Novel sequences encoding hepatitis C virus glycoproteins

Abstract

The present invention concerns a modified nucleic acid molecule comprising a nucleotide sequence coding for a full length hepatitis C virus (HCV) glycoprotein selected from the group consisting of E1 glycoprotein and E1/E2 glycoprotein heterodimer, this molecule having at least one nucleotide alteration, wherein, due to this alteration, at least one RNA splice site selected from the group consisting of RNA splice acceptor and RNA splice donor sites is eliminated from the coding sequence. The invention is also directed to methods for expressing on the surface of a cell and a pseudovirion an HCV glycoprotein, wherein the majority of the glycoprotein is full length. The invention further provides a cell and a pseudovirion expressing such glycoprotein. The invention still further provides a method for determining whether an agent inhibits HCV fusion with and entry into a target cell. The invention also provides an agent that inhibits HCV fusion with and entry into a target cell. The invention further provides methods for treating a subject afflicted with an HCV-associated disorder, for preventing an HCV infection in a subject, and for inhibiting in a subject the onset of an HCV-associated disorder.

1 - 172 . (canceled) 
     
     
         173 . A pseudovirion expressing on its surface a hepatitis C virus (HCV) E1 glycoprotein encoded by a nucleic acid sequence comprising consecutive nucleotides the sequence of which corresponds to a sequence encoding an HCV E1 glycoprotein in which (a) at least one nucleotide has been altered to eliminate an RNA splice acceptor site or an RNA splice donor site, wherein the altered nucleotide is (i) an A886C mutation, (ii) a G888T mutation, or (iii) a G675A mutation, or (b) nucleotides 675-887 have been deleted, the positions of the mutation and the deleted nucleotides being numbered by reference to SEQ ID NO:2, and wherein the majority of the glycoprotein is full length. 
     
     
         174 . The pseudovirion of  claim 173 , wherein at least one nucleotide in the HCV E1-encoding nucleic acid has been altered to eliminate an RNA splice acceptor site and the altered nucleotide is an A886C mutation. 
     
     
         175 . The pseudovirion of  claim 173 , wherein at least one nucleotide in the HCV E1-encoding nucleic acid has been altered to eliminate an RNA splice acceptor site and the altered nucleotide is a G888T mutation. 
     
     
         176 . The pseudovirion of  claim 173 , wherein at least one nucleotide in the HCV E1-encoding nucleic acid has been altered to eliminate an RNA splice donor site and the altered nucleotide is a G675A mutation. 
     
     
         177 . The pseudovirion of  claim 173 , wherein nucleotides 675-887 have been deleted in the HCV E1-encoding nucleic acid. 
     
     
         178 . The pseudovirion of  claim 173 , wherein greater than 70% of the altered HCV glycoprotein expressed on the surface of the pseudovirion is a full length HCV glycoprotein. 
     
     
         179 . The pseudovirion of  claim 173 , wherein greater than 90% of the altered HCV glycoprotein expressed on the surface of the pseudovirion is a full length HCV glycoprotein. 
     
     
         180 . The pseudovirion of  claim 173  expressing on its surface HCV E1, E2, or E1/E2 glycoproteins, wherein the HCV E1 glycoprotein-encoded nucleic acid sequence comprises further consecutive nucleotides, the sequence of which corresponds to a sequence encoding HCV E2 glycoprotein, wherein the HCV E2 glycoprotein-encoding sequence has at least one nucleotide that has been altered to eliminate an RNA splice acceptor site, and wherein the altered nucleotide in the HCV E2 glycoprotein-encoding sequence is an A2138T mutation, the position of the mutation being numbered by reference to SEQ ID NO:2. 
     
     
         181 . An immunogen comprising the pseudovirion of  claim 173 . 
     
     
         182 . A pharmaceutical composition comprising the pseudovirion of  claim 173  and a pharmaceutically acceptable carrier. 
     
     
         183 . The pharmaceutical composition of  claim 182 , further comprising an adjuvant. 
     
     
         184 . A method for determining whether an agent inhibits entry of hepatitis C virus (HCV) into a target cell susceptible to infection by HCV, comprising:
 (a) separately contacting (1) a pseudovirion expressing HCV E1/E2 glycoprotein heterodimer on its surface, wherein a majority of the E1/E2 glycoprotein is full length, which pseudovirion was produced using at least one vector which provides virion packaging functions and expresses a reporter gene, with (2) a target cell in the presence and absence of an agent under conditions which would normally permit entry of the pseudovirion into the target cell in the absence of the agent; and   (b) lysing the contacted target cell and determining whether there is a reduction in reporter gene activity in the presence of the agent compared with reporter gene activity in the absence of the agent;   wherein a reduction in reporter gene activity in the presence of the agent indicates that the agent inhibits entry of HCV into the target cell.   
     
     
         185 . A method for screening a plurality of agents, not known to inhibit entry of hepatitis C virus (HCV) into a target cell susceptible to infection by HCV, to identify at least one agent that inhibits such entry, which method comprises:
 (a) separately contacting (1) a pseudovirion expressing HCV E1/E2 glycoprotein heterodimer on its surface, wherein a majority of the E1/E2 glycoprotein is full length, which pseudovirion was produced using at least one vector which provides virion packaging functions and expresses a reporter gene, with (2) a target cell in the presence and absence of a plurality of agents under conditions which would normally permit entry of the pseudovirion into the target cell in the absence of said plurality of agents;   (b) lysing the contacted target cell and determining whether there is a reduction in reporter gene activity in the presence of the plurality of agents compared with the reporter gene activity in the absence of said plurality of agents; and   (c) if the reporter gene activity is reduced in the presence of the plurality of agents, separately determining which of the agents present in said plurality of agents causes a reduction in reporter gene activity, so as to thereby identify at least one agent that inhibits entry of HCV into a target cell.   
     
     
         186 . A method for treating a subject afflicted with a hepatitis C virus (HCV)-associated disorder, preventing an HCV infection in a subject, or inhibiting the onset of an HCV-associated disorder in a subject, which treatment is effected by inhibiting entry of HCV into a target cell susceptible to infection by said virus, which method comprises administering to the subject an agent, wherein said agent is
 (1) determined to inhibit entry of HCV into a target cell using a method comprising:
 (a) separately contacting a pseudovirion expressing HCV E1/E2 glycoprotein heterodimer on its surface, wherein the majority of the E1/E2 glycoprotein is full length, which pseudovirion was produced using a packaging vector that expresses a reporter gene, with a target cell in the presence and absence of the agent under conditions which would normally permit entry of the pseudovirion into the target cell in the absence of the agent; and 
 (b) lysing the contacted target cell and determining whether there is a reduction in reporter gene activity in the presence of the agent compared with the reporter gene activity in the absence of the agent; 
 wherein a reduction in reporter gene activity in the presence of the agent indicates that the agent inhibits entry of HCV into the target cell; and 
   (2) administered in a therapeutically or prophylactically effective amount to treat the subject.   
     
     
         187 . The method of  claim 186 , further comprising administration of at least one conventional antiviral agent. 
     
     
         188 . A method for preventing a hepatitis C virus (HCV) infection in a subject, or inhibiting the onset of an HCV-associated disorder in a subject, the prevention or inhibition of which is effected by immunizing the subject, which method comprises:
 (a) injecting into the subject a pharmaceutical composition comprising an HCV pseudovirion expressing on its surface a hepatitis C virus (HCV) glycoprotein selected from the group consisting of E1 glycoprotein and E1/E2 glycoprotein heterodimer, wherein the majority of the glycoprotein is full length; and thereby   (b) eliciting a protective HCV immune response in the subject.   
     
     
         189 . The method of  claim 188 , further comprising injecting into the subject a hepatitis C virus (HCV) glycoprotein selected from the group consisting of E1 glycoprotein, E1/E2 glycoprotein heterodimer and immunogenic fragments thereof. 
     
     
         190 . The method of  claim 188 , further comprising injecting into the subject a nucleic acid vector capable of expressing a hepatitis C virus (HCV) glycoprotein selected from the group consisting of E1 glycoprotein, E1/E2 glycoprotein heterodimer and immunogenic fragments thereof. 
     
     
         191 . The method of  claim 188 , further comprising administration of at least one conventional antiviral agent.