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DNA vaccine constructs against enterovirus 71 elicit immune response in mice
BACKGROUND: Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives o...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225814/ https://www.ncbi.nlm.nih.gov/pubmed/17445254 http://dx.doi.org/10.1186/1479-0556-5-6 |
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author | Tung, Wong Siew Bakar, Sazaly Abu Sekawi, Zamberi Rosli, Rozita |
author_facet | Tung, Wong Siew Bakar, Sazaly Abu Sekawi, Zamberi Rosli, Rozita |
author_sort | Tung, Wong Siew |
collection | PubMed |
description | BACKGROUND: Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. METHODS: The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. RESULTS: The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. CONCLUSION: The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine. |
format | Online Article Text |
id | pubmed-3225814 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-32258142011-11-30 DNA vaccine constructs against enterovirus 71 elicit immune response in mice Tung, Wong Siew Bakar, Sazaly Abu Sekawi, Zamberi Rosli, Rozita Genet Vaccines Ther Research BACKGROUND: Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. METHODS: The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. RESULTS: The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. CONCLUSION: The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine. BioMed Central 2007-04-19 /pmc/articles/PMC3225814/ /pubmed/17445254 http://dx.doi.org/10.1186/1479-0556-5-6 Text en Copyright ©2007 Tung et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Tung, Wong Siew Bakar, Sazaly Abu Sekawi, Zamberi Rosli, Rozita DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title | DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title_full | DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title_fullStr | DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title_full_unstemmed | DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title_short | DNA vaccine constructs against enterovirus 71 elicit immune response in mice |
title_sort | dna vaccine constructs against enterovirus 71 elicit immune response in mice |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225814/ https://www.ncbi.nlm.nih.gov/pubmed/17445254 http://dx.doi.org/10.1186/1479-0556-5-6 |
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