Journal of Current Biomedical Reports  jcbior.com 

Volume 1, Number 1, 2020  

1 
 

Original research 

In silico prediction of B cell epitopes of the hemolysis-
associated protein 1 for vaccine design against leptospirosis 

 
Sakineh Poorhosein Fookolaee1, Somayyeh Talebishelimaki1, Mohammad Taha Saadati Rad2,3, 

Mostafa Akbarian Rokni4,* 
 

1 Department of Nursing, Faculty of Nursing and Midwifery, Islamic Azad University, Sari, Iran 
2 Young Researchers and Elite Club, Sari Branch, Islamic Azad University, Sari, Iran 

3 Department of Medicine, Sari Branch, Islamic Azad University, Sari, Iran 
4 Department of Nursing, Faculty of Nursing & Midwifery, Babol University of Medical Sciences, Babol, Iran 

 

Abstract 
Leptospirosis is known as a zoonotic disease of global importance originated from infection with the 
spirochete bacterium Leptospira. Although several leptospirosis vaccines have been tested, the 
vaccination is relatively unsuccessful in clinical application despite decades of research. Therefore, this 
study was conducted to predict B cell epitopes of the hemolysis-associated protein 1 (Hap1) for vaccine 
design against leptospirosis. For prediction of linear epitopes, the sequence of extracellular region of 
Hap1 was submitted to ABCpred, BCPREDs, Bcepred, Bepipred and Ellipro servers. DiscoTope 2.0 and 
B-pred servers were used for prediction of conformational epitopes from the entire PDB structure of 
Hap1 that obtained from the homology modeling method. Further analysis for solvent accessible areas 
and relative solvent accessibility of all the residues on the PDB structures using Naccess program and 
NetSurfP server defined that predicted conformational B cell epitopes had higher solvent accessible and 
their residues were exposed on the surface therefore, immunoinformatics analysis showed that 
hemolysis-associated protein 1 can properly stimulate the B cells immune responses. 

Keywords: Leptospirosis; Hemolysis-associated protein 1; B cell epitope; in silico

1. Introduction 
Leptospirosis is an infection caused by corkscrew-

shaped bacteria called Leptospira. Signs and 
symptoms can range from none to mild such as 
headaches, muscle pains, and fevers to severe with 
bleeding from the lungs or meningitis [1]. If it causes 
jaundice, kidney failure, and bleeding, it is known as 
Will's disease, and if it causes bleeding in the lungs, it 
is also known as severe pulmonary hemorrhage 
syndrome.[2]. Leptospira can be transmitted by both 
wild and domestic animals, and up to 10 different 

                                                           
* Corresponding author:  
Dr. Mostafa Akbarian Rokni, Ph.D 
Department of Nursing, Faculty of Nursing & Midwifery,  
Babol University of Medical Sciences, Babol, Iran  
Tel/Fax: +98 935 8462251 
Email: akbarianmostafa1357@gmail.com 
https://orcid.org/0000-0002-9198-6818 
 
Received: July, 28, 2020 
Accepted: August, 18, 2020 

genetic types of it can cause disease in humans. The 
most common animals that spread the disease are 
rodents [3]. The identification of the common 
immunogenic proteins of Leptospira would be a major 
step toward the development of purer, better-defined, 
and probably more-efficient vaccines. Such vaccines 
would provide cross-protection against a wide range of 
pathogenic Leptospira strains [4] Outer membrane 
proteins such as LipL21 and LipL32, LipL41 are 
expressed only in pathogenic species of Leptospira [5]. 
Recent data indicated that hemolysis-associated 

https://jcbior.com/


Poorhosein Fookolaee et al. 

2 
 

protein 1 (Hap1) (Figure 1) could be a good candidate 
for developing a new generation of vaccines able to 
induce broad protection against leptospirosis disease 
[6]. It has been suggested that this protein is only 
produced by pathogenic leptospires and Hap1 
vaccination induces significant protection compared 
to similar OmpL1 based vaccination [6]. Active 
immunotherapy with the peptide vaccines which are 
designed to be chimeric with multi-epitopes of B cells 
and T helper cells can induce the generation of an 
adaptive immune response [7]. Several experimental 
techniques are currently available for the selection of 
suitable B cell epitopes. The experimental approaches 
applied for detecting immunogenic regions are often 
laborious and resource-intensive. Computational 
techniques for predicting B cell epitopes are fast, 
scalable, and cost-effective, focusing on experimental 
experiments and a better understanding of antigen-
antibody interaction [8]. Recent researches have 
shown there are limitations to the current epitope 
prediction methods. Therefore, enhancing the 
reliability of computational B cell epitope prediction 
methods remains a major challenge in computational 
vaccinology [9]. Nevertheless, prediction results 
produced by multiple computational tools could be 
used to gain a consensus result. Basically, the 
recognition of either small discrete T-cell epitopes or 
large conformational epitopes recognized by soluble 
antibodies and B cells is the key molecular event for the 
immune response to pathogens. B cell epitopes can be 
classified into two types: linear (continuous) and 
conformational (discontinuous). While linear epitopes 
comprise of continuous residues in the sequence, 
conformational epitopes are composed of amino acids 
that are not neighboring in primary sequence and are 
brought into close proximity in the folded protein 
structure [10]. Localization of these epitopes is of 
clinical interest for the development of diagnostic 
tools, vaccines, and cancer immunotherapies. Many 
attempts have been made for predicting the antigenic 
sites from certain features of proteins' primary 
structures. Different parameters such as static 
accessibility, hydrophilicity, and mobility of the short 
segments in polypeptide chains have been associated 
with the position of continuous epitopes in proteins 
[11]. The present research aimed to predict new B cell 
epitopes for the Hap1. Combination approaches were 
used by combining results from the sequence and the 
structure-based methods and the solvent-accessible 

surface area calculating tools. Furthermore, we used 
PEPOP 2.0 application to predict new conformational 
epitopes from PDB structure of Hap1. 

 
2. Materials and Methods 
2.1. Retrieval of protein sequences 
All sequences of Hap1 were retrieved from 

UniProt (www.uniprot.org) in FASTA format. 
2.2. Linear   B   cell   epitopes   prediction    
For the prediction of linear epitopes, the sequence 

of Hap1was submitted to ABCpred, BCPREDs, 
Bcepred, Bepipred, and Ellipro servers [9, 12]. The 
hidden Markov model, Thornton’s method, Support 
Vector Machine classifiers, Recurrent Neural 
Network, and physicochemical properties of amino 
acids were applied to predict linear B cell epitopes. 
Only, the linear peptides which were predicted 
frequently by 3 or more servers were selected. 

2.3. Conformational B cell epitope prediction  
DiscoTope 2.0 and B-pred servers were used for 

the prediction of conformational epitopes from the 
entire PDB structure of Hap1 that obtained from the 
homology modeling method. The DiscoTope method 
incorporates a new spatial neighborhood description 
and a half-sphere exposure as a surface measure based 
on the protein structure and epitope propensity scores 
and predicts residues that can be involved in B-cell 
epitopes. B-pred is a web-based platform for scoring 
and predicting B-cell epitopes based on the structures 
of the potential immunological proteins. The method 
scores the peptides set of a protein-based on the 

Figure 1. Tertiary structure of Hap 1. In this figure the protein is shown 

in the third structure 



Poorhosein Fookolaee et al. 

3 
 

average solvent exposure, by a filter on filtering the 
average local model quality for each peptide [13].  

 
3. Results 
The predicted linear B cell epitopes of the HAP1 

are shown in Table 1. The predicted B cell epitopes 
were ranked according to their score obtained by a 
trained recurrent neural network. A higher score of 
peptide means a higher probability to be an epitope. As 
listed in Table 1, peptides were predicted by 5 servers 
used in the current study. The peptide 
“TGEIGEPGDGDLVSDA” had the highest score 
compared to other epitopes. These peptides reside in 
37-210 regions of the Hap1. Some studies on the 
vaccine are shown in the Table 2.  

Discotope and B-pred predicted 167 B-Cell 
epitope residues out of 3420 total residues 
corresponding to conformational epitopes, 
approximately located in the region of linear B cell 
epitopes (Table 3). Further analysis for solvent 
accessible areas and relative solvent accessibility of all 
the residues on the PDB structures using the Naccess 
program and NetSurfP server defined that predicted 
conformational B cell epitopes had higher solvent 
accessible and their residues were exposed on the 
surface.  

 
4. Discussion 
Although the majority of B cell epitopes appear to 

be conformational, most of the computational 
methods focused on the prediction of sequential 
epitopes. Linear epitope prediction approaches can be 
classified as propensity scale methods, improved 
propensity scale methods and machine learning 
methods [23]. If the tertiary structure of an antigen is 
known, there are improved methods for identifying 
conformational B cell epitopes. Examples are 
Discotope web server and PEPOP. These are based on 
features like amino acid propensity scales and solvent 
accessibility [24]. In this study, linear and 
conformational B cell epitopes of Hap1 were predicted 
using both primary sequence and tertiary structure. 
Based on combination approaches and considering 
lowest identity with the IR  and frequently prediction 
using several tools, the best peptides were the linear B 
cell epitope TGEIGEPGDGDLVSDA (Table 1) and 
conformational B cell epitopes (Table 2). In addition, 
PEPOP also predicted 167 new conformational 
peptides. 

Table 1. Predicted linear B cell epitopes using various servers. 

 

Rank Sequence Start position Score 

1 TGEIGEPGDGDLVSDA 101 0.96 

2 GSVINYYGYVKPGQAP 54 0.94 

3 YRISFTTYKPGEVKGS 210 0.89 

3 DDGDDTYKEERHNKYN 164 0.89 

4 GQAPDGLVDGNKKAYY 66 0.88 

5 EDTIPGTNETVKTLLP 37 0.85 

5 RIKIPNPPKSFDDLKN 183 0.85 

5 FDTWIRVERMSAIMPD 131 0.85 

6 PEEKSMPHWFDTWIRV 122 0.84 

7 YGYVKPGQAPDGLVDG 60 0.83 

7 NETVKTLLPYGSVINY 44 0.83 

7 RHNKYNSLTRIKIPNP 174 0.83 

8 ASVGLLFPPGIPGVSP 228 0.81 

9 AVIAEMGVRMISPTGE 88 0.80 

9 AKAAKAKPVQKLDDDD 149 0.80 

10 QKQAIAAEESLKKAAS 253 0.78 

 



Poorhosein Fookolaee et al. 

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Poorhosein Fookolaee et al. 

5 
 

The predicted epitopes by PEPOP are widely 
distributed within the CRR (Cysteine‐rich region) 
domain and often partly overlapped, consistent with 
the view that PEPOP predicted segmented epitopes 
and the CRR domain displayed a mosaic of 
overlapping epitopes. Considering less identity with 
the IR and high RSA (Relative solvent accessibility) 
score conformational epitopes can be suitable for 
further experimental tests. In the present work, new 
conformational epitope and a linear B cell epitope 
were predicted using various bioinformatics analyses. 
In conclusion, findings of the present work, using the 
bioinformatics analyses could be used in mAbs 
(monoclonal antibodies) production, vaccine design 
and the diagnostic tools. In addition, the current in 
silico approaches are reducing time and minimizing 
the total number of necessary tests to find possible and 
proper epitopes. In the next step, synthesis of 
determined peptides in vitro and in vivo experimental 
studies are essential for assurance of the predicted 
epitopes. Due to the prevalence of this disease in Iran, 
especially in the northern regions of the country, which 
have paddy fields and agricultural lands and many 
people are at risk of contracting this disease, the 
preparation and design of vaccines seems necessary. 

In this research, we used immunoinformatics 
methods to predict appropriate vaccine against 

Leptospira. Selection of suitable epitopes of the Hap1 
as antigens, and utilizing them for raising mAbs 
against the Hap1, with ability of Leptospira inhibition 
would be beneficial in treatment of leptospirosis. To 
the best of our knowledge, for the first time, in this 
study the linear and conformational B cell epitopes of 
the Hap1 were predicted, screened and assessed using 
the well-known bioinformatics comprehensive 
analyses. All sequences were joined to each other by 
proper linkers. Epitopes were evaluated as non-
allergenic, antigenic, soluble, with safety and efficacy. 
These predicted epitopes might be used to design a 
vaccine against Leptospira, could be validated in 
model hosts to verify their efficacy as vaccine. 

Author Contributions  
All authors contributed equally to this manuscript, 

and approved the final version of manuscripts.  
Conflict of Interests  
The authors declare that they have no conflicts of 

interest.  
Ethical declarations  
Not applicable.  
Financial Support  
None. 

 
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Table 3. Conformational B-cell epitopes from full-length protein using 

different servers 

 

Epitope Position 

ASP, GLU, THR, ILE, LYS, GLU 14, 14, 30, 24, 8, 20 

SER 15 

PHE, GLN 13, 27 

PRO 9 

LYS, ASN 8, 15 

LEU, LYS 22, 6 

ASP, GLU, THR, ILE, LYS, GLU 15, 14, 30, 24, 7, 20 

SER 23 

SER 15 

PHE 24 

PRO 8 

LYS, ASN 8, 15 

LEU, LYS 22, 7 

ASP, GLU, THR 14, 14, 30 

LYS, GLU 8, 20 

SER 20 

PHE, GLN 24, 13 

 

http://dx.doi.org/10.5495/wjcid.v6.i4.61
http://dx.doi.org/10.5495/wjcid.v6.i4.61
http://dx.doi.org/10.5495/wjcid.v6.i4.61
https://pubmed.ncbi.nlm.nih.gov/17471039/
https://pubmed.ncbi.nlm.nih.gov/17471039/
https://pubmed.ncbi.nlm.nih.gov/17471039/
https://pubmed.ncbi.nlm.nih.gov/19345023/
https://pubmed.ncbi.nlm.nih.gov/19345023/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/18285490/
https://pubmed.ncbi.nlm.nih.gov/11598056/
https://pubmed.ncbi.nlm.nih.gov/11598056/
https://pubmed.ncbi.nlm.nih.gov/11598056/
https://pubmed.ncbi.nlm.nih.gov/11598056/
https://pubmed.ncbi.nlm.nih.gov/11598056/
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