Identification of linear B-cell epitopesin Trypanosoma vivax using peptidemicroarray 1. Introduction In order to produce a vaccine,classical and conventional vaccinology has been the most common way to achieveit and widely used over the past decades. However, this methodology is time- consuming and in not all the casesresearchers can found successful candidates due to different problems in theorganism cultivation in vitro ordifficulties on the subunit vaccines identification. The term reverse vaccinology (RV) was firstused by Rino Rappuoli and refers to use and take advantage of the genomicsequence of the organism, select between many different proteins the parasitecan express and predict only the potential antigen candidates rather thanworking with it in vitro (1). One of the advantages RV has compared with the classical vaccinology isthe fact that with application of genomic technologies, more proteins that werenot considered previously are now available because all genes can beanalyze. As a consequence, RV give us amuch wider variety of candidates (2).
Several approaches on vaccine design have now apply not only RV but alsostructural biology. Possibleanti-parasite vaccines for human and animals have been studied in the last fewyears i.e against ticks and tick borne diseases (TTBD) in livestock (3). Trypanosoma vivax is a protozoan parasite from the genus Trypanosoma causative of African Animal Trypanosomiasis (AAT), adisease that affects livestock in Africa and South America. A vaccinedevelopment for AAT is crucial since the disease generate tremendous losesevery year decreasing cattle population. It has been estimated that calfmortality is up to 20% in infected regions (4) and can reduce cattle density by 37-70% andproductivity by 50% (5). Moreover, AAT not only has an impact inanimal production but also in crop agriculture since in most SSA countriescattle is used as part of farming systems. T.
vivax cell specific proteins (TVCSP) with possible surfacelocalization have been demonstrated to be unique and expressed in only specificlife stages (6, 7). However, the physiological role aswell as the immune response these proteins are able to develop in the hostremain obscure. The potential application of the TVCSP to identify immunogenicpeptides that can stimulate the host immune response The application of peptides arrayssince their first description in 1992 (8), led researchers to studyprotein-protein interactions, specially mapping immunodominant regions inantigens and seromarker discovery in immunology. Peptide arrays in Trypanosome diagnosis likethe identification of new antigens for Trypanosomacruzi (9) are a good example of how thistechnique employs different hundreds of peptides in order to identify epitopesand new antigens that may be likely conserved among different strains. The objective of this chapter is touse a reverse vaccinology approach to identify T and B cell in silico epitopesand evaluate the immunogenicity of Trypanosomavivax cell surface proteins (TVCSP) in natural infections in order toidentify immunogens across the populations.
The recognition of epitopes will beperformed using peptide microarrays in sera from naturally infected livestock. 2. Methods 2.1 T-celland B-cell epitope predictionIn order toidentify epitopes located in the antigen-presenting cell’s surface, weperformed an in silico prediction of B-cell epitopes and T cell epitopes forMHC-I and MHC-II respectively. All FamX protein sequences were used as input topredict epitopes for MHC class I with the Immune Epitope Database (IEDB) (10)available online (http://tools.iedb.
org).The IEDB recommended prediction method was chosen, giving the advantage thatthis method is a consensus of the artificial neural network (ANN) (11),stabilized matrix method (SMM) (12),combinational libraries (CombLib) (13)and NetMHC-3.0 (14).Six different bovine leucocyte antigen (BoLA) alleles were selected: D18.4(BoLA-1*02301) HD6 (BoLA-6*01301), JSP.1 (BoLA-3*00201), T2A (BoLA-2*01201),T2B (BoLA-6*04101) and T2C (BoLA-3*00101) with an epitope length 8-11 aa. Athreshold of 0.
3 in percentile rank was selected to get best hits only. In thecase of MHC-II, since there are no BoLA alleles available to perform in silicopredictions, we used human leucocyte antigen (HLA) as stated in a previouspublication (15).Seven HLA alleles were selected for the predictions: DRB1*0401, DRB1*0301,DRB1*1401, DRB1*1101, DRB3*0201, DRB1*0801 and DRB3*0101. Two MHC class IIservers with different prediction methods were used: TEPITOPEpan (16)based on the position specific scoring matrix (PSSM) and IEDB server. In bothanalysis, the percentile rank cut-off was set to 3% and peptide length of15-mer.
For MHC-I and MHC-II predicted epitopes, all the peptides wereevaluated for antigenicity with VaxiJen v2.0 (17)selecting parasite as target organism and using a threshold of 0.05. B-cell epitopeswere also predicted in silico for FamX protein sequences.
Linear epitopes forthe analysis were performed with BCPred (18),ABCPred (19)and IEDB (Bepipred Linear Epitope Prediction) servers. For all cases, thethreshold was set to 0.8 and the peptide length up to 20aa for BCPred andABCPred only.
The results from these three servers were compared with the onesobtained with the peptide microarray assay. 2.2 Sera screeningSera from naturally infectedlivestock potentially positive for T.vivaxwas screened with a novel rapid diagnostic test called Very Diag (Ceva-Africa).
Very Diag is a lateral flow immunoassay that can detect in a qualitative wayantibodies against Trypanosoma congolenseand Trypanosoma vivax from sera andbovine whole blood samples. This test is based on the recombinant TvGM6 antigenin order to detect Trypanosoma vivaxand the TcoCB1 to detect Trypanosomacongolense at the same time within a unique test (20).