HIV-2 transmembrane glycoprotein homodimer (gp 80)

Abstract

Characterization of the envelope transmembrane protein of human immunodeficiency virus type 2 (HIV-2) was carried out using murine polyclonal and monoclonal antibodies or patient sera specific for HIV-2 proteins. A 80-Mr glycoprotein (gp80) was produced in HIV-2 infected cells along with three other glycoproteins that were recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of gp140 (gp300). The gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Among these four glycoproteins, only gp80 and gp125 were associated with HIV-2 virions. As the other glycoproteins, gp80 was recognized by all HIV-2 positive sera. A murine polyclonal antibody raised against the purified gp300 recognized all four glycoproteins. On the other hand, a monoclonal antibody raised against a synthetic polypeptide deduced from the sequence of the transmembrane glycoprotein of HIV-2, recognized gp140, gp300 and gp80; thus indicating that gp80 should be related to the transmembrane protein of the envelope. Dimerization of envelope glycoprotein precursor and the transmembrane glycoprotein was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. Dimerization of the envelope precursors might be essential for the processing of these glycoproteins into the mature products extracellular and transmembrane glycoproteins. Furthermore the dimeric form of the transmembrane glycoproteins might be important for the optimal structure of the virus and thus for its infectivity.

What is claimed is:  
     
         1 . HIV-2 virus containing a structural glycoprotein of 80 kDa, wherein said virus is in a purified Form.  
     
     
         2 . A glycoprotein of human immunodeficiency virus type 2 (HIV-2) wherein 
 (A) said glycoprotein is dimeric form of tine transmembrane glycoprotein of HIV-2;    (B) said glycoprotein has an apparent molecular weight of about 80 kDa (gp80); and    (C) said glycoprotein is in a purified form.    
     
     
         3 . A glycoprotein of simian immunodeficiency virus (SIV) or of a simian retroviral variant of SIV, wherein 
 (A) said glycoprotein is dimeric form of the transmembrane glycoprotein of SIV;    (B) said glycoprotein has an apparent molecular weight of about 65 kDa (gp65SIV); and    (C) said glycoprotein is in a purified form.    
     
     
         4 . A non-glycosylated dimeric transmembrane envelope protein of HIV-2 in a purified form.  
     
     
         5 . A labeled antigen-comprising a glycoprotein of human immunodeficiency virus type 2 (HIV-2), wherein 
 (A) said glycoprotein is a dimeric form of the transmembrane protein of HIV-2;    (B) said glycoprotein has an apparent molecular weight of about 80 kDa (gp80); and    (C) said antigen is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.    
     
     
         6 . The labeled antigen of  claim 5 , which is non-glycosylated.  
     
     
         7 . The labeled antigen of  claim 5 , wherein the antigen is in a purified form.  
     
     
         8 . A labeled antigen comprising a glycoprotein of simian immunodeficiency virus (SIV) or of a simian retroviral variant of SIV, wherein 
 (A) said glycoprotein is dimeric form of the transmembrane protein of SIV;    (B) said glycoprotein has an apparent molecular weight of about 65 kDa (gp65 SIV ); and    (C) said antigen is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.    
     
     
         9 . The labeled antigen of  claim 8 , wherein the antigen in a purified form.  
     
     
         10 . An immunological complex between the glycoprotein of  claim 2  and an antibody recognizing said glycoprotein.  
     
     
         11 . An immunological complex between the glycoprotein of  claim 3  and an antibody recognizing said glycoprotein.  
     
     
         12 . An immunological complex between the antigen of  claim 4  and an antibody recognizing said antigen.  
     
     
         13 . An immunological complex between the antigen or  claim 5  and an antibody recognizing said antigen.  
     
     
         14 . An immunological complex as claimed in  claim 10 , wherein the antibody is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.  
     
     
         15 . An immunological complex as claimed in  claim 11 , wherein the antibody is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.  
     
     
         16 . A method for detecting infection of cells by human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: 
 (A) providing a composition comprising cells suspected of being infected with HIV-2;    (B) disrupting cells in the composition to expose intracellular proteins; and    (C) assaying the exposed intracellular proteins for the presence of gp80 glycoprotein of HIV-2.    
     
     
         17 . The method of  claim 16 , wherein said assaying of exposed intracellular proteins is carried out by electrophoresis of said proteins.  
     
     
         18 . The method of  claim 16 , wherein said assaying of exposed intracellular proteins is carried out by polyacrylamide gel electrophoresis of said proteins.  
     
     
         19 . The method of  claim 16 , wherein said assaying of exposed intracellular proteins is carried out by immunoassay with antibodies that are immunologically reactive with gp80 glycoprotein of HIII-2.  
     
     
         20 . The method of  claim 19 , wherein said antibodies are labeled with an immunoassay label.  
     
     
         21 . A method of detecting antigens of human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: 
 (A) providing a composition suspected of containing antigens of HIV-2; and    (B) assaying said composition for the presence of gp80 glycoprotein of HIV-2.    
     
     
         22 . The method of  claim 21 , wherein said assaying of said composition is carried out by electrophoresis of said proteins.  
     
     
         23 . The method of  claim 21 , wherein said assaying of said composition is carried out by polyacrylamide gel electrophoresis of said proteins.  
     
     
         24 . The method of  claim 21 , wherein said assaying of said composition is carried out by immunoassay with antibodies that are-immunologically reactive with gp80 glycoprotein of HIV-2.  
     
     
         25 . The method of  claim 24 , wherein said antibodies are labeled with an immunoassay label.  
     
     
         26 . A method of distinguishing HIV-2 infection from HIV-1 infection in cells suspected of being infected therewith, wherein the method comprises: 
 (A) providing in extract containing intracellular proteins of said cells; and    (B) assaying said extract for the presence of gp80 glycoprotein of HIV-2.    
     
     
         27 . The method of  claim 26 , wherein said assaying of said extract is carried out by electrophoresis of said proteins.  
     
     
         28 . The method of  claim 26 , wherein said assaying of said extract is carried out by polyacrylamide gel electrophoresis of said proteins.  
     
     
         29 . The method of  claim 26 , wherein said assaying of said extract is carried out by immunoassay with antibodies that are immunologically reactive with gp80 glycoprotein of HIV-2.  
     
     
         30 . The method of  claim 29 , wherein said antibodies are labeled with an immunoassay label.  
     
     
         31 . A method of making gp80 glycoprotein of human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: 
 (A) providing a composition containing cells in which HIV-2 is capable of replicating;    (B) infecting said cells with HIV-2;    (C) culturing said cells under conditions to cause HIV-2 to proliferate;    (D) disrupting said cells to expose intracellular proteins; and    (E) recovering gp80 glycoprotein from the resulting exposed intracellular proteins.    
     
     
         32 . A method of making gp80 glycoprotein of HIV-2, wherein the method comprises detergent solubilization of HIV-2 virions.  
     
     
         33 . An in vitro diagnostic method for the detection of the presence or absence of antibodies which bind to an antigen comprising the glycoprotein of  claim 2 , which method comprises: 
 contacting said antigen with a biological fluid for a time and under conditions sufficient for the retroviral antigen and antibodies in the biological fluid to form an antigen-antibody complex; and    detecting the formation of the complex.    
     
     
         34 . The method of  claim 33 , wherein the detecting step further comprises measuring the formation of said antigen-antibody complex.  
     
     
         35 . An in vitro diagnostic method for the detection of the presence or absence of antibodies which are capable of binding to an antigen comprising the glycoprotein of  claim 3 , which method comprises: 
 contacting said antigen with a biological fluid for a time and under conditions sufficient for the retroviral antigen and antibodies-in the biological fluid to form an antigen-antibody complex; and    detecting the formation of the complex.    
     
     
         36 . The method of  claim 35 , wherein the detecting step further comprises measuring the formation of said antigen-antibody complex.  
     
     
         37 . A method for detection of antibodies which specifically bind to antigenic sites of an antigen indicative of the presence of the glycoprotein of  claim 3  in a sample of human body fluid, which comprises: 
 contacting said antigen with antibodies from human body fluid for a time and under conditions sufficient to permit formation of an antigen-antibody complex between said antigen and said antibodies; and  
 measuring the formation of said antigen-antibody complex by immunoassay based on Western Blot technique or ELISA (enzyme linked immunosorbent assay) or indirect immunofluorescent assay.  
 
     
     
         38 . A diagnostic kit for the detection of the presence or absence of antibodies which are capable of binding to a glycoprotein as claimed in  claim 2 , which kit comprises: 
 antigens comprising said glycoprotein; and    means for detecting the formation of immune complex between said antigens and said antibodies;    wherein the antigens and the means are present in an amount sufficient to perform said detection.    
     
     
         39 . The diagnostic kit of  claim 38 , wherein the means for detecting the formation of the immune complex are immunological assay means selected from the group consisting of radioimmunoassay, immunoenzymatic, and immunofluorescent assay means.  
     
     
         40 . A diagnostic kit for the detection of the presence or absence of antibodies which are capable of binding to a glycoprotein as claimed in  claim 3 , which kit comprises: 
 antigens comprising said glycoprotein; and    means for detecting the formation of immune complex between said antigens and said antibodies;    wherein the antigens and the means are present in an amount sufficient to perform said detection.    
     
     
         41 . The diagnostic kit of  claim 40 , wherein the means for detecting the formation of the immune complex are immunological assay means selected from the group consisting of radioimmunoasssay, immunoenzymatic, and immunofluorescent assay means.  
     
     
         42 . A method for the preparation of dimeric form of the transmembrane glycoprotein of human immunodeficiency virus type 2 (HIV-2), wherein the method comprises: 
 (A) providing an extracellular composition containing gp80 glycoprotein of HIV-2; and    (B) dissociating the gp80 glycoprotein into gp36 glycoprotein of HIV-2.    
     
     
         43 . The method of  claim 42 , wherein said gp80 is dissociated in a buffer less than about pH 6.  
     
     
         44 . The method of  claim 43 , wherein the pH is about 4.8.  
     
     
         45 . The method of  claim 42 , wherein said gp80 is dissociated in an ionic detergent.  
     
     
         46 . An immunogenic composition comprising a pharmaceutically effective amount of a glycoprotein of  claim 2  in association with a pharmaceutically acceptable carrier therefor.  
     
     
         47 . An immunogenic composition comprising glycoprotein of  claim 2  or a non-glycosylated protein thereof capable of eliciting antibody production.  
     
     
         48 . A vaccine composition comprising a neutralizing amount of dimeric transmembrane envelope glycoprotein or unglycosylated form thereof of HIV-2 and a pharmaceutically acceptable carrier therefor.  
     
     
         49 . A diagnostic kit for the detection of the presence or absence of antibodies which bind to antigens indicative of human immunodeficiency virus type 2 (HIV-2), wherein said kit comprises 
 glycosylated or non-glycosylated gp80 or mixtures thereof and means for detecting the formation of immune complexes between said antigens and said antibodies,    wherein the antigens and means are present in an amount sufficient to perform said detection.