Malaria antigen screening method

Abstract

The invention provides a method of identifying an antigen from a pathogen or a disease antigen comprising the use of an adenoviral vector array comprising two or more different adenoviral vectors, wherein each adenoviral vector comprises a nucleic acid sequence encoding a different antigen of a pathogen. The adenoviral vectors are administered to antigen presenting cells (APCs) in vitro or to an animal in vivo. The immunogenicity of the antigen is measured by screening for an immune response from effector T lymphocytes in vitro and by screening for the absence of pathogen-induced disease onset in vivo.

1 . A method of identifying an antigen from a pathogen, which method comprises
 (a) preparing an adenoviral vector array comprising two or more different adenoviral vectors, wherein each adenoviral vector comprises a nucleic acid sequence encoding a different antigen of a pathogen,   (b) contacting antigen presenting cells (APCs) with the adenoviral vector array, wherein each different adenoviral vector transduces an APC such that the nucleic acid sequences of the different adenoviral vectors are expressed and the different antigens are produced in the APCs,   (c) incubating the APCs with effector T lymphocytes obtained from a mammal immunized with the pathogen, and   (d) screening for an immune response from the effector T lymphocytes, wherein an immune response from an effector T lymphocyte contacting an APC indicates T lymphocyte recognition of the antigen produced by the APC, whereupon the antigen is identified.   
     
     
         2 . The method of  claim 1 , wherein the pathogen is selected from a  Plasmodium  species, human immune deficiency virus (HIV), severe acute respiratory syndrome (SARS) virus, foot and mouth disease (FMD) virus, and  Mycobacterium tuberculosis.    
     
     
         3 . The method of  claim 3 , wherein the pathogen is selected from the group consisting of  Plasmodium berghei, Plasmodium chabaudi, Plasmodium vinckei, Plasmodium yoelii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae , and  Plasmodium ovale.    
     
     
         4 . The method of any of  claims 1 - 3 , wherein the antigen presenting cells are dendritic cells. 
     
     
         5 . The method of any of  claims 1 - 4 , wherein the immune response is selected from the group consisting of cytokine secretion from effector T lymphocytes, cytotoxicity of effector T lymphocytes, and immune activation of effector T lymphocytes. 
     
     
         6 . The method of  claim 5 , wherein the cytokine is selected from the group consisting of interferon gamma (IFN-γ), TNF-β, TNF-α, GM-CSF, CD40 ligand, Fas ligand, and interleukins. 
     
     
         7 . The method of any of  claims 1 - 6 , wherein each of the multiple adenoviral vectors exhibits reduced native binding to a coxsackievirus and adenovirus receptor (CAR). 
     
     
         8 . The method of  claim 7 , wherein each of the two or more different adenoviral vectors comprises a fiber protein wherein a native CAR-binding site is mutated. 
     
     
         9 . The method of  claim 8 , wherein each of the two or more different adenoviral vectors comprises a fiber protein comprising a nonnative amino acid sequence. 
     
     
         10 . The method of  claim 9 , wherein the nonnative amino acid sequence comprises an RGD sequence. 
     
     
         11 . The method of  claim 9  or  claim 10 , wherein the nonnative amino acid sequence is inserted into an exposed loop of the fiber protein. 
     
     
         12 . A method of identifying an antigen from a pathogen, which method comprises
 (a) providing an adenoviral vector array comprising two or more different adenoviral vectors, wherein each adenoviral vector comprises a nucleic acid sequence encoding a different antigen of a pathogen,   (b) administering each of the adenoviral vectors of the adenoviral vector array to a mammal, such that the nucleic acid sequence is expressed and the antigen is produced in the mammal,   (c) infecting each mammal with the pathogen, and   (d) screening the infected mammal for onset of a disease caused by the pathogen, wherein the absence in the infected mammal of a disease caused by the pathogen indicates that the adenoviral vector encodes an antigen of the pathogen, whereupon the antigen is identified.   
     
     
         13 . The method of  claim 12 , wherein the pathogen is selected from a  Plasmodium  species, human immune deficiency virus (HIV), severe acute respiratory syndrome (SARS) virus, foot and mouth disease (FMD) virus, and  Mycobacterium tuberculosis.    
     
     
         14 . The method of  claim 13 , wherein the pathogen is selected from the group consisting of  Plasmodium berghei, Plasmodium chabaudi, Plasmodium vinckei, Plasmodium yoelii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae , and  Plasmodium ovale.    
     
     
         15 . The method of  claim 12 , wherein the adenoviral vector is administered as part of an unpurified cell lysate comprising the adenoviral vector. 
     
     
         16 . A method of inducing an immune response against a pathogen in a mammal, which method comprises (a) preparing an adenoviral vector comprising a nucleic acid sequence encoding an antigen of a pathogen identified by the method of any of  claims 1 - 15 , and (b) administering the adenoviral vector to a mammal, wherein the antigen is expressed in the mammal to induce an immune response. 
     
     
         17 . The method of any of  claims 1 - 16 , wherein the adenoviral vectors are replication-deficient. 
     
     
         18 . The method of  claim 17 , wherein each of the adenoviral vectors requires complementation of the E1 region of the adenoviral genome for replication. 
     
     
         19 . The method of  claim 17  or  claim 18 , wherein each of the adenoviral vectors requires complementation of the E4 region of the adenoviral genome for replication. 
     
     
         20 . The method of any of  claims 16 - 19 , wherein each of the adenoviral vectors lacks the entire E1 region and at least a portion of the E4 region of the adenoviral genome. 
     
     
         21 . The method of any of  claims 16 - 20 , wherein each of the adenoviral vectors lacks all or part of the E3 region of the adenoviral genome. 
     
     
         22 . A method of identifying a disease antigen, which method comprises
 (a) preparing an adenoviral vector array comprising two or more different adenoviral vectors, wherein each adenoviral vector comprises a nucleic acid sequence encoding a different disease antigen,   (b) contacting antigen presenting cells (APCs) with the adenoviral vector array, wherein each different adenoviral vector transduces an APC such that the nucleic acid sequences of the different adenoviral vectors are expressed and the different antigens are produced in the APCs,   (c) incubating the APCs with effector T lymphocytes obtained from a mammal affected by the disease, and   (d) screening for an immune response from the effector T lymphocytes, wherein an immune response from an effector T lymphocyte contacting an APC indicates T lymphocyte recognition of the antigen produced by the APC, whereupon the disease antigen is identified.   
     
     
         23 . The method of  claim 22 , wherein the disease is cancer. 
     
     
         24 . The method of  claim 22 , wherein the disease is an autoimmune disease. 
     
     
         25 . The method of any of  claims 1 - 24 , wherein the mammal is a mouse. 
     
     
         26 . The method of any of  claims 1 - 24 , wherein the mammal is a human.