Conformationally Specific Viral Immunogens

Abstract

The present invention provides methods of making engineered viral proteins and protein complexes that are useful as vaccine immunogens, engineered viral proteins and protein complexes made using such methods, and pharmaceutical compositions comprising such engineered viral proteins and protein complexes. Such engineered viral proteins and protein complexes may comprise one or more cross-links that stabilize the conformation of an antibody epitope, such as a quaternary neutralizing antibody, and may exhibit an enhanced ability to elicit a protective immune response when administered to a subject as a component of a vaccine.

1 . A method for producing an immunogen, the method comprising: (a) obtaining a viral protein or protein complex in one or more conformations that favor the elicitation of protective immune responses, (b) identifying one or more regions in the tertiary and/or quaternary structure of the viral protein or protein complex in which the introduction of one or more cross-links could stabilize the conformation of an antibody epitope, (c) introducing into the viral protein or protein complex one or more dityrosine cross-links at one or more of the regions identified in step (b) to form an engineered viral protein or protein complex, wherein the engineered viral protein or protein complex has one or more properties selected from the group consisting of:
 i. enhanced ability bind to a neutralizing antibody as compared to the viral protein or protein complex,   ii. enhanced ability bind to a broadly neutralizing antibody as compared to the viral protein or protein complex,   iii. enhanced ability bind to and activate B cell receptors as compared to the viral protein or protein complex,   iv. enhanced ability to elicit an antibody response in an animal as compared to the viral protein or protein complex,   v. enhanced ability to elicit a protective antibody response in an animal as compared to the viral protein or protein complex,   vi. enhanced ability to elicit production of neutralizing antibodies in an animal as compared to the viral protein or protein complex,   vii. enhanced ability to elicit production of broadly neutralizing antibodies in an animal as compared to the viral protein or protein complex,   viii. enhanced ability to elicit a protective immune response in an animal as compared to the viral protein or protein complex, and   ix. enhanced ability to bind to and elicit production of antibodies that recognize quaternary neutralizing epitopes in an animal as compared to the viral protein or protein complex.   
     
     
         2 . The method of  claim 1 , further comprising performing an assay to assess the ability of the engineered viral protein or protein complex to bind to a neutralizing antibody, bind to a broadly neutralizing antibody, bind to and activate B cell receptors, elicit an antibody response in an animal, elicit a protective antibody response in an animal, elicit production of neutralizing antibodies in an animal, elicit production of broadly neutralizing antibodies in an animal, elicit a protective immune response in an animal, and/or elicit production of antibodies that recognize quaternary neutralizing epitopes in an animal. 
     
     
         3 . The method of  claim 1 , wherein at least one dityrosine cross-link originates from a point mutation to tyrosine. 
     
     
         4 . The method of  claim 1 , further comprising introducing into the viral protein or protein complex one or more point mutations to tyrosine at one or more of the regions identified in step (b), and then subsequently introducing one or more dityrosine cross-links to form the engineered viral protein or protein complex. 
     
     
         5 . The method of  claim 1 , wherein the engineered viral protein or protein complex is useful as a vaccine immunogen in a mammalian subject. 
     
     
         6 . The method of  claim 1 , wherein the engineered viral protein or protein complex is useful as a vaccine immunogen in a human subject. 
     
     
         7 . The method of  claim 1 , wherein the viral protein or protein complex is derived from a virus from the group consisting of Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales, Lentiviruses, Human Immunodeficiency Viruses, Retroviruses, Orthomyxoviruses, Paramyxovirus, Influenza viruses, Poxviruses, Flaviviruses, Togaviruses, Coronaviruses, Rhabdoviruses, Bunyaviruses, Filoviruses, Reoviruses, Mononegavirales, Hepadnaviruses, and Hepatitis viruses. 
     
     
         8 . The method of  claim 1 , wherein the viral protein or protein complex is a viral envelope protein or protein complex. 
     
     
         9 . The method of  claim 1 , wherein the engineered viral protein or protein complex is soluble and does not form aggregates during production or when stored at a high concentration. 
     
     
         10 . A pharmaceutical composition comprising: a pharmaceutically effective amount of an engineered viral protein or protein complex made using the method of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         11 . A method for producing an immunogen, the method comprising: (a) obtaining a viral protein or protein complex in one or more conformations that favor the elicitation of protective immune responses, (b) introducing into the viral protein or protein complex one or more cross-links that are stable under physiological conditions, wherein the engineered viral protein or protein complex has one or more of properties selected from the group consisting of:
 i. enhanced ability bind to a neutralizing antibody as compared to the viral protein or protein complex,   ii. enhanced ability bind to a broadly neutralizing antibody as compared to the viral protein or protein complex,   iii. enhanced ability bind to and activate B cell receptors as compared to the viral protein or protein complex,   iv. enhanced ability to elicit an antibody response in an animal as compared to the viral protein or protein complex,   v. enhanced ability to elicit a protective antibody response in an animal as compared to the viral protein or protein complex,   vi. enhanced ability to elicit production of neutralizing antibodies in an animal as compared to the viral protein or protein complex,   vii. enhanced ability to elicit production of broadly neutralizing antibodies in an animal as compared to the viral protein or protein complex,   viii. enhanced ability to elicit a protective immune response in an animal as compared to the viral protein or protein complex, and   ix. enhanced ability to bind to and elicit production of antibodies that recognize quaternary neutralizing epitopes in an animal as compared to the viral protein or protein complex.   
     
     
         12 . The method of  claim 11 , further comprising performing an assay to assess the ability of the engineered viral protein or protein complex to bind to a neutralizing antibody, bind to a broadly neutralizing antibody, bind to and activate B cell receptors, elicit an antibody response in an animal, elicit a protective antibody response in an animal, elicit production of neutralizing antibodies in an animal, elicit production of broadly neutralizing antibodies in an animal, elicit a protective immune response in an animal, and/or elicit production of antibodies that recognize quaternary neutralizing epitopes in an animal. 
     
     
         13 . The method of  claim 11 , wherein the cross-link is targeted to identified and selected positions within the protein or protein complex's tertiary or quaternary structure. 
     
     
         14 . The method of  claim 11 , wherein the cross-links comprise dityrosine cross links. 
     
     
         15 . The method of  claim 13 , wherein at least one dityrosine cross-link originates from a point mutation to tyrosine. 
     
     
         16 . The method of  claim 13 , further comprising introducing into the viral protein or protein complex one or more point mutations to tyrosine at one or more of the positions identified in the tertiary and/or quaternary structure of the viral protein or protein complex such that the introduction of one or more cross-links could stabilize the conformation of an antibody epitope, and then subsequently introducing one or more dityrosine cross-links to form the engineered viral protein or protein complex. 
     
     
         17 . The method of  claim 11 , wherein the engineered viral protein or protein complex is useful as a vaccine immunogen in a mammalian subject. 
     
     
         18 . The method of  claim 11 , wherein the engineered viral protein or protein complex is useful as a vaccine immunogen in a human subject. 
     
     
         19 . The method of  claim 11 , wherein the viral protein or protein complex is derived from a virus belonging the group consisting of Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales, Lentiviruses, Human Immunodeficiency Viruses, Retroviruses, Orthomyxoviruses, Paramyxovirus, Influenza viruses, Poxviruses, Flaviviruses, Togaviruses, Coronaviruses, Rhabdoviruses, Bunyaviruses, Filoviruses, Reoviruses, Mononegavirales, Hepadnaviruses, and Hepatitis viruses. 
     
     
         20 . The method of  claim 11 , wherein the viral protein or protein complex is a viral envelope protein or protein complex. 
     
     
         21 . The method of  claim 11 , wherein the engineered viral protein or protein complex is soluble and does not form aggregates during production or when stored at a high concentration. 
     
     
         22 . A pharmaceutical composition comprising: a pharmaceutically effective amount of an engineered viral protein or protein complex made using the method of  claim 11 , and a pharmaceutically acceptable carrier.