title: Evaluation of approaches to identify the targets of cellular immunity on a proteome-wide scale.
abstract: BACKGROUND
  Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products.
  
  
  RESULTS
  We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems.
  
  
  CONCLUSIONS
  This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health.
authors.affiliation: Queensland Institute of Medical Research, Brisbane, Queensland, Australia.
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Cardoso
authors.forename: Joanne S
authors.initials: JS
authors.lastname: Roddick
authors.forename: Penny
authors.initials: P
authors.lastname: Groves
authors.forename: Denise L
authors.initials: DL
authors.lastname: Doolan
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002478:Cells, Cultured; D006801:Humans; D007111:Immunity, Cellular / Q000502:physiology*; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D051379:Mice; D020543:Proteome / Q000032:analysis*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma
references.citation: Curr Opin Immunol. 2010 Feb;22(1):118-23
references.pmid: 20153156
references.citation: Immunol Rev. 2010 May;235(1):190-205
references.pmid: 20536564
references.citation: Nucleic Acids Res. 1981 Jul 10;9(13):2999-3014
references.pmid: 6269068
references.citation: Cell Res. 2010 Jan;20(1):4-12
references.pmid: 20010916
references.citation: Proc Natl Acad Sci U S A. 1961 Oct 15;47:1588-602
references.pmid: 14479932
references.citation: J Immunol Methods. 2008 Mar 20;332(1-2):129-41
references.pmid: 18243240
references.citation: J Immunol Methods. 2002 Feb 1;260(1-2):157-72
references.pmid: 11792386
references.citation: Infect Immun. 2008 Aug;76(8):3374-89
references.pmid: 18474646
references.citation: Science. 2004 Jul 2;305(5680):86-90
references.pmid: 15232106
references.citation: Nat Rev Immunol. 2008 Aug;8(8):607-18
references.pmid: 18641646
references.citation: Science. 1997 Aug 15;277(5328):973-4
references.pmid: 9281074
references.citation: Malar J. 2010 Aug 24;9:241
references.pmid: 20735847
references.citation: Int J Parasitol. 2011 Jan;41(1):3-20
references.pmid: 20816843
references.citation: Mol Biochem Parasitol. 2006 Aug;148(2):144-60
references.pmid: 16644028
references.citation: J Exp Med. 1990 Mar 1;171(3):763-73
references.pmid: 1689762
references.citation: Anal Chem. 2004 Apr 1;76(7):1844-9
references.pmid: 15053642
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9952-7
references.pmid: 12886016
references.citation: Philos Trans R Soc Lond B Biol Sci. 2001 Dec 29;356(1416):1965-73
references.pmid: 11779399
references.citation: Trends Biotechnol. 2005 Mar;23(3):150-6
references.pmid: 15734558
references.citation: Proteomics. 2010 Jun;10(12):2259-69
references.pmid: 20391532
references.citation: Genome Res. 2002 Mar;12(3):487-92
references.pmid: 11875038
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):547-52
references.pmid: 15647345
references.citation: Proteomics. 2008 Nov;8(22):4680-94
references.pmid: 18937256
references.citation: Vaccine. 2010 Apr 19;28(18):3201-10
references.pmid: 20188680
references.citation: Science. 2002 Aug 9;297(5583):1016-8
references.pmid: 12114528
references.citation: Infect Immun. 2008 Apr;76(4):1702-8
references.pmid: 18268027
references.citation: J Virol. 2004 Jun;78(11):5891-9
references.pmid: 15140987
references.citation: N Biotechnol. 2008 Oct-Dec;25(2-3):126-32
references.pmid: 18786663
references.citation: Blood. 2008 Sep 1;112(5):1557-69
references.pmid: 18725574
references.citation: J Virol. 1987 Feb;61(2):446-53
references.pmid: 3027374
references.citation: Proteomics. 2007 May;7(10):1678-86
references.pmid: 17443847
references.citation: J Virol. 2008 Jul;82(14):7120-34
references.pmid: 18480455
references.citation: FEBS Lett. 1999 Jan 8;442(1):15-9
references.pmid: 9923595
references.citation: Protein Sci. 2003 Jun;12(6):1216-21
references.pmid: 12761392
references.citation: Genome Res. 2004 Oct;14(10B):2076-82
references.pmid: 15489329
references.citation: J Leukoc Biol. 2004 Feb;75(2):163-89
references.pmid: 14525967
references.citation: Curr Pharm Biotechnol. 2010 Apr;11(3):272-8
references.pmid: 20210744
references.citation: Curr Opin Chem Biol. 2002 Jun;6(3):390-8
references.pmid: 12023121
references.citation: J Immunol Methods. 2003 Mar 1;274(1-2):265-70
references.pmid: 12609552
references.citation: Acta Trop. 2010 Jun;114(3):171-6
references.pmid: 19913490
references.citation: Curr Opin Immunol. 2007 Feb;19(1):79-86
references.pmid: 17140786
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6958-63
references.pmid: 20351286
references.citation: J Immunol. 1992 Sep 15;149(6):2103-9
references.pmid: 1517574
references.citation: Mol Biochem Parasitol. 2007 Jan;151(1):100-10
references.pmid: 17125854
references.citation: FEBS Lett. 2000 Oct 6;482(3):185-8
references.pmid: 11024457
references.citation: J Infect Dis. 1997 Jan;175(1):91-7
references.pmid: 8985201
references.citation: Vaccine. 2006 Feb 20;24(8):1124-31
references.pmid: 16202487
references.citation: J Struct Funct Genomics. 2010 Sep;11(3):201-9
references.pmid: 20574660
references.citation: J Immunol. 1997 Oct 1;159(7):3424-33
references.pmid: 9317141
references.citation: Science. 1989 Apr 14;244(4901):182-8
references.pmid: 2649980
references.citation: Annu Rev Immunol. 2005;23:651-82
references.pmid: 15771583
references.citation: Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7648-53
references.pmid: 9636204
references.citation: J Immunol. 2004 Sep 1;173(5):3148-54
references.pmid: 15322175
references.citation: J Immunol. 1991 Feb 1;146(3):1020-5
references.pmid: 1988490
references.citation: J Biol Chem. 1987 Mar 5;262(7):2937-40
references.pmid: 3102479
references.citation: Nature. 2000 Aug 17;406(6797):793-8
references.pmid: 10963610
references.citation: Biochem Biophys Res Commun. 2003 May 23;305(1):1-5
references.pmid: 12732187