Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-15
2003-08-26
Sisson, B. L. (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S073000, C530S350000, C530S387100, C530S388200
Reexamination Certificate
active
06610838
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences encoding antigenic proteins associated with
Borrelia burgdorferi sensu lato
(
Borrelia burgdorferi sensu stricto, Borrelia garinii,
and
Borrelia afzelii;
collectively designated Bb hereinafter), particularly polypeptides associated with virulence; vaccine formulations comprising these polypeptides are also part of the invention. The invention also relates to methods for producing Bb immunogenic polypeptides and corresponding antibodies. Other embodiments of the invention relate to methods for detecting Lyme disease and transformed cells comprising Bb associated nucleic acids.
BACKGROUND OF THE INVENTION
Lyme disease is a multisystem disease resulting from tick transmission of the infectious agent, Bb (Rahn and Malawista, 1991). Although recognised 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 20th 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,
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), OspB and 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 immunisation with OspA (Schaible et al., 1990, Fikrig et al., 1992, Erdile et al., 1993), and therefore, OspA remains one of the main candidates for 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, standardised 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 isolates and a third set recognised all Bb strains (Rosa et al., 1989).
Serological assays for the diagnosis and detection of Lyme disease are thought to offer the best promise for sensitive and specific diagnosis. However, serologic assays generally use whole Bb as antigen and suffer from a low “signal to noise” ratio, i.e., 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 60K “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 (e.g., 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 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 inapparent or asymptomatic infections. This number has been estimated to be as high as 50% (Steere et al., 1986).
There is clearly a need for means of preparing Bb-specific antigens, e.g., for the development of diagnostic tests for Lyme disease. Adequate assays do not exist and should ideally meet several criteria, including (1) expression of an antigen by all pathogenic Bb strains, (2) elicitation of an immune response in all Lyme disease patients, (3) high immunogenicity with a detectable antibody response early in the infection stage, (4) antigens unique to Bb without cross-reactivity to other antigens, and (5) distinction between individuals exposed to non-pathogenic as opposed to pathogenic forms of Bb.
There have been several studies describing low molecular weight proteins from Bb. Katona et al. showed the presence of a major low-molecular weight lipoprotein specific for
B. burgdorferi
and raised the possibility that it was a borrelial equivalent of Braun's lipoprotein (Katona et al., 1992). Another study reported an immunogenic 14 kDa surface protein of
B. burgdorferi
recognised by sera from Lyme disease patients (Sambri et al., 1991). A 14 kDa mitogenic lipoprotein of
B. burgdorferi
was reported by Honarvar et al. (1994).
Sadziene et al. (1994) when analysing an Osp-less
B. burgdorferi
strain identified a 13 kDa surface exposed protein which was designated p13.
OBJECT OF THE INVENTION
It is an object of the invention to provide novel nucleic acid fragments and polypeptide fragments which are useful in the preparation of diagnostics and prophylactic means and compositions relating to infections with Bb. It is a further object to provide novel vaccines and diagnostic means as well as methods for the preparation and use of such vaccines and diagnostic means. Finally, it is an object of the invention to provide tools such as vectors and transformed cells which facilitate the preparation of the polypeptide fragments and the vaccines.
SUMMARY OF THE INVENTION
The inventors have surprisingly found that an antigen from Bb with an apparent molecular weight of 13 kDa (determined by SDS-PAGE, and subsequent visualization such as staining with Coomassie Blue) is highly conserved i
Frommer William S.
Frommer & Lawrence & Haug LLP
Kowalski Thomas J.
Sisson B. L.
Symbicom Aktiebolag
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