Method of attenuating pathogenic mycobacteria and strains of...

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...

Reexamination Certificate

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C424S009100, C424S009200, C424S093100, C424S093200, C424S184100, C424S234100, C435S068100, C435S086000, C435S086000, C435S086000, C435S866000, C435S440000, C435S471000

Reexamination Certificate

active

06403100

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention provides novel attenuated strains of pathogenic mycobacteria and, in particular, of
Mycobacterium tuberculosis
and
M. bovis.
Attenuation is achieved by downregulating or eliminating the expression of the &agr;-crystallin heat shock protein gene (“acr gene”). The invention also provides vaccines and methods of vaccinating mammals for protection against mycobacterial diseases including, in particular, tuberculosis.
2. Relevant Art
Pathogenic mycobacteria are the causative agents of a number of human and animal diseases. For example, tuberculosis is a health problem of considerable importance in the human population. Recent estimates are that as much as one-third of the population of the world is infected with
M. tuberculosis,
that there are 30 million active cases, that there are some 10 million new cases annually, and that tuberculosis causes some 6 percent of all deaths worldwide. See, e.g., Daniel, T., Tuberculosis in Harrison et al., eds.,
Principles of Internal Medicine,
McGraw-Hill, Inc., New York, N.Y. (13th ed., 1994) (hereafter “Harrison's (1994)”; the entirety of Harrison's is hereby incorporated by reference). At present, the only vaccine is an attenuated strain of
M. bovis
designated bacillus Calmette-Guerin (“BCG”). While this vaccine is considered safe, its efficacy is still uncertain. Ibid.
BCG is created by multiple passages of the
M. bovis
organism. These multiple passages are considered necessary to attenuate the pathogenicity of the organism but, as the organism adapts to laboratory passaging, it undergoes undefined and unknown changes from that of the wild type. These changes, in turn, diminish the relevance of the immune response generated by use of BCG to subsequent challenge by wild type, pathogenic bacteria, and are probably due at least in part to the continuing uncertainty over its efficacy. Moreover, since
M. bovis
infects cows, rather than humans, a portion of the immune response it raises is not directed against antigens relevant to human disease.
What is therefore needed is a method of producing attenuated mycobacteria without the multitude of passages currently necessary to produce BCG, so that the immune response generated is more relevant to the challenge posed by wild type, pathogenic bacteria. What is also needed, in particular, is a vaccine for tuberculosis based on mycobacteria less removed from the wild type than are those used to create BCG. What is even more needed is a vaccine which is based on
M. tuberculosis,
and which can therefore be expected to raise an immune response more directly relevant to the antigens presented by wild type
M. tuberculosis.
It would further be desirable to have a vaccine of organisms capable of replicating for a short time in a vaccinated host, to permit the organism to present proteins and other antigens which may only be presented live, metabolizing organisms, but also capable of being completely contained by the host. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
This invention provides a vaccine for protection against tuberculosis. The vaccine comprises pharmaceutically acceptable excipients and Mycobacterium sp. attenuated by having the expression level of the &agr;-crystallin heat shock protein gene reduced by at least 75% of the wild type expression level, said Mycobacterium sp. being present in a concentration effective to provide immunoprotection to a host mammal. In preferred embodiments, expression of the &agr;-crystallin heat shock protein gene is eliminated. In more preferred forms, the gene for the protein is “knocked out.”
In one embodiment, the vaccines have the Mycobacterium sp. in an isotonic salt solution. Preferred dosages are between 1 and around 1,000,000 cells per dose. More preferred dosages are between around 100 and around 100,000 cells per dose. Somewhat more preferred are between around 100 and around 50,000 cells per dose. Even more preferred are between around 100 and around 25,000 cells per dose. Most preferred are between around 100 and around 1,000 cells per dose.
This invention also provides for a method of vaccinating a mammal susceptible to an infection of Mycobacterium sp. The method comprises the administration of an amount of Mycobacterium sp. attenuated by having the expression level of &agr;-crystallin heat shock protein gene reduced by at least 75% of the wild type expression level, said administration of Mycobacterium sp. in an amount effective to provide immunoprotection to a host mammal. Preferred routes of administration are via the nasal passages or via parenteral injection. The preferred dose is between 100 and 100,000 cells, depending, inter alia, on the route of administration.
In addition to vaccines and methods of vaccinating, this invention provides for novel strains of Mycobacterium sp. wherein the expression level of the &agr;-crystallin heat shock protein gene is reduced by at least 75% compared to the wild type expression level. In one embodiment, the strains are housed in a sealed vial where the vial is packaged with instructional material stating a method of injecting the strain into a human to provide the human with immunoprotection from tuberculosis.
In one embodiment of the vaccine, methods, and strains described above, the Mycobacterium species is selected from the group consisting of
M. tuberculosis
and
M. bovis.
Additionally, the invention provides a method of manufacturing attenuated mycobacteria by reducing the expression of the &agr;-crystallin heat shock protein gene by at least 75% compared to the wild type expression level. In one embodiment, the reduction is by deletion of the gene encoding the protein. In preferred embodiments, the mycobacteria manufactured by this method are
M. tuberculosis
or
M. bovis.
Further, the invention provides a method for attenuating virulence of a Mycobacterium sp., wherein the method comprises reducing the expression of the &agr;-crystallin heat shock protein gene by at least 75% compared to the wild type expression level. In one embodiment, the reduction is by deletion of the gene encoding the protein. In preferred embodiments, the mycobacteria whose virulence are attenuated by this method are
M. tuberculosis
or
M. bovis.
DEFINITIONS
“Attenuated” refers to a state of reduced pathogenicity of an organism that in its wild state is pathogenic. It is usually measured by an inhibition, partial or complete, of the ability of a pathogenic organism to colonize, infect, grow, reproduce, or survive in a host. When attenuation is due to a defined genetic change, that change may be a gene deletion, replacement, or alteration, transcriptional inhibition, or translational inhibition.
“Imnmunoprotection” refers to the ability of the immune system of a mammal such as a human to reduce infection of a pathogen such as
M. tuberculosis
or
M. bovis
when challenged with what would otherwise be an infectious dose.
“Isotonic salt solutions” are salt solutions that are osmotically balanced against the cells in which they are contacted. They are preferably at physiologic pHs of between 6.5 and 7.5.
“Instructional material” refer to written, audio or visual means to convey the proper use of material or pharmaceuticals, vaccines or reagents.
“Pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the acr attenuated mycobacteria without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.
The phrase “expression level of &agr;-crystallin heat shock protein gene reduced by at least 75% of the wild type expression level” refers to a reduction of at least 75% in the relative level of expression of &agr;-crystallin heat shock protein between a wild type pathogenic strain and a strain deliberately altered to reduce natural levels of the protein, measured by any scientifically valid technique.

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