Immunogenic compositions and methods of use thereof

Abstract

Embodiments of the disclosure encompass compositions and methods for generating immune responses in an animal or human host. Embodiments of the compositions encompass proteins derived from the surface proteins of bacteria and protozoa, and in particular the flagellum component flagellin, and which have adjunctival properties when administered in conjunction with an immunogen. Embodiments of the compositions of the disclosure are modified to incorporate a heterologous transmembrane-cytoplasmic domain allowing the peptides to be incorporated into virus-like particles. Embodiments of the methods of generating an immunological response in an animal or human comprise exposing the immune system of an animal or human host to an immunogen and a virus-like particle comprising an adjuvant polypeptide including a host cell Toll-like receptor ligand polypeptide having a transmembrane-cytoplasmic tail polypeptide, and a heterologous signal peptide.

1 . An adjuvant polypeptide comprising:
 at least one domain capable of selectively interacting with a Toll-like receptor of an animal or human cell, and wherein the domain is capable of increasing an immune response in a recipient host; and   at least one heterologous region selected from the group consisting of a signal peptide region, a transmembrane-cytoplasmic tail region, and an immunogenic peptide.   
     
     
         2 . The adjuvant polypeptide of  claim 1 , comprising a first region, wherein the first region comprises a signal peptide; a second region, wherein the second region comprises at least one domain capable of selectively interacting with a Toll-like receptor of an animal or human cell, and wherein the domain is capable of increasing an immune response in a recipient host; and a third region, wherein the third region comprises a transmembrane-cytoplasmic tail peptide. 
     
     
         3 . The adjuvant polypeptide of  claim 1 , wherein the at least one domain capable of selectively interacting with a Toll-like receptor of an animal or human cell is derived from a surface protein of a bacterial species, or of a protozoal species. 
     
     
         4 . The adjuvant polypeptide of  claim 3 , wherein the surface protein is a protein of a bacterial or a protozoal flagellum, or a fragment thereof. 
     
     
         5 . The adjuvant polypeptide of  claim 4 , wherein the surface protein is a flagellin of the bacterial species  Salmonella enteritidis.    
     
     
         6 . The adjuvant polypeptide of  claim 1  having the amino acid sequence SEQ ID NO.: 10, or a conservative variant thereof. 
     
     
         7 . The adjuvant polypeptide of  claim 1 , further comprising a peptide linker. 
     
     
         8 . The adjuvant polypeptide of  claim 6 , wherein the peptide linker has an amino acid sequence according to SEQ ID NO.: 18. 
     
     
         9 . The adjuvant polypeptide of  claim 1 , wherein the signal peptide is a signal peptide of a bee melittin polypeptide. 
     
     
         10 . The adjuvant polypeptide of  claim 1 , wherein the transmembrane-cytoplasmic tail is derived from a hemagglutinin A polypeptide of an influenza virus. 
     
     
         11 . The adjuvant polypeptide of  claim 1 , further comprising an immunogenic peptide. 
     
     
         12 . The adjuvant polypeptide of  claim 1 , wherein the immunogenic peptide has an amino acid sequence according to SEQ ID NO.: 19. 
     
     
         13 . A nucleic acid molecule, comprising:
 a region encoding a bacterial flagellin polypeptide, or a fragment thereof, wherein the flagellin polypeptide or fragment thereof comprises at least one domain capable of specifically interacting with a Toll-like receptor of an animal or human cell; and   at least one region selected from the group consisting of: a region encoding a heterologous signal peptide and a region encoding a transmembrane-cytoplasmic tail capable of being incorporated into a virus-like particle or virosome.   
     
     
         14 . The nucleic acid molecule of  claim 13 , comprising:
 a region encoding a bacterial flagellin polypeptide, or a fragment thereof, wherein the flagellin polypeptide or fragment thereof comprises at least one domain capable of specifically interacting with a Toll-like receptor of an animal or human cell;   a region encoding a heterologous signal peptide; and   a region encoding a transmembrane-cytoplasmic tail capable of being incorporated into a virus-like particle or virosome.   
     
     
         15 . The nucleic acid molecule of  claim 13 , wherein the heterologous signal peptide is a bee melittin signal peptide, and the transmembrane-cytoplasmic tail is from an influenza virus hemagglutinin. 
     
     
         16 . The nucleic acid molecule of  claim 13 , comprising:
 a first nucleotide sequence encoding an amino acid sequence from amino acid positions of about 1 to about 305 of sequence SEQ ID NO.: 10, or a conservative variant thereof;   a second nucleotide sequence encoding an amino acid sequence from amino acid positions about 430 to about 565 of sequence SEQ ID NO.: 10, or a conservative variant thereof; and   a third nucleotide sequence disposed between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a region selected from the group consisting of: a region of a bacterial or protozoal surface protein polypeptide, a peptide linker, an immunogenic peptide, and an antigenic peptide.   
     
     
         17 . The nucleic acid molecule of  claim 16 , wherein the first nucleotide sequence is
 according to about position 1 to about position 615 of nucleotide sequence SEQ ID NO.: 9, or a conservative variant thereof; and   the second nucleotide sequence is according to about position 1293 to about position 1695 of nucleotide sequence SEQ ID NO.: 9, or a conservative variant thereof.   
     
     
         18 . The nucleic acid molecule of  claim 16 , comprising the nucleic acid sequence according to SEQ ID NO.: 9, or a conservative variant thereof. 
     
     
         19 . The nucleic acid molecule of  claim 16  having the nucleic acid sequence according to SEQ ID NO.: 9. 
     
     
         20 . The nucleic acid molecule of  claim 13 , wherein the nucleic acid molecule is operably inserted into a nucleic acid expression vector. 
     
     
         21 . The nucleic acid molecule of  claim 20 , wherein the nucleic acid expression vector is selected from the group consisting of: a plasmid, a baculovirus vector, a cosmid, a viral vector, a chromosome, a mini-chromosome, a modified vaccinia Ankara (MVA) vector, a recombinant poxvirus vector, a recombinant adenovirus vector, an alphavirus vector, and a paramyxovirus vector. 
     
     
         22 . An immunogenic composition comprising:
 an adjuvant polypeptide comprising at least one region capable of selectively interacting with a Toll-like receptor protein of a host; and   an immunogen capable of producing an immune response in a recipient host.   
     
     
         23 . The immunogenic composition of  claim 22 , further comprising a virus-like carrier, wherein the virus-like carrier is selected from the group consisting of a virus-like particle and a virosome, and wherein the adjuvant polypeptide and the immunogen are incorporated in the virus-like particle or the virosome. 
     
     
         24 . The immunogenic composition of  claim 22 , wherein the adjuvant polypeptide is a surface polypeptide of a bacterial species or of a protozoal species, or a modified variant of the surface polypeptide. 
     
     
         25 . The immunogenic composition of  claim 24 , wherein the surface polypeptide is a bacterial flagellin. 
     
     
         26 . The immunogenic composition of  claim 25 , wherein the flagellin is of the bacterial species  Salmonella enteritidis.    
     
     
         27 . The immunogenic composition of  claim 24 , wherein the modified variant of the adjuvant polypeptide comprises at least one heterologous peptide region, wherein the at least heterologous region is selected from the group consisting of a signal peptide region and a transmembrane-cytoplasmic tail region. 
     
     
         28 . The immunogenic composition of  claim 22 , wherein the modified variant of the adjuvant polypeptide comprises a first heterologous peptide region, wherein the first heterologous peptide region is a signal peptide, a second heterologous peptide region, wherein the second heterologous peptide region is a transmembrane-cytoplasmic tail peptide, and at least one region capable of selectively interacting with a Toll-like receptor protein of a host. 
     
     
         29 . The immunogenic composition of  claim 25 , wherein the bacterial flagellin polypeptide is a modified bacterial flagellin polypeptide modified by deletion of a region of a full-length bacterial flagellin polypeptide. 
     
     
         30 . The immunogenic composition of  claim 22 , wherein the adjuvant polypeptide further comprises a heterologous peptide region, wherein said region is disposed between two domains of the adjuvant polypeptide, wherein each of said domains is capable of selectively targeting a toll-like receptor protein of a host. 
     
     
         31 . The immunogenic composition of  claim 22 , wherein the adjuvant polypeptide further comprises a heterologous peptide region, wherein said region is antigenic. 
     
     
         32 . The immunogenic composition of  claim 31 , wherein the heterologous peptide region is antigenic and has the amino acid sequence according to SEQ ID NO.: 19. 
     
     
         33 . The immunogenic composition of  claim 23 , wherein the virus-like carrier is a virosome, comprising:
 at least one viral surface envelope glycoprotein expressed on the surface of the virosome; and   at least one adjuvant molecule expressed on the surface of the virosome, wherein the at least one adjuvant molecule comprises a membrane-anchored form of a bacterial or protozoal surface component that is a mammalian toll-like receptor (TLR) ligand molecule.   
     
     
         34 . The immunogenic composition of  claim 33 , wherein the virus-like carrier is a virus-like particle and further comprises a viral core protein capable of self-assembling into a virus-like particle core. 
     
     
         35 . The immunogenic composition of  claim 34 , wherein the viral core protein and the at least one viral surface envelope glycoprotein are from different viruses. 
     
     
         36 . The immunogenic composition of  claim 34 , wherein the viral core protein is selected from the group consisting of: a retrovirus Gag protein, a retrovirus matrix protein, a rhabdovirus M protein, a filovirus viral core protein, a coronavirus M protein, a coronavirus E protein, a coronavirus NP protein, a bunyavirus N protein, an influenza M1 protein, a paramyxovirus M protein, an arenavirus Z protein, a cytomegalovirus (CMV) core protein, a herpes simplex virus (HSV) core protein, Vesicular Stomatitis Virus (VSV) M protein, an Ebola Virus VP40 protein, a Lassa Fever Virus Z protein, and a combination thereof. 
     
     
         37 . The immunogenic composition of  claim 36 , wherein the retrovirus gag protein is selected from the group consisting of; a Human Immunodeficiency Virus (HIV) Gag protein, a Simian Immunodeficiency Virus (SIV) Gag protein, a human foamy virus Gag protein, and a Murine Leukemia Virus (MuLV) Gag protein. 
     
     
         38 . The immunogenic composition of  claim 33 , wherein the at least one viral surface envelope surface glycoprotein is selected from the group consisting of: a retrovirus/lentivirus glycoprotein, a bunyavirus glycoprotein, a coronavirus glycoprotein, an arenavirus glycoprotein, a filovirus glycoprotein, an influenza virus glycoprotein, a paramyxovirus glycoprotein, a rhabdovirus glycoprotein, an alphavirus glycoprotein, a flavivirus glycoprotein, a cytomegalovirus glycoprotein, a herpes virus glycoprotein, and a combination thereof. 
     
     
         39 . The immunogenic composition of  claim 38 , wherein the retrovirus glycoprotein is selected from the group consisting of: a human immunodeficiency virus (HIV) glycoprotein, a simian immunodeficiency virus (SIV) glycoprotein, a simian-human immunodeficiency virus (SHIV) glycoprotein, a feline immunodeficiency virus (FIV) glycoprotein, a feline leukemia virus glycoprotein, a bovine immunodeficiency virus glycoprotein, a bovine leukemia virus glycoprotein, an equine infectious anemia virus glycoprotein, a human T-cell leukemia virus glycoprotein, a mouse mammary tumor virus envelope glycoprotein (MMTV), a human foamy virus glycoprotein, and a combination thereof. 
     
     
         40 . The immunogenic composition of  claim 38 , wherein the viral surface envelope surface glycoprotein is further selected from the group consisting of: an influenza virus glycoprotein, a Respiratory syncytial virus (RSV) glycoprotien, a Lassa Fever virus glycoprotein, an Ebola Virus glycoprotein, a Marburg virus glycoprotein, a VSV glycoprotein, a rabies virus glycoprotein, a hepatitis virus glycoprotein, a herpes virus glycoprotein, a CMV glycoprotein, and a combination thereof. 
     
     
         41 . The immunogenic composition of  claim 23 , wherein the virus-like carrier comprises an influenza hemagglutinin, a matrix protein M1, and a modified bacterial flagellin adjuvant polypeptide, wherein the modified bacterial flagellin adjuvant comprises a heterologous transmembrane-cytoplasmic tail and is incorporated into the virus-like carrier, and wherein the virus-like carrier is a virus-like particle or a virosome. 
     
     
         42 . The immunogenic composition of  claim 22 , further comprising a pharmacologically acceptable carrier. 
     
     
         43 . A method of generating an immunological response in an animal or human host, comprising:
 exposing an animal or human host to an immunogen and a virus-like carrier, wherein the virus-like carrier is a virus-like particle or a virosome, and wherein the virus-like carrier comprises an adjuvant polypeptide comprising a host cell Toll-like receptor ligand polypeptide derived from a bacterial or protozoal flagellum polypeptide, and at least one heterologous peptide selected from the group consisting of: a transmembrane-cytoplasmic tail polypeptide and a heterologous signal peptide;   thereby generating in the recipient host an immune response directed against the immunogen.   
     
     
         44 . The method of  claim 43 , wherein the immunogen is incorporated into the virus-like carrier. 
     
     
         45 . The method of  claim 43 , further comprising:
 delivering to the recipient host or host cell at least one expression vector, wherein the at least one expression vector or a multiplicity of expression vectors comprise at least one polynucleotide encoding at least one polypeptide selected from the group consisting of: a viral core protein, a viral surface envelope glycoprotein, and an adjuvant molecule, wherein each of the polynucleotide or polynucleotides is operably linked to an expression control region;   expressing in the recipient host or host cell at least one viral surface envelope glycoprotein, and at least one adjuvant molecule, thereby assembling a virosome virus-like carrier.   
     
     
         46 . The method of  claim 45 , wherein an expression vector further comprises a polynucleotide encoding a viral core protein, wherein the viral core protein is incorporated into a virus-like particle. 
     
     
         47 . The method of  claim 45 , wherein an expression vector is selected from the group consisting of: a plasmid, a cosmid, a viral vector, an artificial chromosome, a mini-chromosome, a baculovirus vector, a modified vaccinia Ankara (MVA) vector, a recombinant poxvirus vector, a recombinant VSV vector, a recombinant adenovirus expression systems, an alphavirus vector, a paramyxovirus vector, and a combination thereof. 
     
     
         48 . A method of immunizing a host comprising:
 co-expressing in one or more host cells at least one viral surface envelope surface glycoprotein, and at least one adjuvant molecule;   whereby the at least one viral surface envelope glycoprotein and the adjuvant molecule assemble to form a virus-like carrier, and wherein the at least one adjuvant molecule is a mammalian toll-like receptor ligand molecule.   
     
     
         49 . The method of  claim 48 , further comprising co-expressing a viral core protein, wherein the viral core protein is assembled into the virus-like carrier, thereby forming a virus-like particle. 
     
     
         50 . The method of  claim 48 , wherein the at least one adjuvant molecule is a bacterial flagellin molecule. 
     
     
         51 . The method of  claim 48 , wherein the virus-like particle is a chimeric virus-like particle comprising an influenza hemagglutinin, a matrix protein M1, and a modified bacterial flagellin adjuvant polypeptide, wherein the modified bacterial flagellin comprises a heterologous transmembrane-cytoplasmic tail and is incorporated into the chimeric virus-like particle, the chimeric virus-like particle inducing an immune response in the animal or human host and thereby inhibiting the development of an influenza infection in the host.