Immunologically active proteins from Borrelia burgdorferi,...

Details Immunologically active proteins from Borrelia burgdorferi,... Immunologically active proteins from Borrelia burgdorferi,...

2003-03-26

2004-10-26

Swartz, Rodney P (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S009100, C424S009200, C424S184100, C424S185100, C424S200100, C424S234100, C435S009000, C435S069100, C530S300000, C530S350000, C536S023100, C536S023700

Reexamination Certificate

active

06808711

ABSTRACT:

Lyme borreliosis is the most frequent of the infectious diseases of humans which are transmitted by ticks. A substantial proportion of the ticks which serve as vectors for transmitting Lyme borreliosis are infected with the pathogen of Lyme
borreliosis,
i.e. the spirochete
Borrelia burgdorferi.
Depending on the geographic region, the percentage infected can vary from 1% up to 100%.
An infection with
B. burgdorferi
leads to a complex clinical picture, which can be subdivided into different stages.
In some cases, the infection with
B. burgdorferi
can take a subclinical course. However, late sequelae, which are caused by unrecognized or untreated Borrelia infections, frequently present a problem. Particularly because of the dangerous diseases, such as carditis, myositis, iritis, panophthalmitis or neurological manifestations, which can occur when infections are not recognized or not treated, it is important to be able to diagnose a possible infection with
B. burgdorferi
as reliably and accurately as possible.
The pathogen can be detected in patient material, in particular in the early stages. However, it is disadvantageous in this context that culturing
B. burgdorferi
is relatively difficult and therefore as a rule left to specialist laboratories.
It is desirable, therefore, to detect the antibodies in serum and also in cerebrospinal fluid, in the case of neurological manifestations, and in joint aspirates in the case of joint ailments.
For diagnosis, it is important to be able to decide whether an infection only occurred recently, or whether the infection is one which took place some while ago. This distinction can be made immunologically determining the nature of the detected antibodies; as a rule, IgM antibodies suggest that the infection only occurred recently whereas IgG antibodies suggest that the infection took place some while ago.
It is also important for diagnosis that the diagnostic tests are specific, i.e. that no cross-reactions occur with those bacterial pathogens, such as
Treponema pallidum,
which are to certain degree phylogenetically related to the borrelias.
On the other hand, however, it is also of importance for diagnosis that, if at all possible, all the strains of
Borrelia burgdorferi
can be recognized by the proteins or peptides which are employed in the test method.
Since Lyme borreliosis is widespread and since an infection can readily be transmitted by means of tick bite, there is also a substantial need to develop vaccines which ensure immune protection against borrelia infections.
Those proteins which, on the surface of the bacteria, come into contact with the immune system of the infected organism are particularly suitable for developing vaccines.
Two proteins have now been found, within the context of the present invention, which are particularly suitable both for diagnosis and for developing vaccines.
The present invention relates, therefore, to immunologically active proteins from
Borrelia burgdorferi
which are present in a form which is free of other proteins derived from
Borrelia burgdorferi
and which exhibit the sequence of the protein 1829-22A, having the amino acid sequence (SEQ ID NO:1)
MKKFNLIIEALFAILLTACNFGLMEETKIALESSSKDVKNKILQIKKDAE

DKGVNFAAFTSSETGSKVTNGGLALREAKIQAINEVEKFLKRIEEEALKL

KEHGNSGQFLELFDLLLEVLESLEPIGIKGLKDFISEEAKCNPISTSERL

IEVKVQIENKMEEVKRKQNLNKERKSNKGKKKK
or a part sequence thereof having at least 10 consecutive amino acids, or the sequence of the protein 1829-22B, having the amino acid sequence (SEQ ID NO:2)
MIKYNKIILTLTLLASLLAACSLTGKARLESSVKDITNEIEKAIKEAEDA

GVKTDAFTETQTGGKVAGPKIRAAKIRVADLTIKFLEATEEETITFKENG

AGEDEFSGIYDLILNAAKAVEKIGMKDMTKTVEEAAKENPKTTANGIIEI

VKVMKAKVENIKEKQTKNQK
or a part sequence thereof having at least 10 consecutive amino acids.
In accordance with the invention, preference is given to using those part sequences which possess epitopes which are diagnostically and/or therapeutically relevant. In the case of protein 1829-22A, whose sequence is given in the sequence listing under SEQ ID NO:1, the following part sequences are particularly preferred: the region between amino acid 31 (Lys) and amino acid 55 (Asn). Another preferred polypeptide is located between position 60 (Thr) and position 71 (Gly). A further preferred polypeptide is located between amino acid 82 (Gln) and amino acid 108 (Gln). The C-terminal region between amino acid 130 (Gly) and amino acid 183 (Lys) is also particularly preferred.
In the case of protein 1829-22B, which is represented by Seq. ID No. 2, the following part regions are particularly preferred: amino acid 61 (Gln) to amino acid 71 (Ile); amino acid 87 (Glu) to amino acid 108 (Gly); amino acid 121 (Glu) to amino acid 145 (Asn), and the C-terminal region from amino acid 150 (Ile) to amino acid 170 (Lys). The positions of the amino acids are given in the sequence listings. The peptides which exhibit the abovementioned part sequences can either be prepared by means of chemical synthesis or be expressed recombinantly in suitable host systems.
The proteins or peptides according to the invention may be prepared by means of recombinant methods, which has the advantage that no other proteins derived from
B. burgdorferi
are associated with the desired proteins. Alternatively, suitable peptides may also be synthesized in the classical chemical maimer. Such peptides are also free of immunologically inactive impurities. However, it is entirely possible to employ the proteins or peptides according to the invention in test kits or in vaccines together with other proteins which have been isolated from
B. burgdorferi.
The term immunologically active protein which is used within the context of the present invention, encompasses not only protein which comprises the complete amino acid sequence of protein 1829-22A or protein 1829-22B but also parts of these proteins which are at least long enough to encompass at least one linear epitope. In general, the minimum length of a peptide according to the invention which is able to exhibit the property of an epitope is at least 6, preferably 10, particularly preferably 25 and very particularly preferably at least 50 amino acids.
The fact must be taken into consideration that, in the individual strains of
Borrelia burgdorferi,
at least minor changes occur in the amino acid sequence of the protein, depending on the particular strain. The present invention therefore also relates immunologically active proteins or peptides which exhibit high degree of homology with the above-described amino acid sequences.
The immunologically active proteins or peptides according to the invention exhibit an homology of at least 60%, preferably at least 80% and particularly preferably at least 90%, based on proteins 1829-22A and 1829-22B according to the invention. The term homology of 90% is understood as meaning, for example, that, in the homologous peptide,
9
out of 10 amino acids are identical to the corresponding amino acids at the homologous sites in amino acid sequence 1829- 22A or amino acid sequence 1829- 22B.
Within the context of the present invention, those regions of the proteins or peptides according to the invention are particularly important which exhibit epitopes, that is sites in the protein to which antibodies bind specifically. Determining at which sites epitopes are to be expected can be either achieved using computer methods which are known per se, or it is also possible to synthesize defined short peptides having a length of at least 10, preferably at least 25, amino acids. These peptides are then tested with positive sera to determine whether immunological reactions do or do not take place. In this way, it is possible to identify linear epitopes. These proteins or peptides, can be made by recombinant methods, with the peptides, for example, being expressed in microorganisms as fusion proteins, or the peptides can be synthesized by means of classical synthesis (Merrifield technique).
Identifying immunologically relevant epitopes is important not only for diagnosis but also, in particular, for preparing vaccines. For

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