Giuseppe
Emeritus
[Source: PLoS ONE, full text: (LINK). Abstract, edited.]
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Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles
Pele Chong<SUP>1</SUP><SUP>,</SUP><SUP>2</SUP><SUP>*</SUP>, Meng-Shin Guo<SUP>1</SUP>, Fion Hsiao-Yu Lin<SUP>1</SUP>, Kuang-Nan Hsiao<SUP>1</SUP>, Shu-Yang Weng<SUP>1</SUP>, Ai-Hsiang Chou<SUP>1</SUP>, Jen-Ren Wang<SUP>3</SUP>, Shih-Yang Hsieh<SUP>1</SUP>, Ih-Jen Su<SUP>1</SUP>, Chia-Chyi Liu<SUP>1</SUP><SUP>*</SUP>
<SUP></SUP>
1 Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, 2 Graduate Institute of Immunology, China Medical University, Taichung, Taiwan, 3 Department of Medical Laboratory Science and Biotechnology, College of Medicine National Cheng Kung University, Tainan, Taiwan
Abstract
Background
Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.
Principal Finding
In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10<SUP>6</SUP> the tissue culture's infectious dose (TCID<SUB>50</SUB>) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10<SUP>−5</SUP> was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24?28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35?38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176?190.
Conclusion
These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.
Citation: Chong P, Guo M-S, Lin FH-Y, Hsiao K-N, Weng S-Y, et al. (2012) Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles. PLoS ONE 7(11): e49973. doi:10.1371/journal.pone.0049973
Editor: Sylvie Alonso, National University of Singapore, Singapore
Received: August 21, 2012; Accepted: October 15, 2012; Published: November 30, 2012
Copyright: ? 2012 Chong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant (#99A1-VCSP01-014) from the National Science Council (NSC) of Taiwan and a grant (#100A1-VC-PP-10-014) from NHRI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: pelechong@nhri.org.tw (PC); georgeliu@nhri.org.tw (CCL)
-Pele Chong<SUP>1</SUP><SUP>,</SUP><SUP>2</SUP><SUP>*</SUP>, Meng-Shin Guo<SUP>1</SUP>, Fion Hsiao-Yu Lin<SUP>1</SUP>, Kuang-Nan Hsiao<SUP>1</SUP>, Shu-Yang Weng<SUP>1</SUP>, Ai-Hsiang Chou<SUP>1</SUP>, Jen-Ren Wang<SUP>3</SUP>, Shih-Yang Hsieh<SUP>1</SUP>, Ih-Jen Su<SUP>1</SUP>, Chia-Chyi Liu<SUP>1</SUP><SUP>*</SUP>
<SUP></SUP>
1 Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, 2 Graduate Institute of Immunology, China Medical University, Taichung, Taiwan, 3 Department of Medical Laboratory Science and Biotechnology, College of Medicine National Cheng Kung University, Tainan, Taiwan
Abstract
Background
Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.
Principal Finding
In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10<SUP>6</SUP> the tissue culture's infectious dose (TCID<SUB>50</SUB>) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10<SUP>−5</SUP> was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24?28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35?38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176?190.
Conclusion
These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.
Citation: Chong P, Guo M-S, Lin FH-Y, Hsiao K-N, Weng S-Y, et al. (2012) Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles. PLoS ONE 7(11): e49973. doi:10.1371/journal.pone.0049973
Editor: Sylvie Alonso, National University of Singapore, Singapore
Received: August 21, 2012; Accepted: October 15, 2012; Published: November 30, 2012
Copyright: ? 2012 Chong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant (#99A1-VCSP01-014) from the National Science Council (NSC) of Taiwan and a grant (#100A1-VC-PP-10-014) from NHRI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: pelechong@nhri.org.tw (PC); georgeliu@nhri.org.tw (CCL)
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