This application claims the benefit of the filing date of U.S. provisional application 61/,178,261, filed May 14, 2009, which is incorporated by reference herein in its entirety.

This application was supported, in whole or in part, by the National Institutes of Health (grant numbers R43AI072810 and R43AI074092) and the CDC, grant number CKOOO 107. Therefore, the U.S. government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 30, 2010, is named 64557286.txt and is 13,501 bytes in size.

FIELD OF THE INVENTION

This invention relates, e.g., to agents and methods for diagnosing Lyme disease.

BACKGROUND INFORMATION

Lyme disease (sometimes referred to herein as LD or Lyme borreliosis) is a common vector- borne disease that is a significant public health concern. The disease is transmitted by the bite of various species of Ixodes ticks carrying the etiologic agent, a pathogenic Borrelia bacterium (a spirochete). Organisms of the Borrelia burgdorferi sensu lato group belong to the family

Spirochaetaceae, genus Borrelia. There are at least 1 1 species in the B. burgdorferi complex and an unknown but large number of substrains. At least three genospecies of the Borrelia burgdorferi sensu lato group have been identified as pathogens: B. burgdorferi sensu stricto, B. afzelli, and B. garinii. All three of these genospecies are found in Europe, but in North America, B. burgdorferi sensu stricto (sometimes referred to herein simply as B. burgdorferi) is, in general, the only pathogenic species found. The major reservoir of the infection in the United States is the white footed mouse, and the infection can be transmitted to many mammalian species, including various other forms of wildlife, e.g. Eastern chipmunks, and dogs, cats, and humans.

Clinically, Lyme disease is a progressive disease with a wide array of manifestations. Each of the three pathogenic genospecies of the Borrelia burgdorferi sensu lato group is associated with distinct clinical manifestations, suggesting that differences in genospecies may play an important role in the wide array of clinical manifestations observed in Lyme Disease. Early diagnosis and treatment is critical to prevent progression. Late disseminated infection can be associated with permanent damage to the nervous and musculoskeletal systems. Unlike most bacterial diseases that can be defined microbiologically by direct observation or culture of the pathogen, B. burgdorferi is difficult to culture or observe in clinical samples.

Therefore, Lyme disease is diagnosed indirectly. Erythema migrans (EM) is the classic marker for this infection at early stages. However, not all patients infected with pathogenic Borrelia develop EM. In the absence of EM, the current basis for diagnosis is the demonstration of an antibody response against a pathogenic Borrelia in an appropriate clinical setting. Unfortunately, current serologic assays for such antibodies lack sensitivity and affinity for detection of anti-5. burgdorferi antibodies in the early stages of the disease.

OspC was first identified as a seroreactive major outer surface protein in a subset of B. burgdorferi strains (Bissett et al. ( 1987) JCHn Microbiol 25, 2296-301 ; Wilske et al. (1988) Ann N Y Acad Sci 539, 126-143). It is a virulence factor upregulated just prior transmission to the mammalian host and is indispensable for establishing infection. OspC is the major protein expressed on the surface of B. burgdorferi in early infection (Stevenson et al. ( 1995) Infect Immun 6JJ ₁ 4535-9), induces a very early and strong IgM immune response (Wilske et al. (1993) Infect Immun 61, 2182- 91), and therefore has been suggested as an antigen marker for the serodiagnosis of early Lyme disease (Theisen et al. (1993) J Clin Microbiol 3JL 2570-6; Padula et al. (1994) J Clin Microbiol 32, 1733-8; Fung er αl. (1994) Infect Immun 62 _* 3213-21 ; Gerber et al. (1995) J Infect Dis VU, 724-7; Mathiesen et al. (1998) J Clin Microbiol 36, 3474-9; Panelius et al. (2002) J Med Microbiol 5_i, 731-9; Jobe et al. (2008) Clin Vaccine Immunol ϋ, 981-5).

OspC is one of the most diverse and thoroughly studied proteins in the Borrelia proteome. Distinct ospC genotypes are correlated with niche preference in natural reservoir species and invasiveness, pathogenesis and clinical manifestations in humans. Twenty-one known OspC phyletic groups (referred to as OspC genotypes), classified by letters A to U (Qiu et al. (1997) Hereditas Y2J_, 203-16; Wang et al. ( 1999) Genetics JH, 15-30; Qiu et al. (2002) Genetics 16O ₁ 833- 49), are distinguished by at least 8% amino acid sequence divergence. Table 1 summarizes some of the properties, including GenBank accession numbers that provide the sequences, as currently determined, of the 21 OspC genotypes. Given that there is at least 70% homology between all 21 known OspC genotypes, the presence of common epitopes that can be targeted for the development of new immunoprophylatic components has been explored (See, e.g., Earnhart et al. (2007) Clin Vaccine Immunol JL4, 628-34).

¹ A single GenBank sequence of each type is given as an example. This table refers to the GenBank accession numbers, including the sequences, as of the date of filing this application. The accession numbers and the corresponding sequences are incorporated by reference into this application. The SEQ ID NOs in this table refer to nucleic acid sequences encoding some representative OspC proteins. The sequences of the corresponding proteins are provided in the Sequence Listing filed with this application. *B. burgdorferi sensu stricto Groups P through S are only found in Europe.

There is a need for a simple, sensitive and specific diagnostic method for the detection of Lyme disease, particularly at early times after infection.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows OspC seroprofiling of laboratory infected mice. ELISA immunoarrays of five rOspC proteins (B, E, F, I and K) detect anti-OspC antibodies in all infected mice, regardless of the OspC- type of the B. burgdorferi with which the mouse was infected. All other rOspC proteins failed to detect anti-OspC antibodies from at least one B. burgdorferi-m ^' fected mouse (e.g., rOspC-type A did not detect mice infected with B. burgdorferi strains having either OspC-type D or M). Anti-mouse IgG HRP secondary antibody was used. ELISA readings below the detection cutoff (negative) are indicated as light grey bars.

Figure 2 shows variation among naturally-infected white-footed mice in the amount of antibodies detected by each rOspC protein. Each graph represents the frequency distribution of OD values obtained from the reaction of IgG in serum from naturally-infected white-footed mice (P. leucopus) to each type-specific-rOspC protein by ELISA. Serum panel tested positive for B. burgdorferi infection by a serological method.

Figure 3 shows variation among naturally-infected dogs in the amount of antibodies detected by each rOspC protein. Each graph represents the frequency distribution of OD values obtained from the reaction of IgG in serum from naturally-infected dogs (Canis lupus familiaris) to each type- specific-rOspC protein by ELISA. Serum panel tested positive for B. burgdorferi infection by a serological method.

Figure 4 shows variation among naturally-infected humans from North America in the amount of antibodies detected by each rOspC protein. Each graph represents the frequency distribution of OD values obtained from the reaction of IgG in serum from naturally-infected humans (Homo sapiens) to each type-specific-rOspC protein by ELISA. Serum panel tested positive for B. burgdorferi infection by a serological method. Figure 5 shows variation among naturally-infected humans from Europe in the amount of antibodies detected by each rOspC protein. Each graph represents the frequency distribution of OD values obtained from the reaction of IgG in serum from naturally-infected humans {Homo sapiens) to each type-specific-rOspC protein by ELISA. Serum panel tested positive for B. burgdorferi infection by a serological method.

Figure 6 shows the results of a seroprofiling study for vaccine design.

-rU, recombinant proteins from types A through U; SA-sU, serum harvested from mice that were vaccinated with type-specific OspC proteins.

DESCRIPTION

The present inventors have performed a series of comprehensive seroprofiling studies, on 16 of the 21 known OspC types, using serum panels from naturally infected white-footed mice, dogs and humans, and identify herein those OspC types which exhibit the most cross-reactive immunodominant epitopes. These studies are described in detail in the Examples. Briefly, a combination of OspC proteins from the OspC protein genotypes (sometimes referred to herein as OspC protein families or groups) K, B and J are shown to specifically and efficiently recognize antibodies resulting from infections by Lyme disease causative Borrelia found in North American in approximately 100% of the infected subjects that tested seropositive by other methods. A combination of OspC proteins from the OspC protein families K plus E, or K plus F, are shown to specifically and efficiently recognize antibodies resulting from infections by Lyme disease causative Borrelia found in Europe in approximately 100% of the infected subjects that tested seropositive by other methods. These appear to be the minimum number of OspC proteins that are required to detect infection by all of the known forms of Borrelia that cause systemic disease in either North America or Europe, respectively. Common epitopes present in OspC types B, E, F, J and K detect most anti- OspC antibodies present in serum samples from patients infected with B. burgdorferi that previously tested seropositive by other methods.

Diagnostic fragments, including peptides containing immunodominant epitopes, from each of these families of proteins are currently being identified; these peptides can be used instead of the full-length proteins in compositions and methods of the invention. Much of the following discussion relates to these peptides, as the peptides are preferred over full-length proteins as reagents for, e.g., the diagnostic tests of the invention. As used herein, there is no distinction between the length of peptides and polypeptides; and the terms "polypeptide," "protein," and "peptide" are used interchangeably.

The proteins of the invention are antigenic and therefore are useful to detect or diagnose the presence of Lyme disease-causing Borrelia. As described herein, antigenic refers to the ability of a compound to bind products of an immune response, such as antibodies, T-cell receptors or both.

Such responses can be measured using standard antibody detection assays, such as ELISA or standard T-cell activation assays.

The diagnostic assays of the invention are useful to identify those at risk for progressive (disseminated) illness. Antibody detection using antigen preparations of the present invention, incorporating a suitable combination (mixture) of OspC proteins, is much more sensitive than the current, single strain protocols.

Peptides, compositions comprising the peptides (such as diagnostic compositions), kits and methods of the invention offer a number of other advantages, as well. For example, they allow for simple, inexpensive, rapid and accurate detection of Lyme disease, and avoid serologic cross- reactivity with other conditions with "Lyme-like" symptoms, such as myalgias, arthralgias, malaise or fever, including conditions such as syphilis, chronic arthritis, and multiple sclerosis. This allows for an accurate diagnosis. Furthermore, a diagnostic test of the invention {e.g. an ELISA assay) is useful in serum samples that contain anti-OspA antibodies or other antibodies produced in response to a vaccine based on an outer surface protein of Borrelia; the OspC peptides (proteins) in the compositions of the invention do not cross-react with such antibodies, thereby allowing the differentiation of vaccinated individuals from individuals who were naturally infected with B. burgdorferi. In addition, in embodiments in which small peptides are used, the peptides in any combination can be readily combined with other peptides of the combination, or with other diagnostic peptides, e.g. from other Borrelia proteins, into a linear, multi-antigenic peptide for use in a diagnostic assay.

One aspect of the invention is a composition comprising OspC polypeptides (peptides) from Lyme Disease-causing Borrelia species, wherein the composition comprises one or more isolated polypeptides (peptides) from OspC families as follows: (a) at least one of each of K, B, and J; or (b) at least one of each of K and E; or (c) at least one of each of K and F. The polypeptides (peptides) in these compositions are sometimes referred to herein as "polypeptides (peptides) of the invention," and the compositions comprising these combinations of polypeptides (peptides) are sometimes referred to as "compositions of the invention." One or more polypeptides (peptides) from each of the noted families - e.g., 1, 2, 3, 4, 5 or more - may be present in a composition of the invention.

Another aspect of the invention is a diagnostic reagent, comprising a composition of peptides of the invention and, optionally, a system for detecting complexes of the peptides and specific antibodies, and/or a substrate for immobilizing the peptides (e.g., a microwell plate, an Immobilon or nitrocellulose membrane, or latex beads). A diagnostic reagent may comprise a detection system comprising detectable binding partners specific for the peptides and a signal generating reagent. In one embodiment, the binding partner is an antibody against a pathogenic Borrelia which is attached to an enzyme that, in the presence of a suitable substrate, can produce a detectable signal. Another aspect of the invention is a composition comprising a combination of peptides

(polypeptides) of the invention and, optionally, one or more additional peptides (polypeptides) that specifically recognize antibodies to a causative agent of Lyme disease. The additional peptides may be used in conjunction with a combination of peptides of the invention as part of a cocktail; or one or more of the additional peptides may be fused at the N-terminus and/or the C-terminus of one of the peptides of the invention to form a fusion peptide or polypeptide. The terms peptide and polypeptide are used interchangeably herein; for example, an amino acid sequence consisting of three 9-15-mer peptides linked directly to one another can be referred to as either a peptide or a polypeptide.

Another aspect of the invention is a kit for diagnosing Lyme disease in a subject, which comprises a combination of peptides of the invention and optionally comprises one or more additional peptides (polypeptides) as noted above. The peptide(s) may comprise a detectable label, or the kit may include a detection system (e.g. a labeled conjugate and a reagent) for detecting a peptide which is specifically bound to an antibody in the sample. In one embodiment, the kit contains a substrate for immobilizing the peptide, such as a microwell plate, an Immobilon or nitrocellulose membrane, or latex beads.

Another aspect of the invention is a method for diagnosing Lyme disease in a subject suspected of having antibodies against a causative agent of Lyme disease (e.g. for diagnosing exposure to and/or infection by a pathogenic Borrelia, or for detecting an immune response to a Lyme disease-causing Borrelia), comprising contacting a sample from the subject with a composition of peptides (polypeptides) of the invention, under conditions such that anti-OspC antibodies, if present in the sample, bind specifically to the peptides to form specific peptide/antibody complexes; and detecting antibodies that have bound to the OspC peptides (detecting the peptide/antibody complexes, e.g., detecting the amounts of the complexes). In one embodiment, the detection method is an enzyme-linked immunosorbent assay (ELISA); and/or is carried out in vitro. In one embodiment of the invention, an elevated level of antibody {e.g., a statistically significant increase, at least two {e.g., three) standard deviations higher) in the subject compared to a corresponding level of antibody in a control (such as a subject, or a pool of subjects, that exhibit no clinical manifestations of Lyme Disease or that have no known history of Lyme Disease), indicates that the subject has undergone an immune response to a Lyme disease-causing Borrelia {e.g., has been exposed to and/or infected by a pathogenic Borrelia, and/or has Lyme Disease). Another aspect of the invention is a method for eliciting an immune response in an animal

{e.g., immunizing an animal against Lyme disease), comprising administering to the animal an effective amount of a composition comprising one or more isolated polypeptides from OspC families as follows: a) at least one of each of C and K, b) at least one of each of C, K and A, or c) at least one of each of C, K, A and J, and, optionally, an adjuvant.

One aspect of the invention is a combination of isolated peptides (proteins) of the invention {e.g., a composition comprising a combination of isolated peptides (proteins) of the invention), each of which binds specifically to an antibody induced by a causative agent of Lyme disease (a pathogenic Borrelia), e.g. in a sample from a subject having Lyme disease. An antibody "induced by" a pathogenic Borrelia is sometimes referred to herein as an antibody "against" the pathogenic Borrelia. A polypeptide from a particular OspC "family," as used herein, refers to a polypeptide for which the encoding nucleic acid sequence is at least about 98% identical to that of the representative member of the family which is indicated in Table 1. A peptide (having an immunodominant epitope) from a particular OspC family member has a nucleic acid coding sequence that is at least about 98% identical to the sequence from the same region of the representative family member which is indicated in Table 1.

GenBank (www.ncbi.nlm.nih.gov) entries are periodically curated by NCBI staff, e.g. to correct mistakes in sequences. The "Comment" section of a GenBank record indicates when a sequence for a given accession number has been replaced with an updated (corrected) sequence. At any given time, only one sequence is associated with a given GenBank accession number. The sequences provided in this application reflect the sequence as known and listed in GenBank at the time of filing of this application. If these sequences are corrected, the updated sequences are to be considered as part of the instant application. As described herein, the ospC families of the present invention share about 98% identity at the nucleic acid level between strains of the same family and share no more than about 92% identity at the nucleic acid level between strains of different families. Determination of identity excludes any non-ospC sequences. Members of the same ospC family have similar antigenic profiles, e.g. they elicit an immune response against, or bind to antibodies produced in a cell in response to infection by, similar strains of Lyme disease causing Borrelia. The proteins of the present invention unexpectedly elicit immune responses to, and bind to antibodies produced in response to infection by, Lyme disease-causing Borrelia of different genospecies than the genospecies from which the component polypeptides were derived.

The members of a given OspC family will be evident to a skilled worker. For example, in one embodiment of the invention, Borrelia burgdorferi ospC family K comprises strains having the OspC alleles 272, 297, sh-2-82, CS9, OEAI l MUL, OC12 and OC13, wherein the OspC allele OC 12 is characterized by ospC of GenBank accession number AF029871 (e.g., having nucleic acid sequence, SEQ ID NO:5 and amino acid sequence SEQ ID NO: 13). In another embodiment, ospC family B comprises strains having the OspC alleles CS7, 109a, PMi, 160b, LDP73, MI415, 61BV3, VS219, 51405UT, MR623, OC2 and OC3, wherein the OspC allele OC2 is characterized by ospC of GenBank accession number AF029861 (e.g., having nucleic acid sequence, SEQ ID NO:1 and amino acid sequence SEQ ID NO: 9). In another embodiment, ospC family J comprises strains having the OspC alleles MIL, 188a, MI403 and OCI l, wherein the OspC allele OCI l is characterized by ospC of GenBank accession number AF029870 (e.g., having nucleic acid sequence, SEQ ID NO:4 and pep amino acid sequence SEQ ID NO: 1 T). In another embodiment, OspC family E comprises strains having the OspC alleles N40, 88a, 167BJM, SD91, NP 14,28691, 48102UT, and OC5, wherein the OspC allele OC5 is characterized by ospC of GenBank accession number AF029864 (e.g., having nucleic acid sequence, SEQ EDNO:2 and amino acid sequence SEQ EDNO: 10). In another embodiment, OspC family F comprises strains having the OspC alleles 27579, B 156, cawtb32, MI407, B. pacificus strain, and OC6, wherein the ospC allele OC6 is characterized by ospC of GenBank accession number AF029865 (e.g., having nucleic acid sequence, SEQ ID NO:3 and amino acid sequence SEQ ID NO: 11). A variant OspC protein or peptide, having a sequence that is at least 98% identical at the nucleic acid level to the comparable region of a known family member as discussed above, can also be considered to be a member of the family, and thus can also be used in a composition of the invention. A peptide or polypeptide, including a modified form thereof, which "binds specifically" to

("is specific for," binds "preferentially" to) an OspC antibody against a pathogenic Borrelia interacts with the antibody, or forms or undergoes a physical association with it, in an amount and for a sufficient time to allow detection of the antibody. By "specifically" or "preferentially" is meant that the peptide has a higher affinity, e.g. a higher degree of selectivity, for such an antibody than for other, non-OspC, antibodies in a sample. In general, the peptide has an affinity for the OspC antibody of at least about 2-fold higher than for other, non-OspC, antibodies in the sample. The affinity or degree of specificity can be determined by a variety of routine procedures, including, e.g., competitive binding studies.

As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "an" OspC antibody, as used above, includes 2, 3 or more OspC antibodies.

An "isolated" peptide or polypeptide of the invention is in a form other than it occurs in nature, e.g. in a buffer, in a dry form awaiting reconstitution, as part of a kit, etc. In some embodiments, the peptide is substantially purified. The term "substantially purified", as used herein refers to a molecule, such as a peptide, that is substantially free of other proteins, lipids, carbohydrates, nucleic acids and other biological materials with which it is naturally associated. For example, a substantially pure molecule, such as a peptide, can be at least about 60%, by dry weight, preferably at least about 70%, 80%, 90%, 95%, or 99% the molecule of interest.

In one aspect of the invention, the peptides (proteins, polypeptides) of a combination are physically unlinked to one another, and are present in the composition as individual components of a cocktail. In another aspect of the invention, two or more of the peptides of a combination are joined to one another to form a longer peptide (a chimeric peptide). In one embodiment, such joined

(linked) peptides are separated by a spacer. The spacer may consist, for example, of between about one and five {e.g., three) amino acids, preferably uncharged amino acids, e.g., aliphatic amino acids such as GIy or Ala. In one embodiment, the spacer is a triple GIy spacer. A linker may, e.g., provide distance between epitopes of different antigenic peptides.

Polypeptides of the invention, including polypeptides comprising linked peptides, maybe of any suitable length (e.g. between about 20-80 amino acids, or more), and they may contain any desirable number of linear epitopes (e.g. between about 2-5, or more), provided that they can function in a diagnostic or therapeutic (vaccine) method of the invention. For example, between 3 to 5 peptides of about 9-15 amino acids each may be combined, optionally in the presence of suitable spacers, to generate a polypeptide of about 45-50 amino acids. A length of about 50 amino acids can be readily synthesized chemically by current technologies. Other methods may be used to generate longer peptides. The peptides can be linked in any order. For example, a peptide of the invention may lie at the N-terminal end of a multipeptide, at the C-terminal end of a multipeptide, or between other peptides. Optionally, a peptide can contain an N-terminal Cys or Lys residue, e.g., to facilitate the addition of a biotin molecule.

The peptides of the invention may be modified by a variety of techniques, such as by denaturation with heat and/or SDS. A peptide of the invention may be modified to provide an additional N- or C-terminal amino acid sequence suitable for biotinylation, e.g., cysteine or lysine; suitable for chemical lipidation, e.g., cysteine; or the like. Peptides of the invention may be modified by any of a variety of known modifications. These include, but are not limited to, glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formatoin, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, modifications with fatty acids, transfer-RNA mediated addition of amino acids to proteins such as arginylation, etc. Analogues of an amino acid (including unnatural amino acids) and peptides with substituted linkages are also included.

Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, for instance, are described in many basic texts, such as Proteins—Structure and Molecular Properties, 2nd ed., T.E. Creighton, W.H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslationail Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y. Acad. Sci. 663:48-62. Peptides of the invention that consist of any of the sequences discussed herein may be modified by any of the discussed modifications. Such peptides still "consist of the amino acids.

In one embodiment of the invention, a peptide (protein) of the invention, which can be, e.g., a single full-length polypeptide; a peptide having a single immunodominant epitope; or a chimeric peptide in which two or more peptides, each having a different immunodominant epitope, are joined together; is associated with (e.g. coupled, fused or linked to, directly or indirectly) one or more additional moieties. The association can be, for example, via a terminal amino acid linker (such as Lys or Cys) or a chemical coupling agent; and the additional moiety or moieties may be linked to a peptide at its N-terminus, its C-terminus, or both. In one embodiment, a peptide (protein) of the invention is flanked by one or more additional peptides (e.g. from an OspC protein or from another protein of ^' Borrelia), on its N-terminus, its C-terminus, or both. In other embodiments, the additional moiety is, e.g., a detectable label, a fusion partner such as a chemical compound, or a substrate that immobilizes the peptide (e.g. a microwell plate, an Immobilon or nitrocellulose membrane, or latex beads).

A peptide of the invention can be fused to a fusion partner (e.g. a peptide or other moiety) that can be used to improve purification, to enhance expression of the peptide in a host cell, to aid in detection, to stabilize the peptide, etc. Examples of suitable compounds for fusion partners include polyethylene glycol, PEGylation, or other chemicals. Among the many suitable peptide or polypeptide fusion partners are, e.g., β-galactosidase, glutathione-S-transferase, a histidine tag, etc. In some embodiments, a peptide of the invention is provided with a detectable label, such as those described below.

A peptide of the invention can be associated with a substrate that immobilizes the peptide. The substrate can be, e.g., a solid or semi-solid carrier, support or surface. The association can be covalent or non-covalent, and can be facilitated by a moiety associated with the peptide that enables covalent or non-covalent binding, such as a moiety that has a high affinity to a component attached to the carrier, support or surface. For example, the peptide can be associated with a biotin moiety, and the component associated with the surface can be avidin. The peptide can be immobilized on the solid or semi-solid surface or carrier either prior to or after the addition of the sample containing antibody.

A peptide of the present invention can be in the form of a pharmaceutically acceptable salt. Suitable acids and bases that are capable of forming salts with the peptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

A peptide of the invention (including a chimeric polypeptide) can be produced using conventional chemical synthesis techniques, such as those described, e.g., in G. Barony et al., The Peptides: Analysis, Synthesis & Biology, Academic Press, pp. 3-285 (1980). Such chemically synthesized peptides can be obtained from commercial suppliers. Peptides produced by chemical synthesis can be obtained at purities exceeding about 95%. Therefore, there is typically a much reduced likelihood for undesirable cross reactivity with random antibodies than by using peptides obtained by other methods.

Alternatively, a peptide of the invention can be produced recombinantly following conventional genetic engineering techniques. To produce a recombinant peptide of the invention, a nucleic acid encoding the peptide is inserted into a suitable expression system. Generally, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected peptide is operably liked to an expression control sequence permitting expression of the peptide. Numerous types of appropriate expression vectors are known in the art, including, e.g., vectors containing bacterial, viral, yeast, fungal, insect or mammalian expression systems. Methods for obtaining and using such expression vectors are well-known. For guidance in this and other molecular biology techniques used for compositions or methods of the invention, see, e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, New York; Miller et al, Genetic Engineering, 8:277-298 (Plenum Press, current edition), Wu et al, Methods in Gene Biotechnology (CRC Press, New York, NY, current edition), Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, NJ, current edition), and Current Protocols in Molecular Biology, (Ausabel et al, Eds.,), John Wiley & Sons, NY (current edition), and references cited therein.

Suitable host cells or cell lines for the recombinant nucleic acids or vectors of the invention transfection by this method include bacterial cells. For example, various strains of E. coli {e.g., HBlOl, MC 1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, Streptomyces, and other bacilli and the like can also be employed in this method. Alternatively, a peptide of the invention can be expressed in yeast, insect, mammalian, or other cell types, using conventional procedures. Thus, the present invention provides a method for producing a recombinant peptide or polypeptide (e.g., a chimeric peptide), which involves transfecting or transforming, e.g., by conventional means such as electroporation, a host cell with at least one expression vector containing a polynucleotide of the invention under the control of an expression control sequence (e.g. a transcriptional regulatory sequence). The transfected or transformed host cell is then cultured under conditions that allow expression of the peptide or polypeptide. The expressed peptide or polypeptide is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art, including liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention. One skilled in the art can determine the purity of the peptide or polypeptide by using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g. SDS-PAGE); column chromatography (e.g. high performance liquid chromatography (HPLC)), or amino-terminal amino acid analysis.

Included in the invention are a polynucleotide encoding and/or expressing a peptide or polypeptide of the invention, a vector comprising the polynucleotide, and a host cell comprising the polynucleotide acid or vector.

A peptide of the invention may be used in combination with one or more additional peptides or polypeptides from the same or a different protein, from the same or a different pathogenic Borrelia strain, wherein the additional peptide(s) or polypeptide(s) also bind specifically to an antibody against a pathogenic Borrelia. The combination may comprise a cocktail (a simple mixture) of individual peptides or polypeptide, or it may be in the form of a fusion peptide or polypeptide (a multimeric or chimeric peptide). For example, a peptide of the invention may be fused at its N-terminus or C-terminus to another suitable peptide. Two or more copies of a peptide of the invention may be joined to one another, alone or in combination with one more additional peptides. Combinations of fused and unfused peptides or polypeptides can be used. In one embodiment, the additional peptide(s) contain B-cell and/or T-cell epitopes from a protein of a pathogenic Borrelia.

Suitable additional peptides or polypeptides (sometimes referred to herein as "antigenic peptides or polypeptides" or as "agents") can be derived from Borrelia antigens, such as OspA, OspB, DbpA, flagella-associated proteins FlaA(p37) and FlaB(p41 ), BBK32, BmpA(p39), p21 , p39, p66 or p83. See, e.g., Barbour erα/(1984) /«/ecf. Immun.45_, 94-100; Simpson etal. (199O)J. Clin. Microbiol.28, 1329-1337; Hansen etal. (1988) Infect. Immun. 56, 2047-2053; Hansen etal. (1988) Infect. J. Clin. Microbiol. 26, 338-346; Wilske et al. (1986) Zentral, Bakteriol, Parsitenkd, Infektionshkr, Hyg. Abt. 1 Orig. føjtøe, yl 2j6J3, 92-102; Dorward etα/. (199I)J. Clin. Microbiol.29, 1 162-1 170; published NTIS U.S. patent application No. 485,551; European patent application No. 465,204; International Patent Application No. PCT/US91/01500; International Patent Application No. PCT/EP90/02282; International Patent Application No. PCT/DK89/00248; International patent application No. WO92/00055. Polypeptides or peptides derived from other microorganisms can also be used.

A composition comprising peptides or polypeptides of the invention and, optionally, one or more additional agent(s), is particularly well-suited for diagnosing Borrelia infections early after infection (e.g., within one to two weeks after the onset of infection). The expression of OspC has been recognized in early human infection (e.g. an IgM antibody to it appears early after infection). Other proteins whose expression has been recognized early in human infection include BBK32, the flagella-associated protein, FlaB(p41), and, to a lesser extent, BmpA(p39), VIsE and the flagella- associated protein, FlaA(p37). Polypeptides or peptides which derive from those polypeptides are also suitable for assays for early infection. For example, suitable linear epitopes have been identified in FlaB(p41), e.g. residues 120 to 235. See, e.g., Crother etal. ((2003) Infect. Immun. Η, 3419-3428 and Wang et al. (1999)) Clin Microbial Rev \2, 633-653. Other peptides bearing either linear or conformational epitopes are known in the art.

Linear epitopes of OspC proteins have been described previously by others in conjunction with, e.g., diagnostic assays based on single Borrelia proteins. However, they were not disclosed as being a part of a composition comprising additional polypeptides, having broad cross-reactivity, as described herein. These linear epitopes, or comparable peptides from the OscC families K, B, J, E or F, can be also used in compositions of the present invention: PVV AESPKKP (SEQ ID NO:6), reported by Steere et al. (1987) Ann. Intern Med. 107, 725-731 ; ILMTLFLFISCNNS (SEQ ID NO:7), reported by AC Steere (2001) N Engl J Med 345, 115-25; one or more epitopes contained between amino acids 161 and 210, reported by Jobe et al. (2003) Clin Diagn Lab Immunol Jj), 573- 8)]; the peptides described in US Published Patent Application No. 2007/0178117, from the loop 5 region and/or the alpha helix 5 region of OspC; or the OspC peptides described in US Pat. No. 6,716,574. Variants of previously identified epitopes can be readily selected by one of skill in the art, based in part on known properties of the epitopes. For example, a known epitope may be lengthened or shortened, at one or both ends, by about 1-3 amino acids; one, two or more amino acids may be substituted by conservative amino acids; etc. Furthermore, if a region of a protein has been identified as containing a suitable epitope, an investigator can "shift" the region of interest (select different sub-sequences) up to about 5 amino acids in either direction from the endpoints of the original rough region, e.g. to optimize the activity. Methods for confirming that variant peptides are suitable are conventional and routine. Methods for identifying additional epitopes, particularly from variable regions rather than the conserved regions discussed above (e.g. from OspC, BBK32 or DbpA), are conventional.

Another aspect of the invention is a method for diagnosing Lyme disease in a subject (e.g. for diagnosing exposure to and/or infection by a pathogenic Borrelia, or for detecting an immune response to a Lyme disease causing Borrelia), comprising providing a sample, such as a bodily fluid (which would be expected to contain antibodies) obtained from the subject, and assaying it for the presence of an antibody against a causative agent of Lyme disease (e.g. an antibody capable of binding to such an agent), wherein an elevated level of antibody in the subject compared to a corresponding level of antibody in a control (such as a known unaffected subject, the mean or median value from a pool of such subjects, or a preset value that is proportional to the value from an unaffected subject) indicates an infection by the causative agent and/or that the subject has Lyme disease. A "causative agent for Lyme disease," as used herein, includes a pathogenic species of B. burgdorferi, such as the three identified pathogenic species that are discussed above. Other species of Borrelia which have been implicated in Lyme disease, such as, e.g., B. lusitaniae and B. valaisianae, are also included, provided they induce antibodies which can react specifically with a protein or peptide of the invention. It is to be understood that the term "pathogenic Borrelia," as used herein, refers to any such pathogenic genospecies that causes Lyme disease. "Lyme disease," as used herein, refers to an disease which exhibits the characteristics as summarized in Dattwyler, RJ. and Wormser, G. "Lyme borreliosis." in Infectious Diseases Medicine and Surgery (eds.) S. Gorbach and J. Bartlett, 3 ^rd edition, Saunders Pub. New York, New York, 2003 and which is caused by a pathogenic Borrelia.

In one embodiment of a diagnostic method of the invention, a composition comprising peptides of the invention and, optionally, one of more of the above-mentioned additional peptides (e.g. in the form of a cocktail or a fusion peptide or polypeptide) is used in a single tier assay, for detecting early/or and late stage Lyme disease. Such a peptide cocktail or fusion polypeptide can be effective in the diagnosis of Lyme disease as caused by a wide spectrum of pathogenic Borrelia isolates. One embodiment of a diagnostic method of the invention comprises contacting (incubating, reacting) a combination of proteins (peptides) of the invention with a sample of a biological fluid (e.g. serum or CSF) from a subject (e.g. human or other animal) to be diagnosed (a subject exhibiting the clinical symptoms of, or suspected of having, Lyme disease). In the presence of an antibody response to infection with a pathogenic Borrelia, antigen-antibody complexes are formed between the proteins (peptides) and antibodies in the sample. The antigen-antibody complexes are sometimes referred to herein as an antibody-protein or antibody-peptide complexes, peptide- antibody complexes, or antibody-epitope complexes; these terms are used interchangeably. Subsequently the reaction mixture is analyzed to determine the presence or absence of the antigen- antibody complexes. A variety of conventional assay formats can be employed for the detection, such, e.g., as ELISA or lateral flow. The presence of an elevated amount of an antibody-peptide complex indicates that the subject was exposed to and infected with a pathogenic Borrelia capable of causing Lyme disease. In an ELISA assay, a positive response is defined as a value 2 or 3 standard deviations greater than the mean value of a group of healthy controls. In some embodiments, a second tier assay is required to provide an unequivocal sero-diagnosis of Lyme disease.

The subject can be any subject (patient) in which antibodies can be made against the causative agent and detected. Typical subjects include vertebrates, such as mammals, including wildlife (e.g. mice and chipmunks), dogs, cats, non-human primates and humans. In one embodiment, the diagnostic method involves detecting the presence of naturally occurring antibodies against pathogenic Borrelia (e.g. B. Burgdorferi) which are produced by the infected subject's immune system in its biological fluids or tissues, and which are capable of binding specifically to the peptide or combinations of peptides of the invention and, optionally, one or more suitable additional antigenic polypeptides or peptides. Phrases such as "sample containing an antibody" or "detecting an antibody in a sample" are not meant to exclude samples or determinations (detection attempts) where no antibody is contained or detected. In a general sense, this invention involves assays to determine whether an antibody produced in response to infection with a pathogenic Borrelia is present in a sample, irrespective of whether or not it is detected. Conditions for reacting peptides and antibodies so that they react specifically are well-known to those of skill in the art. See, e.g., Current Protocols in Immunology (Coligan etal., editors, John Wiley & Sons, Inc) or the Examples herein. A diagnostic method of the invention comprises analyzing a sample of body fluid or tissue likely to contain antibodies. The antibodies can be, e.g., of IgG, IgE, IgD, IgM, or IgA type. Generally, IgM and/or IgA antibodies are detected, e.g. for the detection of early infection. IgG antibodies can be detected when some of the additional peptides discussed above are used in the method (e.g. peptides for the detection of flagellum proteins). The sample is preferably easy to obtain and may be serum or plasma derived from a venous blood sample or even from a finger prick. Tissue from other body parts or other bodily fluids, such as cerebro-spinal fluid (CSF), saliva, gastric secretions, mucus, etc. are known to contain antibodies and may be used as a source of the sample. Once the peptide antigen and sample antibody are permitted to react in a suitable medium, an assay is performed to determine the presence or absence of an antibody-peptide reaction. Among the many types of suitable assays, which will be evident to a skilled worker, are immunoprecipitation and agglutination assays.

In embodiments of the invention, the assay may comprise (1) immobilizing the antibody(s) in the sample, adding a composition of peptides of the invention, and then detecting the degree of antibody bound to the peptides, e.g. by the peptides being labeled or by adding a labeled substance (conjugate, binding partner), such as a labeled antibody, which specifically recognizes the peptides; (2) immobilizing a composition of peptides of the invention, adding the sample containing an antibody(s), and then detecting the amount of antibody bound to the peptides, e.g. by adding a labeled substance (conjugate, binding partner), such as a labeled antibody, which specifically recognizes the antibody; or (3) reacting the composition of peptides and the sample containing antibody(s) without any of the reactants being immobilized, and then detecting the amount of complexes of antibodies and peptides, e.g. by the peptides being labeled or by adding a labeled substance (conjugate, binding partner), such as a labeled antibody, which specifically recognizes the peptides.

Immobilization of the peptides of the invention can be either covalent or non-covalent, and the non-covalent immobilization can be non-specific (e.g. non-specific binding to a polystyrene surface in e.g. a microtiter well). Specific or semi-specific binding to a solid or semi-solid carrier, support or surface, can be achieved by the peptides having, associated with them, a moiety which enables their covalent or non-covalent binding to the solid or semi-solid carrier, support or surface. For example, the moiety can have affinity to a component attached to the carrier, support or surface. In this case, the moiety may be, e.g., a biotin or biotinyl group or an analogue thereof bound to an amino acid group of the peptide, such as 6-aminohexanoic acid, and the component is then avidin, streptavidin or an analogue thereof. An alternative is a situation in which the moiety has the amino acid sequence His-His-His-His-His-His (SEQ ID NO:8) and the carrier comprises a Nitrilotriacetic Acid derivative (NTA) charged with Ni ⁴⁺ ions. Among suitable carriers, supports or surface are, e.g., magnetic beads or latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene- divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass, gelatin or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or Fab fragments of such antibodies. The protocols for immunoassays using antigens for detection of specific antibodies are well known in art. For example, a conventional sandwich assay can be used, or a conventional competitive assay format can be used. For a discussion of some suitable types of assays, see Current Protocols in Immunology (supra). In a preferred assay, a peptide of the invention is immobilized to the solid or semi-solid surface or carrier by means of covalent or non-covalent binding, either prior to or after the addition of the sample containing antibody.

Devices for performing specific binding assays, especially immunoassays, are known and can be readily adapted for use in the present methods. Solid phase assays, in general, are easier to perform than heterogeneous assay methods which require a separation step, such as precipitation, centrifugation, filtration, chromatography, or magnetism, because separation of reagents is faster and simpler. Solid-phase assay devices include microtiter plates, flow-through assay devices, dipsticks and immunocapillary or immunochromatographic immunoassay devices.

In embodiments of the invention, the solid or semi-solid surface or carrier is the floor or wall in a microtiter well; a filter surface or membrane (e.g. a nitrocellulose membrane or a PVDF (polyvinylidene fluoride) membrane, such as an Immobilon membrane); a hollow fiber; a beaded chromatographic medium (e.g. an agarose or polyacrylamide gel); a magnetic bead; a fibrous cellulose matrix; an HPLC matrix; an FPLC matrix; a substance having molecules of such a size that the molecules with the peptide bound thereto, when dissolved or dispersed in a liquid phase, can be retained by means of a filter; a substance capable of forming micelles or participating in the formation of micelles allowing a liquid phase to be changed or exchanged without entraining the micelles; a water-soluble polymer; or any other suitable carrier, support or surface.

In some embodiments of the invention, the peptides are provided with a suitable label which enables detection. Conventional labels may be used which are capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Suitable detection methods include, e.g., detection of an agent which is tagged, directly or indirectly, with a fluorescent label by immunofluorescence microscopy, including confocal microscopy, or by flow cytometry (FACscan); detection of a radioactively labeled agent by autoradiography; electron microscopy; immunostaining; subcellular fractionation, or the like. In one embodiment, a radioactive element (e.g. a radioactive amino acid) is incorporated directly into a peptide chain; in another embodiment, a fluorescent label is associated with a peptide via biotin/avidin interaction, association with a fluorescein conjugated antibody, or the like. In one embodiment, a detectable specific binding partner for the antibody is added to the mixture. For example, the binding partner can be a detectable secondary antibody which binds to the first antibody. This secondary antibody can be labeled, e.g., with a radioactive, enzymatic, fluorescent, luminescent, or other detectable label, such as an avidin/biotin system.

A "detection system" for detecting a bound peptide, as used herein, may comprise a detectable binding partner, such as an antibody specific for the peptide. In one embodiment, the binding partner is labeled directly. In another embodiment, the binding partner is attached to a signal generating reagent, such as an enzyme that, in the presence of a suitable substrate, can produce a detectable signal. A surface for immobilizing the peptide may optionally accompany the detection system.

In embodiments of the invention, the detection procedure comprises visibly inspecting the antibody-peptide complex for a color change, or inspecting the antibody-peptide complex for a physical-chemical change. Physical-chemical changes may occur with oxidation reactions or other chemical reactions. They may be detected by eye, using a spectrophotometer, or the like.

In one embodiment of the method, the peptide or mixture of peptides, is electro- or dot- blotted onto nitrocellulose paper. Subsequently, the biological fluid (e.g. serum or plasma) is incubated with the blotted antigen, and antibody in the biological fluid is allowed to bind to the antigen(s). The bound antibody can then be detected, e.g. by standard immunoenzymatic methods.

In another embodiment of the method, latex beads are conjugated to the antigen(s) of the invention. Subsequently, the biological fluid is incubated with the bead/peptide conjugate, thereby forming a reaction mixture. The reaction mixture is then analyzed to determine the presence of the antibodies. One preferred assay for the screening of blood products or other physiological or biological fluids is an enzyme linked immunosorbant assay, i.e., an ELISA. Typically in an ELISA, the isolated antigen(s) of the invention is adsorbed to the surface of a microtiter well directly or through a capture matrix (i.e., antibody). Residual, non-specific protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent). The well is then incubated with a biological sample suspected of containing specific anti-pathogenic Borrelia (e.g. B. burgdoferi) antibody. The sample can be applied neat, or more often it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. After incubating for a sufficient length of time to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with an optimal concentration of an appropriate anti- immunoglobulin antibody (e.g., for human subjects, an anti-human immunoglobulin (αHuIg) from another animal, such as dog, mouse, cow, etc.) that is conjugated to an enzyme or other label by standard procedures and is dissolved in blocking buffer. The label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase, glucose oxidase, etc. Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove unbound conjugate, and a suitable substrate for the enzyme is added. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally (measured at an appropriate wave length). The cutoff OD value may be defined as the mean OD+3 standard deviations (SDs) of at least 50 serum samples collected from individuals from an area where Lyme disease is not endemic, or by other such conventional definitions. In the case of a very specific assay, OD+2 SD can be used as a cutoff value.

In one embodiment of an ELISA, a peptide or mixture of peptides of the invention is immobilized on a surface, such as a ninety-six-well ELISA plate or equivalent solid phase that is coated with streptavidin or an equivalent biotin-binding compound at an optimal concentration in an alkaline coating buffer and incubated at 4 ^° C. overnight. After a suitable number of washes with standard washing buffers, an optimal concentration of a biotinylated form of a composition/antigen of this invention dissolved in a conventional blocking buffer is applied to each well; a sample is added; and the assay proceeds as above.

See the Examples for typical conditions for performing ELISA assays.

Another useful assay format is a lateral flow format. Antibody to human or animal antibody or staph A or G protein antibodies is labeled with a signal generator or reporter (i.e. colloidal gold) that is dried and placed on a glass fiber pad (sample application pad). The diagnostic peptide is immobilized on membrane, such as a PVDF (polyvinylidene fluoride) membrane (e.g. an Immobilon membrane (Millipore)) or a nitrocellulose membrane. When a solution of sample (blood, serum, etc) is applied to the sample application pad, it dissolves the colloidal gold labeled reporter and this binds to all antibodies in the sample. This mixture is transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by capillary action. If antibodies against the diagnostic peptide are present, they bind to the diagnostic peptide striped on the membrane generating a signal. An additional antibody specific to the colloidal gold labeled antibody (such as goat anti-mouse IgG) is used to produce a control signal.

It should be understood by one of skill in the art that any number of conventional protein assay formats, particularly immunoassay formats, may be designed to utilize the isolated peptides of this invention for the detection of pathogenic Borelia {e.g. B. burgdorferi) infection a subject. This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats that are known to those of skill in the art.

Reagents for ELISA or other assays according to this invention can be provided in the form of kits. Such kits are useful for diagnosing infection with a pathogenic Borrelia {e.g. a B. burgdorferi), using a sample from a subject {e.g. a human or other animal). Such a diagnostic kit can contain peptides of the invention (and, if desired, additional peptides as discussed above) and, optionally, a system for (means enabling) detection of peptides of the invention bound to antibodies against a protein from a pathogenic Borrelia, and/or a surface to which the peptides can be bound. In one embodiment, a kit contains a mixture of suitable peptides or means for preparing such mixtures, and/or reagents for detecting peptide-antibody complexes.

The kit can include microtiter plates to which the peptide(s) of the invention have been pre- adsorbed, another appropriate assay device, various diluents and buffers, labeled conjugates or other agents for the detection of specifically bound antigens or antibodies, and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens. Other components of a kit can easily be determined by one of skill in the art. Such components may include coating reagents, polyclonal or monoclonal capture antibodies specific for a peptide of the invention, or a cocktail of two or more of the antibodies, purified or semi-purified extracts of these antigens as standards, MAb detector antibodies, an anti-mouse or anti-human antibody with indicator molecule conjugated thereto, an ELISA plate prepared for absorption, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup, etc. In one embodiment, a kit comprises buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide-antibody complex. Such kits provide a convenient, efficient way for a clinical laboratory to diagnose infection by a pathogenic Borrelia, such as a B. burgdorferi.

Another aspect of the invention is a method for eliciting an immune response in an animal against OspC proteins {e.g., for immunizing an animal against Lyme disease), comprising administering to the animal an effective amount of a composition of the invention and, optionally, an adjuvant. Suitable animals include a variety of mammals, including dogs, cats, and humans.

As noted above, in early infection, OspC is the major outer membrane protein expressed by the spirochete. Even though OspC has been demonstrated to have limited surface exposure, OspC is a potent immunogen. Immunization with OspC is protective against tick-transmitted Borrelia infection (Gilmore et al. ( 1999) Infect Immun. 64, 2234 2239). However, because OspC is highly variable in its sequence, the protection is limited to the Borrelia burgdorferi strain expressing the same immunizing OspC encoded by a specific allele. Challenge with heterologous isolates, expressing other OspC alleles results in infection (Probert et al. (1997) J. Infect. Dis. 175, 400-405). OspC is very diverse (see, e.g., Jauris-Heipke et al. (1993), Med. Microbiol. Immunol. 182, 37 50). Livey et al. found thirty-four alleles in seventy-six B. burgdorferi sensu lato isolates (Livey et al. (1995) MoI. Microbiol. 18, 257-269). What is needed is a selection of Borrelia antigens (e.g., OspC antigens from a limited number of families, which induce antibodies that cross-react with OspC proteins expressed by members of other families) that can be used to elicit immune responses to (e.g., to vaccinate against) all or most forms of Borrelia that cause systemic disease. The present inventors have identified combinations of polypeptides or peptides that can be used to elicit immune responses to (e.g., to vaccinate or protect against) most if not all forms of Borrelia that cause systemic disease. For example, the introduction into a subject of (e.g., vaccination with) OspC polypeptides from families C and K and, optionally, A and/or J, can elicit an immune response to (e.g., vaccinate or protect against) infection with Borrelia sensu lato. One aspect of the invention is a method for eliciting a specific immune reaction in an animal

(e.g., immunizing an animal against Lyme disease), comprising administering to the animal an effective amount of a composition comprising one or more isolated polypeptides from OspC families as follows: a) at least one of each of C and K, b) at least one of each of C, K and A, or c) at least one of each of C, K, A and J, and, optionally, an adjuvant. The polypeptides of the present invention elicit specific immune responses to OspC. The combinations of polypeptides elicit immune responses against strains of Lyme disease-causing Borrelia of the same genospecies from which the polypeptides were isolated, as well as a variety of genospecies with which they cross-react. The immune response includes humoral responses, secretory responses, cell-mediated responses and combinations thereof in an animal treated with the compositions of the present invention. In some embodiments of the invention, an immune response can result in at least some level of immunity in the treated animal, including a protective response. It is expected that the treated animal will develop immunity against infection by a variety of Lyme disease causing Borrelia, including Borrelia burgdorferi, Borrelia afzelii and Borrelia garinii.

The term immunity, as used herein, is understood to mean the ability of the treated animal to resist infection, to resist systemic infection, to overcome infection such as systemic infection or to overcome infection such as systemic infection more easily or more quickly when compared to non- immunized or non-treated individuals. Immunity can also include an improved ability of the treated individual to sustain an infection with reduced or no clinical symptoms of systemic infection. The individual may be treated with the proteins of the present invention either proactively, e.g. once a year or maybe treated after sustaining a tick bite.

When used as a vaccine, the combination of proteins and/or peptides of the invention prevents Lyme disease from becoming systemic. The proteins of the present invention can be effective in preventing of Lyme disease as well as having a therapeutic effect on established infection, for example after the tick bite is noticed by the patient. The proteins and chimeric proteins of the present invention are expected to act at the level of the tick as well as the level of the host in preventing both infection and disease due to Borrelia burgdorferi, Borrelia afzelii and/or Borrelia garinii. The present invention allows the development of a worldwide vaccine comprising only three (from OspC families C, K and A) or two (from OspC families C and K) necessary to generate a protective immune response against all pathogenic strains of Borrelia.

An immunogenic composition of the invention can include additional components suitable for in vitro and in vivo use. These additional components include buffers, carrier proteins, adjuvants, preservatives and combinations thereof. For use as a vaccine, a composition of the present invention can include suitable adjuvants, well known in the art, to enhance immunogenicity, potency or half-life of the proteins in the treated animal. Adjuvants and their use are well known in the art (see for example PCT Publication WO 96/40290, the entire teachings of which are incorporated herein by reference). The composition can be prepared by known methods of preparing vaccines. For example, the OspC proteins to be used in the compositions can be isolated and/or purified by known techniques such as by size exclusion chromatography, affinity chromatography, preparative electrophoresis, selective precipitation or combinations thereof. The prepared proteins can be mixed with suitable other reagents as described above, where the chimeric protein is at a suitable concentration. The dosage of protein or chimeric protein will vary from 1 mμg to 500 mμg and depends upon the age, weight and/or physical condition of the animal to be treated. The optimal dosage can be determined by routine optimization techniques, using suitable animal models. The composition to be used as an immunogen (e.g., a vaccine) can be administered by any suitable technique. In one embodiment, administration is by injection, e.g. subcutaneous, intramuscular, intravenous, or intra peritoneal injection. In another embodiment, the composition is administered to mucosa, e.g. by exposing nasal mucosa to nose drops containing the proteins of chimeric proteins of the present invention. In another embodiment, the immunogenic composition is administered by oral administration. In another embodiment of the present invention the chimeric proteins are administered by DNA immunization.

In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

EXAMPLES

Example I - Material and Methods

Borrelia burgdorferi strains. B. burgdorferi isolates were cultured from blood or erythema migrans skin biopsies of human patients seen at the Westchester Medical Center (kindly provided by Dr. Gary Wormser, New York Medical College (NYMC), Valhalla, NY). Fifteen OspC group-specific Borrelia burgdorferi human isolates were typed for OspC phyletic group in Dr. Ira Schwartz laboratory (NYMC, Valhalla, NY) and were kindly provided to us for this study. Low passage B. burgdorferi were grown at 34°C in Barbour- Stoenner-Kelly H (BSK-H) medium supplemented with antibiotic mixture for Borrelia (Sigma-Aldrich, St. Louis, MO). Total DNA was isolated from spirochetes using IsoQuik Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, WA). Patients provided informed consent and experimentation guidelines were followed as approved by the New York Medical College IRB. Infection of mice with B. burdorferi. Viability and number of spirochetes grown to mid- or late-log phase was done by dark field microscopy (Axio Imager, Zeiss, Germany). 10 ⁵ bacteria were used to infect C3H-HeJ mice subcutaneously. Three weeks later mice were bled and the serum was tested for the presence of B. burgdorferi antibodies using the ViraBlot test (VIRAMED Biotech AG). Animal experimentation guidelines were approved by UTHSCs Institutional Animal Care and Use Committee.

Serum panels from naturally infected hosts. For the purpose of seroprofiling we used serologically characterized serum panels only. A panel, n=43, was obtained from the natural reservoir of B. burgdorferi, the white-footed mouse (P. leucopus) and was previously screened for B. burgdorferi infection by C6 ELISA (Immunetics, Boston, MA). A panel, n=38, was obtained from naturally infected dogs with Lyme disease previously tested for B. burgdorferi infection by whole cell sonicate ELISA. A panel, n=25, was obtained from naturally infected humans with Lyme disease from the United States. This panel was obtained from patients presenting with erythema migrans and was previously screened for B. burgdorferi infection by C6 ELISA (Immunetics, Boston, MA). The last panel, n=40, was obtained from naturally infected humans with Lyme disease from Europe. This panel comprises serum from 19 patients presenting with erythema migrans with IgM and IgG antibodies to B. burgdorferi; 1 1 patients with IgM and IgG antibodies to B. burgdorferi and 10 patients with IgM antibodies to B. burgdorferi. These 21 patients did not present with erythema migrans. Patients provided informed consent and experimentation guidelines were followed.

Cloning, expression and purification of recombinant OspC proteins. A 560 bp-fragment of each B. burgdorferi ospC type gene was amplified by PCR. A Nde VBamH I fragment was cloned into pET9c (Novagen, Gibbstown, NJ). Plasmids were sequenced (GENEWIZ, Inc., South Plainfield, NJ) and the sequences of ospC-fragments were confirmed by ClustalW alignment with Genbank published sequences. Recombinant OspC proteins were expressed in Escherichia coli BL21 (DE3) and purified by ion exchange chromatography using Q-Sepharose Fast Flow (GE Healthcare, Sweden). Protein concentration was determined with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). OspC proteins were analyzed on a 15% SDS-PAGE Coomassie stained gel. OspC seroprofiling. OspC-immunoarrays were done using ELISA. Purified recombinant OspC protein was used to coat Nunc MaxiSorp™ flat-bottom ELISA plates (eBioscience, San Diego, CA) and indirect ELISA was performed using serum (1 :100) from C3H mice, P. leucopus, dog, or human. Species-specific IgG secondary antibody was used for mouse, P. leucopus and dog (1 :50,000, Jackson ImmunoResearch, West Grove, PA). For human, anti-human IgM+IgG horseradish peroxidase-conjugated secondary antibody was used (1 :50,000, Jackson ImmunoResearch, West Grove, PA).

Example II - Cloning, expression and purification of group-specific OspC

Sixteen of the 17 ospC genotypes endemic to the US were cloned. The ospC gene from 15 of the 17 genotypes were cloned from B. burgdorferi isolates cultured from blood or erythema migrans skin biopsies of human patients seen at the Westchester Medical Center (Valhalla, NY). These isolates were typed for OspC phyletic group by reverse line blotting in Dr. Ira Schwartz laboratory (NYMC). OspC genotype L was amplified from a plasmid constructed from B. burgdorferi DNA isolated from ticks. OspC genotype O is rare in the northeastern US and was not available. All ospC genes were cloned in an expression vector (pET9c) and sequences confirmed by ClustalW alignment against Genbank standards [Wang et al. (1999) Genetics 151,15-30; Seinost et al. (1999) Infect Immun 67, 3518-24]. Each of the 16 recombinant OspC proteins (A-N, T and U) was expressed in E. coli BL21(DE3)pLys devoid of any markers or tags and purified under native conditions by ion exchange chromatography, and protein purity was analyzed by Coomassie stained SDS-PAGE. All purified recombinant OspC proteins showed a single major band with an apparent molecular mass ranging between 20-25 kDa.

Example HI - OspC screening for diagnostic design

One goal of these studies was to identify proteins that can detect B. burgdorferi anti-OspC antibodies induced by epitopes shared by all OspC types, in order to identify the immunodominant

OspC genotypes. These proteins can be included, e.g., in a diagnostic assay for Lyme disease, such as a multi-antigen diagnostic assay. To accomplish this, we performed two comprehensive seroprofiling studies using 16 purified recombinant OspC types.

In the first trial, the level of OspC-type specific IgG antibody (OD ₄₅ 0) was determined in a serum panel from 15 C3H-HeJ mice infected in the laboratory with each strain of B. burgdorferi previously typed for its ospC phyletic group (Figure 1). OspC type L-specific serum was not generated because this strain was not available. Positive reactions were determined using the OD450 from three serum samples from uninfected mice plus three standard deviations to calculate the cutoff. We observed that recombinant OspC proteins belonging to genotype L detected IgG antibodies induced by 80% of the OspC -typed B. burgdorferi strains; proteins belonging to genotypes A, C, D, H, N and U detected IgG antibodies induced by 87% of the OspC-typed strains; proteins belonging to genotypes G, J, M and T detected IgG antibodies induced by 93% of the OspC-typed strains; and proteins belonging to genotypes B, E, F, I and K detected group-specific IgG antibodies induced by 100% of the OspC-typed strains tested. In the second trial, the diagnostic efficacy of all rOspC protein types was tested by evaluating the level of OspC -type specific antibody in serum obtained from naturally infected hosts: white- footed mouse (Peromyscus leucopus, n=43), dog (Canis lupus familiar is, n=38) and human {Homo sapiens, from the northeastern United States, n=25, and from Europe, n=40). The four serum panels included in this analysis tested positive for B burgdorferi infection by serological methods (B. burgdorferi whole cell sonicate or C6 ELISA). Positive reactions were determined using the OD ₄ 50 from three previously screened negative samples plus three standard deviations to calculate the cutoff. We detected substantial variation among individuals within a species in the proportion of positive reactions to each recombinant OspC protein (Table 2). Using serum from naturally infected white-footed mice (P leucopus), IgG detection ranged between 33% (group T) to 79% (group L). Using serum from dogs with Lyme disease, IgG detection ranged between 13% (group J) to 82% (group B); using serum from human American Lyme disease, IgM+IgG detection ranged from 24% (group M) to 84% (group K) and using serum from human European Lyme disease, IgM+IgG detection ranged from 25% (group U) to 80% (groups E and K). No one rOspC type detected 100% of the B burgdorferi infections in any of the species. However, rOspC types A, B, E, F, I, K and L detected infected hosts from all species (average 68.14%, sd =7.22).

Table 2. Percentage of naturally infected serum samples with anti-OspC antibody

% Positive

Serum panel rA | rB | rC | rD | | rE | rF | [£Gj I rH I rl I rK I rL I I rM| rN | rT | rU

NI P. leucopus 70 61 42 72 7ϋ 74, .51 35 m ^? 58 JS7| 44 33 56

NI Dog 68 82 61 32 66 74 53 24 « 1 13 ΪIII 55 26 63

NI Human US 6& 7,t ^β a ^r 8C 8ft 64 J6 isis- ! 24l ^! 32 i

NI Human EU 65, m & 48 m 60 til 360 35 43 40 25

A B E F I K L rA-rU represent purified recombinant OspC proteins; NI, naturally infected serum panels tested positive for B. burgdorferi infection by serological methods. NI P. leucopus, n=43, is serum panel from naturally infected white-footed mice; NI Dog, n=38, is serum panel from naturally infected dogs with Lyme disease; NI Human US, n=25, is serum panel from human North American patients with signs and symptoms of Lyme disease; NI Human EU, n=40, is serum panel from human European patients with signs and symptoms of Lyme disease; shaded gray, OspC proteins that detect the highest titer of antibodies.

The effectiveness of each rOspC protein as a diagnostic tool is dependent on the probability of detecting anti-Borrelia OspC antibodies in infected hosts well above the limit of detection. Although low sensitivity rOspC proteins successfully identified anti-Borrelia antibodies in some infected animals, the majority of positive sera samples were very near the cutoff of detection C, D, H, J, M, N, T, U in P. leucopus (Fig.2); C, D, G, H, J, N, T, U in dog (Fig.3); H, M, T in human US (Fig.4); and C, D, H, J, M, N, T, U in human EU (Fig. 5). In contrast, much of the positive sera that rOspC types A, B, E, F, I, K and L detected is far above the limit of detection, thus decreasing the risk of false negative assays. For example, rOspC type M detected anti-5. burgdorferi (OspC) antibodies in 67% of infected mice (Table 2), but nearly 60% of those were within 0.2 OD of the limit of detection (Fig.2). rOspC type B also detected anti-5. burgdorferi (OspC) antibodies in 61% of infected mice (Table 2) and only 10% were within 0.2 OD of the limit of detection (Fig. 2). No single rOspC protein identified more than 84% (type K, Table 2) of infected individuals suggesting that a combination of rOspC proteins or peptides from different OspC types could be used to identify anti-Borrelia OspC antibodies.

In all four serum panels, we observed that a number of individuals reacted to all 16 OspC types and that a number of samples did not have antibodies to any OspC. For naturally infected P. leucopus, n=43, 4 (9%) had IgG antibodies that bind to all OspC groups and 1 (2.3%) did not have antibodies to any OspC; for dogs with Lyme disease, n=38, none (0%) had IgG antibodies to all OspC groups and 2 (5.2%) did not have antibodies to OspC of any group; for humans in the Lyme disease American panel, n=25, 5 (20%) had IgM+IgG antibodies to all OspC and all samples had antibodies to all OspC groups; for humans in the Lyme disease European panel, n=40, 7 (18%) had IgM+IgG antibodies to all OspC groups and 5 (13%) did not have antibodies to OspC of any type. In humans, the low percentage of samples with antibodies to all OspC types (-19%) emphasizes the need for inclusion of OspC antigens from at least two groups in a diagnostic assay. The percentage of samples without antibodies to OspC of any type (0- 13%) emphasizes the need for prudence when interpreting negative OspC results, given that we only included serum panels that tested positive for B. burgdorferi infection. In order to identify the most sensitive OspC types, we analyzed the previously screened OspCs against OspC-positive serum (US and EU, Table 3). The combination of rOspC types K and B identified 24 of the 25 (96%) North American human confirmed LD patients with confirmed antibodies to OpsC. The combination of rOspC type K with either type E or type F detected all 35 (100%) European humans with confirmed antibodies to OspC (five European humans with confirmed LD did not have detectable OspC antibodies).

Table 3. Percentage of Lyme disease samples correctly identified by OspC-pairs

% Positive US LD Panel % Positive EU LD Panel

A B E F I K L A B E I I SiHtF L

A 68 88 84 84 80 88 76 A 74 89 97 91 89 94 83

B 72 92 92 92 96 88 B 77 94 91 80 91 91

E 80 80 80 88 88 91 1OO 97 100 97

F 80 80 88 88 F 89 91 94 94

I 76 84 84 I 80 91 91

K 84 88 K 91 97

L 68 L 83

US LD, is serum panel from human North American patients with signs and symptoms of Lyme disease and IgM+IgG antibodies to OspC, n=25; EU LD, is serum panel from human European patients with signs and symptoms of Lyme disease and IgM+IgG antibodies to OspC, n=35.

Example IV - Discussion

One objective of this study was to identify suitable OspC types to be included in a diagnostic assay for Lyme disease {e.g., a multi-antigenic diagnostic assay). Data from our seroprofiling analysis indicate that seven rOspC type proteins detected high anti-OspC antibody titers in infected hosts, regardless of species or the ospC genotype of the infecting B. burgdorferi strain. Although no one rOspC protein identified all humans with multiple signs and symptoms of LD, combinations of as few as two rOspC proteins identified all patients with anti-OspC antibodies. Immuno- crossreactivity between distinct OspC type proteins, apparently due to antibodies targeting shared epitopes, along with the rapid and strong anti-OspC antibody response, indicates that combinations of these immunodominant rOspC proteins are useful for the diagnosis of pathogenic Borreliα infection.

The polymorphism of the OspC gene, the immunoreactivity to the OspC protein and its implications for diagnostic design have been investigated previously by the present inventors and others. It has been reported that OspC alone, when antigen from a single strain of OspC is employed, is not sensitive enough for a robust assay for Lyme disease. In one study, when acute and convalescent-phase serum samples from patients with erythema migrans were tested for reactivity against rOspC by ELISA, the sensitivity of the IgM test was 73% and the specificity was 98% (Fung et al. (1994) Infect lmmun 62, 3213-21). In another study, when serum samples from patients with EM and other symptoms of Lyme disease were tested against a synthetic peptide based in the C- terminal amino acids residues of OspC of B. burgdorferi by ELISA, the sensitivity of the IgM test was 36-45% while the IgG test was <8% (Mathiesen et al. (1998) JCUn Microbiol 36, 3474-9). By contrast, the studies herein show that antibody detection using antigen preparations of the present invention, incorporating a suitable combination (mixture) of OspC proteins, is much more sensitive than such single strain protocols.

Reports demonstrating that OspC immunization is protective against only B. burgdorferi expressing the same OspC type raised the question of OspC-type specificity. OspC-type specificity was further supported by a study of seven recombinant OspC types that found that despite strong sequence conservation in the N- and C-terminus of OspC, the antibody responses to this protein were type specific. That is, serum from mice infected with type A or D strains was immunoreactive in a type-specific manner and there was little or no cross-reactivity with other OspC types. In sharp contradiction, we found, surprisingly, that all 16 rOspC proteins in our library cross-react with a minimum of 12 other OspC proteins. Further, five B. burgdorferi genotypes (B, E, F, I and K) induce OspC antibodies that react to all 16 rOspC-types. Three B. burgdorferi genotypes (D, E and M) induced the most type-specific OspC antibodies, but still cross-react with 8, 9, and 12 rOspC types, respectively. Without wishing to be bound by any particular theory, it is suggested that the difference in conclusion between the former study (Earnhart et al. (2005) Infect lmmun 73, 7869-77) and the current study may be due to methodology; ELISAs are far more sensitive and quantitative than are immunoblots. Our conclusions are also supported by the observation that most patients infected with B. burgdorferi (regardless of strain type) develop anti-OspC antibodies that bind to OspC belonging to genotype A used in commercial serodiagnostic assays in both ELISA and immunoblot formats. Our results suggest major cross-reactivity between OspC antibodies. Although the protective OspC epitopes are genotype-specific, shared OspC epitopes elicit detectable antibody responses for use in diagnostic applications. A combination of only two rOspC proteins identified 59 of the 60 human LD patients that had positive anti-OspC serology from Europe and North America. However, the best combination of rOspC proteins for LD diagnosis differed on the two continents. rOspC types B and K identified 96% of the US LD patients, with one patient's serum reacting only to type J. Over 76% of North American patients reacted positively with type J, suggesting that a diagnostic assay based on these three proteins components will likely decrease false negative results. Combinations of rOspC types E and K as well as F and K identified all European patients that had anti-OspC antibodies. It is noted that these data are not necessarily an indication of overall diagnostic efficacy of an OspC only-based assay, but rather may suggest that the OspC types identified herein are the best antigens to include in a multi-antigen assay for the diagnosis of Lyme disease. The winning of different rOspC combinations on each continent may correlate with differences in the composition of B. burgdorferi genotypes to which European and North American humans are exposed. B. burgdorferi genotypes A-O, T and U are endemic in the United States, genotypes A, B and J are endemic in both continents, while genotypes P, Q, R and S appear to be restricted to Europe (Seinost et al. (1999) Infect Immun 67, 3518-24; Lagal et al. (2003) J CHn Microbiol 4_i, 5059-65).

Although it has been reported that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the variable region and that the immunodominant epitopes of OspC reside in the variable domains of the protein, it would appear from our results, surprisingly, that common epitopes present in OspC types B, E, F, J and K detect all, or nearly all, anti-OspC antibodies present in serum samples from seropositive patients infected with B. burgdorferi.

Example V - OspC seroprofiling for vaccine design

Optimal protein or peptide components of a subunit vaccine for Lyme disease should induce a high titer of type-specific antibody (OD450>0.5) that binds to 100% of OspC types. In order to identify the most immunogenic OspC types, we performed a series of comprehensive seroprofiling studies, using a serum panel from 16 mice immunized in the laboratory with each purified recombinant type-specific OspC protein (rA-rU) to perform an OspC-immunoarray. We determined the titer of antibody (OD450) to all proteins from each type-specific serum of immunized mice (Figure 6). We define a high titer of type-specific OspC antibody as samples with OD450>0.5. We observed that purified recombinant proteins induced a high titer of OspC-type-specific antibodies that reacted to a variable number of OspC proteins. OspC types A, C, J and K had the highest antibody titers against all 16 OspC proteins tested. OspC type K induces the highest titer of antibody to 100% of OspC types. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications (including U.S. provisional application 61 /,178,261, filed May 14, 2009) cited above and in the figures, are hereby incorporated in their entirety by reference.