Immunoinformatics approach to Rift Valley fever virus vaccine design in ruminants

Immunoinformatics approach to Rift Valley fever virus vaccine design in ruminants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunoinformatics approach to Rift Valley fever virus vaccine design in ruminants Elijah Kolawole Oladipo, Oluseyi Rotimi Taiwo, Fashanu Omotoyosi Teniola, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3475857/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Rift Valley fever is a new arboviral illness with serious public health implications. The disease, which is brought on by the Rift Valley fever virus (RVFV), is linked to severe economic losses in animals as well as a number of human ailments. There are currently no authorized live attenuated vaccines for human use, and they have been linked to increased risks of abortion in ruminants. In this study, the use of reverse vaccination was applied in creating a vaccine using a segment for use in ruminants by exploring proteins in the M-glycoprotein segment. Glycoprotein sequences were retrieved from cattle, sheep and goats from endemic countries across Africa and subjected to computational analysis for B and T cell epitope prediction. Highly conserved, immunodominant epitopes were obtained and connected with the ideal linkers for proper conformation. A Bos taurus-specific beta-defensin was also added to increase immunogenicity. The secondary as well as tertiary structures of the vaccine were then determined by SOPMA and alphafold2, respectively. Analysis of the vaccine’s physicochemical properties all yielded favorable results regarding its potential effectiveness and safety. Furthermore, molecular docking analysis was carried out using Toll-like receptors 7 and 8 to show molecular binding interactions. While in-silico analyses have yielded satisfactory results, translational research may be required to validate the vaccines’ effectiveness in animals. Arboviral diseases Molecular docking Reverse Vaccinology Rift Valley fever virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Rift Valley Fever Virus (RVFV) is a vector-borne zoonosis, a negative-stranded and tri-segmented RNA virus which belongs to the genus Phlebovirus and family Bunyaviridae, having negative socio-economic impacts as well as affecting human and animal health 1,2 . The RVFV has been reported to be endemic to Arabian and Peninsula Africa 3,4 although it was earlier identified in Kenya’s Rift Valley in early 1930 5 , thereby giving it its name. Kenya was reported to have experienced the deadliest epi-zoonotic RVFV before the major outbreak in South Africa between 1950 and 1951, which claimed about 500,000 abortions and 100,000 deaths of sheep, while subsequent epizootics were observed in Zambia, Namibia, Mozambique, the Sudan, Zimbabwe and other East African countries 6,7 . Its outbreaks have been associated with periods of greater than average rainfall, although it cannot easily be predicted, thereby suggesting the interplay between the environment and human and animal health 1,2 . RVFV can cause severe disease in newly born ruminants such as sheep, goats, camels and cattle with up to 100% fatality rate with mosquitoes being the main vector of infection although Sandflies have shown the capability of transmitting the virus 4 . However, adults seem less susceptible. In ruminants, the virus causes Rift Valley Fever (RVF), an acute hemorrhagic fever, accompanied by abortion storms in pregnant females 7 . Abortion rates may be up to 100% in pregnant animals 6 . These dire statistics indicate the need for concerted efforts at developing preventive measures against the RVFV's future resurgence. The RVFV genome is tri-segmented with an L, M and S segment. The Large segment (L) encodes the RNA polymerase (RNA dependent L protein), the small segment (S) encodes the Nucleoprotein and the non-structural Nucleoprotein (NSs) while the medium segment (M) encodes four proteins (two structural Glycoproteins (Gn and Gc) and two non-structural glycoproteins (NSm and LGp) 8 . Several key vaccination attempts have been made to develop vaccines for RVFV. These include both conventional methods, such as live attenuated vaccines and inactivated vaccines, and more advanced ones, such as genetically modified live attenuated vaccines, DNA vaccines, viral vectored vaccines, viral replicons vaccines, subunit vaccines, etc. 9 . Live attenuated vaccines induce long-lasting immunity and are generally inexpensive to produce, but they are also linked to an increase in the rate of abortion in pregnant animals and can have negative effects on organs within, especially the liver when an attenuated virus spreads inside hepatic cells, as in a naturally occurring RVFV infection 9 . Other vaccinations have also faced certain difficulties, such as the inability to distinguish between infected and vaccinated animals, viremia, the requirement for booster shots, etc. Multi-epitope subunit vaccines incorporate predicted immunodominant epitope peptides from various immunogenic genes that have been identified through proteomic research 10 . Short immunogenic peptide fragments employed in multi-epitope vaccinations produce potent immune responses while considerably reducing the risk of allergic reactions in the host system. The identification of the immunogenic epitopes obtained from viral glycoprotein or nucleocapsid sequences has greatly benefited the in-silico design of peptide vaccines 11 . These epitopes are analyzed and approved using computational techniques based on their physicochemical characteristics, immunogenicity, and non-allergenicity analysis. Subunit vaccines are cost-effective, non-toxic, and have safer effects on the animals compared to other vaccines that have been developed for RVFV. In this research, we provide a computational design approach to develop an RVFV multi-epitope subunit vaccine, which incorporates several antigenic determinants that are conserved across different RVFV strains. The vaccine design is optimized for increased stability, immunogenicity, and manufacturability, using various adjuvants and carrier proteins, and evaluated in different delivery systems. Overall, this manuscript provides a comprehensive and innovative approach to the development of an RVFV multi-epitope subunit vaccine, highlighting the potential of this approach for the development of potent vaccines against other viral diseases. METHODOLOGY Retrieval of Rift Valley Fever Protein Sequences The National Center for Biotechnology Information's (NCBI) immunoinformatics investigation yielded a total of 60 RVFV amino acid sequences, which included sequences from the L, M, and S genomic segments. These sequences came from nations like Kenya, Madagascar, South Africa, Rwanda, Namibia, Egypt, Zimbabwe, and Nigeria. Antigenicity Prediction The VAXIJEN v2.0 server was used to determine the antigenicity of the Rift Valley fever proteins 11 . Without using alignment, the Vaxijen server estimates a protein's antigenicity based on its physicochemical characteristics. The default threshold value was set at 0.4. Membrane Topology of Selected Proteins The trans-membrane topology of the protein sequences was assessed for their intra-cytoplasmic, extra-cytoplasmic and transmembrane portions using the TMHMM server v2.0 12 . TMHMM predicts transmembrane topology through a hidden Markov model. Proteins with large extra-cytoplasmic regions were considered for further analyses. Multiple Sequence Alignment The Glycoprotein M sequences underwent repeated sequence alignment using the MEGA 11 programme to show conserved regions across the different sequences 13 . The MUSCLE algorithm was specifically used to align the different protein sequences 14 . Prediction of Linear B Cells The ABCpred, SVMTriP, and BepiPreD servers predicted the linear B-cell epitopes. For linear B-cell prediction, ABCpred employs a partly recurrent neural network (Jordan network) with a single hidden layer. The networks comprise 35 residues in a single hidden layer and a changeable optional window length 15 . Support Vector Machine (SVM) TRIP is used to locate linear epitopes based on amino acid features such as secondary structure, flexibility, antigenicity, hydrophilicity, and solvent accessibility in order to predict B-cells 16 . Using a hidden Markov model, BepiPred forecasts B-cell epitopes from antigen sequences 17 . Physicochemical Properties Analysis Analyzing the physicochemical characteristics of the chosen B-cells allowed for an evaluation of their suitability for vaccine development. ExPASy Protparam was used for this 18 . Additionally, utilizing VaxiJen, allerTop, and ToxinPred, these peptides were tested for their allergenicity, antigenicity, and toxicity 11, 19-20 . Prediction of Cytotoxic T Cell Lymphocyte (CTL) The glycoprotein's (M segment) CTL epitope was predicted using NetMHCII PAN 4.1 21 . The server employs artificial neural networks (ANS) to forecast peptide binding to MHC alleles. The technique is taught to examine peptide ligands that have been eluted by mass spectrometry and have a higher binding affinity (BA). The Bovine Leukocyte Antigen (BoLA) was chosen as the reference allele set in this study. The affinity of the glycoprotein peptides for the 20 BoLA alleles BoLA-JSP.1, BoLA-HD6, BoLA-T2c, BoLA-T2b, BoLA-T2a, BoLA-T7, BoLA-D18.4, BoLA-AW10, BoLA-T5, BoLA-1:00901 and BoLA-1:00902 was assessed. The chosen peptide has a length of 9 amino acids. Prediction of Helper T Cell Lymphocyte (HTL) Strongly binding MHC-II helper T cell epitopes were predicted using NetMHCII pan 4.0 21 . Based on machine learning techniques trained on binding affinity or mass spectrometry (-eluted ligands) techniques, this tool forecasts the likelihood that a peptide will present an antigen. The NETMHCII PAN 4.0 was used to predict epitopes 22 . The HTL epitopes were chosen to have a default length of fifteen amino acids, and the binding affinity of overlapping glycoprotein peptides to the following alleles was discovered; The following DRB1 codes are available: DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0104, DRB1_0105, DRB1_0106, DRB1_0107, DRB1_0108, DRB1_0109, DRB1_0110, DRB1_0111, DRB1_0112, DRB1_0113, DRB1_0115, DRB1_0116 Prediction of Interleukin- 4 and Interleukin-10 Inducing Epitopes Helper T Cells, which produce cytokines like interleukins, are crucial in the coordination of the immune response. Therefore, using IL4Pred and IL10Pred, the interleukin 4 and interleukin 10 producing capacity of a subset of strong binding epitopes was evaluated 23,24 . Antigenicity, Toxicity and Allergenicity Prediction The toxicity, antigenicity, and allergenicity of strong CTL peptides and HTL epitopes that induce IL4 and IL10 were examined. The ToxinPred server, according to Gupta et al . 19 , predicts peptide toxicity along with its physical properties, such as molecular weight, charge, hydrophobicity, and amphipathicity. The AllerTOP v2.0 server reportedly uses machine learning methods including auto and cross variance transformation, and amino acid E-descriptors, and predicts the allergenicity of proteins and peptides, according to Dimitrov et al . 20 . Doytchinova et al . 11 claim that the categorization of antigens by VaxiJen v2.0 is solely based on the physicochemical characteristics of the proteins involved. Epitope Conservation Determination B-cell, HTL and CTL were assessed by comparing with multiple sequence alignment to determine that they fall within conserved regions. For the final vaccine design, only completely conserved epitopes were considered. For the vaccine to be efficacious across a broad spectrum, highly conserved epitopes are required 25 . Furthermore, we prioritized selecting epitopes in the extra-cytoplasmic portion of the glycoprotein sequence. Primary Construct Assembly The chosen HTL, CTL, and B cell epitopes served as the building blocks for the vaccine. To increase the final construct's immunogenicity, a stiff EAAAK linker was used to introduce a Bos taurus-specific beta defense adjuvant to the N-terminus of the vaccine. AAY linkers were used to connect additional CTL epitopes. The HTL and B-cell epitopes were joined together using GPGPG linkers. According to Kavoosi et al . 26 , the glycine-rich linker GPGPG serves to increase solubility and promote free movement across adjacent domains. In order to boost the immunogenicity of the vaccine design using the EAAAK linker, a Bos taurus-specific Beta defensin adjuvant was also inserted at the N-terminus 27 . The Proposed Vaccine's Allergenicity, Solubility, and Antigenicity The allergenicity of the vaccine construct was assessed using the AllerTop v.2.0 server, while the antigenicity and solubility of the vaccine construct were assessed using VaxiJen v2.0 and Protein Sol 11,20,28 . Analysis of the Vaccine Construct's Physicochemical Characteristics Secondary and Tertiary Structure Prediction The secondary structure was predicted using the SOPMA server. The server predicts protein secondary structure from amino acid sequences using the self-optimized prediction approach 29 . The Alpha-fold server was used to predict the tertiary structure of the vaccine construct 30 . Alpha fold uses innovative neural networks in combination with the primary amino acid sequence and alignment sequence of homologs to predict the tertiary structure of a particular protein. Conformational Epitopes Prediction from 3D Vaccine Thornton's approach and a residue clustering algorithm on the IEDB database are both used by the ElliPro server to predict conformational epitopes on the 3D structure. According to Ponomarenko et al. 31 , ElliPro offers a framework for the prediction of tertiary structure models from amino sequences. Refinement and Validation of The Tertiary Structure The quality of the 3D structure of the chosen model, protein structure refinement, protein interaction prediction, GPCR applications, and water position prediction were all improved using the GalaxyWEB refine server 32 . By recreating and repackaging protein side chains using dynamic simulation techniques, this server improves 3D protein architectures. This facilitates the production of proteins with appropriate structural relaxation. After that, structural analysis using Ramachandran and Z-score analysis was used to validate the vaccine's structure 33,34 . PROCHECK and ProSA-web were used. The Ramachandra plot, which displays the proportion of the amino acid present in the preferred, permitted, and forbidden zones, is produced by the PROCHECK server. ProSA-web performs mathematical analysis on the entire protein structure quality score and presents values and energy. Codon Optimization and In-silico Cloning To adapt the vaccine for expression in the Escherichia coli strain K-12 sub-strain MG1655 chosen as the expression host, codon optimization was carried out using JCAT 35 . This is crucial for maximizing protein expression during the recombinant vaccine's cloning process. Using the SnapGene v5.1.7 program, the chimeric model was virtually cloned into an appropriate expression vector (E. coli K12 pET-28a (+)) 36 . Molecular Docking A computational procedure called Molecular Docking is used to predict the affinities of the ligand and receptor to create stable complexes. Using the HDOCK server, the final vaccine design was docked against the toll-like receptors 7 (TLR-7) and 8 (TLR8) 37 . RESULTS Antigenicity Prediction Table 1 shows the antigenicity properties of selected proteins. The glycoprotein (M) sequences were seen to be the most antigenic of all the RVFV proteins . Membrane Topology of the Selected Sequences The development of subunit multi-epitope vaccines requires an in-depth knowledge of transmembrane topology. To develop subunit multi-epitope vaccines, epitopes located in the extra-cytoplasmic segments are crucial for efficacy 38 . In this study, the TMHMM tool was used to analyze the topology of the different genome segments. The transmembrane topology of the selected proteins is presented in Table 2. Glycoprotein had a large part of their proteins in the extra-cytoplasmic portion indicating their usability in designing a vaccine against RVF V. Retrieval of Sequences A total of 60 RVFV M segment sequences from cattle, sheep and goat were obtained from the NCBI. The sequences were obtained from a total of eight countries including Kenya, Zimbabwe, South Africa, Egypt, Madagascar and Namibia. Multiple sequence alignment (MEGA) The multiple sequence alignment showed large overlapping and conserved portions (Figure 1). Prediction of Linear B- Cell Epitopes Epitopes with amino acid lengths between 10 and 20 that cut across at least two of the servers used (Bepipred, ABCpred and SvMtrip) were selected. The selected epitopes then had their physicochemical properties assessed for suitability in a vaccine construct. The result is presented in Table 3. Prediction of CTL A total of 130 strong binding CTL peptides were predicted for the selected Bovine Leukocyte Antigen (BoLA) alleles. Based on their binding score or ability, these peptides were selected. Strongly binding CTL peptides were examined for their stability index, toxicity, allergenicity, and antigenicity. Table 4 contains a list of the twenty-seven epitopes from the glycoprotein protein that passed these tests. However, not all the 27 made the final construct. Helper T- Lymphocyte Prediction A total of 97 strong peptides were found to demonstrate strong binding for the selected MHC-II alleles and were further assessed to induce interleukin 4 and interleukin 10 cytokines. After being evaluated for their physico-chemical characteristics, epitopes that showed potential to stimulate the production of the aforementioned cytokines were also tested for antigenicity, allergenicity, and noxiousness. Table 5 shows the Helper T lymphocyte epitopes that scaled through the screening processes. Primary Construct Assembly The vaccine primary construct comprised a Bos taurus specific Beta defensin adjuvant, 6 CTL peptides, 3 HTL peptides and 2 B cell-based peptides. The adjuvant was attached to the first CTL peptides using the EAAAK linker and to subsequent CTL peptides using the AAY linker. The rest of the primary construct was linked using GPGPG linkers. Our final vaccine construct was a 202 amino acid-long primary sequence (Figure 2). Prediction of Physico-Chemical Properties RVFV Vaccine The antigenicity index for a chimeric model with 0.4 points was determined using Vaxijen v2.0 server output and it was found to be quite high (0. 6926). The multi-epitope protein construct was also anticipated to be dispersible upon overexpression using Protein Sol. A grand total of 202 amino acids protein had a theoretical isoelectric point (Pl) of 9.44, and also a relative molar mass (Mr) of 21.4 kDa in accordance with the ExPasy Protparam web server. The sequence also had 26 cations (Arg + Lys) and 12 anions (Asp + Glu) residues, respectively. Assembled sequence was also shown to be sturdy with a stability index of 20.37. The multi-epitope has a DIVIDEND score of -0.121 along with an acyclic index of 81.19, which indicates that it is hydrophilic in nature (Figure 3) and thermo-stable. Codon Optimization The JCAT server, reverse translation and codon optimization (ECAI) optimizations for the adaptation of E. coli strain K-12 sub-strain MG1655 were performed 35 . The improved GC content after codon optimization was 53.3% at a Codon Adaptation Index (CAI) of 1.0. In order to successfully integrate the RVF construct into the E. coli expression system, a high GC content is a sign of stability. The restriction enzymes Xho1 and EcoR1 were utilized as the cloning site to clone the back-translated nucleotide into the E. coli expression system. An effective insertion of the segment into the pET28a (+) vector resulted in the production of a 6096 bp clone. In-silico cloning of the vaccine in an Escherichia coli pET28a (+) vector is presented in Figure 4. Prediction of Secondary and Tertiary Vaccine Structure The server SOPMA analyzed the secondary structure of the intended protein, and the findings were as follows: Alpha helix (31.55%), Random coil (44.92%), Beta turn (5.35%), and Extended strand (18.18%) while the tertiary structure is presented in Figure 5a. Tertiary Structure Refinement and Validation Results from the PROCHECK assessment of the stoichiometry quality of the structure revealed the residue and overall structural computation. Ramachandran’s plot analysis indicated that 95.7 % of protein remains were located in their preferred regions (Figure 5c). Furthermore, results from ProSA predict implied oversights of 3D model protein, predicted a negative score of -4.85, which is a favorable indicator of model quality (Figure 5b). Prediction of Discontinuous Epitopes Ellipro server predicted an aggregate of 91 conformational epitopes from 187 amino acid residues on the vaccine tertiary construct 31 . Also, the Jmol viewer allows for the uptake and foresight of immune epitopes in a given protein sequence or structure. Conformational epitopes with protrusion index scores greater than 0.7 are shown in Figure 5(a-c). Molecular Docking Results from HDock predicted 10 models docking between the vaccine construct and Toll-like receptor 7. The rankings were based on Docking Score, Confidence Score and Ligand RMSD (Å) (Figure 7 and Table 7). Immune Response Incentive The server C-ImmSim simulates immune findings indicated in a system of effective immune systems and an extended duration of the vaccine candidate. The vaccine design, as shown in Figure 8, produces a strong and consistent main and secondary immunological system. The main immune response was defined by the level of the IgM antibodies after an interval of 4-5 days of antigenic exposure. The basic immune response was distinguished by enhanced B-cell generated and also in the increase of IgM, IgG1 + IgG2 expression and IgM + IgG antibodies. The advanced vaccine construct did not only evoke a strong B-cell increase; it also led to the multitude of memory B-cells. Specific interleukin concentration, a high level of IFN-γ for a long interval, CD4 activity, T-helper lymphocyte count, and B lymphocyte count were revealed. DISCUSSION RVF is a zoonotic illness spread by mosquitoes. It poses a lethal threat to humans and livestock in many sub-Saharan countries, such as South Africa, Egypt, Madagascar, Mauritania, and Senegal. The disease has been documented in the Arabian Peninsula as well as in the Middle East, and India 39 . In animals, the disease has a high fatality rate, where it causes epizootics of abortion and fetal death and high mortality in neonatal ruminants. The virus also causes infection in humans, although with a lower mortality rate. The Smithburn live attenuated RVFV has been extensively adopted in different countries for prevention; however, like many live attenuated vaccines, there are challenges with safety due to reversal to virulence characterized by vaccine-induced teratogenesis, abortion, and fetal death. As a result, it is not recommended for usage in developing nations 40 . Furthermore, the vaccine faces a challenge due to the risk of genetic reassortment 41 . Therefore, there is a need for novel approaches to designing safe and effective vaccines against this disease. Advances in Whole Genome Sequencing (WGS) and proteomics have increased our understanding of viral proteins, which are being used in vaccine design via a reverse vaccinology approach. The use of peptide vaccines has emerged as an outstanding alternative to conventional approaches to vaccine design 42 . Sub-unit epitope-based vaccines provide an excellent alternative when considering safety, practicability, and cost-effectiveness. Moreover, the potency and immunogenicity of these vaccines can be boosted through selected engineering of target epitopes 43 . By applying recent developments in immunoinformatics and computational technologies, scientists can selectively construct vaccines with minimal side effects and allergenicity 44 . Consequently, a number of vaccines have been designed against various diseases such as SARS-CoV-2, Ebola, hepatitis C, and cholera by applying this approach 45,46,47,48 . Our objective in this study was to design a safe, non-allergenic, and non-toxic subunit vaccine that is capable of eliciting optimal immunity against RVF in ruminants. We selected the M glycoprotein gene after immunoinformatics analysis and extensive literature on Rift Valley fever vaccines. The M segment was chosen due to its high antigenicity and the presence of large extra-cytoplasmic segments. Furthermore, experimental vaccines developed using this protein have yielded good results. In different studies, RVFV vaccines developed using the glycoprotein showed excellent immune responses and demonstrated full protection against experimental heterologous challenge infections in sheep and cattle 49,50 . The vaccine was also shown to differentiate naturally infected from vaccinated animals (DIVA) compatible 51,52 also demonstrated in an immunological study the pivotal role of the Gn and Gc segments in antibody production and the immune response against RVFV. Furthermore, the M segments basically constitute surface proteins, so they have a great potential for inducing the production of neutralizing antibodies 53,54 . The glycoprotein gene was screened for linear B-cells, CD-4 helper T lymphocytes (HTL), and CD-8 cytotoxic T-lymphocytes (HTL), which were all integrated into the eventual vaccine construct. Research by Dodd et al . 55 has shown the role B-cells and CD-4 Helper T cells play in mediating viral clearance. Specifically, CD-4 cells were shown to play major roles in maintaining robust IgG and neutralizing antibody responses as well as controlling viral replication in vivo . CD-4 activates B cells, and signals isotype class switching, affinity maturation, and antibody production through its effect on antibody-mediated immunity. They also mediate cytotoxicity by stimulating CD-8 cells 55 . The final vaccine construct was made up of 11 epitopes, comprising 6 CTL peptides, 3 HTL peptides, and 2 linear B cell peptides. All peptides had their physicochemical properties assessed to ensure that they were antigenic, non-allergenic, and non-toxic. HTL peptides were also evaluated for their ability to induce interleukin 4 (IL-4) and interleukin 10 (IL-10). These peptides were linked with AAY and GPGPG linkers. Linkers are vital in vaccine design as they ensure that protein amino acid residues are situated in a conformation similar to their natural state. A 38-amino-acid-long Bos taurus beta-defensin was further added to increase the vaccine's immunogenicity with the help of a rigid EAAAK linker. Analysis of the vaccines’ antigenicity, allergenicity, and other physico-chemical properties all showed favorable results. The vaccine was found to be antigenic, non-allergenic, and non-toxic, indicating a favorable safety potential. Further physicochemical analysis indicated the vaccine to be stable, basic, and soluble. The potency of the vaccine to induce an immune response was further evaluated by molecular docking analysis and immune simulation. Two toll-like receptors; TLR-7 and TLR-8, which have affinity for viral RNA ligands, were selected. The vaccine construct had more binding affinity to the TLR-8 with a binding score of -324 compared with the TLR-7, which had a binding score of -278. Furthermore, immune simulation with C-ImmSim showed sustained immune response for up to 350 days after injecting with 100 units. After 350 days, the IgG+IgM had dropped to a basal level of about 5000 antibody titers from a peak of about 30,000 that had been reached during the first 30 days of administration. CONCLUSION Rift Valley fever is an evolving arboviral disease, which requires concerted efforts at prevention and control. To this end, novel vaccines that are able to combine potency and safety are needed, both in animals and humans. In this study, we present an immunoinformatics approach to the development of a multi-epitope subunit RVFV vaccine for use in ruminants. The M glycoprotein gene was selected because of its high antigenicity and experimental evidence of its efficacy in vaccines. Six CTL epitopes, three HTL and 2 B-cell epitopes were joined together in a chimeric construct and subjected to analysis. In-silico analysis demonstrated the immunogenicity and safety of the vaccine construct; however, further in-vitro and in vivo analyses are required to validate the study. Declarations Funding The authors declare that they received no funds, grants, or other support during the preparation of this manuscript. Competing Interests As the authors of this manuscript, we have no relevant financial or non-financial interests to disclose. Authors’ Contributions Conceptualization, EKO; experimental design: EKO; data collection and analysis, ORT, FOT , APT, AMB, OIO, BVO, JIT; result interpretation: EKO, ORT; manuscript draft, review and editing: EKO, ORT, IOA . 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ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC Bioinformatics. 2008;9:1-8 32. Heo L, Park H, Seok C. GalaxyRefine: Protein structure refinement driven by side-chain repacking. Nucleic Acids Research. 2013;41(W1):W384-W388 33. Laskowski R, MacArthur M, Thornton J. PROCHECK: Validation of protein-structure coordinates. 2006 34. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research. 2007;35(suppl_2):W407-W410 35. Grote A, Hiller K, Scheer M, Münch R, Nörtemann B, Hempel DC, Jahn D. JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Research. 2005;33(suppl_2):W526-W531 36. Asghari A, Nourmohammadi H, Majidiani H, Shariatzadeh ,A Shams M, Ontazeri FM. In silico analysis and prediction of immunogenic epitopes for pre-erythrocytic proteins of the deadly Plasmodium falciparum. Infect. Genet. Evol. 2021;104985 101016/jmeegid2021104985 37. Yan Y, Zhang D, Zhou P, Li B, Huang SY. HDOCK: a web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy. Nucleic Acids Research. 2017;45(W1) W365-W373 38. Jardine JG, Ota T, Sok D, Pauthner M, Kulp DW, Kalyuzhniy O, Nemazee D. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science. 2015;349(6244):156-161 39. Gerken KN, LaBeaud AD, Mandi H, L’Azou Jackson M, Breugelmans JG, King CH. Paving the way for human vaccination against Rift Valley fever virus: A systematic literature review of RVFV epidemiology from 1999 to 2021. PLoS Neglected Tropical Diseases. 2022;16(1):e0009852 40. Botros B, Omar A, Elian K, Mohamed G, Soliman A, Salib A, Earhart K. Adverse response of non‐indigenous cattle of European breeds to live attenuated Smithburn Rift Valley fever vaccine. Journal of Medical Virology. 2006;78(6):787-791 41. 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Rep. 2018;8(1):16107 47. Kar T, Narsaria U, Basak S, Deb D, Castiglione F, Mueller DM, Srivastava AP. A candidate multi-epitope vaccine against SARS-CoV-2. Scientific Reports. 2020;10(1):10895 48. Oladipo EK, Akindiya OE, Oluwasanya GJ, Akanbi GM, Olufemi SE, Adediran DA, Babalola MO. Bioinformatics analysis of structural protein to approach a vaccine candidate against Vibrio cholerae infection Immunogenetics. 2022;1-16 49. Faburay B, Wilson WC, Gaudreault NN, Davis AS, Shivanna V, Bawa B, Richt JA. A recombinant Rift Valley fever virus glycoprotein subunit vaccine confers full protection against Rift Valley fever challenge in sheep. Scientific Reports. 2016;6(1):27719 50. Wilson WC, Faburay B, Trujillo JD, Ragan I, Sunwoo SY, Morozov I, Richt JA. Preliminary evaluation of a recombinant Rift Valley fever virus glycoprotein subunit vaccine providing full protection against heterologous virulent challenge in cattle. Vaccines. 2021;9(7):748 51. Faburay B, Lebedev M, McVey DS, Wilson W, Morozov I, Young A, Richt JA. A glycoprotein subunit vaccine elicits a strong Rift Valley fever virus neutralizing antibody response in sheep. Vector-borne and Zoonotic Diseases. 2014;14(10):746-756 52. Wright D, Allen ER, Clark MH, Gitonga JN, Karanja HK, Hulswit RJ, Warimwe GM. Naturally acquired Rift Valley fever virus neutralizing antibodies predominantly target the Gn glycoprotein. Iscience. 2020;23(11):101669 53. Lorenzo G, Martín-Folgar R, Hevia E, Boshra H, Brun A. Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(-/-) mice induced by different DNA vaccination regimens. Vaccine. 2010;28:2937–2944 54. Doyle JD, Barbeau DJ, Cartwright HN, McElroy AK. Immune correlates of protection following Rift Valley fever virus vaccination. NPI Vaccines. 2022;7(1):129 55. Dodd KA, McElroy AK, Jones ME, Nichol ST, Spiropoulou CF. Rift Valley fever virus clearance and protection from neurologic disease are dependent on CD4+ T cell and virus-specific antibody responses. Journal of Virology. 2013;87(11):6161-6171 Tables Table 1 Antigenicity properties of selected proteins Genome Segment Protein Antigenicity L Polymerase Non-antigen M Glycoprotein Antigen S Nucleocapsid Non-antigen Table 2 Transmembrane topology of selected protein highlighted in bold Genome Segment Protein Protein Segment Position L Polymerase 1-2091 Inside M Glycoprotein 1-16 Signal peptide 17-584 Outside 585-603 TMHelix 604-675 Inside 676-685 TMHelix 686-1159 Outside 1160-1178 TMHelix 1179-1197 Inside N Nonstructural 1-265 Inside Nucleocapsid 1-245 Inside Table 3 Selected predicted linear B-cell-based epitopes Peptide Antigenicity Allergen city DSLREEEMPE Antigenic Non- allergic VYLDKLDLKTEENLLPD Antigenic Non- allergic SYWTGSFSPKCLSSRRCHLV Antigenic Non- allergic SKLKTKMKGVCEVGVQALKK Antigenic Non- allergic Table 4 Predicted CTL epitopes. Peptides selected for the final vaccine construct are highlighted in bold Peptides Antigenicity score Instability status Toxicity status Allergenicity status SLKKGSYPL 1.3734 Stable Non- toxic Non-allergic KIKTVSSEL 1.4693 Stable Non- toxic Non-allergic GNPCMKEKL 0.0854 Stable Non- toxic Non-allergic TMAGIAMTV 1.9979 Stable Non- toxic Non-allergic NCIDWVHKL 0.6161 Stable Non- toxic Non-allergic RAPNLVSYK 0.1302 Stable Non- toxic Non-Allergic RQMTGASLK 1.7603 Stable Non- toxic Non-allergic EVVPFAVFK 0.4533 Stable Non- toxic Non-allergic IQVSGVWKK 1.5642 Stable Non- toxic Non-allergic SAHGNPCMK 0.9450 Stable Non- toxic Non-allergic RVADNINQV 0.5002 Stable Non- toxic Non-allergic VVFAEDPHL 1.4568 Stable Non- toxic Non-allergic ITSTGTGTL 0.0479 Stable Non- toxic Non-allergic LSAKPIQRV 0.1587 Stable Non- toxic Non-allergic FKNSKKVYL 1.5282 Stable Non- toxic Non-allergic LKIAPRKVL 0.6219 Stable Non- toxic Non-allergic VQADLTLMF 1.8723 Stable Non- toxic Non-allergic HKGQYKGTM 0.6591 Stable Non- toxic Non-allergic TGFKISSAV 1.6721 Stable Non- toxic Non-allergic TKDQCQILH 0.5748 Stable Non- toxic Non-allergic VQADLTLM 1.8470 Stable Non- toxic Non-allergic GDIGVHMA 0.2486 Stable Non- toxic Non-allergic TKDQCQIL 0.4858 Stable Non- toxic Non-allergic LEKGKFPL 1.6611 Stable Non- toxic Non-allergic RETMAGIA 0.3986 Stable Non- toxic Non-allergic LKIAPRKV 0.2515 Stable Non- toxic Non-allergic RQMTGASL 1.5040 Stable Non- toxic Non-allergic Table 5 Selected HTL epitopes Peptides Antigenicity status Allergenicity status Toxicity status Stability status QSAHYLNNDGKMASV Antigenic Non-allergic Non-toxic Stable FLKIKTVSSELSCRE Antigenic Non-allergic Non-toxic Stable VLKCLKIAPRKVLNP Antigenic Non-allergic Non-toxic Stable Table 6 Conformational epitopes predicted from the vaccine’s tertiary construct No. Predicted discontinuous epitope Number of residues Score 1. A:V152, A:L153, A:N154, A:P155, A:G156, A:P157, A:G158, A:P159, A:G160, A:S161, A:K162, A:L163, A:T165, A:K166, A:K168 15 0.773 2. A:K194, A:T195, A:E196, A:E197, A:N198, A:L199, A:L200, A:P201, A:D202 9 0.733 3. A:D1, A:F2, A:A3, A:S4, A:C5, A:H6, A:T7, A:N8, A:G9, A:G10, A:I11, A:C12, A:L13, A:P14, A:N15, A:R16, A:C17, A:P18, A:G19, A:H20, A:G25, A:I26, A:C27, A:F28, A:R29, A:P30, A:R31, A:V32, A:K33, A:C34 30 0.711 4. A:K180, A:G181, A:P182, A:G183, A:P184, A:G185, A:V186, A:Y187 8 0.697 5. A:P117, A:G118, A:P119, A:G120, A:F121, A:E135, A:G136, A:P137, A:G138, A:P139, A:G140, A:V141, A:L142, A:K143, A:C144, A:L145, A:K146 17 0.669 6. A:G81, A:T82, A:G83, A:T84, A:L85, A:A86, A:A87, A:Y88, A:R89, A:Q90, A:M91, A:T92, A:G93, A:A94, A:S95, A:K97, A:A98 17 0.639 7. A:K125, A:T126, A:V127, A:S128, A:S129, A:E130 6 0.633 Table 7 Molecular docking for Toll-like receptors Toll-like Receptor Docking score Confidence Score Ligand rmsd (Å) TLR-7 -278.26 0.9286 46.14 TLR-8 -324.28 0.9703 94.62 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3475857","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":246991719,"identity":"d31c9708-d80e-4d04-8fe5-56fa25aa3b9a","order_by":0,"name":"Elijah Kolawole 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solubility\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/0fbe2fdc174647b0c1f2b76f.png"},{"id":49433863,"identity":"341b5ec7-f5d7-425c-a1bb-405126e1807c","added_by":"auto","created_at":"2024-01-10 19:22:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301797,"visible":true,"origin":"","legend":"\u003cp\u003eIn-silico cloning of the vaccine in an \u003cem\u003eEscherichia coli \u003c/em\u003epET28a (+) vector (The vaccine is highlighted in red)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/180d70ddda6b0a2b42cbdcb4.png"},{"id":49433868,"identity":"eafa8e08-a11d-4fd3-a446-582befa4d99e","added_by":"auto","created_at":"2024-01-10 19:22:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":465355,"visible":true,"origin":"","legend":"\u003cp\u003ea = Refined tertiary structure, b = ProSA evaluation of refined tertiary structure, c = Ramachandran evaluation of refined tertiary structure\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/485678694bfd45d9e2f57677.png"},{"id":49434576,"identity":"db14b78d-49bb-4d4b-a882-1cd18ee595f2","added_by":"auto","created_at":"2024-01-10 19:38:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":631995,"visible":true,"origin":"","legend":"\u003cp\u003e3D image showing conformational epitopes\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/29dc71a8b57ae41e76a00156.png"},{"id":49434375,"identity":"defc3e19-d3ab-4554-b0b9-3d1349315ac7","added_by":"auto","created_at":"2024-01-10 19:30:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1091931,"visible":true,"origin":"","legend":"\u003cp\u003ea = Molecular docking of the construct and TLR-7, b = Molecular docking of the construct and TLR-8\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/4aba52ddb61918fd4aab1a54.png"},{"id":49433870,"identity":"5527de86-a7e8-4028-b997-3e717fb1b651","added_by":"auto","created_at":"2024-01-10 19:22:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":235599,"visible":true,"origin":"","legend":"\u003cp\u003eImage showing immune response incentive\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/437be102d1cd080cf21984d7.png"},{"id":49434706,"identity":"b1308ae4-a07d-412b-be4b-72cbddc633f6","added_by":"auto","created_at":"2024-01-10 19:46:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3262816,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3475857/v2/71b85ee3-39c7-43c8-8e22-43e4eb2b0374.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eImmunoinformatics approach to Rift Valley fever virus vaccine design in ruminants\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRift Valley Fever Virus (RVFV) is a vector-borne zoonosis, a negative-stranded and tri-segmented RNA virus which belongs to the genus\u003cem\u003e\u0026nbsp;Phlebovirus\u0026nbsp;\u003c/em\u003eand family \u003cem\u003eBunyaviridae,\u0026nbsp;\u003c/em\u003ehaving negative socio-economic impacts as well as affecting human and animal health\u003csup\u003e1,2\u003c/sup\u003e.\u0026nbsp;The RVFV has been reported to be endemic\u0026nbsp;to Arabian and Peninsula Africa\u003csup\u003e3,4\u003c/sup\u003e although it was earlier identified in Kenya\u0026rsquo;s Rift Valley in early 1930\u003csup\u003e5\u003c/sup\u003e, thereby giving it its name.\u0026nbsp;Kenya was reported to have experienced the deadliest epi-zoonotic RVFV before the major outbreak in South Africa between 1950 and 1951, which claimed about\u0026nbsp;500,000 abortions and 100,000 deaths of sheep, while subsequent epizootics were observed in Zambia, Namibia, Mozambique, the Sudan, Zimbabwe and other East African countries\u003csup\u003e6,7\u003c/sup\u003e. \u0026nbsp;Its\u0026nbsp;outbreaks have been associated with periods of greater than average rainfall, although it cannot easily be predicted, thereby suggesting the interplay between the environment and human and animal health\u003csup\u003e1,2\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRVFV\u0026nbsp;can cause severe disease in newly born ruminants such as sheep, goats, camels and cattle with up to 100% fatality rate\u0026nbsp;with mosquitoes being the main vector of infection although Sandflies have shown the capability of transmitting the virus\u003csup\u003e4\u003c/sup\u003e. However, adults seem less susceptible. In ruminants, the virus causes Rift Valley Fever (RVF), an acute hemorrhagic fever, accompanied by abortion storms in pregnant females\u003csup\u003e7\u003c/sup\u003e. Abortion rates may be up to 100% in pregnant animals\u003csup\u003e6\u003c/sup\u003e. \u0026nbsp;These dire statistics indicate the need for concerted efforts at developing preventive measures against the RVFV\u0026apos;s future resurgence.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe RVFV genome is tri-segmented with an L, M and S segment. The Large segment (L) encodes the RNA polymerase (RNA dependent L protein), the small segment (S) encodes the Nucleoprotein and the non-structural Nucleoprotein (NSs) while the medium segment (M) encodes four proteins (two structural Glycoproteins (Gn and Gc) and two non-structural glycoproteins (NSm and LGp)\u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral key vaccination attempts have been made to develop vaccines for RVFV. These include both conventional methods, such as live attenuated vaccines and inactivated vaccines, and more advanced ones, such as genetically modified live attenuated vaccines, DNA vaccines, viral vectored vaccines, viral replicons vaccines, subunit vaccines, etc.\u003csup\u003e9\u003c/sup\u003e. Live attenuated vaccines induce long-lasting immunity and are generally inexpensive to produce, but they are also linked to an increase in the rate of abortion in pregnant animals and can have negative effects on organs within, especially the liver when an attenuated virus spreads inside hepatic cells, as in a naturally occurring RVFV infection\u003csup\u003e9\u003c/sup\u003e. \u0026nbsp;Other vaccinations have also faced certain difficulties, such as the inability to distinguish between infected and vaccinated animals, viremia, the requirement for booster shots, etc.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMulti-epitope subunit vaccines incorporate predicted immunodominant epitope peptides from various immunogenic genes that have been identified through proteomic research\u003csup\u003e10\u003c/sup\u003e. Short immunogenic peptide fragments employed in multi-epitope vaccinations produce potent immune responses while considerably reducing the risk of allergic reactions in the host system. The identification of the immunogenic epitopes obtained from viral glycoprotein or nucleocapsid sequences has greatly benefited the in-silico design of peptide vaccines\u003csup\u003e11\u003c/sup\u003e. These epitopes are analyzed and approved using computational techniques based on their physicochemical characteristics, immunogenicity, and non-allergenicity analysis. Subunit vaccines are cost-effective, non-toxic, and have safer effects on the animals compared to other vaccines that have been developed for RVFV.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this research, we provide a computational design approach to develop an RVFV multi-epitope subunit vaccine, which incorporates several antigenic determinants that are conserved across different RVFV strains. The vaccine design is optimized for increased stability, immunogenicity, and manufacturability, using various adjuvants and carrier proteins, and evaluated in different delivery systems.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, this manuscript provides a comprehensive and innovative approach to the development of an RVFV multi-epitope subunit vaccine, highlighting the potential of this approach for the development of potent vaccines against other viral diseases.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cp\u003e\u003cstrong\u003eRetrieval of Rift Valley Fever Protein Sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Center for Biotechnology Information\u0026apos;s (NCBI) immunoinformatics investigation yielded a total of 60 RVFV amino acid sequences, which included sequences from the L, M, and S genomic segments. These sequences came from nations like Kenya, Madagascar, South Africa, Rwanda, Namibia, Egypt, Zimbabwe, and Nigeria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigenicity Prediction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe VAXIJEN v2.0 server was used to determine the antigenicity of the Rift Valley fever proteins\u003csup\u003e11\u003c/sup\u003e. Without using alignment, the Vaxijen server estimates a protein\u0026apos;s antigenicity based on its physicochemical characteristics. The default threshold value was set at 0.4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMembrane Topology of Selected Proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trans-membrane topology of the protein sequences was assessed for their intra-cytoplasmic, extra-cytoplasmic and transmembrane portions using the TMHMM server v2.0\u003csup\u003e12\u003c/sup\u003e. TMHMM predicts transmembrane topology through a hidden Markov model. Proteins with large extra-cytoplasmic regions were considered for further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiple Sequence Alignment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Glycoprotein M sequences underwent repeated sequence alignment using the MEGA 11\u0026nbsp;programme\u0026nbsp;to show conserved regions across the different sequences\u003csup\u003e13\u003c/sup\u003e. The MUSCLE algorithm was specifically used to align the different protein sequences\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Linear B Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ABCpred, SVMTriP, and BepiPreD servers predicted the linear B-cell epitopes. \u0026nbsp;For linear B-cell prediction, ABCpred employs a partly recurrent neural network (Jordan network) with a single hidden layer. The networks comprise 35 residues in a single hidden layer and a changeable optional window length\u003csup\u003e15\u003c/sup\u003e. Support Vector Machine (SVM) TRIP is used to locate linear epitopes based on amino acid features such as secondary structure, flexibility, antigenicity, hydrophilicity, and solvent accessibility in order to predict B-cells\u003csup\u003e16\u003c/sup\u003e. Using a hidden Markov model, BepiPred forecasts B-cell epitopes from antigen sequences\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical Properties Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalyzing the physicochemical characteristics of the chosen B-cells allowed for an evaluation of their suitability for vaccine development. ExPASy Protparam was used for this\u003csup\u003e18\u003c/sup\u003e. Additionally, utilizing VaxiJen, allerTop, and ToxinPred, these peptides were tested for their allergenicity, antigenicity, and toxicity\u003csup\u003e11, 19-20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Cytotoxic T Cell Lymphocyte (CTL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe glycoprotein\u0026apos;s (M segment) CTL epitope was predicted using NetMHCII PAN 4.1\u003csup\u003e21\u003c/sup\u003e. The server employs artificial neural networks (ANS) to forecast peptide binding to MHC alleles. The technique is taught to examine peptide ligands that have been eluted by mass spectrometry and have a higher binding affinity (BA). The Bovine Leukocyte Antigen (BoLA) was chosen as the reference allele set in this study. The affinity of the glycoprotein peptides for the 20 BoLA alleles BoLA-JSP.1, BoLA-HD6, BoLA-T2c, BoLA-T2b, BoLA-T2a, BoLA-T7, BoLA-D18.4, BoLA-AW10, BoLA-T5, BoLA-1:00901 and BoLA-1:00902 was assessed. The chosen peptide has a length of 9 amino acids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Helper T Cell Lymphocyte (HTL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrongly binding MHC-II helper T cell epitopes were predicted using NetMHCII pan 4.0\u003csup\u003e21\u003c/sup\u003e. Based on machine learning techniques trained on binding affinity or mass spectrometry (-eluted ligands) techniques, this tool forecasts the likelihood that a peptide will present an antigen. The NETMHCII PAN 4.0 was used to predict epitopes\u003csup\u003e22\u003c/sup\u003e. The HTL epitopes were chosen to have a default length of fifteen amino acids, and the binding affinity of overlapping glycoprotein peptides to the following alleles was discovered; The following DRB1 codes are available: DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0104, DRB1_0105, DRB1_0106, DRB1_0107, DRB1_0108, DRB1_0109, DRB1_0110, DRB1_0111, DRB1_0112, DRB1_0113, DRB1_0115, DRB1_0116\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Interleukin- 4 and Interleukin-10 Inducing Epitopes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHelper T Cells, which produce cytokines like interleukins, are crucial in the coordination of the immune response. Therefore, using IL4Pred and IL10Pred, the interleukin 4 and interleukin 10 producing capacity of a subset of strong binding epitopes was evaluated\u003csup\u003e23,24\u003c/sup\u003e. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigenicity, Toxicity\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand Allergenicity Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe toxicity, antigenicity, and allergenicity of strong CTL peptides and HTL epitopes that induce IL4 and IL10 were examined. The ToxinPred server, according to Gupta \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e19\u003c/sup\u003e, predicts peptide toxicity along with its physical properties, such as molecular weight, charge, hydrophobicity, and amphipathicity. The AllerTOP v2.0 server reportedly uses machine learning methods including auto and cross variance transformation, and amino acid E-descriptors, and predicts the allergenicity of proteins and peptides, according to Dimitrov \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e20\u003c/sup\u003e. Doytchinova \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e11\u003c/sup\u003e claim that the categorization of antigens by VaxiJen v2.0 is solely based on the physicochemical characteristics of the proteins involved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpitope Conservation Determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB-cell, HTL and CTL were assessed by comparing with multiple sequence alignment to determine that they fall within conserved regions. For the final vaccine design, only completely conserved epitopes were considered. For the vaccine to be efficacious across a broad spectrum, highly conserved epitopes are required\u003csup\u003e25\u003c/sup\u003e. Furthermore, we prioritized selecting epitopes in the extra-cytoplasmic portion of the glycoprotein sequence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Construct Assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chosen HTL, CTL, and B cell epitopes served as the building blocks for the vaccine. To increase the final construct\u0026apos;s immunogenicity, a stiff EAAAK linker was used to introduce a Bos taurus-specific beta defense adjuvant to the N-terminus of the vaccine. AAY linkers were used to connect additional CTL epitopes. The HTL and B-cell epitopes were joined together using GPGPG linkers. According to Kavoosi \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e26\u003c/sup\u003e, the glycine-rich linker GPGPG serves to increase solubility and promote free movement across adjacent domains. In order to boost the immunogenicity of the vaccine design using the EAAAK linker, a Bos taurus-specific Beta defensin adjuvant was also inserted at the N-terminus\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Proposed Vaccine\u0026apos;s Allergenicity, Solubility, and Antigenicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe allergenicity of the vaccine construct was assessed using the AllerTop v.2.0 server, while the antigenicity and solubility of the vaccine construct were assessed using VaxiJen v2.0 and Protein Sol\u003csup\u003e11,20,28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the Vaccine Construct\u0026apos;s Physicochemical Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary and Tertiary Structure Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe secondary structure was predicted using the SOPMA server. The server predicts protein secondary structure from amino acid sequences using the self-optimized prediction approach\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Alpha-fold server was used to predict the tertiary structure of the vaccine construct\u003csup\u003e30\u003c/sup\u003e. Alpha fold uses innovative neural networks in combination with the primary amino acid sequence and alignment sequence of homologs to predict the tertiary structure of a particular protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConformational Epitopes Prediction from 3D Vaccine\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThornton\u0026apos;s approach and a residue clustering algorithm on the IEDB database are both used by the ElliPro server to predict conformational epitopes on the 3D structure. According to Ponomarenko \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e, ElliPro offers a framework for the prediction of tertiary structure models from amino sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRefinement and Validation of\u0026nbsp;The Tertiary Structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quality of the 3D structure of the chosen model, protein structure refinement, protein interaction prediction, GPCR applications, and water position prediction were all improved using the GalaxyWEB refine server\u003csup\u003e32\u003c/sup\u003e. By recreating and repackaging protein side chains using dynamic simulation techniques, this server improves 3D protein architectures. This facilitates the production of proteins with appropriate structural relaxation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter that, structural analysis using Ramachandran and Z-score analysis was used to validate the vaccine\u0026apos;s structure\u003csup\u003e33,34\u003c/sup\u003e. PROCHECK and ProSA-web were used. The Ramachandra plot, which displays the proportion of the amino acid present in the preferred, permitted, and forbidden zones, is produced by the PROCHECK server. ProSA-web performs mathematical analysis on the entire protein structure quality score and presents values and energy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCodon Optimization and In-silico Cloning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo adapt the vaccine for expression in the Escherichia coli strain K-12 sub-strain MG1655 chosen as the expression host, codon optimization was carried out using JCAT\u003csup\u003e35\u003c/sup\u003e. This is crucial for maximizing protein expression during the recombinant vaccine\u0026apos;s cloning process. Using the SnapGene v5.1.7 program, the chimeric model was virtually cloned into an appropriate expression vector (E. coli K12 pET-28a (+))\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA computational procedure called Molecular Docking is used to predict the affinities of the ligand and receptor to create stable complexes. Using the HDOCK server, the final vaccine design was docked against the toll-like receptors 7 (TLR-7) and 8 (TLR8)\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eAntigenicity Prediction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 shows the antigenicity properties of selected proteins. The glycoprotein (M) sequences were seen to be the most antigenic of all the RVFV proteins\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMembrane Topology of the Selected Sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe development of subunit multi-epitope vaccines requires an in-depth knowledge of transmembrane topology. To develop subunit multi-epitope vaccines, epitopes located in the extra-cytoplasmic segments are crucial for efficacy\u003csup\u003e38\u003c/sup\u003e. In this study, the TMHMM tool was used to analyze the topology of the different genome segments. The transmembrane topology of the selected proteins is presented in Table 2. Glycoprotein had a large part of their proteins in the extra-cytoplasmic portion indicating their usability in designing a vaccine against RVF\u003cstrong\u003eV.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRetrieval of Sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 60 RVFV M segment sequences from cattle, sheep and goat were obtained from the NCBI. The sequences were obtained from a total of eight countries including Kenya, Zimbabwe, South Africa, Egypt, Madagascar and Namibia.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiple sequence alignment (MEGA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe multiple sequence alignment showed large overlapping and conserved portions\u0026nbsp;(Figure 1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Linear B- Cell Epitopes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpitopes with amino acid lengths between 10 and 20 that cut across at least two of the servers used (Bepipred, ABCpred and SvMtrip) were selected. The selected epitopes then had their physicochemical properties assessed for suitability in a vaccine construct. The result is presented in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of CTL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 130 strong binding CTL peptides were predicted for the selected Bovine Leukocyte Antigen (BoLA) alleles. Based on their binding score or ability, these peptides were selected. Strongly binding CTL peptides were examined for their stability index, toxicity, allergenicity, and antigenicity. Table 4 contains a list of the twenty-seven epitopes from the glycoprotein protein that passed these tests. However, not all the 27 made the final construct.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHelper T- Lymphocyte Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 97 strong peptides were found to demonstrate strong binding for the selected MHC-II alleles and were further assessed to induce interleukin 4 and interleukin 10 cytokines. After being evaluated for their physico-chemical characteristics, epitopes that showed potential to stimulate the production of the aforementioned cytokines were also tested for antigenicity, allergenicity, and noxiousness. Table 5 shows the Helper T lymphocyte epitopes that scaled through the screening processes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Construct Assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vaccine primary construct comprised a \u003cem\u003eBos taurus\u0026nbsp;\u003c/em\u003especific\u003cem\u003e\u0026nbsp;\u003c/em\u003eBeta defensin adjuvant, 6 CTL peptides, 3 HTL peptides and 2 B cell-based peptides. The adjuvant was attached to the first CTL peptides using the EAAAK linker and to subsequent CTL peptides using the AAY linker. The rest of the primary construct was linked using GPGPG linkers. Our final vaccine construct was a 202 amino acid-long primary sequence (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Physico-Chemical Properties RVFV Vaccine\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antigenicity index for a chimeric model with 0.4 points was determined using Vaxijen v2.0 server output and it was found to be quite high (0. 6926). The multi-epitope protein construct was also anticipated to be dispersible upon overexpression using Protein Sol. A grand total of 202 amino acids protein had a theoretical isoelectric point (Pl) of 9.44, and also a relative molar mass (Mr) of 21.4 kDa in accordance with the ExPasy Protparam web server. The sequence also had 26 cations (Arg + Lys) and 12 anions (Asp + Glu) residues, respectively. Assembled sequence was also shown to be sturdy with a stability index of 20.37. The multi-epitope has a DIVIDEND score of -0.121 along with an acyclic index of 81.19, which indicates that it is hydrophilic in nature (Figure 3) and thermo-stable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCodon Optimization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe JCAT server, reverse translation and codon optimization (ECAI) optimizations for the adaptation of \u003cem\u003eE. coli\u003c/em\u003e strain K-12 sub-strain MG1655 were performed\u003csup\u003e35\u003c/sup\u003e. The improved GC content after codon optimization was 53.3% at a Codon Adaptation Index (CAI) of 1.0. In order to successfully integrate the RVF construct into the \u003cem\u003eE. coli\u003c/em\u003e expression system, a high GC content is a sign of stability. The restriction enzymes Xho1 and EcoR1 were utilized as the cloning site to clone the back-translated nucleotide into the \u003cem\u003eE. coli\u003c/em\u003e expression system. An effective insertion of the segment into the pET28a (+) vector resulted in the production of a 6096 bp clone. In-silico cloning of the vaccine in an \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003epET28a (+) vector is presented in Figure 4.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Secondary and Tertiary Vaccine Structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe server SOPMA analyzed the secondary structure of the intended protein, and the findings were as follows: Alpha helix (31.55%), Random coil (44.92%), Beta turn (5.35%), and Extended strand (18.18%) while the tertiary structure is presented in Figure 5a.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTertiary Structure Refinement and Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults from the PROCHECK assessment of the stoichiometry quality of the structure revealed the residue and overall structural computation. Ramachandran\u0026rsquo;s plot analysis indicated that 95.7 % of protein remains were located in their preferred regions (Figure 5c). Furthermore, results from ProSA predict implied oversights of 3D model protein, predicted a negative score of -4.85, which is a favorable indicator of model quality (Figure 5b).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Discontinuous Epitopes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEllipro server predicted an aggregate of 91 conformational epitopes from 187 amino acid residues on the vaccine tertiary construct\u003csup\u003e31\u003c/sup\u003e. Also, the Jmol viewer allows for the uptake and foresight of immune epitopes in a given protein sequence or structure. Conformational epitopes with protrusion index scores greater than 0.7 are shown in Figure 5(a-c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults from HDock predicted 10 models docking between the vaccine construct and Toll-like receptor 7. The rankings were based on Docking Score, Confidence Score and Ligand RMSD (\u0026Aring;) (Figure 7 and Table 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune Response Incentive\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe server C-ImmSim simulates immune findings indicated in a system of effective immune systems and an extended duration of the vaccine candidate. The vaccine design, as shown in Figure 8, produces a strong and consistent main and secondary immunological system. The main immune response was defined by the level of the IgM antibodies after an interval of 4-5 days of antigenic exposure. The basic immune response was distinguished by enhanced B-cell generated and also in the increase of IgM, IgG1 + IgG2 expression and IgM + IgG antibodies. The advanced vaccine construct did not only evoke a strong B-cell increase; it also led to the multitude of memory B-cells. Specific interleukin concentration, a high level of IFN-\u0026gamma; for a long interval, CD4 activity, T-helper lymphocyte count, and B lymphocyte count were revealed.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eRVF is a zoonotic illness spread by mosquitoes. It poses a lethal threat to humans and livestock in many sub-Saharan countries, such as South Africa, Egypt, Madagascar, Mauritania, and Senegal. The disease has been documented in the Arabian Peninsula as well as in the Middle East, and India\u003csup\u003e39\u003c/sup\u003e. In animals, the disease has a high fatality rate, where it causes epizootics of abortion and fetal death and high mortality in neonatal ruminants. The virus also causes infection in humans, although with a lower mortality rate. The Smithburn live attenuated RVFV has been extensively adopted in different countries for prevention; however, like many live attenuated vaccines, there are challenges with safety due to reversal to virulence characterized by vaccine-induced teratogenesis, abortion, and fetal death. As a result, it is not recommended for usage in developing nations\u003csup\u003e40\u003c/sup\u003e. Furthermore, the vaccine faces a challenge due to the risk of genetic reassortment\u003csup\u003e41\u003c/sup\u003e. Therefore, there is a need for novel approaches to designing safe and effective vaccines against this disease.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdvances in Whole Genome Sequencing (WGS) and proteomics have increased our understanding of viral proteins, which are being used in vaccine design via a reverse vaccinology approach. The use of peptide vaccines has emerged as an outstanding alternative to conventional approaches to vaccine design\u003csup\u003e42\u003c/sup\u003e. Sub-unit epitope-based vaccines provide an excellent alternative when considering safety, practicability, and cost-effectiveness. Moreover, the potency and immunogenicity of these vaccines can be boosted through selected engineering of target epitopes\u003csup\u003e43\u003c/sup\u003e. By applying recent developments in immunoinformatics and computational technologies, scientists can selectively construct vaccines with minimal side effects and allergenicity\u003csup\u003e44\u003c/sup\u003e. Consequently, a number of vaccines have been designed against various diseases such as SARS-CoV-2, Ebola, hepatitis C, and cholera by applying this approach\u003csup\u003e45,46,47,48\u003c/sup\u003e. Our objective in this study was to design a safe, non-allergenic, and non-toxic subunit vaccine that is capable of eliciting optimal immunity against RVF in ruminants.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe selected the M glycoprotein gene after immunoinformatics analysis and extensive literature on Rift Valley fever vaccines. The M segment was chosen due to its high antigenicity and the presence of large extra-cytoplasmic segments. Furthermore, experimental vaccines developed using this protein have yielded good results. In different studies, RVFV vaccines developed using the glycoprotein showed excellent immune responses and demonstrated full protection against experimental heterologous challenge infections in sheep and cattle\u003csup\u003e49,50\u003c/sup\u003e. The vaccine was also shown to differentiate naturally infected from vaccinated animals (DIVA) compatible\u003csup\u003e51,52\u003c/sup\u003e also demonstrated in an immunological study the pivotal role of the Gn and Gc segments in antibody production and the immune response against RVFV. Furthermore, the M segments basically constitute surface proteins, so they have a great potential for inducing the production of neutralizing antibodies\u003csup\u003e53,54\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe glycoprotein gene was screened for linear B-cells, CD-4 helper T lymphocytes (HTL), and CD-8 cytotoxic T-lymphocytes (HTL), which were all integrated into the eventual vaccine construct. Research by Dodd \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e55\u003c/sup\u003e has shown the role B-cells and CD-4 Helper T cells play in mediating viral clearance. Specifically, CD-4 cells were shown to play major roles in maintaining robust IgG and neutralizing antibody responses as well as controlling viral replication \u003cem\u003ein vivo\u003c/em\u003e. CD-4 activates B cells, and signals isotype class switching, affinity maturation, and antibody production through its effect on antibody-mediated immunity. They also mediate cytotoxicity by stimulating CD-8 cells\u003csup\u003e55\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe final vaccine construct was made up of 11 epitopes, comprising 6 CTL peptides, 3 HTL peptides, and 2 linear B cell peptides. All peptides had their physicochemical properties assessed to ensure that they were antigenic, non-allergenic, and non-toxic. HTL peptides were also evaluated for their ability to induce interleukin 4 (IL-4) and interleukin 10 (IL-10). These peptides were linked with AAY and GPGPG linkers. Linkers are vital in vaccine design as they ensure that protein amino acid residues are situated in a conformation similar to their natural state. A 38-amino-acid-long \u003cem\u003eBos taurus\u003c/em\u003e beta-defensin was further added to increase the vaccine\u0026apos;s immunogenicity with the help of a rigid EAAAK linker.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of the vaccines\u0026rsquo; antigenicity, allergenicity, and other physico-chemical properties all showed favorable results. The vaccine was found to be antigenic, non-allergenic, and non-toxic, indicating a favorable safety potential. Further physicochemical analysis indicated the vaccine to be stable, basic, and soluble.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe potency of the vaccine to induce an immune response was further evaluated by molecular docking analysis and immune simulation. Two toll-like receptors; TLR-7 and TLR-8, which have affinity for viral RNA ligands, were selected. The vaccine construct had more binding affinity to the TLR-8 with a binding score of -324 compared with the TLR-7, which had a binding score of -278. Furthermore, immune simulation with C-ImmSim showed sustained immune response for up to 350 days after injecting with 100 units. After 350 days, the IgG+IgM had dropped to a basal level of about 5000 antibody titers from a peak of about 30,000 that had been reached during the first 30 days of administration.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eRift Valley fever is an evolving arboviral disease, which requires concerted efforts at prevention and control. To this end, novel vaccines that are able to combine potency and safety are needed, both in animals and humans. In this study, we present an immunoinformatics approach to the development of a multi-epitope subunit RVFV vaccine for use in ruminants. The M glycoprotein gene was selected because of its high antigenicity and experimental evidence of its efficacy in vaccines. Six CTL epitopes, three HTL and 2 B-cell epitopes were joined together in a chimeric construct and subjected to analysis. In-silico analysis demonstrated the immunogenicity and safety of the vaccine construct; however, further in-vitro and in vivo analyses are required to validate the study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare that they received no funds, grants, or other support during the preparation of this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAs the authors of this manuscript, we have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConceptualization, EKO; experimental design: EKO; data collection and analysis,\u0026nbsp;\u003c/em\u003eORT,\u0026nbsp;\u003cem\u003eFOT\u003c/em\u003e, APT, AMB, OIO, BVO, JIT; result interpretation: EKO, ORT; manuscript draft, review and editing: EKO, ORT, IOA\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;All authors commented on the previous versions of the manuscript, read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnyamba A, Linthicum KJ, Small J, Britch SC, Pak ES de La Rocque \u003cem\u003eet al\u003c/em\u003e. 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ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research. 2007;35(suppl_2):W407-W410\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e35. Grote A, Hiller K, Scheer M, M\u0026uuml;nch R, N\u0026ouml;rtemann B, Hempel DC, Jahn D. JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Research. 2005;33(suppl_2):W526-W531\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e36. Asghari A, Nourmohammadi H, \u0026nbsp;Majidiani H, Shariatzadeh ,A Shams M, Ontazeri FM. In silico analysis and prediction of immunogenic epitopes for pre-erythrocytic proteins of the deadly \u003cem\u003ePlasmodium falciparum.\u0026nbsp;\u003c/em\u003eInfect. Genet. Evol. 2021;104985\u0026nbsp;\u003ca href=\"https://doi.org/10.1016/j.meegid.2021.104985\" target=\"_blank\"\u003e101016/jmeegid2021104985\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e37. Yan Y, Zhang D, Zhou P, Li B, Huang SY. HDOCK: a web server for protein\u0026ndash;protein and protein\u0026ndash;DNA/RNA docking based on a hybrid strategy. Nucleic Acids Research. 2017;45(W1) W365-W373\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e38. Jardine JG, Ota T, Sok D, Pauthner M, Kulp DW, Kalyuzhniy O, Nemazee D. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science. 2015;349(6244):156-161\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e39. Gerken KN, LaBeaud AD, Mandi H, L\u0026rsquo;Azou Jackson M, Breugelmans JG, King CH. Paving the way for human vaccination against Rift Valley fever virus: A systematic literature review of RVFV epidemiology from 1999 to 2021. PLoS Neglected Tropical Diseases. 2022;16(1):e0009852\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e40. Botros B, Omar A, Elian K, Mohamed G, Soliman A, Salib A, Earhart K. Adverse response of non‐indigenous cattle of European breeds to live attenuated Smithburn Rift Valley fever vaccine. Journal of Medical Virology. 2006;78(6):787-791\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e41. Sall AA, Zanotto PDA, Sene OK, Zeller HG, Digoutte JP, Thiongane Y, Bouloy M. Genetic reassortment of Rift Valley fever virus in nature. Journal of Virology. 1999;73(10):8196-8200\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e42. World Health Organization. Guide line for production and quality control of synthetic peptide vaccine. WHO Technical Report Series. 1999;No989\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e43. Shey RA, Ghogomu SM, Esoh KK \u003cem\u003eet al.\u003c/em\u003e In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases. Sci. Rep. 2019;9(1):4409\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e44. Seib KL, Zhao X, Rappuoli R. Developing vaccines in the era of genomics: a decade of reverse vaccinology. Clin. Microbiol. Infect. 2012;18(Suppl 5):109\u0026ndash;16\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e45. Dash R, Das R, Junaid M\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e. In silico-based vaccine design against Ebola virus glycoprotein. Adv. Appl. Bioinform. Chem. 2017;10:11\u0026ndash;28\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e46. Ikram A, Zaheer T, Awan FM \u003cem\u003eet al.\u003c/em\u003e Exploring NS3/4A NS5A and NS5B proteins to design conserved subunit multi-epitope vaccine against HCV utilizing immunoinformatics approaches. Sci. Rep. 2018;8(1):16107\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e47. Kar T, Narsaria U, Basak S, Deb D, Castiglione F, Mueller DM, Srivastava AP. A candidate multi-epitope vaccine against SARS-CoV-2. Scientific Reports. 2020;10(1):10895\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e48. Oladipo EK, Akindiya OE, Oluwasanya GJ, Akanbi GM, Olufemi SE, Adediran DA, \u0026nbsp;Babalola MO. Bioinformatics analysis of structural protein to approach a vaccine candidate against Vibrio cholerae infection Immunogenetics. 2022;1-16\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e49. Faburay B, Wilson WC, Gaudreault NN, Davis AS, Shivanna V, Bawa B, Richt JA. A recombinant Rift Valley fever virus glycoprotein subunit vaccine confers full protection against Rift Valley fever challenge in sheep. Scientific Reports. 2016;6(1):27719\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e50. Wilson WC, Faburay B, Trujillo JD, Ragan I, Sunwoo SY, Morozov I, Richt JA. Preliminary evaluation of a recombinant Rift Valley fever virus glycoprotein subunit vaccine providing full protection against heterologous virulent challenge in cattle. Vaccines. 2021;9(7):748\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e51. Faburay B, Lebedev M, McVey DS, Wilson W, Morozov I, Young A, Richt JA. A glycoprotein subunit vaccine elicits a strong Rift Valley fever virus neutralizing antibody response in sheep. Vector-borne and Zoonotic Diseases. 2014;14(10):746-756\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e52. Wright D, Allen ER, Clark MH, Gitonga JN, Karanja HK, Hulswit RJ, Warimwe GM. Naturally acquired Rift Valley fever virus neutralizing antibodies predominantly target the Gn glycoprotein. Iscience. 2020;23(11):101669\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e53. Lorenzo G, Mart\u0026iacute;n-Folgar R, Hevia E, Boshra H, Brun A. Protection against lethal Rift Valley fever virus (RVFV) infection in transgenic IFNAR(-/-) mice induced by different DNA vaccination regimens. Vaccine. 2010;28:2937\u0026ndash;2944\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e54. Doyle JD, Barbeau DJ, Cartwright HN, McElroy AK. Immune correlates of protection following Rift Valley fever virus vaccination. NPI Vaccines. 2022;7(1):129\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e55. Dodd KA, McElroy AK, Jones ME, Nichol ST, Spiropoulou CF. Rift Valley fever virus clearance and protection from neurologic disease are dependent on CD4+ T cell and virus-specific antibody responses. Journal of Virology. 2013;87(11):6161-6171\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 1 Antigenicity properties of selected proteins\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 22pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGenome Segment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 22pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eProtein\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 22pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenicity\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePolymerase\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-antigen\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGlycoprotein\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 19.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigen\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 20.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 20.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNucleocapsid\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 20.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-antigen\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 2 Transmembrane topology of selected protein highlighted in bold\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGenome Segment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eProtein\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eProtein Segment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePosition\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePolymerase\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1-2091\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"border: medium;padding: 0in 5.4pt;height: 1.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" style=\"border: medium;padding: 0in 5.4pt;height: 1.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGlycoprotein\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1-16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eSignal peptide\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e17-584\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eOutside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e585-603\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTMHelix\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e604-675\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e676-685\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTMHelix\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e686-1159\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eOutside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1160-1178\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTMHelix\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1179-1197\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;height: 1.35pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eN\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNonstructural\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1-265\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNucleocapsid\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1-245\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInside\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 3 Selected predicted linear B-cell-based epitopes\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 27.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePeptide\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 27.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenicity\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 27.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAllergen city\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eDSLREEEMPE\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eVYLDKLDLKTEENLLPD\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eSYWTGSFSPKCLSSRRCHLV\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eSKLKTKMKGVCEVGVQALKK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 4 Predicted CTL epitopes. Peptides selected for the final vaccine construct are highlighted in bold\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePeptides\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenicity score\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eInstability status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eToxicity status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAllergenicity status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eSLKKGSYPL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.3734\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eKIKTVSSEL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.4693\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGNPCMKEKL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.0854\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTMAGIAMTV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.9979\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNCIDWVHKL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.6161\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eRAPNLVSYK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.1302\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-Allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eRQMTGASLK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.7603\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eEVVPFAVFK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.4533\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eIQVSGVWKK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.5642\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eSAHGNPCMK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.9450\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;background: white;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eRVADNINQV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.5002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eVVFAEDPHL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.4568\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eITSTGTGTL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.0479\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eLSAKPIQRV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.1587\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eFKNSKKVYL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.5282\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eLKIAPRKVL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.6219\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eVQADLTLMF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.8723\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eHKGQYKGTM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.6591\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTGFKISSAV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.6721\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;background: white;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTKDQCQILH\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.5748\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;background: white;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eVQADLTLM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.8470\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;background: white;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eGDIGVHMA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.2486\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTKDQCQIL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.4858\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eLEKGKFPL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.6611\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eRETMAGIA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.3986\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eLKIAPRKV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.2515\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: medium;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border: medium;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83.35pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eRQMTGASL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.75pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.5040\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 2pt;height: 17.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 2pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon- toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94.5pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 1pt;height: 17.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 5 Selected HTL epitopes\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003ePeptides\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenicity status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAllergenicity status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eToxicity status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStability status\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eQSAHYLNNDGKMASV\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eFLKIKTVSSELSCRE\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eVLKCLKIAPRKVLNP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAntigenic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-allergic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eNon-toxic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5pt;height: 14.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eStable\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 6 Conformational epitopes predicted from the vaccine\u0026rsquo;s tertiary construct\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;height: 25.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eNo.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;height: 25.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003ePredicted discontinuous epitope\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;height: 25.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eNumber of residues\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;height: 25.55pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eScore\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 28.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 28.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:V152, A:L153, A:N154, A:P155, A:G156, A:P157, A:G158, A:P159, A:G160, A:S161, A:K162, A:L163, A:T165, A:K166, A:K168\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 28.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 28.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:#F0F0F6;'\u003e0.773\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e2.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:K194, A:T195, A:E196, A:E197, A:N198, A:L199, A:L200, A:P201, A:D202\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e0.733\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e3.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:D1, A:F2, A:A3, A:S4, A:C5, A:H6, A:T7, A:N8, A:G9, A:G10, A:I11, A:C12, A:L13, A:P14, A:N15, A:R16, A:C17, A:P18, A:G19, A:H20, A:G25, A:I26, A:C27, A:F28, A:R29, A:P30, A:R31, A:V32, A:K33, A:C34\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:#F0F0F6;'\u003e0.711\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e4.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:K180, A:G181, A:P182, A:G183, A:P184, A:G185, A:V186, A:Y187\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e0.697\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 0.7in;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e5.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 0.7in;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:P117, A:G118, A:P119, A:G120, A:F121, A:E135, A:G136, A:P137, A:G138, A:P139, A:G140, A:V141, A:L142, A:K143, A:C144, A:L145, A:K146\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 0.7in;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;height: 0.7in;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e0.669\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e6.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:G81, A:T82, A:G83, A:T84, A:L85, A:A86, A:A87, A:Y88, A:R89, A:Q90, A:M91, A:T92, A:G93, A:A94, A:S95, A:K97, A:A98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e0.639\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e7.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003eA:K125, A:T126, A:V127, A:S128, A:S129, A:E130\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;background:white;'\u003e0.633\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTable 7 Molecular docking for Toll-like receptors\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width:6.5in;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eToll-like Receptor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eDocking score\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eConfidence Score\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eLigand rmsd (\u0026Aring;)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTLR-7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e-278.26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: medium;padding: 5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.9286\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border: 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Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Arboviral diseases, Molecular docking, Reverse Vaccinology, Rift Valley fever virus","lastPublishedDoi":"10.21203/rs.3.rs-3475857/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3475857/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRift Valley fever is a new arboviral illness with serious public health implications. The disease, which is brought on by the Rift Valley fever virus (RVFV), is linked to severe economic losses in animals as well as a number of human ailments. There are currently no authorized live attenuated vaccines for human use, and they have been linked to increased risks of abortion in ruminants.\u003c/p\u003e\n\u003cp\u003eIn this study, the use of reverse vaccination was applied in creating a vaccine using a segment for use in ruminants by exploring proteins in the M-glycoprotein segment. Glycoprotein sequences were retrieved from cattle, sheep and goats from endemic countries across Africa and subjected to computational analysis for B and T cell epitope prediction. Highly conserved, immunodominant epitopes were obtained and connected with the ideal linkers for proper conformation. A \u003cem\u003eBos taurus-specific\u003c/em\u003e beta-defensin was also added to increase immunogenicity. The secondary as well as tertiary structures of the vaccine were then determined by SOPMA and alphafold2, respectively.\u003c/p\u003e\n\u003cp\u003eAnalysis of the vaccine’s physicochemical properties all yielded favorable results regarding its potential effectiveness and safety. Furthermore, molecular docking analysis was carried out using Toll-like receptors 7 and 8 to show molecular binding interactions. \u0026nbsp;While in-silico analyses have yielded satisfactory results, translational research may be required to validate the vaccines’ effectiveness in animals.\u003c/p\u003e","manuscriptTitle":"Immunoinformatics approach to Rift Valley fever virus vaccine design in ruminants","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-01-10 19:22:11","doi":"10.21203/rs.3.rs-3475857/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-11-07 17:13:45","doi":"10.21203/rs.3.rs-3475857/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9956fe8f-2925-49a9-b0b5-cfeb9c06db31","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-09T17:49:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-10 19:22:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-3475857","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3475857","identity":"rs-3475857","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}