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Unlocking livestock development potential through science, influence and capacitydevelopment ILRI APM, Addis Ababa, 15-17 May 2013Immunoinformatics & MHC-Tetramers,Revolutionary Technologies forVaccine DevelopmentThis document is licensed for use under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License May 2013Nicholas Svitek, Andreas Martin Hansen, Lucilla Steinaa, Rosemary Saya, Elias Awino, Morten Nielsen, Søren Buus, Vishvanath NeneIntroductionEast Coast fever (ECF) is a tick-borne lethal disease of cattle in eastern, central and southern Africa that is caused by the apicomplexan parasite Theileria parva . An approved vaccinecurrently exists based on the infection and treatment method (ITM) whereby cattle are given a lethal dose of the parasite concomitantly with a high dose of oxytetracycline. Eventhough it induces a lifelong immunity based on a strong cytotoxic CD8+ T cell (CTL) response against homologous strains, it has several drawbacks as it can induce a carrier state, it isexpensive to produce, and necessitates a liquid nitrogen cold chain to be delivered to the field. The development of a recombinant vaccine can help solve these problems.GoalsTo generate a comprehensive map of T. parva antigens and discover new antigens from the parasite that can be included in a wide-spectrum recombinant vaccine.MethodologyUsing NetMHCpan for the identification of new antigens and CTL epitopes from T. parva and utilization of Bovine Leucocyte Antigen (MHC) class I tetramers to confirm the identityof the new CTL epitopes and evaluate the CTL immune response towards these T. parva antigens in cattle.ResultsBovine MHC-tetramers were successfully generated and were efficient in binding to T. parva specific CTL. Bovine MHC-tetramers are sufficiently sensitive to measure immuneresponse from Tp1+-CTL directly isolated from infected/vaccinated cattle. Moreover, the MHC-tetramer & NetMHCpan technologies allowed identification of the precise and correctCTL epitope sequence from a known T. parva antigen (Tp2).ConclusionImmunoinformatics and MHC-tetramers are precise and sensitive technologies that can lead to identification of new antigens and CTL epitopes from T. parva or any other infectiousdiseases of cattle where CTL play a role.Partners FundingUniversity of Vermont, USDA-ARS, University of Copenhagen, Danish Technical University BREAD Program of the NSF (USA) and the Bill & Melinda Gates FoundationAnimal 1day 8 p.i.Animal 1day 16 p.i.Animal 2day 8 p.i.Animal 2day 17 p.i.CD8+Antigen Known CTL epitope BoLA allele FP1 Alternative epitope FP2Tp2 27SHEELKKLGML37 N*04101 0.133 29EELKKLGML37 0.009CD8+Tetramer +Control Tp227-37CTLhttp://flow.csc.mrc.ac.uk/?page_id=852CD8+CD8+/Tp1+Antigen-specificCD8+ T CellTCRbiotinInfected LymphocyteT. parva proteinT. parva peptide presentedby MHC class I moleculeT CD8+= CTL(killing)Graham SP, et al., PNAS, 2006 & Graham SP, et al., Infect Immun., 2008A) Mechanism of Immunity duringITM VaccinationB) List of Known T. parva Antigens& CTL Peptide-Epitopes Identified byConventional MethodsA) MHC-Tetramers Mimic Natural“MHC-CD8+ Killer Cell Interaction”B) Identification of MHC-Tetramer Positive CD8+ Killer Cellswith the Flow CytometerC) Identification with Tetramer of Tp1+ CTL from ITM-Vaccinated CattleA) Prediction with NetMHCpan of an AlternativeCTL Epitope from the Tp2 AntigenTp1+Tp1+CD8+ImmunoinformaticsComputer algorithmstrained on biological data(NetMHCpan)Tp229-37C) Immunoinformatics to Speed Up the Identification ofAntigens & CTL Peptide-EpitopesFigure 1 Paradigm of Cellular Immunity to Theileria parvaFigure 2 MHC class I Tetramers to Identify T. parva CTL Peptide-Epitope Positive CellsFigure 3 MHC Class I Tetramers & Immunoinformatics to Identify the Correct CTL Peptide-Epitope Sequenceanti-CD8Tp1-tetramerKillingT. parva genomeC) Confirmation of the Alternative Epitope by theUse of MHC-Tetramers.Prediction of parasite peptidesthat binds tobovine MHC class I moleculesAntigen CTL epitope BoLA allele FPTp1 214VGYPKVKEEML224 N*01301 0.070Tp2 27SHEELKKLGML37 N*04101 0.133Tp2 49KSSHGMGKVGK59 N*01201 0.017Tp2 96FAQSLVCVL104 BoLA-T2c 0.036Tp2 98QSLVCVLMK106 N*01201 0.036Tp4 328TGASIQTTL336 N*00101 0.086Tp5 87SKADVIAKY95 BoLA-T5 0.017Tp7 206EFISFPISL214 BoLA-T7 0.058Tp8 379CGAELNHFL387 N*00101 0.168Tp9 199AKFPGMKKSK208 N*02301 0.113FP: the false-positive fraction score for NetMHCpantrained with BoLA peptide binding data (the fraction ofpeptides with a predicted binding affinity stronger thanthe predicted affinity of the know CTL epitope). A FPvalue closer to 0 shows stronger binding affinity.00.10.20.30.40.50.60.70.80.940,000 nM 8,000 nM 1,600 nM 320 nM 64 nM 12.8 nM 2.56 nM 0.512 nM 0.102 nM no peptideTp2.29-37 (Alt)Tp2.27-37O.D.(@450nm)[Peptide]B) Confirmation of the Higher Binding Affinity by theAlternative Tp2 CTL Peptide-EpitopeAntigen CTL epitope BoLA (MHC) class ITp1 214VGYPKVKEEML224 N*01301Tp2 27SHEELKKLGML37 N*04101 (T2b)Tp2 49KSSHGMGKVGK59 N*01201 (T2a)Tp2 96FAQSLVCVL104 BoLA-T2cTp2 98QSLVCVLMK106 N*01201 (T2a)Tp4 328TGASIQTTL336 N*00101Tp5 87SKADVIAKY95 BoLA-T5Tp7 206EFISFPISL214 BoLA-T7Tp8 379CGAELNHFL387 N*00101Tp9 199AKFPGMKKSK208 N*02301T. parva infects bovine lymphocytes. MHC class I molecules binds toand presents specific T. parva CTL peptide-epitopes to cytotoxic Tlymphocytes (CTL). If the CTL is specific for that epitope, it will kill theinfected cell.Initially, ELISpot and in vitro cytotoxicity assays lead to the discoveryof these T. parva CTL peptide-epitopes presented by these specificMHC class I molecules.Since using ELISpot and cytotoxicity assays are arduous, time consuming and expensive for initial screening ofnovel antigens and CTL epitopes, the recourse to immunoinformatics like NetMHCpan predicting peptidebinding to MHC class I molecules can speed up the identification of CTL peptide-epitopes. This can lead to anarrower list of T. parva peptides that can be potential CTL epitopes to test in ELISpot and cytotoxicity assays.To evaluate the NetMHCpan technology, the known T. parva CTL peptide-epitopes were evaluated in thesystem and for most of them, they were predicted as binders to their respective MHC class I molecules.The MHC class I tetramer is a technology that can be used to identify CTL that are specific for a particular T. parva peptide-epitope. It is based on uniting together4 recombinant MHC class I molecules that are bound to a specific T. parva CTL peptide-epitope and to a fluorochrome of which the fluorescence can be measuredby flow cytometry upon excitation with a laser.The MHC class I tetramer was generated with the Tp1 T. parva CTL peptide-epitope and the BoLA-N*01301 MHC class I molecule bound to PE (the fluorochrome). The Tp1+-specific CTL can beidentified in vaccinated animals at around day 16 post-inoculation with the parasite.Analysing known T. parva antigens with NetMHCpan lead to the identification of analternative, shorter, CTL peptide-epitope from the Tp2 antigen with predicted strongerbinding affinity to the MHC class I molecule than the one previously identified byconventional methods.FP1: the false-positive fraction score using NetMHCpan for the known Tp2 CTL peptide-epitope.FP2: the false-positive fraction score using NetMHCpan for the alternative Tp2 CTL peptide-epitope. A FP value closer to 0 shows stronger binding affinity.Evaluating peptide binding to the MHC class I molecule revealed that the predictedalternative epitope from the Tp2 antigen is the correct epitope since the initiallydiscovered Tp2 peptide-epitope did not show any binding to the molecule.Using the MHC class I tetramer technology with the known and the alternative CTLpeptide-epitopes from the Tp2 antigen, and a cell line specific for the Tp2 CTL peptide-epitopes demonstrated that the sequence recognized by the bovine CTL is precisely thealternative Tp2 CTL peptide-epitope; confirming again the correct prediction byNetMHCpan.TCR = T Cell Receptor,which will recognizeepitope by bindingto the peptide-MHCclass I complexCD8+ CTLspecific for Tp1CD8+ CTL