Methods and reagents for the analysis and purification of polysaccharides

Abstract

The disclosure provides fusion proteins comprising a carbohydrate recognition domain of an innate immunity receptor and a heterologous polypeptide. The fusion proteins of the disclosure may be used, for example, to fingerprint polysaccharide compositions and to purify polysaccharide compositions. Polysaccharide compositions include those isolated from Ganoderma lucidum (Reishi). The methods and reagents of the disclosure may also be used to identify innate immunity receptors and cell types that bind to polysaccharide compositions (including polysaccharide compositions associated with pathogens), whereupon modulators of the identified receptors can then be obtained. The fusion proteins also may be used to inhibit the interaction between a polysaccharide composition and an innate immunity receptor on a cell surface. The methods and reagents of the disclosure are used in one example to determine that the DLVR1 innate immunity receptor on macrophages interacts with Dengue virus (DV), and that DLVR1 is responsible for DV-mediated secretion of proinflammatory cytokines from macrophages. The disclosure also provides DVLR1 antibodies that prevent the secretion of proinflammatory cytokines by DV-infected macrophages.

1 . A fusion protein comprising: 
 (a) a carbohydrate recognition domain of an innate immunity receptor;    and    (b) a heterologous polypeptide.    
     
     
         2 . The fusion protein of  claim 1  wherein said heterologous polypeptide comprises an immunoglobulin or a fragment of an immunoglobulin.  
     
     
         3 . The fusion protein of  claim 2  wherein said immunoglobulin is IgG.  
     
     
         4 . The fusion protein of  claim 1  wherein said heterologous polypeptide is IgG Fe.  
     
     
         5 . The fusion protein of  claim 1  wherein said heterologous polypeptide is human IgG1 Fe.  
     
     
         6 . The fusion protein of  claim 5  wherein said human IgG1 Fe is a variant that does not bind to Fe receptors.  
     
     
         7 . A method for determining whether a specific carbohydrate component is present in a composition comprising a polysaccharide, the method comprising: 
 (a) contacting said polysaccharide with a fusion protein comprising: 
 (i) a carbohydrate recognition domain of an innate immunity receptor, wherein said carbohydrate recognition domain is capable of binding to said specific carbohydrate component; and  
 (ii) a heterologous polypeptide; and  
   (b) determining whether said fusion protein has bound to said polysaccharide.    
     
     
         8 . The method of  claim 7  wherein said composition comprising said polysaccharide is immobilized on a solid support, and wherein step (b) is accomplished by determining whether said heterologous polypeptide is present on said solid suppport, wherein the presence of said heterologous polypeptide is indicative of the presence of said specific carbohydrate in said composition.  
     
     
         9 . The method of  claim 8  wherein said heterologous polypeptide is an immunoglobulin or a fragment of an immunoglobulin.  
     
     
         10 . A kit comprising: 
 (a) a fusion protein comprising: 
 (i) a carbohydrate recognition domain of an innate immunity receptor; and  
 (ii) a heterologous polypeptide; and  
   (b) reagents for detecting the presence of said heterologous polypeptide on said solid support.    
     
     
         11 . A method for determining whether an innate immunity receptor binds to a pathogen, the method comprising: 
 (a) contacting said pathogen with a fusion protein comprising: 
 (i) a carbohydrate recognition domain of said innate immunity receptor, wherein said carbohydrate recognition domain is capable of binding to a specific carbohydrate component; and  
 (ii) a heterologous polypeptide;  
   (b) determining whether said fusion protein has bound to said pathogen.    
     
     
         12 . The method of  claim 11  wherein said fusion protein is immobilized on a solid support, and wherein step (b) is accomplished by using an antibody specific for said pathogen.  
     
     
         13 . A method of inhibiting the interaction between an innate immunity receptor on the surface of a cell and a polysaccharide that binds to a carbohydrate recognition domain of said innate immunity receptor, the method comprising contacting said cell with a fusion protein comprising: 
 (a) said carbohydrate recognition domain of said innate immunity receptor; and    (b) a heterologous polypeptide.    
     
     
         14 . A method of inhibiting the interaction between an innate immunity receptor on the surface of a cell and a pathogen-associated polysaccharide that binds to a carbohydrate recognition domain of said innate immunity receptor, the method comprising contacting said cell with an antibody antagonist of said innate immunity receptor.  
     
     
         15 . The method of  claim 14  wherein said pathogen is selected from the group consisting of an enveloped virus, a fungal cell, and a bacterial cell.  
     
     
         16 . The method of  claim 14  wherein said interaction is inhibited in vivo by administering a pharmaceutical composition comprising said antibody to an organism suspected of coming into contact with said pathogen.  
     
     
         17 . The method of  claim 14  wherein said antibody is a monoclonal antibody.  
     
     
         18 . A method of treating an organism infected with an enveloped virus, the method comprising administering to said organism an agent that inhibits the activity of DVLR1.  
     
     
         19 . The method of  claim 18  wherein said organism is a human.  
     
     
         20 . The method of  claim 19  wherein said virus is influenza virus.  
     
     
         21 . The method of  claim 19  wherein said virus is a member of the Flaviviridae family.  
     
     
         22 . The method of  claim 21  wherein said virus is a member of the Flavivirus genus or the Hepacivirus genus.  
     
     
         23 . The method of  claim 22  wherein said virus is a member of the Flavivirus genus selected from the group consisting of West Nile Virus, Japanese encephamyelitis virus, yellow fever virus, tick-borne encephamyelitis virus, and Dengue virus.  
     
     
         24 . The method of  claim 23  wherein said virus is Dengue virus.  
     
     
         25 . The method of  claim 24  wherein said agent is an antibody against DVLR1.  
     
     
         26 . The method of  claim 24  wherein said agent is a monoclonal antibody against DVLR1.  
     
     
         27 . The method of  claim 24  wherein said agent is a humanized antibody against DVLR1.  
     
     
         28 . The method of  claim 24  wherein said agent is a fragment of an antibody against DVLR1.  
     
     
         29 . The method of  claim 24  wherein said agent is a mediator of RNA inteference.  
     
     
         30 . The method of  claim 29  wherein said agent is an siRNA comprising sequence from DVLR1.  
     
     
         31 . An isolated antibody, or epitope-binding fragment thereof, that binds to DVLR1 on CD14+ macrophages and which inhibits Dengue virus-mediated TNF-α secretion from CD14+ macrophages.  
     
     
         32 . A purified monoclonal antibody, or epitope-binding fragment thereof, which binds to a DVLR1 epitope bound by a monoclonal antibody selected from the group consisting of monoclonal antibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5, wherein said antibody or fragment thereof inhibits TNF-α secetion by macrophages after infection with Dengue virus.  
     
     
         33 . A pharmaceutical composition comprising the monoclonal antibody, or epitope-binding fragment thereof, of  claim 32  and a pharmaceutically acceptable excipient.  
     
     
         34 . A monoclonal antibody, or epitope-binding fragment thereof, the monoclonal antibody selected from the group consisting of monoclonal antibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5.  
     
     
         35 . A pharmaceutical composition comprising the monoclonal antibody, or epitope-binding fragment thereof, of  claim 34  and a pharmaceutically acceptable excipient.