Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1995-06-06
2001-09-04
Swartz, Rodney P. (Department: 1645)
Drug, bio-affecting and body treating compositions
Lymphokine
C424S085100, C424S093200, C435S243000
Reexamination Certificate
active
06284238
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates, in general, to granulocytic Ehrlichia. In particular, the present invention relates to a human promyelocytic leukemia cell line infected with granulocytic Ehrlichia, a method of continually growing granulocytic Ehrlichia, vaccines comprising granulocytic Ehrlichia or granulocytic Ehrlichia antigens, methods of preventing ehrlichiosis in an animal, antibodies to granulocytic Ehrlichia, and methods for detecting granulocytic Ehrlichia or antibodies to granulocytic Ehrlichia in an animal.
2. Relted Art
A wide range of virus, bacteria and parasites are transmitted to mammals by ticks. In the United States, Lyme disease is the most frequently reported tick borne disease. The etiologic agent of Lyme disease,
B. burgdofeni
, is transmitted by ticks of the genus Ixodes (Mather et al., JAVMA 205:186-188 (1994)).
Another tick borne disease, ehrlichiosis, is caused by the genera of bacteria known as Ehrlichia (for review of Ehrlichia and Ehrlichia diseases see, Rikihisa,
Clin. Microbiol. Reviews
4(3):286-308 (1991)). The various species of Ehrlichia are all members of the family of Rickettsiaceae. Unlike
B. burgdorferi
, Ehrlichia do not require a mammalian host to maintain themselves in the wild. However, if a mammalian host is involved, most Ehrlichia species infect monocytes of the blood and in several cases have been isolated in monocyte cell culture (see, for example, Dawson et al., U.S. Pat. No. 5,192,679 issued Mar. 9, 1993 which claims a canine monocyte macrophage cell line DH82 infected with
E. canis
).
Ehrlichia equi
is the causative agent of equine ehrlichiosis (Rikihisa,
Clin. Microbiol. Reviews
4(3):286-308 (1991)). Although the transmission mechanism has not been defined, it appears to be unusual in that it infects granulocytes and not monocytes.
E. equi
has a broad range of hosts. Experimental infection of horses, burros, sheep goats, dogs, cats, monkeys, and baboons has been reported (Lewis,
Vet. Parasitol
. 2:61-74 (1976)). In Europe, another granulocytic Ehrlichia,
E. phagocytophila
, causes tick borne fever in sheep, cattle, and bison, and is vectored by
I. ricinis
(Ristic, M. et al., in
Bergey's Manual of Systemic Bacteriology
, Kreig, N. et al., eds., Williams and Wilkins, Baltimore (1984), pp. 704-709). Although these organisms are carried by different ticks and are reported on different continents, they are indistinguishable from one another by serologic or PCR methods (Chen et al.,
J. Clin. Microbiol
. 32:589-595 (1994)). Recently, several fatal cases of human granulocytic Ehrlichosis have been reported in Wisconsin, Minnesota, and Connecticut as well as a non-fatal human case in Florida (Chen et al.,
J. Clin. Microbiol
. 32:589-595 (1994)). These cases of ehrlichiosis were caused by an Ehrlichia that was indistinguishable from
E. equi
and
E. phagocytophila
by PCR methods (Bakken, J. S. et al.,
JAMA
272:212-218 (1994); Rynkiewicz and Liu, New Engl. J. Med. 330:292-293 (1994)).
SUMMARY OF THE INVENTION
Unlike
E. canis
, there are no reports of cell culture conditions for the isolation of
E. equi, E. phagocytophila
, or human granulocytic Ehrlichia, nor is there an animal model for transmission. Both the cell culture and transmission model are essential in the development of appropriate antibiotic therapy and in vaccine development, as well as in the development of reliable diognostic products.
The invention further provides a method of continually growing granulocytic Ehrlichia comprising infecting a human promyelocytic leukemia cell line with granulocytic Ehrlichia and cultivating the infected cell line in a suitable culture medium.
Thus, the present invention provides a human promyelocytic leukemia cell line infected with granulocytic Ehrlichia.
The invention also provides vaccine comprising granulocytic Ehrlichia together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the granulocytic Ehrlichia is present in an amount effective to elicit protective immune responses in an animal to granulocytic Ehrlichia.
The invention further provides a vaccine comprising an granulocytic Ehrlichia antigen(s) together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the antigen is present in an amount effective to elicit protective antibodies in an animal to granulocytic Ehrlichia.
The invention also provides a method of preventing ehrlichiosis in an animal comprising administering to the animal the above-described vaccine.
The invention further provides a method for identifying granulocytic Ehrlichia in an animal comprising analyzing tissue or body fluid from the animal for a nucleic acid, protein, polysaccharide, or antibody specific to granulocytic Ehrlichia.
The invention also provides a substantially pure granulocytic Ehrlichia antigen having an approximate molecular weight selected from the group consisting of 21 kDa, 23 kDa, 40 kDa, 47 kDa, 85 kDa, 130 kDa, and 145 kDa.
The invention further provides a substantially pure polypeptide comprising the above-described antigen.
The invention also provides an isolated nucleic acid molecule coding for a polypeptide comprising the above-described antigen.
The invention further provides an antibody having binding affinity to the above-described antigen.
The invention also provides an antibody having binding affinity to the above-described antigen and/or polypeptide.
The invention further provides a nucleic acid probe for the detection of the presence of granulocytic Ehrlichia nucleic acid in a sample from an individual comprising a nucleic acid molecule sufficient to specifically detect under stringent hybridization conditions the presence of the above-described molecule in the sample.
The invention also provides a method of detecting granulocytic Ehrlichia nucleic acid in a sample comprising:
a) contacting the sample with the above-described nucleic acid probe, under conditions such that hybridization occurs, and
b) detecting the presence of the probe bound to granulocytic Ehrlichia nucleic acid.
The invention further provides a kit for detecting the presence of granulocytic Ehrlichia nucleic acid in a sample comprising at least one container means having disposed therein the above-described nucleic acid probe.
The invention also provides a diagnostic kit for detecting the presence of granulocytic Ehrlichia in a sample comprising at least one container means having disposed therein the above-described antibody.
The invention also provides a diagnostic kit for detecting the presence of antibodies to Ehrlichia in a sample comprising at least one container means having disposed therein the above-described antigen(s).
Further objects and advantages of the present invention will be clear from the description that follows.
REFERENCES:
patent: 4759927 (1988-07-01), Dutta
patent: 5192679 (1993-03-01), Dawson et al.
Winjum et al “Invitro proliferation of a canine granulocatic Ehrlichia” Vet. Microbiol. 34:355-362 1993.*
Goodman, J. L. et al., “Direct Cultivation of the Causative Agent of Human Granulocytic Ehrlichiosis,”New England J. Med.334(4):209-215 (Jan. 1996).
Anderson et al., “Ehrlichia chaffeensis, a New Species Associated with Human Ehrlichiosis,”J. Clin. Micro.29(12):2838-2842 (1991).
Bakken et al., “Human Granulocytic Ehrlichiosis in the Upper Midwest United States,”JAMA272(3):212-218 Jul. (1994).
Brouqui, P. et al., “Cytopathic Effect, Plaque Formation, and Lysis ofEhrlichia chaffeensisGrow on Continuous Cell Lines,”Infect. Immun.62(2):405-411 Feb. (1994).
Chen et al., “Identification of a Granulocytotropic Ehrlichia Species as the Etiologic Agent of Human Disease,”J. Clin. Microbiol.32(3):589-595 Mar. (1994).
Collins et al., “Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture,”Nature270: 347-349 (1977).
Eremeeva et al., “Differentiation among Spotted Fever Group Rickettsiae Species by Analysis of Restriction Fragment Length Poymorphism of PCR-Amplified DNA,”J. Clin. Micro.32(3):803-810 Mar. (1994).
Holland et al., “Is
Coughlin Richard T.
Gingrich-Baker Cindy
Aquila Biopharmaceuticals Inc.
Pennie & Edmonds LLP
Swartz Rodney P.
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