Bifunctional protein anchors

Abstract

The disclosure relates to the areas of immunology and vaccine delivery. More specifically, it relates to a bacterial vaccine delivery technology with built-in immunostimulatory properties which allow the immobilization of any antigen of interest, without prior antigen modification. Provided is an antigen-loaded immunogenic carrier complex comprising at least one bifunctional polypeptide attached to an immunogenic carrier, the bifunctional polypeptide comprising a peptidoglycan binding domain (PBD) through which the polypeptide is attached to the carrier, fused to an antigen binding domain (ABD) to which at least one antigen of interest is bound. Also described is a pharmaceutical (e.g., vaccine) composition comprising an antigen-loaded immunogenic carrier complex.

1 - 7 . (canceled) 
     
     
         8 . A method for providing an antigen-loaded immunogenic carrier complex comprising: at least one bifunctional polypeptide attached to an immunogenic carrier, wherein the bifunctional polypeptide comprises a peptidoglycan binding domain (PBD) through which the bifunctional polypeptide is attached to the carrier, fused to an antigen binding domain (ABD) able to bind an antigen of interest, wherein the PBD comprises a peptide selected from the group consisting of a LysM domain, a peptide retrieved from a homology search in a peptide database with a LysM domain in the C-terminus of  Lactococcus lactis  cell wall hydrolase AcmA (“AcmA LysM domain”) and a peptide having at least 70% sequence identity to an AcmA LysM domain, provided that the PBD is able to a Gram-positive microorganism's cell wall; and wherein at least one antigen of interest is bound to the ABD, the method comprising:
 providing an immunogenic carrier; 
 providing a bifunctional polypeptide comprising a peptidoglycan binding domain (PBD) fused to an antigen binding domain (ABD) allowing attachment of said polypeptide to said immunogenic carrier; 
 contacting said immunogenic carrier and said polypeptide; and 
 contacting said polypeptide with an antigen of interest. 
 
     
     
         9 . The method according to  claim 8 , wherein providing an immunogenic carrier comprises preparing non-viable spherical peptidoglycan particles from a Gram-positive bacterium. 
     
     
         10 . The method according to  claim 9 , wherein
 providing said bifunctional polypeptide comprises selecting an antigen binding domain from a random peptide or antibody library.   
     
     
         11 . The method according to  claim 10 , comprising producing the bifunctional polypeptide in a host cell by recombinant expression of a nucleic acid construct encoding said polypeptide. 
     
     
         12 . The method according to  claim 11 , wherein said host cell secretes the polypeptide in culture medium. 
     
     
         13 - 17 . (canceled) 
     
     
         18 . The method according to  claim 10 , wherein selecting the ABD from a random peptide or antibody library utilizes phage display technology. 
     
     
         19 . The method according to  claim 8 , wherein the bifunctional polypeptide comprises an ABD selected from a random peptide or antibody library. 
     
     
         20 . The method according to  claim 19 , comprising producing the bifunctional polypeptide in a host cell by recombinant expression of a nucleic acid construct encoding the polypeptide. 
     
     
         21 . The method according to  claim 20 , wherein the host cell secretes the polypeptide in culture medium. 
     
     
         22 . The method according to  claim 19 , wherein selecting an ABD from a random peptide or antibody library utilizes phage display technology. 
     
     
         23 . The method according to  claim 8 , comprising producing the bifunctional polypeptide in a host cell by recombinant expression of a nucleic acid construct encoding the polypeptide. 
     
     
         24 . The method according to  claim 23 , wherein the host cell secretes the polypeptide in culture medium. 
     
     
         25 . A method of providing an antigen-loaded immunogenic carrier complex, the method comprising:
 contacting an immunogenic carrier with a bifunctional polypeptide comprising a peptidoglycan binding domain fused to an antigen binding domain (ABD) so as to attach the bifunctional polypeptide to the immunogenic carrier; and   contacting the bifunctional polypeptide with an antigen of interest.   
     
     
         26 . The method according to  claim 25 , wherein the immunogenic carrier comprises non-viable spherical peptidoglycan particles from a Gram-positive bacterium. 
     
     
         27 . The method according to  claim 25 , wherein the ABD has been selected by phage display. 
     
     
         28 . The method according to  claim 25 , further comprising:
 producing the bifunctional polypeptide in a host cell by recombinant expression of a nucleic acid construct encoding the polypeptide before contact with the immunogenic carrier.   
     
     
         29 . The method according to  claim 28 , wherein the host cell secretes the polypeptide in culture medium associated with the host cell. 
     
     
         30 . A method of providing an antigen-loaded immunogenic carrier complex, the method comprising:
 recombinantly expressing a bifunctional polypeptide comprising a peptidoglycan binding domain fused to an antigen binding domain (ABD) in a host cell by expressing a nucleic acid construct encoding the bifunctional polypeptide;   contacting an immunogenic carrier with the bifunctional polypeptide so as to attach the bifunctional polypeptide to the immunogenic carrier, wherein the immunogenic carrier comprises non-viable spherical peptidoglycan particles from a Gram-positive bacterium; and   contacting the polypeptide with an antigen of interest.   
     
     
         31 . The method according to  claim 25 , wherein the ABD has been selected by phage display.