AU686407B2

AU686407B2 – 66 kDa antigen from Borrelia
– Google Patents

AU686407B2 – 66 kDa antigen from Borrelia
– Google Patents
66 kDa antigen from Borrelia

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AU686407B2

AU686407B2
AU28632/95A
AU2863295A
AU686407B2
AU 686407 B2
AU686407 B2
AU 686407B2
AU 28632/95 A
AU28632/95 A
AU 28632/95A
AU 2863295 A
AU2863295 A
AU 2863295A
AU 686407 B2
AU686407 B2
AU 686407B2
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borrelia
seq
polypeptide
ile
ser
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1994-06-20
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AU2863295A
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Alan George Barbour
Sven Bergstrom
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Symbicom AB

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Symbicom AB
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1994-06-20
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1995-06-19
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1998-02-05

1995-06-19
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1996-01-15
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1998-02-05
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1998-02-05
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2015-06-19
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Classifications

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria

C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria

C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

A61P31/04—Antibacterial agents

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira

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

A61K2039/53—DNA (RNA) vaccination

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

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K2319/00—Fusion polypeptide

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K2319/00—Fusion polypeptide

C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

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

Description

WO 95/35379 PCTIUS95/07665 1 66 kDa ANTIGEN FROM BORRELIA Mention Is made of U.S. Serial Nos. 079,601 filed 23 June 1993, 137,175 filed 26 October 1993, 262,220 filed 20 June 1994, 320,416 filet 3 October 1994, and 375,993 filed 20 January 1995, each Incorporated herein by reference; and, this application is a continuation-in-part of U.S. Serial No. 262,220.
The present invention relates to nucleic acid fragments encoding antigenic proteins associated with Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii; denoted Bb herein), particularly polypeptides associated with virulence of the bacteria. The invention also relates to methods for producing Bb immunogenic polypeptides and corresponding antibodies.
Other embodiments of the invention relate to compositions and methods for detecting Lyme disease and also vaccines against infections with Borrelia burgdorferi sensu lato are a part of the invention as is methods of immunizing animals against diseases caused by’these infections. Vectors and transformed cells comprising Bb-associated nucleic acids are also included.
DESCRIPTION OF RELATED ART Lyme disease is a multisystem disease resulting from tick transmission of the infectious agent, Bb (Rahn and Malawista, 1991). Although recognized as a clinical entity within the last few decades (Steere et al., 1977), case reports resembling Lyme disease date back to the early part of the century. Cases of the disease have been reported in Europe, Asia and North America (Schmid, 1985). Despite a relatively low total incidence compared to other infectious diseases, Lyme disease represents a significant health problem because of its potentially severe cardiovascular, neurologic and arthritic complications, difficulty in diagnosis and treatment and high prevalence in some geographic regions.
Bb is not a homogeneous group but has a variable genetic content, which may in turn affect its virulence, pattern of pathogenesis and immunogenicity. Lyme borreliosis associated borreliae are so far taxonomically placed into three species,
I
I~LIIIIIII— WO 95135379 PCT/US95/07665 2 Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (Burgdorfer et al. 1983, Baranton et al.
1992, Canica et al. 1993). It is well documented that considerable genetic, antigenic and immunogenic heterogeneity occurs among them, as well as among the strains within the separate species (Baranton et al. 1992, Canica et al. 1993, Zingg et al. 1993, Wilske et al. 1993, Adam et al. 1991, Marconi and Garon 1992). The major evidence of this phenomenon is provided by the molecular studies of the plasmid-encoded outer surface protein A (OspA), B (OspB), and C (OspC) (Barbour et al. 1984, Jonsson et al. 1992, Wilske et al.
1993, Marconi et al. 1993). In different animal models efficient protection is achieved by passive and active immunization with OspA (Simon et al., 1991 Fikrig et al., 1992, Erdile et al., 1993), therefore, OspA remains one of the main candidates for a Borrelia vaccine. It is unclear, however, whether inter- and intra-species heterogeneity of OspA, as well as other competitors for immunoprophylaxis, allow efficient cross-protection (Fikrig et al. 1992, Norris et al., 1992). Furthermore, it was recently suggested that certain protective antibodies produced early in the course of Borrelia infection are unrelated to OspA (Norton Hughes et al., 1993, Barthold and Bockenstedt, 1993).
Its virulence factors, pathogenetic mechanisms and means of immune evasion are unknown. At the level of patient care, diagnosis of the disease is complicated by its varied clinical presentation and the lack of practical, standardized diagnostic tests of high sensitivity and specificity.
Antimicrobial therapy is not always effective, particularly in the later stages of the disease.
Variation among Bb strains and species and the changes resulting from in vitro passage add to the problems of developing vaccines or immunodiagnostics from either the whole organism or specifically associated proteins. Using a PCR assay, it was found that one set of oligonucleotide primers was specific for North American Bb isolates, another for most European SWO 95135379 PCT/S95/07065 3 isolates and a third set recognized all Bb strains (Rosa et al., 1989).
Serological assays for the diagnosis and detection of Lyme disease are thought to offer the most promise for sensitive and specific diagnosis. However, serologic assays generally use whole Bb as the antigen and suffer from a low «signal to noise» ratio, a low degree of reactivity in positive samples, particularly early in the disease, as compared to negative samples. This problem results in high numbers of false negatives and the potential for false positives. Background reactivity in negative controls may be due in part to conserved antigens such as the 41K flagellin and the «Common Antigen». These Bb proteins possess a high degree of sequence homology with similar proteins found in other bacteria. Therefore normal individuals will often express anti-flagellar and anti-60K antibodies. Unique, highly reactive Bb antigens for serological assays are therefore desirable but heretofore unavailable.
Diagnosis of Lyme disease remains a complex and uncertain endeavour, due to lack of any single diagnostic tool that is both sensitive and specific. Clinical manifestations and history are the most common bases for diagnosis. However, there is a pressing need for specific, sensitive, reproducible and readily available confirmatory tests. Direct detection offers proof of infection but is hampered by the extremely low levels of Bb that are typically present during infection, as well as the inaccessibility of sites that tend to be consistently positive heart and bladder). Culture, although sensitive, is cumbersome and requires 1-3 weeks to obtain a positive result. PCR appears to offer promise in terms of direct detection (Lebech et al., 1991) and indeed Goodman et al (1991) have reported detection of Bb DNA in the urine of patients with active Lyme disease using a PCR method. However, it is unlikely that PCR assays will become commonly used in clinical laboratories because of the r r WO 95/35379 11CIMUS95/07665 4 degree of skill required for its use and the high risk of DNA contamination.
Another problem in detection of Lyme disease is the substantial number of humans exposed to Bb who develop unapparent or asymptomatic infections. This number has been estimated as high as 50% (Steere et al., 1986).
There is clearly a need for means of preparing Bb-specific antigens, for the development of diagnostic tests for Lyme disease or vaccines against Lyme disease. Adequate assays do not exist and should ideally meet several criteria, including expression of an antigen by all pathogenic Bb strains, elicitation of an immune response in all Lyme disease patients, high immunogenicity with a detectable antibody response early in the infection stage, antigens unique to Bb without cross reactivity to other antigens and, distinction between individuals exposed to nonpathogenic as opposed to pathogenic forms of Bb.
Problems similar to those relating to diagnosis exist when attempting to prepare a vaccine against diseases caused by Bb. Successful single antigen vaccines have until now not been prepared, possibly due to the inter-strain and interspecies antigenic variation. As mentioned above, OspA has been the main candidate for the immunogenic constituent of a single antigen vaccine, but time has proven that in order for such a vaccine to be efficient it has to contain OspA from at least three different Bb species (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii).
A number of investigators have reported the presence of proteins with molecular weights in the region between 60 and 75 kDa. Many of these proteins are also recognised by antibodies in patient sera when analyzed by Western blots. (Barbour 1984, Luft et al., 1989). Protease treatment of Borrelia burgdorferi cells (Barbour et al. 1984) showed that a minor protein with an apparent molecular weight of 66 kDa was WO 95/35379 PCT/US95/07665 accessible to proteolytic cleavage, and hence probably associated with the outer envelope. Coleman and Benach (1987) isolated a protein with apparent molecular weight of 66 kDa from an outer envelope fraction isolated from Borrelia burgdorferi B31. However, direct amino acid sequencing of Bb proteins with the apparent molecular weights 66-, 68-, 71-, and 73-kDa revealed these proteins to have high sequence similarity with the E. coli heat-shock proteins (Luft et al., 1991) making them less suitable for the use in prophylaxis and serodiagnosis.
SUMMARY OF THE INVENTION The inventors have surprisingly found that an antigen from Bb with an apparent molecular weight of 66 kDa (determined by SDS-PAGE, and staining with Coomassie Blue) is highly conserved in the three strains B. burgdorferi sensu stricto B31, B. garinii IP90, and B. afzelii ACAI, whereas this antigen cannot be found in Borrelia species related to relapsing fever and avian borreliosis. The disclosed antigens therefore are excellent candidates for vaccines and diagnostics relating to infections with Bb.
Thus, the present invention addresses one or more of the foregoing or other problems associated with the preparation and use of Bb specific antigens, particularly those antigens which are associated with virulence and which are useful for developing detection and diagnostic methods for Lyme disease as well as vaccines against Lyme disease. The invention involves the identification of such antigens, as well as the identification and isolation of Bb nucleic -cid sequences that encode Bb antigens or antigenic polypeptides derived therefrom. These sequences are useful for preparing expression vectors for transforming host cells to produce recombinant antigenic polypeptides. It is further proposed that these antigens will be useful as vaccines or as immunodiagnostic agents for Bb associated diseases such as Lyme disease in particular.
WO 95/35379 PCT/US95/07665 6 The DNA disclosed herein was isolated from the bacterium Borrelia burgdorferi sensu lato hereafter designated as Bb..
The microorganism is a spiral-shaped organism approximately 0.2 micron in diameter and ranging in length from about 10-30 microns. Like other spirochetes, it possesses an inner membrane, a thin peptidoglycan layer, an outer membrane, and periplasmic flagella which lie between the inner and outer membranes. Bb is obligate parasite found only in association with infected animals and arthropod vectors in endemic areas.
Bb-like organisms have also been identified in birds raising the possibility that birds could also serve as an animal reservoir. While some Bb isolates have been cloned, most isolates have not been cloned and most likely represent mixtures of differ, .t variants even at the time of culture origination.
Bb has similarities with other relapsing fever organisms such as B. hermsii. Bb has a single chromosome with two unusual features, linear conformation and small size (approximately 900 kilobase pairs). Fresh isolates of Bb contain up to four linear plasmids and six circular supercoiled plasmids. The plasmid content of different Bb isolates is highly variable.
For example, in one study only two of thirteen strains had similar plasmid profiles. Some plasmids are lost during in vitro passage which may correlate with loss of virulence.
Outer surface proteins OspA and OspB are encoded on the 49 kbp linear plasmid. The 66 kDa membrane-associated proteins discovered by the inventors are encoded on the Bb chromosome.
In order to identify DNA segments encoding the 66 kDa proteins, purified protein was isolated from B. afzelii ACAI, by preparative SDS-PAGE for subsequent use in amino acid sequencing. The peptide was transferred to polyvinylene diffusable membranes, sequence analysis was performed using standard sequencing techniques (Matsudaira, 1987). An 8 amino acid sequence was identified (SEQ ID NO: Codons for the amino acid sequence were selected by reverse translation based on conclusion that codons containing A or T were favoured
I
I PCT/US95107665 WO 95/35379 7 and knowledge of published DNA sequences for several Bb proteins. A choice favouring A or T containing codons was based on the observation that the G C content of Bb DNA is only 28-35% (Burman et al. 1990). A 24 nucleotide segment was synthesized having the structure in SEQ ID NO: 2 (corresponding to amino acids 6-33): GAA AAA GAT ATW TTT AAA ATW AAT 3′ (SEQ ID NO: 2) -wherein W denotes the bases A or T, i.e. the 24 nucleotide segment exists in 4 variants.
DNA libraries were prepared by restriction enzyme digestion of DNA prepared from B. burgdorferi B31, B. afzelii ACAI and B. garinii The 24 residue oligonucleotide probe was used as a probe to screen the DNA library prepared from B. garinii Ip90 to identify DNA encoding the 66 kDa protein isolated from chis Bb species.
A 592 bp DNA fragment coding for part of the 66 kDa protein from B. garinii Ip90 was used as a probe to screen DNA libraries prepared from B. burgdorferi B31 and B. afzelii ACAI to identify DNA encoding the 66 kDa protein from these Bb species.
Antigenicity of the 66 kDa protein was determined. Antiserum collected from rabbits injected with the 66 kDa protein prepared from B. garinii Ip90 was shown to react with the 66 kDa proteins, as detected on immunoblots of B. garinii as well as B. burgdorferi B31 and B. afzelii ACAI. No reactive spots were detected in normal rabbit serum. This result should lead to straightforward production of monoclonal antibodies reactive with the 66 kDa polypeptides from one strain of one species exclusively as well as from two or all three species. Antibodies could be produced and used for screening strains for protein expression, for determining WO 95/35379 PCT/US95/07665 8 structural location and for examining bactericidal activity of antibodies against these proteins.
The nucleic acid segments of the present invention thus encode amino acid sequences associated with Bb. Some of these amino acid sequences are antigenic. The nucleic acid sequences are also important for their ability to selectively hybridize with complementary stretches of Bb gene segments.
Varying conditions of hybridization may be desired, depending on the application envisioned and the selectivity of the probe toward the target sequence. Where a high degree of selectivity is desired, one may employ relatively stringent conditions to form the hybrids, such as relatively low salt and/or high temperature conditions. Under these conditions, little mismatch between the probe and template or target strand is tolerated. Less stringent conditions might be employed where, for example, one desires to prepare mutants or to detect mutants when significant divergence exists.
In clinical diagnostic embodiments, nucleic acid segments of the present invention may be used in combination with an appropriate means, such as a label, to determine hybridization with DNA of a pathogenic organism. Typical methods of detection might utilize, for example, radioactive species, enzyme-active or other marker ligands such as avidin/biotin, which are detectable directly or indirectly. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as alkaline phosphatase or peroxidase rather than radioactive or other reagents that may have undesirable environmental effects. Enzyme tags, for example, often utilize colorimetric indicator substrates that are readily detectable spectrophotometrically, many in the visible wavelength range. Luminescent substrates could also be used for increased sensitivity.
Hybridizable DNA segments may include any of a number of segments of the disclosed DNA. For example, relatively short WO 95135379 PCTUS95/076165 9 segments including 12 or so base pairs may be employed, or, more preferably when probes are desired, longer segments including 20, 30 or 40 base pairs, depending on the particular applications desired. Shorter segments are preferred as primers in such applications as PCR, while some of the longer segments are generally preferable for blot hybridizations. It should be pointed out, however, that while sequences disclosed for the DNA segments of the present invention are defined by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and SEQ ID NO: 13, a certain amount of variation or base substitution would be expected, as may be found in mutants or strain variants, but which do not significantly affect hybridization characteristics. Such variations, including base modifications occurring naturally or otherwise, are intended to be included within the scope of the present invention.
While the 66 kDa Bb antigen has been disclosed in terms of specific amino acid sequences from three strains of Bb, it is nonetheless contemplated that the amino acid sequence will be found to vary even further from species to species and isolate to isolate. Moreover, it is quite clear that changes may be made in the underlying amino acid sequence through e.g., site-directed mutagenesis of the DNA coding sequence, in a way that will not negate its antigenic capability.
The invention also relates to at least partially purified antigenic Bb proteins or polypeptides capable of eliciting an in vivo immunogenic response in animals which are later challenged with Bb. These proteins may comprise all or part of the amino acid sequence encoded by the herein disclosed DNA. Particularly preferred antigenic proteins have the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 14. These proteins, as well as their epitopes, will be useful in connection with vaccine development, and as antigen(s) in immunoassays for detection of Bb antibodies in biological fluids such as serum, seminal or vaginal fluids, urine, saliva, body exudates and the like.
WO 95/35379 PCT/US9507665 In other aspects, the invention concerns recombinant vectors such as plasmids, phage or viruses, which comprise DNA segments in accordance with the invention, for use in replicating such sequences or even for the expression of encoded antigenic peptides or proteins. Vectors or plasmids may be used to transform a selected host cell. In preparing a suitable vector for transforming a cell, desired DNA segments from any of several Bb sources may be used, including genomic fragments, cDNA or synthetic DNA. In practice of the present invention, an expression vector may incorporate at least part of the DNA sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13 encoding one or more epitopic segments of the disclosed 66 kDa antigens.
Expression vectors may be constructed to include any of the DNA segments disclosed herein. Such DNA might encode an antigenic protein specific for virulent strains of Bb or even hybridization probes for detecting Bb nucleic acids in samples. Longer or shorter DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the 66 kDa proteins expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA. A wide variety of expression vectors is possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resistance genes which may be useful in identifying transformed cells.
Recombinant vectors such as those described are particularly preferred for transforming bacterial host cells. Accordingly, a method is disclosed for preparing transformed bacterial host cells that includes generally the steps of selecting a suitable bacterial host cell, preparing a vector containing a desired DNA segment and transforming the selected bacterial host cell. Several types of bacterial host cells may be employed, including Bb, E. coli, B. subtilis, and the like as well as other suitable pro- and eukaryotic host cells.
I I WO 95/35379 PCT/US95/107665 11 Transformed cells may be selected using various techniques, including screening by differential hybridization, identification of fused reporter gene products, resistance markers, anti-antigen antibodies and the like. After identification of an appropriate clone, it may he selected and cultivated under conditions appropriate to the circumstances, as for example, conditions favouring expression or, when DNA is desired, replication conditions.
Another aspect of the invention involves the preparation of antibodies and vaccines from the antigenic 66 kDa proteins or epitopic regions of that protein encoded by the disclosed DNA. The invention thus relates to one or more antibodies, monoclonal or polyclonal, that may be generated in response to the 66 kDa Bb proteins or their epitopes. It is expected that the sensitivity and specificity of antibody response to this 66 kDa proteins and their epitopes will be superior to the response that has been obtained from other Bb antigens that are not associated with virulence. Previous work with several Bb antigens isolated from both virulent and avirulent strains indicated low sensitivity when immunoiJuorescence and ELISA assays were employed, especially during early stages of infection.
In both immunodiagnostics and vaccine prepartion, it is often possible and indeed more practical to prepare antigens from segments of a known immunogenic protein or polypeptide.
Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide. Potential antigenic or immunogenic regions may be identified by any of a number of approaches, Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods (1981) hydrophobicity analysis (see, Jameson and Wolf, 1988; Kyte and Doolittle, 1982; or U.S. Patent No.
4,554,101). Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue from these values average hydrophilicities can be calculated and regions of greatest hydrophilicity determined. Using one or more of WO 95/35379 PCT/US95/07665 12 these methods, regions of pr-dicted antigenicity may be derived from the amino acit sequence assigned to the 66 kDa polypeptide. Regions from the 66 kDa antigens having a high likelihood of being epitopes include the sequences corresponding to positions 175-190, 285-305, 365-385, and 465-490.
It is contemplated that the antigens and immunogens of the invention will be useful in providing the basis for one or more assays to detect antibodies against Bb. Previous assays have used whole Bb as the antigen. Sera from normal individuals not exposed to Bb often contain antibodies that react with Bb antigens, in particular antigens that have epitopes in common with other bacteria. It is necessary to adjust assay conditions or the diagnostic threshold of reactivity to avoid false positive reactions due to these cross-reactive antibodies in normal sera. These adjustments may in turn decrease the sensitivity of the assay and lead to false negative reactions, particularly in the early stages of Bb inrection. Assays using the disclosed 66 kDa proteins or antigenic polypeptides thereof, are expected to give superior results both in sensitivity and selectivity when compared to assays that use whole Bb or even purified flagella in either an indirect ELISA or an antibody capture ELISA format. Western immunoblots based on reactions with such antigens (whole Bb, flagella and the like) have been difficult to interpret due to the presence of antibodies in sera from unexposed individuals. These antibodies cross-react with Bb antigens, most particularly the 41 kDa flagellin and the 60 kDa common antigen protein. Generally, assays which use whole organisms or purified flagella tend to contain antigens with epitopes that will cross react with other bacterial antigens. For example, the N and C terminal regions of the Bb flagellin possess 52-55% sequence identity with the Salmonella typhimurium and Bacillus subtilis sequences (Wallich et al., 1990), exemplifying the highly conserved nature of flagellin structure. The 60 kDa Bb protein is likewise 58 homologous with the E. coli protein (Shanafelt et al., 1991). Such cross WO 95/35379 PCT/US95/07665 13 reactivity is not likely with the 66 kDa antigen, which is apparently unique to Bb.
It is further anticipated that a recombinant derived 66 kDa Bb protein will be particularly preferred for detecting Bb infections. Unexposed individuals should have a low reactivity to one or more epitopes of the 66 kDa proteins thereby making it possible to use lower dilutions of serum and increase sensitivity. Using a combination of more than one of these unique antigens may also enhance sensitivity without sacrificing specificity.
Preferred immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISAS), immunoblot techniques, and the like, known in the art. However, it readily appreciated that utility is not limited to such assays, and useful embodiments include RIAs and other nonenzyme linked antibody binding assays or procedures.
Yet another aspect of the invention is a method of detecting Bb nucleic acid in a sample. The presence of 3b nucleic acid in the sample may be indicated by the presence of the polypeptide products which it encodes. The method therefore includes detecting the presence of at least a portion of any of the polypeptides herein disclosed. Suitable detection methods include, for example, immunodetection reagents, PCR amplification, and hybridization.
Yet another aspect of the invention includes one or more primers capablc of priming amplification of the disclosed DNA of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13. Such primers are readily generated taking into account the base sequence of the DNA segment of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13, the disclosed DNA, or deriving a base sequence from the amino acid sequence of a purified polypeptide encoded by the DNA. Primers are analogous to hybridization probes, but are generally relatively short DNA segments, usually about 7-20 nucleotides.
WO 95/35379 PMTUS95/07665 14 Methods of diagnosing Lyme disease are also included in the invention. In one embodiment, an antibody-based method includes obtaining a sample from a patient suspected of having Lyme disease, exposing that sample to one or more epitopes of the Bb protein which is encoded by the DNA disclosed and finally determining a reactivity of the antibody with one or more epitopes of a Bb protein that may be in the sample. The reactivity measured is indicative of the presence of Lyme disease. Typical samples obtainable from a patient include human serum, plasma, whole blood, cerebrospinal fluid, seminal or vaginal fluids, exudates and the like.
Several variations of antibody-based methods are contemplated for development; for example, an indirect ELISA using the 66 kDa proteins or other Bb proteins as an antigen. The 66 kDa proteins may be produced in large quantities by recombinant DNA vectors already disclosed and purified. Optimal concentration of the antigen could be determined by checker board titration and diagnostic potential of the 66 kDa proteins assay examined further by testing serum from mice at different stages of infection and infected with different strains of Bb. These results could indicate the relative time course for sera conversion for each of the assays and would also show whether infection with different strains :auses variation in anti-66 kDa protein titers.
Likewise, reactive epitopes of the 66 kDa polypeptides are contemplated as useful either as antigens in an ELISA assay or to inhibit the reaction of antibodies toward intact 60 kDa proteins bound to a well. Epitopic peptides could be generated by recombinant DNA techniques previously disclosed or by synthesis of peptides from individual amino acids. In either case, reaction with a given peptide would indicate presence of antibodies directed against more epitopes. In addition to its diagnostic potential, this method is seen as being particularly effective in characterizing monoclonal antibodies against the 66 kDa proteins and other virulence associated proteins.
WO 95/35379 PCTUS95/07( In further aspects, the present invention concerns a kit for the detection of Bb antigens, the kit including, alternatively, an antibody reactive with 66 kDa antigenic proteins or a protein or peptide which includes an epitope thereof, together with means for detecting a specific immunoreaction between an antibody and its corresponding antigen. Examples of suitable means include labels attached directly to the antigen or antibody, a secondary antibody having specificity for human Ig, or protein A or protein G. Alternatively, avidin-biotin mediated Staphylococcus aureus binding could be used. For example, the monoclonal antibody may be biotinylated so as to react with avidin complexed with an enzyme or fluorescent compound.
A particular embodiment of the invention concerns kits for detection of antibodies against the described Bb 66 kDa antigens, epitopes thereof as represented by portions of the amino acid sequences, or closely related proteins or peptides, such as epitopes associated with other virulenceassociated proteins detected by comparison of low-passage, virulent and high-passage, avirulent strains of Bb. The antigen for the kit(s) consists of the Bb 66 kDa proteins or portions thereof produced by a recombinant DNA vector in E.
coli or another bacterial or nonbacterial host. Alternatively, the antigen may be purified directly from Bb or manufactured as a synthetic peptide. Samples for the assays may be body fluids or other tissue samples from humans or animals.
The presence of reactive antibodies in the samples may be demonstrated by antibody binding to antigen followed by detection of the antibody-antigen complex by any of a number of methods, including ELISA, RIA, fluorescence, agglutination or precipitation reactions, nephelometry, or any of these assays using avidin-biotin reactions. The degree of reactivity may be assessed by comparison to control samples, and the degree of reactivity used as a measure of present or past infection with Bb. The assay(s) could also be used to monitor reactivity during the course of Lyme disease, to determine the efficacy of therapy.
IWO 95/35379 PCT/US95/07665 16 In still further embodiments, the invention contemplates a kit for the detection of Bb nucleic acids in the sample, wherein the kit includes one or more nucleic acid probes specific for the 66 kDa genes, together with means for detecting a specific hybridization between such a probe and Bb nucleic acid, such as an associated label.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hence, the invention relates to an isolated nucleic acid fragment comprising a nucleotide sequence which encodes a polypeptide exhibiting a substantial immunological reactivity with a rabbit antiserum raised against a 66 kDa polypeptide derived from Borrelia garinii said rabbit antiserum exhibiting substantially no immunological reactivity with whole cell preparations (prepared as described herein) from at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica.
It is preferred that the 66 kDa polypeptide used for rising the rabbit antiserum and derived from Borrelia garinii comprises the amino acid sequence 1-600 in SEQ ID NO: 8.
By the term «nucleic acid fragment» as used herein is meant a fragment of DNA or RNA, but also of PNA (cf. Nielsen P E et al., 1991), having a length of at least two joined nucleotides. It will be understood, that although the disclosed nucleic acid fragments of the present invention are DNA fragments, it may be desirable to employ a RNA fragment in e.g. a viral vector, the genome of which is natively composed of RNA. For the purposes of preparing e.g. probes for hybridization assays as described below, PNA fragments may prove useful, as these artificial nucleic acids have been demonstrated to exhibit very dynamic hybridization properties.
C I WO 95/35379 PCT/US95/07665 17 The term «a substantial immunological reactivity» is meant to designate a marked immunological binding between an antibody/antiserum on the one hand, and on the other an antigen, under well-defined conditions with respect to physicochemical parameters as well as concentrations of antigens and antibodies. Thus, a substantial immunological reactivity should be clearly distinguishable from a non-specific interaction between an antibody/antiserum and an antigen. This distinction can for instance be made by reacting the antibody/antiserum with a known concentration of an antigen which has previously been shown not to react with the antibody/antiserum, and then using this reaction as a negative control. A positive control could suitably be the reaction between the antibody/antiserum and the same concentration of the antigen used for the immunisation resulting in the production of the antibody/antiserum. In such an assay, an antigen resulting in a relative signal of at least 10% (calculated as Sm*(Sp-Sn)-100, where Sm is the measured signal, Sp the positive control signal, and S n the negative control signal) is regarded as having a substantial immunological reactivity. An antigen exhibiting «substantially no immunological reactivity» therefore is defined as an antigen giving a signal of less than Although the data presented herein demonstrates that there is no cross-reactivity between antigens from Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica and the disclosed polypeptides, it is conceivable that a few isolates of these bacteria will exhibit some cross-reactivity. As can be deduced from the above it is expected that the cross-reactivity will be less than 5% (since there is no reactivity with at least 95% of randomly chosen Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica), and according to the invention this cross-reactivity may be even lower, such as at the most 4% and 3%, preferably at the most such as According to the invention the cross-reactivity is most preferred at most such as In such a case there will be no substantial
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SWO 95/35379 ‘CT/US95/07665 18 immunological reactivity between the rabbit antiserum mentioned above and whole cell preparations of Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica.
The above-cited considerations concerning cross-reactivity apply for all cross-reactions between on the one hand the polypeptides/DNA fragments of the invention and on the other hand material from Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.
When using the term «cross-reactivity» is herein meant the phenomenon that two species exhibit a common feature which is detected in a reaction. In the present context the term cross-reactivity is used for similar reactions in antigenantibody interactions as well as in hybridization interactions.
Nucleic acid fragments of the invention useful as hybridisation probes and/or primers are not necessarily those fragments encoding immunologically useful polypeptides.
Therefore the invention also relates to nucleic acid fragments which hybridises readily with either a DNA fragment having the nucleotide sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with a DNA fragment complementary thereto, but exhibits no substantial hybridization with genomic DNA from at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica when the hybridization conditions are highly stringent.
Preferred nucleic acid fragments of the invention are DNA fragments, especially those which have nucleotide sequences with a sequence homology of at least 70% with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with subsequences thereof. However, the degree of homology may be even
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WO 95/35379 PCT/US95/07665 19 higher such as at least 75%, 80%, 85%, 87%, and 89%. It is preferred that the degree of homology is at least 90%, such as 92%, 94% or 95%, and especially preferred are DNA fragments with a sequence homology of at least 96% with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13. Especially for high accuracy hybridization assays, a total homology is necessary, and therefore preferred. Other preferred nucleotide acid fragments of the invention are those which encode a polypeptide of the invention (cf. the below discussions concerning these polypeptides and their degree of homology with the amino acid sequences disclosed herein) which has an amino acid sequence exhibiting a sequence homology of at least 50% with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ NO: 10, or SEQ ID NO: 14 or with subsequences thereof.
The terms «homology» and «homologous» are, with respect to DNA fragments, intended to mean a homology between the nucleotides in question between which the homology is to be established, in the match with respect to identity and position of the nucleotides of the DNA fragments. With respect to polypeptides and fragments thereof described herein, the terms are intended to mean a homology between the amino acids in question between which the homology is to be established, in the match with respect to identity and position of the amino acids of the polypeptides.
Considerations «similar to those given above for the immunological reactivity and cross-reactivity of antigens can be applied for the distinction between a nucleic acid fragment which «hybridizes readily» and a fragment which «exhibits substantially no hybridization» under high stringency conditions.
The term «highly stringent» when used in conjunction with hybridisation conditions is as defined in the art, i.e. 0 C under the melting point Tm, cf. Sambrook et al, 1989, pages 11.45-11.49.
WO 5/3537 9 PCT/US95/07665 Interesting nucleic acid fragments of the invention encode a polypeptide comprising an amino acid sequence comprised in a polypeptide present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, and/or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica. This encoded polypeptide may according to the invention comprise at least a part of an amino acid sequence of a 66 kDa protein which is present in Bb, and it is preferred that the polypeptide encoded by the nucleic acid fragment of the invention is a G6 kDa protein present in whole cell preparations, and preferably this 66 kDa protein is also present in fraction B (as discussed in thu examples).
It is especially preferred that the encoded polypeptide further is a natively surface exposed protein of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii
ACAI.
By the terms «present» and «substantially absent», when referring to amino acid sequences and polypeptides in bacteria, are meant that the concentration of the amino acid sequence/polypeptide in a bacterium where it is «present» is at least 100 times higher than in a bacteriuin where it is substantially absent. However, it is preferred that the ratio of the concentrations are at least 1000, and more preferred at least 10,000, 100,000 or even higher. It is especially preferred that there can be observed no concentration of the amino acid sequence/polypeptide in the bacterium from where it is substantially absent.
It will be understood from the above that various analogues and subsequences of the nucleic acids disclosed herein are interesting aspects of the invention, as are nucleic acid fragments encoding fused polypeptides including polypeptides encoded by nucleic acid fragments of the invention.
I 1 WO 95/35379 PCT/US95/07065 21 The term «analogue» with regard to the nucleic acid fragments of the invention is intended to indicate a nucleotide sequence which encodes a polypeptide identical or substantially identical to a polypeptide encoded by a nucleic acid fragment of the invention (SEQ ID NO’s: 4, 6, 8 and 14).
It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia, to the preference of the organisms in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of a nucleic acid fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the nucleic acid fragment in question.
Also, the term «analogue» is used in the present context to indicate a nucleic acid fragment or a nucleic acid sequence of a similar nucleotide composition or sequence as the nucleic acid sequence encoding the amino acid sequence having the immunological properties discussed .bove, allowing for minor variations which do not have an .adverse effect on the biological function and/or immunogenicity as compared to the disclosed polypeptides, or which give interesting and useful novel binding properties or biological functions and immunogenicities etc. of the analogue. The analogous nucleic acid fragment cr nucleic acid sequence may be derived from an animal or a human or may be partially or completely of synthetic origin as described herein. The analogue may also be derived through the use of recombinant nucleic acid techniques.
Furthermore, the terms «analogue» and «subsequence» are intended to allow for variations in the sequence such as substitution, insertion (including introns), addition, deletion and rearrangement of one or more nucleotides, which variations do not have any substantial effect on the polypeptide encoded by a nucleic acid fragment or a subsequence thereof. The term «substitution» is intended to mean the WO 95/35379 PCTUS95/07665 22 replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides, «addition» is understood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence, «insertion» is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence, «deletion» is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it, and «rearrangement» is intended to mean that two or more nucleotide residues have been exchanged with each other.
A preferred method of preparing variants of the 66 kDa antigens disclosed herein is site-directed mutagenesis. This technique is useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, derived from the 66 kDa antigen sequences, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing-considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sec. of sufficient size and sequence complexity to form a stab.-e duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al., 1983). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 .WO 95135370 PCIT/US95/07665 23 phage (Messing et al., 1981). These phage are readily commercially available and their use is generally well known to those skilled in the art.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes the 66 kDa antigens. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al.
(1978). This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E.
coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original nonmutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected 66 kDa genes using site-directed mutagenesis is provided as a means of producing potentially useful species of the 66 kDa genes and is not meant to be limiting as there are other ways in which sequence variants of the 66 kDa genes may be obtained.
For example, recombinant vectors encoding the desired 66 kDa genes may be treated with mutagenic agents to obtain sequence variants (see, a method described by Eichenlaub, 1979) for the mutagenesis of plasmid DNA using hydroxylamine.
Another example is the possibility of introducing point mutations in the disclosed sequences by use of PCR techniques, wherein the primers used include mismatches which, after the completion of the PCR cycles, appear in the amplification products. The thus obtained amplified mutated nucleic acid fragments can thereafter be introduced into suitable vectors and used for the production of recombinantly transformed host cells.
WO 95/33379 PCT/US9S107665 24 Analogues/subsequences of the disclosed nucleic acid fragments which also form part of the invention are nucleic acid fragments which are fused to at least one other nucleic acid fragment which encodes a protein enhancing the immunogenicity of the fused protein relative to a protein without the encoded fusion partner. Such encoded proteins may e.g. be lipoproteins, e.g. the outer membrane lipoprotein from E.
coli and OspA from Bor:elia burgdorferi sensu lato; viral proteins, e.g. from Hepatitis B surface antigen, Hepatitis B core antigen, and the influenza virus non-structural protein NS1; immunoglob-lin binding proteins, e.g. protein A, protein G, and the synthetic ZZ-peptide; T-cell epitopes; or B-cell epitopes.
Other nucleic acid fragments to form part of a nucleic acid fragment of the invention encoding a fusion polypeptide are those encoding polypeptides which facilitates expression and/or purification of the fused peptide. Such encoded polypeptides could according to the invention be bacterial fimbrial proteins, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide; the maltose binding protein; gluthatione S-transferase; f-galactosidase; or polyhistidine.
Other nucleic acid fragments of the invention of special interest are those encoding at least one epitope present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of «t least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica. Preferred are epitopes of a 66 kDa protein present in whole cell preparations of Bb.
By the term «epitope» is meant the spatial part of an antigen responsible for the specific binding to the antigen-binding part of an antibody. It goes without saying that the identification of epitopes of the disclosed antigens will 1acili- WO 95/35379 PCTIUS95/0765 tate the production of polypeptides which exhibit marked antigenicity thus making them interesting with respect to diagnosis of Borreliosis and vaccination against infections with Bb.
The identification of epitopes can be performed in several ways. One possibility is to make a hydrophobicity plot as described herein, and thereafter selecting the special linear sequences of the polypeptide and investigate their immunogenicity. As mentioned herein, several regions of the disclosed polypeptides are regarded as interesting. Thus, nucleic acids encoding polypeptides substantially identical to the amino acid sequences 175-190, 285-305, 365-385, or 465-490 in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 also form part of the invention; such nucleic acid fragments may also be part of nucleic acid fragments encoding fusion polypeptides comprising multiple copies of at least one of epitope, as such fusion polypeptides should exhibit superior immunological utility in diagnostics as well as in vaccines.
Another way of simply identifying epitopes is to digest a polypeptide antigen with a known amino acid sequence with endo- and exopeptidases. The obtained fragments are tested against antibodies directed against the whole polypeptide, and by way of deduction, the precise location of the linear epitopes can be determined. A variation of this method involves the recombinant production of subfragments (cf. the above) of the full-length polypeptide followed by the same test procedure.
Another part of the invention relates to a substantially pure polypaptide exhibiting a substantial immunological reactivity with an antiserum from rabbits immunised with a 66 kDa polypeptide derived from Borrelia garinii IP90, said rabbit antiserum exhibiting substantially no immunological reactivity with whole cell preparations from at least 95% of randomly selected B. hermsii, B. crocidurae, B. anserina, or B.
hispanica. It is preferred that the 66 kDa polypeptide used
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WOI 95/35379) PCT/US95/07665 26 for rising the rabbit antiserum and derived from Borrelia garinii IP90 comprises the amino acid sequence 1-600 in SEQ ID NO: 8.
It will be understood that such a polypeptide may be encoded by a DNA fragment of the invention and that the polypeptides encoded by the DNA fragments of the invention also form part of the invention.
By the term «polypeptide» is herein understood a molecule comprising at least two amino acids joined by a peptide bond.
The term polypeptide thus indicate small peptides (less than amino acid residues), oligopeptides (between 10 and 100 amino acid residues), proteins (the functional entity including at least one peptide and/or prosthetic groups and/or glycosylation and/or lipidation etc.) as well as traditional polypeptides (more than 100 amino acid residues).
Interesting polypeptides according to the invention are those prepared by the well known methods of liquid or solid phase peptide synthesis utilizing the successive coupling of the individual amino acids of the polypeptide sequence. Alternatively, the polypeptide can be synthesized by the coupling of individual amino acids forming fragments of the polypeptide sequence which are later coupled so as to result in the desired polypeptide. These methods thus also constitute another interesting part of the invention.
Preferred polypeptides of the invention are recombinant polypeptides, normally prepared by a process comprising inserting a nucleic acid fragment of the invention in an expression vector, transforming a host organism or a host cell (normally a host organism or host cell which does not natively express the polypeptide of the invention) with the vector, I WO 95/35379 PCT/US95/07665 27 culturing the transformed host cell under conditions facilitating the expression of the polypeptide by the host organism or host cell, harvesting the polypeptide, and optionally subjecting the polypeptide to post-translational modification(s), and performing an at least partial purification of the polypeptide.
The need for post-translational modifications exists because certain polypeptides are prepared in the above-described manner lacking for instance a fatty-acylation of an amino acid residue, or the polypeptide have for some reason been prepared in an elongated version which should be cleaved before the polypeptide will prove functional.
Thus, according to the invention the post-translational modifications involves lipidation, glycosylation, cleavage, or elongation of the polypeptide. In some instances, the host cell or cell line also processes the translation product so as to obtain a processed polypeptide.
The present invention thus also relates to the use of the nucleic acid fragments of the invention in the construction of vectors and in host cells. The following is a general discussion relating to such use and the particular considerations in practising this aspect of the invention.
In general, of course, prokaryotes are preferred for the initial cloning of nucleic sequences of the invention and constructing the vectors useful in the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by way of example, strains such as E. coli K12 strain 294 (ATCC No.
31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting.
WO 95/35379 PCT/US95/07665 28 Prokaryotes are also preferred for expression. The aforementioned strains, as well as E. coli W3110 lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
Those promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiase, or common baker’s yeast is the most commonly used among
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I L WO 95/35379 PCT/US95/07665 29 eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980).
This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic of the yeast host e’ll genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequence, associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeastcompatible promoter, origin of replication and termination sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from verte- WO 95/35379 PCT/US95/07665 brate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines.
Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compat’ -e with the host cell systems.
An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
WO 95/35379 PCT/US95/07665 31 In the light of the above discussion, the methods for recombinantly producing the polypeptide of the invention are also a part of the invention, as are the vectors carrying and/or being capable of replicating the nucleic acids according to the invention in a host cell or a cell-line. According to the invention the expression vector can be e.g. a plasmid, a cosmid, a minichromosome, or a phage. Especially interesting are vectors which are integrated in the host cell/cell line genome after introduction in the host.
Another part of the invention are transformed cells (useful in the above-described methods) carrying and capable of replicating the nucleic acid fragments of the invention; the host cell can be a microorganism such as a bacterium, a yeast, or a protozoan, or a cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Especially interesting are cells from the bacterial species Escherichia, Bacillus and Salmonella, and a preferred bacterium is E. coli.
Yet another part of the invention relates to a stable cell line producing a polypeptide according to the invention, and preferably the cell line carries and expresses a nucleic acid of the invention.
Returning to the polypeptides of the invention: Also polypeptides which comprises an amino acid sequence exhibiting a sequence homology of at least 50% with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ NO: 10, or SEQ ID NO: 14 or with subsequences thereof are interesting embodiments of the polypeptides of the invention. However this homology should normally be higher, such as at least 60%, 70%, 80%, 85%, or even 90%. Preferred polypeptides have a homology of at least 92%, such as at least 95%, 97%, 98%, 99%, or even 100%. Other preferred polypeptides of the invention are those which are encoded by a nucleic acid fragment of the invention (cf. the discussions above concerning these nucleic acid fragments and their degree of homology with the nucleic acid sequences WO 95/35379 PC’/iUSJ95/07665 32 disclosed herein) which has a nucleic acid sequence exhibiting a sequence homology of at least 70% with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with subsequences thereof.
A very important part of the invention is vaccines for conferring increased resistance to infection with Bb.
Thus, an important part of the invention relates to vaccines comprising an amount of a polypeptide according to the invention, the amount of the polypeptide being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being, optionally in combination with a pharmaceutically acceptable carrier or vehicle and the vaccine optionally further comprising an adjuvant. Of course, also vaccines comprising polypeptide fragments encoded by the nucleic acid fragments of the invention are a part of the invention, as such polypeptide fragments as mentioned above also form part of the invention.
By the term «conferring substantially increased resistance to infections» is meant that the administration of the vaccine to the animal has the effect that disease caused by infections with at least one strain of bacteria is avoided or at least that the risk of catching the disease is significantly reduced.
Part of the present invention contemplates vaccine preparation and use. General concepts related to methods of preparation and use are discussed in the following as applicable to preparations and formulations with the polypeptides of the invention.
Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorpor- WO 95/35379 PCT/US95/07665 33 ated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, iagnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be WO 95/35379 PCT/US95107665 34 derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual’s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 1 Ag to 500 gg, especially in the range from about 10 Ag to 50 Ag. Suitable regimes for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 700 to 101 0 C for 30 second to 2 minute periods respectively. Aggregation by reactivating with WO 95/35379 PCT/US95/07665 pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gramnegative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monooleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens.
The assays may be performed by labelling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384.and 3,949,064, as illustrative of these types of assays.
It is contemplated that the vaccines of the invention should be effective in activating both arms of the immune system.
Thus, vaccines capable of eliciting a cell-mediated immune reaction are also a part of the invention.
One such vaccine of the invention is a live vaccine comprising a non-pathogenic microorganism carrying and being capable of expressing a nucleic acid fragment of the invention, the live vaccine being effective in conferring increased resistance to infection with Borrelia burgdorferi sensu lato in an animal, including a human being. The nonpathogenic microorganism could for instance be a bacterium such as a strain of Mycobacterium bovis BCG. The live vaccine WO 95/35379 PCT/US95/07665 36 could for instance express a multitude of the polypeptides of the invention, thereby making it more immunogenic.
Another way of eliciting a cell-mediated response is to employ an adjuvant as described above. However, recent research have revealed a new an exciting possibility, wherein a DNA fragment is introduced into non-replicating cells of the vaccinated animal, whereafter the translational product is exposed on the cell-surface thereby eliciting a cellmediated response. These methods are reviewed in Ulmer et al., 1993, which hereby is included by reference.
Therefore, also a part of the invention is a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigens by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigens being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being.
It is also possible that a vaccine according to the invention comprising other Borrelia antigens may prove useful, as a more efficient immunological response could be elicited. Such a combination vaccine could for instance contain OspA, OspB, OspC, OspD, and/or PC. In this regard, also combination vaccines comprising at least two different pol-pa3ptides according to the invention are interesting.
Methods of actively immunizing animals, including mammals such as human beings against infections with Bb are also parts of the invention. The methods generally consist of the administration to the animal of an immunogenically effective amount of the vaccines of the invention. Methods for passive immunisation comprising administering to the animal an immunogenically effective amount of an antibody of the invention (as described below) are also included in the invention.
WO 95/35379 PCT/VS95/076i65 37 An important part of the invention relates to at least partially purified antibodies, polyclonal or monoclonal, reacting substantially specifically with a protein according to the invention, or proteins encoded by the nucleic acid fragments of the invention. According to the invention, monoclonal antibodies are preferred.
The phrase «reacting substantially specifically» is intended to indicate that the antibody will show no substantial immunological reactivity (as defined above) with other antigens which might possibly be present in an embodiment of the present invention where the antibody is used.
The antibodies of the invention are prepared by methods wellknown to the skilled person.
Other important parts of the present invention are compositions adapted for the determination of Bb in animals (including mammals, e.g. humans). Accordingly, methods of determining the presence of Bb are also a part of the invention.
A diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in an animal, including a human being, or in a sample, the composition comprising an amount of the polypeptide of the invention effective to detectably react with antibodies present in the animal or in the sample, the antibodies being directed against Borrelia burgdorferi sensu lato, the composition optionally comprising a detectable label, is also a part of the invention. Similar compositions including the nucleic acid fragments of the invention or the antibodies of the invention are also a part of the invention, as will be apparent from the claims.
The phrase «to detactably react with» is intended to mean a reaction between two substances in an assay, the reaction being significant enough so as to give a signal in the assay which is clearly different from a negative signal. Thus, the detectable reaction is highly dependent on the type of detec- WO 95/35379 ;PCT/VS9107065 38 tion means used. Very sensitive methods like ELISAG and RIAs will detect reactions involving few molecules, whereas more insensitive reactions will demand that the reaction involves many molecules.
Methods of determining the presence of Bb antibodies or components of Bb in samples or in animals are also parts of the invention, as is a method of determining the presence of antibodies directed against Borrelia burgdorferi sensu lato in an animal, including a human being, or in a sample, comprising administering the polypeptide of the invention to the animal or incubating the sample with the polypeptide of the invention, and detecting the presence of bound antibody resulting from the administration or incubation. Likewise, a method of determining the presence of a Borrelia burgdorferi sensu lato antigen in an animal, including a human being, or in a sample, comprising administering an antibody of the invention to the animal or incubating the sample with the antibody, and detecting the presence of bound antigen resulting from the administration or incubation, forms part of the invention. Finally a method of determining the presence of Borrelia burgdorferi sensu lato nucleic acids in an animal, including a human being, or in a sample, comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the presence of hybridized nucleic acids resulting from the incubation, is also included in the invention.
Finally, diagnostic kits for the diagnosis of on-going or previous Bb infection forms part of the invention. The diagnostic kits of the invention comprises an antibod, a nucleic acid, or a polypeptide according to the invention in combination with a means for detecting the interaction with the relevant substance reacting with these substances of the invention; the choice of these detection means is discussed elsewhere herein.
WO 9’5/35379) Vc’IYUS95/07665 39 In both the diagnostic methods, compositions, and kits the antibodies, nucleic acids or polypeptides according to the invention may optionally be coupled to solid or semi-solid carriers, as is well-known in the art.
As will appear from the examples, the present invention at s to the utility of Bb associated antigenic proteins as ac -iostic or preventive tools in Lyme disease. Proteins have been identified as associated only with virulent isolates of Bb, providing a basis for several types of diagnostic tests for infections with Bb and for Lyme disease, including immunodiagnostic and nucleic acid identification, such as those based on amplification procedures (PCR etc.).
It is contemplated that several assays for the presence of Bb or for Lyme disease may be developed using any of the polypeptides of the invention, the corresponding nucleic acid fragments encoding the protein, functionally similar proteins and their epitopes, or by detection of other appropriate nucleic acids. These methods are similar in principle to those previously described (Magnarelli et al., 1989; Magnarelli et al., 1984; and Craft et al., 1984). Reactive epitopes representing portions of the 66 kDa protein sequences could be utilized in an analogous manner.
Another promising assay is the microcapsule agglutination technique (MCAT) (Arimitsu et al., 1991). In this procedure, microscopic polystyrene beads are coated with Bb antigen and incubated with dilutions of patient serum. After overnight incubation at 4 0 C, the agglutination patterns are determined.
Using whole Bb as antigen, the MCAT has been shown to be highly discriminatory between Lyme disease patients and healthy individuals, with little overlap in agglutination titer, although false positive reactions have been observed with rheumatoid arthritis patients (Anderson et al., 1988) and leptospirosis samples (Barbour, 1988). An assay using 66 kDa protein alone or in combination with other antigens such WVO 5/35379 PCT/US95/07665 as the 94 kDA, 30 kDa and 21 kDa antigens should be feasible.
Such combinations may increase sensitivity of the assay.
Also contemplated within the scope of the present invention is the use of the disclosed nucleic acid fragments as hybridization probes. While particular examples are provided to illustrate such use, the following provides general background for hybridization applications taking advantage of the disclosed nucleic acid sequences of the invention.
I e invention has disclosed a DNA segment encoding an antigenic Bb protein. Detection of that DNA or various parts thereof is expected to provide the basis for a useful assay.
One method of detecting the 66 kDa antigen genes is based on selective amplification of known portions of the gene. A particular method utilizes PCR amplification, using any of a number of primers that could be prepared from knowledge of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and SEQ ID NO: 13. Generally, such primers are relatively short, 7-28 base pairs in length, and may be derived from the respective sense or anti-sense strands of the disclosed DNA segment. Synthesis of these primers may utilize standard phosphoramidite chemistry (Beaucage et al., 1981).
As mentioned, in certain aspects, the DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA or PNA) sequences having the ability to specifically hybridize to Bb gene sequences. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of the sequence, e.g., SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13 or derived from flanking regions of these genes. The ability of such nucleic acid probes to specifically hybridize to the Bb gene sequences lend them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, either uses .WO 95/35379 P(;;rWUS95/07665 41 are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs.
To provide certain of the advantages in accordance with the invention, the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence, such as that shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13. A size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. Thus, one will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant production.
The present invention will find particular utility as the basis for diagnostic hybridization assays for detecting Bbspecific RNA or DNA in clinical samples. Exemplary clinical samples that can be used in the diagnosis of infections are thus any samples which could possibly include nucleic acid, including samples from tissue, blood serum, urine or the like. A variety of tissue hybridization techniques and systems are known which can be used in connection with the hybridization aspects of the invention, including diagnostic assays such as those described in Falkow et al., U.S. Patent 4,358,535 which is hereby incorporated by I WO 95/35379 PCI’US95/07665 42 Accordingly, the nucleotide sequences of the invention are important for their ability to selectively form duplex molecules with complementary stretches of Bb gene segments.
Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degree of selectivity of the probe toward the target sequence. Fcr applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temrerature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50 0 C to 70 0 C. These conditions are particularly selective, and tolerate little, if any, mis 1 ratch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex. In these circumstances, one would desire to employ conditions such as 0.15 M-0.9 M salt, at temperatures ranging from 20 0 C to 550C. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In clinical diagnostic embodiments, nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization.
A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as alkaline phosphatase or peroxidase, instead of radioactive or other WO 95/35379 PCT/US95/07665 43 environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples. Luminescent substrates, which give off light upon enzymatic degradation, could also be employed and may provide increased sensitivity.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids amniotic fluid cerebrospinal fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove non-specifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
Plasmids pJB-101, pJB-102, and pJB-104 have been deposited on the 16 June 1994 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the numbers DSM 9253, DSM 9254, and DSM 9255, respectively, under the terms and conditions of the Budapest Treaty.
WO 95/35379 PCT/US95/07665 44 LEGENDS TO THE FIGURES Figs. 1A and lB. Effect of proteases on B. afzelii ACAI cells.
A: Coomassie blue-stained PAGE of the bacterial lysates after the cells were incubated with buffer alone, lane 1; trypsin, lane 2; or proteinase K, lane 3.
B: PAGE of the subcellular fraction of membrane components (Fraction B) recovered from the cells treated in three different ways described in section A. Arrows indicate the position of 66 kDa protein. Mw: molecular weight, kD: kiloda- Iton.
Figs. 2A and 2B. Comparison of phenotypic expression of the 66 kDa protein in Borrelia species.
A: Coomassie blue-stained PAGE of the whole cell proteins of B. burgdorferi B31, lane 1; B. afzelii ACAI, lane 2; B garinii Ip90, lane 3; B. hermsii, lane 4; B. crocidurae, lane and B. anserina, lane 6.
B: Reactivity of Borrelia proteins against rabbit anti-66 kDa protein antibody in Western blot. Borrelia species are numbered as in section A. Arrow indicates the position of the 66 kDa protein. Mw: molecular weight, kD: kilodalton.
Figs. 3A and 3B. Southern blot analysis of DNA.
A: DNA separated by pulse-field agarose gel electrophores’s.
Lane 1, DNA prepared from B. burgdorferi B31. Lane 2, DNA prepared from Borrelia afzelii ACAI. Lane 3, DNA prepared from Borrelia garinii B: DNA subsequently transferred to a Hybond-N membrane and cross-linked with UV-light and probed at 55°C with a radiolabelled DNA probe derived by PCR amplification of the 66 kDa gene from Borrelia garinii Ip90. Lane 1, Lane 2 and Lane 3 same as above.
Figs. 4A and 4B. Western blot analysis of recombinant 66 kDa protein expressed in E. coli.
A: Proteins prepared from uninduced E. coli.
WO 95/35379 PC’TUS95/07665 B: Proteins prepared from induced E. coli.
Proteins were separated by 12.5% SDS-PAGE and subsequently transferred to an Immobilon-P membrane by electroblotting.
Non-specific binding was blocked by immersing the filter in 5% BSA. The proteins were visualised by using the rabbit anti-66 kDa serum as primary antibody and an alkaline phosphatase conjugated anti-rabbit IgG secondary antibody with a subsequent developing reaction using the substrate BCIP. Lane 1, proteins prepared from whole cells. Lane 2, proteins from the supernatant obtained after sonication. Lane 3, proteins obtained by extraction of the pellet after sonication with 2M urea. Lane 4, proteins obtained by further extraction of the pellet after sonication with 8M urea. Lane fraction B prepared from B. garinii Figs. 5A, 5B, and 5C. Plot of antigenicity index of the 66 kDa protein.
The plots were made using the Jameson-Wolf algorithm provided in the MacVector software package.
A: 66 kDa protein from B. burgdorferi B31.
B: 66 kDa protein from B. afzelii ACAI.
C: 66 kDa protein from B. garinii
EXAMPLES
Bacterial strains and culture conditions. Borrelia strains used in this study were the following: strain B31 of B.
burgdorferi, a tick isolate from North America (ATCC 35210); strain ACAI of B. afzelii, a human skin isolate from Sweden (Asbrink et al. 1984); strain Ip90 of B. garinii, a tick isolate from the Asian Russia (Kryuchechnikov et al. 1988); strain B. burgdorferi B313, a mutant of B. burgdorferi B31 lacking OspA and OspB (Sadziene et al. 1993).
Also used were three relapsing fever borreliae species, B.
hermsii, B. crocidurae, and B. hispanica, and B. anserina, the causative agent of avian borreliosis.
WO 95/35379 PCTIUS95/07665 46 Borreliae were grown in BSK II medium (Barbour 1984) and the cells were harvested in late-log phase by centrifugation at 5,000 rpm for 20 min.
The Escherichia coli strains Dh5a and BL21 were used for transformation with the recombinant plasmids in, respectively, DNA cloning and gene expression experiments. E. coli strains were grown in Luria broth medium (Gibco BRL, Gaithersburg, MD) supplemented, when required, with carbenicillin (Sigma, St. Louis, MO) at 50 pg/ml.
EXAMPLE 1.
Preparation of Borrelia proteins, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot.
1.1 Preparation of Borrelia proteins.
For the whole-cell protein preparations, bacteria harvested from 200 ml of BSK II medium were washed twice with phosphate-buffered saline-5mM MgCl2, (PBS-Mg). The pellet was suspended in 2 ml of PBS, sonicated and the supernatant was collected after centrifugation at 10,000 rpm for 30 min. In some experiments whole-cell lysate was obtained by boiling washed bacteria for 3 min in SDS-PAGE sample buffer.
The subcellular fraction of borreliae outer membrane components (designated Fraction B) was prepared as described elsewhere (WO 90/04411). Briefly, cells harvested from 1.5 1 of the culture were washed three times with 10 mM Tris-HCl (pH 150 mM NaC1 and 5 mM MgCl 2 (TSM buffer). Octyl-f- D-glucopyranoside (OGP) (Sigma St. Louis, MO) was added to a final concentration of 2% in 10 ml TSM buffer and the suspension was incubated at 37 0 C for 60 min. The cell lysate was centrifuged and the supernatant was incubated at 56 0 C for min. The precipitate was removed by centrifugation at 20,000 rpm for 30 min at 37 0 C, and the supernatant was dialysed WO 95/35379 PCT/US95/07665 47 against water at 4 0 C for 2 days. The precipitate (Fraction B) formed in the dialysis bag was recovered by centrifugation at 20,000 rpm for 30 min at 250C.
1.2 Separation of proteins by SDS-PAGE.
Bacterial proteins were separated by 12.5% SDS-PAGE essentially according to Laemmli (1970). Subsequently, gels were either stained with Coomassie Blue R-250 (CB) (Sigma, St Louis, MO) or were subjected to Western blotting.
1.3 Western blotting.
The proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) by electroblotting at 0.8 mA/cm 2 for 1 h.
The nonspecific binding was blocked by immersing the filter for 2 h into 5% bovine serum albumin (BSA) (Sigma, St. Louis, MO) in PBS, containing 0.05% Tween-20 (PBS-T). Primary or secondary antibodies were diluted with 2.5% BSA in PBS-T, and both incubations of the filter for 1 h was followed by washing in PBS-T. In a developing reaction the substrate for the alkaline phosphatase conjugate was 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma, St. Louis, MO).
EXAMPLE 2.
Preparation of antiserum against 66 kDa.
2.1 Purification of 66 kDa The 66 kDa protein was purified by 12.5% SDS-PAGE of Fraction B obtained from the B. garinii IP90 spirochaetes. The appropriate band was visualized by staining the gel with 0.05% CB in water without fixation in MeOH and Acetic acid. The protein band contained approximately 100 Ag of 66 kDa.
WO 95/35379 ‘C’T/US9S/07d7d 48 2.2 Immunization of rabbits.
Approximately 100 Ag of the 66 kDa protein prepared as described above was homogenised and used in each of four immunizations of one rabbit performed in one and two (for the last immunization) months intervals. Seven serum samples were obtained during a 5 months period, and serum was diluted 1:1,000 when used for Western blot analysis.
EXAMPLE 3.
Cell surface proteolysis of Borrelia cells.
3.1 Protease treatment of borreliae cells.
Cell surface proteolysis of B. afzelii ACAI cells was conducted as previously described (Barbour et al. 1984). Briefly, washed spirochaetes were resuspended in PBS-Mg at a concentration of 2 x 10 9 cells/ml. To 950 Al of the cell suspension was added 50 il of one of the following: distilled water, proteinase K (Sigma, St Louis, Mo) (4 mg/ml in water) or trypsin (Gibco BRL, Gaithesburg, MD) (1 mg/ml in 10 3
M
HC1). After incubation for 40 min at 20 0 C the proteolytic treatment was stopped by the addition of 10 Al from a solution of the peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, MO) (50 mg of PMSF per 1 ml of isopropanol), and the cells were centrifuged and washed twice with PBS-Mg. The pellets were resuspended in TSM buffer. Onethird of the cell suspension of each preparation was subjected to the whole cell protein extraction by boiling in SDS-PAGE sample buffer. The remaining part of the suspensions were used to prepare the subcellular fraction of the borrelial outer membrane components, Fraction B, as described above.
3.2 Analysis of the protease treated Borrelia cells.
The SDS-PAGE result of the protease treated B. afz’~lii ACAI cells is presented in Fig. 1. As seen in the CB stained WO 95/35379 PCT/US95/0765 49 protein profiles of the whole-cell lysates (Fig. 1A), proteinase K affected considerably the minor protein with an apparent molecular weight of 66 kDa. The protein composition of the subcellular fractions of outer membrane components (Fraction B) recovered from protease treated and untreated spirochaetes, was also investigated (Fig. 1B). The 66 kDa protein was shown to constitute a substantial part of the Fraction B, obtained from the protease untreated cells. In the Fraction B derived from the spirochaetes proteolysed with trypsin or proteinase K, the 66 kDa protein was, respectively, reduced in amount or entirely absent. The finding that protease treatment reduces the amount of the 66 kDa protein clearly shows that the 66 kDa protein is surface exposed, and most probably associated with the outer membrane of the Borrelia.
EXAMPLE 4.
Expression of the 66 kDa protein in different Borrelia species 4.1 SDS-PAGE analysis.
The CB stained SDS-PAGE of the whole-cell protein preparations of Lyme disease borreliae and other Borrelia species is shown in Fig. 2A. The 66 kDa protein was present in the whole-cell preparation of B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90. The PAGE revealed no major differences among the borrelial strains in respect of either apparent molecular weight or expression level of the 66 kDa protein. In the analogous preparations of B. hermsii, B.
crocidurae, and B. anserina no visible band corresponding to the 66 kDa protein was detectable. In addition to being present in fraction B from B. afzelii ACAI (cf. example 6), the 66 kDa protein was recovered also in the Fraction B of B.
burgdorferi B31 and B. garinii Ip90, however, it was absent in the Fraction B obtained from B. crocidurae and B. hispanica (data not shown).
WO 95/35379 P071,J8951)R07665 4.2 Western blotting.
In Western blot analysis (Fig. 2B), the 66 kDa protein of B.
burgdorferi B31, B. afzelii ACAI, and B. narinii Ip90 reacted similarly with the rabbit antiserum, raised against the 66 kDa protein of the latter strain. There was no apparent reactivity of the antiserum with B. hermsii, B. crocidurae, B. anserina (Fig. 2B), and B. hispanica (data not shown) proteins.
The rabbit antiserum raised against the 66 kDa protein of B.
garinii Ip90, in Western blots reacted equally against 66 kDa protein of B. burgdorferi B31 and B. Afzelii ACAI indicating that 66 kDa protein is highly conserved among Lyme disease associated borreliae.
These data indicate that 66 kDa protein is unique among Lyme disease borreliae. Conversely, it was shown recently that the ospC gene homologues and OspC-related proteins are present in Borrelia species not associated with Lyme borreliosis (Marconi et al. 1993).
EXAMPLE In vitro growth inhibition of borreliae by antibodies against the 66 kDa protein The in vitro growth inhibition of borreliae by antibodies against the 66 kDa protein was performed as described elsewhere (Sadziene et al. 1993). Briefly, borreliae were grown to the concentration of approximately 108 cells/ml, as counted in a Petroff-Hauser chamber by phase-contrast microscopy. The concentration of the cells was adjusted to 2 x 107 cells/ml by adding fresh medium. 100 il of the diluted culture was placed in flat-bottomed wells of 96-well microtiter plates, and the rabbit antiserum against the 66 kDa protein prepared as described above diluted twofold in BSK II medium was added. The serum obtained from the same rabbit before the WO 95/35379 PCT/VS95/07005f6 51 immunization was used for negative control. The plates were then incubated for 72 h at 34 0 C, and the inhibitory titer of, the antiserum was evaluated by comparing the cell counts with the negative control. Complement was inactivated in all sera by heat-treatment at 56 0 C for 30 min.
The effect of the rabbit monospecific polyclonal anti-66 kDa protein antibodies on in vitro growth of borreliae was examined. The growth inhibition, occurring after adding the antibodies into the culture, resulted in reduced cell counts and appearing of mainly not motile spirochaetes, carrying large surface blebs. For all Lyme disease associated Borrelia strains included in the assay, in vitro growth was inhibited by the antibodies against the 66 kDa protein. The inhibitory titer of the antiserum was 1:8, 1:4,and 1:4 for, respectively, B. burgdorferi B31, B. afzelii ACAI, and B. garinii The inhibitory titer of the antiserum was 1:16 when the growth inhibition test was performed on the B. burgdorferi B31 mutant B313 lacking OspA and OspB.
The antiserum raised against the 66 kDa protein of B. garinii Ip90 was able to inhibit the in vitro growth of all three Lyme disease associated Borrelia strains used in the assay.
This further indicates that the 66 kDa protein is highly conserved among Lyme disease associated borreliae and hence is an antigen being a potential vaccine candidate and a diagnostic tool.
EXAMPLE 6.
Isolation and N-terminal amino acid sequencing of the 66 kDa protein.
6.1 Amino acid sequencing.
The Fraction B of strain ACAI of B. afzelii was electrophoresed and transferred to Fluorotrans transfer membrane (Pall, WO 95/35379 PCiU895/07005 52 East Hills, NY). The protein bands were visualized by staining the membrane with 0.1% CB in 50% methanol. After destaining with 50% methanol, the 66 kDa protein band was cut from the membrane and N-terminal amino acid sequence analysis was performed on a 477A sequenator (Applied Biosystems, Foster City, CA) at UmeA University.
N-terminal amino acid sequence of the 66 kDa protein, recovered from the Fraction B of B. afzelii ACAi, is presented, SEQ ID NO: 1.
6.2 Design of o:igonucleotide probe.
The sequence of the 8 amino acid fragment was used to design the oligonucleotide sequence, SEQ ID NO: 2. The choice of A and T nucleotides in the wobble positions was reasoned by the preferential utilisation of codons with A and T nucleotj’.es in Borrelia genome (Burman et al. 1990).
EXAMPLE 7.
Preparation of Bb DNA libraries.
7.1 Extraction of DNA.
The spirochaetes harvested from 400 ml of culture, were washed twice with 50 mM Tris-HC1 (pH=7.4) and resuspended in ml of buffer containing 50 mM Tris-HCl sucrose, and 50 mM EDTA. The cells were lysed by adding SDS to a final concentration of lysozyme (Sigma, St. Louis, MO) (1.5 mg/ml), proteinase K (Sigma, St. Louis, MO) (0.1 mg/ml), and RNAase A (Sigma, St Louis, MO) (10 Ag/ml). The DNA was extracted with buffered phenol and ethanol precipitated.
WO 95/35379 PCT/US95/07665 53 7.2 Construction of a genomic DNA library.
Restriction enzymes were obtained from Boehringer, Mannheim, Germany. 100 ng of borrelial genomic DNA prepared as described above was completely digested using EcoRI, XbaI, and PstI restriction endonucleases separately or in combination. For the partial digestions, 1 U of HindIII restriction endonuclease was incubated with 100 ng of genomic DNA for min. at 37 0 C. Twenty nanograms of appropriately digested pUC18 (Pharmacia, Uppsala, Sweden) vector was used for ligations.
EXAMPLE 8.
Cloning and sequencing of the gene encoding the 66 kDa protein.
8.1 Screening of genomic library prepared from B. garinii The recombinant plasmids were transformed into competent E.
coli Dh5a cells. Initially, B. garinii Ip90 HindIII digested genomic DNA library was screened with the designed degenerated oligonucleotide probe: 5′-GAA AAA GAT ATW TTT AAA ATW AAT-3′ (SEQ ID NO: 2) synthesized on the basis of the N-terminal amino acid sequence of the 66 kDa protein obtained in Example 5 (corresponding to amino acids 6-13). A recombinant plasmid designated (pJB-100) recovered from one positive E. coli clone was sequenced. A gene fragment containing 592 bp including the ATG start codon followed by a discontinued open reading frame (ORF) was identified. The full-length 66 kDa protein gene was retrieved from B. garinii Ip90 EcoRI/XbaI genomic DNA library in the same vector by probing with the radiolabelled 66 kDa protein gene fragment within BamHI and HindIII restriction sites on pJB-100. A recombinant plasmid WO 95/35379 PCT/US95/07665 54 designated pJB-101 derived from another positive E. coli clone, harboured a 4.1 kb DNA insert. The sequencing of the 66 kDa protein gene proceeded until the TAA stop codon was detected. The clones were sequenced by the dideoxy chain termination method, using /y- 35 S/dATP (Amersham, Buckinghamshire, UK) and the Pharmaci. T7 sequencing kit according to the procedure described by the manufacturer (Pharmacia, Uppsala, Sweden). The sequence fragments were assembled using the GENEUS software for VAX computer.
8.2 Screening of genomic library prepared from B. burgdorferi B31 and B. afzelii ACAI.
The 592 bp 66 kDa protein gene fragment within BamHI and HindIII restriction sites was recovered from plasmid preparation and radiolabelled by random primer technique. It was then used to screen B. burgdorferi B31 and B. afzelii ACAI genomic DNA libraries. A recombinant plasmid designated pJB- 102 was found to harbour a 2.4 kb insert comprising a segment of the 66 kDa protein gene from B. burgdorferi B31 lacking the coding sequence for the signal peptide and a recombinant plasmid designated pJB-105 was found to harbour a 1.5 kb insert comprising the DNA encoding the initial Met and the following 17 amino acids. Together with the DNA sequence found in pJB-102, the full sequence encoding the 66 kDa protein from B31 was then established. A recombinant plasmid designated pJB-104 was found to harbour a 10 kb insert comprising the complete 66 kDa protein gene from B. afzelii ACAI. Both strands of the full-length genes coding for the 66 kDa protein in different Lyme disease Borrelia species were sequenced as described above.
8.3 Sequence analysis.
Sequence analyses were performed using the University of Wisconsin GCG Sequence Analysis Software Version 7.2 for VAX computer, MacVector (IBI, Newhaven CT) for Macintosh computers, and PC-Gene (Genofit) for XT/AT personal computers.
WO 95/35379 ‘CT/US95/07665 Search in protein sequence databases was performed at the NCBI using the BLAST network service.
The nucleotide sequence of the 66 kDa protein gene of B.
burgdorferi B31, B. afzelii ACAI, B. garinii Ip90, as well as neighbouring regions are shown in SEQ ID NO: 3, SEQ ID NO: SEQ ID NO: 7, and SEQ ID NO: 13. The ATG start codon was followed by an ORF of 1857, 1860 and 1866 nucleotides for strains B31, ACAI, and Ip90, respectively. A consensus ribosomal binding site (RBS), GGAAGG, could be detected upstream of the start codon. Further upstream, sequences closely resembling the «-10″-region (-TATTAT-) and the (-TTGAAT-) were located at positions -185 and -209, respectively. The B31 clone did not contain the ATG start codon and the sequence coding for the signal sequence, but contained the sequence coding for the complete processed protein. The 66 kDa protein gene terminated at a TAA triplet, which was followed by AT rich region containing putative stem and loop structures.
The deduced amino acid sequence of the 66 kDa protein of B.
burgdorferi B31, B. afzelii ACAI and B. garinii Ip90 is presented in SEQ ID NO: 4 and SEQ ID NO: 14, SEQ ID NO: 6 and SEQ ID NO: 8, respectively. The computer analysis predicted the potential leader peptidase I cleavage site between amino acid residues at position 21, and the N-terminal peak was found on the hydrophobicity plot (data not shown) in all three cases. The processed 66 kDa protein from the strains B31, ACAI and Ip90 consisted of, respectively, 597, 598 and 600 amino acids with a calculated molecular weight of 65,802 kDa, 65,796 kDa and 65.944 kDa. The amino acid sequence of the 66 kDa protein from B. burgdorferi B31 was 92.7% and 91.5% identical to the sequences from, respectively, B.
afzelii ACAI and B. garinii Ip90. When compared with each other, the two latter strains showed 93.9% identity.
The level of similarity and identity between the deduced amino acid sequence of the 66 kDa protein from different WO 95/35379 PCT/US95/07665 56 borrelia strains further shows that this protein can be useful as a vaccine against Lyme disease as well as a target for diagnostic use.
8.4 Antigenicity plot Potential antigenic regions of the deduced amino acid sequences of the 66 kDa proteins from Borrelia burgdorferi sensu stricto B31, Borrelia afzelii ACAI, and Borrelia garinii IP90 were identified by calculation of the antigenic index using the algorithm of Jameson and Wolf (1988). The results are shown in Fig. 5. Proposed epitopic regions having a high antigenic index are e.g. the amino acid sequences corresponding to positions 175-190, 285-305, 365-385, and 465-490.
The 66 kDa proteins were examined for the sequence similarity to other known proteins in database libraries. There were no other sequences related significantly to the 66 kDa proteins.
EXAMPLE 9 Localization of the 66 kDa protein gene.
9.1 Separation of DNA by pulse-field agarose gel electrophoresis.
For the pulse-field AGE, the genomic DNA prepared from B.
burgdorferi B31, B. afzelii ACAI and B. garinii Ip90 was recovered in 1% agarose blocks as previously described (Ferdows and Barbour, 1989). One-dimensional and pulse-field AGE were performed in 0.7% and 1% agarose, respectively, in TBE buffer. For the pulse-field AGE pulse times were 1 s for 9 h and then 5 s for 9 h at a constant current of 180 mA.
WO 95/35379 PCTI/S95/07605 57 9.2 Southern blotting.
Following depurination, denaturation and neutralization of the gels, the DNA was transferred to Hybond-N membrane (Amersham, Buckinghamshire, UK) by the method of Southern (Sambrook et al. 1989), and cross-linked with UV light.
Filters were prehybridized and hybridized for, respectively, 1 h and 4 h, and washed. The temperature was 37 0 C for probing with degenerate oligonucleotide, end-labelled with /y- 32 P/dATP (Amersham, Buckinghamshire, UK), and 55 0 C for probing with DNA fragment, radiolabelled by random primer technique (Amersham, Buckinghamshire, UK).
The hybridizing band corresponded to the position of the 1 Mbp linear chromosome of Lyme disease borreliae, cf. Fig. 3.
There was no significant signs of hybridization with the DNA from relapsing fever Borrelia species, B. hermsii, B. crocidurae, and B. hispanica (data not shown).
Furthermore, the 66 kDa protein gene being localized to the chromosome of borreliae shows a higher degree of conservation among Lyme disease associated borreliae contrary to the plasmid-encoded major outer surface proteins A, B, and C which exhibit a significant species and strain dependent genetic and antigenic polymorphism (Barbour 1986, Jonsson et al. 1992, Wilske et al. 1993).
EXAMPLE Expression of the 66 kDa protein from B. burgdorferi B31 in E. coli.
Two oligonucleotide primers, ATA TTT GCT GCA GCA GAT-3′ SEQ ID NO: 11 CTA AAG GAA TTC TTT TGC-3′ SEQ ID NO: 12 WO 95/35379 PCT/US95/07665 58 were designed to anneal to the 5′ end (devoid of the leader peptide sequence) and the 3’ end of the 66 kDa protein gene from B. burgdorferi B31. The primers contained, respectively, PstI and EcoRI restriction sites, and were used to amplify the 66 kDa protein gene in the PCR. PCR amplification was performed using Ampli-Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT). The PCR product was then treated with the mentioned restriction enzymes, purified by AGA and ligated into the T7 based expression vector pRSET (Invitrogen, San Diego, CA). The recombinant plasmid was then used to transform E. coli BL21 cells. E. coli BL21 cells containing the insert were grown and induced with by adding isopropyl-f-Dthiogalactopyranoside (IPTG) (Sigma, St. Louis, MO) to a final concentration of 1 mM to express the introduced 66 kDa protein gene. The 66 kDa protein gene product was subsequently identified by SDS-PAGE and Western blot with rabbit antiserum raised against the 66 kDa protein. Fig. 4 show the southern blot.
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WO 95/35379 PCT/US95/07665 SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: Symbicom AB STREET: c/o Astra Hassle AB, Tvistev&gen 48 CITY: UmeA COUNTRY: Sweden POSTAL CODE (ZIP): S-907 36 (ii) TITLE OF INVENTION: New 66kDa antigen from Borrelia (iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid
STRANDEDNESS:
TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Borrelia afzelii STRAIN: ACAI (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ala Asp Ala Leu Lys Glu Lys Asp Ile Phe 1 5 10 Pro Asp Phe Gly INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Lys Ile Asn Pro Gly Ile WO 95/35379 PCT/US95/07665 68 (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GAAAAAGATA TW TTAAAAT WAAT 24 INFORMATION FOR SEQ ID NO: 3: Wi SEQUENCE CHARACTERI..CS: LENGTH: 2075 base pairs TYPE: nucleic acid STRAflDEDNESS: single TOPOLOGY: lintear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Borrelia burgdorferi STRAIN. B31 (vii) IMMEDIATE SOURCE: CLONE: pJB-102 (ix) FEATURE: NPAME/KEY: CDS LOCATION:109. .1914 (ix) FEATURE: NAME/KEY: sigpeptide LOCATION:l09. 120 OTHER INFORMATION:/partial /label= partial (ix) FEATURE: NAME/KEY: matjpeptide LOCATION:121. .1911 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCTGGCGGAA GGGGGATGTG CTGCAAGGCG A’ITAAGTTGG GTAACGCCAG GG’FITrCCCA GTCACGACGT TGTAAAACGA, CGGCCAGTGC CAAGCTTGCA TGCCTGCA GCA ATA IT 117 Ala Ile Phe -4 GCA GCA GAC GCA ‘rrA AAG GAA AAA GAT ATA TTTI AAA ATA AAC CCA TGG 165 Ala Ala Asp Ala Leu Lys Glu Lys Asp Ile Phe Lys Ile Asn Pro Trp 1 5 10 ATG CCA ACA ‘FIT GGA ‘IT GAA AAC ACA, AGT GAA TTIC AGA ‘ITA GAT ATG 213 Met Pro Thr Phe Gly Phe Glu Asn Thr Ser Glu Phe Arg Leu Asp Met 25 WO 95/35379 PCTIUS95/07665 GAC GAG CTT GTT CCT GGG TTT GAA AAC AAA AGC AAA ATT ACC ATT AAG Asp Giu Leu Val Pro Gly Phe Glu Asn Lys Ser Lys Ile Thr Ile Lys
CTT
Leu
TCA
Ser
AAA
Lys
AAT
Asn T1T Phe
AGC
Ser
ATT
Ile
TAC
Tyr 160
ACG
Thr
ACA
Thr
AAA
Lys
ACA
Thr
AAA
Lys
GCT
Ala
GGC
Gly
ATG
Met
AAT
Asn
ACT
Thr
CTT
Leu 145
AAA
Lys
GGT
Gly
TAT
Tyr
AAT
Asn
CCT
Pro 225 CCA TIT GAA GCT Pro Phe Giu Ala TAC ATT AAG GTA Tyr Ile Lys Vai GAT CAA TFT AAA Asp Gin Phe Lys 85 TAC GAT TTT TT Tyr Asp Phe Phe 100 AAA GAG TCT TTA Lys Giu Ser Leu 115 TAC TAT GGA TTC Tyr Tyr Giy Phe 130 GCA AGA GGT ACT Ala Arg Gly Thr CTC CCA AAA CTC Leu Pro Lys Leu 165 AAC AGA AAT CAA Asn Arg Asn Gln 180 CAA GGA ATC CTT Gin Gly Ile Ldu 195 CTA CTT GAT CAA Leu Leu Asp Gin 210 TT GAA TTA AAT Phe Giu Leu Asn AAT CCC Asn Pro 55 GAA GAT Glu Asp 70 ATT GAC Ile Asp ATT AAA Ile Lys ITr AGT Phe Ser CCA AGC Pro Ser 135 TCT AAA Ser Lys 150 GAC CTT Asp Leu GAG AAT Glu Asn TAT GGA Tyr Gly AAC GAA Asn Glu 215 ‘FFT GGC Phe Gly 230 GAA TTA GGC AAA GAC Glu Leu Gly Lys Asp CT GCA CTA AAA GCG Leu Ala Leu Lys Ala GTG GGA GAT ATT ACA Vai Gly Asp Ile Thr 90 ATA AGT ACT ATG ACA Ile Ser Thr Met Thr 105 TTT GCA CCT ATG ACT Phe Ala Pro Met Thr 120 AAT GAT AGG GCA GTA Asn Asp Arg Ala Val 140 AAC ATA GGA ACA ATT Asn Ile Giy Thr Ile 155 ACA ‘IT GCA ATA GGG Thr Phe Ala Ile Gly 170 GAC AAA GAC ACT CCA Asp Lys Asp Thr Pro 185 A’T CAA GCA ACA TGG Ile Gin Ala Thr Trp 200 GAT ACT AAA TCT GTA Asp Thr Lys Ser Vai 220 TTG TCA GGA GCC TAT Leu Ser Gly Ala Tyr 235
GAT
Asp
GAA
Glu
GCC
Ala
GAT
Asp
GGA
Gly 125
AGA
Arg
GAG
Gin
GGA
Gly
TAC
Tyr
AAA
Lys 205
ATT
Ile
CCA
Pro
GGC
Gly
CAA
Gin
‘ITT
Phe 110
TTT
Phe
GGG
Gly
CTG
Leu
ACA
Thr
AAT
Asn 190
CCA
Pro
GCA
Ala
‘TC
Phe
AAA
Lys
ATC
Ile
GAC
Asp
AAA
Lys
ACA
Thr
GGA
Gly
GGC
Gly 175
AAA
Lys
ATA
Ile
GAA
Glu 309 357 405 453 501 549 597 645 693 741 789 GGA AAC GAG Gly Asn Glu ACA TTC AAT AAT TCA TCA ATA ACA TAC TCT TTA AAA GAT AAA TCC GTA Thr 240 Phe Asn Asn Ser Ser Ile Thr Tyr Ser Leu 245 250 Lys Asp Lys Ser Val 255 WO 95/35379 PTU9176 PC’r/US95/07665 GTT GGC AAC GAT Val Gly Asn Asp TTA TTG Leu Leu 260 AGO CCA ACT Ser Pro Thr TOA AAT TOT Ser Asn Ser GOA TOT TFI’ Ala Ser Phe AAA AAT ACC Lys Asn Thr 290 GGA GOT Gly Ala 275 AAA TAT AAG LYS Tyr LYS GGA TTA ACA AAA Giy Leu Thr Lys GCA ATT TTA Ala Ile Leu 270* ATA AAO GAT Ile Asn Asp 285 GGA ATA GAT Gly Ile Asp TAT OTT ATT TTG Tyr Leu Ile Leu CAA ATG Gin Met 295 GGA AOT GAT Gly Thr Asp Trr Phe 300 1029 COT TIT GOA AGO Pro Phe Ala Ser 305 GAT TTT TOT ATA TIT GGA CAC ATO Asp Phe Ser Ile Phe Gly His Ile 310 315 TOA AAA GOCA GOG Ser Lys Ala Ala AAA GOT GAA ATA Lys Ala Giu Ile 1077
AAT
Asn 320 TTO AAA AAA GAA Phe Lys Lys Giu
ACA
Thr 325 000 TCA GAT OCT Pro Ser Asp Pro AAO AAA Asn Lys 330 1125 TI’r GAT OCA AAT Phe Asp Pro Asn GGO AAT GOT OTT Gly Asn Ala Leu 340 TCA ACA GGA GCA Ser Thr Gly Ala AAT TTO AGO Asn Phe Ser 345 AAA AAO ACA Lys Asn Thr GAA TTG Giu Leu 350 1173 1221 GGO ATT GOA Gly Ile Ala GAT ACC GGT Asp Thr Giy 370 TTr Phe 355
AGT
Ser 360 ATA GGT TFI’ GOT Ile Gly Phe Ala TGG AAT AAA Trp Asn Lys 365 GAA AAA GAA TOO TGG GOG ATT.XAA. GGA TOT GAT TOO TAO Giu Lys Giu Ser Trp Ala Ile Lys Gly Ser Asp Ser Tyr 375 380 AGT ACA Ser Thr 385 GGA ATA Giy Ile 400 AGA OTO TTT GGA Arg Leu Phe Gly AGO TAT GGA CAA Ser Tyr Gly Gin 405 GAA CAA GAO AAA AAA TOT GGA GTT GOA TITG Giu Gin Asp Lys Lys Ser Gly Vai Ala Leu 390 395 AAO OTT TAO AGA TOT AAA SAT ACA GAA AAA Asn Leu Tyr Arg Ser Lys Asp Thr Giu Lys 1269 1317 1365 AGA TTA AAA ACC Arg Leu Lye Thr TOT GAA AAT GCA TTT CAA AGO TTA AAT Ser Giu Asn Aia Phe Gin Ser Leu Asn 425 GTT GAA Val Giu 430 1413 ATT TOA AGO Ile Ser Ser TGG ATA ACA Trp Ile Thr 450 GAA GAO AAO Giu Asp Asn AAA AAA GGG Lye Lye Giy 440 TAO GAT ATT Tyr Asp Ile 455 ATT ATA AAT Ile Ile Asn GGA TTA GGA Giy Leu Gly 445 1461 TOT ATO GGT OTT Ser Ile Gly Leu TTA AGA CAA AAA TOT GTA Leu Arg Gin Lys Ser Vai 460 1509 GAA AA Giu Asn 465 TAT COT ACA ACA Tyr Pro Thr Thr
ATT
Ile 470 TOA AGO ACC AOT Ser Ser Thr Thr
GAA
Giu 475 AAO AAT CAA AOT Asn Asn Gin Thr 1557 WO 95135379 GAA CAA AGT Glu Gin Ser 480 GAA GAT GCA Glu Asp Ala CCA ATA GCA Pro Ile Ala GCA TAC ATT Ala Tyr Ile 530 ATT TAT TTA Ile Tyr Leu 545 ACA ATT TCT Thr Ile Ser 560 AAA AAC ACA Lys Ann Thr ATA GCC TAC PCT/US95/07665 TCA ACA Ser Thr ATG AAA Me.t Lys 500 TCC ATT Ser Ile 515 TI’A GGA Leu Gly AAA ACA Lys Thr CTT GGA Lenl Gly AAT AAT Asn Asn 580 AGC GGA ACA AAG Thr Lys GGC TTG Gly Leu ACA GAA Thr Glu TCT AAT Ser Asn 535 CTT AGC Leu Ser 550 GAT TCA Asp Ser GCT ATT
ACC
Thr
GCC
Ala
GCA
Ala 520
AAA
Lys
CTT
Leu
AAT
Asn
GGA
ACA ACC Thr Thr 490 1TA TAT Leu Tyr 505 TAT GTA Tyr Val CTC TCA Leu Ser GAA AAA Glu Lys AAC ATT Asn Ile 570 AGT GCT CTG ACA Leu Thr TAT GCA Tyr Ala 510 TAC AT Tyr Ile 525 GCT ACA Ala Thr AGA TTT Arg Phe CTT GCT Leu Ala CAA TTC
TIT
Phe 495
A’IT
Ile
GGA
Gly
AAA
Lys
ACA
Thr
AAT
Asn 575
AAA
1605 1653 1701 1749 1797 1845 1893 Ala Ile Gly Ser Ala Phe Leu Gin Phe Lys 585 590 TAA CAGCAAAAGA AGGGCTITGI CCCTCTITT 1944 Ile Ala Tyr Ser Gly Ser 595 TTATC ITrAA AAACAATTGG GATACCTTA TATITCITC CITGCAAATT IT rCATAAG CATCITGAAT TITTATAAAT TTATCATTTG CATCTTFTG TCTACAGGA TCATITGCAA ACTTATCAGG A INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 601 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ala Ile Phe Ala Ala Asp Ala Leu Lys Giu Lys Asp Ile Phe Lys Ile -4 1 5 Asn Pro Trp Met Pro Thr Phe Gly Phe Giu Asn Thr Ser Glu Phe Arg is 20 Leu Asp Met Asp Giu Leu Val Pro Giy Phe Giu Asn Lys Ser Lys Ile 35 2004 2064 2075 WO 95/35379 PCT/U$95/07665 72 Thr Ile Lys Leu Lys Pro Phe Giu Ala Asn Pro Giu Leu Gly Lys Asp Asp Glu Ala Asp Gly 125 Arg Gin Giy Lys 205 Ile Giy Lys Ala Ile 285 Giy Lys Pro Giy Gin Phe 110 Phe Gly Leu Thr Asn 190 Pro Ala Asn Ser Ile 270 Asn Ile Ala Pfie Ser Lys L~ys Ile Asn Asp Phe Lys Ser Thr Ile Giy Tyr 160 Gly Thr 175 Lys Thr Ile Lys Giu Thr Glu Thr 240 Val Val 255 Leu Ala Asp Lys Asp Pro Ala Asn s0 Ala Tyr Gly Asp Met Tyr Asn Lys Thr Tyr 130 Leu Ala 145 Lys Leu Giy Asn Tyr Gin Asn Leu 210 Pro Phe 225 Phe Asn Gly Asn Ser Phe Asn Thr 290 Phe Ala 305 Phe Lys Ile Gin Asp Giu Tyr Arg Pro Arg Gly 195 Leu Giu Asn Asp Gly 275 Tyr Ser Lys Val Lys 85 Phe Leu Phe Thr Leu 165 Gin Leu Gin Asn Ser 245 Leu Lys Ile Phe Thr Glu 70 Ile Ile Phe Pro Ser 150 Asp Glu Tyr Asn Phe 230 Ile Ser Tyr Leu Ser 310 Pro 55 Asp Asp Lys Ser Ser 135 Lys Leu Asn Giy Giu 215 Gly Thr Pro Lys Gin 295 Ile Ser Leu Vai Ile Phe i2 0 Asn Asn Thr Asp Ile 200 Asp Leu Tyr Thr Leu 280 Met Phe Asp Ala Gly Ser 105 Ala Asp Ile Phe Lys 185 Gin Thr Ser Ser Leu 265 Gly Gly Gly Pro Leu Lys Asp Ile Thr Met Pro Met Arg Ala Gly Thr 155 Ala Ile 170 Asp Thr Ala Thr Lys Ser Giy Ala 235 Leu Lys 250 Ser Asn Leu Thr Thr Asp His Ile 315 Asn Lys 330 Ala Thr Thr Thr Val 140 Ile Gly Pro Trp Val 220 Tyr Asp Ser Lys Phe 300 Ser Lys 320 325 Ala Giu Ile Phe Asp Pro Asn Gly Asn Ala Leu Asn Phe Ser Lys Asn 335 340 345 WO 95/35379 73 Thr Giu Leu Gly Ile Ala Phe Ser Thr Gly Ala PCTIVS95/07665 Ser Ile Gly Phe Ala 350 355 360 Trp 365 Asp Val Thr Asn Gly 445 Lys Asn Leu Tyr 525 Ala Arg Leu Gin Asn Ser Ala Giu Val 430 Leu Ser Gin Thr Ala 510 Ile Thr Phe Ala Phe 590 Asp Ser Gly 400 Arg Ile Trp Giu Giu 480 Giu Pro Ala Ile Thr 560 Lys Ile Thr Thr 385 Ile Leu Ser Ile Asn 465 Gin Asp Ile Tyr 545 Ile Asn Ala Giy 370 Arg Ser Lys Ser Thr 450 Tyr Ser Ala Ala Ile 530 Leu Ser Thr Giu Leu Tyr Thr Tyr 435 Ser Pro Ser Met Ser 515 Leu Lys Leu Asn Ser 595 Lys Phe Gly Ile 420 Giu Ile Thr Thr Lys 500 Ile Gly Thr Giy Asn 580 Gly Giu Gly Gin 405 Ser Asp Gly Thr Ser 485 Leu Ser Pro Gly Trp 565 Aia Ser Ser Trp 375 Giu Gin 390 Asn Leu Giu Asn Asn Lys Leu Tyr 455 Ile Ser 470 Thr Lys Giy Leu Thr Giu Ser Asn 535 Leu Ser 550 Asp Ser Aia Ile Ala Ile Asp Lys Tyr Arg Ala Phe 425 Lys Gly 440 Asp Ile Ser Thr Thr Thr Ala Leu 505 Ala Tyr 520 Lys Leu Leu Giu Asn Asn Giy Ser 585 Lys Lys Ser 410 Gin Ile Leu Thr Thr 490 Tyr Val Ser Lys Ile 570 Ala Giy Ser 395 Lys Ser Ile Arg Giu 475 Pro Leu Vai Ser Leu 555 Ile Phe Ser 380 Gly Asp Leu Asn Gin 460 Asn Asn Asp Pro Asp 540 Ile Giu Leu INFORMA.TION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 2264 base pairs TYPE: nucleic acid STRANfDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 95/35379 PCT/US9.5/07605 74 (vi) ORIGINAL SOURCE: ORGANISM: Borrelia afzeii STRAIN: ACA-I (vii) IMMDIATE SOURCE: CLONE: pJB-104 (ix) FEATURE: NAME/KEY: CDS LOCATION:303. .2162 (ix) FEATURE: NAME/KEY: sigpeptide LOCATION:303. .365 (ix) FEATURE: NAME/KEY: matpeptide LOCATION:366. .2159 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCAAAAACAA TAACTTACGC TTTATACTAC ATICTAGCAA CAGGA’ITACT GGTTi’ATTTA GTATAAAT1’A ATCATI’TAAA ATAAATAAGA TrAGTTGAC.A ATACAATI’AA TCTrATITAT 120 AAA’ITTGAAT AGTATAAAAT CACAAATACC AATATGATAT TGAATTIT=A TCTAATAG’IT 180 TTAATATI’GT ATACATGTTA ‘ITATGTACAA TAAGTAATAT GTA’ITATATA TATA’rrATTA 240 AGACGTI’AA AAAATAACTA AAACTAATAA AAAGTTTATA GTI’ACAACAG GAAGGTATAA 300 TTr ATG AAA AAT CAT ATT TTA TAT AAA ‘ITA ATT ATA TIT TFIA ACC ACA 347 Met Lys Ann His Ile Leu Tyr Lys Leu Ile Ile Phe Leu Thr Thr -21 -20 -15 TCT GCA GCA ATA Tr GCA GCA. GAC GCA TI’A AAG GAA AAA GAT ATA IT 395 Ser Ala Ala Ile Phe Ala Ala Asp Ala Leu Lys Giu Lys Asp Ile Phe 1 5 AAA ATA AAC CCG TGG ATA CCG AC-A TIT GGA Trr GAA AAC ACA AGT GAA 443 Lys Ile Asn Pro Trp Ile Pro Thr Phe Gly Phe Glu Asn Thr Ser Giu 20 TTC AGA TTT GAT ATG GAT GAA CTT GTC CCT GGG TFT GAA AAC AAA AGT 491 Phe .Arg Phe Asp Met Asp Glu Leu Val Pro Gly Phe Glu Asn Lys Ser 35 AAA A’IT ACT A’IT AAA CTT AAA CCA TIT GAA ACT AAT CCA GAA TTA GGC 539 Lys Ile Thr Ile Lys Leu Lys Pro Phe Glu Thr Asn Pro Glu Leu Giy 50 AAA GAC GAT CCA Ti’T TCA GCT TAC ATT AAA GTG GAA GAT CTIT GCA TTA 587 Lys Asp Asp Pro Phe Ser Ala Tyr Ile Lys Val Glu Asp Leu Ala Leu 65 WO 95/35379 PCT/US95/07665 AAA GOA GAA GGC AAA AAA GAO GCT CAA TTC AAA ATC GAT GTA GGA GAT Lys Ala Giu Gly Lys Lys Asp Ala Gln Phe Lys Ile Asp Val Gly Asp 80 85 ATA ACA GCC CAA ATT AAT Ile Thr Ala G2n Ile Asn ATA TAO GAT TTT TIW ATT AAA ATA AGT ACT Ile Tyr Asp Phe Phe Ile Lys Ile Ser Thr 100 105 ATG ACG GAT Met Thr Asp ATG ACT GGA Met Thr Gly 125 GAO TTT AAT AAA Asp Phe Asn Lys TCT TTA TT AGO Ser Leu Phe Ser ‘ITT GOG OCT Phe Ala Pro 120 AAT GAT AGA Asn Asp Arg TTC AAA AGO ACT Phe Lys Ser Thr TAT GGA TTC Tyr Gly Phe OCA AGT Pro Ser 135 GCA GTA Ala Val 140 AGA GGG ACA ATT Arg Gly Thr Ile GCA AGA GGT ACT Ala Arg Gly Thr
TCT
Ser 150 AAA AAO ATA GGA Lys Asn Ile Gly ATT CAA OTG GGA Ile Gin Leu Gly AAA CTC CCA CAA Lys Leu Pro Gin
ATO
Ile 165 GAC CTT ACA TTr Asp Leu Thr Phe
GCA
Ala 170 ATA GGA GGA ACA Ile Gly Gly Thr
GGO
Gly 175 ACA GGT AAT AGA Thr Giy Asn Arg
AAT
Asn
ISO
CAA GAG AAT GAO Gin Giu Asn Asp AAA GAO Lys Asp 185 ACT OCA TAO Thr Pro Tyr ACA TOG AAG Thr Trp Lys 205
AAT
Asn 190 AAA ACC TAT CAA Lys Thr Tyr Gin GGA ATO Gly Ile 195 OTT TAT GGA Leu Tyr Gly ATT CAA GCA Ile Gin Ala 200 GAT ACT CAA Asp Thr Gin OCA ATA AAA AAT Pro Ile Lys Asn
ATA
Ile 210 OTT GAT CAA AAC Leu Asp Gin Asn
GAA
Glu 215 1019 TCT GTA Ser Val 220 ATT GOA GAA ACA Ile Aia Giu Thr
OCT
Pro 225 TTT GAA TTA AAO Phe Giu Leu Asn
TTT
Phe 230 GGC TTA TOA GGA Gly Leu Ser Gly
GCT
Ala 235 TAT GGA AAT GAA Tyr Gly Asn Glu
ACA
Thr 240 TTC AAT AAT TCA Phe Asn Asn Ser ATA ACA TAO TCT Ile Thr Tyr Ser
TTA
Leu 250 1067 1115 1163 AAA GAT AAA TCC Lys Asp Lys Ser OTA ATT Leu Ile 255 GGT AAC GAT Gly Asn Asp TTA AGC CCA ACT Leu Ser Pro Thr TTA TCA Leu Ser 265 AAT TOT GCA Asn Ser Ala ACA AAA ATO Thr Lys Ile 285
ATT
Ile 270 TTG GCA TOT ‘nIT Leu Ala Ser Phe GCT CAA TAT Ala Gin Tyr AAG OTT GGA TTA Lys Leu Gly Leu 280 CAA ATO GOT ACT Gin Met Gly Thr 295 1211 1259 AAT AAT AAA AAT Asn Asn Lys Asn TAT OTT ATT TTA Tyr Leu Ile Leu WO 95/35379 PCT/US95/07665 GAT TT Asp Phe 300 GGA ATA GAT CCT Gly Ile Asp Pro GCA AGC GAT TTT Ala Ser Asp Phe GTA TIT GGA CAC Val Phe Gly His 1307
ATC
Ile 315 TCA AAA GCA Ser Lys Ala GCA AAT Ala Asn 320 TTG AAA AAA GGA ATA TCT TTA OAT CCT Leu Lys Lys Gly Ile Ser Leu Asp Pro 325
AGT
Ser 330 1355 AAA AAA GCC Lys Lys Ala GAG GAT Glu Asp 335 ATA T=T GAT Ile Phe Asp CCA AAT Pro Asn 340 GGC AAT GCC CT Gly Asn Ala Leu AAT TTC Asn Phe 345 1403 AAT AAA AAT ACA Asn Lys Asn Thr 350 GAA CTA GGC ATT Glu Leu Gly Ile TTT TCA ACA OGA Phe Ser Thr Gly GCA AGC ATA Ala Ser Ile 360 TGG AAA GTT Trp Lys Val 1451 1499 GGG CIT GCT Gly Leu Ala 365 TGG AAT AAA Trp Asn Lys GAC OAC Asp Asp 370 GGT OAA AAA OAA Oly Glu Lys Glu
TCT
Ser 375 AAA GGA Lys Gly 380 TCT GAT TCC TAC Ser Asp Ser Tyr AGT ACA AGA CTA TTT GGA Ser Thr Arg Leu Phe Oly 385 390 OAA CAA OAC AAA Glu Gin Asp Lys
-,AA
Lys 395 TCT OGA GTT GCA Ser Gly Val Ala
TTA
Leu 400 OGA ATA AGC TAT Gly Ile Ser Tyr
GGG
Gly 405 CAA AAT CTT TAC Gin Asn Leu Tyr
AGA
Arg 410 TCT AAA OAT ACA Ser Lys Asp Thr
GAA
Glu 415 AAA AGA TTA AAA Lys Arg Leu Lys
ACC
Thr 420 ATA TCT GAA AAT Ile Ser Glu Asn OCA =rr Ala Phe 425 CAA AGC TTA Gin Ser Leu CTT ATO AAT Leu Met Asn 445 GTT GAA ATT TCA Val Giu Ile Ser
AGC
Ser 435 TAT OAA OAC AAT AAA AAG GGG Tyr Giu Asp Asn Lys Lys Gly OGA CTG GOT TOG ATA ACA TCT ATC Gly Leu Gly Trp Ile Thr Ser Ile GGT CTT Gly Leu 455 TCA ACC Ser Thr 470 TAT OAT ATT Tyr Asp Ile TTA AGT OCT Leu Ser Ala 1547 1595 1643 1691 1739 1787 1835 1883 1931 TTA AGA Leu Arg 460 AAT GAO Asn Glu 475 CAA AAA TCT OTA Gin Lys Ser Val AAC TAT CCT ACA Asn Tyr Pro Thr AAC AAT CAA Asn Asn Gin
OCT
Ala 480 OGA CAA AGT TCA Gly Gin Ser Ser
ACA
Thr 485 GGC ACA CAA GCC Oly Thr Gin Ala
ATA
Ile 490 ACA CCT AAT CTA Thr Pro Asn Leu
ACA
Thr 495 TT GAA OAC OCA Phe Glu Asp Ala
ATG
Met 500 AAA CTA GGC ATA Lys Leu Giy Ile OCT TTA Ala Leu 505 TAT CTT GAT Tyr Leu Asp OCA ATT CCA ATA OAA TCC ATT TCA ACA Ala Ile Pro Ile Glu Ser Ile Ser Thr GAA OCA TAT Glu Aia Tyr 520 WO 95/35379 PCT/US95/07665 77 GTA GTA CCA TAT ATT GGA GCA TAC CTT TTA GGA CCT TCT AAT AAA ATA 1979 Val Val Pro Tyr Ile Gly Ala Tyr Leu Leu Gly Pro Ser Asn Lys Ile 525 530 535 TCA AGC GAT GCT ACA AAA ATT TAT TTA AAA ACA GGA CTT AGT CTT GAA 2027 Ser Ser Asp Aa Thr Lys Ile Tyr Leu Lys Thr Gly Leu Ser Leu Glu 540 545 550 AAA CTA ATA AGA TT ACA ACA ATT TCT CTT GGA TGG GAT TCA AAT AAT 2075 Lys Leu Ile Arg Phe Thr Thr Ile Ser Leu Gly Trp Asp Ser Asn Asn 555 560 565 570 ATT ATA GAA CTT GCT AAT AAA AAC GCA AAT AAT GCT GCT ATT GGC AGT 2123 Ile Ile Glu Leu Ala Asn Lys Asn Ala Asn Asn Ala Ala Ile Gly Ser 575 580 585 GCT TTC TTG CAA TTC AAA ATA GCC TAC AGC GGA AGC TAA CAGCAAAAGA 2172 Ala Phe Leu Gin Phe Lys Ile Ala Tyr Ser Gly Ser 590 595 AGGGCCAAAA GCCCTTCTTT TTTATCTTTA AAAACAAATT AATCAATTAA TTACTTAATA 2232 TTTCTTTCTT’r TGCAAATCPT TTCATAAGCA TC 2264 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 619 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Lys Asn His Ile Leu Tyr Lys Leu Ile Ile Phe Leu Thr Thr Ser -21 -20 -15 Ala Aia Ile Phe Ala Ala Asp Ala Leu Lys Glu Lys Asp Ile Phe Lys 1 5 Ile Asn Pro Trp Ile Pro Thr Phe Gly Phe Glu Asn Thr Ser Glu Phe 20 Arg Phe Asp Me: Asp Glu Leu Val Pro Gly Phe Glu Asn Lys Ser Lys 35 Ile Thr Ile Lys Leu Lys Pro Phe Glu Thr Asn Pro Glu Leu Gly Lys 50 Asp Asp Pro Phe Ser Ala Tyr Ile Lys Val Giu Asp Leu Ala Leu Lys 65 70 Ala Glu Gly Lys Lys Asp Ala Gin Phe Lys Ile Asp Val Gly Asp Ile 85 Thr Ala Gin Ile Asn Ile Tyr Asp Phe Phe Ile Lys Ile Ser Thr Met 100 105 wmmi WO 95/35379 Thr Asp Phe Asp 110 Thr Gly Phe Lys 125 Val Arg Gly Thr 140 Ile Gin Leu Giy Gly Gly Thr Gly 175 Pro Tyr Asn Lys 190 Trp Lys Pro Ile 205 Val Ile Ala Giu 220 Tyr Gly Asn Giu Asp Lys Ser Leu 255 Ser Ala Ile Leu 270 Lys Ile Asn Asn 285 Phe Gly Ile Asp 300 Ser Lys Ala Ala Lys Ala Giu Asp 335 Lys Asn Thr Glu 350 Leu Ala Trp Asn 365 Gly Ser Asp Ser 380 PCIUS95/07605 Phe Ser Ile 160 Thr Thr Lys Thr Thr 240 Ile Ala Lys Pro Asn 320 Ile Leu Lys Tyr Asn Thr Leu 145 Lys Gly Tyr Asn Pro 225 Phe Gly Ser Asn Phe 305 Leu Phe Gly Asp Ser 385 Ly3 Giu 115 Tyr Tyr 130 Ala Arg Leu Pro Asn Arg Gin Gly 195 Ile Leu 210 Plie Giu Asn Asn Asn Asp Phe Gly 275 Thr Tyr 290 Ala Ser Lys Lys Asp Pro Ile Ala 355 Asp Gly 370 Thr Arg Ser Gly Gly Gin Asn 180 Ile Asp) Leu Ser Leu 260 Ala Leu Asp Gly Asn 340 Phe Giu Leu Leu Phe Thr Ile 165 Gin Leu Gin Asn Ser 245 Leu Gin Ile Phe Ile 325 Gly Ser Lys Phe *Phe Pro Ser 150 Asp Giu Tyr Asn Phe 230 Ile Ser Tyr Leu Ser 310 Ser Asn Thr Giu Gly 390 Ser Ser 135 Lys Leu Asn Gly Glu 215 Gly Thr Pro Lys Gin 295 Val Leu Ala
G
1 y Ser 375 Glu Phe 120 Asn Asn Thr Asp Ile 200 Asp Leu Tyr Thr Leu 280 Met Phe Asp Leu Ala 360 Trp Gin Ala Pro Asp Arg Ile Gly Phe Ala 170 Lys Asp 185 Gin Ala Thr Gin Ser Gly Ser Leu 250 Leu Ser 265 Gly Leu Gly Thr Gly His Pro Ser 330 Asn Phe 345 Ser Ile Lys Val Asp Lys Met Ala Thr 155 Ile Thr Thr Ser Ala 235 Lys Asn Thr Asp Ile 315 Lys Asn Gliy Lys Lys 395 Ser Gly Val Ala Leu Gly Ile Ser Tyr Gly Gin Asn Leu Tyr Arg Ser 400 405 410 WO 95/3537! Lys Asp %’CTIUS9,1107665 Thr Giu Lys Arg Lou Lys Thw Ile 8cr Giu Asn Ala Phe Gin 415 Met Arg 460 Giu Pro Leu Vai Ser 540 Leu Ile Phe Leu Asn 445 Gin Asn Asn Asp Pro 525 Asp Ile Giu Leu Asn 430 Gly Lys Asn Leu Tyr 510 Tyr Ala Arg Leu Gin 590 Vai Lou Ser Gin Thr 495 Ala Ile Thr Phe Ala 575 Phe Giu Gly Val Ala 480 Phe Ile Giy Lys Thr 560 Aen Lys Ile Ti-p Giu 465 Gly Glu Pro Ala Ile 545 Thr Lys Ser Ile 450 Aen Gin Asp Ile Tyr 530 Tyr Ile Asn Ser 435 Thr Tyr Ser Aia Giu 515 Leu Leu Ser Ala Tyr Ser Pro Ser Met 500 5cr Lou Lye Lou Asn.
580 Giu Ile Thr Thr 485 Lye Ile Gly Thr Gly 565 Asn Asp Giy Ser 470 Gly Leu 5cr Pro Giy 550 Trp, Ala Asn Leu 455 Thr Thr Gly Thr Ser 535 Leu Asp Ala 425 Lye Lye 440 Tyr Asp Lou Ser Gin Ala Ile Ala 505 Giu Ala 520 Asn Lye Ser Lou Ser Aen Ile Gly 585 Gly Ile Ala Ile 490 Lou Tyr Ile Giu Asn 570 Ser Leu Lou Asn 475 Thr Tyr Val Ser Lys 555 Ile Ala Ile Ala Tyr Ser Giy 595 INFOR~MATION FOR SEQ ID NO: 7: (i)SEQUENCE CHARACTERISTICS: LENGTH: 2547 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Borriia garinii STRAIN: 1p90 (vii) IMMEDIATE SOURCE: CLONE: (ix) FEATURE: NAME/KEY: CDS LOCATION:380. .2245 WO 95135379 PTU9/76 PCT/lUS95/07665 (ix) FEATUR.E: NAME/KEY: sigpeptide LOCATION:380. .442 (ix) FEATURE: NAME/KEY: zatypeptide LOCATION:443. 2242 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AAGCTITTGT CAAAAACAAT ACCITACGCT TTATACTACA ‘ITCTAGCAAC AGGATTGCTA GTI’IATITAG TATAAATrAA TCATITAAAA TAAATAAGAT TAATTrACAA TAAAATrAAT C~r’I~ATAGATTTGAATA ATATAAAAAT CATAAAATAA TAATATGATC TGAATI7TT ACCTAATATT TTAATATTAT ATACATGTTA TATATATAT1’ ATrATATGCA TAATAGCATG TATATAATAT A’ITTTAGCAT AATAGCMTGT ATATAATATA ITTTAGCATA ATAGCATGTA TATAATATAT ‘rrTATTAATG CGTTAATAA ATAACTAGAA CTAATAAAAA GTITATAGTr ACAACAGGAA GGTATAATT ATG AAA .AAT CAT ATT TI’A TAT AAA TTA ATT ATA Met Lys Asn His Ile LeU Tyr Lys Leu Ile Ile -21 -20 IT ‘IWA ACT ACA TCT GTA GCA ATA TIT GCA GCA GCA GAT AAA TTA AAG Phe Leu Thr Thr Ser Val Ala Ile Phe Ala Ala Ala Asp Lys Leu Lys -s 1 GAA GAA GAT ATA TTT AAA ATA AAT CCA TGG ATA CCT ACA TTT GGA ATT Giu Giu Asp Ile Phe Lys Ile Asn Pro Trp Ile Pro Thr Phe Gly le 120 180 240 300 360 412 460 GAA AAC AC-A AGT Giu Asn Thr Ser TTT GAA AAC AAA Phe Glu Asn Lys GAG TI’C AGA CTT GAT ATG GAT GAG Glu Phe Arg Leu Asp Met Asp Giu G7T CCT GGA Vai Pro Gly AGC AAA ATT Ser Lys Ile AAT CCC Asn Pro GAA GAT Giu Asp GAA TI’A GGC AAZ AC Giu Leu Gly Lys Asp 60 CTT GCA TTA AAA GCG Lau Ala Leu Lys Ala ACT ATI’ AAA CTT Thr Ile Lys Leu GAC CCA TTC TCA Asp Pro Phe Ser 65 GAA GGT AAA AAA Glu Gly Lys Lys so CCA TIT GAA GTT Pro Phe Glu Val TAC ATT AAG GTA Tyr Ile Lys Vai ATI’ GAC GTA Ile Asp Val GGA GAC ATA ACA GCC CAA ATI’ AAT Gly Asp le Thr Ala Gin Ile Aen 95 GGG GAT CCA TTr AAA Gly Asp Pro Phe Lys ATA TAC GAT TIT TIT Ile Tyr Asp Phe Phe 100 A7AT AAA GAA TCT TrA Asn Lys Giu Ser Leu 115 ATT AAG ATA AGC ACT ATG ACM GAT TIT GAC TT Ile Lys Ile Ser Thr Met Thr Asp Phe Asp Phe 105 110 WO 95135379 TFI AGT TIT GCG CCC Phe Ser Phe Ala Prc 120 CCA AGC AAA GAC AGz Pro Ser Lys Asp Arc 135 TCT AAA AAC ATA GGP Ser Lys Asn Ile Gly 155 GAC CTT ACA TTT GCA Asp Leu Thr Phe Ala 170 GAG AAT GAC AAA GAC Glu Asn Asp Lys Asp 185 TAT GGG GTT CAA GCA Tyr Gly Val Gin Ala 200 AAC GAA GAT AAT CGA Asn Glu Asp Asn Arg 215 TTT GGC TTA TCA GGA Phe Gly Leu Ser Gly 235 ATA ACA TAC TCT TTA Ile Thr Tyr Ser Leu AGT CCA ACT TTA TCA Ser Pro Thr Leu Ser 265 TAT AAG CIT GGA TTA Tyr Lys Leu Giy Leu 280 TTA CAA ATG GGT ACC Leu Gin Met Gly Thr 295 TCT GTA TIT GGA CAC Ser Val Phe Gly His PCT1US95107665 ATG ACC GGA TTC AAA Met Thr Giy Phe Lys 125 L ATA GTA AGA GGA ACA f Ile Val Arg Gly Thr 140 ACA ATT CAA ATG GGA Thr Ile Gin Met Gly 160 ATA GGG GGA ACA GGC Ile Gly Giy Thr Gly 175 ACT CCA TAC AAT AAA Thr Pro Tyr Asn Lys 190 ACA TGG AAG CCA ATA Thr Trp Lys Pro Ile 205 TCT GTA ATT GCA GAA Ser Val Ile Ala Glu 220 GCT TAT GGA AAT AAA.
Ala Tyr Gly Asn Lys 240 AAA GAT AAA TCT GTA I Lys Asp Lys Ser Val 255 AAT TCT GCA ATI TTA Asn Ser Ala Ile Leu 270 ACA AAA ATC AAC AAT Tffr Lys Ile Asn Asn 285 GAT TrT GGA ATA GAT Asp Phe Gly Ile Asp I 300 ATC TCA AAA GCA GCA 2 Ile Ser Lys Ala Ala 2 320
AGC
Ser A’Il le 145
TAC
Tyr
ACA
Thr
ACC
Thr
AAA
Lys
ACA
Thr 225
ACA
Thr
GTT
Val
GCA
Ala
AAA
Lys
CCT
Pro 305
AT
sn ACT TAC TAC Thr Tyr Tyr 130 CIT GCA AGA Leu Ala Arg AAG CTC CCA Lys Leu Pro GGT AAC AGA Gly Asn Arg 180 TAT AAA GGA Tyr Lys Gly 195 AAT CTA CTT Asn Leu Leu 210 CCT TTT GAA Pro Phe Glu TTC AAT AAT Phe Asn Asn GGT AAC GAT Gly Asn Asp 260 TCT TT GGA Ser Phe Gly 275 AAT ACC TAT Asn Thr Tyr 290 TTT GCA AGC Phe Ala Ser TCT AAA AAA Ser Lys Lys GGA TTr Gly Phe GGT GOT Gly Ala 150 CAA ATA Gin lie 165 AAT CAA Asn Gin ATA CTI Ile Leu GAT AAA Asp Lys TTA AAT Leu Asn 230 TCA TCA Ser Ser 245 TTA TTG Leu Leu GCT CAA Ala Gin CTT ATT Leu Ile GAT TTI %sp Phe 310 3GA ATA ;ly Ile 844 892 940 988 1036 1084 1132 1180 1228 1276 1324 1372 1420 1468 315 TCC GTA GAT CCT ATT AAA AAA GCC GAA GAT ATA TTT GAT CCA AAT GGC Ser Val Asp Pro Ile Lys Lys Ala Glu Asp Ile Phe Asp Pro Asn Gly 330 335 340 WO 95/35379 AAT GCT CT! Asn Ala Leu 345 PCT1US9507665 AAT TTC AGT AAA Asn Phe Ser Lye
AAT
Asn 350 ACA GAG CTG Thr Giu Leu GGC AT Gly Ile 355 GCA Trr TCA Ala Phe Ser 1516 ACA GGA Thr Gly 360 GAA TCT Glu Ser 375 GCA AGC ATA GGG Ala Ser Ile Gly
CT!
Leu 365 CTC TGG AAT AAA GAC Leu Trp Aen Lye Asp GAC GGT GAA AAA Asp Gly Giu Lye TGG AAG GT Trp Lye Val
AAG
Lye 380 GGA GCT GAT TCC Gly Ala Asp Ser AGT ACA AGA CTA Ser Thr Arg Leu TTr Phe 390 1564 1612 1660 GGA GAA CAA GAC Gly Giu Gin Asp
AAA
Lye 395 AAA TCT GGA GTT Lye Ser Gly Val TTA GGA ATA AGT Leu Gly Ile Ser TAT GGA Tyr Oly 405 CAA AAT CT Gin Aen Leu TCC GAA AAT Ser Glu Asn 425
TAT
Tyr 410 AGA TCC AAA GAT Arg Ser Lye Asp
ACA
Thr 415 GAA AAA AGA TTA Glu Lye Arg Leu AAA ACC ATA Lye Thr Ile 420 AGC TAT GAA Ser Tyr Olu 1708 GCA TTT CAA AGC TTA AAT GTT GAA ATC Ala Phe Gin Ser Leu Asn Val Glu Ile 430 1756 GAC AAC Asp Asn 440 AAA AAA GGA CT ATG AAC GGA CTA GGA Lye Lye Gly Leu Met Asn Gly Leu Oly 445 ATA ACA TCT ATC Ile Thr Ser Ile 1804 CT! TAT GAT AT Leu Tyr Asp Ile
TA
Leu 460 AGA CAA AAA TCT Arg Gin Lye Ser
GTA
Val 465 GAA AAC TAT CCC Olu Aen Tyr Pro
ACA
Thr 470 ACA ACA AGC TCA Thr Thr Ser Ser OCT OCT GAT OCA AAC AAT Ala Ala Asp Ala Aen Asn 475 480 CAA GCC GGA CAA Gin Ala Giy Gin AGT TCA Ser Ser 485 1852 1900 1948 GGA Gly AGC ACA CAA Ser Thr Gin 490 OCT ACA ACC CCT AAT CTA ACA T!T GAA Ala Thr Thr Pro Asn Leu Thr Phe Olu 495 GAC GCA ATG Asp Ala Met 500 ATA GAA TCC Ile Glu Ser AAA CTC GGT Lye Leu Gly 505 ATA GCT TTA TAT Ile Ala Leu Tyr
CT
Leu 510 GAT TAT GCA AT Asp Tyr Ala Ile 1996 AT TCA Ile Ser 520 ACA GAA GCA TAT Thr Glu Ala Tyr
GTA
Val 525 GTA CCT TAT ATT Val Pro Tyr Ile GCA TAC CT TTA Ala Tyr Leu Leu 2044 2092 GGG Gly 535 CAT TTT AAT AAA Hie Phe Asn Lye
ATC
Ile 540 TCA AGC GAT GCT Ser Ser Asp Ala AAA ATT TAT Lye Ile Tyr TTA AAG Leu Lye 550 ACA GGA CT AGT Thr Gly Leu Ser
CT!’
Leu 555 GAA AAA CTA ATA AGA Glu Lye Leu Ile Arg 560 TIT ACA ACA Phe Thr Thr AT TCT CT Ile Ser Leu 565 2140 WO 95135379 PCT/US95/07665 GGC TGG GAT TCA AAT AAC ATT ATA GAA CTT GCT AAT AAA AAC ACA AAT Gly Trp Asp Ser Asn Asn Ile Ile Giu Leu Ala Asn Lye Asn Thr Asn 570 575 580 AAT GCT GCC ATT GGT AGT GOT TTC TTG CAA TTC AAA ATA GCC TAC AGT Asn Ala Ala Ile Gly Ser Ala Phe Leu Gin Phe Lye Ile Ala Tyr Ser 585 590 595 GGA AGC TAA AAGCAAAAGA AGGGCTrTAG GCCCTTC7Tr TTTTATC7TT Gly Ser 600 AAAAACAAAT TAATATTAAT TACTTTATAT TTCTITCTIT GCAAATCTI’ TCATAAGCAT CTTGAATrT AATAAATITA TCATTGCAT C*TITTGCCT TACAGGATCA TTTGCAAACC TGTCAGGATG ATATTTTATA ACAAGACTFT TATAAGCCTT TrTAATTCk TCATOACTAG CACTATAGAC TAACCCCAAA ACACTATAGG GATTTACAAT TTTAATATTA ATATCTFTAT AAGCTrCATA ACCATCAGAT TC 2188 2236 2285 2345 2405 2465 2525 2547 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 621 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Lye Asn His -21 Val Ala Ile Phe Lys Ile Asn Pro Phe Arg Leu Asp Lye Ile Thr Ile Ile Leu Tyr Lye Leu Ile Ile Phe Leu Thr Thr Ser Ala Ala Ala Asp Lye Leu Lys 5 Glu Glu Asp Ile Phe Trp Ile Pro Thr Gly Ile Glu Asn Thr Ser Glu Asn Lye Ser Met Asp Glu Pro Gly Phe Lye Leu Pro Phe Giu Val Aen Pro Giu Leu Gly Lye Asp Lye Ala Asp Pro Phe Glu Gly Lye Ala Tyr Ile Gly Asp Pro Lye Val 70 Phe Lye 85 Glu Asp Leu Ala Leu Ile Asp Val Gly Asp Ile Thr Ala Gin Ile Asn Ile Tyr Asp Phe Phe Ile Lye 100 Ile Ser Thr 105 WO 95/35379 Met Thr Ai
I:
Met Thr G: 125 Ile Val A3 140 Thr Ile G1 Ile Gly G] Thr Pro T i9 Thr Trp Lj 205 Ser Val 11 220 Ala Tyr Gi Lys Asp Ly Asn Ser Al.
27 Thr Lys Ii 285 Asp Phe Gi 300 Ile Ser Ly Lys Lys Al.
Ser Lys As Giy Leu Lei 365 PCTIUS9SIO766S
LC
Ln Ly I0 y .5 a 0 e y s a n 0 .1 Phe Asp -Phe Lys Gly Thr Met Gly 160 *Thr Gly Asn Lys Pro Ile Ala Giu Asn Lys 240 Ser Val 255 Ile Leu Asn Asn Ile Asp Ala Ala 320 Giu Asp 335 Thr Giu Trp Asn Phe Ser Ile 145 Thr Thr Lys Thr 225 Thr Vai Ala Lys Pro 305 Asn Ile Lieu Eqs [‘yr 385 *Asn Lys 115 Thr Tyr 130 Leu Ala Lys Leu Giy Asn T.yr LYS 195 Asn Leu 210 Pro Phe Phe Asn Gly Asn Ser Phe 275 Asn Thr 290 Phe Ala Ser Lys Phe Asp Gly Ile 355 Asp Asp 370 Ser Thr Giu Ser Leu *Tyr Giy Phe *Arg Gly Ala 150 Pro Gin Ile 165 Arg Asn Gin 180 Gly Ile Leu Leu Asp Lys Giu Leu Asn 230 Asn Ser Ser 245 Asp Leu Leu 260 Gly Ala Gin Tyr Leu Ile Ser Asp Phe 310 Lys Giy Ile 325 Pro Asn Gly 340 Ala Phe Ser Gly Giu Lyes Arg Leu PheC 390 Phe Pro 135 Ser Asp Giu Asn 215 Phe Ile Ser Tyr Leu 295 Ser Ser 4,sn trhr ;iu 375 ;iy Ser Phe 120 Ser Lye Lye Asn Leu Thr Asn Asp 185 Gly Val 200 Giu Asp Gly Leu Thr Tyr Pro Thr 265 Lys Leu 280 Gin Met Val Phe Val Asp Ala Leu 345 Giy Ala 360 Ser Trp Giu Gin Al Asi Ile Phe 170 Lys Gird Ser Ser 250 Leu Gly Giy Gly Pro 330 1&sn Ser Lys zsp aPro 2Arg Gly 155 Ala Asp Ala Arg Gly 235 Leu Ser Leu Thr His 315 Ile Phe Ile Val Lye 395 Lys Gly Ala Asp Ser 380 Lys Ser Gly Vai Ala 400 Leu Gly Ile Ser Tyr Gly Gin Aen Leu 405 Tyr Arg 410 mmummow WO 95135379 PCTIUS95/07665 Ser Lys Asp Thr Giu Lys Arg Leu Lys Thr Ile Ser Giu Asn Ala Phe 415 Gin Leu Leu 460 Ala Thr Leu Tyr Ile 540 Giu Asn Ser Leu Asn 430 Asn Giy Gin Lys Ala Asn Pro Asn 495 Leu Asp 510 Val Pro Ser Asp Leu Ile Ile Giu 575 Phe Leu 590 Giu Giy Vai 465 Gin Thr Ala Ile Thr 545 Phe Ala Phe Ile Trp 450 Giu Ala Phe Ile Giy 530 Lys Thr Asn Lys 420 Ser Tyr Giu Thr Ser Ile Tyr Pro Thr 470 Gin Ser Ser 485 Asp Ala Met 500 Ile Giu Ser Tyr Leu Leu Tyr Leu Lys 550 Ile Ser Leu 565 Asn Thr Asn 580 Ala Tyr Ser Asp Asn 440 Gly Leu 455 Thr Thr Gly Ser Lys Leu Ile Ser 520 Gly His 535 Thr Giy Giy Trp Asn Ala Gly Ser 600 425 Lys Lys Tyr Asp Ser Ser Thr Gin 490 Giy Ile 505 Thr Giu Phe Asn Leu Ser Asp Ser 570 Ala Ile INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 64 base pairs TYPE: nucleic acid STRANDE15MESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Borriia burgdorferi STRAIN: B31 (vii) IMdMEDIATE SOURCE: CWONR: pJB-lOS (ix) FEATURE: NAME/MY: CDS LOCAT)N:14. .61 WO 95135379 PCT/US95/07665 86 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION:14..61 OTHER INFORMATION:/partial /label= partial (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GGAGGGTATA ATT ATG AAA AGC CAT ATT TTA TAT AAA TTA ATC ATA TTT 49 Met Lys Ser His Ile Leu Tyr Lys Leu Ile Ile Phe 605 610 TTA ACC ACA TCT GCA 64 Leu Thr Thr Ser 615 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Lys Ser His Ile Leu Tyr Lys Leu Ile Ile Phe Leu Thr Thr Ser 1 5 10 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCAATATTTG CTGCAGCAGA T 21 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 95/35379 87 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: PCT1UJS95/07665 GGCCTAAAGG AATTCTTI’G C INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 2031 base pairs TYPE: nucleic acid STRAN’DEDNESS: single TOPOLOGY: linear (ii) MOLECUJLE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Borrelia burgdorferi STRAIN: B31 (ix) FEATURE: NIAME/KEY: CDS LOCATION:14. .1870 (ix) FEATURE: NTAME/KEY: sigpeptide LOCATION:14. .76 (ix) FEATURE: NAMdE/KEY: matpeptide LOCATION:77. .1867 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GGAGGGTATA ATI’ ATG AAA AGC CAT ATT TTA TAT AAA TI’A ATC ATA TT Met Lys Ser His Ile Leu Tyr Lys Leu Ile Ile Phe -21 -20 -15 TI’A ACC ACA TCT GCA GCA ATA ‘FIT GCA GCA Leu Thr Thr Ser Ala Ala Ile Phe Ala Ala GAC GCA TTA AAG GA.A AAA Asp Ala Leu Lys Glu Lys GAT ATA ‘FIT AAA ATA AAC CCA Asp Ile Phe Lys Ile Asn Pro ACA AGT GAA ITC AGA TTA GAT Thr Ser Glu Phe Arg Leu Asp ATG CCA ACA ‘FIT Met Pro Thr Phe GGA ‘FFT GAA AAC Gly Phe Glu Asn CCT GGG I’IT GAA Pro Gly Phe Glu ATG GAC GAG CTTI Met Asp Glu Leu AAC AAA AGC AAA Asn Lys Ser Lys GAA TI’A GGC AAA Glu Leu Gly Lys ATT ACC Ile Thr GAC GAT Asp Asp ATI’ AAG C’FT AAA Ile Lys Leu Lys ‘FIT GAA GCT AAT Phe Glu Ala Asn CCA ‘FrC TCA Pro Phe Ser TAC A’Fr AAG GTA Tyr Ile Lys Val GAA GAT Glu Asp WO 9513537 CTT GCA 9 PCTIS95/0766 CTA AAA GCG GAA GGC AAA AAA GGC GAT CAA TTT AAA AT GAC Leu Ala Leu Lys Ala Glu Gly Lys Lys 80 Gly Asp Gin
GTG
Val
ATA
Ile
TTT
Phe 120
AAT
Asn
AAC
Asn
ACA
Thr
GAC
Asp
ATT
Ile 200
GAT
Asp
TTG
Leu
TAC
Tyr
ACT
Thr
GAT
Asp
ACT
Thr
CCT
Pro
AGG
Arg
GGA
Gly TIT GCA Phe Ala 170 AAA GAC Lys Asp 185 CAA GCA Gin Ala ACT AAA Thr Lys TCA GGA Ser Gly TCT TTA Ser Leu 250 TTA TCA Leu Ser 265 ATT ACA GCC Ile Thr Ala ATG ACA GAT Met Thr Asp ATG ACT GGA Met Thr Gly 125 GCA GTA AGA Ala Val Arg 140 ACA ATr CAG Thr Ile Gin 155 ATA GGG GGA Ile Gly Gly ACT CCA TAC Thr Pro Tyr ACA TGG AAA Thr Trp Lys 205 TCT GTA ATT Ser Val Ile 220 GCC TAT GGA Ala Tyr Gly 235 AAA GAT AAA Lys Asp Lys AAT TCT GCA Asn Ser Ala
CAA
Gin
TTT
Phe 110
TTT
Phe
GGG
Gly
CTG
Leu
ACA
Thr
AAT
Asn 190
CCA
Pro
GCA
Ala
AAC
Asn
TCC
Ser
ATT
Ile 270 ATC AAT Ile Asn 95 SAC TIT Asp Phe AAA AGC Lys Ser ACA ATT Thr Ile GGA TAC Gly Tyr 160 GGC ACG Gly Thr 175 AAA ACA Lys Thr ATA AAA Ile Lys GAA ACA Glu Thr GAG ACA Glu Thr 240 GTA GTT Val Val 255 U TA GCA Leu Ala
ATG
Met
AAT
Asn
ACT
Thr
CTT
Leu 145
AAA
Lys
GGT
Gly
TAT
Tyr
AAT
Asn
CCT
Pro 225
TTC
Phe
GGC
Gly
TCT
Ser TAC SAT Tyr Asp AAA SAG Lys Glu 115 TAC TAT Tyr Tyr 130 GCA AGA Ala Arg CTC CCA Leu Pro AAC AGA Asn Arg CAA GGA Gin Gly 195 CTA CTT Leu Leu 210 TTT SAA Phe Glu AAT AAT Asn Asn AAC SAT Asn Asp ‘IT GGA Phe Gly 275 Phe Lys Ile Asp TTT TTT ATT AAA Phe Phe Ile Lys 100 TCT TTA ‘IT AGT Ser Leu Phe Ser GGA TTC CCA AGC Gly Phe Pro Ser 135 GGT ACT TCT AAA Gly Thr Ser Lys 150 AAA CTC SAC CT Lys Leu Asp Leu 165 AAT CAA SAG AAT Asn Gin Glu Asn 180 ATC CTT TAT GGA Ile Leu Tyr Gly SAT CAA AAC GAA Asp Gin Asn Glu 215 TTA AAT ‘IT GGC Leu Asn Phe Gly 230 TCA TCA ATA ACA Ser Ser Ile Thr 245 TTA TTG AGC CCA Leu Leu Ser Pro 260 GCT AAA TAT AAG Ala Lys Tyr Lys 433 481 529 577 625 673 721 769 817 865 913 CTT GSA TTA ACA AAA ATA AAC SAT AAA AAT ACC TAT CIT ATT ‘TG CAA Leu Gly Leu Thr Lys 280 Asn Asp Lys Asn Thr 290 Tyr Leu lie Leu Gin 295 WO 95/35379 ATG GSA A PCTIUS95/07665 CT SAT TIT GSA ATA GAT CCT TTT GCA AGC GAT ‘FIT TCT ATA 1009 Met Gly Thr Asp Phe Gly Ile Asp Pro Phe Ala Ser Asp Phe Ser Ile 300 305 ‘FIT GGA CAC ATC TCA AAA GCA Phe Gly His Ile Ser Lys Ala 315 GAT OCT AAC AAA AAA GOT GAA Asp Pro Asn Lys Lys Ala Glu 330 AAT TTC AGC AAA AAC ACA GAA Asn Phe Ser Lys Asn Thr Glu 345 350 AGT ATA GGT ‘FIT GCT TGG AAT Ser Ile Gly Phe Ala Trp Asn 360 365 GCG ATT AAA GGA TCT GAT TCC Ala Ile Lys Gly Ser Asp Ser 380 GAC AAA AAA TOT GSA GTI’ GOA Asp Lys Lys Ser Gly Val Ala 395 TAC AGA TOT AAA GAT ACA GAA Tyr Arg Ser Lys Asp Thr Giu 410 GCA TTT CAA AGC TTA AAT G’T Ala Phe Gin Ser Leu Asn Vai 425 430 AAA GGG A’IT ATA AAT GSA TTA Lys Gly Ile Ile Asn Gly Leu 440 445 SAT A’IT ‘FrA ASA CAA AAA TOT Asp Ile Leu Arg Gin L3Fs Ser 460 AGC ACC ACT GAA AAC AAT CAA Ser Thr Thr Giu Asn Asn Gin 475 ACC ACA ACC COT AAT OTS ACA Thr Thr Thr Pro Asn Leu Thr 490 GOG AAT ‘FTC AAA Ala Asn Phe Lys 320 ATA ‘FIT GAT OCA Ile Phe Asp Pro 335 ‘FTG GGO A’IT G0k Leu Gly Ile Ala AAA SAT ACC GGT Lys Asp Thr Giy 370 TAO AGT ACA NGC2A Tyr Ser Thr Ai, 385 TTG GGA ATA AGO Leu Gly Ile Ser 400 AAA AGA ‘FTA AAA Lys Arg Leu Lys 415 GAA A’IT TCA AGO Giu Ile Ser Ser GSA TGG ATA ACA Gly Trp Ile Thr 450 GTA GAA AAO TAT Val Glu Asn Tyr 465 ACT GAA CAA AGT Thr Glu Gin Ser 480 ‘FIT SAA SAT GCA Phe Giu Asp Ala 495 AAA GAA Lys Giu AAT GGC Asn Gly 340 ‘FIT TCA Phe Ser 355 GAA AAA Giu Lys OTO ‘FIT Leu Phe TAT GSA Tyr Giy ACC ATA Thr Ile 420 TAT SAA Tyr Giu 435 TOT ATO Ser Ile COT ACA Pro Thr TOA ACA Ser Thr ATG AAA Met Lys 500 310 ACA COO TCA Thr Pro Ser 325 AAT GOT OTI’ Asn Ala Leu ACA GSA GOA Thr Giy Ala SAA TOO TGG Giu Ser Trp 375 GSA GAA CAA Gly Giu Gin 390 CAA AAO CTT- Gin Asn Leu 405 TOT SAA AAT Ser Giu Asn SAC AAO AAA Asp Asn Lys GGT OTT TAO Gly Leu Tyr 455 ACA A’FT TOA Thr Ile Ser 470 AGO ACA AAG Ser Thr Lys 485 OTO GGO ‘FTG Leu Gly Leu 1057 1105 1153 1201 1249 1297 1345 1393 1441 1489 1537 1585 GOC ‘FTA Ala Leu 505 TAT OTT SAT TAT Tyr Leu Asp Tyr
GCA
Ala 510 AT’ OCA ATA GOA le Pro Ile Ala
TOO
Ser 515 A’FT TCA ACA GAA Ile Ser Thr Glu 1633 WO 95/35379 GCA TAT GTA GTA CCT TAC ATT GGA GCA TAC ATT TTA Ala Tyr Val Val Pro Tyr Ile Gly Ala Tyr Ile Leu 520 525 530 AAA CTC TCA AGC GAT GCT ACA AAA ATT TAT TTA AAA Lye Leu Ser Ser Asp Ala Thr Lye Ile Tyr Leu Lye 540 545 CTT GAA AAA CTA ATA AGA ‘IT ACA ACA ATT TCT CTT Leu Glu Lys Leu Ile Arg Phe Thr Thr Ile Ser Leu 555 560 AAT AAC ATT ATA GAA CTT GCT AAT AAA AAC ACA AAT Asn Asn Ile Ile Glu Leu Ala Asn Lye Asn Thr Asn 570 575 GGA AGT GCT TTC TTG CAA TTC AAA ATA GCC TAC AGC Gly Ser Ala Phe Leu Gin Phe Lys Ile Ala Tyr Ser 585 590 595 CAGCAAAAGA AGGGCTTTGG CCCTTCTTTT TTATCTTTAA AAA TATTTCTTTC CTTGCAAATT TITTCATAAG CATCTTGAAT TT CATCTITITG TCTTACAGGA TCATTTGCAA ACTTATCAGG A INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 618 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Met Lye Ser His Ile Leu Tyr Lys Leu Ile Ile Phe -21 -20 -15 Ala Ala Ile Phe Ala Ala Asp Ala Leu Lye Glu Lys 1 Ile Asn Pro Trp Met Pro Thr Phe Gly Phe Glu Asn Arg Leu Asp Met Asp Glu Leu Val Pro Gly Phe Glu Ile Thr Ile Lys Leu Lys Pro Phe Glu Ala Asn Pro 50 Asp Asp Pro Phe Ser Ala Tyr Ile Lye Val Glu Asp 65 Ala Glu Gly Lys Lye Gly Asp Gin Phe Lye Ile Asp PCT/US95/07665 GGA CCT TCT AAT Gly Pro Ser Asn 535 ACA GGA CTT AGC Thr Gly Let Ser 550 GGA TGG GAT TCA Gly Trp Asp Ser 565 AAT GCT GCT ATT Asn Ala Ala Ile 580 GGA AGC TAA Gly Ser CAATTGG GATTACCTTA ATAAAT TTATCATTTG 1681 1729 1777 1825 1870 1930 1990 2031 WO 95/3531 Thr Al a Thr Asp Thr Gly 125 Val Arg 140 Ile Gin Gly Gly Pro Tyr Trp Lys 205 Vai Ile 220 Tyr Gly Asp Lye Ser Aia Lys Ile 285 Phe Giy 300 Ser Lys Lys Ala Asn Thr Ala Trp 365 Ser Asp 380 PCTUS95IO7665 Asn Met Tyr Asp Phe Phe 91.
Ile Gin Ile Phe Asp 110 Phe Lys Gly Thr Leu Gly Thr Gly 175 Asn Lys 190 Pro Ile Ala Giu Asn Giu Ser Vai 255 Ile Leu 270 Asn Asp Ile Asp Ala Ala Glu Ile 335 Giu Leu 350 Asn Lye Ser Tyr 100 Phe Ser Ile Tyr 160 Thr Thr Lys Thr Thr 240 Val Ala Lye Pro Asn 320 Phe Gly Asp Ser Asn Thr Leu 145 Lye Gly Tyr Asn Pro 225 Phe Gly Ser Aen Phe 305 Phe Asp Ile Thr Thr 385 Lye Tyr 130 Ala Leu Asn Gin Leu 210 Phe Asn Asn Phe Thr 290 Ala Lye Pro Ala Giy 370 Arg Giu 115 Tyr Arg Pro Arg Gly 195 Leu Giu Asn Asp Gly 275 Tyr Ser Lye Asn Phe 355 Giu Leu Ser Gly Gly Lye Asn 180 Ile Asp Leu Ser Leu 260 Ala Leu Asp Giu Gly 340 Ser Lys Phe Leu Phe Phe Pro Thr Ser Leu Asp 165 Gin Giu Leu Tyr Gin Asn Asn Phe 230 Ser Ile 245 Leu Ser LYe Tyr Ile Leu Phe Ser 310 Thr Pro 325 Asn Ala Thr Gly Giu Ser Gly Glu 390 Lye Ser Ser 135 Lye Leu Asn Gly Glu 215 Gly Thr Pro Lye Gin 295 Ile Ser Leu Ala Trp 375 Gin Ile Phe 120 Asn Asn Thr Asp Ile 200 Asp Leu Tyr Thr Leu 280 Met Phe Asp Asn Ser 360 Ala Asp Ser 105 Ala Asp Ile Phe Lye 185 Gin Thr Ser Ser Leu 265 Gly Gly Gly Pro Phe 345 Ile Ile Lys Thr Pro Arg Gly Al a 170 Asp Ala Lys Gly Leu 250 Ser Leu Thr His Asn 330 Ser Gly Lys Lye Me t Met Ala Thr Ile Thr Thr Ser Ala 235 Lye Asn Thr Asp Ile 315 Lye Lys Phe Gly Ser 395 WO 95/35379 WO 9535379PCTIIJS95/07665 Gly Val Asp Thr Leu Asn Asn Gly 445 Gin Lys 460 Asn Asn Asn Leu Asp Tyr Pro Tyr 525 Asp Ala 540 Ile Arg Glu Leu Leu Gin Ala Leu Gly 400 Giu Lys Arg 415 Vai Giu Ile 430 Leu Gly Trp Ser Val Giu Gin Thr Giu 480 Thr Phe Glu 495 Ala Ile Pro 510 Ile Gly Ala Thr Lys Ile Phe Thr Thr 560 Ala Asn Lys 575 Phe Lys Ile 590 Ile Ser Tyr Gly Gin Aim Leu Tyr Arg Ser j.ys 405 410 Leu Ser Ile Aen 465 Gin Asp Ile Tyr 545 Ile Asn Ala Thr Tyr 435 Ser Pro Ser Met Ser 515 Leu Lys Leu Asn Ser 595 Ile Ser Gi 420 Giu Asp As Ile Gly Le Thr Thr Ii 47 Thr Ser Th 485 Lys Leu Gi, 500 Ile Ser Th Gly Pro Se.
Thr Gly Le, 55 Gly Trp As] 565 Asn Ala Al, 580 Giy Ser Ala Phe 425 Lys Gly 440 Asp Ile Ser Thr Thr Thr Ala Leu 505 Ala Tyr 520 Lys Leu Leu Glu Asn Asn Gly Ser 585 Gin Ile Leu Thr Thr 490 Tyr Val Ser Lys Ile 570 Ala Ser Ile Arg Glu 475 Pro Leu Val Ser Leu 555 Ile Phe WO 95/35379 PCT/US95/07665 93 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page ,line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution Deutsche SammJi.ng von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code and country) Maschroder Weg lb D-38124 Braunschweig Federal Republic o2 Germany- Date of deposit Accession Number 16 June 1994 DSM 9253 C. ADDITIONAL INDICATIONS (leave blank ifno applicable) This information is continued on an additional sheet As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent -is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if dl indications are not for all drignated Stale) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the genera nature of heindications ‘Accession Number ofDeposit») For receiving Office use only For International Bureau use only l This sheet was received with the international application This sheet was received by the International Bureau on: Authorized offir s Authorized officer val, Divisionmm ,U»lF’.’rataiolai Division Form PCT/RO/134 (July 1992)
I
WO 95/35379 PCTUS9.5IO7665 94 INJCATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred t nthe description on page line B3. IDENT’IFICATION OF DEPOSIT Further deposits are identified on an additional sheet r Name of depositary institution Deutsche Saxnmlung von Mikroorganismen und Zeilkulturen GmbH (DSM) Address of depositary institution (includingpostal code and couniry) Maschroder Weg lb D-38124 Braunschweig Federal Republic of Germany, C. ADDITONAL INDICATIONS (huwve blank if not applicable’) This information is continued on an additional sheet As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifte indicadons are nor for all dsi gnated Slates E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (spe~cify diegeneral nature of uleindications eg., ‘Accession Number of Deposit») For recciving Offirc-. use only For International Bureau use only [2’This sheet was received with the international application JThsheet ;4ras received by the international Bra n 0(2Fiss [Authorized of: r m~nl ii~ Form PCr/RO1134 (July 1992) Authorized officer
I
WO 95/35379 PCT/US95/07665 INDICATIONS RELATING TO A DEPOSITED MICRO ,GANISM (PCT Rule l3bis) A. The indications made below relate to the mnicroorganism referred to in the description on page 43 line B. IDENT IFICATION OF DEPOSIT Further deposits are identified on an additional sheet E Name of depositary institution Deutsche «anmmlung von Mikroorganismen und Zeilkulturen GmbH (DSM) Address of depositary institution (incdudingpostal code and cuntry) Maschroder Weg lb D-38124 Braunschweig Federal Republic of Germany, Date of deposit Accession Number 16 June 1994 DSM 9255 C. AD DITIONAL INDICATIONS (lev b~kmnk if oux applicable) Thbis information is continued on an additional sheet As regards the respective Patent Of fices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been ref 1sed or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if Ili indicatons are naor arol ii ignaled Slaw c) E. SEPARATE FUJRN1ShING OF INDICATIONS (leave blank if nog applicable) The indications listed beiow will be submitted to the Internationa I Bureau la ter (specify Legencrai nature ofiseindicauions eag., ‘Accesuion N’um~ber ofDgpasi:) For receiving Office use only or International Bureau use only fI~T is sheet waS received with the international application I 0EThis sheet was received by the International Bureau on: Authorized officer I Yvd .,06mFw Division Form PCr/ROI134 (July 1992) Authorized officer

Claims (13)

1. An isolated nucleic acid fragment which comprises a nucleotide sequence which encodes a polypeptide exhibiting a substantial immunological reac- tivity with a rabbit antibody raised against a 66 kDa polypeptide derived from Borrelia garinii IP90 which comprises the amino acid sequence 1-600 of SEQ ID NO: 8, said rabbit antibody exhibiting substantially no immunological reactivity with whole cell preparations from at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica, and/or hybridises readily with either a DNA fragment having the nucleotide sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with a DNA fragment complemen- tary thereto, but exhibits no substantial hybridization with genomic DNA from at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia a-serina, or Borrelia hispanica when the hybridization conditions are highly stringent.

2. The nucleic acid fragment according to claim 1, which encodes a polypeptide compris–.g an amino acid sequence comprised in a polypeptide present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, and/or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, or Borrelia hispanica.

3. The nucleic acid fragment according to claim 1 or 2, which encodes a polypeptide comprising at least a part of an amino acid sequence of a 66 kDa protein present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii RA$\4 IP90, and/or Borrelia afzelii ACAI but substantially absent 3 C AMENDED SHEET ‘T 0\ 13149PCI.C02/PK/199607 12 from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.

4. The nucleic acid fragment according to any of the prece- ding claims, which encodes a polypeptide comprising at least one epitope present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Bbrrelia anserina, and Borrelia hispanica. The nucleic acid fragment according to any of the prece- ding claims, which encodes a polypeptide comprising at least one epitope of a 66 kDa protein present in whole cell pre- parations of Borrelia burgdorferi B31, Borrelia garinii or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.

6. A nucleic acid fragment according to any of the preceding claims, which comprises a nucleic acid fragient encoding a polypeptide which has the same amino acid sequence as positions 175-190, 285-305, 365-385, or 465-490 in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 14.

7. The nucleic acid fragment according to any of the prece- ding claims, which encodes a 66 kDa protein present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of ran- domly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica.

8. The nucleic acid fragment according to claim 7, wherein the encoded protein is present in fraction B from Borrelia 13149PCI.C02/PK/199607 12 AMEN.rDED SHEET qq burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI.

9. The nucleic acid fragment according to claim 8, wherein the encoded protein natively is a surface exposed protein of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI. The nucleic acid fragment according to any of the prece- ding claims, which encodes a polypeptide which has an amino acid sequence’exhibiting a sequence homology of at least with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ NO: 10, or SEQ ID NO: 14 or with subsequences thereof.

11. The nucleic acid fragment according to any of the prece- ding claims, wherein the nucleotide sequence has a sequence homology of at least 70% with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with subsequences thereof.

12. A nucleic acid fragment according to any of the preceding claims, which comprises the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or subsequences thereof.

13. A nucleic acid fragment according to any of the preceding claims, which encodes a fusion polypeptide.

14. The nucleic acid fragment according to claim 13 encoding a fusion polypeptide comprising as a fusion partner a polypeptide fragment which enhances the immunogenicity of the fusion polypeptide relative to the immunogenicity of a polypeptide not comprising said second fusion partner or which facilitates the expression of the fusion polypeptide in a host cell and/or the subsequent purification of the polypeptide. AMENDE SHEET QWrO I49PCI.CO2/K I9960712 AMENDED SHEET The nucleic acid fragment according to claim 13 or 14, which encodes a fusion polypeptide comprising as a fusion partner a polypeptide fragment which has the same amino acid sequence as positions 175-190,

285-305, 365-385, or 465-490 in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 14, is a lipoprotein selected from the outer membrane lipoprotein from E. coli and OspA from Borrelia burgdor- feri sens6 lato, is a viral protein selected from Hepatitis B surface antigen, Hepatitis B core antigen, and the influenza virus non-structural protein NS1, is a immunoglobulin binding protein selected from protein A, protein G, and the ZZ-peptide, is a T-cell epitope, is a B-cell epitope, is a bacterial fimbrial protein selected from the pilus components pilin and papA, and/or is the maltose binding protein, gluthatione S- transferase, 0-galactosidase, or poly-histidine. 16. The nucleic acid fragment according to any of the prece- ding claims, which is a DNA fragment. 17. A substantially pure polypeptide exhibiting a substantial immunological reactivity with a rabbit antibody raised against a 66 kDa polypeptide derived from Borrelia garinii which comprises the amino acid sequence 1-600 of SEQ ID NO: 8, said rabbit antibody exhibiting substantially no A.4 I mmunological reactivity with whole cell preparations from at AMENDED SHEET r O 3149PC1,C02/PKI199607 12 least 95% of randomly selected B. hermsii, B. crocidurae, B. anserina, or B. hispanica, said substantially pure polypeptide being free from other Borrelia-derived antigens. 18. The polypeptide according to claim 17, which comprises an amino acid sequence comprised in a polypeptide present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of ran- domly selected Borrelia hermsii, Borrelia crocidurae, Borrelia ansefina, or Borrelia hispanica. 19. The polypeptide according claim 17 or 18, which comprises at least a part of the amino acid sequence of a 66 kDa protein present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica. A polypeptide according to any of claims 17-19 comprising at least one epitope present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell pre- parations of at least 95% of randomly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica. 21. A polypeptide according to any of claims 17-20, which comprises at least one epitope of a 66 kDa protein present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI but substantially absent from whole cell preparations of at least 95% of ran- domly selected Borrelia hermsii, Borrelia crocidurae, Borrelia anserina, and Borrelia hispanica. 22. The polypeptide according to claim any of claims 17-21, which comprises at least one of the amino acid sequences 175- 13149PCI.C02/PK/199607 12 AMENDED SHEET 7 AMENDED SHEET 0 f 190, 285-305, 365-385, or 465-490 in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 14. 23. The polypeptide according to any of claims 17-22, which has an amino acid sequence identical to a 66 kDa protein present in whole cell preparations of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI. 24. The polypeptide according to claim 23, wherein the 66 kDa protein is present in fraction B from Borrelia burgdorferi B31, Borrelialgarinii IP90, or Borrelia afzelii ACAI. 25. The polypeptide according to claim 23 or 24, wherein the 66 kDa protein is a surface exposed protein of Borrelia burgdorferi B31, Borrelia garinii IP90, or Borrelia afzelii ACAI. 26. The polypeptide according to any of claims 17-25, which has an amino acid sequence exhibiting a sequence homology of at least 50% with SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 14 or with subsequences thereof. 27. A polypeptide according to any of claims 17-26, which is encoded by a nucleotide sequence exhibiting a sequence homology of at least 70% with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13 or with subsequences thereof. 28. The polypeptide accord-ing to claim 27 which comprises the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 14. 29. The polypeptide according to claim 28, which is encoded by a DNA fragment comprising the nucleotide sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 13. A fusion polypeptide comprising as a first fusion partner the polypeptide according to any of claims 17-29. 1’ r 13149PCI .CO2/PK/199607 12 A ENDED -‘cET I 31. The fusion polypeptide according to claim 30, which comprises as a second fusion partner a polypeptide fragment which enhances the immunogenicity of the fusion polypeptide relative to the immunogenicity of a polypeptide not compri- sing said second fusion partner or which facilitates the expression of the fusion polypeptide in a host cell and/or the subsequent purification of the polypeptide. 32. The fusion polypeptide according to claim 30 or 31, wherein a second fusion partner is a polypeptide which has the same amino acid sequence as positions 175- 190, 285-305, 365-385, or 465-490 in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 14, which is a lipoprotein selected from the outer membrane lipoprotein from E. coli and OspA from Borrelia burgdor- feri sensu lato, which is a viral protein selected from Hepatitis B sur- face antigen, Hepatitis B core antigen, and the influenza virus non-structural protein NS1, which is a immunoglobulin binding protein selected from protein A, protein G, and the synthetic ZZ-peptide, which is a T-cell epitope, which is a B-cell epitope, which is a bacterial fimbrial protein selected from the pilus components pilin and papA, and/or which is the maltose binding protein, gluthatione S- transferase, 0-galactosidase, or poly-histidine. 33. A method of preparing a polypeptide as defined in any of RA claims 1-16, comprising ‘T 0o 13149PC1.CO2/PK/199607 12 AMENDED AMENDED JlET inserting the nucleic acid fragment according to any of claims 1-16 in an expression vector, transforming a host organism or a host cell with the vector, culturing the transformed host cell under conditions facilitating the expression of the polypeptide by the host organism or host cell, and harvesting the polypeptide, and optionally subjecting the polypeptide to post-translational modification(s); or synthesising the polypeptide by solid-phase peptide synthesis or by liquid-phase peptide synthesis. 34. A method according to claim 33, wherein the expression vector is selected from a plasmid, a cosmid, a minichromoso- me, or a phage; the host cell is a microorganism selected from a bacterium, a yeast, or a protozoan, or a cell derived from a multicellular organism selected from a fungus, an insect cell, a plant cell, or a mammalian cell; and the post- translational modifications involves lipidation or glycosylation. A vaccine comprising an amount of the polypeptide accor- ding to any of claims 17-32 or of the polypeptide prepared by the method according to claim 33 or 34, the amount of the polypeptide being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being, in combination with a pharmaceutically acceptable carrier or vehicle and the vaccine optionally further comprising an adjuvant. 36. A live vaccine comprising a non-pathogenic microorganism R carrying and being capable of expressing the nucleic acid TOV 13149PC1.CO2/PK/199607 12 fragment according to any of claims 1-16, the live vaccine being effective in conferring increased resistance to infec- tion with Borrelia burgdorferi sjnsu lato in an animal, including a human being. 37. A vaccine according to claim 35 which is a combination vaccine, further comprising at least one further Borrelia antigen. 38. A combination vaccine according to claim 37, wherein the at least one further Borrelia antigen is selected from the group consisting of OspA, OspB, OspC, OspD, and PC. 39. A combination vaccine comprising at least two different polypeptides according to any of claims 17-32 or at least two polypeptides prepared by the method according to claim 33 or 34, the vaccine comprising an amount of the polypeptides effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being, in combination with a pharmaceutically acceptable carrier or vehicle, the ;-.ccine optionally further comprising an adjuvant. 40. A vaccine comprising the nucleic acid fragment according to any of claims 1-16, the vaccine effecting in vivo expres- sion of antigens by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigens being effective to confer substantially increased resistance to infections with Borrelia burgdorferi sensu lato in an animal, including a human being. 41. An at least partially purified antibody raised against and reacting substantially specifically with the polypeptide according to any of claims 17-32, the antibody being polyclonal or monoclonal. 42. The antibody according to claim 41, which is a monoclonal 4″ ntibody. 13149PC1.CO2/PK/199607 12 [o0 43. A diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in a sample, the composition comprising the polypeptide according to claim any of claims 17-32 or the polypeptide prepared by the method according to claim 33 or 34, the amount of the polypeptide being effective to detectably react with antibodies present in the sample, the antibodies being directed against Borrelia burgdorferi sensu lato, the composition optionally comprising a detectable label. 44. A diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in a sample the composition comprising an amount of the nucleic acid fragment according to any of claims 1-16 which is effective to detectably bind to a nucleic acid fragment from Borrelia burgdorferi sensu lato present in the sample, the composition optionally comprising a detectable label. A diagnostic composition adapted for the determination of Borrelia burgdorferi sensu lato in a sample, the composition comprising an amount of the antibody according to claim 41 or 42 which is effective to detectably react with an antigen from Borrelia burgdorferi sensu lato present in the sample, the composition optionally comprising a detectable label. 46. A method of immunizing an animal, including a human being against infections with Borrelia burgdorferi sensu lato, the method comprising administering to the animal an immunogenically effective amount of the vaccine according to any of claims 35-40. 47. A method of passively immunizing an animal, including a human being against infections with Borrelia burgdorferi sensu lato, the method comprising administering to the animal an immunogenically effective amount of the antibody according to claim 41 or 42. 13149PC1.C02/PK199607 12 AIS’ E: T 1Q 48. A method of determining the presence of antibodies directed against Borrelia burgdorferi sensu lato in a sample, comprising incubating the sample with the polypeptide according to any of claims 17-32 or with the polypeptide prepared by the method according to claim 33 or 34, and detecting the presence of bound antibody resulting from the administration or incubation. 49. A method of determining the presence of a Borrelia burg- dorferi sensu lato antigen in a sample, comprising incubating the sample with the antibody according to claim 41 or 42, and detecting the presence of bound antigen resulting from the administration or incubation. A method of determining the presence of Borrelia burgdor- feri sensu lato nucleic acids in a sample, comprising incubating the sample with the nucleic acid fragment according to any of claims 1-16, and detecting the presence of hybridized nucleic acids resulting from the incubation. 51. A replicable expression vector carrying the nucleic acid fragment according to any of claims 1-16, the vector being capable of replicating in a host organism or cell line. 52. The vector according to claim 51, which is selected from the group consisting of a plasmid, a phage, a cosmid, a mini- chromosome or a virus. 53. A vector according to claim 51 or 52 which, when introduced in a host cell, is integrated in the host cell genome. 54. A transformed cell carrying and capable of replicating the nucleic acid fragment according to any of claims 1-16. A transformed cell according to claim 54, which is a microorganism selected from a bacterium, a yeast, a proto- >7 zoan, or a cell derived from a multicellular organism 13149PC1.C02/PK/199607 12 AMENDED SHEET 107 selected from a fungus, an insect cell, a plant cell, or a mammalian cell. 56. A transformed cell according to claim 55 which is a bacterium of the genus Escherichia, Bacillus or Salmonella. 57. A transformed cell according to claim 56, which is an E. coli cell. 58. A stable cell line producing :-he polypeptide according to any of claims’17-32. 59. A stable cell line according to claim 58, which carries and expresses the nucleic acid fragment according to any of claims 1-16. A diagnostic kit comprising an antibody according to claim 41 or 42 and a means for detecting the reaction between the antibody and antigen bound thereto, a- polypeptide according to any of claims 1»-32 and a means for detecting the reaction between the polypeptide and antibody bound thereto, or a nucleic acid fragment according to any of claims 1-16 and a means for detecting the binding between the nucleic acid fragment and nucleic acid bound thereto. 13149PC1.C02/PK1199607 12

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