Osp a proteins of Borrelia burgdorferi subgroups, encoding...

Details Osp a proteins of Borrelia burgdorferi subgroups, encoding... Osp a proteins of Borrelia burgdorferi subgroups, encoding...

1999-04-01

2004-01-13

Minnifield, Nita (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...

C424S184100, C424S234100, C424S235100, C530S300000, C530S350000

Reexamination Certificate

active

06676942

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel antigens, to methods for their production, to compositions containing them and to their use in the prevention, treatment and diagnosis of Lyme Disease in humans and other animals. In particular the present invention discloses novel serotypes/genotypes of the outer surface protein (Osp A) from the spirochete
Borrelia burgdorferi,
the causative agent of Lyme disease, and vaccine and diagnostic reagents based on
B. burgdorferi
of more than one subgroup.
BACKGROUND OF THE INVENTION
Lyme disease in humans is a chronic progressive disease caused by
B. burgdorferi,
which is transmitted to humans mainly by Ixodes ticks. The disease attacks many organs, notably the skin, heart, liver, central and peripheral nervous system, kidneys as well as the musculoskeletal system.
Lyme disease itself is the most common vector borne infection in the USA and has been reported in every continent except Antarctica.
A number of groups have isolated and proposed the major surface protein (Osp A) of
B. burgdorferi,
as being a potential vaccine candidate for use against Lyme disease. For example, International patent application published under WO90/04411 (SIMBICOM) discloses the cloning and expression of an Osp A protein derived from
B. burgdorferi
B31 and its use as a vaccine, M. M. Simon and colleagues have cloned and expressed Osp A from
B. burgdorferi
ZS7 (European Patent Application No. 90117943.2 published under No. 0418 827), and demonstrated its protective capacity to induce antibodies in passively immunised SCID mice (PNAS: 87 1990, 3768-3772). Flavell and colleagues (Science (1991) 250 p553-556) have cloned and expressed the gene for Osp A from
B. burgdorferi,
N40 and have demonstrated its protective efficacy in C3H/He mice.
All isolates of
B. burgdorferi
identified above appear to be closely related. However we have now identified six subgroups of
B. burgdorferi
by analysing 55 spirochete isolates from different geographical areas and sources with series of immunological, biochemical and molecular genetic techniques. The finding of different subgroups of
B. burgdorferi
isolates have important implications for both effective vaccination and diagnosis of Lyme disease. Since ELISA diagnostic assays directed against a strain of which species N40 is a member would not or only partially cross react with species from other subgroups. Equally a vaccine based on only one Osp A for example from N40 would not provide optimal protection against
B. bugdorferi
from a different subgroup.
BRIEF SUMMARY OF THE INVENTION
The present inventors have identified five additional subgroups of
B. burgdorferi
based on their Southern blotting and PCR amplification of Osp A sequences, and differences in the amino acid and nucleic acid sequences themselves and also in reactivity against monoclonal antibodies to Osp A proteins.
DETAILED DESCRIPTION OF THE INVENTION
The surprising discovery that
B. burgdorferi
exhibits such heterogeneity has important implications for vaccines against, and diagnostics reagents for the detection of, Lyme disease, since vaccines or diagnostic based on an Osp A from one group of
B. burgdorferi
may not detect or protect against infection of
B. burgdorferi
from a second group. Indeed the present inventors have shown that whereas protection can be afforded by anti Osp A antibodies generated by recombinant Osp A or viable or killed organisms within a group of closely related strains, no or only partial, protection is observed if challenge is made with an organism from a different group.
A first subtype having representative ZS7, B31, N40 is referred to herein as group I (alternative nomenclature refers to group I strains as group A).
A second subgroup hereinafter group II (alternatively known as group B) is herein disclosed and is exemplified by the species ZQ1. This species has been deposited at the DSM Deutsche Sammlung von Mikroorganismen Und Zellkulturen of Mascheroderweg 91B D-3300 Braunschweig on Jul. 11, 1991 and given the accession No. DSM6606. This group is distinct in a number of ways from group I.
Firstly, plasmid analysis of strain ZQ1 when compared with representatives from group I shows that ZQ1 has at least two unique plasmids of 18 and 14 Kd. Furthermore plasmid of 16 Kb found in group I strains is absent from strain ZQ1. Secondly when examined by polyacrylamide gel electrophoresis Osp A from ZQ1 has an apparent molecular weight of 32 Kd.
Thirdly various monoclonal antibodies specific for Osp A of group I, such as the antibodies designated LA2 L26 LA28 LA33 do not react with Osp A from group II. LA2 and LA26 antibody are known and have been described in European patent application No. EP0418827. The hybridoma producing LA2 has been deposited under accession No. ECACC 89 09 1302 on Sep. 13, 1989 at the European Collection of animal cell cultures, Public health laboratory services Porton Down Wiltshire SP40J9. The hybridoma producing LA26 has been deposited at the same culture collection under accession No. 9005406 on Jun. 28, 1990.
However an other monoclonal antibody, LA31.1, does react with the Osp A species of subgroups I and II suggesting common epitopes among Osp A species of different subgroups do exist.
We have also characterised Osp A DNA from group II strains by PCR amplification. Two pairs of Osp A primers were used, these being designated prOsp A1-prOsp A4 and prOsp A1-prOsp A2.
The primers have the following sequences:
prOsp A1: 5′-GGGAATAGGT CTAATATTAG CC-3′  (SEQ.ID NO:13)
prOsp A2: 5′-TGCCTGAATT CCAAGCTGCA-3′  (SEQ. ID NO:14)
prOsp A4: 5′-GCAGTTAAAG TTCCTTCAAG AACATAGC-3′  (SEQ. ID NO:15)
and correspond to nucleotides at positions 138-160 (prOsp A1), 611-638 (prOsp A4), and 759-778 (prOsp A2), of ZS7 Osp A respectively. Total DNA of Group II,
B. burgdorferi
are distinguishable from that of Group I in that group II will not permit successful PCR (polymerase chain reaction) amplification when prOsp A1 and prOsp A2 are used as primers whereas total DNA from group I will permit a successful reaction. In contrast, when prOsp A1 and prOsp A4 are used as primers, then both DNA's of group I and group II will undergo polymerase chain reaction.
These results show that group II,
B. burgdorferi
have a different Osp A sequences to group I Osp As.
Equally, group II Osp A DNA, can be distinguished from group I Osp A DNA by Southern blotting utilising Osp A ZS7 sequence as a probe. Digesting genomic DNA of
B. burgdorferi
group II with the restriction enzyme Hind III shows a different banding pattern than that of group I strain.
For example a group I strain reveals two hybridisation fragments of 1.2 kb and 0.3 kb whereas a group II strain expressed two fragments of 0.9 kb and 0.4 kb.
Also, passive immunisation of SCID mice with immune sera to Osp A from group II will not protect against
B. burgdorferi
group I challenge.
Finally when compared to the sequence of Osp A ZS7 the DNA and amino acid sequence of ZQ1 strain Osp A Sequence ID 1 and 2 (i.e. a group II strain), demonstrates the substantial variation between a group I and group II strain at both the DNA and amino acid levels.
The surprising inability to protect against heterologous challenge means that a vaccines based on a single Osp A from a group I organism may be ineffective against infection with
B. burgdorferi
from group II organism and vice versa. Consequently there is a need for a vaccine which will protect against group II infection.
Accordingly there is provided DNA sequence encoding an Osp A derived from a
B. burgdorferi
characterised in that polymerase chain reaction amplification will take place utilising prOsp A1-A4 primers, but not prOsp A1-A2, and the invention further provides purified or isolated Osp A encoded thereby.
Preferably the Osp A of the present invention will not react with monoclonal antibody LA2 or LA26. Most preferably the Osp A of the present invention will have a relative molecular weight of 32 kDa as determined by SDS electr

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