Cloning, sequencing and expresssion of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens

Abstract

A method of preventing or inhibiting infection by a parasite or virus in vivo comprises administering to a human in need thereof a parasite or virus mitogen in a sub-mitogenic amount sufficient to induce a protective immune response against the parasite or virus in the human.

1 - 78 . (canceled)  
     
     
         79 . A purified nucleic acid molecule 
 (a) selected from SEQ ID NOS: 8, 9, 10, and 11;    (b) that encodes a peptide selected from SEQ ID NOS: 1, 2, 3, and 4;    (c) that hybridizes to either strand of a denatured, double-stranded DNA comprising the nucleic acid molecule of (a) or (b) under conditions of moderate stringency;    (d) derived by in vitro mutagenesis from SEQ ID NOS: 8, 9, 10, and 11;    (e) degenerate from SEQ ID NOS: 8, 9, 10, and 11 as a result of the genetic code;    (f) that encodes Tc45 polypeptide, an allelic variant of Tc45 polypeptide, or a homolog of Tc45 polypeptide;    (g) that encodes an eukaryotic protein with an amino acid racemase activity;    (h) that encodes an eukaryotic protein with a proline racemase activity;    (i) that encodes an eukaryotic protein, which is recognized by antibodies raised against an eukaryotic protein having proline racemase activity;    (j) that has at least 80% of identity with the sequence of an eukaryotic gene encoding a protein with a racemase activity; and/or    (k) that is a fragment of a polynucleotide containing at least 50 nucleotides of the sequence of the proline racemase gene of  T. cruzi  or hybridizing under stringent conditions with a polynucleotide according to any one of (g), (h), (i) or (j).    
     
     
         80 . A recombinant vector that directs the expression of a nucleic acid molecule of  claim 79 .  
     
     
         81 . A purified polypeptide 
 (a) that is encoded by a nucleic acid molecule of  claim 79;     (b) that has a molecular weight of approximately 45 kDa as determined by SDS-PAGE, which is post translationally modified or not;    (c) that is an eukaryotic protein with proline racemase activity;    (d) that is a protein of (c), which is a P38 to P45 kDa protein;    (e) that is a P38 to P45 kDa protein according to (d), which is a parasite protein;    (f) that is a P38 to P45 kDa protein according to (e), wherein the parasite is  T. cruzi;      (g) that is a purified eukaryotic amino acid racemase having a molecular weight of 38 kDa to 45 kDa more or less 10%; and/or    (h) that is a Tc45 polypeptide.    
     
     
         82 . Purified polyclonal or monoclonal antibodies that bind to a polypeptide of  claim 81 .  
     
     
         83 . A host cell transfected or transduced with the vector of  claim 80 .  
     
     
         84 . A method for the production of Tc45 polypeptide comprising culturing a host cell of  claim 83  under conditions promoting expression, and recovering the polypeptide from the host cell or the culture medium.  
     
     
         85 . The method of  claim 84 , wherein the host cell is selected from the group consisting of bacterial cells, parasite cells and eukaryotic cells.  
     
     
         86 . A recombinant vector as claimed in  claim 80 , which is a plasmid deposited at CNCM under the Accession Number 1-2221 or 1-2344.  
     
     
         87 . An immunological complex comprising a Tc45 polypeptide and an antibody as claimed in  claim 82 .  
     
     
         88 . A method of detecting a parasite in a biological sample, said method comprising: 
 (a) contacting parasite DNA of the biological sample with a primer or a probe, which hybridizes with the nucleic acid molecule of  claim 79;     (b) amplifying a nucleotide sequence using said primer or said probe; and    (c) detecting a hybridized complex formed between said primer or probe and the DNA.    
     
     
         89 . A method of detecting a parasite in a biological sample, said method comprising: 
 (a) contacting the parasite extract or the biological sample with antibodies as claimed in  claim 82;  and    (b) detecting the resulting immunocomplex.    
     
     
         90 . A kit for detecting a parasite, said kit comprising: 
 (a) a polynucleotide probe, which hybridizes with the nucleic acid molecule of  claim 79;  and    (b) reagents to perform a nucleic acid hybridization reaction.    
     
     
         91 . A kit for detecting a parasite comprising: 
 (a) purified antibodies as claimed in  claim 82;     (b) standard reagents for performing an immune reaction; and    (c) detection reagents.    
     
     
         92 . An in vitro method of screening for active molecules capable of inhibiting a polypeptide encoded by a nucleic acid molecule as claimed in  claim 79 , said method comprising the steps of: 
 (a) contacting the active molecules with said polypeptide;    (b) testing the capacity of the active molecules, at various concentrations, to inhibit the activity of the polypeptide; and    (c) choosing the active molecule that provides an inhibitory effect of at least 80% on the activity of the said polypeptide.    
     
     
         93 . A process of preparation of a purified eukaryotic protein as claimed in claim  79 (g) or claim  81 (c) with a racemase activity comprising: 
 (a) selecting a gene encoding a protein having a racemase activity;    (b) transforming a host with a recombinant vector containing the gene;    (c) culturing the host and producing the protein encoded by the gene; and    (d) separating the purified eukaryotic protein with the racemase activity from the culture; or separating the purified eukaryotic protein recognized by antibodies raised against said protein as claimed in claim  79 (g) or claim  81 (c).    
     
     
         94 . A process for detecting a  T. cruzi  infection by contacting purified P45 protein and fragments or peptides thereof, which are recognized by antibodies raised against a polypeptide as claimed in  claim 81 , with serum of a patient suspected to be infected.  
     
     
         95 . An immunizing composition containing at least a purified protein as claimed in  claim 81  or a fragment thereof, in an amount sufficient to induce an immune response in vivo or to induce the inhibition of a mitogenic polyclonal immunoresponse in vivo, wherein the immunizing composition optionally contains a pharmaceutically acceptable carrier therefor.  
     
     
         96 . A vaccine composition against a  T. cruzi  infection containing the purified P38 to P45 kDa protein or a fragment thereof according to  claim 81 .  
     
     
         97 . A process for screening a molecule capable of inhibiting the amino acid racemase activity of a eukaryotic protein comprising: 
 (a) contacting the purified eukaryotic racemase protein with standard doses of a molecule to be tested;    (b) measuring inhibition of racemase activity; and    (c) selecting the molecule.    
     
     
         98 . A method of inhibiting a eukaryotic protein with an amino acid racemase activity, which comprises treating a patient by administering an effective amount of a molecule that can be selected by the process of  claim 97  that inhibits said eukaryotic protein.  
     
     
         99 . Method according to  claim 98 , wherein the parasite is  T. cruzi.    
     
     
         100 . A method for producing an eukaryotic recombinant amino acid racemase as claimed in claim  79 (g) or claim  81 (c) comprising: 
 (a) culturing a bacterial or a eukaryotic host harboring an over-expression system including an insert containing a polynucleotide sequence encoding an eukaryotic amino acid racemase;    (b) separating the recombinant eukaryotic amino acid racemase from the host proteins; and    (c) purifying the eukaryotic amino acid racemase.    
     
     
         101 . A method according to  claim 100 , wherein the amino acid racemase is a proline racemase.  
     
     
         102 . A method according to  claim 100 , wherein the recombinant bacterial host contains an insert derived from the insert contained in the strain deposited at CNCM under Accession number I-2344.  
     
     
         103 . A method for the production of D-amino acid using a purified eukaryotic amino acid racemase comprising: 
 (a) incubating L-amino acid with the recombinant eukaryotic amino acid racemase;    (b) separating the D-amino acid produced in (a); and    (c) purifying the D-amino acid.    
     
     
         104 . A method of preventing or inhibiting infection by a virus or a protozoan parasite in vivo, wherein the method comprises administering to a subject in need thereof a virus or a protozoan parasite mitogen in a sub-mitogenic amount sufficient to induce a protective immune response against the virus or the protozoan parasite in the subject, wherein the virus or protozoan parasite mitogen is optionally administered to the subject in admixture with a pharmaceutically acceptable carrier.  
     
     
         105 . The method of  claim 104 , wherein the virus mitogen is an animal or human virus mitogen in natural or recombinant form.  
     
     
         106 . The method of  claim 105 , wherein the subject is a human.  
     
     
         107 . The method of  claim 106 , wherein the mitogen is a protozoan parasite mitogen of  Plasmodium berghei  in natural or recombinant form, or of  plasmodium falciparum  or  plasmodium vivax  in natural or recombinant form.  
     
     
         108 . A method of detecting a eukaryotic protein as claimed in  claim 81  in a sample comprising: 
 (a) contacting the sample with antibodies raised against an amino acid racemase; and    (b) detecting the resulting immunocomplex.    
     
     
         109 . A molecule for preventing or treating a parasite or a virus infection, wherein said molecule can be selected by the process of  claim 97  and inhibits a parasite or a virus racemase activity.