Plasmodium falciparum AMA-1 protein and uses thereof

Abstract

In this application is described the expression and purification of a recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain. The method of the present invention produces a highly purified protein which retains folding and disulfide bridging of the native molecule. The recombinant AMA-1 is useful as a diagnostic reagent, for use in antibody production, and as a protein for use alone, or as part of, a vaccine to prevent malaria.

1 . A nucleotide fragment encoding  P. falciparum  AMA-1 ectodomain protein consisting of amino acids 83-531 of AMA-1.  
     
     
         2 . The nucleotide fragment according to  claim 1  wherein said fragment is from  P. falciparum  3D7, Genbank Accession no. U65407.1.  
     
     
         3 . The nucleotide fragment of  claim 2 , further comprising of a 6-histidine tag on the carboxy terminal end of the encoded protein.  
     
     
         4 . The nucleotide fragment of  claim 3 , further comprising of a 6-histidine tag on the amino terminal end of the encoded protein.  
     
     
         5 . The nucleotide fragment of  claim 4 , said fragment defined in SEQ ID NO:1.  
     
     
         6 . A recombinant vector comprising the nucleotide sequence of  claim 5 .  
     
     
         7 . The vector of  claim 6  wherein said vector is pWRMAL-AMA1/E.  
     
     
         8 . A recombinant vector comprising the nucleotide fragment of  claim 1 .  
     
     
         9 . An isolated  P. falciparum  AMA-1 ectodomain protein consisting of amino acids 83-531 of AMA-1.  
     
     
         10 . An isolated AMA-1 protein according to  claim 9  having the amino acid sequence defined in SEQ ID NO:2.  
     
     
         11 . A host cell transformed with the vector according to  claim 7 .  
     
     
         12 . The host cell of  claim 11  wherein said host cytoplasm is oxidative.  
     
     
         13 . The host cell of  claim 12  wherein said host cell is Origami DE3.  
     
     
         14 . The host cell of  claim 12  wherein said host cell is Tuner DE3.  
     
     
         15 . A method for isolating and purifying recombinant  P. falciparum  AMA-1 protein comprising: 
 growing a host cell containing a recombinant vector expressing  P. falciparum  AMA-1 protein according to  claim 8  in a suitable culture medium,    causing expression of said vector under suitable conditions for production of soluble AMA-1 protein,    lysing said host cells and recovering said AMA-1 protein, and    refolding said AMA-1 protein such that it reacquires its native folding.    
     
     
         16 . The method of  claim 15  wherein said expression of said vector is by induction with IPTG at a temperature range of 25° C.-30° C.  
     
     
         17 . The method of  claim 15  wherein said induction is at 28° C.  
     
     
         18 . The method of  claim 15  wherein lysing of cells is in the presence of a mild detergent.  
     
     
         19 . The method of  claim 18  wherein said mild detergent is sarkosyl.  
     
     
         20 . The method of  claim 15  further comprising removal of  E. coli  proteins.  
     
     
         21 . The method of  claim 20  wherein said removal of  E. coli  proteins is by application to a Ni-NTA column, followed by anion exchange chromatography, followed by cation exchange chromatography.  
     
     
         22 . The method of  claim 15  wherein said refolding is in the presence of about 1 mM reduced glutathione and about 0.25 mM oxidized glutathion.  
     
     
         23 . An isolated protein according to  claim 9 , wherein said protein retains its native disulfide bridges.  
     
     
         24 . The isolated protein of  claim 23  wherein said purified protein is at least 95% pure.  
     
     
         25 . An isolated protein according to  claim 23 , wherein said purified protein is at least 96% pure.  
     
     
         26 . An isolated protein according to  claim 23  wherein said purified protein is at least 97% pure.  
     
     
         27 . An isolated protein according to  claim 23  wherein said purified protein is at least 98% pure.  
     
     
         28 . An isolated protein according to  claim 23  wherein said purified protein is at least 99% pure.  
     
     
         29 . A method for in vitro diagnosis of malaria antibodies in a biological sample, comprising 
 (i) contacting said biological sample with a composition comprising a AMA-1 protein according to  claim 22  under appropriate conditions which allow the formation of an immune complex, wherein said peptide is labeled with a detectable label, and    (ii) detecting the presence of said immune complexes visually or mechanically.    
     
     
         30 . A kit for determining the presence of malaria antibodies in a biological sample, comprising: 
 at least one peptide or protein composition according to  claim 23 ,    a buffer or components necessary for producing a buffer;    means for detecting immune complexes formed betweem the peptide and antibodies present in the sample.    
     
     
         31 . A method for in vitro monitoring malaria infection or prognosing the response to treatment of patients suffering from malaria infection comprising: 
 incubating a biological sample from a patient with malaria infection with an AMA-1 protein according to  claim 23  or a suitable part thereof under conditions allowing the formation of an immunological complex,    removing unbound components,    calculating the anti-AMA-1 titers present in said sample    and monitoring the natural course of malaria infection, or prognosing the response to treatment of said patient on the basis of the amount anti-AMA-1 titers found in said sample at the start of treatment and/or during the course of treatment.    
     
     
         32 . A kit for monitoring malaria infection or prognosing the response to treatment of patients suffering from malaria infection comprising: 
 at least one AMA-1 protein or a suitable part thereof according to  claim 23 ,    a buffer or buffer components    means for detecting the immune complexes formed between the peptide and antibodies present in the sample, and    optionally, a means for determining the amount of immune complex formed.    
     
     
         33 . An antibody produced against the recombinant AMA-1 protein according to  claim 23 .  
     
     
         34 . The antibody of  claim 33  wherein said antibody is monoclonal or polyclonal.  
     
     
         35 . A method for in vitro diagnosis or detection of malaria antigen present in a biological sample, comprising: 
 (i) contacting said biological sample with an antibody specific for the protein of  claim 23 , preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex,    (ii) removing unbound components,    (iii) incubating the immune complexes formed with heterologous antibodies which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies conjugated to a detectable label under appropriate conditions,    (iv) detecting the presence of said immune complexes visually or mechanically.    
     
     
         36 . A kit for in vitro detection of a malaria antigen present in a biological sample, comprising: 
 at least one antibody which reacts with the recombinant protein of  claim 23 , wherein said antibody is preferentially immobilized on a solid substrate,    a buffer, or components necessary for producing the buffer, enabling a binding reaction between these antibodies and the malaria antigens present in the biological sample, and    a means for detecting the immune complexes formed in the preceding binding reaction.    
     
     
         37 . An immunogenic composition comprising the isolated  P. falciparum  AMA-1 of  claim 23 .  
     
     
         38 . The composition of  claim 37  further comprising an adjuvant.  
     
     
         39 . A vaccine against malaria comprising  P. falciparum  AMA-1 according to  claim 23 .  
     
     
         40 . The vaccine of  claim 39  further comprising an adjuvant.  
     
     
         41 . The vaccine of  claim 40  wherein said adjuvant is montanide.  
     
     
         42 . A method for inducing in a subject an immune response against malaria infection comprising administering to said subject a composition comprising an immunologically effective amount of  P. falciparum  AMA-1 of  claim 23  in an acceptable diluent.  
     
     
         43 . The method of  claim 42  wherein said composition further comprises an adjuvant.  
     
     
         44 . The method of  claim 43  wherein said adjuvant is montanide.  
     
     
         45 . A method for inducing a protective immune response to malaria in a mammal, comprising 
 administering a composition comprising a protein according to  claim 23  in an amount effective to induce an immune response in said mammal.    
     
     
         46 . The method according to  claim 45  wherein the composition further comprises an adjuvant.  
     
     
         47 . The method according to  claim 46  wherein said adjuvant is montanide.  
     
     
         48 . A multivalent vaccine for protection against infection with more than one strain of  P. falciparum  comprising a  P. falciparum  protein according to  claim 23  from more than one strain of  P. falciparum , said  P. falciparum  selected from the group consisting of 3D7, FVO and CAMP.  
     
     
         49 . The multivalent vaccine of  claim 48 , further comprising an adjuvant.