Immuno-Efficacy of Multi-Epitope Chimeric Peptides Against Foot and Mouth Disease Virus: Potential Vaccine Candidates for Newly Emerged Serotype O and A

Artificially designed, chimeric peptide-based recombinant vaccines are novel approaches to combat the phylogenetically diverse Foot and Mouth Disease (FMD) Virus (FMDV) strains. Among seven distinct serotypes, only serotype O and A are dominantly circulating in Bangladesh and neighbouring countries of Asia, where transboundary transmission, recurrent outbreaks and emergence of novel lineages FMDV are highly prevalent. The objective of this study was to develop multi-epitope recombinant peptides, procuring immunogenicity against circulating diverse subtypes of FMDV serotype O and A. Two chimeric peptides, named B1 (41.0 kDa) and B3 (39.3 kDa), have been designed to incorporate potential B-cell and T-cell epitopes selected from multiple FMDV strains, including previously reported and newly emerged sub-lineages. After expression, characterization and immunization of guineapigs with considerable antigen load of B1 and B3 followed by the serological assays revealed the significant protective immunogenicity, developed from the higher (100 μg) doses of both antigens, against most of the currently prevalent serotype O and A strains of FMDV. The efficient expression, antigenic stability, and multivalent immunogenic potency of the chimeric peptides strongly indicate their credibility as novel vaccine candidates for FMDV serotypes O and A circulating in Bangladesh and surrounding territories. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 September 2021 doi:10.20944/preprints202109.0463.v1


Introduction
Foot-and-mouth disease (FMD) is a highly contagious disease of transboundary importance that affects more than 70 species of wild and domesticated cloven-hoofed animals globally [1]. It causes a devastating economic loss in food security, job loss and trade restrictions to the livestock industries in the endemic countries worldwide [2]. Furthermore, FMD is a matter of global animal health concern due to its transboundary nature and recent invasive outbreaks in the FMD-free countries like the United Kingdom in 2001 [3,4], Japan in 2010 [5][6][7], China 2010 [8] and South Korea 2010 [9]. subtypes like lineages, sublineages and topotypes [10]. Serotypes O, A, and Asia1 have had a worldwide dominant distribution along with continuous evolution and phylogenic differentiation [11]. Specifically, serotype O viruses are responsible for the majority of the outbreaks globally, followed by serotype A. Thus these two serotypes warrant special efforts to reduce the global burden of FMD [12]. Also, these two serotypes encounter a higher rate of mutations resulting in the evolutionary diversification of taxonomic groups.
The capsid of FMDV is composed of four structural proteins (VP1, VP2, VP3 and VP4). VP1 comprises the crucial epitopes for viral entry and subsequent lysis of infected host cells [13].
Notably, the G-H loop (aa positions 134-160) and the B-C loop (aa position: 40-60) of VP1 are critical for eliciting neutralizing antibodies against FMDV in the infected hosts [14]. The highly conserved RGD (Arg-Gly-Asp) motif within the VP1 G-H loop plays a significant role in viral entry into host cells and protective immunity in the host [15][16][17]. Due to rapid mutations in the surface regions of FMDV, especially within the immunogenically critical loops of VP1, an antigenic mismatch occurs between vaccine and field strains within the same serotypes, even some times, within the same lineages and sub-lineages that ultimately result in vaccine failure [18,19].
Recently, Al Amin et al. reported the development of trivalent inactivated FMD vaccine composed of newly emerged global strains of each serotype [20][21][22][23]. Several limitations of inactivated whole virus vaccine pose potential threats towards its acceptability. The possibility of virus escape during production, inactivation and subsequent storage and transport conditions imposes potential hazards for inactivated virus vaccine manufacturers [24]. Removing non-structural peptides (NSPs) from purified antigen preparations is crucial for pre-and post-vaccination differentiation of infected and vaccinated animals. Reportedly, the cross-protection deficiencies among FMDV serotypes, intraserotype diversity and antigenic mismatch can contribute to emergence of vaccine-escape field strains [18,25], which necessitates the frequent reconditioning of vaccine strains to defend the most recently emerged strains of FMDV in susceptible areas. Emergence of phylogenetically distinct subgroups and their country-wide transmission with poor-quality surveillance give rise to frequent vaccine failure in Bangladesh [26]. The emergence of two novel sub-lineages Ind2001BD1 and Ind2001BD2 of serotype O, and a novel lineage BD-18 (G-IX) [22,27] strongly indicates the necessity of FMD vaccine development with potential immunogenicity against both of the recently emerged and previously reported endemic strains of dominant serotype O and A. Interestingly, serotype O strains of FMDV which are genotypically very closely related to the Ind2001BD1 sublineage, have been reported from China, revealing the transboundary prevalence of the newly emerged strains [28]. A report of a recent FMD outbreak in vaccinated cattle in Eastern Saudia Arabia showed the prevalence of serotype O strains of 98% similarity with the novel sub-lineage [29], indicating lack of sero-protection from the vaccine strains differing by 11% sequence dissimilarities. In serotype A, the most antigenically divergent type, vaccine mismatch found between the vaccine strain and the field viruses from the same sub-lineage C (a subgroup of VP3 Δ59 variant) under the lineage A/ASIA/G-VII in Bangladesh [18]. Moreover, previously in India, vaccine mismatch due to antigenic drift in serotype A field viruses from the existing Indian vaccine strain IND 40/2000 of the same serotype was reported [30]. Thus, to combat the repetitive incidence of FMDV by diverse strains of serotype O and serotype A, it is necessary to develop a single platform comprising the immunogenic capacity against most of the circulating strains throughout this territory [31].
FAO and OIE jointly proposed a 5-stage Progressive Control Pathway for Foot-and-mouth disease (PCP-FMD) for assisting and facilitating FMD endemic countries or regions like Bangladesh. The Government of Bangladesh (GoB) aims to reduce the impact of FMD progressively and towards eradicating the disease through strategic implementation of mass vaccination by inactivated whole virus. Recent advancements in FMD vaccine development strategy in the south Asian territories includes recombinant subunit vaccine based on VP1 sequence of vaccine strains isolated more than a decade ago [31]. Incorporation of protective antigen sequence in various types of vectors have been performed using pandemic strains which are evolutionarily distant from the FMDV strains circulating since last ten years. The lack of vaccine strain amelioration along with underestimation of their antigenic heterogeneity has led to vaccine failure and transboundary transmission of FMDV [26].
For these reasons, many research groups worldwide are investigating scopes of developing alternative effective vaccine approaches for FMD [32]. Epitope-based vaccines are emerging as promising candidates for future vaccines due to the absence of any risks of virus replication, the capability to function as DIVA vaccines, devoid of any residual peptides from whole viruses, and the combination of multiple epitopes on a single polypeptide platform [33]. As previously mentioned, VP1 possesses multiple conformational B-cell epitopes located in the G-H loop, B-C loop and C-terminal (aa positions: 190-210 approximately), it is highly crucial for the selection of antigenic fragments to design multiple epitope-based vaccine candidates [34,35]. These epitopes are located in receptor binding sites of VP1 and can elicit humoral responses in infected animals.
Several artificial vaccines based on epitopes from the G-H loop of VP1 of FMDV have shown neutralizing efficiency in guinea pigs, swine, etc. [36][37][38]. However, none of these endeavors incorporated the epitopes from the novel lineages and sub-lineages that emerged in Bangladesh and spread over many other countries.
Thus, in this study, we have designed two complete artificial chimeric peptides, named as B1 and B3, composed of the surface epitope fragments, GH loop and C terminal of VP1 capsid peptides, of circulating FMDV serotype O (B1) and A (B3) strains, representing the different clusters of their phylogroups. The recombinant peptides possess conformational B-cell epitopes, like GH loop and C-terminal, and two crucial T-cell epitopes that are assumed to enhance T-cell dependent immunity and cytotoxic T-cell proliferation. Also, two universal T cell epitopes, PADRE (Panhuman leukocyte Antigen DR-binding Epitope) and Invasin, have been included to further enhance T cell activity and memory cell development for long term immunity [36,39,40]. The chimeric peptides have been efficiently expressed in a prokaryotic system with significant yield and conformational stability. Guinea pig models have been immunized with the vaccines formulated with the chimeric peptides B1 and B3. Serological assays exhibited broad neutralizing efficacy of the vaccines of B1 and B3 against the viruses, including the newly emerged strains in the recent years, of serotypes O and A, respectively, in Bangladesh.  selected as conformational B-cell epitopes to construct two multi-epitope recombinant peptides, namely B1 and B3. As illustrated in tables 1 and 2, six G-H loop fragments and three C-terminal fragments were oriented along with two T-cell epitopes, one from the N-terminal of VP1 and another from 3A peptides of FMDV strains. In both B1 and B3 constructs, two universal T-cell epitopes, 12 amino acid long PADRE (Pan-human leukocyte Antigen DR-binding Epitope) and 16 amino acids long Invasin (from Yersinia), were included at 5' and 3' of both chimeras. The GEDG spacer sequence differentiated each epitope of B1 for proper spatial orientation of all immunogenic fragments. In B3, four spacer sequences were GEDC, and the rest eight spacers were GEDG. Subsequently, the DNA sequence encoding both B1 and B3 were obtained by reverse genetics tools. Codon optimization is very crucial for the heterologous expression of eukaryotic pathogenic peptides through the bacterial system. B1 and B3 encoding DNA were optimized for codon usage using the OptimumGene algorithm to ensure the influences of the factors like codon usage bias, mRNA secondary structure, ribosomal binding sites, repeat sequences and restriction sites etc. Optimization of these parameters ensures a significant level of heterologous expression of both B1 and B3.

Prediction and analysis secondary and tertiary structures of B1 and B3
The Mfold Web-based software (mfold.rna.albany.edu) was used to analyze the structures of folding messengers RNAs of the chimeric genes [42], whereas the RNAfold (rna.tbi.univie.ac.at) and Genesee (www.genebee.msu.su/services/ rna2_reduced.html)online server revealed the energetic stability of the predicted structures. Results were confirmed by CentroidFold Web Server, which indicated the mRNA was stable enough for effectual translation in the prokaryotic host [43].
GOR-IV (gor.bb.iastate.edu/) [44] and Jpred 4 (compbio.dundee.ac.UK/jpred/) [45] online servers were used for prediction of secondary structure of B1and B3 peptide. In order to analyze the sequence and predict the peptides structures and functions, the percentage of random coils, alpha-helices and beta-sheets, the solvent accessibility were evaluated by the PredictPeptides server [46].
3D structures of B1 and B3 were predicted based on amino acid sequences using the PONDEROSA-C/S, a server-based software package for automated peptides 3D structure determination. Results were examined by using Ponderosa Analyzer. The relevant results were viewed with PyMOL molecular graphics systems [47,48]. 3D structures were evaluated using Ramachandran Plot Analysis [49]. Various physical and chemical parameters of B1 and B3 were computed (Supplementary table-4) using ExPASy -ProtParam tool [50].

Transformation and expression of B1 and B3
The codon-optimized DNA constructs of B1 and B3 were then commercially synthesized from GenScript (www.genscript.com) with two restriction sites EcoR1 and Hindlll, respectively, at 5'

Extraction, Purification and Quantification of chimeric B1 and B3
The next day, the harvested cells were subjected to one freeze-thaw cycle followed by sonication

Characterization of Expressed B1 and B3 Peptides
The molecular weights of purified B1 and B3 were determined by 12.5% SDS-PAGE using the

Immunization of Guinea Pigs
Forty-nine guinea pigs were divided into two groups, nine in the control group and forty in the experimental group. Forty GPs were further divided into eight groups (A-H), five in each, whereas nine GPs of the control group were divided into three (X, Y and Z) groups, three in each (Supplementary Table- antigens in each group. All the animals were sacrificed on day-28, and blood was collected aseptically by direct cardiac puncture. Serum samples were prepared from the blood samples of each animal by centrifugation and were stored until further use.

Detection of anti-B1 and anti-B3 antibody in GP serum
The presence of antibodies in the polyclonal GP serum was determined by western blotting the B1 and B3 antigens. The WB analysis of B1 was performed using both anti-B1 sera and serum of control group-X. Similarly, B3 was blotted by anti-B3 serum and control group-Y serum. Goat anti-Guinea Pig IgG antibody (Invitrogen, USA) was used as secondary antibody and SuperSignal WestPico PLUS Chemiluminescent Substrate and ChemiDoc Imaging System were used for image development.

Virus Neutralization Tests
To (TCID50) of FMDV in 50% of the wells. All control serum was also tested for determining the SN50 titer similarly. To detect the protective titer for FMDV strains, the SN50 titer above 1.2 has been considered effective, which was shown in previous reports recognizing the anti-FMDV immune response of guinea pigs as a model for evaluation of vaccine potency in cattle [52,53].

Statistical Analysis
Statistical analyses of all the raw data of the experiment were performed in R software version 4.0.5 [54] and the graphs were built in ggplot2, an open-source R package [55]. We used the oneway ANOVA test to compare the antibody titers raised among the different vaccination doses.
When the overall difference across the overall groups was significant, we computed Tukey HSD (Tukey Honest Significant Differences, R function: TukeyHSD()) for performing multiple pairwise-comparison between the means of groups. The function TukeyHSD() takes the fitted ANOVA as an argument. Analyses were carried out at a 95% confidence level and p-values less than 0.05 were considered as statically significant.  (Fig. 1A). For T-cell epitopes, two epitopes were included, one of which is in the Nterminal region (aa 25-36 for type-O and aa 29-37 for type-A) of VP1 (Figure 2A), and the other one is in the 3A protein region of FMDV (Table 1 and 2). The GEDG spacer between each epitope enhanced the acidic and basic amino acid ratio in the final B1 construct, stabilizing the solubility and cytosolic conformation.  Invasin TAKSKKFPSYTATYQF Universal T-cell epitopes [36] optimization also nullified the unfavorable peaks to prolong the mRNA half-life and disrupt stemloop structures to enhance the ribosomal binding and stability. were in highly preferrd observaton as green crosses, 12.60 % resdues were in preferrd observaton as brown crosses and rest 6.82 % were in questionable observation as red crosses.

Prediction of Secondary and Tertiary Structures of the Recombinant Peptides
The 'Mfold' server was used to evaluate the minimum free energy for chimeric mRNA of codonoptimized sequences. The results showed that the minimum free energy of the best predicted structure for the optimized B1and B3 constructs were -448.80 kcal/mol and -466.30 kcal/mol. The first nucleotides at 5' did not have a long stable hairpin or pseudoknot. Therefore, the binding of ribosomes to the translation initiation site and the following translation process can be readily accomplished in the target host. These outcomes were in agreement with data obtained from the 'RNAfold' web server. Several online programs were used to predict the secondary structures of the chimeric peptides, and GOR-IV achieved the best result. Results indicated that total residues of B1 were made up of 77 strands (19.74%), 96 helices (24.62%) and 217 random coils (55.64%); whereas B3 contains 161 helices (42.04%), 47 extended strands (12.27%) and 175 random coils (45.69%).

Prediction of 3D Structure and Physicochemical Parameters of the Recombinant Peptides
B1 and B3 were composed of 390 and 383 amino acid residues encoded by 1170 and 1149 nucleotides sequences, respectively. The presence of the RGD motif on the surface of both recombinant peptides was evident when the 3D structures were analyzed using PyMOL. The presence of cysteine residues enhanced tertiary structure stability by forming disulfide bridges ( Figure-2B and 2E). 3D Model of recombinant peptides was analyzed by Ramachandran Plot ( Figure 2C and 2F), which showed more than 80% amino acid residues of both B1 and B3 reside

Expression, Purification and Characterization of the chimeric peptides B1 and B3
The SDS-PAGE analysis of total cell extract of transformed and induced E. coli BL-21(DE3) cells revealed that both B1 and B3 had been expressed in the insoluble fraction of IPTG-induced cells.
After purification and desalting, the molecular weight of both the chimera B1 and B3, confirmed by SDS-PAGE, was determined at 41.0 kDa and 39.0 kDa, respectively ( Figure 3A). The 6X-Histidine tag on the C-terminal of the recombinant peptides was detected by Western blot ( Figure   3B) using an anti-His mouse monoclonal antibody (Thermo, USA).

Multi-epitope Chimeric Proteins: New Savior for FMD Vaccination Strategy
The development of subunit vaccine emerged as the most rational and cost-effective method for FMD vaccine [32]. The approach of multi-epitope based chimeric peptides is considered as the best strategy for preventing FMD for their capability of incorporating multiple epitope fragments in a single platform. A heterologous prokaryotic system for recombinant peptides expression has become prevalent and standardized due to the simple cultivation method, low maintenance cost and easy extraction protocols [56]. While using the prokaryotic system for eukaryotic peptides expression, one of the significant constraints is codon optimization and the solubility of the expressed peptides [57][58][59]. Also, the proper three-dimensional configurations of epitope fragments must permit the maximum exposure for effective antigen presentation and processing [60]. The artificial recombinant peptides must mimic the spatial arrangement of surface antigens to develop immune responses specific to target pathogens [61]. These factors have been emphasized in the novel design and orientations of both B1 and B3, respective vaccine candidates for FMDV type O and A circulating in Bangladesh.

Rational Design, Orientation and Efficient Expression of Artificial Chimera B1 and B3
Computational analysis of B1 and B3 constructs revealed that insertion of hydrophobic GG spacer sequence results formation of two recombinant peptides with much higher isoelectric points, 9.92 process. The shortages of disulfide links between the GH loops and slightly higher isoelectric points have caused partial intracellular degradation of B3, which resulted in a lower yield of B3 than that of B1. Lower yield and cytoplasmic instability due to high pI has also been observed in the expression of another B1-like peptides (B2), with only change in the spacer region (GG).
Notably, the isoelectric point of B2 was detected at 9.92 in silico. This peptide was also expressed but, after purification and SDS-PAGE detection, the peptides was found degraded into smaller fragments indicating cytoplasmic instability, proteolytic degradation and extracellular deterioration in the purification steps.
Antigenic Stability and Protective Immunogenicity developed by B1 and B3 The characterization and identification of purified B1 and B3 peptides through SDS-PAGE and Western Blot analysis revealed the validation of computationally predicted molecular weights and antigenic conformation. The arrangement of multiple epitopes in a single polypeptide must be designed to maintain the antigenicity similar to the infecting pathogen [62][63][64]. To enhance the cell-mediated immune responses, significant T-cell epitopes have been incorporated in both B1 and B3 constructs. These constructs are the very first design containing both universal and FMDV-originated T-cell epitopes in the single platforms along with B-cell epitopes. Although, the cytokine assays for quantitative detection of anti-viral immune responseindicators, like IFN-γ, IL-2, IL-4 could not be accomplished during this study due to poor availability of immunoassay kits for guinea pig cytokine assays during the COVID-19 pandemic.
Several reports showed that the inclusion of CpG oligonucleotide enhances the immunogenicity of recombinant peptides-based vaccine candidates [65]. These modifications can be explored to enhance the efficacy of the B1 and B3 antigens in future studies.

Conclusion
To control and restrict the transboundary transmission and prevalence of FMD, caused by type-O strains closely related to Ind2001BD1 sub-lineage in Bangladesh and neighboring territories, the application of multiple epitopes based recombinant vaccines will enhance immune diversity targeting most prevalent genotypes. This study presents the novel designs for FMDV vaccine candidates comprised of protective antigens from the most recent sub-lineage, highly capable of  Figure-2: Map of pET 21a(+) expression vector with selectable marker.  Suppl. Figure-3: Insertion sequences and amino acid sequences of B1 and B3.