Recombinant plasmodium falciparum merozoite surface proteins 4 and 5 and their use

Abstract

Accordingly, the invention provides constructs in which the nucleic acids encoding Plasmodium falciparum MSP4 and MSP5, and the resulting polypeptides, have been modified. More particularly, this invention provides constructs encoding recombinant MSP4 and MSP5 polypeptides, which are expressed as soluble, secreted polypeptides in a baculovirus-insect cell expression system. It was surprisingly found that the recombinant polypeptides contain an EGF-like domain at the C-terminus that is properly folded in the polypeptide.

What is claimed is: 
     
         1 . A purified nucleic acid molecule comprising the DNA sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 28, 29, or 8. 
     
     
         2 . A purified nucleic acid molecule encoding an amino acid sequence comprising the sequence of SEQ ID NOS: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, or 22. 
     
     
         3 . A purified nucleic acid molecule that hybridizes to either strand of a denatured, double-stranded DNA comprising the nucleic acid sequence of any one of  claims 1  or  2  under conditions of moderate stringency in 50% formamide and 6×SSC, at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SOS. 
     
     
         4 . The purified nucleic acid molecule as claimed in  claim 3 , wherein said isolated nucleic acid molecule is derived by in vitro mutagenesis from SEQ ID NOS: 1-8. 
     
     
         5 . A purified nucleic acid molecule degenerate from SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 as a result of the genetic code. 
     
     
         6 . A purified nucleic acid molecule, which encodes recombinant MSP4 or MSP5 polypeptide, an allelic variant of recombinant MSP4 or MSP5 polypeptide DNA, or a homolog of recombinant MSP4 or MSP5 polypeptide DNA. 
     
     
         7 . A recombinant vector that directs the expression of a nucleic acid molecule selected from the group consisting of the purified nucleic acid molecules of  claims 1 ,  2 ,  5 , and  6 . 
     
     
         8 . A recombinant vector that directs the expression of a nucleic acid molecule of  claim 3 . 
     
     
         9 . A recombinant vector that directs the expression of a nucleic acid molecule of  claim 4 . 
     
     
         10 . A purified polypeptide encoded by a nucleic acid molecule selected from the group consisting of the purified nucleic acid molecules of  claims 1 ,  2 ,  5 , and  6 . 
     
     
         11 . A purified polypeptide according to  claim 10  in non-glycosylated form. 
     
     
         12 . A purified polypeptide encoded by a nucleic acid molecule of  claim 3 . 
     
     
         13 . A purified polypeptide according to  claim 12  in non-glycosylated form. 
     
     
         14 . A purified polypeptide encoded by a nucleic acid molecule of  claim 4 . 
     
     
         15 . A purified polypeptide according to  claim 14  in non-glycosylated form. 
     
     
         16 . Purified antibodies that bind to a polypeptide of  claim 10 . 
     
     
         17 . Purified antibodies according to  claim 16 , wherein the antibodies are monoclonal antibodies. 
     
     
         18 . Purified antibodies that bind to a polypeptide of  claim 12 . 
     
     
         19 . Purified antibodies according to  claim 18 , wherein the antibodies are monoclonal antibodies. 
     
     
         20 . Purified antibodies that bind to a polypeptide of  claim 14 . 
     
     
         21 . Purified antibodies according to  claim 20 , wherein the antibodies are monoclonal antibodies. 
     
     
         22 . A host cell transfected, transduced or infected with the vector of  claim 7 . 
     
     
         23 . A method for the production of recombinant MSP4 or MSP5 polypeptide comprising culturing a host cell of  claim 22  under conditions promoting expression, and recovering the polypeptide from the culture medium. 
     
     
         24 . The method of  claim 23 , wherein the host cell is selected from the group consisting of insect cells, bacterial cells, yeast cells, plant cells, and animal cells. 
     
     
         25 . A host cell transfected, infected or transduced with the vector of  claim 8 . 
     
     
         26 . A method for the production of recombinant MSP4 and MSP5 polypeptides comprising culturing a host cell of  claim 25  under conditions promoting expression, and recovering the polypeptide from the culture medium or cellular pellets. 
     
     
         27 . The method of  claim 26 , wherein the host cell is selected from the group consisting of insect cells, bacterial cells, yeast cells, plant cells, and animal cells. 
     
     
         28 . A host cell transfected, infected or transduced with the vector of  claim 9 . 
     
     
         29 . A method for the production of recombinant MSP4 and MSP5 polypeptide comprising culturing a host cell of  claim 28  under conditions promoting expression, and recovering the polypeptide from the culture medium or cellular aggregates. 
     
     
         30 . The method of  claim 29 , wherein the host cell is selected from the group consisting of insect cells, bacterial cells, yeast cells, plant cells, and animal cells. 
     
     
         31 . A recombinant vector selected from the group consisting of I-3512, I-3513, I-3514, I-3694, I-3695 and I-3696. 
     
     
         32 . An immunological complex comprising a recombinant MSP4 and MSP5 polypeptide and an antibody that specifically recognizes said polypeptide. 
     
     
         33 . A method for detecting infection by malaria parasite, wherein the method comprises providing a composition comprising a biological material suspected of being infected with malaria parasite, and assaying for the presence of native MSP4 and/or MSP5 polypeptide of malaria parasite that binds to an antibody specific for recombinant MSP4 or MSP5 polypeptide. 
     
     
         34 . The method of  claim 33 , wherein it is used to detect  Plasmodium falciparum.    
     
     
         35 . The method of  claim 33 , wherein it is used to detect  Plasmodium vivax.    
     
     
         36 . The method of  claim 33 , wherein the native MSP4 or MSP5 polypeptide is assayed by electrophoresis or by immunoassay with antibodies that are immunologically reactive with the recombinant MSP4 and MSP5 polypeptide. 
     
     
         37 . An in vitro diagnostic method for the detection of the presence or absence of antibodies, which bind to an antigen comprising recombinant MSP4 or MSP5 polypeptide, wherein the method comprises contacting the antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an antigen-antibody complex, and detecting the formation of the complex. 
     
     
         38 . The method of  claim 35 , which further comprises measuring the formation of the antigen-antibody complex. 
     
     
         39 . The method of  claim 35 , wherein the formation of antigen-antibody complex is detected by immunoassay based on Western blot technique, ELISA, indirect immunofluorescence assay, or immunoprecipitation assay. 
     
     
         40 . A diagnostic kit for the detection of the presence or absence of antibodies, which bind to recombinant MSP4 or MSP5 polypeptide or mixtures thereof, wherein the kit comprises an antigen comprising recombinant MSP4 or MSP5 polypeptide or mixtures of recombinant MSP4 and MSP5 polypeptides, and means for detecting the formation of immune complex between the antigen and antibodies, wherein the means are present in an amount sufficient to perform said detection. 
     
     
         41 . An immunogenic composition comprising at least one recombinant MSP4 or MSP5 polypeptide or a mixture thereof in an amount sufficient to induce an immunogenic or protective response in vivo, and a pharmaceutically acceptable carrier therefore. 
     
     
         42 . The immunogenic composition of  claim 41 , wherein said composition comprises a sufficient amount of at least one recombinant MSP4 or MSP5 polypeptide to induce neutralizing antibodies in vivo. 
     
     
         43 . The immunogenic composition of  claim 41  comprising at least the recombinant polypeptide MSP4p40. 
     
     
         44 . The immunogenic composition of  claim 41  comprising at least the recombinant polypeptide MSP4p30. 
     
     
         45 . The immunogenic composition of  claim 41  comprising at least the recombinant polypeptide MSP4p20. 
     
     
         46 . The immunogenic composition of  claim 41  comprising at least the recombinant polypeptides MSP5p45 and MSP5p35. 
     
     
         47 . The immunogenic composition of  claim 39  comprising at least the recombinant polypeptide PvMSP4p20. 
     
     
         48 . A polynucleotide selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. 
     
     
         49 . A polynucleotide selected from SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 30. 
     
     
         50 . The immunogenic composition of  claim 41  comprising at least a recombinant polypeptide chosen among PfMSP4p21ss1 and PfMSP4p21ss2. 
     
     
         51 . The immunogenic composition of  claim 39  comprising at least the recombinant polypeptides MSP4p40 and MSP4p20. 
     
     
         52 . The immunogenic composition of  claim 39  further comprising an antigen comprising the recombinant MSP1p19 polypeptide. 
     
     
         53 . A vaccine composition comprising at least one recombinant MSP4 or MSP5 polypeptide or a mixture thereof in an amount sufficient to induce a protective response in vivo, and a pharmaceutically acceptable carrier therefore, wherein the at least one recombinant MSP4 or MSP5 polypeptide is chosen among MSP4p40, MSP4p30, MSP4p20, MSP4p21ss1, MSP4p21ss2, MSP5p45, MSP5p35 and PvMSP4p20. 
     
     
         54 . A method for in vitro diagnosing a  Plasmodium  infection in a sample of biological material from a human suspected to be infected by the parasite, wherein the method comprises the following steps:
 contacting an antigen comprising recombinant MSP4 or MSP5 polypeptide with the sample for a time and under conditions sufficient for the antigen and antibodies in the sample to form an antigen-antibody complex, and   detecting the formation of the complex.   
     
     
         55 . The method as claimed in  claim 54 , which further comprises measuring the formation of the antigen-antibody complex. 
     
     
         56 . The method as claimed in  claim 54 , wherein the formation of antigen-antibody complex is detected by immunoassay based on Western blot technique, ELISA, indirect immunofluorescence assay, or immunoprecipitation assay. 
     
     
         57 . The method of  claim 36 , wherein the native MSP4 or MSP5 polypeptide is assayed by immunoassay with antibodies secreted by hybridoma deposited at C.N.C.M. on Nov. 18, 2005 under Accession number I-3517, I-3518, I-3519 or I-3520. 
     
     
         58 . Purified antibodies according to  claim 17 , wherein they are produced from hybridoma deposited at C.N.C.M. on Nov. 16, 2005 under Accession number I-3517, I-3518, I-3519 or I-3520 
     
     
         59 . Hybridoma deposited at C.N.C.M. on Nov. 16, 2005 under Accession number I-3517, I-3518, I-3519 or I-3520. 
     
     
         60 . A method for detecting a malaria parasite, wherein the method comprises providing a composition comprising a biological material suspected of being infected with malaria parasite, and assaying for the presence of native MSP4 and/or MSP5 polypeptide of malaria parasite that binds to an antibody specific for recombinant MSP4 or MSP5 polypeptide or a mixture of at least one antibody specific for recombinant MSP4 polypeptide and at least one antibody specific for recombinant MSP5 polypeptide. 
     
     
         61 . The method of  claim 60 , wherein the recombinant MSP4 or MSp5 polypeptide is selected among the purified polypeptides of  claim 10 . 
     
     
         62 . The method of  claim 60 , wherein the antibody specific for recombinant MSP4 or MSP5 polypeptide is selected among the purified antibodies of  claim 58 . 
     
     
         63 . The method of  claim 60 , wherein it is used to detect  Plasmodium falciparum  or  Plasmodium vivax.