Sains Malaysiana 51(9)(2022): 2985-2997


Construction of Multi-Epitopes Vaccine Candidate against SARS-CoV-2 D614G Variant

(Pembinaan Calon Vaksin Epitop Pelbagai terhadap Varian D614G SARS-CoV-2)



 1School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

2Universiti Sains Malaysia (USM)-RIKEN Interdisciplinary Collaboration for Advanced Sciences (URICAS), 11700 Gelugor, Penang, Malaysia

3Department of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia


Diserahkan: 28 November 2021/Diterima: 23 Mac 2022


ǂ These authors contributed equally to this work



COVID-19 caused by the SARS-CoV-2 virus has become a real threat due to the emergence of new variants which are more deadly with higher infectivity. Vaccine constructs that target specific SARS-CoV-2 variants are needed for stemming COVID-19 fatality. The spike (S) glycoprotein is the major antigenic component that triggers the host immune response. Reverse vaccinology strategy was applied to the S protein of COVID-19 variant D614G to identify highly ranked antigenic proteins. In this study, a multi-epitope synthetic gene was designed using computational strategies for the COVID-19 D614G variant. The SARS-CoV-2 D614G variant protein sequence was retrieved from the NCBI database. The prediction of linear B-cell epitopes was carried out using Artificial Neural Network (ANN)-based ABCpred and BepiPred 2.0 software. The top 15 highly antigenic epitopes sequences were then selected. Propred 1 and Propred servers were used to identify major histocompatibility complex (MHC) class I and class II binding epitopes within pre-determined B-cell epitopes to predict T-cell epitopes. The top 5 MHC class I and class II were selected. Further in-silico testing for its solubility, allergenicity, antigenicity, and other physiochemical properties was analyzed using Bpred. The constructed gene was subjected to assembly PCR and the gene product was confirmed by Sanger sequencing. The findings from this study suggested that a highly antigenic specific region of the SARS-CoV-2 D614G variant can be predicted in-silico and amplified using the assembly PCR method. The designed synthetic gene was shown to elicit specific humoral and cell-mediated immune responses towards the SARS-CoV-2 variants.


Keywords: Assembly PCR; D614G variant; gene construct; multi-epitopes; SARS-CoV-2 spike



COVID-19 yang disebabkan oleh virus SARS-CoV-2 telah menjadi ancaman sebenar kerana kemunculan varian baharu yang lebih mematikan dengan jangkitan yang lebih tinggi. Pembinaan vaksin yang mensasarkan varian SARS-CoV-2 tertentu diperlukan untuk membendung kematian disebabkan COVID-19. Glikoprotein lonjakan (S) adalah komponen antigen utama yang mencetuskan tindak balas imun perumah. Strategi vaksinologi terbalik diterapkan pada protein S daripada varian D614G COVID-19 untuk mengenal pasti protein antigen kelas tinggi. Dalam kajian ini, gen sintetik epitop pelbagai direka bentuk menggunakan strategi komputasi untuk varian D614G COVID-19. Urutan protein varian SARS-CoV-2 D614G diambil daripada pangkalan data NCBI. Ramalan epitop sel B linear dilakukan dengan menggunakan perisian ABCpred dan BepiPred 2.0 berasaskan rangkaian neural tiruan (ANN). Lima belas urutan epitop antigen teratas kemudian dipilih. Perisian Propred 1 dan Propred digunakan untuk mengenal pasti epitop pengikat kelas I dan kelas II kompleks kehistoserasian utama (MHC) dalam epitop sel B yang telah ditentukan untuk meramalkan epitop sel T. Lima kelas I dan kelas II MHC teratas dipilih. Ujian in-sillico lebih lanjut untuk kelarutan, kealergenan, keantigenan dan sifat fisiokimia lain dianalisis menggunakan Bpred. Gen yang dibina dikenakan himpunan PCR dan produk gen tersebut disahkan oleh penjujukan Sanger. Hasil daripada kajian ini menunjukkan bahawa kawasan yang sangat khusus antigen varian SARS-CoV-2 D614G dapat diramalkan dalam silika dan dikembangkan menggunakan kaedah himpunan PCR. Gen sintetik yang dibangunkan menunjukkan penghasilan tindak balas imun khusus humoral dan sel yang dimediasi khusus terhadap varian SARS-CoV-2.


Kata kunci: Epitop pelbagai; himpunan PCR; pembinaan gen; peningkatan SARS-CoV-2; varian D614G



Bhattacharya, M., Sharma, A.R., Patra, P., Ghosh, P., Sharma, G., Patra, B.C., Lee, S.S. & Chakraborty, C. 2020. Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach. Journal of Medical Virology 92(6): 618-631.

CDC. 2022. Malaria Vaccine Recommended for Broader Use by WHO: Best Thing Since Bed Nets. who.html

CDC. 2021. SARS-CoV-2 Variant Classifications and Definitions. variant-info.html#Consequence

Cheng, J., Randall, A.Z., Sweredoski, M.J. & Baldi, P. 2005. SCRATCH: A protein structure and structural feature prediction server. Nucleic Acids Research 33(Web Server issue): W72-W76.

Di, L. 2015. Strategic approaches to optimizing peptide ADME properties. The AAPS Journal 17(1): 134-143.

Dimitrov, I., Bangov, I., Flower, D.R. & Doytchinova, I. 2014. AllerTOP v.2 - A server for in silico prediction of allergens. Journal of Molecular Modeling 20(6): 2278.

Doytchinova, I.A. & Flower, D.R. 2007. Identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties. Vaccine 25(5): 856-866.

Dutta, N.K., Mazumdar, K., Lee, B.H., Baek, M.W., Kim, D.J., Na, Y.R., Park, S.H., Lee, H.K., Kariwa, H. & Park, J.H. 2008. Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine. Immunology Letters 118(1): 65-71.

Galloway, S.E., Paul, P., MacCannell, D.R., Johansson, M.A., Brooks, J.T., MacNeil, A., Slayton, R.B., Tong, S., Silk, B.J., Armstrong, G.L. & Biggerstaff, M. 2021. Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020 - January 12, 2021. MMWR Morb Mortal Wkly Rep 70(3) 95-99.

Guan, P., Doytchinova, I.A., Zygouri, C. & Flower, D.R. 2003. MHCPred: A server for quantitative prediction of peptide-MHC binding. Nucleic Acids Research 31(13): 3621-3624.

Gurung, A.B. 2020. In silico structure modelling of SARS-CoV-2 Nsp13 helicase and Nsp14 and repurposing of FDA approved antiviral drugs as dual inhibitors. Gene Reports 21: 100860-100860.

Hebditch, M., Carballo-Amador, M.A., Charonis, S., Curtis, R. & Warwicker, J. 2017. Protein-sol: A web tool for predicting protein solubility from sequence. Bioinformatics (Oxford, England) 33(19): 3098-3100.

Heo, L., Park, H. & Seok, C. 2013. GalaxyRefine: Protein structure refinement driven by side-chain repacking. Nucleic Acids Research 41(Web Server issue): W384-W388.

Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, S., Herrler, G., Wu, N.H., Nitsche, A., Muller, M.A., Drosten, C. & Pöhlmann, S. 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181(2): 271-280.

Hou, Y. J., Chiba, S., Halfmann, P., Ehre, C., Kuroda, M., Dinnon III, K.H., Leist, S.R., Schafer, A., Nakajima, N., Takahashi, K. & Lee, R.E. 2020. SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo. Science (New York) 370(6523): 1464-1468.

Khateeb, J., Li, Y. & Zhang, H. 2021. Emerging SARS-CoV-2 variants of concern and potential intervention approaches. Critical Care 25(1): 244.

Korber, B., Fischer, W.M., Gnanakaran, S., Yoon, H., Theiler, J., Abfalterer, W., Hengartner, N., Giorgi, E.E., Bhattacharya, T., Foley, B. & Hastie, K.M. 2020. Tracking changes in SARS-CoV-2 Spike: Evidence that D614G increases infectivity of the COVID-19 Virus. Cell 182(4): 812-827.

Koyama, T., Platt, D. & Parida, L. 2020. Variant analysis of SARS-CoV-2 genomes. Bulletin of the World Health Organization 98(7): 495-504.

Kringelum, J.V., Lundegaard, C., Lund, O. & Nielsen, M. 2012. Reliable B cell epitope predictions: Impacts of method development and improved benchmarking. PLOS Computational Biology 8(12): e1002829.

Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L. & Wang, X. 2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581(7807): 215-220.

Laskowski, R.A., MacArthur, M.W. & Thornton, J.M. 2006. PROCHECK: Validation of protein-structure coordinates. In International Tables for Crystallography. The Netherlands: Kluwer Academic Publishers.

Ma, J. 2020. Coronavirus: China’s first confirmed COVID-19 case traced back to November 17 South China Morning Post. society/article/3074991/coronavirus-chinas-first-confirmed-covid-19-case-traced-back.

Musto, H., Naya, H., Zavala, A., Romero, H., Alvarez-Valín, F. & Bernardi, G. 2006. Genomic GC level, optimal growth temperature, and genome size in prokaryotes. Biochemical and Biophysical Research Communications 347(1): 1-3.

Nain, Z., Karim, M.M., Sen, M.K. & Adhikari, U.K. 2020. Structural basis and designing of peptide vaccine using PE-PGRS family protein of Mycobacterium ulcerans - An integrated vaccinomics approach. Molecular Immunology 120: 146-163.

Naz, A., Awan, F.M., Obaid, A., Muhammad, S.A., Paracha, R.Z., Ahmad, J. & Ali, A. 2015. Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: A reverse vaccinology based approach. Infection, Genetics and Evolution 32: 280-291.

Nelson-Sathi, S., Umasankar, P.K., Sreekumar, E., Nair, R.R., Joseph, I., Nori, S.R.C., Philip, J.S., Prasad, R., Navyasree, K.V., Ramesh, S. & Pillai, H. 2022. Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction. BMC Molecular and Cell Biology 23(1): 1-12.

Patra, P., Mondal, N., Patra, B.C. & Bhattacharya, M. 2020. Epitope-based vaccine designing of nocardia asteroides targeting the virulence factor mce-family protein by immunoinformatics approach. International Journal of Peptide Research and Therapeutics 26(2): 1165-1176.

Plante, J.A., Liu, Y., Liu, J., Xia, H., Johnson, B.A., Lokugamage, K.G., Zhang, X., Muruato, A.E., Zou, J., Fontes-Garfias, C.R. & Mirchandani, D. 2021. Spike mutation D614G alters SARS-CoV-2 fitness. Nature 592(7852): 116-121.

Rehman, S.U., Nadeem, A., Javed, M., Hassan, F-U., Luo, X., Khalid, R.B. & Liu, Q. 2020. Genomic identification, evolution and sequence analysis of the heat-shock protein gene family in buffalo. Genes 11(11): 1388.

Saha, R., Ghosh, P. & Burra, V.L.S. 2021. Designing a next generation multi-epitope based peptide vaccine candidate against SARS-CoV-2 using computational approaches. 3 Biotech 11(2): 47.

Saha, S. & Raghava, G.P.S. 2006. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins: Structure, Function, and Bioinformatics 65(1): 40-48.

Sanchez-Trincado, J.L., Gomez-Perosanz, M. & Reche, P.A. 2017. Fundamentals and methods for T- and B-Cell epitope prediction. Journal of Immunology Research 2017: 2680160.

Schindewolf, C. & Menachery, V.D. 2019. Middle East respiratory syndrome vaccine candidates: Cautious optimism. Viruses 11(1): 74.

Singh, H. & Raghava, G.P.S. 2003. ProPred1: Prediction of promiscuous MHC Class-I binding sites. Bioinformatics 19(8): 1009-1014.

Suzuki, H. & Morton, B.R. 2016. Codon adaptation of plastid genes. PLoS ONE 11(5): e0154306.

Verma, J. & Subbarao, N. 2021. Insilico study on the effect of SARS-CoV-2 RBD hotspot mutants' interaction with ACE2 to understand the binding affinity and stability. Virology 561(1): 107-116.

Walls, A.C., Park, Y.J., Tortorici, M.A., Wall, A., McGuire, A.T. & Veesler, D. 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181(2): 281-292.

Wilkins, M.R., Gasteiger, E., Bairoch, A., Sanchez, J-C., Williams, K.L., Appel, R.D. & Hochstrasser, D.F. 1999. In Protein Identification and Analysis Tools in the ExPASy Server. Methods Mol. Biol. 112: 531-552.

World Health Organization (WHO). 2021. Draft Landscape and Tracker of COVID-19 Candidate Vaccines. Accessed on 25 May 2021.

Xu, J.B., Zhao, S.Z., Teng, T.S., Abdalla, A.E., Zhu, W., Xie, L.X., Wang, Y.L. & Guo, X.Q. 2020. Systematic comparison of two animal-to-human transmitted human coronaviruses: SARS-CoV-2 and SARS-CoV. Viruses 12(2): 244.

Zhang, J., Zeng, H., Gu, J., Li, H., Zheng, L. & Zou, Q. 2020. Progress and prospects on vaccine development against SARS-CoV-2. Vaccines 8(2): 153.

Zhang, L., Jackson, C.B., Mou, H., Ojha, A., Peng, H., Quinlan, B.D., Rangarajan, E.S., Pan, A., Vanderheiden, A., Sulthar, M.S. & Li, W. 2020. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nature Communications 11(1): 6013.


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