Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-11-30
2004-03-30
Duffy, Patricia A. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023700, C435S183000
Reexamination Certificate
active
06713617
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the discovery of a new gene, which is the first isolated and cloned, that encodes an amino acid eukaryotic racemase. The invention covers, particularly, the Tc45 gene encoding a
Trypanosoma cruzi
-derived B-cell mitogen. The encoded protein also is a eukaryotic proline racemase. The invention also relates to a process of production of D-amino acid using an eukaryotic amino acid racemase. This invention also relates to the use of the protein encoded by the Tc45 gene to induce a protective immune response against
T. cruzi
infection in a human. This invention also relates to methods of using other parasite mitogens and viral mitogens for inducing protective immunity against the corresponding parasitic or viral infections in humans.
The process of production of an D-amino acid by using a L-amino acid source comprises the use of an eukaryotic amino acid racemase specific for the amino acid of interest, the said racemase being produced from a recombinant expression system containing a vector having a polynucleotide sequence encoding the said enzyme. In prokaryotic hosts, the racemases are known to be implicated in the synthesis of D-amino acids and/or in the metabolism of L-amino acids. Therefore, the presence of free D-amino acids in tumors and in progressive autoimmune and degenerative diseases suggests the biological importance of eukaryotic amino acid racemases. It is well known that proteins or peptides containing D-amino acids are resistant to proteolysis by host enzymes. In addition, such proteins containing D-amino acids, at least one D-amino acid residue, can display antibiotic or immunogenic properties.
Isolation and characterization of molecules playing a key role in parasite metabolism, or in their interactions with the host immune defense, are fundamental for the development of rational strategies for vaccination and therapy. Attempts to provide effective immunity to parasites are limited by poor specific immune responses to parasite antigenic molecules in early phases of infection. Lymphocyte polyclonal activation is a generalized mechanism of immune evasion amongst pathogens. Such “parasite evasion” owes, at least in part, to the release of mitogenic or superantigenic moieties that inhibit host specific responses by triggering polyclonal, parasite non-specific lymphocyte activation. The resulting non-specific immune responses are associated with immunosuppression and autoimmunity, as observed in human and experimental infections by the protozoan parasite
Trypanosoma cruzi
, the etiological agent of Chagas disease
2-6
.
To date, there is no effective treatment or vaccine against
Trypanosoma cruzi
infection and Chagas disease pathology. Attempts to isolate immunodominant protective epitopes have failed
1
. Using a mouse model of
T. cruzi
infection it has previously been shown that reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy
2, 7-9
. As we have suggested, and has been demonstrated by Arala-Chaves
31
for
Candida albicans
infections, mitogenic moieties can be used as vaccination targets to induce specific neutralization of the mitogen, thus aborting the microorganism “strategy” to deviate immune responses into non-specific polyclonal activation and immunosuppression. Understanding the mechanisms underlying “non-specific” lymphocyte activation may open the way for their neutralization, and thus allow for effective immune responses against infectious agents. There is a need in the art for a molecule that could be an appropriate target for such attempts.
Furthermore, there is a growing interest in the biological role of D-amino acids, either as free molecules or within polypeptide chains in human brain, tumors, anti-microbial and neuropeptides, as well as in “protein fatigue”
32
, suggesting widespread biological implications. Research on D-amino acids in living organisms has been hampered by their difficult detection. However, recent purification of a serine racemase from mammalian brain
33
indicates conservation throughout evolution. There also exists a need in the art for racemases that are specific for known compounds.
SUMMARY OF THE INVENTION
This invention aids in fulfilling these needs in the art. More particularly, this invention relates to the characterization of a parasite molecule implicated in polyclonal responses that may serve as a novel target for vaccination and therapy. After identifying a protein with B-cell mitogenic properties in culture supernatants of infective parasite forms, the corresponding gene was cloned and its genomic organization was characterized. The protein has been characterized as a cofactor independent proline racemase, with strong homology to the proline racemase isolated from
Clostridium sticklandii
, thus providing the first report on an eukaryotic amino acid racemase gene.
In particular, this invention provides a purified peptide comprising an amino acid sequence (SEQ ID NOS: 1, 2, 3 and 4) encoded by the Tc45 gene. This invention also provides polypeptide fragments derived from SEQ ID NOS: 7,8, 9, 10 and 11 containing at least 10 amino acids.
This invention additionally provides purified polynucleotides comprising the nucleic acid sequences of the Tc45 gene (SEQ ID NOS: 7, 8, 9, 10 and 11). This invention also provides nucleic acid fragments derived from SEQ ID NOS: 7, 8, 9, 10 and 11 containing 15 to 40 nucleotides.
Additionally, the invention includes a purified polynucleotide that hybridizes specifically under conditions of moderate stringency with a polynucleotide of SEQ ID NOS: 7, 8, 9, and 10.
SEQ. ID 7 represents the full nucleotide sequence encoding the
Trypanosoma cruzi
proline racemase and N-terminal signal sequence and the 5′ and 3′ flanking non-coding regions.
The SEQ ID 8 represents the full nucleotide sequence and its corresponding polypeptide sequences [including the N terminal signal sequence and the 3′ non-coding flanking region] coding for a proline racemase of
T. cruzi.
The construct as disclosed in SEQ ID 8 deleted of the 3′ non-coding flanking region and inserted in the PET28 vector (NOVAGEN), transformed in
E. coli
DH5 &agr; was deposited at the CNCM under the accession number I-2344.
A derived construct of I-2344 deleted of nucleotide sequence corresponding to the signal peptide coding sequences, which is described in SEQ ID 3, is used for the production of a recombinant active proline racemase in
E. coli
. The
E. coli
DH 5&agr; containing the plasmid with an insert of 239 base pairs deposited at CNCM under accession number I-2221, was obtained after amplification of the region by PCR technique with the primers SEQ ID Nos. 12 and 13. The insert was cloned into p TOPO II commercialized by INVITROGEN and then transformed in
E. coli.
The invention further includes polynucleotide fragments comprising at least 10 nucleotides capable of hybridization under conditions of moderate stringency conditions with any one of the nucleotide sequences enumerated above.
In another embodiment of the invention, a recombinant DNA sequence comprising at least one nucleotide sequence enumerated above and under the control of regulatory elements that regulate the expression of racemase activity in a host is provided.
The invention also includes a recombinant host cell comprising a polynucleotide sequence enumerated above or the recombinant vector defined above.
In still a further embodiment of the invention, a method of detecting parasitic strains that contain the polynucleotide sequences set forth above is provided.
Additionally, the invention includes kits for the detection of the presence of parasitic strains that contain the polynucleotide sequences set forth above.
The invention also contemplates antibodies recognizing peptide fragments or polypeptides encoded by the polynucleotide sequences enumerated above.
Still further, the invention provides for a screening method for active molecules for the treatment of infections due to parasites, particularly
T. Cruz
Arala-Chaves Mario
Arala-Chavez Rul Mario Dores
Cosson Alain
Coutinho Antonio
DeGrave Wim
Arala-Chavez Rul Mario Dores
Duffy Patricia A.
Fernandes de Almeida Eduardo Mansinho
Fernandes de Almeida Sara Isabel Mansinho
Finnegan Henderson Farabow Garrett & Dunner LLP
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