Particles of HCV envelope proteins: use for vaccination

Abstract

The present invention is based on the finding that the envelope proteins of HCV induce a beneficial immune response in chronically HCV-infected chimpanzees. The immunization can preferentially be carried out using HCV envelope proteins in the form of particles which are produced in a detergent-assisted manner. The envelope proteins when presented as such to chronic HCV carriers are highly immunogenic and stimulate both the cellular and humoral immune response.

1 . An oligomeric particle consisting essentially of HCV envelope proteins and having a diameter of 1 to 100 nanometer.  
     
     
         2 . Oligomeric particle according to  claim 1  having a diameter of 2 to 40 nanometer.  
     
     
         3 . Oligomeric particle according to any of claims  1  or  2 , in which the amino acid sequence of the HCV envelope protein is a consensus sequence derived from an isolate-, subtype-, strain-, or genus-consensus sequence.  
     
     
         4 . Oligomeric particle according to any of  claims 1  to  3 , possibly containing non-HCV epitopes.  
     
     
         5 . Oligomeric particle according to any of  claims 1  to  4 , wherein at least one cysteine residue of said envelope protein is alkylated.  
     
     
         6 . Oligomeric particle according to  claim 5 , wherein said cysteine residue is alkylated by means of active halogens, ethylenimine or N-(iodoethyl)trifluoroacetamide.  
     
     
         7 . Oligomeric particle according to any of  claims 1  to  4 , wherein at least one cysteine residue of said envelope protein is mutated to a natural amino acid, preferentially selected from the group consisting of methionine, glutamic acid, glutamine and lysine.  
     
     
         8 . Oligomeric particle according to any of  claims 1  to  7 , wherein said envelope proteins are HCV E1 proteins or parts thereof.  
     
     
         9 . Oligomeric particle according to any of  claims 1  to  7 , wherein said envelope proteins are HCV E1s proteins or parts thereof.  
     
     
         10 . Oligomeric particle according to any of  claims 1  to  7 , wherein said envelope proteins are HCV E2 proteins or parts thereof.  
     
     
         11 . Oligomeric particle according to any of  claims 1  to  7 , wherein said envelope proteins are SEQ ID No 13 and/or SEQ ID No 14, or parts thereof.  
     
     
         12 . Oligomeric particle according to any of  claims 1  to  11 , wherein said envelope proteins are encoded by an isolate nucleotide consensus sequence, subtype nucleotide consensus sequence, species nucleotide consensus sequence, or genus nucleotide consensus sequence, or parts thereof.  
     
     
         13 . Oligomeric particle according to any of  claims 1  to  7 , wherein said envelope proteins are a mixture of HCV E1, HCV E1s, HCV E2 proteins and/or SEQ ID No 13 and/or SEQ ID No 14, or parts thereof.  
     
     
         14 . Oligomeric particle according to any of  claims 1  to  13 , wherein said envelope proteins, or parts thereof, are derived from different HCV strains or genotypes.  
     
     
         15 . Oligomeric particle according to any of  claims 1  to  14 , wherein said envelope proteins, or parts thereof, are a mixture consisting of HCV envelope proteins from one strain or genotype of HCV and at least one other strain or genotype of HCV.  
     
     
         16 . Oligomeric particle according to any of  claims 1  to  15 , obtainable by a method characterized by the following steps: 
 I purifying HCV envelope proteins in solution, 
 possibly including the use of an optionally first detergent,  
 possibly including the use of a disulphide bond cleavage agent,  
 possibly including the use of an alkylating agent,  
 
 II replacing said solution of said purified HCV envelope proteins with a detergent or salt, resulting in oligomeric particles,  
 III purifying the oligomeric particles formed after said replacing, 
 possibly including further reducing the concentration of the detergent or salt of step II.  
 
 
     
     
         17 . Oligomeric particle according to  claim 16 , wherein said optionally first detergent is Empigen-BB, wherein the detergent of step II is CHAPS, octylglucaside, Tween, or any other detergent, and wherein said salt is betaine.  
     
     
         18 . Oligomeric particle according to any of claims  16  or  17 , wherein said Empigen-BB is used at a concentration of 1% to 10% and wherein said CHAPS or Tween is used at a concentration of 0.01% to 10%, or said betaine is used at a concentration of 0.01% to 10%.  
     
     
         19 . Method according to any of  claims 16  to  18  to produce oligomeric particles according to any of  claims 1  to  14 .  
     
     
         20 . Method to produce an oligomeric particle according to any of  claims 1  to  14 , characterized by the following steps: 
 I purifying HCV envelope proteins in solution, 
 possibly including the use of an optionally first deterrent,  
 possibly including the use of a disulphide bond cleavage agent,  
 possibly including the use of an alkylating agent,  
 
 II replacing said solution of said purified HCV envelope proteins with a detergent or salt, resulting in oligomeric particles.  
 III purifying the oligomeric particles formed after said replacing, 
 possibly including further reducing the concentration of the detergent or salt of step II.  
 
 
     
     
         21 . Method to produce an oligomeric particle according to  claim 20 , wherein said optionally first detergent is Empigen-BB, wherein the detergent of step II is CHAPS, octylglucaside, Tween, or any other detergent, and wherein said salt is betaine.  
     
     
         22 . Method to produce an oligomeric particle according to any of claims  20  or  21 , wherein said Empigen-BB is used at a concentration of 1% to 10% and wherein said CHAPS or Tween is used at concentrations of 0.01% to 10%, or said betaine is used at a concentration of 0.01%o to 10%.  
     
     
         23 . Composition comprising an oligomeric particle according to any of  claims 1  to  22 .  
     
     
         24 . Composition according to  claim 23 , which also comprises HCV core, P7, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and/or NS5B protein, or parts thereof.  
     
     
         25 . Composition according to  claim 24  wherein said NS3 protein, or parts thereof, have an amino acid sequence given by SEQ ID No 1 or SEQ ID No 2.  
     
     
         26 . Use of the oligomeric particle according to any of  claims 1  to  25  for the manufacture of an HCV vaccine composition.  
     
     
         27 . Use of the oligomeric particle according to any of  claims 1  to  25  for inducing immunity against HCV in chronic HCV carriers.  
     
     
         28 . Use of the oligomeric particle according to  claim 27  for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously to or after any other therapy.  
     
     
         29 . Use of the oligomeric particle according to  claim 27  for inducing immunity against HCV in HCV-infected individuals prior to or after liver transplantation, or after presumed infection.  
     
     
         30 . Use of the oligomeric particle according to any of  claims 1  to  28  for prophylactically inducing immunity against HCV.  
     
     
         31 . Use of the oligomeric particle according to any of  claims 1  to  28  for inducing immunity against HCV, characterized in that said oligomeric particle or said composition is used as a part of a series of time and compounds.  
     
     
         32 . The oligomeric particle or the composition according to any of  claims 1  to  25  for use as an HCV vaccine.  
     
     
         33 . The oligomeric particle or the composition according to any of  claims 1  to  25  for inducing immunity against HCV in chronic HCV carriers.  
     
     
         34 . The oligomeric particle or the composition according to  claim 33  for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously to or after any other therapy.  
     
     
         35 . The oligomeric particle or the composition according to any of  claims 1  to  25  for inducing immunity against HCV in HCV-infected individuals prior to or after liver transplantation, or after presumed infection.  
     
     
         36 . The oligomeric particle or the composition according to any of  claims 1  to  25  for prophylactically inducing immunity against HCV.  
     
     
         37 . The oligomeric particle or the composition according to any of  claims 1  to  25  for inducing immunity against HCV, characterized in that said oligomeric particle is part of a series in time and compounds.  
     
     
         38 . A purified single HCV envelope protein.  
     
     
         39 . The purified single HCV envelope protein according to  claim 38 , wherein said envelope protein is E1 or E1s.  
     
     
         40 . Composition containing the purified single HCV envelope protein according to any of claims  38  or  39 .  
     
     
         41 . The composition according to  claim 40  for use as an HCV vaccine.  
     
     
         42 . Use of the composition according to  claim 40  as an HCV vaccine.  
     
     
         43 . Use of the composition according to  claim 40  for the manufacture of an HCV vaccine.  
     
     
         44 . Specific antibodies generated against the oligomeric particle according to any of  claims 1  to  25 , or against the purified single HCV envelope protein according to any of  claims 38  to  40 .  
     
     
         45 . Use of the specific antibody according to  claim 44 , to treat or prevent HCV infection.  
     
     
         46 . Kit for detecting HCV antigens, comprising the specific antibody according to  claim 44 .  
     
     
         47 . Kit for detecting HCV antibodies present in a biological sample, comprising the oligomeric particle according to any of  claims 1  to  25 , or the purified single HCV envelope protein according to any of  claims 38  to  40 , in a suitable container.  
     
     
         48 . Kit for detecting HCV related T cell response, comprising the oligomeric particle according to any of  claims 1  to  25 , or the purified single HCV envelope protein according to any of  claims 38  to  40 .  
     
     
         49 . Immunoassay for detecting HCV antibody, which immunoassay comprises: 
 (1) providing the oligomeric particle according to any of  claims 1  to  25 , or the purified single HCV envelope protein according to any of  claims 38  to  40 , or parts thereof,    (2) incubating a biological sample with said oligomeric particle, or said HCV envelope protein under conditions that allow formation of antibody-antigen complex,    (3) determining whether said antibody-antigen complex comprising said oligomeric particle or said HCV envelope protein is formed.