Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6652-8


Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71 Yong-Qiang Deng 1 & Jie Ma 1 & Li-Juan Xu 1 & Yue-Xiang Li 1,2 & Hui Zhao 1 & Jian-Feng Han 1 & Jiang Tao 1 & Xiao-Feng Li 1 & Shun-Ya Zhu 1 & E-De Qin 1 & Cheng-Feng Qin 1,2

Received: 26 March 2015 / Revised: 23 April 2015 / Accepted: 26 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibodybased treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208– 222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with Yong-Qiang Deng and Jie Ma contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6652-8) contains supplementary material, which is available to authorized users. * Cheng-Feng Qin [email protected] 1

Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China


Graduate School, Anhui Medical University, Hefei 230032, China

human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism. Keywords Human enterovirus 71 . VP1 protein . SP70 peptide . Neutralizing antibody

Introduction Human enterovirus 71 (EV71) belongs to a member of the Enterovirus genus of the Picornaviridae family. It has one single serotype but can be phylogenetically classified into three genogroups (A, B, and C) and 11 subgenotypes (A, B1 to B5, and C1 to C5) (Zhang et al. 2010). EV71 is a major causative agent of hand, foot, and mouth disease (HFMD), which most frequently affects young adults and children (Solomon et al. 2010). EV71 infections have been closely associated with neurologic complications such as aseptic meningitis, fatal encephalitis, poliomyelitis-like paralysis, and even deaths. Over the past decade, the magnitude and frequency of EV71 outbreaks have substantially increased and now being an important public health issue, especially in the AsiaPacific regions (McMinn 2002; Wang et al. 2013a). A few inactivated EV71 vaccine products have finished the final stage of clinical trials (Zhu et al. 2013, 2014). However, a vaccine is not yet available, and there is currently no effective antiviral treatment for severe EV71 infections (Bek and McMinn 2010). EV71 viruses are small, non-enveloped, icosahedral particles that contain a single-stranded, positive-sense, polyadenylated genome RNA (McMinn 2012). The crystal

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structure of EV71 virion shows that 60 copies each of three viral structural proteins VP1–VP3 form an outer capsid and 60 copies of VP4 attach to the inner surface of the capsid (Plevka et al. 2012; Wang et al. 2012). Among these capsid proteins, the VP1 protein is exposed on the virion surface and is believed to be the major contributor in adsorption, uncoating, assembly, and neutralization. Especially, the VP1 protein contains many well-characterized linear and conformational neutralizing epitopes, such as SP55 and SP70 peptides, which span amino acids 163–177 and 208–222, respectively (Foo et al. 2007b; Li et al. 2009). Thus, VP1 has usually been considered as an ideal target for eliciting a protective immune response against EV71 infection (Chang et al. 2010, 2011; Li et al. 2009; Ye et al. 2014). Antibody-based immunotherapy represents an alternative option for the control of EV71 infection. Previously, polyclonal antibodies or intravenous immunoglobulin with high-titer neutralization antibodies show good efficacy in treatment of EV71 in mouse model, and some of them have been administrated in clinical use (Cao et al. 2011; Foo et al. 2007a, b, 2008). To date, a number of EV71-specific mAbs with neutralization capability have been well described, and most of them target the VP1 protein (Chang et al. 2010, 2011; Ku et al. 2012; Li et al. 2009; Lim et al. 2012; Man-Li et al. 2012). Recently, the cryo-electron microscopy structure of a complex between EV71 and mAb MA28-7 identified strainspecific neutralizing epitopes located on a surfaceexposed amino acid residue VP1-145 and in the positively charged patches (VP1-98, 242, and 244) around the fivefold axis (Lee et al. 2013). However, antibody-based neutralization mechanism remains largely unknown, and only a few neutralization determinants on EV71 virions have been identified. In this study, we generated an EV71-specific neutralizing mAb 2G8 that could protect Balb/C suckling mice from lethal EV71 infection. Fine epitope mapping and reverse genetic analyses demonstrated the critical roles of the VP1 protein for 2G8 binding and neutralization. Importantly, recombinant EV71 with K218A or L220A mutation escaped the neutralizing of human sera from natural EV71 infection. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.

Materials and methods Cells and viruses Human rhabdomyosarcoma (RD) cells and Africa green monkey kidney Vero cells were maintained in Dulbecco’s modified essential medium supplemented with 10 % fetal

bovine serum (FBS) plus 100 U of penicillin and 100 μg of streptomycin/ml. EV71 strain AH/08/06 (GenBank No. HQ611148), classified as subtype C4, was isolated from an HFMD patient during an HFMD outbreak in 2008 in Anhui, China (Han et al. 2010). The viral stocks were prepared in RD cells, and virus titers (50 % tissue culture infectious doses (TCID50)) were determined by using the Reed-Muench method. Monoclonal antibody preparation Six-week-old female BALB/c mice were subcutaneously immunized twice at 3-week intervals with 400 μl of heatinactivated EV71 emulsified in Freund’s complete or incomplete adjuvant (Sigma). Three days after the final immunization, spleen cells from the mice and mouse myeloma SP2/0 cells were fused and maintained according to the standard procedure (Deng et al. 2011). Hybridomas were screened for secretion of anti-EV71-specific mAbs by indirect enzymelinked immunosorbent assay (ELISA) using EV71 virus as antigen. The hybridomas that secreted specific mAbs were then cloned twice by limited dilution of the cells. The isotype of each mAb was determined by IsoStrip (Roche Diagnostics). Indirect immunofluorescence assay Indirect immunofluorescence assays were performed as follows. Briefly, Vero cells were infected with EV71 and CA16 and fixed with ice-cold acetone. Cells were incubated with a 100-fold dilution of mAbs or normal mouse serum for 60 min and washed three times in 0.05 % Tween containing phosphate-buffered saline (PBST). Cells were then treated with FITC-conjugated anti-mouse secondary antibody (1:200) in PBS for 30 min and rinsed five times with PBST. Positive cells were detected under a fluorescent microscope. All steps were performed at room temperature. Western blotting The yeast-expressed virus-like particles (VLPs) (Li et al. 2013) and Escherichia coli-expressed VP1, VP2, and VP3 proteins of EV71 were separated using 12 % SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with PBST containing 5 % skimmed milk and then reacted with the appropriate mAb 2G8 for 1 h. Subsequently, membranes were washed three times with PBST and incubated with alkaline phosphataseconjugated goat anti-mouse secondary antibody (1:1000). Following incubation for 1 h, the membranes were washed three times with PBST and visualized with NBT/BCIP substrate (KPL). All steps were performed at room temperature.

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Peptide ELISA For peptide ELISA, 96-well microtiter plates were coated with 400 ng of the various synthetic peptides synthesized by BoTai Biochem Ltd, including two synthetic SP55 and SP70 peptides, and a series of alanine-scanning mutant SP70 peptides in a PBS buffer overnight at 4 °C. Plates were blocked for 2 h with PBST containing 5 % skimmed milk. Subsequently, plates were incubated in turn with 2G8 or control in triplicate and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5000) (Thermo Scientific) for 1 h. Plates were washed and sequentially incubated with 3,3′,5,5′tetramethylbenzidine (TMB) substrate (Promega). The colorimetric reaction was stopped by adding 2 M H2SO4, and emission (450 nm) was read using a microplate reader (Beckman). All steps were performed at room temperature. The means and standard deviations of two independent assays are reported. Binding activities, expressed as a percentage of the reactivity on the wild-type (WT) peptide, are shown on the Y-axis, and the mutated residues are shown on the X-axis. Dot blotting Candidate epitope residues identified by ELISA also were verified by dot blotting. Briefly, 10 μg of WT and alaninescanning mutant SP70 peptides were doted to a nitrocellulose membrane (Pall). The blot was then processed as Western blotting as described above. Signals were visualized by TMB substrate (KPL). All steps were performed at room temperature. Microneutralization assay In vitro neutralization assays were performed according to the standard procedure (Han et al. 2010). Briefly, twofold serial dilutions of mAbs were added to approximately 10 TCID50 of EV71 and incubated at 37 °C for 1 h. Following this incubation, the virus-mAbs mixture were incubated in duplicate with RD cells in 96-well plates and incubated at 37 °C for 1 h. Then, 100 μl of Dulbecco’s modified essential medium (DMEM) containing 2 % FBS was added to wells and incubated at 37 °C with 5 % CO2. After 3 days, the cells were observed to evaluate the appearance of cytopathic effects (CPE). These results were expressed as percentage of neutralization (% neutralization) with standard deviations from two independent experiments. Construction and recovery of recombinant EV71 mutant viruses A series of mutations within the VP1 protein were cloned into the full-length infectious cDNA clone of EV71 strain A12 by using fusion PCR technique as previously described (Wang

et al. 2013b), and the primers used in this study is listed in Table S1. Then, the mutated DNA fragment was cut and ligated into the plasmid with BamH I and Nru I (New England Biolabs). Then, the constructed mutant plasmids were linearized by Mlu I (New England Biolabs) and used as templates for in vitro transcription by using the T7 RiboMAX™ Express Large Scale RNA Production System (Promega) according to the manufacturer’s protocol. RNA transcripts were transfected into RD cells with Lipofectamine 2000 (Invitrogen), and rescued viruses were harvested 3–4 days after transfection. All plasmids mentioned above were confirmed by DNA sequencing. Pre- and post-adsorption inhibition assay Neutralization of EV71 before or after adsorption to RD cells (1 h at 4 °C) was performed using 103 plaque-forming unit (PFU) of EV71 and 2G8 (1:10) as described previously (Ku et al. 2013). In the post-adsorption assay, after washing away the unbound viruses, mAbs were incubated for additional 1 h. All cells were washed three times and cultured in DMEM containing 2 % FBS. After 3 days, the cells were observed to evaluate the appearance of CPE as described above. Animal challenge experiments Groups of 1-day-old BALB/C suckling mice were injected intracerebrally (i.c.) with 20 μl of EV71 (106 TCID50), and 12 h later, 100 μl of heat-treated mAb 2G8 (1:10) was administrated intraperitoneally (i.p.). The control group received the same volume of PBS. The animals were monitored daily for clinical signs of infection, including ruffled hair, hunched back, paralysis, and death, for 3 weeks. Survival curve was graphed by GraphPad Prism version 5 (GraphPad Software). Sequence analysis and molecular modeling Amino acid sequences encoding VP1 genes of all EV71 subgenotypes and CA16 used in this study were retrieved from GenBank. Sequence analysis was performed using MEGALIGN in Lasergene software (DNAstar). The probable epitope mapping of mAb 2G8 was analyzed by using Pymol ( based on the crystal structures of VP1 protein (PDB code: 4AED). Statistical analysis All statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software). For survival analysis, KaplanMeier survival curves were analyzed by the log-rank test in comparison to the PBS control. For neutralization assays, an unpaired Student’s t test was used. Neutralization titers that resulted in 50 % neutralization were determined using non-

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linear regression analysis. Significance was accepted when the P value was

Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71.

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently ava...
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