Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-12-17
2002-01-29
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C435S243000, C435S261000, C435S252100, C424S269100
Reexamination Certificate
active
06342230
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to vaccine against a rickettsial disease called heartwater, to a rickettsia used in the preparation of said vaccine, to a method of producing a said vaccine, and to a method of vaccinating mammals and, in particular, domestic and wild ruminants.
BACKGROUND OF THE INVENTION
Rickettsiae are non-motile bacteria which depend on the intracellular milieu of host cells for growth and are capable of infecting mononuclear and endothelial cells. As a result, they were initially considered to occupy a special taxonomic niche between viruses and bacteria but are now classified within the bacteria.
Rickettsiae are transmitted to mammals by arthropod vectors such as lice, ticks, and mites. They are responsible for a variety of diseases including typhus, Rocky Mountain spotted fever, and rickettsial pox. In southern Africa, tick bite fever and heartwater are common rickettsial diseases. As a result of their dependence on the intracellular milieu of their host cells for growth, it is difficult to treat rickettsial infections. To enable a host mammal to develop immunity against this infection, bacteriostatic antibiotics are used. These include the tetracycline group of antibiotics and the chloramphenicols.
Heartwater is an economically important disease of domestic ruminants caused by the rickettsia
Cowdria ruminantium
and transmitted by Amblyomma tick species. Heartwater is a constraint to livestock production because it can cause mortality rates of 20-80% in susceptible animals.
Currently, there is only one method of vaccination of domestic animals against heartwater. This method involves infection of the animal intravenously with live virulent
Cowdria ruminantium
rickettsiae followed by treatment with tetracycline antibiotics when clinical disease is manifested. The treated animal then develops immunity to heartwater. However, if treatment is too early the animal fails to become immunized and, if it is treated too late, death can occur. This method of vaccination is both laborious and expensive and is accompanied by considerable risk and uncertainty. Hence a vaccine that is easy to administer, is inactivated and induces protection against mortality would have a major impact on livestock production in areas of the world that are affected by this disease, namely sub-Saharan Africa and the eastern Caribbean. Such a vaccine would have wide-scale application if it did not require stringent handling, transportation, and storage conditions.
BRIEF SUMMARY OF THE INVENTION
The subject invention provides materials and methods for preventing, or reducing the severity of, heartwater. In accordance with this invention, there is provided a vaccine against heartwater comprising cultured inactivated
Cowdria ruminantium
rickettsiae mixed with a suitable adjuvant. The vaccine of the subject invention can be injected into an animal and, once injected, induces immune responses which protect the animal from severe heartwater when exposed to infection with
Cowdria ruminantium
rickettsiae.
In a preferred embodiment of the subject invention, there is further provided for the rickettsia to be the Mbizi strain of
Cowdria ruminantium
. The rickettsia of the subject invention was deposited with the National Bank for Industrial Micro-organisms and Cell Culture in Sofia, Bulgaria (NBIMCC) located at 125, Tsarigradskochausse Blvd., Block 2, 1113 Sofia, Bulgaria under accession number 3568 on Nov. 2, 1998.
In a specific embodiment, the rickettsia are cultured, using methods known in the art, in bovine endothelial cells and harvested from the cultured supernate by centrifugation. The harvested rickettsiae are inactivated by suspension in a solution of &bgr;-propiolactone in phosphate buffered saline at a temperature of 4° C. for a period of 2.5 hours. The inactivated rickettsiae can be stored frozen at a temperature of between −40° C. to −80° C., prior to mixing with an adjuvant.
In a preferred embodiment, the pathologically inactivated rickettsiae are mixed with an adjuvant. The concentration of rickettsiae is approximately 300 &mgr;g protein per milliliter of phosphate buffered saline mixed with about 1 ml of adjuvant. The vaccine may be stored on ice until ready for use. The vaccine can be injected, subcutaneously, preferably in the shoulder region of the animal.
The invention further extends to a method of producing a vaccine for use against heartwater. In a specific embodiment, this method comprises the steps of:
(a) isolating and culturing pathologically active rickettsiae in a suitable culture medium;
(b) harvesting the rickettsiae from the culture medium once the concentration of rickettsiae in said culture medium has reached a predetermined minimum or until the happening of a predetermined event;
(c) rendering the rickettsiae pathologically inactive; and
(d) mixing the pathologically inactive rickettsiae with a suitable adjuvant.
The invention also provides for a method of immunizing mammals against heartwater comprising inoculating an animal, preferably cattle, alternatively sheep, goats, deer, springbok and antelope, further alternatively other susceptible ruminants, subcutaneously, preferably in the shoulder region, with a vaccine as described above and reinoculating the animal after the first inoculation, preferably between 4 and 8 weeks after the first inoculation.
DETAILED DISCLOSURE OF THE INVENTION
The subject invention provides materials and methods for inducing a protective immune response against heartwater. In accordance with this invention, there is provided a vaccine against heartwater comprising cultured inactivated
Cowdria ruminantium
rickettsiae mixed with a suitable adjuvant. The vaccine of the subject invention can be injected into an animal and, once injected, induces immune responses which protect the animal from severe heartwater when exposed to infection with
Cowdria ruminantium
rickettsiae.
Specifically exemplified herein is the use of the Mbizi strain of
Cowdria ruminantium
. Preferably, the Mbizi strain is formulated with an appropriate adjuvant and then used as a vaccine composition.
The Mbizi strain of
Cowdria ruminantium
accession No. 3568, has been deposited for the purposes of this patent application under conditions that assure that access to this culture is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.
In a specific embodiment, the rickettsiae are cultured, using methods known in the art, in bovine endothelial cells and harvested from the cultured supernate by centrifugation. The pelletised organisms are then resuspended and washed in phosphate buffered saline after which they are rendered pathologically inactive by washing for 2.5 hours in a 0.4% by volume of &bgr;-propiolactone in phosphate
Barbet Anthony F.
Burridge Michael J.
Mahan Suman M.
Smith Gillian E.
Graser Jennifer E.
Saliwanchik Lloyd & Saliwanchik
University of Florida
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