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Identification of Novel Multi Epitopes Vaccine against the Capsid Protein (ORF2) of Hepatitis E Virus

Mashair A. A. Nouri, Yassir A. Almofti*, Khoubieb Ali Abd-elrahman, Elsideeq E. M. Eltilib

Department of Molecular Biology and Bioinformatics, College of Veterinary Medicine, University of Bahri, Khartoum- Sudan

Abstract: Hepatitis E virus (HEV) is non-enveloped, small virus with a positive RNA sense in the family Hepeviredae genus Orthohepevirus. More than 20 million individuals annually infected by HEV with increased mortality rate ranged from 8% to 20% in pregnant women. The aim of the present study was to design multi peptides vaccine against HEV using immunoinformatic tools that elicited humoral and cellular immunity. The capsid protein sequences of HEV were retrieved from NCBI and subjected to various immunoinformatics tools from IEDB to assess their conservancy, surface accessibility and antigenicity as promising epitopes against B cells. Moreover the binding affinity of the conserved predicted epitopes was analyzed against MHC-I and MHC-II alleles of the T cells. The predicted epitopes were further assessed for their population coverage. For B-cell 32, 23 and 12 epitopes were predicted as linear conserved epitopes, surface accessibility and antigenic respectively. However the best B cell epitopes that overlapped the prediction tools were ¹⁶⁵PLQD¹⁶⁸, ²¹⁹PTSVD²²³, 452PTPSPAPS⁴⁵⁹, ⁵⁵⁶GYPYNY⁵⁶¹ and ⁶¹⁵DYPA⁶¹⁹. For T cell, the MHC-I alleles interacted with 37 conserve epitopes. Four epitopes (³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁸⁹SSAGGQLFY³⁹⁷ and 394QLFYSRPVV⁴⁰²) interacted with MHC class-I with high affinity and specificity and hence were proposed as vaccine candidates. Moreover seven epitopes out of 125 predicted epitopes (were 205YAISISFWP²¹³, ²⁹⁹LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹, ³⁶⁷GIALTLFNL³⁷⁵, ³⁶⁸IALTLFNLA³⁷⁶, 379LLGGLPTEL³⁸⁷ and ³⁹⁴QLFYSRPVV⁴⁰²) were proposed as vaccine since they demonstrated high affinity to MHC-II alleles. The epitopes ³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷ and 394QLFYSRPVV402 were recognized interacting with both MHC-I and MHC-II alleles. The population coverage epitopes set for MHC-I and MHC-II alleles was 78.97% and 99.99%, respectively. While the epitopes set for all T cell proposed epitopes was 100%. Thirteen epitopes were predicted eliciting B and T cells and proposed as vaccine candidates against HEV. However these proposed epitopes require clinical trials studies to ensure their efficacy as vaccine candidates.

Keywords: Hepatitis E Virus, capsid protein, Immune epitope database (IEDB), epitope, B-cell, T-cell

Received August 14, 2019; Revised September 20, 2019; Accepted October 07, 2019

Citation: Mashair A. A. Nouri, Yassir A. Almofti, Khoubieb Ali Abd-elrahman, Elsideeq E. M. Eltilib, Identification of Novel Multi Epitopes Vaccine against the Capsid Protein (ORF2) of Hepatitis E Virus. American Journal of Infectious Diseases and Microbiology, 7 (1). 26-42. January 2019. doi:10.12691/ajidm-7-1-5

1. Introduction

Hepatitis E Virus (HEV) causes hepatitis infection in human and it is the major cause of acute hepatic infection in developed countries of Asia, Africa and Latin America [1,2,3,4]. In industrialized countries including Japan, Europeans countries and United State sporadic cases of locally acquired Hepatitis E were record [5,6]. In these countries a significant proportion of healthy individuals were found to be seropositive for Hepatitis E antibody. Among them 20% have been reported in United State [6]. HEV have four genotypes that causes sporadic cases of hepatitis E in human [3,7,8]. Genotype 1 and 2 only infect human mainly in developing countries, while genotype 3 and 4 infect animal and considered as zoonotic [3,7,9,10,11].

World Health organization (WHO) in 2014 reported that there were 20 million Hepatitis E infections annually [8,12]. In developing countries and epidemic setting the mortality rate in general population range from 0.2% to 4% but increased to 8% to 20% in pregnant women (genotype1) [7,13]. Hepatitis E virus (HEV) infection is self-limiting disease but may progress to fetal disease when combined with pregnancy and chronic liver problem (genotype 1, 2) [14]. Genotypes 3 and 4 the hepatitis progress in chronic liver problem but not in pregnancy [14]. Chronic Hepatitis E virus Infection is now considered clinical problem associated with sever mortality and morbidity in organ transplant [11]. The acute HEV infection diagnosis by detection of anti-HEV IgM and/ or HEV RNA in sera [15].

HEV transmitted by oral and fecal route mainly through contaminated water [16,17]. The infection mostly through consuming contaminated food that contains HEV [3]. Hepatitis E virus is non-enveloped, small virus with a positive sense RNA [1,17]. Hepatitis E virus belongs to the genius Orthohepevirus family Hepeviredae [3,4,13,17]. The virus contains three opening reading frames genes (ORFs). ORF1 encodes poly protein, ORF2 encodes capsid protein, ORF3 encodes phosphoprotein [13,17].

The first line of defense against HEV infection is basic sanitation but typical intervention did not prevent additional infection in southern Sudan and Uganda. Thus effective and safe vaccine is needed. All major HEV genotypes in humans have the same serotypes so the vaccine development could be facilitated [18,19]. Also the vaccine that prepared to specific country would be effected for all countries [18]. In China HEV vaccine (commercial name of Hecolin) was used since 2012 but the long-term efficacy of this vaccine has not determined [19].

The genome of HEV comprise of 7.2 kDa. The ORF2 encode for a 660-aa long viral capsid protein [20], and it is located at the 3 end of the viral genome. Some studies exhibited outstanding antibody response directed against immunodominant antigenic epitopes contained in ORF2 capsid protein. The recombinant HEV ORF2 protein was used as a vaccine. It has shown perfect efficacy for protecting against hepatitis E disease and good immunogenicity.

The aim of this study was to design an in silico Hepatitis E virus vaccine by using bioinformatics approach. This was performed by predicting peptides from the capsid protein (ORF2) that elicit both T and B-cells.

2. Materials and Methods

2.1. Protein Sequence Retrieval

A total of 73 capsid protein sequences of HEV were retrieved from NCBI database on 17 Aug 2017 [21]. These sequences were retrieved from various parts of the world. The retrieved capsid protein strains and their accession numbers and area of collection were listed in Table 1.

Table 1. The accession numbers , area of collection and date of collection of HEV strains

2.2. Phylogenetic Evolution

Phylogenetic tree of the retrieved sequences of the capsid proteins of HEV was created using MEGA7.0.26 (7170509) software [22]. The protein tree was constructed using maximum likelihood parameter in the software.

2.3. Determination of Conserved Regions in the Capsid Protein

the retrieved sequences were used as scheme to obtain conserved regions using multiple sequence alignment (MSA). Sequence alignment was used to illustrate the conserved regions within the capsid protein by using ClustalW as perform in the BioEdit program version 7.2.5 [23].

2.4. Conserved Epitopes Prediction

To determine the conserves regions among HEV capsid protein the targeted peptides were obtained by using different prediction tools from Immune Epitope Database (IEDB) (www.iedb.org) [24,25]. The reference sequence of the capsid protein was submitted to different B and T cells prediction tools in IEDB [26].

2.5. B-cell Epitope Prediction

Bepipred prediction tool from immune epitope database at (http://tools.iedb.org/bcell/result/) [27] was used as linear B-cell epitopes prediction tool with threshold value 0.353.

Emini surface accessibility prediction tool (http://tools.iedb.org/bcell/result/) [28] of the IEDB was used to predict surface accessible epitopes with threshold value of 1.000. Kolaskar and Tongaonkar antigenic method (http://tools.iedb.org/bcell/result/) [29] with a threshold value 1.031 was used to determine the antigenic epitopes.

2.6. Prediction of T-cell Epitopes

2.6.1. MHC Class I Binding Predictions

Epitopes binding to the Major histo-compatibility class I (I) molecules was predicted by the IEDB I prediction tools (http://tools.iedb.org/mhci/).The binding of cleaved epitopes to molecules was proposed by artificial neural network () method [30,31]. Peptide length for all selected epitopes was set to 9 amino acids (mers). The conserve predicted epitopes that bound to alleles were at score equal to or less than 300 half-maximal inhibitory concentration (IC50) [32].

2.6.2. MHC Class II Binding Predictions

Epitopes binding to the Major histo-compatibility class II (MHC- II) molecules was predicted by the IEDB MHC-II prediction tools (http://tools.iedb.org/mhcii/) [33,34].

The peptides fragment from protein antigen bound to the professional antigen presenting cells that the MHC-II molecule were expressed on its surface [34]. The neural network-based alignment method (NN-align) was used [35,36,37,38]. All conserved epitopes bound to variable alleles HLA-DR, HLA-DQ, HLA-DP with different lengths were determined [33].

2.7. Homology Modeling

Homology modeling was used for constructing the three dimensional (3D) structure of the reference sequence of the capsid protein. Raptor X structure prediction server (http://raptorx.uchicago.edu/StructurePrediction/predict/) was used for this purpose. The 3D structure was then treated with Chimera software 1.8 to display the position of proposed epitopes [39,40,41].

2.8. Population Coverage

Conserved epitopes that bound to MHC-I and MHC-II from capsid protein were submitted for population coverage against the whole world population. For calculation of MHC-I, MHC-II interacted alleles the IEDB population coverage tool was used (http://tools.iedb.org/population/result/) [42].

3. Results

3.1. Phylogenetic Evolution

Phylogenetic tree demonstrated the evolutionary divergence within the retrieved sequences of the capsid protein. As shown in Figure 1 retrieved strains showed molecular divergence in their common ancestors.

3.2. Sequences Alignment

Sequence alignment of all retrieved strains was performed using ClustalW that presented by Bioedit software. As shown in Figure 2 the retrieved sequences demonstrated conservancy when sequences were aligned. The conserved regions were recognized by identity of amino acid sequences among the retrieved sequences.

3.3. Prediction of B-cell Epitopes

The reference sequence of the capsid protein was subjected to Bepipred linear epitopes, Emini surface accessibility and Kolaskar and Tongaonkar antigenicity in IEDB. The thresholds for these software were shown in Figure 3. Bepipred predicted 32 linear epitopes. Emini surface accessibility predicted 23 epitopes on the surface. While Kolaskar and Tongaonkar antigenicity method predicted 12 antigenic epitopes. The predicted epitopes were shown in Table 2. Most importantly eight epitopes were found to be both on surface and antigenic using Emini surface accessibility and kolaskar antigenicity methods, respectively. The best B cell predicted epitopes that were on surface and antigenic with best score and overlapped the B cell prediction methods were ¹⁶⁵PLQD¹⁶⁸ and 615DYPA⁶¹⁹ ⁵⁵⁶GYPYNY⁵⁶¹ ⁴⁵²PTPSPAPS⁴⁵⁹ and ²¹⁹PTSVD²²³. The three D structure of these predicted epitopes in the capsid protein was shown in Figure 4.

Figure 1. Evolutionary divergence analysis of capsid protein of the HEV. The retrieved strains showed divergence in their common ancestors

Figure 2. Multiple sequence alignment (MSA) of the retrieved strains using Bioedit software and ClustalW. Dots indicated the conservancy and letters in cubes showed the alteration in amino acid

Table 2. B cell predicted epitopes. The table demonstrated the predicted conserved epitopes with their surface accessibility score (a) and antigenicity score (b)

Figure 3. Prediction of B-cell epitopes using (a) Bepipred linear epitope, threshold value 0.353 (b) Emini surface accessibility, threshold value 1.000 and (c) Kolaskar & Tongaonkar antigenicity methods, threshold value 1.031. Yellow areas above the threshold (red line) are suggested to be a part of B cell epitope, while green areas are not

Figure 4. Position of proposed conserved B cell epitopes in structural level of the capsid protein. Five epitopes were illustrated in this figure to interact with B cell. These epitopes showed conservancy, high score in surface accessibility and antigenicity using IEDB software. The ball like structures represent the predicted epitopes. The position of these epitopes was according to their position in the capsid protein

3.4. Prediction of Epitopes Interacted with MHC-I Alleles

Based on ANN-align and IC50 less than 300 in IEDB, MHC-1 binding prediction tool predicted 37 epitopes that interacted with the cytotoxic T cell as they strongly linked with multiple alleles. MHC-1 predicted epitopes were shown in Table 3. Four epitopes interacted with MHC class-I with high affinity and specificity and hence were proposed as vaccine candidates. These epitopes were ³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁸⁹SSAGGQLFY³⁹⁷ and 393QLFYSRPVV⁴⁰². The 3D structure of these four proposed epitopes was shown in Figure 5 and Figure 6.

Table 3. Conserve peptides with bind alleles of MHCI, IC50 , start and end of peptides

Figure 5. T cell proposed epitopes that interact with MHC-I alleles. These epitopes are ³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁹⁴QLFYSRPVV⁴⁰² and ³⁸⁹SSAGGQLFY³⁹⁷. The first three epitopes were found interacting with both MHC-1 and MHC-II alleles. The latter interacted only with MHC-I alleles and it was shown in figure (6). The ball like structures represent the predicted epitopes. The positions of these proposed epitopes was according to their position in the capsid protein of HEV

Figure 6. T cell proposed epitopes that interact with MHC-II alleles. These epitopes were ²⁰⁵YAISISFWP²¹³, ²⁹⁹LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹, 368IALTLFNLA³⁷⁶, ³⁶⁷GIALTLFNL³⁷⁵, 379LLGGLPTEL³⁸⁷ and ³⁹⁴QLFYSRPVV⁴⁰². The last three epitopes were shown in Figure 5. The epitope ³⁸⁹SSAGGQLFY³⁹⁷ also interacted with MHC-1 alleles. The ball like structures represent the predicted epitopes. The positions of these proposed epitopes was according to their position in the capsid protein of HEV

3.5. Prediction of Epitopes that Interacted with MHC Class II

Based on NN-align method with half-maximal inhibitory concentration (IC50) less than 3000, the reference sequence of Hepatitis E capsid protein was analyzed. The analysis depend on the IEDB MHC-II binding prediction tool based on NN-align. A total of 125 epitopes were predicted to interact with MHC-II alleles. Among them seven epitopes were proposed as vaccine since they demonstrated high affinity to MHC-II alleles. These epitopes were 205YAISISFWP²¹³, ²⁹⁹LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹, ³⁶⁷GIALTLFNL³⁷⁵, ³⁶⁸IALTLFNLA³⁷⁶, 379LLGGLPTEL³⁸⁷ and ³⁹⁴QLFYSRPVV⁴⁰². These epitopes and their interacted alleles were shown in Table 4. The position and the 3D structures of these epitopes in the capsid protein were demonstrated in Figure 5 and Figure 6.

Table 4. List of most promising epitopes that had binding affinity with MHC-II alleles with thei peptide sequence, IC50 and percentile rank of capsid protein of HEV

3.6. Population Coverage

The predicted epitopes from the capsid protein that interacting with MHC-I and II alleles were subjected to population coverage analysis. The results of population coverage of all predicted epitopes against the world for MHC class I and II were listed in Table 5. The population coverage of all MHC-I and II predicted epitopes was 96.93% and 99.99% respectively.

Table 5. The (%) of the population coverage of all predicted epitopes of MHC-I and MHC-II

In addition to that the proposed epitopes that demonstrated higher affinity to interact with MHC-I and MHC-II alleles and that bound to different sets of alleles were selected for population coverage analysis. Table 6 demonstrated the population coverage percentages for each proposed epitope and epitopes sets. For MHC-I the epitopes ³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁹³QLFYSRPVV⁴⁰² and ³⁸⁹SSAGGQLFY³⁹⁷interacted with most frequent MHC-I alleles and they demonstrated high percentage against the whole world population coverage with epitope set 78.97%. Strikingly the first three epitopes were found interacted with MHC-1 and MHC- II. While the latter epitope interacted only with most frequent MHC-I alleles. For MHC- II epitopes, in addition to the overlapped epitopes in MHC-1 (³⁶⁷GIALTLFNL³⁷⁵, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁹³QLFYSRPVV⁴⁰²), four epitopes namely ²⁰⁵YAISISFWP²¹³, ²⁹⁹LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹ and ³⁶⁸IALTLFNLA³⁷⁶ were also interacted with high affinity to MHC-II alleles with high population coverage percentage with epitope set 99.99%. The overall MHC-1 and MHC-II whole world population coverage epitope set for all suggested epitopes was 100%.

Table 6. The population coverage (PC) of MHC-I and MHC-II for the proposed epitopes. The population coverage of MHC-I and MHC-II combined alleles was calculated for all proposed epitopes

4. Discussion

The spread of lethal hepatitis E infection over the world lead to increase the care about vaccination process. Therefore the aim of present study was to propose highly potential immunogenic epitopes eliciting the B and T cells and work as vaccine candidates against the lethal infection of Hepatitis E Virus. This was performed by using the capsid protein as immunogenic target to exploit epitopes that could be used as a vaccine. Previous studies focused on ORF2 protein as an important immunogenic part of HEV that elicited B and T cells since the genetic analysis of ORF2 has demonstrated over 85% similarity among genotypes 1–4 HEV strains that infect humans [43]. Moreover, the identiﬁcation of both conformational and linear neutralizing epitopes in HEV capsid protein suggests that this protein is the target for neutralizing antibodies [44,45,46]. Consequently, most HEV vaccine candidates are based on recombinant expressed full length or truncated HEV-capsid protein (ORF2) [44].

The first HEV-ORF2 protein vaccine employed was a truncated ORF2 protein (aa 394-607) of Chinese genotype 1HEV isolate. However this vaccine demonstrated poor immunogenicity in mice when administered with alum adjuvant [47]. Another developed ORF2 truncated region (HEV239, aa 368-606 of the ORF2 product), showed more immunogenicity than the aa 394-607 ORF2 truncated product [47,48] .the latter was approved in China in 2012 as the only HEV vaccine available worldwide under the new trade name Hecolin. In this study multiple epitopes were predicted within the Hecolin, the truncated protein (HEV239, aa 368-606 of the ORF2 product). For instance the epitopes ⁴⁵²PTPSPAPS⁴⁵⁹ and ⁵⁵⁶GYPYNY⁵⁶¹ that elicited the B cell and ²⁹⁹LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹, ³⁶⁷GIALTLFNL³⁷⁵, 368IALTLFNLA³⁷⁶, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁸⁹SSAGGQLFY³⁹⁷ and 394QLFYSRPVV⁴⁰² that elicited the T cell were located within these regions. This result indicated that our predicted epitopes may have the potentiality to act with high immunogenicity.

Moreover, one study evaluated the antibody cross-reactivity against capsid proteins of various HEV genotypes, the putative neutralizing region (aa 452-617) of ORF2 products of various HEV genotypes was employed in an indirect ELISA to measure patient sera reactivity toward ORF2 aa 452-617 from various HEV genotypes [49]. They showed that patient sera demonstrated variable immune reactivity against the antigens tested [49]. Finally, they concluded that aa 483-533 region demonstrated the highest antigenic potential. Compared to our proposed epitopes two B cell epitopes were found within the region of ORF2 aa 452-617. Namely ⁴⁵²PTPSPAPS⁴⁵⁹ and ⁵⁵⁶GYPYNY⁵⁶¹ epitopes and to some extend the epitope 615DYPA⁶¹⁹. This reslt strengthen the potentiality of these epitopes to elicit the humoral immunity. As Behloul et al [49] concluded that aa 483-533 region demonstrated the highest antigenic potential, only one epitopes from this study ⁵¹⁸KVTLDGRPL⁵²⁶ was found located within 483-533 region and it was not proposed as a vaccine candidate due to its low population coverage compared to the other proposed epitopes (Table 3 and Table 5).

Strikingly, the majority of our proposed vaccine epitopes were located closed to each other spanning the region 205-402 of the capsid protein (660 aa). For instance the positions of these epitopes were as follows; ¹⁶⁵PLQD¹⁶⁸, ²⁰⁵YAISISFWP²¹³, ²¹⁹PTSVD²²³, 299LLDFALELE³⁰⁷, ³⁴¹LTTTAATRF³⁴⁹, ³⁶⁷GIALTLFNL³⁷⁵, ³⁶⁸IALTLFNLA³⁷⁶, ³⁷⁹LLGGLPTEL³⁸⁷, ³⁸⁹SSAGGQLFY³⁹⁷ and 394QLFYSRPVV⁴⁰². The mature HEV capsid protein requires proteolytic processing and the ﬁnal product, lacking the ﬁrst 111 amino acid (aa) and the last 52 aa of full-length ORF2 product, forms virus-like particles [44,50,51]. Crystal structure determinations suggest that HEV capsid protein has three domains designated Shell domain (S aa 129-319), Middle domain (M, aa 320-455), and Protruding domain (P, aa 456–606) [52]. Accordingly we can speculated that S and M domains are more immunogenic since they contain the majority of the immunogenic epitopes and the other proposed epitopes located within the protrusion domain (⁴⁵²PTPSPAPS⁴⁵⁹, ⁵⁵⁶GYPYNY⁵⁶¹ and ⁶¹⁵DYPA⁶¹⁹).

For the vaccine to be beneficial it should cover large world population. Here in this study the proposed epitopes were subjected for population coverage against the whole world population. In this essence the proposed epitopes that demonstrated higher affinity to interact with MHC-I and MHC-II alleles and that bound to different sets of alleles were selected for population coverage analysis (Table 6). The proposed epitopes demonstrated favorable epitope sets of 78.97% and 99.99% of MHC-1 and MHC-II, respectively, in the whole world population. The overall MHC-1 and MHC-II whole world population coverage epitope set for all suggested epitopes was 100%. The result indicated that the proposed epitopes as a vaccine candidates could cover large population of the world and effectively interacted with the human common alleles worldwide. This result further strengthen the proposed epitopes to work as a vaccine candidates against HEV.

In the history of vaccine development such as live-attenuated vaccine of microbial pathogens (viruses, bacteria, etc.) have been used for stimulation of immune responses that protect the host against infections. therefore vaccination has proven to be essential in prevention of various deadly infectious diseases. However this type of vaccine might contain unnecessary proteins particles for the stimulation of protective immunity and may lead to allergenic responses. This has led to shift towards a new era of vaccine development via reverse vaccinology. The peptide vaccines containing only epitopes capable of inducing positive, eligible T cell and B cell mediated immune responses were developed for multiple viruses such as vaccine for hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papilloma virus (HPV), also there are many vaccines under processing. [53,54,55].

5. Conclusion

As the viral infections are wide spread over the world, it is difficult to treated zoonotic virus diseases that threatened human life. Thus there is a need to create vaccines more accurate, specific and have low side effect. To our knowledge this was the first study proposed multi epitopes vaccine for Hepatitis E Virus among the different peptides tested that elicited both B cell and T cell and it is expected to be more targeted to stimulate the immune system and be immunogenic and less allergic than conventional biochemical vaccines.

Acknowledgments

Authors would like to thank the staff members of College of Veterinary Medicine, University of Bahri, Sudan for their cooperation and support.

Competing Interest

The authors declare that they have no competing interests.

Fund

No fund was received.

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