Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1998-07-15
2001-12-04
Saoud, Christine J. (Department: 1647)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093200, C435S440000, C435S471000, C435S252300
Reexamination Certificate
active
06326001
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to vaccines used in veterinary applications and, more particularly, to a live, recombinant, attenuated vaccine for disease states that are caused by organisms that include capsule where the presence of the capsule is required for virulence but not immunoprotection. The invention has specific application to a recombinantly produced vaccine that has been engineered such that it lacks capsule.
2. Description of the Prior Art
Vaccines are preparations used to prevent specific diseases in animals and humans by inducing immunity. This is accomplished by exposing a patient to an antigen for a particular disease which, in turn, causes the immune system of the patient to produce large quantities of antibody. The presence of the antibody in the patient's blood protects the patient from a later attack by the disease causing agent. Vaccines may either be composed of subunits of the agent, or the live or killed agent itself For example, poliomyelitis, commonly referred to as “polio”, is typically prevented by either administering a live, attenuated oral poliovirus vaccine, which is common practice for treating children, or by administering a killed or inactivated poliovirus vaccine, which is the usual practice for treating adults since they are generally at higher risk for contracting polio from the live vaccine. If a live vaccine is to be used, its virulence must be attenuated in some way; otherwise the virus in the vaccine will cause the disease it is intended to protect against.
A number of diseases are caused by encapsulated bacteria wherein the capsule, which is the gum-like layer of polysacharide or polypeptide exterior to the cell wall of these bacteria, is required for pathogenesis. Swine pleuropneumonia is one example, and virulence factors for
Actinobacillus pleuropneumoniae
, the bacterium which causes the disease, include capsular polysaccharide, endotoxin, and protein exotoxins. Swine pleuropneumonia is one of the major respiratory diseases affecting swine production throughout the world, and accounts for millions of dollars in annual losses to the industry in the United States alone.
U.S. Pat. No. 5,429,818 to Inzana, which is herein incorporated by reference, discloses that non-encapsulated mutants of
Actinobacillus pleuropneumoniae
are a virulent and capable of providing excellent protection against subsequent exposure to the virulent bacteria. The non-capsulated mutants described in Inzana were prepared by ethylmethanesulfunate mutagenesis. However, such procedures have the disadvantages that some spontaneous or chemically induced mutants may not be stable, and the nature of the mutation(s) is (are) unknown.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a safe and effective, live, attenuated, recombinant vaccine for diseases caused by bacteria and fungi which are normally encapsulated and where the capsule is required for virulence but not immunoprotection.
It is another object of this invention to genetically engineer certain bacteria or fungi to lack capsule such that they are rendered a virulent and the genetic nature of the mutation is known.
It is yet another object of this invention to provide a safe and effective, live, attenuated, recombinant vaccine for pleuropneumonia.
According to the invention, a recombinant, live, attenuated strain of
Actinobacillus pleuropneumoniae
which has been genetically engineered to lack capsule has been produced. Since the capsule is required for virulence, but not immunoprotection, the strain will be useful as a vaccine against swine pleuropneumonia. The vaccine was produced by cloned plasmid vector that cannot replicate in
A. pleuropneumoniae
. The capsule export and synthesis genes of
A. pleuropneumoniae
serotype 5 were sequenced. A large deletion was made in the cloned synthesis genes for the capsule, and genes encoding for kanamycin resistance and sucrose sensitivity were then cloned into the deleted site to serve as marker genes. This suicide vector was inserted into a virulent
A. pleuropneumoniae
serotype 5 strain using electroporation in order to obtain a homologous recombination event by double cross over between homologous regions of the chromosome and plasmid. Four isolates were obtained, and each lacked iridescence suggesting a lack of capsule. The lack of capsule and the deleted region of the capsule genes was confirmed in one strain by dot blotting and Southern blotting, respectively. The presence of the marker genes in the recombinant strain was also confirmed. No other change in any other phenotypic properties could be identified, and the marker genes were not found in other regions of the chromosome. The recombinant strain, referred to as J45-100, was very serum sensitive, had reduced virulence in pigs at ten times the 50% lethal dose for the parent strain, and should provide protection for swine against pleuropneumonia.
This invention will be useful for producing vaccines against any encapsulated organism that produces toxins or other virulence factors where the capsule is required for virulence but not immunoprotection. All that will be required will be to clone the genes encoding for capsule synthesis for the organism, and then delete and replace the section of the cloned gene with a marker gene on a suicide vector, and then introduce the vector into the desired organism and screen for a genetically modified organism that lacks capsule. The invention should be useful in producing vaccines for additional bacteria infectants including, but not limited to,
Pasteurella multocida, Pasteurella haemolytica
, and
Pseudomonas aeruginosa
, as well as fungi such as
Cryptococcus neofornans
which is a pathogen associated with acquired immune deficiency syndrome (AIDS) in cats and humans.
REFERENCES:
patent: 5429818 (1995-07-01), Inzana
patent: WO9310815 (1993-06-01), None
Benjamini, Immunology: A short course, Wiley-Liss, pp. 4-5, 1991.*
Webster's New World Dictionary, immunity, p. 675, 1991.
Inzana Thomas J.
Ward Christine
McGuireWoods LLP
Saoud Christine J.
Turner Sharon
Virginia Tech Intellectual Properties Inc.
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