Recombinant process for preparing a complete malaria antigen gp190/MSP1

Abstract

Recombinant manufacturing process for a complete malaria antigen gp190/MSP1 The invention concerns a recombinant manufacturing process for the complete gp190/MSP1 surface protein of Plasmodium, in particular P. falciparum, as well as the complete DNA sequence of this protein and suitable host organisms for expression of the sequence, by means of which the protein in its entirety can for the first time be synthesized outside the parasite. The invention opens up for the first time the possibility of manufacturing the gp190/MSP1 surface protein in sufficient quantity; in addition there is the matter of making gp190/MSP1 available as a vaccine. Finally the invention sets out a process for the stabilization of AT-rich genes (FIG. 1 ).

1 - 41 . (canceled)  
     
     
         42 . A nucleic acid comprising a nucleotide sequence encoding a merozoite surface protein-1 (MSP-1) of a  Plasmodium,  wherein the encoded MSP-1 protein has a molecular weight in a range of 190 kD to 220 kD, wherein the encoded MSP-1 protein has a signal peptide and an attachment signal, and wherein the adenine and thymine (AT) content of the nucleotide sequence encoding the MSP-1 protein is less than the AT content of the naturally occurring nucleotide sequence encoding the MSP-1 protein.  
     
     
         43 . The nucleic acid of  claim 42 , wherein the  Plasmodium  is a strain of  Plasmodium falciparum.    
     
     
         44 . The nucleic acid of  claim 43 , wherein the strain of  Plasmodium falciparum  is strain PFB-1.  
     
     
         45 . The nucleic acid of  claim 42 , wherein the AT content of the nucleotide sequence encoding the MSP-1 protein is reduced from about 74% to about 55%.  
     
     
         46 . The nucleic acid of  claim 42 , wherein the MSP-1-encoding nucleotide sequence is set forth in SEQ ID NO:2.  
     
     
         47 . The nucleic acid of  claim 42 , wherein the nucleotide sequence encodes an MSP-1 protein having the amino acid sequence consisting of amino acids 1-1639 of SEQ ID NO:3.  
     
     
         48 . The nucleic acid of  claim 42 , wherein the MSP-1-encoding nucleotide sequence lacks recognizable splice donor and splice acceptor sites.  
     
     
         49 . A nucleic acid comprising a nucleotide sequence encoding a merozoite surface protein-1 (MSP-1) of a  Plasmodium,  wherein the encoded MSP-1 protein has a molecular weight in a range of 190 kD to 220 kD, wherein the encoded MSP-1 protein has a signal peptide and lacks an attachment signal, and wherein the adenine and thymine (AT) content of the nucleotide sequence encoding the MSP-1 protein is less than the AT content of the naturally occurring nucleotide sequence encoding the MSP-1 protein.  
     
     
         50 . The nucleic acid of  claim 49 , wherein the Plasmodium is a strain of  Plasmodium falciparum.    
     
     
         51 . The nucleic acid of  claim 50 , wherein the strain of  Plasmodium falciparum  is strain PFB-1.  
     
     
         52 . The nucleic acid of  claim 49 , wherein the AT content of the nucleotide sequence encoding the MSP-1 protein is reduced from about 74% to about 55%.  
     
     
         53 . The nucleic acid of  claim 49 , wherein the nucleotide sequence encodes an MSP-1 protein having the amino acid sequence consisting of amino acids 1-1621 of SEQ ID NO:3.  
     
     
         54 . The nucleic acid of  claim 49 , wherein the MSP-1-encoding nucleotide sequence lacks recognizable splice donor and splice acceptor sites.  
     
     
         55 . A nucleic acid comprising a nucleotide sequence encoding a merozoite surface protein-1 (MSP-1) of a  Plasmodium,  wherein the encoded MSP-1 protein has a molecular weight in a range of 190 kD to 220 kD, wherein the encoded MSP-1 protein lacks a signal peptide and an attachment signal, and wherein the adenine and thymine (AT) content of the nucleotide sequence encoding the MSP-1 protein is less than the AT content of the naturally occurring nucleotide sequence encoding the MSP-1 protein.  
     
     
         56 . The nucleic acid of  claim 55 , wherein the  Plasmodium  is a strain of  Plasmodium falciparum.    
     
     
         57 . The nucleic acid of  claim 56 , wherein the strain of  Plasmodium falciparum  is strain PFB-1.  
     
     
         58 . The nucleic acid of  claim 55 , wherein the AT content of the nucleotide sequence encoding the MSP-1 protein is reduced from about 74% to about 55%.  
     
     
         59 . The nucleic acid of  claim 55 , wherein the nucleotide sequence encodes an MSP-1 protein having the amino acid sequence consisting of amino acids 20-1621 of SEQ ID NO:3.  
     
     
         60 . The nucleic acid of  claim 55 , wherein the MSP-1-encoding nucleotide sequence is set forth in SEQ ID NO:7.  
     
     
         61 . The nucleic acid of  claim 55 , wherein the MSP-1 protein further comprises six histidine residues at the amino terminus of the protein.  
     
     
         62 . The nucleic acid of  claim 55 , wherein the MSP-1-encoding nucleotide sequence lacks recognizable splice donor and splice acceptor sites.  
     
     
         63 . A vector comprising the nucleic acid of  claim 42 ,  claim 49 , or  claim 55 .  
     
     
         64 . The vector of  claim 63 , wherein the vector is an expression vector.  
     
     
         65 . The vector of  claim 63 , further comprising cleavage sites for endonucleases at the 5′ and 3′ ends of the MSP-1-encoding nucleotide sequence, said endonuclease cleavage sites being absent from the remainder of the nucleotide sequence and from the vector.  
     
     
         66 . The vector of  claim 63 , wherein the MSP-1-encoding nucleotide sequence is operably linked to a promoter.  
     
     
         67 . The vector of  claim 66 , wherein the promoter is an inducible promoter.  
     
     
         68 . The vector of  claim 63 , wherein the vector is a viral vector.  
     
     
         69 . The vector of  claim 68 , wherein the vector is a vaccinia vector.  
     
     
         70 . The vector of  claim 68 , wherein the vector is an adenovirus vector.  
     
     
         71 . A host cell comprising the vector of  claim 63 .  
     
     
         72 . The host cell of  claim 71 , wherein the host cell is a prokaryotic cell.  
     
     
         73 . The host cell of  claim 72 , wherein the host cell is  Escherichia coli.    
     
     
         74 . The host cell of  claim 71 , wherein the host cell is an avirulent strain of  Salmonella.    
     
     
         75 . The host cell of  claim 71 , wherein the host cell is a eukaryotic cell.  
     
     
         76 . The host cell of  claim 75 , wherein the host cell is a HeLa cell.  
     
     
         77 . The host cell of  claim 75 , wherein the host cell is a CHO cell.  
     
     
         78 . The host cell of  claim 75 , wherein the host cell is a yeast cell.  
     
     
         79 . An organism comprising the vector of  claim 63 , wherein the organism is a non-virulent strain of  Toxoplasma  or  Leishmania.    
     
     
         80 . The organism of  claim 79 , wherein the organism is  Toxoplasma gondii.    
     
     
         81 . A composition comprising: 
 a) a merozoite surface protein-1 (MSP-1) of a  Plasmodium,  wherein the MSP-1 protein has a molecular weight in a range of 190 kD to 220 kD; and    b) an adjuvant.    
     
     
         82 . The composition of  claim 81 , wherein the adjuvant is aluminum oxide.  
     
     
         83 . A composition comprising the vector of  claim 63 .  
     
     
         84 . A composition comprising the host cell of  claim 71 .  
     
     
         85 . A composition comprising the organism of  claim 79.