Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection
- Guo-hui Chang1Email author,
- Yan-jun Luo†1,
- Xiao-yan Wu†1,
- Bing-yin Si†1,
- Lei Lin†1 and
- Qing-yu Zhu†1
© Chang et al; licensee BioMed Central Ltd. 2010
Received: 6 April 2010
Accepted: 26 May 2010
Published: 26 May 2010
Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death.
In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus.
The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.
Enterovirus 71 (EV71) is a viral pathogen within the Picornaviridae family and causes clinical diseases in humans with manifestations such as herpangina, aseptic meningitis, encephalitis, pulmonary edema and hand, foot and mouth disease (HFMD). EV71-infected children can develop severe neurological complications that lead to rapid clinical deterioration and death . Since the first instances in 1969 , several large epidemics of HFMD have been reported in the Asia-Pacific region, especially in Southeast Asia [3–6]. In China, between 1999-2009, HFMD outbreaks caused by EV71 have affected more than 500,000 young children and resulted in more than 200 deaths in cities, such as Beijing, Shenzhen, Guangdong [7–9]. In fact, after the eradication of the poliovirus , EV71 has been regarded as the most important neurotropic enterovirus.
Since there is no EV71 vaccine available and treatment is very limited, a humanized monoclonal antibody might be a viable treatment option against EV71 infection in humans. Anti-EV71 MAbs which have specificity and neutralizing activity could be a promising candidate to be humanized and used for treatment of EV71 infection.
EV71 contains a positive-stranded RNA enclosed by capsid proteins VP1, VP2, VP3 and VP4. VP1 is composed of 297 amino acids and has been shown to be immunogenic . It has been reported that the synthetic peptides SP55 and SP70, which contain amino acids 163-177 and 208-222 of VP1, respectively, can elicit neutralizing antibody against EV71 infection [12, 13]. Also, immunization using a recombinant VP1 protein of EV71 was shown to confer protection against lethal EV71 infection in newborn mice, indicating that VP1 contains important antigenic sites that contribute to the neutralization of the virus [14, 15].
In this study, we generated several MAbs by immunizing mice with purified EV71 virus, strain Henan2 (Hn2). These MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. We identified a monoclonal antibody, clone 4E8 with strong neutralizing activity against EV71. The MAb 4E8 specifically reacted with synthetic peptides which containing amino acids 240-250 and 250-260 of VP1 by ELISA assay. The in vivo protection test showed 4E8 can partialy protect the mice against the lethal challenge of Hn2 virus.
50% lethal dose (LD50) assay
Neutralizing antibody assay
In vitro neutralization assays of monoclonal antibodies*
Convalscent patient serum
Normal mouse serum
Identification of monoclonal antibody epitopes by peptide-coated ELISA
Epitope-specificity assay of monoclonal antibody by ELISA*
Normal mouse serum
Amino acid sequence analysis of EV71 Hn2 VP1
Passive protection test in mice
The genus Enterovirus, belongs to the family Picornaviridae, and consists of 66 different subtypes, including polioviruses (PVs), coxsackievirus group A (CVA) and coxsackievirus group B (CVB), echoviruses, and enteroviruses [17, 18]. EV-71 was first described in 1969 during an outbreak of HFMD in California that was associated with central nervous system (CNS) complications. EV71 infections are generally mild, such as HFMD and herpangina, but occasionally lead to severe diseases with CNS involvement and clinical symptoms such as aseptic meningitis, poliomyelitis-like paralysis and possibly fatal encephalitis in neonates . Passive transfer of specific antibodies has been shown to reduce the severity of viral infections, including Japanese encephalitis infection , varicella infection  and coxsackievirus infection . High-quality MAbs which have specificity, avidity and neutralizing activity might be a viable treatment option for EV71 infection in humans.
Previous studies have shown the neutralizing epitope of EV71 was mainly located at the VP1 protein. So we selected synthetic VP1 peptides as the antigen to screen a set of MAbs that we had generated by immunization with inactived EV71 isolated strain Hn2. Among the MAbs we characterised, four were shown to have neutralizing activity, and two can react specifically with the E.coli expressed VP1 protein and synthetic peptides of VP1. Experiments are currently in progress to see if the neutralizing MAbs that do not react with VP1 may neutralize the virus by alternative routes, for example, by preventing uncoating of the virus by binding to neighbouring capsid pentamers or by forming aggregates of the viruses as has been observed in other picornaviruses [23, 24].
Computerized antigenicity prediction method as used in this study can predict antigenic determinants with about 75% accuracy. Four of the seven epitopes predicted by this method in the EV71 VP1 protein are located at the N-terminus, and only one at the C-terminus. This result is consistent with reports that show the region spanning amino acids 66-132 of VP1 contains the major dimerization domain and cross-reactive enterovirus epitopes . However, there are also reports show that the region between amino acids 132 and 297 is indispensable, and contains important conformational epitope which can react with the convalescent-phase sera . In this study, we have shown that the MAbs 4C6 and 4E8 can specifically react with peptides P25(aa240-250) and P26(aa250-260), one of which overlaps with the predicted C-terminus epitope. Whether the two peptides form a conformational epitope or just a linear neutralizing epitope requires futher research. Nevertheless, it is important that we have shown the 4E8 MAb was able to protect suckling BALB/c mice from a lethal dose of EV71 Hn2 virus, and, therefore, could be a promising candidate to be humanized and used for treatment of EV71 infection.
In this study, we generated a set of MAbs by immunization BALB/c mice with inactived EV71 isolated strain Hn2. Among the MAbs we characterised, four were shown to have neutralizing activity, and two can react specifically with the E.coli expressed VP1 protein and synthetic peptides P25(aa240-250) and P26(aa250-260) of VP1. It is important that we have shown the 4E8 MAb was able to protect suckling BALB/c mice from a lethal dose of EV71 Hn2 virus, and, therefore, could be a promising candidate to be humanized and used for treatment of EV71 infection.
Materials and methods
Cells and viruses
Rhabdomyosarcoma cells (RD cell) and Vero cells (derived from African green monkey kidney) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) plus 2 mM L-glutamine, 100U of penicillin, and 100 μg of streptomycin per ml. The EV71 Hn2 strain was isolated from the anal swabs of one HFMD patient from Henan province, P.R.China in 2009. The patient had the typical clinical symptoms of central nervous system (CNS) involvement, such as fever, exanthemas, aseptic meningitis, and stiffness of the neck . The Hn2 strain was C4 genotype and the complete sequence of Hn2 was submitted to GenBank as accession no. GQ994992.
Virus purification and titer determination
The isolated Hn2 virus were purified by three rounds of plaque assay in RD cells, then propagated in Vero cells. The infected cells were harvested and completely lysed by three cycles of freeze-thaw. The virus from the tissue culture was purified by precipitation with 7% polyethylene glycol 8000 and then centrifuged onto a 30% sucrose cushion at 25,000 g for 4 h. Virus titers were determined as the median end-point of the tissue culture infectious dose (TCID50). Serially diluted virus samples (from 10-2 to 10-9) were added to Vero cells in 96-well plates, and eight wells were used at each dilution. The 96-well plates were incubated for 7 days at 37°C, and the TCID50 values were measured by determining CPE. The TCID50 values were calculated by the method of Reed-Muench .
Determination of LD50 in mice
To determine the 50% lethal dose (LD50), specific-pathogen-free, two-day-old BALB/c mice were inoculated intraperitoneally with 100 μl of serially diluted Hn2 virus. The survival of the mice was monitored daily, and the LD50 was calculated as described by Reed-Muench .
Production of monoclonal antibody
The purified Hn2 virus was inactivated by heating at 56°C for 30 min. Groups of 5 adult (4 weeks old) female BALB/c mice were immunized intraperitoneally in a 50% emulsion of Freund's complete adjuvant with 10 μg heat-inactivated EV71 virus. Two booster doses, again in 50% emulsion with Freund's incomplete adjuvant, were given at 2 weeks intervals. Two weeks after the last immunization, blood samples were obtained from the tail and tested by enzyme-linked immunosorbent assay (ELISA) using purified Hn2 virus as antigen. The fusion of spleen cells with mouse myeloma cell was done as described . For the production of MAbs, BALB/c mice (4-6 weeks old) were injected intraperitoneally with 106 hybridoma cells per mouse. Ten days later, the ascitic fluid was drained by using an 18-gauge needle. The ascites were centrifuged at 1,000 g for 10 min to remove cells, MAbs were purified from collected supernatants by precipitation with 50% saturated ammonium sulfate (pH 7.0) and dialysised against 0.04M phosphate buffer (pH 6.8). The MAbs were further purified by using protein A agarose columns (Bio-Rad Laboratories).
In vitro neutralizing antibody assay
The presence of neutralizing MAbs was determined by in vitro assay in Vero cell. Monoclonal antibody samples were incubated at 56°C for 30 min to inactivate the complement. Briefly, 50 μl of two-fold antibody dilutions were mixed with equal volumes of 100TCID50 of Hn2 virus, and then the mixture was added to monolayers of Vero cells in 96-well plates. All serum samples were tested in 8 wells. After 3 days of growth, the titer of neutralizing antibody was read as the highest dilution that gave complete protection from CPE.
Peptide ELISA assay
Synthetic peptides spanning the entire sequence of the Hn2 VP1 protein (GenBank accession no. GQ994992) were synthesized by Invitrogen Company (Beijing, P.R.China). Each peptide contains 10 amino acid residues and the levels of antibody reactive to each synthetic peptide were measured by ELISA assay. The 96-well plates were coated overnight at 4°C with 50 μl of 0.1M carbonate buffer (pH 9.6) containing 500 ng of synthetic peptide or E.coli expressed EV71 VP1 protein (supplied by Professor Hanzhong Wang, Wuhan Institute of Virology, Chinese Academy of Science). After blocking with 2% bovine serum albumin (BSA), the plates were incubated with 50 μl of MAb at the indicated dilutions at 37°C for 1 h and then washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody (SantaCruze, USA) was used as secondary antibody at 37°C for 1 h and the reaction was developed by TMB substrate (KPL, Gaithersburg, MD). The absorbance at 450 nm was measured by an ELISA plate reader (Tecan Sunrise, USA). Each assay was performed three times independently.
Passive protection test in mice
Two-day-old BALB/c mice were obtained from the Laboratory Animal Center, Academy of Military Medical Sciences, Beijing. Institutional guidelines for animal care and use were followed throughout the experiments. Groups of mice were injected intraperitoneally with 100 μl (100LD50) of Hn2 virus, followed 24 h later by 100 μl of heat-treated (56°C, 30 min) MAb. Mice in the control group were injected with the same volume of normal mice serum. The mice were monitored for clinical symptoms, paralysis and death up to day 21 post-inoculation.
Computational analysis of VP1 sequence
Nucleotide sequence and the amino acid sequences were edited and aligned using CLC Main Workbench 5.5 software. The antigenicity of the Hn2 VP1 protein were analyzed using the method of Kolaskar and Tongaonkar , and antigen epitopes were predicted using the amino acid sequences of the Hn2 VP1 as query sequences in the Web: http://www.mifoundation.org
This work is supported by Research Grant SKLPBS0919 from State Key Laboratory of Pathogen and Biosecurity.
We thank Prof. Stuart Siddell for help in editing.
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