Mycobacterium tuberculosis specific proteins and genes,...

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

Reexamination Certificate

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

C424S009100, C424S009200, C424S184100, C424S234100, C435S069100, C435S253100, C435S863000, C530S300000, C530S350000

Reexamination Certificate

active

06596281

ABSTRACT:

This invention relates to
Mycobacterium tuberculosis
, other bacterial pathogens whose antigenicity is not caused by a single protein or component, and parasites, including detection thereof, diagnosis of infection and disease, and preparation of vaccines:
BACKGROUND
One of the important goals of research on
Mycobacterium tuberculosis
, the causative agent of tuberculosis (TB), is the identification of mycobacterial antigens that induce protective T-cell responses and/or stimulate humoral immunity during tubercular infection. Antigens in the former class constitute potential candidates for the development of effective vaccines, while those in the latter group can be tested as new, improved tools for diagnosis of TB.
Similarly, numerous other bacterial pathogens have pathogenicity that, as with TB, is not caused by a single protein, as is the case also with parasites generally. Antigens produced by these pathogens are also potential candidates for the development of effective vaccines.
Proteins that are actively secreted by
M. tuberculosis
have attracted considerable attention as potent immunogens. The observation that only live, dividing mycobacteria efficiently induce protective immunity (7, 22) led to the hypothesis that proteins that are actively secreted by
M. tuberculosis
during growth are key in generating protective T-cell responses (4, 23). Indeed, experimental vaccines based on culture filtrate proteins have been shown to induce some levels of protective immunity in animal models of. TB (5, 14, 15, 26). Secreted proteins of
M. tuberculosis
are also potent inducers of antibody production (13).
The identification and immunochemical characterization of individual components of
M. tuberculosis
culture filtrates is a crucial step toward understanding the role of the secreted proteins in inducing immune responses during the course of TB. More than 30 proteins are present in filtrates from short-term (4-5 day) cultures (3), prior to any substantial contamination of the filtrates by intracellular components released by autolysis of aging cells. Only about ten actively secreted proteins have been identified using antibodies from immunized animals (1); most of them have been characterized by gene cloning and nucleotide sequencing (2, 6, 9, 11, 17-20, 29, 34). Some of the known secreted proteins induce cellular immune responses (35); however, strong human T-cell responses to secreted protein fractions involve yet uncharacterized antigens in the cell filtrate (8, 29).
An aspect of this invention is an isolated DNA sequence encoding the amino acid sequence of the MPT63 antigen, a protein secreted by
M. tuberculosis
, that is specific for mycobacterial species that belong to the
M. tuberculosis
complex, as well as recombinant polypeptide sequences encoded by that DNA.
Another aspect of this invention is an isolated DNA sequence encoding the amino acid sequence of the MTC28 antigen, another protein secreted by
M. tuberculosis
that is similarly specific, as well as purified natural and/or recombinant polypeptide sequences encoded by that DNA.
Another aspect of this invention is a “cocktail” of purified natural and recombinant protein antigens or polypeptides for immunodiagnostics or vaccines, as well as DNA cocktails for vaccines.
Other aspects of this invention are in vitro and in vivo methods of detection of immune responses using the protein or polypeptide cocktails and DNA cocktails of this invention.
SUMMARY OF INVENTION
The gene for the protein MPT63 has been isolated and sequenced (SEQ ID NO:1). That gene can be incorporated into a plasmid and expressed in
E. coli
to produce purified MPT63 protein, whose sequence (SEQ ID NO:2) has been deduced. Additional expression systems will be apparent to persons skilled in the art.
The gene for the protein MTC28 has been isolated and sequenced (SEQ ID NO:3). That gene can be similarly expressed to produce purified MTC28 protein, whose sequence (SEQ ID NO:4) has been deduced.
Both MPT63 and MTC28 are proteins secreted by
M. tuberculosis. Both are specific to the
M. tuberculosis
complex, which includes
M. tuberculosis, M. bovis, M. microti
, and
M. africanum.
This invention includes the MPT63 amino acid sequence shown in
FIG. 1
(SEQ ID NO:2) and the MTC28 amino acid sequence shown in
FIG. 2
(SEQ ID NO:3). A preferred embodiment is the mature recombinant MPT63 protein which is the polypeptide extending from the A in the underlined AYPIT to the C-terminal P in
FIG. 1
, and the mature recombinant MTC28 protein which extends from the D following the underlined portion to the C-terminal R in FIG.
2
. Also preferred are antigenic polypeptides derived from the sequences shown in FIG.
1
and
FIG. 2
, whether produced by natural, recombinant or synthetic (including chemical synthesis) means or other means known in the art. The invention also includes variants of these polypeptides that retain their antigenic and immunogenic properties.
This invention also includes vaccines that contain a recombinant MPT63 polypeptide or a recombinant MTC28 polypeptide. In preferred embodiments the vaccine includes either mature recombinant protein.
This invention includes a method of eliciting an immune response and/or protective immunity against
M. tuberculosis
or another member of the
M. tuberculosis
complex in a vertebrate, said method including administering to the vertebrate a recombinant MPT63 or MTC28 polypeptide, whereby said polypeptide elicits immune responses against the Mycobacterium in the vertebrate.
This invention includes an isolated nucleic acid having the sequence shown in
FIG. 1
(SEQ ID NO:1) or
FIG. 2
(SEQ ID NO:3). Other embodiments can be derived by making silent substitutions, those that do not change the amino acid sequence encoded by the nucleic acid, in the nucleic acid sequence. In preferred embodiments these nucleic acids are made by modifying the sequence by mutagenesis, recombination or synthetic (including chemical synthesis) means or other means known in the art. Also preferred are embodiments wherein the nucleic acid does not contain the entire nucleic acid sequence shown in
FIG. 1
(SEQ ID NO:2) or
FIG. 2
(SEQ ID NO:3), with or without silent substitutions.
A DNA vaccine according to this invention includes a vector, preferably a plasmid vector, and one or more isolated nucleotide sequences each encoding the MPT63, MTC28 polypeptide, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences for expression in a cell of a vertebrate. The DNA vaccine may include the regulatory sequences of CMV immediate-early promoter and/or intron A, or other non-retroviral sequences.
This invention also includes methods of eliciting an immune response and/or protective immunity by administering to a vertebrate such a DNA vaccine, whereby expression of said nucleotide sequences in said cell elicits immune responses against the Mycobacterium.
In preferred methods of this invention the vertebrate is a human. A DNA vaccine according to this invention may be administered to a vertebrate through a route of administration selected from the group consisting of inhalation, intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous. A preferred embodiment is a method wherein the DNA vaccine is administered by contacting the DNA vaccine with a mucosal surface of the vertebrate. A preferred embodiment is a method wherein the DNA vaccine is microsphere encapsulated, and is administered by contacting the microsphere-encapsulated DNA vaccine with a mucosal surface of the vertebrate. A preferred embodiment is a method wherein the DNA vaccine is coated onto gold beads for administration to the vertebrate by particle bombardment delivery. A preferred embodiment is a method wherein the gold beads are approximately 1 pm to 2 pm in diameter. A preferred embodiment is a method wherein the protective immunity is homologous, homotypic, heterotypic, or heterologous.
This invention includes the use of mature MPT63 or MTC28 polypeptide or a fragment(s) thereof in d

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Mycobacterium tuberculosis specific proteins and genes,... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mycobacterium tuberculosis specific proteins and genes,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mycobacterium tuberculosis specific proteins and genes,... will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-3066412

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.