor mouse as previously described (de Silva, A. M., et al. J Infect. Dis. 177:395-400 (1998); Barthold, S. W. Infect Immun. 67:36-42 (1999)).
  • the donor mouse adult C3H HeJ was prepared by infection with 104 virulent B. burgdorferi B31.
  • mice After 10 days, the mouse was anesthetized, and an ear was excised and cut into 2 mm x 2 mm tissue slices. These tissue slices were immediately implanted into the mice that were to be challenged. The implant procedure involved anesthetizing the mice, followed by aseptically making a small (4mm) incision on the back of the mouse. The donor tissue was then placed beneath the skin, and the wound was closed. Three weeks after challenge, mice were sacrificed and tissue biopsies were cultured in BSKI-I media containing 50 ⁇ g/ml of rifampin and 100 ⁇ g/ml of phosphomycin. The cultures were examined every 3 days for growth of B. burgdorferi, and cultures were classified as negative only after 5 weeks of incubation. Bartliold, et al (Infect Immun. 63:2255-2261 (1995)) have shown that
  • OspA is not expressed by host-adapted B. burgdorferi during mouse infection.
  • Table 3 six naive mice challenged with host-adapted organisms all developed local and disseminated infection as shown by positive culture results of their skin (616), ear (616), and joint tissues (6/6).
  • 4 of the 6 Oms66 immunized mice were completely protected following implant challenge using host-adapted organisms. All four of these animals showed no infection as judged by negative cultures of their skin, ear, joint, spinal cord, and bladder tissues.
  • these results demonstrate significant protection against host-adapted B. burgdorferi B31 following immunization with native Oms66.
  • This example demonstrates the vaccination of humans with an Oms66 polypeptide of the invention.
  • Humans most likely to be exposed to ticks carrying Lyme disease are the primary candidates for vaccination with the polypeptides of the invention.
  • Candidates include inhabitants of rural areas where occurrence of Lyme disease is high, such as in the northeast and northwest regions of the United States.
  • Other candidates include outdoor enthusiasts such as hikers or campers as well as people whose occupations might expose them to Lyme-disease carrying ticks.
  • the preferred dose form for adult humans is 25 ⁇ g, administered subcutaneously as a solution in phosphate buffer followed by one or more booster doses at 4 week intervals.
  • the does can include an aluminum adjuvant.

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Abstract

L'invention concerne la mise au point d'un vaccin destiné à créer une immunoprotection contre Borrelia burgdorferi qui est l'agent responsable de la maladie de Lyme. L'invention concerne notamment des vaccins comprenant un polypeptide Oms66.
PCT/US2001/011243 2000-04-13 2001-04-05 Vaccins destines a proteger contre borrelia burgdorferi WO2001078650A2 (fr)

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AU2001259042A AU2001259042A1 (en) 2000-04-13 2001-04-05 Vaccines for protection against borrelia burgdorferi

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US6054296A (en) * 1988-10-24 2000-04-25 Symbicom Ab 66 kDa antigen from Borrelia

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