HIV Antigens and Antibodies

Abstract

The present invention relates to a method for reducing the occurance and/or severity of viral infections. The method embodies procedures for expanding HIV from the blood of HIV antibody negative donors and deriving a non-infectious virus particle product that is antigenic. The procedures for deriving the antigenic, non-infectious virus particle product are optimally designed to maintain the integrity of the envelope proteins while maximizing the depletion of capsid proteins and RNA. The resulting virus particle product, when introduced into humans or non-human animals, enables the production of antibodies that target the natural envelope macromolecular structure that is required for infectivity. The present invention can be applied to producing virus stocks from the blood of HIV-seronegative donors, for deriving non-infectious virus particles that retain intact envelope proteins, for producing anti-viral antibodies, and for administering anti-virus antibodies to patients.

What is claimed is: 
     
         1 . A composition of matter comprising isolated antigen binding proteins (ABPs), wherein said isolated ABPs selectively bind to an epitope on an HIV-1 trimeric envelope glycoprotein subunit (TEGS). 
     
     
         2 . The composition of matter of  claim 1 , wherein said TEGS is prepared by a process comprising:
 obtaining infectious HIV-1 virus particles from human CD4+ cell culture grown in serum-free media;   contacting said infectious HIV-1 virus particles with agents that selectively remove from said particle viral RNA and viral capsid protein while retaining viral envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein; and   isolating protein from said contacted infectious HIV-1 virus particles wherein said isolated protein comprises non-infectious complexes comprising a trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA.   
     
     
         3 . The composition of matter of  claim 2 , wherein said infectious HIV-1 virus particles are Fiebig I/II isolates or founder virus. 
     
     
         4 . The composition of matter of any of  claims 2 - 3 , wherein said agents comprise cyclodextrin. 
     
     
         5 . The composition of matter of any of  claims 1 - 4 , wherein said ABPs neutralize infectious HIV-1 particles. 
     
     
         6 . The composition of matter of any of  claims 1 - 5 , wherein said ABPs neutralize infectious HIV-1 particles from at least one HIV-1 R5 strain and at least one HIV-1 X4 strain. 
     
     
         7 . The composition of matter of any of  claims 1 - 6 , wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment comprising a heavy chain CDR selected from the group consisting of SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73, and a light chain CDR selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74. 
     
     
         8 . The composition of matter of any of  claims 1 - 6 , wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment having a nucleotide sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112. 
     
     
         9 . The composition of matter of any of  claims 1 - 6 , wherein said ABPs compete in binding to TEGS with a reference antibody or antibody fragment having a nucleotide sequence comprising at least a portion of one of the SEQ ID NOs in Table 2. 
     
     
         10 . The composition of matter of  claim 7 , wherein said reference antibody or antibody fragment is selected from the group consisting of: recombinant antibody, chimeric antibody, humanized antibody, single-chain antibody, synthetic antibody, CF antibody and fragments thereof. 
     
     
         11 . The composition of matter according to any of  claims 1 - 10 , wherein said ABPs are selected from the group consisting of: monoclonal antibody, antibody fragment, recombinant antibody, chimeric antibody, humanized antibody, single-chain antibody, synthetic antibody, CF antibody and fragments thereof. 
     
     
         12 . The composition of matter according to any of  claims 1 - 10 , wherein said ABPs are polyclonal antibodies. 
     
     
         13 . The composition of matter of  claim 12 , wherein said ABPs are human polyclonal antibodies. 
     
     
         14 . The composition of matter of  claim 1 , wherein said ABPs are bispecific antibodies. 
     
     
         15 . The composition of matter according to any of  claims 1 - 14 , wherein said ABPs are derived from a phage display. 
     
     
         16 . An immunotherapeutic method comprising administering to a subject a therapeutic amount of said composition of matter of any of  claims 1 - 12 . 
     
     
         17 . A method of producing said composition of matter of  claim 1 , said method comprising:
 obtaining a sequence of said ABPs; and   synthesizing said ABPs based on said obtained sequence, said synthesis occurring in a cell-free protein expression system.   
     
     
         18 . A method for preparing a non-infectious antigenic composition capable of producing neutralizing HIV-1 antibodies comprising:
 obtaining infectious HIV-1 virus particles from human CD4+ cell-culture grown in serum-free media;   contacting said infectious HIV-1 virus particles with agents that selectively remove from said particle viral RNA and viral capsid protein while retaining viral envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein; and   isolating protein from said contacted infectious HIV-1 virus particles wherein said isolated protein comprises non-infectious complexes comprising a trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA.   
     
     
         19 . The method of  claim 18 , wherein said infectious HIV-1 virus particles are Fiebig I/II isolates or founder virus. 
     
     
         20 . The method of any of  claims 18 - 19 , wherein said agents comprise cyclodextrin. 
     
     
         21 . An antigenic composition produced according to said method of any one of  claims 18 - 20 . 
     
     
         22 . An antigenic composition capable of producing neutralizing HIV-1 antibodies comprising a non-infectious HIV-1 trimeric envelope glycoprotein subunit, said subunit comprising HIV-1 envelope, gp120, and gp41 proteins that are not chemically fixed or cross-linked and substantially free of HIV-1 capsid protein, reverse transcriptase and RNA, wherein said composition is substantially free of serum proteins. 
     
     
         23 . The antigenic composition of  claim 22  that is capable of eliciting antibodies that neutralizes infectious HIV-1 particles. 
     
     
         24 . The antigenic composition of  claim 22  that is capable of eliciting antibodies that neutralizes infectious HIV-1 particles from at least one HIV-1 R5 strain and at least one HIV-1 X4 strain. 
     
     
         25 . The antigenic composition of  claim 22 , wherein said antigenic composition is capable of detecting in an immunoassay anti-TEGS antibodies in serum from an animal immunized with said composition. 
     
     
         26 . A method comprising administering an amount of said antigenic composition of any of  claims 22 - 24  to a subject wherein a neutralizing HIV-1 antibody is produced. 
     
     
         27 . The method of  claim 26 , wherein said subject is non-human. 
     
     
         28 . The method of  claim 26 , wherein said subject is human. 
     
     
         29 . The method of  claim 28 , wherein said human subject has a homozygous deletion of 32 base pairs in a gene encoding CCR5 coreceptor for HIV-1. 
     
     
         30 . The method of any of  claims 26 - 29 , further comprising recovering polyclonal antibodies from said subject wherein said polyclonal antibodies neutralize HIV-1. 
     
     
         31 . The method of any of  claims 26 - 29 , further comprising preparing a cell line comprising B cells isolated from said subject, and said cell line producing neutralizing HIV-1 antibody. 
     
     
         32 . The method of  claim 31 , wherein said cell line is a hybridoma, and further comprising growing said hybridoma in cell culture, and recovering a neutralizing HIV-1 antibody from said cell culture. 
     
     
         33 . The method of  claim 31 , wherein said cell line is a hybridoma, and further comprising isolating therefrom a nucleic acid encoding at least one CDR from a gene encoding a neutralizing HIV-1 antibody. 
     
     
         34 . The method of  claim 31 , wherein said cell line comprises immortalized cells or transformed cells. 
     
     
         35 . A method of producing ABPs that neutralize HIV-1, comprising culturing a cell comprising a gene encoding at least part of said ABPs under conditions wherein said gene is expressed and said ABPs are recovered. 
     
     
         36 . The method of  claim 35 , wherein said gene comprises at least one CDR having a sequence obtained from a nucleic acid isolated according to  claim 33 . 
     
     
         37 . The method of  claim 35 , wherein said ABP is selected from the group consisting of: chimeric antibody, humanized antibody, single-chain antibody, and fragments thereof. 
     
     
         38 . In an immunoassay method for the detection of anti-HIV-1 antibodies, the improvement comprising using at least one antigen therein which is an HIV-1 trimeric envelope glycoprotein subunit isolated from an immunogenic composition according to any of  claims 21 - 24 . 
     
     
         39 . An immunoassay method according to  claim 38 , wherein said antigen is bound to an immunoassay support. 
     
     
         40 . An immunoassay method according to  claim 38 , wherein said antigen is in solution. 
     
     
         41 . An immunoassay method according to  claim 40 , wherein said antigen comprises a detectable label. 
     
     
         42 . A method for preparing an immunogenic, inactivated virus composition, comprising:
 obtaining an infectious virus particle, said particle comprising RNA, an envelope protein, a capsid protein, a reverse transcriptase, a gp120 protein, and a gp41 protein; and   contacting said infectious virus particle with agents that selectively remove from said particle said RNA and said capsid protein and said reverse transcriptase, while retaining said envelope protein in a non-denatured conformation, wherein said agents do not chemically fix or cross-link said envelope protein, thereby producing an immunogenic, inactivated virus composition.   
     
     
         43 . The method of  claim 42 , wherein said immunogenic, inactivated virus composition comprises a trimeric envelope glycoprotein subunit (TEGS), said subunit comprising said envelope, said gp120, and said gp41 proteins. 
     
     
         44 . The method of  claim 42 , wherein said infectious virus particle is a Fiebig I/II isolate or transmitted founder virus. 
     
     
         45 . The method of  claim 42 , wherein said infectious virus particle is obtained from a mammalian subject that lacks antibodies against said infectious virus particle. 
     
     
         46 . The method of  claim 45 , wherein said infectious virus particle is selected from the group consisting of an HIV particle, an FIV particle, and an EIAV particle. 
     
     
         47 . The method of  claim 46 , wherein said infectious virus particle is an HIV particle. 
     
     
         48 . The method of  claim 44 , wherein said infectious virus particle comprises an HIV-1 or an HIV-2 particle. 
     
     
         49 . The method of  claim 42 , wherein said agents comprise cyclodextrin 
     
     
         50 . The method of  claim 42 , wherein said agents comprise Benzonase. 
     
     
         51 . The method of any one of  claims 42 - 49 , wherein said infectious virus particle comprises a plurality of distinct HIV isolates. 
     
     
         52 . The method of  claim 51 , wherein said distinct HIV isolates are selected from the group consisting of distinct HIV types, distinct HIV groups, and distinct HIV clades. 
     
     
         53 . The method of  claim 43 , wherein said preparation of said TEGS further comprises:
 obtaining a DNA sequence of said TEGS;   cloning said DNA sequence into an expression vector; and   expressing said cloned DNA sequence to obtain TEGS proteins.   
     
     
         54 . A product produced according to said method of any one of  claims 42 - 52 . 
     
     
         55 . The product of  claim 54 , wherein said product is capable of eliciting antibodies against said trimeric envelope glycoproteins in a mammalian subject inoculated with said product. 
     
     
         56 . The product of  claim 55 , wherein said antibodies are virus neutralizing antibodies 
     
     
         57 . A method of quantifying antibodies present in a sample from a subject, comprising: contacting said sample with said product of  claim 54 , and determining said specific binding of antibodies in said sample to said product. 
     
     
         58 . A method of generating an immune response in a subject, comprising: administering an immunogenic amount of said product of  claim 54  to said subject. 
     
     
         59 . The method of  claim 58 , wherein said subject is genetically resistant to viral infection. 
     
     
         60 . The method of  claim 58 , wherein said subject is receiving antiretroviral therapy. 
     
     
         61 . The method of  claim 58 , wherein said immune response comprises a neutralizing response against said infectious virus particle. 
     
     
         62 . A method of generating an antibody, comprising: administering to a subject an immunogenic amount of said product of  claim 54 , and isolating said antibody, wherein said antibody specifically binds to said product of  claim 54 . 
     
     
         63 . A method of generating a monoclonal antibody, comprising:
 administering to a subject an immunogenic amount of said product of  claim 54 ;   producing a hybridoma using B cells isolated from said immunized subject, wherein said hybridoma produces a monoclonal antibody that specifically binds to said product of  claim 54 ; and   isolating said monoclonal antibody.   
     
     
         64 . A monoclonal antibody that specifically binds to said product of  claim 54 . 
     
     
         65 . The monoclonal antibody of  claim 64 , wherein said antibody is capable of neutralizing in vivo said infectious virus particle. 
     
     
         66 . A polyclonal antibody preparation that specifically binds to said product of  claim 54 . 
     
     
         67 . The polyclonal antibody preparation of  claim 66 , wherein said antibody preparation is capable of neutralizing in vivo said infectious virus particle. 
     
     
         68 . An immunotherapeutic method, comprising administering to a subject a therapeutic amount of said monoclonal antibody of any of  claims 64 - 67 .