Osp-C derived peptide fragments

Details Osp-C derived peptide fragments Osp-C derived peptide fragments

2001-10-10

2004-04-06

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007100, C435S007200, C435S007320, C435S007920, C435S007930, C435S007940, C435S007950

Reexamination Certificate

active

06716574

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel method for the diagnosis of Lyme borreliosis, or more specifically a method for detecting antibodies directed against the OspC protein of
Borrelia burgdorferi
sensu lato. Further, the invention pertains to an immunological agent which comprises a specific peptide fragment derived from the C-terminus of OspC and uses of this immunological agent in the diagnosis of Lyme borreliosis as well as for vaccination purposes. The invention finally relates to novel polypeptide fragments derived from the C-terminus of OspC as well as to short peptides derived from this region.
BACKGROUND OF THE INVENTION
The tickborne spirochaete
Borrelia burgdorferi
is the etiological agent of Lyme borreliosis, which is at present the most common vector-borne human disease in Europe and North America. Lyme borreliosis is a common tick-borne disease which is caused by one of the three genospecies of
B. burgdorferi
sensu lato:
B. burgdorferi
sensu stricto,
B. garinii
, and
B. afzelii
. The clinical manifestations are diverse and may involve the skin, central nervous system, heart, and joints. The symptomatology can be divided into three stages: The first stage: skin lesion; the second stage: meningitis, arthritis, and myocarditis; the third stage: chronic meningitis, chronic arthritis, and chronic skin lesion.
It is desirable to have access to an assay with a high diagnostic sensitivity already in the first stage of Lyme borreliosis, in order to diagnose and treat patients before they develop severe symptoms of the later stages of Lyme borreliosis.
Laboratory diagnosis of Lyme borreliosis has been possible since the discovery of
B. burgdorferi
in 1982. However, the ultimate diagnostic assay has not yet been developed. Laboratory confirmation of Lyme borreliosis still relies mainly on the detection of antibodies to
B. burgdorferi
. Assays based on whole cell
B. burgdorferi
extracts lack diagnostic specificity due to antibodies cross-reacting with antigens from a wide range of bacterial species. Western blotting (WB) has proved difficult to perform due to strain differences, the complexity of the band patterns, and inherent problems in standardization of Western blotting in general. Efforts have therefore mainly been directed towards identification of single immunodominant antigens, either in the native form or as recombinant proteins, which can be purified and used as test antigens.
According to Western blot studies there are only two
B. burgdorferi
antigens that meet the essential criterium of eliciting an early and strong antibody response in the majority of patients. These are the
B. burgdorferi
flagellum and the outer surface protein C (OspC). Whereas the performance of EIA's using purified native
B. burgdorferi
flagellum is well documented, the reported experience with OspC EIA's is still limited.
Other routes to the specific diagnosis of Lyme borreliosis have been suggested. A fraction of membrane related proteins and lipids known as “fraction B” disclosed in EP-A-445,135 has been demonstrated to exhibit an improved diagnostic specificity, but the provision of fraction B requires that
Borrelia burgdorferi
sensu lato is cultured and subsequently treated in a series of steps.
A high prevalence of IgM anti-OspC antibodies has been found in patients in the two first stages of Lyme borreliosis by means of Western blotting, using native and recombinant OspC (rOspC) and by means of ELISA, using rOspC (Fung B. P. et al. (1994); Gerber M. A. et al. (1995); Wilske B. et al. (1994); Padula S. J. et al. (1994)).
SUMMARY OF THE INVENTION
In general, it has been concluded by the present inventors that the sensitivity of diagnosis of the early stages of Lyme borreliosis could be increased by combining the results from an immunoassay based on the detection of anti OspC antibodies and the results from the current available immunoassays for the flagellum.
More specifically, the present inventors have reached the conclusions that certain C-terminal fragments of OspC comprise an epitope which is essential in the human immune system's recognition of OspC. Additionally, it has been found that the serodiagnostic sensitivity of said C-terminal fragments is surprisingly high when compared to that of full-length OspC.
These conclusions have been reached after immunological experiments which surprisingly have revealed that 1) a synthetic peptide derived from the C-terminus of OspC of
B. burgdorferi
sensu lato exhibits an immunological sensitivity in detecting sera from human Lyme borreliosis patients which is at least 85% of the sensitivity of full length recombinant
B. burgdorferi
sensu lato derived OspC (rOspC
fl
) when used in similar assays, and 2) a recombinant
B. burgdorferi
sensu lato OspC truncate which lacks the 7 carboxyterminal amino acids (rOspC
t
) exhibits, when compared to full length recombinant OspC (rOspC
fl
), a very poor, if any, immunological reactivity with sera from patients suffering from Lyme borreliosis.
These immunological experiments were part of scientific work which aimed at producing an immunoassay based on recognition by antisera of recombinant OspC. However, in the first attempt, a diagnostic sensitivity of less than 5% was achieved in early stage of Lyme borreliosis (this involves the first and second stage of Lyme borreliosis), cf. Example 1. Here, the deduced amino acid sequence of three OspC proteins representing each of three
B. burgdorferi
genospecies (
B. burgdorferi
sensu stricto,
B. garinii
, and
B. afzelii
) were used as test antigens. However, the recombinant proteins all lacked the seven C-terminal amino acid residues, because these had not yet been determined for the three pertinent isolates of
Borrelia burgdorferi
sensu lato.
In the second attempt the entire recombinant OspC proteins (rOspc
fl
) from all three strains were produced, including the last seven amino acid residues, which had then been deduced. Further, the deduced amino acid sequence in the C-terminus of the OspC protein was identical for the genospecies of
B. garinii
and
B. afzelii
used in the first attempt, whereas the
B. burgdorferi
sensu stricto genospecies had a valine residue instead of an alanine residue in position 205. By employing the rOspC
fl
proteins as test antigens in an ELISA, diagnostic sensitivities were achieved of 44% for IgM in the first stage of Lyme borreliosis and 48% for IgM in the second stage of Lyme borreliosis in a set of preliminary tests. The diagnostic sensitivity for borreliosis was identical for all three genospecies. Therefore, a more comprehensive testing of the immunological reactivity of rOspC
fl
and of synthetic C-terminus derived peptides were performed, cf. Example 2.
On the background of these findings, it was concluded that the seven carboxy-terminal amino acid residues comprise, constitute, or form part of an antigenic epitope which is essential in the human immunological recognition of OspC and it was therefore conceived that this epitopic region can be the basis for novel and improved diagnostic means.
It was further investigated to what degree each of the single amino acids contributes to the immune reactivity of the C-terminus of OspC, and it was found that the last 5 amino acids can only be varied to a very limited degree, whereas e.g. alanine substitutions in other amino acids in the C-terminus had no or little impact on immune reactivity.
A number of advantages can be provided by using short OspC fragments as part of an immunological agent in the diagnosis of early stage Lyme borreliosis. Most important, an immunoassay, such as an ELISA, which is based on a synthetic peptide—as opposed to using full-length or near-full-length OspC—simplifies the preparation and purification steps of the components of the assay and thus helps standardize the assay.
Further, the use of a short peptide in an immunoassay may lead to a decrease in the cross-reactivity with antibodies raised against other antigens as a consequence of the abolishment of a large number of

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