Identification can stimulate the host immune response

Identification of linear B-cell epitopes
in Trypanosoma vivax using peptide


1.     Introduction

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In order to produce a vaccine,
classical and conventional vaccinology has been the most common way to achieve
it and widely used over the past decades. 
However, this methodology is time- consuming and in not all the cases
researchers can found successful candidates due to different problems in the
organism cultivation in vitro or
difficulties on the subunit vaccines identification.  The term reverse vaccinology (RV) was first
used by Rino Rappuoli and refers to use and take advantage of the genomic
sequence of the organism, select between many different proteins the parasite
can express and predict only the potential antigen candidates rather than
working with it in vitro (1). 
One of the advantages RV has compared with the classical vaccinology is
the fact that with application of genomic technologies, more proteins that were
not considered previously are now available because all genes can be
analyze.  As a consequence, RV give us a
much wider variety of candidates (2). 
Several approaches on vaccine design have now apply not only RV but also
structural biology.  Possible
anti-parasite vaccines for human and animals have been studied in the last few
years 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), a
disease that affects livestock in Africa and South America. A vaccine
development for AAT is crucial since the disease generate tremendous loses
every year decreasing cattle population. It has been estimated that calf
mortality is up to 20% in infected regions (4)  and can reduce cattle density by 37-70% and
productivity by 50% (5).  Moreover, AAT not only has an impact in
animal production but also in crop agriculture since in most SSA countries
cattle is used as part of farming systems.  T.vivax cell specific proteins (TVCSP) with possible surface
localization have been demonstrated to be unique and expressed in only specific
life stages (6, 7). However, the physiological role as
well as the immune response these proteins are able to develop in the host
remain obscure. The potential application of the TVCSP to identify immunogenic
peptides that can stimulate the host immune response


The application of peptides arrays
since their first description in 1992 (8), led researchers to study
protein-protein interactions, specially mapping immunodominant regions in
antigens and seromarker discovery in immunology.  Peptide arrays in Trypanosome diagnosis like
the identification of new antigens for Trypanosoma
cruzi (9) are a good example of how this
technique employs different hundreds of peptides in order to identify epitopes
and new antigens that may be likely conserved among different strains.


The objective of this chapter is to
use a reverse vaccinology approach to identify T and B cell in silico epitopes
and evaluate the immunogenicity of Trypanosoma
vivax cell surface proteins (TVCSP) in natural infections in order to
identify immunogens across the populations. The recognition of epitopes will be
performed using peptide microarrays in sera from naturally infected livestock.



2.     Methods


2.1  T-cell
and B-cell epitope prediction

In order to
identify epitopes located in the antigen-presenting cell’s surface, we
performed an in silico prediction of B-cell epitopes and T cell epitopes for
MHC-I and MHC-II respectively. All FamX protein sequences were used as input to
predict epitopes for MHC class I with the Immune Epitope Database (IEDB) (10)
available online (
The IEDB recommended prediction method was chosen, giving the advantage that
this 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. A
threshold of 0.3 in percentile rank was selected to get best hits only. In the
case of MHC-II, since there are no BoLA alleles available to perform in silico
predictions, we used human leucocyte antigen (HLA) as stated in a previous
publication (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 II
servers with different prediction methods were used: TEPITOPEpan (16)
based on the position specific scoring matrix (PSSM) and IEDB server. In both
analysis, the percentile rank cut-off was set to 3% and peptide length of
15-mer. For MHC-I and MHC-II predicted epitopes, all the peptides were
evaluated for antigenicity with VaxiJen v2.0 (17)
selecting parasite as target organism and using a threshold of 0.05.


B-cell epitopes
were also predicted in silico for FamX protein sequences. Linear epitopes for
the analysis were performed with BCPred (18),
ABCPred (19)
and IEDB (Bepipred Linear Epitope Prediction) servers. For all cases, the
threshold was set to 0.8 and the peptide length up to 20aa for BCPred and
ABCPred only. The results from these three servers were compared with the ones
obtained with the peptide microarray assay.


2.2  Sera screening

Sera from naturally infected
livestock potentially positive for T.vivax
was 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 way
antibodies against Trypanosoma congolense
and Trypanosoma vivax from sera and
bovine whole blood samples. This test is based on the recombinant TvGM6 antigen
in order to detect Trypanosoma vivax
and the TcoCB1 to detect Trypanosoma
congolense at the same time within a unique test (20).