Detection/Measurement Of Malaria Infection Disease Utilizing Natural Immunity By Hemozoin Induction, Screening Of Preventative Or Therapeutic Medicine For Malaria Infection Disease, And Regulation Of Natural Immunity Induction

Abstract

The instant invention is to provide a method for detecting and measuring malaria infection utilizing the induction by hemozoin (HZ); a method for screening a vaccine for malaria infection and a preventative or therapeutic agent for malaria infection using the method for detecting and measuring; and a means for regulating the induction of innate immunity using the HZ, synthetic HZ, or derivatives thereof as an adjuvant or immunostimulant. Malaria infection is detected and measured of by detecting and measuring HZ-induced, TLR9-mediated, and MyD88-dependent innate immune activity. The detection and measurement of malaria infection can be used to diagnose malaria infection. The method for detecting and measuring is also used for screening a vaccine for malaria infection and a preventative or therapeutic agent for malaria infection. Further, HZ, synthetic HZ, or derivatives thereof are used as an adjuvant or immunostimulant to regulate HZ-induced innate immune induction.

1 . A method for detecting and measuring malaria infection, wherein a TLR9-mediated, MyD88-dependent innate immune activity induced by hemozoin that is hydrophobic heme polymers produced as a hemoglobin metabolite by malaria parasites, is detected and measured. 
     
     
         2 . The method for detecting and measuring malaria infection according to  claim 1 , wherein the detection and measurement of an innate immune activity is a detection and measurement of an immune-cell activation. 
     
     
         3 . The method for detecting and measuring malaria infection according to  claim 2 , wherein the detection and measurement of an immune-cell activation is a detection and measurement of a cytokine production by immune cells. 
     
     
         4 . The method for detecting and measuring malaria infection according to  claim 3 , wherein the cytokine is one or more cytokines selected from TNF-α, IL-12p40, MCP-1, IL-6, and IFNα. 
     
     
         5 . The method for detecting and measuring malaria infection according to  claim 2 , wherein the immune-cell is a spleen-cell or dendritic-cell. 
     
     
         6 . A method for screening or testing the quality of a vaccine for malaria infection, wherein a method for detecting and measuring malaria infection according to  claim 1  is used as a method for evaluating a test vaccine. 
     
     
         7 . A method for screening a preventative or therapeutic agent for malaria infection, wherein a TLR9-mediated, MyD88-dependent innate immune activity induced by hemozoin that is hydrophobic heme polymers produced as a hemoglobin metabolite by malaria parasites, synthetic hemozoin, or derivatives thereof, is detected and measured in the presence of a test substance. 
     
     
         8 . A method for screening a preventative or therapeutic agent for malaria infection, wherein a method for detecting and measuring an innate immune activity is a method for detecting and measuring an innate immune activity according to  claim 2 . 
     
     
         9 . An adjuvant for regulating an induction of TLR9-mediated, MyD88-dependent innate immunity, which consists of hemozoin that is hydrophobic heme polymers produced as a hemoglobin metabolite by malaria parasites, synthetic hemozoin, or derivatives thereof. 
     
     
         10 . An immunostimulant for inducing TLR9-mediated, MyD88-dependent innate immunity, comprising as an active ingredient hemozoin that is hydrophobic heme polymers produced as a hemoglobin metabolite by malaria parasites, synthetic hemozoin, or derivatives thereof. 
     
     
         11 . A method for screening a preventative or therapeutic agent for malaria infection, wherein a cytokine production level in the case of administering a test substance and hemozoin, synthetic hemozoin, or derivatives thereof to MyD88 +/+  and TLR9 +/+  nonhuman animal and a cytokine production level in the case of administering a test substance and hemozoin, synthetic hemozoin, or derivatives thereof to MyD88 −/−  and/or TLR9 −/−  nonhuman animal, are measured and evaluated. 
     
     
         12 . The method for screening a preventative or therapeutic agent for malaria infection according to  claim 11 , wherein the cytokine production level is measured as a serum cytokine level by ELISA. 
     
     
         13 . The method for screening according to  claim 11 , wherein the nonhuman animal is a mouse. 
     
     
         14 . A method for screening a preventative or therapeutic agent for malaria infection, wherein a cytokine production level in cells in the case of administering a test substance and hemozoin, synthetic hemozoin, or derivatives thereof to MyD88 +/+  and TLR9 +/+  gene-expressing cells and a cytokine production level in cells in the case of administering a test substance and hemozoin, synthetic hemozoin, or derivatives thereof to MyD88 −/−  and/or TLR9 −/−  gene-expressing cells, are compared and evaluated. 
     
     
         15 . The method for screening a preventative or therapeutic agent for malaria infection according to  claim 14 , wherein the cells are spleen cells or dendritic cells. 
     
     
         16 . The method for screening a preventative or therapeutic agent for malaria infection according to  claim 14 , wherein the cytokine production level in cells is measured as a cytokine level in a cell-culture supernatant by ELISA. 
     
     
         17 . The method for screening a preventative or therapeutic agent for malaria infection according to  claim 11 , wherein the measurement of the cytokine production level is a measurement of the production level of any one or more cytokines selected from TNF-α, IL-12p40, MCP-1, IL-6, and IFNα. 
     
     
         18 . A method for using protein (a) or (b) as defined below as a receptor for a ligand consisting of hemozoin, synthetic hemozoin, or derivatives thereof:
 (a) a protein consisting of the amino acid sequence shown by SEQ ID No: 2;   (b) a protein consisting of an amino acid sequence in which one or a few amino acids are deleted, substituted, or added in the amino acid sequence shown by SEQ ID No:2, the protein being responsive to hemozoin, synthetic hemozoin, or derivatives thereof.   
     
     
         19 . The method for using according to  claim 18 , wherein the protein is encoded by a DNA comprising the nucleotide sequence shown by SEQ ID No: 1 or a complementary sequence thereof. 
     
     
         20 . The method for using according to  claim 18 , wherein the protein is encoded by a DNA which hybridizes under a stringent condition with a DNA comprising the nucleotide sequence shown by SEQ ID No: 1 or a complementary sequence thereof. 
     
     
         21 . A method for using hemozoin, synthetic hemozoin, or derivatives thereof as a ligand for receptor protein (a) or (b) as defined below:
 (a) a protein consisting of the amino acid sequence shown by SEQ ID No: 2;   (b) a protein consisting of an amino acid sequence in which one or a few amino acids are deleted, substituted, or added in the amino acid sequence shown by SEQ ID No: 2, the protein being responsive to hemozoin, synthetic hemozoin, or derivatives thereof.   
     
     
         22 . The method for using according to  claim 21 , wherein the protein is encoded by a DNA comprising the nucleotide sequence shown by SEQ ID No: 1 or a complementary sequence thereof. 
     
     
         23 . The method for using according to  claim 21 , wherein the protein is encoded by a DNA which hybridizes under a stringent condition with a DNA comprising the nucleotide sequence shown by SEQ ID No: 1 or a complementary sequence thereof, the protein being responsive to hemozoin. 
     
     
         24 . A TLR9 agonist which contains hemozoin, synthetic hemozoin, or derivatives thereof as an active ingredient. 
     
     
         25 . The TLR9 agonist according to  claim 24 , which activates MyD88. 
     
     
         26 . The TLR9 agonist according to  claim 25 , which induces an immune response through the TLR-MyD88-dependent pathway. 
     
     
         27 . The TLR9 agonist according to  claim 26 , wherein the immune response through the TLR-MyD88-dependent pathway is a production of any one or more cytokines selected from TNF-α, IL-12p40, MCP-1, IL-6, and IFNα. 
     
     
         28 . The TLR9 agonist according to  claim 24 , which activates TLR9 selectively. 
     
     
         29 . The TLR9 agonist according to  claim 28 , which does not activate TLR2, TLR4, TLR7, or TRIF. 
     
     
         30 . The TLR9 agonist according to  claim 24 , which is used as an agent for controlling innate immunity. 
     
     
         31 . A method for using hemozoin, synthetic hemozoin, or derivatives thereof as a ligand for TLR9. 
     
     
         32 . The method for using according to  claim 31 , which comprises a method for inducing an immune response through the TLR9-MyD88-dependent pathway. 
     
     
         33 . The method for using according to  claim 32 , wherein the immune response through the TLR9-MyD88-dependent pathway is a production of any one or more cytokines selected from TNF-α, IL-12p40, MCP-1, IL-6, and IFNα. 
     
     
         34 . The method for using according to  claim 31 , which comprises a method for controlling innate immunity.