Development of a high-throughput assay for measuring serum neutralizing antibody against enterovirus 71

Huang, Ming-Hsi; Chiang, Pai-Shan; Luo, Shu-Ting; Liou, Guan-Yuan; Lee, Min-Shi

doi:10.1016/j.jviromet.2009.12.015

Cited by 27 publications

(19 citation statements)

References 16 publications

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“…Because EV71 is mainly a human pathogen and has no animal model resembling natural EV71 infection in humans, our work on Anx2 so far has been based solely on the available in vitro infection and neutralization assays; these assays are also commonly applied in the research and development of EV71 vaccines (15,20,28,50). While Anx2 is widely expressed in cell lines commonly used for EV71 amplification in the laboratory, immunohistochemical study of fatal EV71 infection has demonstrated that EV71 antigen was selectively detected only in human neurons at the time of death (5,52).…”

Section: Discussionmentioning

confidence: 99%

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Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity

Yang

Chou

et al. 2011

J Virol

160

131

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Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose.Enterovirus type 71 (EV71) is a member of the Enterovirus genus of the family Picornaviridae and is one of the causative viral agents of hand, foot, and mouth disease (HFMD) (6,7,14,16,41). HFMD is largely a common self-limited childhood illness but may have complications of severe to fatal neurological symptoms in some children (1,5,6,16,21). In the past decade, the frequency of EV71 outbreaks associated with severe neurological illness appeared to have increased in the Pacific region, most notably in China, where large outbreaks have been occurring annually since 2007 (24, 53). While the epidemiological or virological mechanism underlying this regional focus of severe EV71 infection remains largely unknown, the impact of EV71 infection is a global concern, as evidenced by the increase in virological surveillance and studies of EV71 infection in many regions of the world (29,46,53).EV71 is composed of a single-stranded, positive-polarity RNA molecule surrounded by a nonenveloped, pentameric icosahedral capsid (3, 35), which consists of 60 copies of the four structural proteins VP1 to VP4. While there is no animal model for EV71 infection in humans, intraperitoneal injection of EV71 is lethal to suckling mice. Suckling mice born to mothers previously immunized with VP1 subunit vaccines acquire resistance to lethal EV71 challenge (8, 50), and administration of a VP1-based antigen, either protein or DNA, to mice could elicit a neutralizing antibody against EV71 infection in vitro (8,42,44). The serum collected from EV71-infected individuals during the convalescent pha...

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Section: Discussionmentioning

confidence: 99%

“…7A) and was used to determine EV71 infectivity. HepG2 and HepG2-Anx2 cells were infected with various EV71 titers (MOI, 0.001, 0.01, and 0.1) and were given three different periods (15,30, and 60 min) to allow adsorption of the virus onto the cell surface (Fig. 7B).…”

Section: Vol 85 2011 Annexin II Enhances Enterovirus 71 Infectivitymentioning

confidence: 99%

Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity

Yang

Chou

et al. 2011

J Virol

160

131

View full text Add to dashboard Cite

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“…Serum neutralizing antibody titers were detected using TCID 50 assay according to the standard protocol [28]. Serum samples from immunized rabbits were inactivated for 30 min at 56°C, and then diluted two-fold serially in DMEM with an initial dilution of 1∶8.…”

Section: Methodsmentioning

confidence: 99%

Monitoring Antigenic Variations of Enterovirus 71: Implications for Virus Surveillance and Vaccine Development

et al. 2014

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Enterovirus 71 (EV71) causes life-threatening epidemics in Asia and can be phylogenetically classified into three major genogroups (A∼C) including 11 genotypes (A, B1∼B5, and C1∼C5). Recently, EV71 epidemics occurred cyclically in Taiwan with different genotypes. In recent years, human studies using post-infection sera obtained from children have detected antigenic variations among different EV71 strains. Therefore, surveillance of enterovirus 71 should include phylogenetic and antigenic analysis. Due to limitation of sera available from children with EV71 primary infection, suitable animal models should be developed to generate a panel of antisera for monitoring EV71 antigenic variations. Twelve reference strains representing the 11 EV71 genotypes were grown in rhabdomyosarcoma cells. Infectious EV71 particles were purified and collected to immunize rabbits. The rabbit antisera were then employed to measure neutralizing antibody titers against the 12 reference strains and 5 recent strains. Rabbits immunized with genogroup B and C viruses consistently have a lower neutralizing antibody titers against genogroup A (≧8-fold difference) and antigenic variations between genogroup B and C viruses can be detected but did not have a clear pattern, which are consistent with previous human studies. Comparison between human and rabbit neutralizing antibody profiles, the results showed that ≧8-fold difference in rabbit cross-reactive antibody ratios could be used to screen EV71 isolates for identifying potential antigenic variants. In conclusion, a rabbit model was developed to monitor antigenic variations of EV71, which are critical to select vaccine strains and predict epidemics.

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“…The third cell-based neutralization method is the immunological method. Immunological methods have been widely used in neutralization tests, such as ELISA (35)(36)(37) and the immunofluorescence assay (38,39). These tests rely on antibodies to recognize the infected cells that express viral proteins.…”

Section: Discussionmentioning

confidence: 99%

Development of an Enzyme-Linked Immunosorbent Spot Assay To Measure Serum-Neutralizing Antibodies against Coxsackievirus B3

Yang

Tang

et al. 2014

Clin. Vaccine Immunol.

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Coxsackievirus B3 (CVB3) is the most common pathogen that induces acute and chronic viral myocarditis in children. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) and the plaque reduction neutralization test (PRNT) are the most common methods for measuring neutralizing antibody titers against CVB3 in blood serum samples. However, these two methods are inefficient for CVB3 vaccine clinical trials, which require the testing of a large number of serum specimens. In this study, we developed an efficient neutralization test based on the enzyme-linked immunospot (Nt-ELISPOT) assay for measuring CVB3-neutralizing antibodies. This modified ELISPOT assay was based on the use of a monoclonal antibody against the viral capsid protein VP1 to detect the cells that are infected with CVB3, which, after immunoperoxidase staining, are counted as spots using an automated ELISPOT analyzer. Using the modified ELISPOT assay, we characterized the infection kinetics of CVB3 and divided the infection process of CVB3 on a cluster of cells into four phases. The stability of the Nt-ELISPOT was then evaluated. We found that over a wide range of infectious doses (10 2 to 10 6.5 ؋ 50% tissue culture infectious dose [TCID 50 ] per well), the neutralizing titers of the sera were steady as long as they were tested during the log phase or the first half of the stationary phase of growth of the spots. We successfully shortened the testing period from 7 days to approximately 20 h. We also found that there was a good correlation (R 2 ‫؍‬ 0.9462) between the Nt-ELISPOT and the Nt-CPE assays. Overall, the Nt-ELISPOT assay is a reliable and efficient method for measuring neutralizing antibodies in serum.

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Development of a high-throughput assay for measuring serum neutralizing antibody against enterovirus 71

Cited by 27 publications

References 16 publications

Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity

Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity

Monitoring Antigenic Variations of Enterovirus 71: Implications for Virus Surveillance and Vaccine Development

Development of an Enzyme-Linked Immunosorbent Spot Assay To Measure Serum-Neutralizing Antibodies against Coxsackievirus B3

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