Malaria vaccine

Abstract

A fusion protein, from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 hybrid protein and experiments showed that the GLURP-part of the hybrid increased the overall antibody response. Immunizations with the hybrid protein consistently generated a stronger antibody response against the individual GLURP and MSP3 domains than a mixture of the two recombinant molecules injected at one site or the individual recombinant molecules injected simultaneously at two different sites. The difference was most pronounced for the MSP3-specific antibody response suggesting that T cell epitopes located in the GLURP RO-region provide help for B-cell epitopes in the MSP3 region. Moreover, when the animals were injected with a mixture of GLURP and MSP3, individual mice tended to mount a predominant antibody response against either molecule: in some animals GLURP was immunodominant whereas in other animals MSP3 was the dominant immunogen. Additionally, the hybrid was also more antigenic than the individual recombinant proteins since the ELISA-titer of naturally occurring IgG antibodies, in clinically

1 .- 15 . (canceled) 
     
     
         16 . A malaria vaccine comprising a fusion protein that comprises a  Plasmodium falciparum  Glutamate-rich protein (GLURP) genetically coupled to at least one other  Plasmodium falciparum  derived protein selected from the group consisting of circumsporozoite protein (CS), merozoite surface protein 1 (MSP1), merozoite surface protein 3 (MSP3), merozoite surface protein 2 (MSP2), merozoite surface protein 4 (MSP4), merozoite surface protein 5 (MSP5), merozoite surface protein 6 (MSP6), apical membrane antigen (AMA1), Pf155/ring-infected erythrocyte surface antigen (Pf155/RESA), Rhoptry-associated protein (RAP1), Erythrocyte-binding antigen 175 (EBA-175),  Plasmodium falciparum  erythrocyte membrane protein (pfEMP1), exported protein 1 (EXP1), Liver-stage antigen 1 (LSA1), Liver-stage antigen 3 (lSA3), Pf25, Pf45/48, Pf230, Pf27, Pf16, and Pf28 or a homologue/immunogenic portion of said fusion protein. 
     
     
         17 . The malaria vaccine according to  claim 16 , wherein the  Plasmodium falciparum  derived protein that is genetically coupled to GLURP is MSP3. 
     
     
         18 . The malaria vaccine according to  claim 16 , wherein said fusion protein comprises SEQ ID NO: 1. 
     
     
         19 . An immunogenic polypeptide comprising the sequence of SEQ ID NO: 1, wherein said sequence comprises a conservative substitution of one or more amino acids. 
     
     
         20 . The immunogenic polypeptide according to  claim 19 , further comprising at least one other  Plasmodium falciparum  derived protein selected from the group consisting of circumsporozoite protein (CS), merozoite surface protein 1 (MSP1), merozoite surface protein 3 (MSP3), merozoite surface protein 2 (MSP2), merozoite surface protein 4 (MSP4), merozoite surface protein 5 (MSP5), merozoite surface protein 6 (MSP6), apical membrane antigen (AMA1), Pf155/ring-infected erythrocyte surface antigen (Pf155/RESA), Rhoptry-associated protein (RAP1), Erythrocyte-binding antigen 175 (EBA-175),  Plasmodium falciparum  erythrocyte membrane protein (pfEMP1), exported protein 1 (EXP1), Liver-stage antigen 1 (LSA1), Liver-stage antigen 3 (lSA3), Pf25, Pf45/48, P1230, Pf27, Pf16, and Pf28. 
     
     
         21 . A method of making a fusion protein comprising:
 providing a construct that expresses a fusion protein comprising a GLURP and at least one other  Plasmodium falciparum  derived protein selected from the group consisting of circumsporozoite protein (CS), merozoite surface protein 1 (MSP1), merozoite surface protein 3 (MSP3), merozoite surface protein 2 (MSP2), merozoite surface protein 4 (MSP4), merozoite surface protein 5 (MSP5), merozoite surface protein 6 (MSP6), apical membrane antigen (AMA1), Pf155/ring-infected erythrocyte surface antigen (Pf155/RESA), Rhoptry-associated protein (RAP1), Erythrocyte-binding antigen 175 (EBA-175),  Plasmodium falciparum  erythrocyte membrane protein (pfEMP1), exported protein 1 (EXP1), Liver-stage antigen 1 (LSA1), Liver-stage antigen 3 (lSA3), Pf25, Pf45/48, Pf230, Pf27, Pf16, and Pf28;   transforming a  Lactococcus  with said construct; and   growing said  Lactococcus  under conditions that expresses said fusion protein.   
     
     
         22 . The method of  claim 21 , further comprising isolating said fusion protein. 
     
     
         23 . The method of  claim 21 , wherein said fusion protein comprises GLURP and MSP3. 
     
     
         24 . The method of  claim 21 , wherein said fusion protein comprises SEQ. ID. NO.: 1 or a homologue or an immunogenic fragment thereof. 
     
     
         25 . The method of  claim 24 , wherein said homologue or immunogenic fragment thereof comprises at least one B-cell epitope and at least one T-cell epitope of GLURP together with at least one B-cell epitope of Msp3. 
     
     
         26 . The method of  claim 25 , wherein said B-cell epitope of GLURP is selected from the group consisting of P1, P3 and P4, and said B-cell epitope of MSP3 is a b-peptide epitope. 
     
     
         27 . The method of  claim 24 , wherein said homologue or immunogenic fragment thereof induces:
 i) a proliferation of T-lymphocytes withdrawn from an animal or human that has previously been infected with tuberculosis, or a release of IFN-γ, IL-12, TNF-α, IL-4, IL-5, IL-6, IL-10 or TGF-β from said T-lymphocytes; or   ii) an in vivo humoral response determined by production of antibodies in an infected individual, the presence of which are determined by an ELISA technique or a Western blot, wherein the homologue or the immunogenic fragment thereof is absorbed to either a nitrocellulose membrane or a polystyrene surface;   
       wherein the response under i) or ii) is higher than two standard deviations above background. 
     
     
         28 . A recombinant expression system comprising an isolated nucleic acid that comprises a sequence encoding a fusion protein that comprises a  Plasmodium falciparum  Glutamate-rich protein (GLURP) genetically coupled to at least one other  Plasmodium falciparum  derived protein selected from the group consisting of circumsporozoite protein (CS), merozoite surface protein 1 (MSP1), merozoite surface protein 3 (MSP3), merozoite surface protein 2 (MSP2), merozoite surface protein 4 (MSP4), merozoite surface protein 5 (MSP5), merozoite surface protein 6 (MSP6), apical membrane antigen (AMA1), Pf155/ring-infected erythrocyte surface antigen (Pf155/RESA), Rhoptry-associated protein (RAP1), Erythrocyte-binding antigen 175 (EBA-175),  Plasmodium falciparum  erythrocyte membrane protein (pfEMP1), exported protein 1 (EXP1), Liver-stage antigen 1 (LSA1), Liver-stage antigen 3 (lSA3), Pf25, Pf45/48, P1230, Pf27, Pf16, and Pf28. 
     
     
         29 . The recombinant expression system of  claim 28 , wherein said sequence encodes a fusion protein that comprises GLURP and MSP3. 
     
     
         30 . The recombinant expression system of  claim 28 , wherein said sequence encodes SEQ ID NO. 1 or a homologue or an immunogenic fragment thereof. 
     
     
         31 . The recombinant expression system of  claim 28 , wherein said sequence comprises SEQ. ID. NO.:2 or a homologue or an immunogenic fragment thereof. 
     
     
         32 . The recombinant expression system of  claim 30 , wherein said homologue or immunogenic fragment thereof comprises at least one B-cell epitope and at least one T-cell epitope of GLURP together with at least one B-cell epitope of Msp3. 
     
     
         33 . The recombinant expression system of  claim 32 , wherein said B-cell epitope of GLURP is selected from the group consisting of P1, P3 and P4, and said B-cell epitope of MSP3 is a b-peptide epitope. 
     
     
         34 . The recombinant expression system of  claim 30 , wherein said homologue or immunogenic fragment thereof induces:
 i) a proliferation of T-lymphocytes withdrawn from an animal or human that has previously been infected with tuberculosis, or a release of IFN-γ, IL-12, TNF-α, IL-4, IL-5, IL-6, IL-10 or TGF-β from said T-lymphocytes; or   ii) an in vivo humoral response determined by production of antibodies in an infected individual, the presence of which are determined by an ELISA technique or a Western blot, wherein the homologue or the immunogenic fragment thereof is absorbed to either a nitrocellulose membrane or a polystyrene surface;   
       wherein the response under i) or ii) is higher than two standard deviations above background. 
     
     
         35 . The recombinant expression system of  claim 28 , further comprising a pH and growth phase regulated promoter operably linked to said isolated nucleic acid. 
     
     
         36 . A method of malaria immunization in an individual in need thereof, comprising administering to said individual an amount of the vaccine of  claim 16  sufficient to immunize said individual against malaria. 
     
     
         37 . The method of  claim 36 , wherein said vaccine inhibits parasite growth in a monocyte-dependent manner. 
     
     
         38 . The method of  claim 36 , wherein said vaccine is administered orally. 
     
     
         39 . The method of  claim 36 , wherein said vaccine is administered parenterally. 
     
     
         40 . The method of  claim 36 , wherein the vaccine of  claim 17  is administered. 
     
     
         41 . The method of  claim 36 , wherein the vaccine of  claim 18  is administered. 
     
     
         42 . The method of  claim 36 , wherein said vaccine comprises the immunogenic polypeptide of  claim 19 .