Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-25
2001-09-18
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C424S184100, C424S190100, C436S501000, C436S517000, C436S518000, C436S536000
Reexamination Certificate
active
06291190
ABSTRACT:
Tuberculosis is an ancient human scourge that continues to be an important public health problem worldwide. It is an ongoing epidemic of staggering proportions. Approximately one in every three people in the world is infected with
Mycobacterium tuberculosis,
and has a 10% lifetime risk of progressing from infection to clinical disease. Although tuberculosis can be treated, an estimated 2.9 million people died from the disease last year.
There are significant problems with a reliance on drug treatment to control active
M. tuberculosis
infections. Most of the regions having high infection rates are less developed countries, which suffer from a lack of easily accessible health services, diagnostic facilities and suitable antibiotics against
M. tuberculosis
. Even where these are available, patient compliance is often poor because of the lengthy regimen required for complete treatment, and multidrug-resistant strains are increasingly common.
Prevention of infection would circumvent the problems of treatment, and so vaccination against tuberculosis is widely performed in endemic regions. Around 100 million people a year are vaccinated with live bacillus Calmette-Guerin (BCG) vaccine. BCG has the great advantage of being inexpensive and easily administered under less than optimal circumstances, with few adverse reactions. Unfortunately, the vaccine is widely variable in its efficacy, providing anywhere from 0 to 80% protection against infection with
M. tuberculosis
.
BCG has an interesting history. It is an attenuated strain of
M. bovis
, a very close relative of
M. tuberculosis
. The
M. bovis
strain that became BCG was isolated from a cow in the late 1800's by a bacteriologist named Nocard, hence it was called Nocard's bacillus. The attenuation of Nocard's bacillus took place from 1908 to 1921, over the course of 230 in vitro passages. Thereafter, it was widely grown throughout the world, resulting in additional hundreds and sometime thousands of in vitro passages. Throughout its many years in the laboratory, there has been selection for cross-reaction with the tuberculin skin test, and for decreased side effects. The net result has been a substantially weakened pathogen, which may be ineffective in raising an adequate immune response.
New antituberculosis vaccines are urgently needed for the general population in endemic regions, for HIV-infected individuals, as well as health care professionals likely to be exposed to tubercle bacilli. Recombinant DNA vaccines bearing protective genes from virulent
M. tuberculosis
are being developed using shuttle plasmids to transfer genetic material from one mycobacterial species to another, for example see U.S. Pat. No. 5,776,465. Tuberculosis vaccine development should be given a high priority in current medical research goals.
Relevant literature
Mahairas et al. (1996)
J Bacteriol
178(5):1274-1282 provides a molecular analysis of genetic differences between
Mycobacterium bovis
BCG and virulent
M. bovis
. Subtractive genomic hybridization was used to identify genetic differences between virulent
M. bovis
and
M. tuberculosis
and avirulent BCG. U.S. Pat. No. 5,700,683 is directed to these genetic differences.
Cole et al. (1998)
Nature
393:537-544 have described the complete genome of
M. tuberculosis
. To obtain the contiguous genome sequence, a combined approach was used that involved the systematic sequence analysis of selected large-insert clones as well as random small-insert clones from a whole-genome shotgun library. This culminated in a composite sequence of 4,411,529 base pairs, with a G+C content of 65.6%. 3,924 open reading frames were identified in the genome, accounting for ~91% of the potential coding capacity.
Mycobacterium tuberculosis
(M.tb.) genomic sequence is available at several internet sites.
SUMMARY OF THE INVENTION
Genetic markers are provided that distinguish between strains of the
Mycobacterium tuberculosis
complex, particularly between avirulent and virulent strains. Strains of interest include
M. bovis, M. bovis
BCG strains,
M. tuberculosis
(
M. tb.
) isolates, and bacteriophages that infect mycobacteria. The genetic markers are used for assays, e.g. immunoassays, that distinguish between strains, such as to differentiate between BCG immunization and
M. tb.
infection. The protein products may be produced and used as an immunogen, in drug screening, etc. The markers are useful in constructing genetically modified
M. tb
or
M. bovis
cells having improved vaccine characteristics.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Specific genetic deletions are identified that serve as markers to distinguish between avirulent and virulent mycobacteria strains, including
M. bovis, M. bovis
BCG strains,
M. tuberculosis
(
M. tb.
) isolates, and bacteriophages that infect mycobacteria. These deletions are used as genetic markers to distinguish between the different mycobacteria. The deletions may be introduced into
M. tb.
or
M. bovis
by recombinant methods in order to render a pathogenic strain avirulent. Alternatively, the deleted genes are identified in the
M. tb.
genome sequence, and are then reintroduced by recombinant methods into BCG or other vaccine strains, in order to improve the efficacy of vaccination.
The deletions of the invention are identified by comparative DNA hybridizations from genomic sequence of mycobacterium to a DNA microarray comprising representative sequences of the
M. tb.
coding sequences. The deletions are then mapped to the known
M. tb.
genome sequence in order to specifically identify the deleted gene(s), and to characterize nucleotide sequence of the deleted region.
Nucleic acids comprising the provided deletions and junctions are used in a variety of applications. Hybridization probes may be obtained from the known
M. tb.
sequence which correspond to the deleted sequences. Such probes are useful in distinguishing between mycobacteria. For example, there is a 10% probability that an
M. tb.
infected person will progress to clinical disease, but that probability may vary depending of the particular infecting strain. Analysis for the presence or absence of the deletions provided below as “M. tb variable ” is used to distinguish between different
M. tb
strains. The deletions are also useful in identifying whether a patient that is positive for a tuberculin skin test has been infected with
M. tb
or with BCG.
In another embodiment of the invention, mycobacteria are genetically altered to delete sequences identified herein as absent in attenuated strains, but present in pathogenic strains, e.g. deletions found in BCG but present in
M. tb
H37Rv. Such genetically engineered strains may provide superior vaccines to the present BCG isolates in use. Alternatively, BCG strains may be “reconstructed ” to more closely resemble wild-type
M. tb
by inserting certain of the deleted sequences back into the genome. Since the protein products of the deleted sequences are expressed in virulent mycobacterial species, the encoded proteins are useful as immunogens for vaccination.
The attenuation (loss of virulence) in BCG is attributed to the loss of genetic material at a number of places throughout the genome. The selection over time for fewer side-effects resulting from BCG immunization, while retaining cross-reactivity with the tuberculin skin test, has provided an excellent screen for those sequences that engender side effects. The identification of deletions that vary between BCG isolates identifies such sequences, which may be used in drug screening and biological analysis for the role of the deleted genes in causing untoward side effects and pathogenicity.
Identification of
M. Tuberculosis
Complex Deletion Markers
The present invention provides nucleic acid sequences that are markers for specific mycobacteria, including
M. tb., M. bovis
, BCG and bacteriophage. The deletions are listed in Table 1. The absence or presence of these marker sequences is characteristic of the indicated isolate, or strain. As such, they provide a unique charac
Behr Marcel
Schoolnik Gary
Small Peter
Wilson Michael A.
Bozicevic Field & Francis LLP
Guzo David
Leffers, Jr. Gerald G.
Sherwood Pamela J.
The Board of Trustees of the Leland Stanford Junior University
LandOfFree
Molecular differences between species of the M. tuberculosis... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Molecular differences between species of the M. tuberculosis..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Molecular differences between species of the M. tuberculosis... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2480789