Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-12-13
2003-12-09
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S009100, C424S009200, C424S139100, C424S164100, C424S184100, C424S185100, C424S190100, C424S192100, C424S200100, C435S004000, C435S007100, C435S007200, C435S007320, C435S320100, C435S440000, C435S471000, C436S501000, C530S300000, C530S350000
Reexamination Certificate
active
06660274
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of pharmaceutical and diagnostic compositions useful in the diagnosis, treatment and prophylaxis of Lyme borreliosis. More specifically, the invention provides an isolated natural surface antigen of Borellia and antibodies thereto for use in the diagnosis, treatment or prevention of Lyme disease.
BACKGROUND OF THE INVENTION
Borrelia burgdorferi
(sensu lato) is a generic term which encompasses several Borrelia species associated with, and believed to be, the causative agent of Lyme borreliosis (Lyme disease):
B. burgdorferi
sensu stricto,
B. garinii,
and
B. afzelii.
This disease is transmitted by the bite of various species of Ixodes ticks carrying the spirochete. The main reservoir of the infection in the United States is the white footed mouse,
Peromyscus leucopus,
and the infection can be transmitted to many mammalian species including dogs, cats, and man [J. G. Donahue, et al,
Am. J. Trop. Med. Hyg.,
36:92-96 (1987); R. T. Green, et al,
J. Clin. Micro.,
26:648-653 (1988)]. Despite the presence of an active immune response, the disease persists for years in patients. Such persistence is postulated to be the result, at least in part, of antigenic variation in the bacterial proteins [J. R. Zhang et al,
Cell,
89:275-285 (1997)].
The diagnosis of Lyme disease in humans and animals has been compromised by the lack of definitive serology leading to rapid and accurate testing. Current diagnostic tests suffer from low sensitivity and specificity, as illustrated by a recent survey of diagnostic laboratories' performance issued by the Wisconsin State Laboratory of Hygiene [L. Bakken et al,
J. Clin. Microbiol.,
35:537 (1997)]. A simple, sensitive and specific diagnostic composition and method for early detection of Lyme disease is needed in the art.
Publications relating to proteins and polypeptides of
Borrelia burgdorferi
have suggested their use as diagnostic or pharmaceutical agents. Such proteins and polypeptides include outer surface proteins A and B (OspA and OspB), flagellin, and other proteins designated P21, P39, P66, and P83 according to their estimated molecular weights [A. G. Barbour et al,
Infect. Immun
45:94-100 (1984); W. J. Simpson et al,
J. Clin. Microbiol.,
28 1329-1337 (1990); K. Hansen et al,
Infect Immun.,
56:2047-2053 (1988); K. Hansen et al,
Infect. J. Clin. Microbiol.,
26 338-346 (1988); B. Wilske et al,
Zentral, Bakteriol. Parasitenkd. Infektionshkr. Hyg. Abt.
1 Orig. Reihe. A. 263:92-102 (1986); D. W. Dorward et al,
J. Clin. Microbiol.,
29:1162-1170(1991); published NTIS U.S. patent application No. 485,551; European patent application No. 465,204, published Jan. 8, 1992; International Patent Application No. PCT/US91/01500, published Sep. 19, 1991; International Patent Application No. PCT/EP90/02282, published Jul. 11, 1991; International Patent Application No. PCT/DK89/00248, published May 3, 1990; International patent application No. WO92/00055, published Jan. 9, 1992].
A preferred protein candidate for a vaccine is OspA [M. Philipp et al,
J. Spirochetal and Tick
-
borne Diseases,
3:67-79 (1996)]. The expression of OspA is either abrogated or down-regulated when the spirochetes are en route from the tick's midgut to the salivary glands, as blood feeding is taking place [A. DeSilva et al,
J Exp. Med.
183:271-275 (1996)]. This phenomenon generates potential problems that may diminish the OspA vaccine's efficacy. Spirochetal attrition may occur only within the tick midgut [A. DeSilva et al, cited above] and not upon infection of the vertebrate host. Because the saliva of
Ixodes scapularis
contains a decomplementing factor [T. Mather et al, “Ixodes saliva: vector competence for
Borrelia burgdorferi
and potential vaccine strategies”, in VII International Congress on Lyme Borreliosis, San Francisco, Calif. (1996)], spirochetal attrition within the tick's midgut might occur via a mechanism involving only antibody and not complement.
Although the mode of action of antibody-dependent killing is not fully understood, it appears to be a less efficient mechanism of killing than that mediated by antibody and complement, acting together [M. Sole et al,
Infect. Immunol.,
66:2540-2546 (1998)]. Thus, it may permit evasion from the midgut of those spirochetes that have a low surface density of OspA. Indeed, although OspA is an abundant
B. burgdorferi
protein, only a minor fraction of OspA molecules is exposed on the outer surface of the spirochete [D. Cox et al,
Proc. Natl. Acad. Sci.,
93:7973-7978 (1996)]. Thus, small variations in the absolute number of OspA surface molecules may cause significant differences in the spirochete attrition rate and make it possible for a fraction of the resident spirochetes to abscond to the salivary glands.
OspA escape mutants will readily avoid killing altogether, and if they are infectious to the vertebrate host, they will contribute to diminish the OspA vaccine efficacy even further. In clonal populations of
B. burgdorferi
which are allowed to grow in vitro, the prevalence of mutants that resist killing by anti-OspA antibody ranges between 10
−5
and 10
−2
[A. Sadziene et al,
J. Exp. Med,
176:799-809 (1992)]. If such frequencies are reproduced in a feeding nymph, in which spirochete numbers may reach a mean of 7,848 within 15 hours of attachment [A. DeSilva et al,
Am. J. Trop. Med. Hyg.,
53:397-404 (1995)] then several mutants may be present in a single tick. Typical mutant phenotypes include those that express neither OspA nor OspB and, frequently, expressors of a chimeric molecule composed of an N-terminal fragment of OspA fused to a C-terminal fragment of OspB. These deletion mutants have been found in multiple strains of
B. burgdorferi
[P. Rosa et al,
Mol. Microbiol.,
6:3031-3040 (1992)] and in several tick isolates from California [T. Schwan et al,
J. Clin. Microbiol.,
31:3096-3108 (1993)]. Chimeric (deletion) mutants, which are able to resist killing with anti-OspA antibody alone, are killed by the combined action of antibody and complement. Since complement appears to be nonfunctional within the tick [T. Mather et al, cited above], and OspA, and probably its chimeric form as well, are not expressed within the vertebrate host shortly after infection, chimeric OspA escape mutants could infect the vertebrate host. Further, it is estimated that up to 2% of Ixodid ticks are only partially fed and are therefore still questing [Y. Lobet, personal communication]. If such ticks have taken their incomplete meal from a
B. burgdorferi
-infected host, they will have spirochetes in their salivary glands which do not express OspA and which will readily infect an OspA-vaccinated host.
No booster effect has been observed [M. Philipp et al, cited above] nor should one be expected upon spirochetal challenge of an OspA-vaccinated host. Hence, the OspA vaccine may require repeated administrations to maintain effective antibody titers.
There is a thus a need in the art for additional, and improved, methods and compositions for prevention of Lyme disease in humans and animals, and for treatment thereof.
SUMMARY OF THE INVENTION
The present invention satisfies the need in the art by providing methods and compositions for prevention of Lyme Disease based on spirochetal antigens that are expressed by the spirochete during its residence in the vertebrate host. Such methods and compositions may also be employed for the treatment of Lyme Disease.
In one aspect, the invention provides an isolated
Borrelia burgdorferi
sensu lato surface antigen which is expressed in vivo by the spirochete in the vertebrate host. The antigen, referred to herein as P39.5, is further characterized as having a relative molecular mass of 39.5 kDa. Fragments of this antigen are also useful in the compositions and methods of this invention.
In another as
Howson and Howson
Swartz Rodney P
The Administrators of the Tulane Educational Fund
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