Abstract

Rift Valley fever virus (RVFV) is a single strand, negative sense, an envelope spherical particle, of size 80 - 120 nm, segmented RNA virus that belongs to Genus: Phlebovirus of Bunyaviridae family. The clinical manifestations of the disease among animals are abortion and death of newborns. While in humans, although the disease is mild or asymptomatic, there are several reports of high fatality rates. The M segment of RVF virus Genome which encodes the envelope glycoprotein has been used to design a vaccine for immunization against this virus. we aimed to design a novel peptide-based vaccine for RVFV using immunoinformatic approach to predict highly conserved epitopes against glycoprotein receptor Gn and Gc of M protein, that can mediate immune response which can use later to produce a new vaccine that could replace the conventional vaccine. A total of 118 sequences of M protein of RVFV were retrieved from NCBI database and stored as FASTA format for immunoinformatics analysis. ClustalW multiple alignment using BioEdit sequence alignment editor (v7.0.9) was performed to the retrieved sequences to identify the conserved region compared to M protein RVFV reference sequence under gene bank accession number [YP_003848705.1]. The B and T cell epitopes prediction is done by immune epitope database (IEDB). (IEDB) predicted B cell epitopes by Bepipred linear epitope prediction analysis and T cell epitopes using Major Histocompatibility Complex class I and ll binding prediction tool based on Stabilized Matrix Method (SMM). Allergenicity for the Helper T cell epitopes (HTL) predicted using AllerTop software. TAP transporter and Proteasomal cleavage for Cytotoxic T cell (CTL) were predicted from (IEDB). The population coverage over the world was determined. The four best predicted CTL namely (⁸³⁶HTYLQSVRK⁸⁴⁴, ⁶⁷²IPRYSTYLM⁶⁸⁰, ¹⁰⁸⁵ILHFTVPEV¹⁰⁹³ and ⁸³⁴FVHTYLQSV⁸⁴²) were docked with HLA-B*35 and suggested to be universal peptide vaccine for immunization against RVFV. The typical overlapping between the MHC Class I epitope (⁸³⁴FVHTYLQSV⁸⁴²) and MHC Class II (⁸³⁴FVHTYLQSV⁸⁴²) suggest the possibility to presenting these antigens to immune system via both MHC class I and II pathways. In conclusion; the four CTL epitopes are selected as vaccine candidates to develop safer and easier to manufacture without need of culture vaccine for prophylactic method against this virus. We recommend to confirm our result by doing additional in vivo and in vitro complementary steps to support this novel predicted vaccine.

1. Introduction

Rift Valley fever virus (RVFV) is a single strand, negative sense, an envelope spherical particle, of size 80 - 120 nm, segmented RNA virus that belongs to Genus: Phlebovirus of Bunyaviridae family ¹. It contains three Genomic segments; termed as “L” the larger, “M” medium and “S “small segments. The L encode the RNA-dependent RNA polymerase (RdRp), M segment encodes enveloped glycoprotein (Gn,Gc) and non-structural protein (NSm), and the S which encode nucleocapsid protein "N" and non- structural protein (NSs) ^{2, 3, 4}. The M segment encodes the envelope glycoprotein has been used to design a vaccine for immunization against this virus. The virus is a zoonotic virus primary infect animal (ruminant) such as: cattle, buffalo, sheep, goats and camels and cause disease lead to high mortality and abortion rate in in infected livestock which get infection when bite by infected mosquito, subspecies (Aedes, culex, Anopheles) result in economic loss ^{3, 5, 6}. Transmission in humans occurs either from direct transmission from mosquito, or by exposure to infected animal blood, body fluid or tissue, also transmission can be by aerosol during slaughter or maintenance of the infected animal, or by veterinary and obstetric procedures, but there is no human to human transmission has been reported ^{3, 6, 7}. This virus has the ability to infect and cause illness in humans. The incubation period take from 2 to 6 days followed by serial of many different diseases, in usual cases the infection is asymptomatic or mild illness that characterized by self-limiting febrile illness, with some exception of about 1-2% of the reported cases develop severe complication, such as hepatitis, encephalitis, retinitis which may lead to blindness, and hemorrhagic fever occurs in less than 1% of cases characterized by profound Coagulopathy, disseminated Intravascular coagulation and multi-organ dysfunction, lead to about 50% mortality rate for those develop hemorrhagic fever, The fatality ratios are 10-20% among hospitalized patients ^{2, 7, 8}.

RFV virus was disseminated and infected thousands human and animals in many various parts of Africa, as well as Arabian Peninsula with the risk of spreading more to Asian, European and American Region ³. It first identified in Kenya in the 1930s, and be restricted to the eastern region of Africa until occurred in South Africa in 1951 result in abortion of 500,000 fetus and death of 100,000 animal ,then virus spread to west Africa in 1987s. Between the periods of 1977-1979 it was caused epidemic in Egypt result in massive animal death and 200,000 human infections at died of at least 594 from hospitalized cases were predicted. The infection with a virus was documented for the first time outside Africa in the Saudi Arabia and Yemen. In 2000; about 2,000 human infected and at least 245 deaths. In 2007; RVFV outbreaks have been reported in several eastern African countries, in additional to those African countries; it Affects islands in the Indian Ocean and in Sudan ^{7, 9}.

Animal vaccination can prevent death and abortion in livestock and eliminate human infection by controlling one of the main source of human infection. Animal vaccines are available but there are no any commercial available human peptide vaccines for RVFV ^{5, 6}. Formalin-inactivated vaccine for both human and animal are used in endemic country but have drawback of requiring more than vaccine for immunity, use of pathogenic strain, and high cost in manufacturing. The live attenuated vaccine is under development vaccine for both human and veterinary but less effective because the effective vaccine may induce long-Term protective immunity and these vaccines are not induced these long-term Immunities. Another recombinant protein vaccination for ruminant are under development for future controlling of livestock ^{6, 10}.

Because that reason for no availability of human peptide vaccine; our study aimed to design universal peptide vaccine for RVFV from the envelope glycoproteins using computational method to predict epitope so including T helper cells mediated immune response, which can be used latter to produce new vaccine that could replace the conventional vaccine depending on in Silico approaches and information in databases. We consider this study discriminative because there are no any human peptide-based vaccines for RVFV strain.

2. Material and Method

A total of 118 sequences of M protein of RVFV were retrieved from the national center for biotechnology information (NCBI) (https://www.ncbi.nlm.nih.gov/) ¹⁰ for immunoinformatics analysis.

Clustal W multiple alignment was performed to the retrieved sequences through BioEdit sequence alignment editor (v7.0.9) ¹¹, to identify the conserved region compared to M protein RVFV reference sequence under gene bank accession number [YP_003848705.1].

B and T cells’ epitopes were predicted using The Immune Epitope Data Base (IEDB) (https://www.iedb.org/) ¹².

Immune epitope data base analysis resources (https://tools.iedb.org/bcell/) ¹³ was used to predict B cell epitopes by Bepipred linear epitope prediction analysis after submitted the reference sequences.

Cytotoxic T cell epitopes were predicted using Major Histocompatibility Complex class I (MHC class I) binding prediction tool (https://tools.iedb.org/mhci/) ¹⁴ based on Stabilized Matrix Method (SMM) ^{15, 16}. Only epitopes that found in conserved region and have IC50 less than 50 was selected and saved for further analysis.

Helper T cell epitopes were predicted using MHC class II prediction tool (https://tools.iedb.org/mhcii/) ¹⁷ based on SMM ¹⁸. Only 100% conserved epitope with IC50 <50 were selected and saved for further analysis.

MHC class I (HLA-A and HLA-B) and MHC class II (DR, DP and DQ) alleles were used.

Tap score and proteasomal score was determined for each selected epitopes by using proteasomal cleavage/TAP transport /MHC class I combined predictor (https://tools.iedb.org/processing/) ¹⁹.

Allergic properties of epitopes were determined using AllerTop v.2 (https://www.ddg-pharmfac.net/AllerTOP/) ²⁰. Only non-allergic epitopes were saved.

Three dimension (3D) structure was visualized using UCSF-Chimera visualization tool 1.8 ²¹ after getting protein data base bank (pdb) of reference sequences by using Raptor-X software (https://raptorx.uchicago.edu/StructurePrediction/predict/) ²².

Human allele/receptor (HLA-B*35:1) was chosen according to the study done by Gras S. and his colleagues in 2010 whose reported that, the binding of influenza epitope NP (418-426) to B7 family (HLA-B*3501/03/0702) ²³ and other one by Weiskopf D. et al. (2013) whose found the B*3501 has high response frequency and magnitude ²⁴. RVFV epitopes have been selected according to high binding affinity with more than one allele and TAP binding. Their homology modeling in 3D structure has been done with PEP STR MOD-structure prediction of peptide containing natural non-natural and modified residue (https://osddlinux.osdd.net/raghava/pepstrmod/nat_ss.php) ²⁵. While the HLA-B*35:1 3D structure (PDB:3KLR) that bind with NP(418-426) epitope of influenza virus ²³ was retrieved from RCSB protein data bank for docking analysis (https://www.rcsb.org/pdb/home/home.do) ²⁶ and then detecting the influenza ligand using UCSF-chimera ²¹ before docking applied.

A molecular docking technique was applied using two softwares MTiAutoDock 1.0 ²⁷ and patchDock Beta 1.3 version (https://bioinfo3d.cs.tau.ac.il/PatchDock/) ²⁸ to model the interaction of the 3D-modeled epitopes with human allele. Then visualization has done by the UCSF-chimera visualization tool 1.8 ²¹ and PYMOL Molecular Graphic system, version 1.8 schrodinger LLC (https://www.pymol.org) ²⁹.

The fraction of individuals from the whole world predicted to respond to a given set of epitopes with known MHC restriction was calculated using IEDB population coverage calculation tool (https://tools.iedb.org/tools/population/iedb_input) ³⁰.

3. Result

The percentage of the peptides conservancy in this study was 100%.

M protein analyzed using IEDB MHC-1 Binding prediction tool to predict Cytotoxic T cell epitope interacted with different types of selected MHC class I alleles in man. Based on consensus SMM method with IC50≤ 50; 24 conserved peptides were predicted to interact with different selected human MHC1 alleles. The 9-mer epitope ⁸³⁶HTYLQSVRK⁸⁴⁴ shows high affinity to interact with (HLA-A*30:01, HLA-A*68:01 and HLA-A*03:01) with IC50 = 35.64, 35.64 and 39.98 respectively. Epitope ⁶⁷²IPRYSTYLM⁶⁸⁰ shows high affinity to interact with (HLA-B*07:02 and HLA-B*35:01) with IC50 of 14.15 and 48.37 respectively, epitope ¹⁰⁸⁵ILHFTVPEV¹⁰⁹³ shows high affinity to interact with HLA-A*02:03 and HLA-A*02:01 with IC50 of 10.4 and 29.93 respectively, and the epitope ⁸³⁴FVHTYLQSV⁸⁴² shows higher affinity to interact with (HLA-A*02:06 and HLA-A*02:03) alleles with IC50 = 9.52 and 9.53. Results are listed in Table 1 and Figure 1 displays the epitopes at the structural level.

There were 14 predicted conserved HTL epitopes found to interact with Human MHC-II alleles by high affinity illustrated in Table 2. The 9-mer peptide (core) ⁸³⁴FVHTYLQSV⁸⁴² showed high affinity to interact with (HLA- DPA1*01:03/DPB1*02:01) with IC50=48 are selected and modeling as a show in Figure 2. These epitopes that bind with different set of MHC Class II alleles by binding affinity ≤ 50 were subjected to AllerTOP 2.0 software to avoid production of IgE antibodies as possible. ³³⁹AHEVVPFAV³⁴⁷, ⁸³³LFVHTYLQS⁸⁴¹ and ⁶⁷⁵YSTYLMLLL⁶⁸³ were found to be allergic while ⁸³⁴FVHTYLQSV⁸⁴² was found to be non-allergic.

There were an overlapping of three epitopes from MHC class 1 with that of MHC class 2, ³⁴¹EVVPFAVFK³⁴⁹ overlaps with ³³⁹AHEVVPFAV³⁴⁷ from MHC class 2, 672IPRYSTYLM680 overlaps with ⁶⁷⁵YSTYLMLLL⁶⁸³ from MHC class 2 while ⁸³⁴FVHTYLQSV⁸⁴² overlaps with two epitopes from MHC class 2 (⁸³³LFVHTYLQS⁸⁴¹ and ⁸³⁴FVHTYLQSV⁸⁴²). These overlapping are illustrated in Table 3.

The four predicted CTL peptides that interacted with selected human’s MHC-1 alleles: ⁸³⁶HTYLQSVRK⁸⁴⁴, ⁶⁷²IPRYSTYLM⁶⁸⁰, ¹⁰⁸⁵ILHFTVPEV¹⁰⁹³ and ⁸³⁴FVHTYLQSV⁸⁴² were used as "ligands" to detect their interaction with selected HLA (B*35:01) as "receptor" by docking Techniques using offline and online soft wares. Based on polar contacts and the binding energy in kcal/mol unit, the lowest binding energy (kcal/mol) was selected to obtain best binding (pose) and to predict real CTL epitopes as possible, Table 4 and Figure 3.

Population coverage analysis to MHC Class I and II which conducted among different 16 geographical areas illustrated in Table 5. The population coverage for MHCI show high percentage in Africa which coverage 68.78% of east African population, 64.97% for central Africa and 81.13%, 80.24%, 73.44% in west, north and south of Africa respectively, while MHCII show low coverage in those area, less than 10%. On the other hand the other part of the world such as Europe, northern America and east Asia show high coverage of MHCI and MHCII, except in central America which show low coverage for both Class I & II.

4. Discussion

Peptide vaccine is alternative approach for immunization instead of traditional approach (such as live attenuated and inactivated vaccine) to guarantees safer and easier to produce vaccine without difficulties in manufacture ³¹. In our study, we aimed to design universal peptide based vaccine against RVF virus, particularly against glycoprotein receptor" Gn and Gc" using immunoinformatic approach. One type of T-cell called Cytotoxic T (CD8+) play a crucial role in fighting viral infection ³¹. Before the peptide recognize by Cytotoxic T cell, peptide undergo number of processes involved in the MHC-I presentation pathway start from cleavage of the large MHC- 1 presented peptide by proteasomes then binding of these peptides with transporter associated with antigen presenting (TAP) and transported to Endoplasmic reticulum, within the Endoplasmic reticulum the peptide with correct size, and proper amino acid sequence bind to MHC-I forming receptor- peptide complex transferred to the surface and recognized by Cytotoxic T lymphocyte ³². We used stabilized matrix base method (SMM) to predict peptide binding with MHC class I, transporter associated with antigen presenting (TAP) and proteasomal cleavage based on the study done by Peters B. and Sette A. in 2005 ¹⁵. Half-maximum inhibitory concentration (IC50) score which measure the binding affinity of a peptide with MHC-1 is used alternatively to percentile Rank based on the study done by Paul et al. in 2013 whose reported the use of IC50 score as the threshold for peptide selection ³³. For TAP transporter we interpreted our result as lower IC50 corresponds to higher TAP binding based on the study done by reported that the amino acid well suited for TAP binding corresponds to low IC50 ³⁴. We used IEDB Recommended method first because the IEDB software didn’t give us Result by SMM.

method, then we chose SMM method after uploading the result by delete all other methods and remain SMM method, we selected four conserved epitope which has high binding affinity with (MHC I and TAP transporter) and interacted with more than allele: (⁸³⁶HTYLQSVRK⁸⁴⁴) interacted with three alleles (HLA-A*30:03, HLA- A*68:01, HLA-A*03:01) and the peptides (⁶⁷²IPRYSTYLM⁶⁸⁰), (¹⁰⁸⁵ILHFTVPEV¹⁰⁹³), (⁸³⁴FVHTYLQSV⁸⁴²), which interacted with alleles (HLA-B*07:02, HLA-B*35:01), (HLA-A*02:03, HLA-A*02:01) and (HLA- A*02:06, HLA-A*02:03) respectively. To suggest the presence of real interaction between CTL epitopes and the selected allele; the four selected peptides were docked with their allele receptor (HLA-B*3501), we used this allele based on two studies, the first study done by Weiskopf D in 2013 reported that B*3501 has high response frequency and magnitude ²⁴, while the second study done in 2010 by Gras S reported the binding of influenza epitope NP (418-426) to B7 family (HLA-B*3501/03/0702) ²³. Because we did not find protein sequence and PDB ID of HLA-B*3501 in NCBI database/structure we obtain PDB ID from crystal structure of HLA-B*3501 that bind to influenza epitope NP (418-426) ²³. The docking result showed that the epitope (⁸³⁶HTYLQSVRK⁸⁴⁴) has high binding affinity with the binding energy -8.72 kcal/mol and binding energy (-8.7, -8.71, -8.47) kcal/mol with the epitopes (⁶⁷²IPRYSTYLM⁶⁸⁰), (¹⁰⁸⁵ILHFTVPEV¹⁰⁹³), (⁸³⁴FVHTYLQSV⁸⁴²) respectively. Another pathway of immune response against virus is through binding of epitope with MHC Class II to recognize by CD4 T-helper and produce antibody, help in Cytotoxic T lymphocyte activity to fight infected cell, as well as induce innate immunity by activating phagocytic cell ³⁵. We found that the epitope (⁸³⁴FVHTYLQSV⁸⁴²) had high affinity to bind with (HLA-DPA1*01:03/DPB1*02:01) allele. Several overlaps observed between MHC class-I and II in T-cell epitope, and that indicate the possibility of antigen presentation to immune system via MHC class I and II ³⁶. Particularly the epitope (⁸³⁴FVHTYLQSV⁸⁴²) which showed typical overlapping between MHC class I and II and predicted as probable non-allergic when compare with other epitopes (³³⁹AHEVVPFAV³⁴⁷, ⁶⁷⁵YSTYLMLL⁶⁸³, ⁸³³LFVHTYLQS⁸⁴¹) that had predicted as allergic by allergenicity prediction test. Although the IEDB predicted highly effective T cell epitopes But it shows some limitation because it failed to predict B cell epitopes.

Based on the fact that HLA gene is extremely polymorphic and over thousand different human alleles are known, and those alleles are different through many geographic areas around the world ³⁷. We conducted population coverage analysis to MHC Class I and II among different 16 geographical areas. The population coverage for MHC I show high percentage in endemic area which coverage 68.78% of east African population, 64.97% for central Africa and 81.13%, 80.24%, 73.44% in west, north and south of Africa respectively, in addition to high coverage in other areas of the world except in central America which show low coverage, about 2.18%. MHCII show low coverage than MHCI, the highest coverage show in areas at risk to developing future outbreak such as Europe coverage 15.19%, then northern America is 13.55% and 10.21% in east Asia, the other parts of the world show less than 10% population coverage.

In Summary: the best predicted epitopes to be Novel human peptide vaccine against M protein of RVFV are: CTL epitopes: ⁸³⁶HTYLQSVRK⁸⁴⁴, ⁶⁷²IPRYSTYLM⁶⁸⁰, ¹⁰⁸⁵ILHFTVPEV¹⁰⁹³ and especially ⁸³⁴FVHTYLQSV⁸⁴² which is also HTL epitope.

5. Conclusion

Our result based on the predictive and analytic tool (IEDB-AR) that usage for prediction of CTL and THL epitopes from protein sequence of glycoprotein coding M protein of RVFV. Then the CTL epitopes are selected as vaccine candidates to develop safer and easier to manufacture without need of culture vaccine for prophylactic method against this virus. We can support our novel universal predicted human vaccine by using further additional in vivo and In vitro complementary steps to confirm our findings.

Acknowledgments

Authors would like to thank Dr. Hisham NourEldayem Altaib Faculty of medical laboratories sciences, Sudan University of science and Technology for his advice.

Competing Interests

The authors declare that they have no competing interests.

References