Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses


Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR
(5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant
protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher
IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced
elicitation of the neutralizing antibody responses.

Share and Cite:

N. Mat-Rahim and S. AbuBakar, "Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses," World Journal of Vaccines, Vol. 4 No. 1, 2014, pp. 33-43. doi: 10.4236/wjv.2014.41006.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] J. Qiu, “Enterovirus 71 Infection: A New Threat of Global Public Health?” The Lancet Neurology, Vol. 7, No. 10, 2008, pp. 868-869.
[2] S. AbuBakar, H. Y. Chee, N. Shafee, K. B. Chua and S. K. Lam, “Molecular Detection of Enteroviruses from an Outbreak of Hand, Foot and Mouth Disease in Malaysia in 1997,” Scandinavian Journal of Infectious Diseases, Vol. 31, No. 4, 1999, pp. 331-335.
[3] P. C. McMinn, “An Overview of the Evolution of Enterovirus 71 and Its Clinical and Public Health Significance,” FEMS Microbiology Reviews, Vol. 26, No. 1, 2002, pp. 91-107.
[4] Y. T. Lin, L. Y. Chang, S. H. Hsia, Y. C. Huang, C. H. Chiu, C. Hsueh, S. R. Shih, C. C Liu and M. H. Wu, “The 1998 Enterovirus 71 Outbreak in Taiwan: Pathogenesis and Management,” Clinical Infectious Diseases, Vol. 34, Suppl. 2, 2002, pp. S52-S57.
[5] L. J. Herrero, C. S. Lee, R. J. Hurrelbrink, B. H. Chua, K. B. Chua and P. C. McMinn, “Molecular Epidemiology of Enterovirus 71 in Peninsular Malaysia, 1997-2000,” Archives of Virology, Vol. 148, No. 7, 2003, pp. 1369-1385.
[6] H. F. Chen, M. H. Chan, B. L. Chiang and T. S Jeng, “Oral Immunization of Mice Using Transgenic Tomato Fruit Expressing VP1 Protein From Enterovirus 71,” Vaccine, Vol. 24, No. 15, 2006, pp. 2944-2951.
[7] S. AbuBakar, I. C. Sam, J. Mohd Yusof, M. K. Lim, S. Misbah, N. A. MatRahim and P. S. Hooi, “Enterovirus 71 Outbreak, Brunei,” Emerging Infectious Disease, Vol. 15, No. 1, 2009, pp. 79-82.
[8] P. C. McMinn, “Recent Advances in The Molecular Epidemiology and Control of Human Enterovirus 71 Infection,” Current Opinion in Virology, Vol. 2, No. 2, 2012, pp. 199-205.
[9] M. A. Pallansch and M. S. Oberste, “Enterovirus 71 Encephalitis: A New Vaccine on the Horizon?” The Lancet, Vol. 381, No. 9871, 2013, pp. 976-977.
[10] C. H. Chiu, C. Chu, C. C. He and T. Y. Lin, “Protection of Neonatal Mice From Lethal Enterovirus 71 Infection by Maternal Immunization With Attenuated Salmonella Enterica Serovar Typhimurium Expressing VP1 of Enterovirus 71,” Microbes and Infections, Vol. 8, No. 7, 2006, pp. 1671-1678.
[11] D. G. W. Foo, S. Alonso, V. T. K. Chow and C. L. Poh, “Passive Protection Against Lethal Enterovirus 71 Infection in Newborn Mice by Neutralizing Antibodies Elicited by a Synthetic Peptide,” Microbes and Infections, Vol. 9, No.11, 2007, pp. 1299-1306.
[12] X. Li, C. Mao, S. Ma, X. Wang, Z. Sun, Y. Yi, M. Guo, X. Shen, L. Sun and S. Bi, “Generation of Neutralizing Monoclonal Antibodies Against Enterovirus 71 Using Synthetic Peptides,” Biochemical and Biophysical Research Communication, Vol. 390, No. 4, 2009, pp. 1126-1128.
[13] X. Ye, Z. Ku, Q. Liu, X. Wang, J. Shi, Y. Zhang, L. Kong, Y. Cong and Z. Huang, “Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice Through Divergent Mechanisms,” Journal of Virology, Vol. 88, No. 1, 2014, pp. 72-81.
[14] H. Y. Li, J. F. Han, C. F. Qin and R. Chen, “Virus-Like Particles for Enterovirus 71 Produced From Saccharomyces cerevisiae Potently Elicits Protective Immune Responses in Mice,” Vaccine, Vol. 31, No. 32, 2013, pp. 3281-3287.
[15] C. W. Chen, Y. P. Lee, W. F. Wang and C. K. Yu, “Formaldehyde-Inactivated Human Enterovirus 71 Vaccine is Compatible for Co-Immunization With a Commercial Pentavalent Vaccine,” Vaccine, Vol. 29, No. 15, 2011, pp. 2772-2776.
[16] A. Cheng, C. P. Fung, C. C. Liu, Y. T. Lin, H. Y. Tsai, S. C. Chang, A. H. Chou, J. Y. Chang, R. H. Jiang, Y. C. Hsieh, I. J. Su, P. C. H. Chong and S. M. Hsieh, “A Phase I, Randomized, Open-Label Study to Evaluate The Safety and Immunogenicity of an Enterovirus 71 Vaccine,” Vaccine, Vol. 31, No. 20, 2013, pp. 2471-2476.
[17] R. W. Ellis, “Technologies for the Design, Discovery, Formulation and Administration of Vaccines,” Vaccine, Vol. 19, No.17-19, 2001, pp. 2681-2687.
[18] H. Shimizu, B. Thorley, F. J. Paladin, K. A. Brussen, V. Stambos, L. Yuen, A. Utama, Y. Tano, M. Arita, H. Yoshida, T. Yoneyama, A. Benegas, S. Roesel, M. Pallansch, O. Kew and T. Miyamura, “Circulation of Type 1 Vaccine-Derived Poliovirus in the Philippines in 2001,” Journal of Virology, Vol. 78, No. 24, 2004, pp. 13512-13521.
[19] O. M. Kew, R. W. Sutter, B. M. de Gourville, W. R. Dowdle and M. A. Pallansch, “Vaccine Derived PolioViruses and the End Game Strategy for Global Polio Eradication,” Annual Review of Microbiology, Vol. 59, 2005, pp. 587-635.
[20] S. Mamishi, S. Shahmahmoudi, H. Tabatabaie, S. Teimourian, B. Pourakbari, Y. Gherisari, M. Yeganeh, A. Salavati, A. R. Esteghamati, M. M. Gooya, R. Nategh and N. Parvaneh, “Novel BTK Mutation Presenting With Vaccine-Associated Paralytic Poliomyelitis,” European Journal of Pediatrics, Vol. 167, No. 11, 2008, pp. 1335-1338.
[21] M. Giese, “DNA-Antiviral Vaccines: New Developments and Approaches—A Review,” Virus Genes, Vol. 17, No. 3, 1998, pp. 219-232.
[22] C. N. Wu, Y. C. Lin, C. Fann, N. S. Liao, S. R. Shih and M. S. Ho, “Protection Against Lethal Enterovirus 71 Infection in Newborn Mice By Passive Immunization With Subunit VP1 Vaccines and Inactivated Virus,” Vaccine, Vol. 20, No. 5-6, 2001, pp. 895-904.
[23] S. T. Wong, S. AbuBakar, Z. Sekawi and R. Rosli, “DNA Vaccine Constructs against Enterovirus 71 Elicit Immune Response in Mice,” Genetic Vaccines and Therapy, 2007, Vol. 5, p. 6.
[24] W. W. Leitner, H. Ying and N. P. Restifo, “DNA and RNA-Based Vaccines: Principles, Progress and Prospects,” Vaccine, Vol. 18, No. 9-10, 1999, pp. 765-777.
[25] S. Manoj, L. A. Babiuk and S. D. Littel-van den Hurk, “Approaches to Enhance the Efficacy of DNA Vaccines,” Critical Reviews in Clinical Laboratory Sciences, Vol. 41, No. 1, 2004, pp.1-39.
[26] J. Běláková, M. Horynová, M. K?upka, E. Weigl and M. Ra?ka, “DNA Vaccines: Are They Still Just a Powerful Tool For The Future?” Archivum Immunologiae et Therapia Experimentalis, Vol. 55, No. 6, 2007, pp. 387-398.
[27] N. Y. Sardesai and D. B. Weiner, “Electroporation Delivery of DNA Vaccines: Prospects for Success,” Current Opinion in Immunology, Vol. 23, No. 3, 2011, pp. 421-429.
[28] K. Oosterhuis, J. H. van den Berg, T. N. Schumacher and J. B. Haanen, “DNA Vaccines and Intradermal Vaccination By DNA Tattooing,” Current Topic in Microbiology and Immunology, Vol 351, 2012, pp. 221-250.
[29] S. A. Kalams, S. D. Parker, M. Elizaga, B. Metch, S. Edupuganti, J. Hural, S. De Rosa, D. K. Carer, K. Rybczyk, I. Frank, J. Fuchs, B. Koblin, D. H. Kim, P. Joseph, M. C. Keefer, L. R. Baden, J. Eldridge, J. Boyer, A. Sherwat, M. Cardinali, M. Allen, M. Pensiero, C. Butler, A. S. Khan, J. Yan, N. Y. Sardesai, J. G. Kublin, D. B. Weiner and the NIAID HIV Vaccine Trials Network, “Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination With Plasmid Interleukin 12 and Impact of Intramuscular Electroporation For Delivery,” The Journal of Infectious Diseases, Vol. 208, No. 5, 2013, pp. 818-829.
[30] E. J. Yager, C. Stagnar, R. Gopalakrishnan, J. T. Fuller and D. H. Full, “Optimizing Particle-Mediated Epidermal Delivery of an Influenza DNA Vaccine in Ferrets,” In: S. Sudowe and A. B. Reske-Kunz, Eds., Biolistic DNA Delivery: Methods and Protocols, Methods in Molecular Biology, Springer Science+Business Media, Vol. 940, 2013, pp. 223-237.
[31] V. Douin, S. Bornes, L. Creancier, P. Rochaix, G. Favre, A. C. Prats and B. Couderc, “Use and Comparison of Different Internal Ribosomal Entry Sites (IRES) in Tricistronic Retroviral Vectors,” BMC Biotechnology, 2004, Vol. 4, p. 16.
[32] J. C. Lee, T. Y. Wu, C. F. Huang, F. M. Yang, S. R. Shih and J. T. A. Hsu, “High-Efficiency Protein Expression Mediated By Enterovirus 71 Internal Ribosome Entry Site,” Biotechnology and Bioengineering, Vol. 90, No. 5, 2005, pp. 656-662.
[33] C. Jünemann, Y. Song, G. Bassili, D. Goergen, J. Henke and M. Niepmann, “Picornavirus Internal Ribosome Entry Site Elements Can Stimulate Translation of Upstream Genes,” The Journal of Biological Chemistry, Vol. 282, No. 1, 2007, pp. 132-141.
[34] J. Attal, M. C. Theron and L. M. Houdebine, “The Optimal Use of IRES (Internal Ribosome Entry Site) in Expression Vectors,” Genetic Analysis: biomolecular engineering, Vol. 15, No. 3-5, 1999, pp. 161-165.
[35] L. K. Johansen and C. D. Morrow, “Inherent Instability of Poliovirus Genomes Containing Two Internal Ribosome Entry Site (IRES) Elements Supports a Role For the IRES in Encapsidation,” Journal of Virology, Vol.74, No. 18, 2000, pp. 8335-8342.
[36] E. Martínez-Salas, R. Ramos, E. Lafuente and S. López de Quinto, “Functional Interactions in Internal Translation Initiation Directed by Viral and Cellular IRES Element,” The Journal of General Virology, Vol. 82, No. 5, 2001, pp. 973-984.
[37] E. Martínez-Salas, S. López de Quinto, R. Ramos and O. Fernández-Miragall, “IRES Elements: Features of The RNA Structure Contributing to Their Activity,” Biochimie, Vol. 84, No. 8, 2002, pp. 755-763.
[38] E. Y. Dobrikova, P. Flores and M. Gromeier, “Structural Determinants of Insert Retention of Poliovirus Expression Vectors with Recombinant IRES Elements,” Virology, Vol. 311, No. 2, 2003, pp. 241-253.
[39] K. Choi, J. H. Kim, X. Li, K. Y. Peak, S. H. Ha, S. H. Ryu, E. Wimmer and S. K. Jang, “Identification of Cellular Proteins Enhancing Activities of Internal Ribosomal Enty Sites by Competition With Oligodeoxynucleotides,” Nucleic Acids Research, Vol. 32, No. 4, 2004, pp. 1308-1317.
[40] N. Shafee and S. AbuBakar, “Immunization With DNA Vectors Consisting of Selected Dengue 2 Virus Genes Stimulated Antibody Responses in Mice,” International Journal of Virology, Vol. 2, No. 1, 2006, pp. 14-20.
[41] V. Vorndam and M. Beltran, “Enzyme-Linked Immunosorbent Assay-Format Microneutralization Test for Dengue Viruses,” The American Journal of Tropical Medicine Hygiene, Vol. 66, No. 2, 2002, pp. 208-212.
[42] A. Jaegly, F. Mouthon, J. M. Peyrin, B. Camugli, J. P. Deslys and D. Dormont, “Search For a Nuclear Localization Signal in the Prion Protein,” Molecular and Cellular Neuroscience, Vol. 11, No. 3, 1998, pp. 127-133.
[43] A. B. Sachs, “Cell Cycle-Dependent Translation Initiation: IRES Elements Prevail,” Cell, Vol. 101, No. 3, 2000, pp. 243-245.
[44] M. J. Gale, S. L. Tan and M. G. Katze, “Translational Control of Viral Gene Expression in Eukaryotes,” Microbiology and Molecular Biology Review, Vol. 64, No. 2, 2000, pp. 239-280.
[45] K. M. Kean, “The Role of mRNA 5’-Noncoding and 3’- End Sequences on 40S Ribosomal Subunit Recruitment, and How RNA Viruses Successfully Compete With Cellular mRNAs To Ensure Their Own Protein Synthesis,” Biology of the Cell, Vol. 95, No. 3-4, 2003, pp. 129-139.
[46] A. M. Borman, P. L. Mercier, M. Girard and M. Kean, “Comparison of Picornaviral IRES-Driven Internal Initiation of Translation in Cultured Cells of Different Origins,” Nucleic Acids Research, Vol. 25, No. 5, 1997, pp. 925-932.
[47] A. Tanghe, O. Denis, B. Lamvrecht, V. Motte, T. van de Berg and K. Huygen, “Tuberculosis DNA Vaccine Encoding Ag85A Is Immunogenic and Protective When Administered By Intramuscular Needle Injection But Not by Epidermal Gene Gun Bombardment,” Infection and Immunity, Vol. 68, No. 7, 2000, pp. 3854-3860.
[48] C. K. Yu, C. C. Chen, C. L. Chen, J. R. Wang, C. C. Liu, J. J. Yan and I. J. Su, “Neutralizing Antibody Provided Protection Against Enterovirus Type 71 Lethal Challenge in Neonatal Mice,” Journal of Biomedical Science, Vol. 7, No. 6, 2000, pp. 523-528.
[49] R. Buglione-Corbett, K. Pouliot, R. Marty-Roix, K. West, S. Wang, E. Lien and S. Lu, “Serum Cytokine Profiles Associated With Specific Adjuvants Used in a DNA Prime-Protein Boost Vaccination Strategy,” PLoS ONE, Vol. 8, No. 9, 2013, Article ID: e74820.
[50] P. Muir, U. Kammerer, K. Korn, M. N. Mulders, T. Poyry, B. Weissbrich, R. Kandolf, G. M. Cleator and A. M. van Loon, “Molecular Typing of Enteroviruses: Current Status and Future Requirements,” Clinical Microbiology Reviews, Vol. 11, No. 1, 1998, pp. 202-227.
[51] Y. C. Chung, M. S. Ho, J. C. Wu, W. J. Chen, J. H. Huang, S. T. Chou and Y. H. Hu, “Immunization with Virus-Like Particles of Enterovirus 71 Elicits Potent Immune Responses and Protects Mice Against Lethal Challenge,” Vaccine, Vol. 26, No. 15, 2008, pp. 1855-1862.
[52] K. Fujii, N. Nagata, Y. Sato, K. C. Ong, K. T. Wong, S. Yamayoshi, M. Shimanuki, H. Shitara, C. Taya and S. Koike, “Transgenic Mouse Model for The Study of Entero-virus 71 Neuropathogenesis,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 36, 2013, pp. 14753-14758.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.