Rabies is zoonotic disease distributed globally and caused by 11 viral species belonging to single-stranded RNA virus from the Lyssavirus genus related to the family Rhabdoviridae [1,2]. The genus Lyssavirus includes classical rabies virus (Rabies virus (RABV), Lagos bat virus (LBV), Duvenhage virus (DUVV), Mokola virus (MOKV) and Shimoni bat virus (SHIBV) were isolated in Africa, while LBV, MOKV, DUVV and SHIBV exist exclusively in Africa [3]. Complex phylogeny of Lagos bat virus was demonstrated in recent studies [4]. The original isolate of LBV in Nigeria since 1956 was genetically distant from other LBV isolates found up to date. The two viruses originating from Senegal (1985) and found in France (was introduced via Togo or Egypt; 1999) makes another phylogenetic lineage and they were found similar to each other, where a third lineage is formed by isolates from Ethiopia, Zimbabwe, the Central African Republic and South Africa from 1974 to 2006 and the genetic distances between these lineages are greater than those found with other lyssavirus species [4]. This virus is able to infect all mammals and the disease causes thousands of human deaths every year mainly in Asia and Africa where the virus circulates endemically in domestic dogs (Canis lupus familiaris) [2]. The majority of these deaths (99%) are caused by bites from rabid dogs, causing continuous fear and threat to the suspected communities because the disease is invariably fatal but also preventable if managed earlier after infection, the role of domestic dogs as main vectors of rabies is recognized with little data about the dog-associated RABV variant than wild life variants such as skunk and raccoon RABVs circulating in North America [5]. These viruses are responsible for a meningo encephalomyelitis in mammals,transmission of the viruses to a healthy mammal occurs mainly through bite or scratch by an infected mammal (the saliva is the infectious material), bats are considered as the natural hosts of 10 of these viral species, however dogs are the main source of infection in humans with an estimate of 55,000 deaths per annum worldwide among which about 56% occurs in Asia and 44% in Africa [6]. The importance of geographical factors are well documented but are often neglected when measuring the economic load of the disease [7,8]. Since 2005 more data has become available and the global disease situation changed markedly with good control efforts in some parts of the world that leads to rabiesfree countries but increased number of cases in other parts of the world was recorded [9-12]. Although there is no effective treatment after declaration of the disease, there is an effective management against RABV and related lyssaviruses when applied as soon as possible after exposure, it consists of local treatment of the wound, administration of rabies immunoglobulin (if indicated) and vaccinations against rabies that is able to prevent the onset of symptom and death [13]. Moreover, even with well-studied and documented epidemic expansions of wildlife RABV, there is little to know about the spread of rabies in endemic areas [14]. The replication potentials of various lyssaviruses looks different in a given host and the genome organization is highly conserved [15,16]. While the envelope components matrix protein M and glycoprotein G are required for virus release and virus infectivity, respectively [17]. Based on receptor binding and the apoptosis-inducing properties of the sole RABV surface antigen, Glycoprotein G has been designated as the major pathogenicity determinant of genotype 1 lyssaviruses [18,19]. However, many examples of glycoprotein G-independent mechanisms of virus-cell interaction exist, among which those involved in escape from antiviral innate immunity and to be important for in vivo virus replication [20-25]. The nucleotide substitution rate of lyssaviruses is estimated to be around 10−4 per site per year [26]. The RNA-dependent RNA-polymerase together with phosphoprotein (P), functions as the transcriptase and replicase complex, the glycoprotein G is the only outer membrane protein responsible for virus entry and inducing protective immune responses [27]. The role of vaccines is to generate an immune response in order to protect the vaccinated individual upon future exposures to the disease [28]. There is considerable differences in Individuals immune systems that in some cases individual’s immune system will not respond adequately to protect against second exposure and this is one of the reasons explaining the failure of immunization [28]. Vaccine production that depends on biochemical experiments are time consuming, expensive and not always work, furthermore this type of vaccines might constitutes a few hundred of unnecessary proteins for the induction of immunity causing many reactogenic or allergic responses [29,30]. Therefore, Insilco prediction of epitopes of appropriate protein residues would help in production of peptide based vaccines with powerful immunogenic and minimal allergenic effect [29]. Our aim is to design a vaccine for Lagos Rabies virus using peptides of its glycoprotein G as an immunogen to stimulate protective immune response.

Protein sequence retrieval

A total of 26 strains of rabies Lagos virus strains’ glycoprotein were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/protein). Database in November 2016. These 26 strains sequences retrieved are from different areas around the world (include 10 collected from South Africa, 6 from Nigeria, 3 from Senegal, 3 from Kenya, one from Zimbabwe, one from central Africa public and two samples were collected from collaborative laboratories).

Retrieved glycoprotein sequences with their accession number, date of collection and areas of collection were listed in Table 1.

Table 1: Virus strains retrieved, their Accession numbers, date of collection and area of collection.

Conserved regions determination

The retrieved sequences were aligned to obtain conserved regions using multiple sequence alignment (MSA), sequences aligned using Clustal-W as Applied in the Bio-Edit program, version 7.0.9.1 (Hall, 1999) to find out the conserved regions in all 26 retrieved Rabies glycoprotein G sequences [31]. Then the candidate epitopes were analyzed by different prediction tools from Immune Epitope Database IEDB analysis resource (http://www.iedb.org/) [32].

B-cell epitope prediction

The reference sequence of glycoprotein was subjected to different B cell tests [33].

Prediction of linear B-cell epitopes: Bepipred test from immune epitope database (http://tools.iedb.org/bcell/result/) [34], was used as linear B-cell epitopes prediction from the conserved region with a default threshold value of 0.148.

Prediction of surface accessibility: By using Emini surface accessibility prediction tool of the immune epitope database (IEDB) (http://tools.iedb.org/bcell/result/) [35]. The surface epitopes were predicted from the conserved region with the default threshold value 1.0.

Prediction of epitopes antigenicity: Using Kolaskar and Tongaonkar antigenicity method to determine the antigenic sites with a default threshold value of 1.042, (http://tools.iedb.org/bcell/result/) [36].

MHC class I binding predictions

Analysis of peptide binding to MHC1 molecules was assessed by the IEDB MHC I prediction tool at http://tools.iedb.org/mhc1. The attachment of cleaved peptides to MHC molecules was predicted using artificial neural network (ANN) method [37,38] . Prior to prediction, all epitope lengths were set as 9 mers, all conserved epitopes that bind to MHC1 alleles at score equal or less than 500 half-maximal inhibitory concentrations (IC₅₀) were selected for further analysis [37].

MHC class II binding predictions

Analysis of peptide binding to MHC II molecules was assessed by the IEDB MHC II prediction tool at (http://tools.iedb.org/mhcii/ result/ [37]. For MHC II binding predication, human allele references set were used. MHC II groove has the ability to bind different lengths peptides that makes prediction more difficult and less accurate [38]. We used artificial neural networks to identify both the binding affinity and MHC II binding core epitopes. All conserved epitopes that bind to many alleles at score equal or less than 1000 half-maximal inhibitory concentration (IC₅₀) were selected for further analysis [39].

Population coverage calculation

All proposed MHC I and MHC II epitopes from Lagos rabies virus glycoprotein G was assessed for population coverage to the whole world population with the selected MHC I and MHC II binding alleles using IEDB population coverage calculation tool at http://tools.iedb.org/ tools/population/iedb_input [40].

Homology modeling

The reference sequence of Lagos rabies glycoprotein to Raptor X on 21/12/2016, the 3D structure of glycoprotein was received on 22/12/2016 and then treated with chimera software to show the position of proposed peptides [41-45].

B-cell epitope prediction

The reference sequence of Lagos rabies virus glycoprotein (GP) was subjected to Bepipred linear epitope prediction, Emini surface accessibility and Kolaskar and Tongaonkar antigenicity methods in IEDB, to determine the epitope linearity, being in the surface and to test the immunogenicity respectively Tables 2,3 and Figures 1-4.

Figure 1: Bepipred Linear Epitope Prediction; Yellow areas above threshold (red line) are proposed to be a part of B cell epitopes and the green areas are not.

Figure 2: Emini surface accessibility prediction; Yellow areas above threshold (red line) are proposed to be a part of B cell epitopes and the green areas are not.

Figure 3: Kolaskar and Tongaonkar antigenicity prediction; Yellow areas above threshold (red line) are proposed to be a part of B cell epitope while green areas are not.

Figure 4: Structural position of the most promising conserved B cell epitopes of glycoprotein G of Lagos rabies virus.

Prediction of Cytotoxic T-lymphocyte epitopes and interaction with MHC

The reference sequence of glycoprotein G of Lagos rabies virus was analyzed using IEDB MHC-1 binding prediction tool to predict T cell epitopes suggested to interact with different types of MHC I alleles based on ANN- align with (IC₅₀) ≤ 500, the list of conserved epitopes and their corresponding MHC-1 alleles are shown in Table 4 and Figure 5.

Table 2: List of conserved peptides with their surface accessibility score and antigenicity score.

Figure 5: Structural Position of the two most promising conserved epitopes at glycoprotein G of Lagos rabies virus that interact with MHC I alleles.

Table 3: List of conserved peptide that passes Bepipred, Emini surface and Koalaskar tests with their length and scores

Table 4: List of conserved epitopes and their corresponding MHC-1 alleles along with their position in glycoprotein, IC₅₀ and percentile rank.

Prediction of T helper cell epitopes and interaction with MHC II alleles

The reference sequence of glycoprotein G of Lagos rabies virus was analyzed using IEDB MHC II binding prediction tool based on NNalign with half-maximal inhibitory concentration (IC₅₀) ≤ 1000; the list of all epitopes and their correspondent binding MHC II alleles were shown in (supplementary Table 1) while the list most promising three epitopes that had Binding affinity with MHC II alleles along with their positions in the glycoprotein G of Lagos rabies virus were shown in Table 5 and Figure 6.

Table 5: List of most promising three epitopes that had Binding affinity with MHC II alleles along with their positions in the glycoprotein G of Lagos rabies virus, peptide sequence, binding alleles, IC₅₀ and rank.

Figure 6: Molecular position of the most promising three epitopes of Lagos rabies virus that binds MHC II alleles.

Population coverage analysis

Population coverage test was performed to detect the world coverage of all epitopes binds to MHC1 alleles, MHC11 alleles and combined MHC1 and MHC11 as well as selected most promising epitopes for each test.

Population coverage for MHC1: Population coverage results for all epitopes binding to MHC1 alleles of Lagos rabies virus was shown in supplementary Table (II) while the population coverage results of the most promising three peptides was shown on Table 6.

Table 6: Population coverage of most promising 3 epitopes binds to MHC1 alleles and MHC11 alleles of Lagos rabies virus along with their world population coverage.

Population coverage for MHC11: The population coverage results of all peptides binding to MHC11 alleles with their coverage and total HLA hits were shown in supplementary Table (III) and the results of most promising three epitopes were shown on Table 6.

Population coverage for both MHC1 and MHC11 alleles: This test was performed to the most promising epitope alone (FVGYVTTTF) and it was found to bind 13 alleles with population coverage of 97.30% Figure 7.

Figure 7: Molecular position of the most promising epitope for population coverage that binds both MHC1 and MHC11 alleles.

In the present study we proposed for the first time different selected peptides that could be recognized by B cell and T cell to produce antibodies against Lagos rabies virus. The resulting peptide vaccine is expected to be more antigenic and less allergic than the conventional biochemical vaccine.

The reference sequence of Lagos rabies virus glycoprotein G was subjected to Bepipred linear epitope prediction test, Emini surface accessibility test and Kolaskar and Tongaonkar antigenicity test in IEDB, to determine the binding to B cell, being in the surface and to test the immunogenicity respectively.

For Bepipred test there was 22 conserved epitopes that have the binding affinity to B cell while there are 11 epitopes predicted on the surface according to Emini surface accessibility test and 10 epitopes being antigenic as detected by Tongaonkar antigenicity test while only three epitopes (VPGYGKAYT, FYPS and QKV) found to overlap all performed B Cell tests.

Also the reference sequence of Lagos rabies virus glycoprotein G was analyzed using IEDB MHC1 binding prediction tool to predict T cell epitopes interacting with different types of MHC I alleles. Based on Artificial neural network (ANN) with half-maximal inhibitory concentration (IC₅₀) ≤ 500; 23 conserved peptides were predicted to interact with different MHC-1 alleles. The peptide FVGYVTTTF from 93 to 101 had higher affinity to interact with 9 alleles (HLA-A*03:01, HLA-A*03:01, HLA-A*03:01, HLA-A*03:01, HLA-A*03:01, HLA-A*11:01, HLA-A*11:01, HLA-A*11:01, HLA-A*23:01), followed by VTYTNFVGY from 88 to 96 that binds 7 alleles (HLA-B*27:05, HLA-B*15:02, HLA-B*35:01, HLA-B*40:01, HLA-B*44:03, HLA-B*53:01, HLA-B*58:01). These two peptides could be thought about as a possible peptide vaccine for Lagos rabies virus.

The reference glycoprotein (GP) strain was analyzed using IEDB MHC II binding prediction tool based on NN-align with half-maximal inhibitory concentration (IC₅₀) ≤ 1000; there were 39 conserved predicted epitopes found to interact with MHC-II alleles. The peptides FVGYVTTTF, FKRKHFKPT and FRKLVPGYG had the affinity to bind the highest number of MHC11 alleles.

World population coverage results for total epitopes binding to MHC1 alleles was 88,42% and the most promising peptides was FVGYVTTTF, VTYTNFVGY and VTYTNFVGY with world population coverage 56.71% and total HLA hits 3,7 and 7 respectively.

The world population coverage results for all epitopes that have binding affinity to MHC11 alleles was 99.97% while world population coverage of the most promising three epitopes FKRKHFKPT, FRKLVPGYG and FVGYVTTTF was 99.68 % with HLA hits 7,7 and 13 respectively.

The peptide FVGYVTTTF exhibits exceptional population coverage results for both MHC1 and MHC11 alleles of 97.3% with total HLA hits 13 different alleles.

Conclusion

We conclude that to our knowledge this was the first study to propose peptide vaccine for Lagos rabies virus which is expected to be more immunogenic and less allergic than traditional biochemical vaccines, among the different peptides tested for both B cell and T cell this study proposed an interesting T cell epitope (FVGYVTTTF) that have very strong binding affinity to both MHC1 and MHC11 alleles and it was found to bind 13 different alleles with world population coverage 97.3%, which indicates strong potential to formulate peptide vaccine for Lagos Rabies virus.

Recommendations

We recommend further studies to propose a peptide vaccines for the other strains of rabies virus especially Rabies lyssavirus, there will be possibility to find common conserved promising epitopes for multiple strains. Also we recommend in vitro and in vivo evaluation of the most promising peptides.