Recombinant Mycobacterium Encoding A Heparin-Binding Hemagglutinin (HBHA) Fusion Protein And Uses Thereof

Abstract

Recombinant Mycobacteria (rMyc) which contain sequences encoding a heparin-binding hemagglutinin (HBHA) fission protein are provided, as are methods of making and using the rMyc and the fusion protein. The fusion protein includes an amino terminal mycobacterial antigen Ag85B leader peptide and transcription of the fusion protein is driven by an Ag85B promoter sequence. The recombinant fusion protein is produced in abundance by the rMyc, is post-translationally methylated, and is highly antigenic.

1 - 19 . (canceled) 
     
     
         20 . A method of determining whether a subject has a latent tuberculosis infection, comprising:
 detecting the presence or absence of immune reactivity to a fusion protein comprising an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding hemagglutinin (HBHA) protein or antigenic fragment thereof in said patient, wherein the presence of immune reactivity indicates that said subject has a latent tuberculosis infection.   
     
     
         21 . The method of  claim 20 , wherein said step of detecting comprises:
 obtaining a biological sample from said subject, and   detecting said immune reactivity in said biological sample.   
     
     
         22 . The method of  claim 20 , wherein said biological sample is a sputum sample or a serum sample. 
     
     
         23 . The method of  claim 20 , wherein said step of detecting comprises:
 intradermally injecting said subject with said fusion protein, and   detecting immune reactivity at a site of intradermal injection.   
     
     
         24 . A method of eliciting an immune response against  Mycobacterium tuberculosis  in a subject in need thereof, comprising:
 administering to said subject an amount of a fusion protein comprising an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding hemagglutinin (HBHA) protein or antigenic fragment thereof sufficient to elicit an immune response in said subject; or   administering to said subject a  Mycobacterium  comprising a nucleic acid fusion sequence encoding an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding hemagglutinin (HBHA) protein or antigenic fragment thereof, wherein said fusion sequence is operably linked to a promoter, and wherein said  Mycobacterium  is administered in an amount sufficient to elicit an immune response in said subject.   
     
     
         25 . The method of  claim 24 , wherein said immune response is production of one or more of B cells, antibodies and T cells. 
     
     
         26 . The method of  claim 24 , wherein said immune response is a protective immune response. 
     
     
         27 - 37 . (canceled) 
     
     
         38 . A method of preparing a composition comprising a heparin-binding hemagglutinin (HBHA) protein comprising:
 growing a culture of  Mycobacterium  comprising a nucleic acid fusion sequence encoding an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding hemagglutinin (HBHA) protein or antigenic fragment thereof, wherein said fusion sequence is operably linked to a promoter;   obtaining said rHBHA protein from said culture;   purifying said rHBHA protein; and   combining said purified rHBHA protein with a physiologically acceptable carrier.   
     
     
         39 . The method of  claim 38 , wherein said growing is carried out in shake culture or by fermentation. 
     
     
         40 . The method of  claim 38 , further comprising adding at least one agent selected from the group consisting of: one or more antigens that are not HBHA, one or more adjuvants, and one or more immunogenicity enhancers, to said composition. 
     
     
         41 - 44 . (canceled) 
     
     
         45 . The method according to  claim 20 , wherein said antigenic fragment is comprised within the last 30 to 50 carboxyl terminal amino acids of SEQ ID NO:4. 
     
     
         46 . The method according to  claim 45 , wherein said antigenic fragment is or comprises SEQ ID NO: 5.