Attenuated strains of mycobacteria

Details Attenuated strains of mycobacteria Attenuated strains of mycobacteria

2000-07-07

2003-01-07

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...

C424S093200, C424S200100, C424S243100, C435S243000, C435S253100, C435S441000, C435S471000, C530S300000, C530S350000, C530S800000, C530S825000, C536S023100, C536S023700

Reexamination Certificate

active

06503514

ABSTRACT:

FIELD OF INVENTION
The present invention relates to the field of molecular immunology and, in particular, to attenuated strains of Mycobacterium and immunogenic preparations comprising the same.
BACKGROUND TO THE INVENTION
Tuberculosis (TB) is a major cause of mortality throughout the world, particularly in developing countries. There are about 8 to 9 million new cases of clinical disease reported every year and the number of deaths is estimated to be about 3 million. In the U.S. the trend of steady decline in TB has reversed and the problem is compounded by increasing numbers of drug-resistant strains. The tuberculosis complex is a group of four mycobacterial species that are genetically closely related. The three most important members are
Mycobacterium tuberculosis
, the major cause of human TB;
Mycobacterium africanum
, a major human pathogen in some populations; and
Mycobacterium bovis
, the cause of bovine TB. None of these mycobacteria is restricted in being pathogenic for a single-host species.
In addition to being an important human disease, TB is also a major veterinary problem in many countries. Infection of cattle with
M. bovis
results in bovine TB and all animals showing any signs of infection are systematically slaughtered. The economic losses are thus extensive, and furthermore, cattle can serve as a reservoir for human disease.
In a majority of cases of infection, inhaled tubercle bacilli are ingested by phagocytic alveolar macrophages and are either killed or grow intracellularly to a limited extent in local lesions called tubercules. In this way the infection is limited and the primary sites of infection are walled off without any symptoms of disease being observed. Such individuals have a lifetime risk of about 10% for developing active disease. In a latter eventuality, bacilli spread from the site of infection in the lung, through the lung and via lymphatics or blood to other parts of the body producing characteristic solid caseous (cheese-like) necrosis in which bacilli survive. If the necrotic reaction expands breaking into a bronchus, or in the worst case, if the solid necrosis liquefy, a rapid proliferation of the bacilli occurs. The pathological and inflammatory processes set in motion then produce the characteristic weakness, fever, chest pain, cough and bloody sputum which are the hallmarks of active TB.
Effective treatment of TB with antibiotics exists. However, this is expensive and requires prolonged administration of a combination of drugs. There is a problem in compliance with the drug administration regime because of the extended time periods involved and this has contributed to the appearance of drug-resistant strains. There is a recognized vaccine for TB which is an attenuated form of
M. bovis
, known as BCG (bacille Calmette Guérin). This strain was developed in 1921 and the basis for its attenuation is still not known (ref. 1—throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). The efficacy of BCG as a TB vaccine is a subject of controversy and has been estimated in various trials to be anywhere between 0 and 70%.
The molecular basis for the virulence and pathogenesis of
M. tuberculosis
has not been extensively described. Some virulence factors, particularly those related to the sigma factors have been recently identified (ref. 2).
M. tuberculosis
can enter non-phagocytic cells in culture, such as HeLa cells (ref. 3) and once inside can multiply and survive. Recently, a protein encoded by a DNA fragment (1535 bp long) from a strain of
M. tuberculosis
(H37Ra) was reported to mediate the entry of the bacterium and its survival in mammalian cells (ref. 4). This DNA fragment when introduced into a non-pathogenic strain of
E. coli
is able to confer invasiveness to the bacterium, and survival for up to 24 hours in human macrophages. The mce (mycobacterial cell entry) gene was mapped to an Open Reading Frame (ORF) extending from position 182 to 810 on the 1535 bp DNA fragment mentioned above and encodes a protein of molecular weight between 22 and 27 kDa. Subsequent work has shown the gene described in ref. 4 is not a full length gene.
In copending U.S. patent application Ser. No. 08/677,970 filed Jul. 10, 1996, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, there is described the isolation and characterization of genes encoding proteins of mycobacteria associated with cell binding and cell entry and the protein encoded thereby. This gene is referred to herein as the Mycobacterial cell entry (mce) gene and the encoded protein the Mycobacterial cell entry protein (Mcep).
As described therein, restriction enzymes digestion of
M. bovis
BCG DNA and subsequent Southern blot analysis using a PCR amplified product as a probe, indicated the presence of the mce gene on 1.5 kb and 5 kb fragments of Sal I and Xho I digests. The corresponding bands were cut from the Southern blots and ligated into suitably digested pUC18 and pBluescript II (sk+) plasmids respectively. The libraries obtained after transformation of competent
E. coli
libraries were probed for the mce gene by hybridization and two positive clones were identified. The clones were grown, plasmids isolated and analyzed to yield two plasmids: pBCGcepX containing an approximately 5 kb DNA insert hybridizing to mce gene-specific probe and another plasmid pBCGcepS containing a 1.5 kb Sal I insert also hybridizing to the mce-specific probe.
The inserts of plasmids pBCGcepX and pBCGcepS were sequenced by the primer walking technique. The sequence data from the inserts was aligned and it was found that the 1.5 kb Sal I insert of plasmid pBCGcepS was an internal fragment of the larger pBCGcepX clone. The complete sequence of the 4740 bp DNA fragment clones into pBCGcepX is shown in
FIG. 4
of application Ser. No. 08/677,970. Plasmid pBCGcepX has been deposited with ATCC under deposit number 97511 on Apr. 11, 1996.
Mycobacterial infection may lead to serious disease. It would be advantageous to provide attenuated strains of Mycobacterium wherein the mycobacterial cell entry gene is disabled, and immunogenic preparations including vaccines comprising the same.
SUMMARY OF INVENTION
The present invention provides attenuated strains of Mycobacteria which are useful in immunogenic compositions. In accordance with one aspect of the present invention, there is provided an attenuated strain of Mycobacterium wherein the mycobacterial cell entry (mce) gene is functionally disabled. By functionally disabling the mce gene, the ability of the Mycobacterium to invade and infect cells is removed. This attenuation permits the novel strains provided herein to be used in immunogenic compositions for administration to a host to generate an immune response.
The mce gene may be functionally disabled by an insertion into the gene such as to disrupt the mycobacterial cell entry function thereof. The mce gene also may be functionally disabled by deleting at least a part of the gene from the wild-type strain. In addition, mutagenesis of the mce gene may be used to attenuate the wild-type strain.
The mutant strain of Mycobacterium may be prepared by any convenient procedure. Homologous recombination conveniently may be used to replace the mce gene of the wild-type strain of Mycobacterium by a double cross-over event with a disabled mce gene.
The present invention is broadly applicable to strains of Mycobacterium, particularly a species of the tuberculosis complex, including
M. tuberculosis
and
M. bovis.
In another aspect of the invention, there is provided a method of forming an attenuated strain of Mycobacterium, which comprises effecting allelic exchange of a mutant mycobacterial cell entry (mce) gene which is functionally disabled fo

Affiliated with

Also associated with

Rating

Attenuated strains of mycobacteria does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Attenuated strains of mycobacteria, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Attenuated strains of mycobacteria will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3016479