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Generation of murine monoclonal antibodies which cross-neutralize human enterovirus genogroup B isolates
Journal of Virological Methods 173 (2011) 189–195
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Generation of murine monoclonal antibodies which cross-neutralize human enterovirus genogroup B isolates Hsuen-Wen Chang ∗ , Chia-Chyi Liu, Min-Han Lin, Hui-Min Ho, Ya-Ting Yang, Yen-Hung Chow, Pele Chong, Charles Sia ∗ Vaccine Research and Development Center, National Health Research Institutes, Zhunan Township, Miaoli County 350, Taiwan
a b s t r a c t Article history: Received 5 November 2010 Received in revised form 26 January 2011 Accepted 1 February 2011 Available online 18 February 2011 Keyword: EV71 monoclonal antibodies
A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59preimmunized BALB/c (H-2d ) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus. © 2011 Elsevier B.V. All rights reserved.
1. Introduction EV71 is a positive-stranded RNA virus within the Picornaviridae family. Following the first reported case of EV71 infection in 1969 in California, USA (Schmidt et al., 1974), the virus has been infecting periodically human subjects around the globe (Samuda et al., 1987; Ho et al., 1999; Gilbert et al., 1988; AbuBakar et al., 1999; Yang et al., 2009; Ryu et al., 2010). Infection with EV71, together with some of its close genetic counterparts, coxsackievirus 16 (CA16), and CA10 (Pulli et al., 1995), are known to cause the mild exanthematous symptom that may manifest itself as the hand-footand-mouth disease (HFMD)/herpangina in infants and children. Although subclinical coxsackievirus infection is not a public health concern, periodic outbreaks of EV71 infection in the Asia-Pacific region over the past 10–12 years have caused severe symptoms of pulmonary edema/hemorrhage that lead to the deaths of significant numbers of infected children (Chonmaitree et al., 1981; McMinn, 2003). Among EV71 genogroups, viruses of genogroups B and C have been isolated in the outbreaks that occurred from 1986 to 2008 in Taiwan. In the large 1998 outbreak, 78 children have died among 405 patients with severe complications (Lin et al., 2002a). The epidemic activity between 1999 and 2002 is associated mainly
∗ Corresponding authors. Tel.: +886 37 246166; fax: +886 37 583009. E-mail addresses: [email protected] (H.-W. Chang), [email protected] (C. Sia). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.02.003
with genogroup B viruses (Wang et al., 2002; Lin et al., 2002b). In 2006 and 2007, EV71 with genotype B5 is identified, and has reappeared in the 2008 outbreak in the island (Huang et al., 2009). Emerging EV71 infection has been discussed as a potential threat to global public health (Qiu, 2008). In the context of this possibility, effective antiviral drugs to treat EV71 infection, and an efficacious prophylactic vaccine to control virus outbreaks have not been available in the past, and are not expected to be available in the near future. For EV71 outbreaks that have occurred in China over the past 10 years, administration of pooled human gamma globulins has been used to treat patients with EV71-associated encephalitis. Most likely due to low titers of EV71-specific neutralizing antibodies in the pooled human gamma globulin preparations that are used, the practice has not achieved entirely satisfactory results (Liu et al., 2009). Along this line of treatment regimen, the use of humanized monoclonal antibodies that exhibit potent virus neutralizing activities could provide an attractive approach for the prophylaxis of severe EV71 infections. To this end, an immunodominant epitope designated, SP70, predicted by Foo et al. (2007) to be exposed on the surface of the VP1 capsid protein has been conjugated to keyhole limpet hemocyanin (KLH), and used by Li et al. (2009) for immunization to generate murine monoclonal antibodies that neutralize an EV71 isolate of genotype C, which has circulated in China for therapeutic development. Selection of an appropriate immunogen formulation to deliver to the immune system can influence crucially the specificity of the antibodies that are generated. To raise monoclonal antibodies
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capable of cross-neutralizing EV71 genotype B viruses that have circulated in Taiwan in recent years, mice were inoculated with a live Taiwan isolate, EV71/59 with genotype B4, to allow B lymphocytes of the animals to be primed against a broader spectrum of antigenic determinants present within the native viral antigens. Hybridomas producing the antibodies with the desired properties could then be isolated via cloning. The characteristics of the monoclonal antibodies produced by four hybridoma clones are described. 2. Materials and methods 2.1. Mice and immunization Six-to-seven week-old female Balb/c (H-2d ) mice purchased from the National Laboratory Animal Center (Taiwan), and maintained in the animal care facility certified by our institution’s animal care and use committee were immunized individually three times, each time with 106 plaque forming units (PFUs) of the sucrose gradient purified EV71/E59 virus using the intraperitoneal route. Ten days after the final booster, the animals were bled, and serum antibodies were determined using a heat inactivated EV71/E59 (HI-EV71/E59) virus-coated direct enzyme linked immunosorbent assay (ELISA). The mouse that had mounted the strongest antibody responses against the virus was given a final boost with the same dose of the live virus before its spleen was removed 7 days later for fusion. 2.2. Production, purification and characterization of viruses VERO cells obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in VP-SFM serum-free medium (Gibco/Invitrogen, Carlsbad, CA, USA). The EV71/E59 isolate was obtained from the Taiwan Centers for Disease Control (CDC). N0781-TW-01 of genotype B4, and N2838-TW-03 of genotype B5 were provided by Professor Jen-Ren Wang, Department of Medical Technology, National Chen Kung University, Tainan, Taiwan. Batch production of the viruses was done by inoculating each virus at a multiplicity of infection (MOI) of 10−5 into a culture of 1.5–2.0 × 108 VERO cells in a 850 cm2 roller bottle (Corning, NY, USA). The culture bottle was mounted onto a roller rack (Wheaton, Millville, NJ, USA) set to rotate at 0.33 rpm kept in a room at 37 ◦ C to promote virus growth. The culture supernatant harvested 5 days later was filtered through a 0.65 m filter (Sartorius, Hayward, CA, USA) to remove cell debris before it was finally concentrated to around 50.0 mL by passing it through a Minimate 100 K tangential flow filtration (TFF) capsule (Pall Life Sciences, Ann Arbor, MI, USA). The concentrated sample was layered onto a 10–50% continuous sucrose gradient (Merck, West Point, PA, USA), and centrifuged at 32,000 rpm for 3 h. The fraction banded at 35–37% of the gradient was collected, and dialyzed against three exchanges of 1.0 L 1× PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). The titer of the purified virus preparation was determined using the immunoplague-forming assay. 2.3. Generation of EV71/E59-specific monoclonal antibodies 1.0 × 107 red blood cell free splenocytes prepared from a selected experimental mouse were fused with 2 × 107 NS-1 cells (ATCC, Rockville, MD, USA) according to the well established method described previously (Kohler and Milstein, 1975) using Hybri Max [(p7306, PEG 3000–3700 Da in dimethyl sulfoxide (DMSO)) purchased from Sigma/Aldrich (St Louis, MO, USA). Culture supernatants were collected from hybridomas that grow well in HT (hypoxanthine and thymidine) medium (Sigma/Aldrich, St Louis, MO, USA). A hybridoma line producing EV71/E59-specific antibodies that neutralized the immunizing virus was cloned at
Table 1 15-mer peptide panel used to characterize the fine specificities of N1, N3, N4 and N6 monoclonal antibodies. Peptide code
Amino acid location
Peptide sequence
VC1 VC 2 VC 3 VC 4 VC 5 VC 6 VC 7 VC 8 VC 9 VC 10 VC 11 VC 12 VC 13 VC 14 VC 15 VC 16 VC 17 VC 18 VC 19 VC 20 VC 21 VC 22 VC 23 VC 24 VC 25 VC 26 VC 27 VC 28 VC 29 VC 30 VC 31 VC 32 VC 33 VC 34 VC 35 VC 36 VC 37 VC 38 VC 39 VC 40 VC 41 VC 42 VC 43 VC 44 VC 45 VC 46 VC 47 VC 48 VC 49 VC 50 VC 51 VC 52 VC 53 VC 54 VC 55 VC 56 VC 57
566–580 571–585 576–590 581–595 586–600 591–605 596–610 601–615 606–620 611–625 616–630 621–635 626–640 631–645 636–650 641–655 646–660 651–665 656–670 661–675 666–680 671–685 676–690 681–695 686–700 691–705 696–710 701–715 706–720 711–725 716–730 721–735 726–740 731–745 736–750 741–755 746–760 751–765 756–770 761–775 766–780 771–785 776–790 781–795 786–800 791–805 796–810 801–815 806–820 811–825 816–830 821–835 826–840 831–845 836–850 841–855 846–860
GDRVADVIESSIGDS DVIESSIGDSVSRAL SIGDSVSRALTRALP VSRALTRALPAPTGQ TRALPAPTGQDTQVS APTGQDTQVSSHRLD DTQVSSHRLDTGKVP SHRLDTGKVPALQAA TGKVPALQAAEIGAS ALQAAEIGASSNASD EIGASSNASDESMIE SNASDESMIETRCVL ESMIETRCVLNSHST TRCVLNSHSTAETTL NSHSTAETTLDSFFS AETTLDSFFSRAGLV DSFFSRAGLVGEIDL RAGLVGEIDLPLEGT GEIDLPLEGTTNPNG PLEGTTNPNGYANWD TNPNGYANWDIDITG YANWDIDITGYAQMR IDITGYAQMRRKVEL YAQMRRKVELFTYMR RKVELFTYMRFDAEF FTYMRFDAEFTFVAC FDAEFTFVACTPTGE TFVACTPTGEVVPQL TPTGEVVPQLLQYMF VVPQLLQYMFVPPGA LQYMFVPPGAPKPDS VPPGAPKPDSRESLA PKPDSRESLAWQTAT RESLAWQTATNPSVF WQTATNPSVFVKLSD NPSVFVKLSDPPAQV VKLSDPPAQVSVPFM PPAQVSVPFMSPASA SVPFMSPASAYQWFY SPASAYQWFYDGYPT YQWFYDGYPTFGEHK DGYPTFGEHKQEKDL FGEHKQEKDLEYGAC QEKDLEYGACPNNMM EYGACPNNMMGTFSV PNNMMGTFSVRTVGT GTFSVRTVGTSKSKY RTVGTSKSKYPLVIR SKSKYPLVIRIYMRM PLVIRIYMRMKHVRA IYMRMKHVRAWIPRP KHVRAWIPRPMRNQN WIPRPMRNQNYLFKA MRNQNYLFKANPNYA YLFKANPNYAGNSIK NPNYAGNSIKPTGAS GNSIKPTGASRTAIT
The individual peptides were synthesized based on the protein sequence of an EV71 isolate, TW/2086/98, of genotype C2 obtained from the CDC (Taiwan) by Kelowna International Scientific Inc. (San Chung City, Taiwan). Each of the peptides had been high-performance liquid chromatography (HPLC)-purified to >90.0% purity before use. Peptide VC43 recognized by each of the monoclonal antibodies is highlighted in bolded letters.
0.3 cells per well in culture medium [CM: Dubecco’s modified Eagle’s medium (DMEM), Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10.0% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), and 1.0% penicillin/streptomycin mix (Gibco/Invitrogen, Carlsbad, CA, USA)] in the presence of 5.0 × 105 irradiated (2000 rads) BALB/c (H-2d ) splenocytes as feeders. Hybridomas that grew were expanded, characterized to
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Fig. 1. Sequence determination of the CDRs in the light (L) and heavy (H) chains expressed in the hybridomas producing the N1, N3, N4 and N6 antibodies. Primer pairs and PCR conditions used are described in Section 2.
confirm their clonality, and the properties of the monoclonal antibodies they produced. 2.4. Ascites production and purification Ascitic fluids produced by 5 × 105 of the individual hybridoma clones inoculated into individual >6-month-old pristane-treated BALB/c mice was delipidated by mixing it with 0.04 mL of 10% dextran sulphate solution (Sigma/Aldrich, St Louis, MO, USA) and 1.0 mL of 1.0 M calcium chloride (Sigma/Aldrich, St Louis, MO, USA) at 1:0.04:1 (v/v) ratio. The mixture was centrifuged 5 min later at 10,000 × g for 10 min (centrifuge model: Legend Micro 17R, Sorvall Thermo Scientific). The supernatant fraction was collected, and dialyzed in 1.0 L of 1× Tris buffered saline (TBS), pH 7.2 (137 mM NaCl and 10 mM Tris, Sigma/Aldrich, St Louis, MO, USA) in dH2 O overnight before it was loaded onto a protein G column using AKTA prime (GE Healthcare, Salt Lake City, USA). Antibodies bound to protein G were eluted with a 10.0 mM glycine–HCl (Sigma/Aldrich, St Louis, MO, USA), pH 3.0 buffer. Eluates collected in 1.0 mL fractions were dialyzed immediately against 1.0 L of 1× PBS. pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). 2.5. Analysis of the VH and VL regions of the EV71/E59-specific monoclonal antibodies RNA extracted from each of the hybridoma clones using the RNeasy kit purchased from Qiagen (Valencia, CA, USA) was reverse transcribed with the reagents supplied in the Transcriptor High Fidelity cDNA synthesis kit (Roche, IN, USA). Amplification of the CDRs of the chain expressed by the individual hybridomas was performed with polymerase chain reaction (PCR) using a forward primer, 5 -GGA TAC AGT TGG TGC AGC ATC-3 (forward), and a degenerate reverse primer 5 -GAY ATT GTG MTS ACM CAR WCT-3 using the cycling program: 94 ◦ C for 1 min, 94 ◦ C for 1.5 min, 50 ◦ C for 2.0 min, 72 ◦ C for 3.0 min, 72 ◦ C for 1.0 min for 40 cycles. The PCR products obtained were treated with BciVI restriction enzyme (Fermentas, St Leon Rot, Germany) to digest the aberrant chain DNA derived from the NSI fusion partner. Digested PCR products were electrophorized in a 2.0% agarose gel (Sigma/Aldrich, St Louis, MO, USA) to resolve the DNA fragments. DNA band of approximately
360 bp was extracted using a gel fragments extraction kit (Geneaid, Sijhih City, Taiwan), and sent to Mission Bioteck (Taiwan, Nangang, Taipei, Taiwan) for sequence determination. CDRs of the VH (hypervariable region of the heavy chain) chain were amplified using a collection of 9 degenerate forward primers and, a single reverse primer (5 -AGG GGC CAG TGG ATA GAC-3 ). The forward primers used were: OVH1 (5 -SAG GTC CAG CTG CAG CAG YYT GG-3 ), OVH2 (5 -CAG GTR CAG CTG AAG SAG TCA GG-3 ), OVH3 (5 -GAK GTG CAG CTT CAG CAG TCR GG-3 ), OVH5-1 (5 -GAV GTG AWG CTG GTG GAG TCT GA-3 ), OVH5-2 (5 -GAV GTG AWG CTG GTG GAG TCT GG-3 ), OVH11 (5 -GAA GTG CAG CTG TTG GAG ACT GG-3 ), OVH14-1 (5 -GAG GTT CAG CTG CAG CAG TCT GG-3 ), OVH14-2 (5 -GAG GTT CAG CTG CAG CAG TCT GT-3 ), and OVH15 (5 -CAG GTT CAC CTA CAA CAG TCT GG-3 ). The PCR program used to generate a DNA fragment of approximately 300 bp was set as follows: 92 ◦ C for 3 min, 92 ◦ C for 1 min, 68 ◦ C for 30 s, 72 ◦ C for 1 min, 72 ◦ C for 10 min for 25 cycles. The expected PCR product was extracted from the 2% agarose gel, and processed for sequence determination. 2.6. SDS–PAGE and Western blot Twenty microliters of the heat-inactivated (10 min in a 85 ◦ C water bath) sucrose-gradient purified EV71/E59 virus measured to contain 0.12 g of total protein determined with the BCA protein assay (Pierce, Rockford, IL, USA) was mixed with 5.0 L of 5× concentrated loading dye (Bionovas Biotech Co., Ltd., Toronto, Canada), and electrophorized in a 10% SDS–polyacrylamide gel in 1× Tris–glycine SDS-running buffer (Amersham Biosciences, Piscataway, NJ, USA). The resolved viral proteins were transferred onto a PVDF membrane (Biorad, Hercules, CA, USA) by using the Mini Trans-Blot Cell (Biorad). Protein-containing membrane was blocked in 5.0% skim milk (Anchor, New Zealand) prepared in PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA), then washed, and placed in the Mini-Protene II Multiscreen Apparatus (Biorad, Hercules, CA, USA). 2.0 g of the individual test monoclonal antibodies (N1, N3, N4, or N6) prepared in assay buffer (1.0% skim milk in PBS, pH 7.2) was added to the reaction chamber. The membrane was then treated with 0.5 mL of 1:10,000 dilution (in assay buffer) of a horse radish peroxidase (HRP)-conjugated donkey anti-mouse
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been high-performance liquid chromatography (HPLC)-purified to >90% purity. 2.8. Epitope mapping ELISA
Fig. 2. Reactivity of protein G column purified monoclonal antibodies (N1, N3, N4 and N6) against a purified preparation of HI-EV71/E59 in western blot. The assay was performed with 2.0 mg/mL of the individual monoclonal antibodies against 1.2 g/mL of the electrophorized purified EV71/E59 virus preparation.
antiserum (Jackson ImmunoResearch Lab., West Grove, PA, USA). After 0.5 h incubation, the membrane was washed, blotted dry on paper towels, and 100.0 L of Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) was then layered onto the membrane. The membrane was exposed for 0.5, 1.5 and 10 min on an X-ray film (Kodak, Rochester, NY, USA) to detect binding.
Two different formats involving the use of MaxiSorp, and CovaLink NH ELISA plates purchased from NUNC (Napperville, IL, USA) were used to immobilize the test peptides. Individual test peptide reconstituted at 2.0 mg/mL in 5.0% DMSO (Sigma/Aldrich, St Louis, MO, USA) was diluted to 10.0 g/mL in pH 9.6 carbonate–bicarbonate coating buffer (Sigma/Aldrich, St Louis, MO, USA), and coated in pools of five adjacent peptides, or individually onto each well of the MaxiSorp plate. Peptide coating onto CovaLink NH plates was done by following the procedures described by Sondergard-Andersen et al. (1990). Following overnight incubation (for MaxiSorp plate), or 30 min incubation at room temperature (for CovaLink plate), the plates were washed, and blocked in 3.0% skim milk prepared in PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). Varying concentrations of the individual purified monoclonal antibodies prepared in the assay buffer (1.0% skim milk in PBS pH 7.2) were then added to each of the test wells. 100.0 L of a HRP-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Lab., West Grove, PA, USA) at 1 in 10,000 dilution prepared in assay buffer was added 1 h later for detection. The wells were washed, and 50 (L of TMB peroxidase substrate (SureBlueTM , KPL, Gaithersburg, MD, USA) was added to the individual assay wells. Further colour development was stopped by adding 50.0 L of 2.0 N H2 SO4 (Sigma/Aldrich, St Louis, MO, USA) to each well 20 min later. Absorbance readings were recorded at 450 nm with an ELISA reader (Spectra Max M2 model, Sunnydale, CA, USA).
2.7. Peptide synthesis 2.9. Immuno-plaque forming inhibition assay A total of 57 15-mer peptides in overlap of 10 amino acids spanning the entire length of the VP1 capsid protein of an EV71 isolate designated, TW/2086/98, of genotype C2 were synthesized by Kelowna International Scientific Inc. (San Chung City, Taiwan) (Table 1) to characterize the fine specificities of the N1, N3, N4 and N6 monoclonal antibodies directed against the VP1 capsid protein of EV71/E59. The individual peptides used in the present study had
2.5 × 105 rhabdomyosarcoma (RD) cells (ATCC, Rockville, MD, USA) resuspended in 1.0 mL of CM were cultured in individual wells of a 24-well tissue culture plate (Corning Life Sciences, Corning, NY, USA). The cells were kept overnight in a 37 ◦ C incubator equilibrated with 5.0% CO2 to form a monolayer. The culture supernatant was removed, and 200.0 L of CM containing 100 PFUs of each test
Fig. 3. Epitope mapping studies. Maxisorp plates, and CovaLink plates were used to coate the 15-mer VP1 peptides. A concentration of 10.0 g/mL of the individual monoclonal antibodies was used in each test. Results shown represented average absorbance for each pair of the duplicate assays performed with the pooled peptides coated on MaxiSorp plate (A), CovaLink plate (B); and individual peptides coated on these plates (C and D), respectively.
H.-W. Chang et al. / Journal of Virological Methods 173 (2011) 189–195
100
A (N1) 80 60 40 20 0
?
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?
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3
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2
1
% of Virus Neutralization
% of Virus Neutralization
100
C (N4) 80 60 40 20 0
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2
1
Antibody conc (log10 μg/ml) 100
B (N3) 80 60 40 20 ?
3
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2
% of Virus Neutralization
100
% of Virus Neutralization
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3
Antibody conc (log10 μg/ml)
0
193
D (N6) 80 60 40 20 0
1
?
Antibody conc (log10 μg/ml)
% of Virus Neutralization
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3
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2
1
Antibody conc (log10 μg/ml)
100
E (pooled) 80 60 40 20 0
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3
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2
1
Antibody conc (log10 μg/ml)
Fig. 4. Determination of virus neutralizing activity of the individual, and pooled EV71/E59-specific monoclonal antibodies. Results showed % neutralization of 100 PFUs of the individual purified EV71 viruses: EV71/E59 (), N0781-TW-01 (), or N2838-TW-03 (䊉) tested against varying concentrations of each of the monoclonal antibodies (A–D), and a pooled sample of the antibodies (E). An murine IgG2a subclass monoclonal antibody (clone #20102, R&D Systems, Minneapolis, MN, USA) was also assayed as control ().
virus, that had been preincubated (overnight) with varying concentrations of the individual monoclonal antibodies all belonging to the IgG2a subclass, or a mixture of the four monoclonal antibodies were added to each RD cell-containing culture wells. Cultures containing no added monoclonal antibodies, or with the addition of a monoclonal IgG2a antibody (clone #20102, R&D Systems, Minneapolis, MN, USA) were also assayed as positive, and negative controls, respectively. Following 1 h incubation at 37 ◦ C, 0.5 mL of 1.1% methanol cellulose (Merck, West Point, PA, USA) was overlaid onto the cells. The cultures were kept in a 37 ◦ C incubator equilibrated with 5.0% CO2 for 2 days. Culture supernatant was removed, and 0.5 mL of a 3.7% formalin (Merck, West Point, PA, USA) solution prepared in PBS (pH 7.2) was added to the cells. Formalin was removed the next day, and 100.0 L of 1 in 5000 dilution (prepared in assay buffer) of an anti-mouse EV71-specific monoclonal antibody, MAB 979 (Chemicon, Temecula, CA, USA) was then added to individual test wells. Plates were washed 1 h later, and 100.0 L of an anti-mouse IgG-HRP conjugate antibody (Serotec, Kidling-
ton, Oxford, UK) at 1 in 50,000 dilution (prepared in assay buffer) was added for detection. The plates were washed, and 100.0 L of TMB substrate (KPL, Gaithersburg, MD, USA) was added to each assay well. The reaction was allowed to develop for 30 min in the dark. Plagues seen as black spots under an optical microscope were counted. Virus titer was expressed as mean PFUs per mL of the two highest diluted EV71/E59 preparations tested. 3. Results 3.1. CDR sequence analysis Sequence data obtained showed that the hybridomas secreting the N1 and N4 antibodies shared the same CDR1 and CDR2, but a different CDR3, while the N3 and N6 antibody producing hybridomas expressed a different set of chains with the same CDR1 and CDR2, but a distinct CDR3 (Fig. 1). For the heavy chains expressed in the hybridomas, it was not found whether they contained different
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CDRs (data not shown). These observations support that the N1, N3, N4 and N6 antibodies are produced by distinct hybridoma clones. 3.2. Western blot analysis of purified ascitic fluids As depicted in Fig. 2, the individual monoclonal antibodies recognized a viral protein of approximately 36 kDa resolved from the sucrose gradient purified EV71/E59 virus sample. Recombinant VP1 expressed in baculovirus (Chung et al., 2006) was reported to have this molecular mass. Interestingly, the individual EV71/E59specific monoclonal antibodies also bound a protein moiety, Pr, slightly larger than VP1 (approximately 38 kDa) that was also separated in the EV71/E59 preparation. The nature of this protein is under further investigation. 3.3. Epitope mapping studies Results obtained from both ELISA formats revealed that pool 9 contained peptides recognized by each of the monoclonal antibodies (Fig. 3A and B). Fine epitope analysis performed by coating each of the peptides in pool 9 onto the MaxiSorp and CovaLink NH ELISA plates identified a peptide designated VC43, encompassing the amino acid sequence, FGEHKQEKDLEYGAC, was the target recognized by each of the monoclonal antibodies (Fig. 3C and D). 3.4. Virus neutralizing activities of monoclonal antibodies Results obtained from these studies showed that the individual monoclonal antibodies exhibited potent neutralizing activity against the EV71/E59 isolate used for immunization, as well as N0781-TW-01 also with genotype B4, and N2838-TW-03 of genotype B5 that had been found commonly in the more recent outbreaks in Taiwan. At 3log10 g/mL, N3 and N6 monoclonal antibodies completely (100.0%) blocked the infectivity of the RD cells by each of the viral isolates tested (Fig. 4B and D). At this concentration, N1 and N4 monoclonal antibodies also neutralized fully the EV71/E59 and N0781-TW-01 viruses, but neutralized N2838TW-03 infectivity by 81.0% and 88.0%, respectively (Fig. 4A and C). The monoclonal antibodies were noted to act cooperatively in neutralizing the EV71/E59 isolate. Complete inhibition of EV71/E59 virus infectivity was achieved at a markedly lower concentration (2.04log10 g/mL) of a pooled sample of the antibodies (Fig. 4E). 4. Conclusions The objective of the present study was to raise murine monoclonal antibodies that exhibit strong neutralizing activity against EV71 isolates with genotype B found commonly in the recent outbreaks in Taiwan, which could serve as targets for therapeutic development. Using live EV71/E59 for immunization, four hybridoma clones producing monoclonal antibodies found to belong to IgG2a subclass that recognize distinct antigenic sites within a common epitope (VC43) at position 211–225 of VP1 are isolated. The monoclonal antibodies exhibit modest differential virus neutralizing activity against viruses of genogroup B. At a higher concentration of 3log10 g/mL, monoclonal antibodies N3 and N6 are capable of inhibiting completely infection of 100 PFUs of the EV71/E59 virus used for immunization, N0781-TW-01 with the same genotype, as well as N2838-TW-03 of genotype B5 that has remerged in the 2008 outbreak in Taiwan (Huang et al., 2009). Although monoclonal antibodies N1 and N4 at these concentrations also neutralized completely the genotype B4 viruses, they are slightly less effective than the N3 and N6 antibodies in neutralizing the N2838-TW-03 isolate. The findings that the monoclonal antibodies act cooperatively in neutralizing the EV71/E59 virus
illustrates they recognized distinct structural sites in the VC43 epitope. Suckling ICR and BALB/c mice are susceptible to infection with high doses of live EV71 with genotype C. One-day-old mice infected with a mouse-adapted genotype C virus would develop neurological disorders similar to the symptoms seen in subjects with severe EV71 infections (Wang et al., 2004). This mouse model has been used by Chang et al. (2010) to show a monoclonal antibody against an inactivated EV71/Hn2 substrain of genotype C4 is capable of protecting newborn BALB/c mice from lethal challenge with the same isolate. To date, similar studies demonstrating that newborn mice would also develop human-like symptoms upon infection with EV71 of genogroup B have not been reported. Inoculation of high doses of EV71/E59, N0781-TW-01, or N2838-TW-03 used in the present study has not been found to cause severe infection in suckling ICR and BALB/c mice. In this setting, development of mice transgenic for the PSGL-1 and SCARB2 human EV71 receptors (Nishimura et al., 2009; Yamayoshi et al., 2009) that would acquire human-like symptoms upon infection with EV71 within genogroups B and C could provide potentially a suitable small animal model to assess the protective efficacy of the monoclonal antibodies described above. Acknowledgment This study was supported by the intramural grant #99A1VCPP02-014, from the National Health Research Institutes, Taiwan. References AbuBakar, S., Chee, H.Y., Al-Kobaisi, M.F., Xiaoshan, J., Chua, K.B., Lam, S.K., 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. 61, 1–9. Chang, G.-H., Luo, Y.-J., Wu, X.-.Y., Si, B.-Y., Lin, L., Zhu, Q.-Y., 2010. Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection. Virol. J. 7, 106–112. Chonmaitree, T., Menegus, M.A., Schervish-Swierkosz, E.M., Schwalenstocker, E., 1981. Enterovirus 71 infection: report of an outbreak with two cases of paralysis and a review of the literature. Pediatrics 67, 489–493. Chung, Y.C., Huang, J.H., Lai, C.W., Sheng, H.C., Shih, S.R., Ho, M.S., Hu, Y.C., 2006. Expression, purification and characterization of enterovirus-71 virus-like particles. World J. Gastroenterol. 12, 921–927. Foo, D.G., Alonso, S., Phoon, M.C., Ramachandran, N.P., Chow, V.T., Poh, C.L., 2007. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Res. 125, 61–68. Gilbert, G.L., Dickson, K.E., Waters, M.J., Kennett, M.L., Land, S., Sneddon, A.M., 1988. Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement. Pediatr. Infect Dis. J. 7, 484–488. Ho, M., Chen, E.R., Hsu, K.H., Twu, S.J., Chen, K.T., Tsai, S.F., Wang, J.R., Shih, S.R., 1999. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N. Engl. J. Med. 341, 929–935. Huang, S.W., Hsu, Y.W., Smith, D.J., Kiang, D., Tsai, H.P., Lin, K.H., Wang, S.M., Liu, C.C., Su, I.J., Wang, J.R., 2009. Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J. Clin. Microbiol. 47, 3653–3662. Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497. Li, X., Mao, C., Ma, S., Wang, X., Sun, Z., Yi, Y., Guo, M., Shen, X., Sun, L., Bi, S., 2009. Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides. Biochem. Biophys. Res. Commun. 390, 1126–1128. Lin, T.Y., Chang, L.Y., Hsia, S.H., Huang, Y.C., Chiu, C.H., Hsueh, C., Shih, S.R., Liu, C.C., Wu, M.H., 2002a. The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin Infect Dis 34 (Suppl. 2), S52–57. Lin, T.Y., Twu, S.J., Ho, M.S., Chang, L.Y., Lee, C.Y., 2002b. Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg. Infect. Dis. 9, 291–293. Liu, X.J., Li, W., Zhang, Y.Q., Liu, Y.M., Liu, L.Z., 2009. Clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 infection. Zhongguo Dang Dai Er Ke Za Zhi 11, 967–969. McMinn, P.C., 2003. Enterovirus 71 in the Asia-Pacific region: an emerging cause of acute neurological disease in young children. Neurol. J. Southeast Asia 8, 57–63. Nishimura, Y., Shimojima, M., Tano, Y., Miyamura, T., Wakita, T., Shimizu, H., 2009. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat. Med. 15, 794–797. Pulli, T., Koskimies, P., Hyypia, T., 1995. Molecular comparison of coxsackie A virus serotypes. Virology 212, 30–38.
H.-W. Chang et al. / Journal of Virological Methods 173 (2011) 189–195 Qiu, J., 2008. Enterovirus 71 infection: a new threat to global public health? Lancet Neurol. 7, 868–869. Ryu, W.S., Kang, B., Hong, J., Hwang, S., Kim, J., Cheon, D.S., 2010. Clinical and etiological characteristics of enterovirus 71-related diseases during a recent 2-year period in Korea. J. Clin. Microbiol. 48, 2490–2494. Sondergard-Andersen, J., Lauritzen, E., Lind, K., Holm, A., 1990. Covalently linked peptides for enzyme-linked immunosorbent assay. J. Immunol. Methods 131, 99–104. Samuda, G.M., Chang, W.K., Yeung, C.Y., Tang, P.S., 1987. Monoplegia caused by Enterovirus 71: an outbreak in Hong Kong. Pediatr. Infect. Dis. J. 6, 206–208. Schmidt, N.J., Lennette, E.H., Ho, H.H., 1974. An apparently new enterovirus isolated from patients with disease of the central nervous system. J. Infect. Dis. 129, 304–309.
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Generation of murine monoclonal antibodies which cross-neutralize human enterovirus genogroup B isolates Hsuen-Wen Chang ∗ , Chia-Chyi Liu, Min-Han Lin, Hui-Min Ho, Ya-Ting Yang, Yen-Hung Chow, Pele Chong, Charles Sia ∗ Vaccine Research and Development Center, National Health Research Institutes, Zhunan Township, Miaoli County 350, Taiwan
a b s t r a c t Article history: Received 5 November 2010 Received in revised form 26 January 2011 Accepted 1 February 2011 Available online 18 February 2011 Keyword: EV71 monoclonal antibodies
A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59preimmunized BALB/c (H-2d ) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus. © 2011 Elsevier B.V. All rights reserved.
1. Introduction EV71 is a positive-stranded RNA virus within the Picornaviridae family. Following the first reported case of EV71 infection in 1969 in California, USA (Schmidt et al., 1974), the virus has been infecting periodically human subjects around the globe (Samuda et al., 1987; Ho et al., 1999; Gilbert et al., 1988; AbuBakar et al., 1999; Yang et al., 2009; Ryu et al., 2010). Infection with EV71, together with some of its close genetic counterparts, coxsackievirus 16 (CA16), and CA10 (Pulli et al., 1995), are known to cause the mild exanthematous symptom that may manifest itself as the hand-footand-mouth disease (HFMD)/herpangina in infants and children. Although subclinical coxsackievirus infection is not a public health concern, periodic outbreaks of EV71 infection in the Asia-Pacific region over the past 10–12 years have caused severe symptoms of pulmonary edema/hemorrhage that lead to the deaths of significant numbers of infected children (Chonmaitree et al., 1981; McMinn, 2003). Among EV71 genogroups, viruses of genogroups B and C have been isolated in the outbreaks that occurred from 1986 to 2008 in Taiwan. In the large 1998 outbreak, 78 children have died among 405 patients with severe complications (Lin et al., 2002a). The epidemic activity between 1999 and 2002 is associated mainly
∗ Corresponding authors. Tel.: +886 37 246166; fax: +886 37 583009. E-mail addresses: [email protected] (H.-W. Chang), [email protected] (C. Sia). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.02.003
with genogroup B viruses (Wang et al., 2002; Lin et al., 2002b). In 2006 and 2007, EV71 with genotype B5 is identified, and has reappeared in the 2008 outbreak in the island (Huang et al., 2009). Emerging EV71 infection has been discussed as a potential threat to global public health (Qiu, 2008). In the context of this possibility, effective antiviral drugs to treat EV71 infection, and an efficacious prophylactic vaccine to control virus outbreaks have not been available in the past, and are not expected to be available in the near future. For EV71 outbreaks that have occurred in China over the past 10 years, administration of pooled human gamma globulins has been used to treat patients with EV71-associated encephalitis. Most likely due to low titers of EV71-specific neutralizing antibodies in the pooled human gamma globulin preparations that are used, the practice has not achieved entirely satisfactory results (Liu et al., 2009). Along this line of treatment regimen, the use of humanized monoclonal antibodies that exhibit potent virus neutralizing activities could provide an attractive approach for the prophylaxis of severe EV71 infections. To this end, an immunodominant epitope designated, SP70, predicted by Foo et al. (2007) to be exposed on the surface of the VP1 capsid protein has been conjugated to keyhole limpet hemocyanin (KLH), and used by Li et al. (2009) for immunization to generate murine monoclonal antibodies that neutralize an EV71 isolate of genotype C, which has circulated in China for therapeutic development. Selection of an appropriate immunogen formulation to deliver to the immune system can influence crucially the specificity of the antibodies that are generated. To raise monoclonal antibodies
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capable of cross-neutralizing EV71 genotype B viruses that have circulated in Taiwan in recent years, mice were inoculated with a live Taiwan isolate, EV71/59 with genotype B4, to allow B lymphocytes of the animals to be primed against a broader spectrum of antigenic determinants present within the native viral antigens. Hybridomas producing the antibodies with the desired properties could then be isolated via cloning. The characteristics of the monoclonal antibodies produced by four hybridoma clones are described. 2. Materials and methods 2.1. Mice and immunization Six-to-seven week-old female Balb/c (H-2d ) mice purchased from the National Laboratory Animal Center (Taiwan), and maintained in the animal care facility certified by our institution’s animal care and use committee were immunized individually three times, each time with 106 plaque forming units (PFUs) of the sucrose gradient purified EV71/E59 virus using the intraperitoneal route. Ten days after the final booster, the animals were bled, and serum antibodies were determined using a heat inactivated EV71/E59 (HI-EV71/E59) virus-coated direct enzyme linked immunosorbent assay (ELISA). The mouse that had mounted the strongest antibody responses against the virus was given a final boost with the same dose of the live virus before its spleen was removed 7 days later for fusion. 2.2. Production, purification and characterization of viruses VERO cells obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in VP-SFM serum-free medium (Gibco/Invitrogen, Carlsbad, CA, USA). The EV71/E59 isolate was obtained from the Taiwan Centers for Disease Control (CDC). N0781-TW-01 of genotype B4, and N2838-TW-03 of genotype B5 were provided by Professor Jen-Ren Wang, Department of Medical Technology, National Chen Kung University, Tainan, Taiwan. Batch production of the viruses was done by inoculating each virus at a multiplicity of infection (MOI) of 10−5 into a culture of 1.5–2.0 × 108 VERO cells in a 850 cm2 roller bottle (Corning, NY, USA). The culture bottle was mounted onto a roller rack (Wheaton, Millville, NJ, USA) set to rotate at 0.33 rpm kept in a room at 37 ◦ C to promote virus growth. The culture supernatant harvested 5 days later was filtered through a 0.65 m filter (Sartorius, Hayward, CA, USA) to remove cell debris before it was finally concentrated to around 50.0 mL by passing it through a Minimate 100 K tangential flow filtration (TFF) capsule (Pall Life Sciences, Ann Arbor, MI, USA). The concentrated sample was layered onto a 10–50% continuous sucrose gradient (Merck, West Point, PA, USA), and centrifuged at 32,000 rpm for 3 h. The fraction banded at 35–37% of the gradient was collected, and dialyzed against three exchanges of 1.0 L 1× PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). The titer of the purified virus preparation was determined using the immunoplague-forming assay. 2.3. Generation of EV71/E59-specific monoclonal antibodies 1.0 × 107 red blood cell free splenocytes prepared from a selected experimental mouse were fused with 2 × 107 NS-1 cells (ATCC, Rockville, MD, USA) according to the well established method described previously (Kohler and Milstein, 1975) using Hybri Max [(p7306, PEG 3000–3700 Da in dimethyl sulfoxide (DMSO)) purchased from Sigma/Aldrich (St Louis, MO, USA). Culture supernatants were collected from hybridomas that grow well in HT (hypoxanthine and thymidine) medium (Sigma/Aldrich, St Louis, MO, USA). A hybridoma line producing EV71/E59-specific antibodies that neutralized the immunizing virus was cloned at
Table 1 15-mer peptide panel used to characterize the fine specificities of N1, N3, N4 and N6 monoclonal antibodies. Peptide code
Amino acid location
Peptide sequence
VC1 VC 2 VC 3 VC 4 VC 5 VC 6 VC 7 VC 8 VC 9 VC 10 VC 11 VC 12 VC 13 VC 14 VC 15 VC 16 VC 17 VC 18 VC 19 VC 20 VC 21 VC 22 VC 23 VC 24 VC 25 VC 26 VC 27 VC 28 VC 29 VC 30 VC 31 VC 32 VC 33 VC 34 VC 35 VC 36 VC 37 VC 38 VC 39 VC 40 VC 41 VC 42 VC 43 VC 44 VC 45 VC 46 VC 47 VC 48 VC 49 VC 50 VC 51 VC 52 VC 53 VC 54 VC 55 VC 56 VC 57
566–580 571–585 576–590 581–595 586–600 591–605 596–610 601–615 606–620 611–625 616–630 621–635 626–640 631–645 636–650 641–655 646–660 651–665 656–670 661–675 666–680 671–685 676–690 681–695 686–700 691–705 696–710 701–715 706–720 711–725 716–730 721–735 726–740 731–745 736–750 741–755 746–760 751–765 756–770 761–775 766–780 771–785 776–790 781–795 786–800 791–805 796–810 801–815 806–820 811–825 816–830 821–835 826–840 831–845 836–850 841–855 846–860
GDRVADVIESSIGDS DVIESSIGDSVSRAL SIGDSVSRALTRALP VSRALTRALPAPTGQ TRALPAPTGQDTQVS APTGQDTQVSSHRLD DTQVSSHRLDTGKVP SHRLDTGKVPALQAA TGKVPALQAAEIGAS ALQAAEIGASSNASD EIGASSNASDESMIE SNASDESMIETRCVL ESMIETRCVLNSHST TRCVLNSHSTAETTL NSHSTAETTLDSFFS AETTLDSFFSRAGLV DSFFSRAGLVGEIDL RAGLVGEIDLPLEGT GEIDLPLEGTTNPNG PLEGTTNPNGYANWD TNPNGYANWDIDITG YANWDIDITGYAQMR IDITGYAQMRRKVEL YAQMRRKVELFTYMR RKVELFTYMRFDAEF FTYMRFDAEFTFVAC FDAEFTFVACTPTGE TFVACTPTGEVVPQL TPTGEVVPQLLQYMF VVPQLLQYMFVPPGA LQYMFVPPGAPKPDS VPPGAPKPDSRESLA PKPDSRESLAWQTAT RESLAWQTATNPSVF WQTATNPSVFVKLSD NPSVFVKLSDPPAQV VKLSDPPAQVSVPFM PPAQVSVPFMSPASA SVPFMSPASAYQWFY SPASAYQWFYDGYPT YQWFYDGYPTFGEHK DGYPTFGEHKQEKDL FGEHKQEKDLEYGAC QEKDLEYGACPNNMM EYGACPNNMMGTFSV PNNMMGTFSVRTVGT GTFSVRTVGTSKSKY RTVGTSKSKYPLVIR SKSKYPLVIRIYMRM PLVIRIYMRMKHVRA IYMRMKHVRAWIPRP KHVRAWIPRPMRNQN WIPRPMRNQNYLFKA MRNQNYLFKANPNYA YLFKANPNYAGNSIK NPNYAGNSIKPTGAS GNSIKPTGASRTAIT
The individual peptides were synthesized based on the protein sequence of an EV71 isolate, TW/2086/98, of genotype C2 obtained from the CDC (Taiwan) by Kelowna International Scientific Inc. (San Chung City, Taiwan). Each of the peptides had been high-performance liquid chromatography (HPLC)-purified to >90.0% purity before use. Peptide VC43 recognized by each of the monoclonal antibodies is highlighted in bolded letters.
0.3 cells per well in culture medium [CM: Dubecco’s modified Eagle’s medium (DMEM), Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10.0% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), and 1.0% penicillin/streptomycin mix (Gibco/Invitrogen, Carlsbad, CA, USA)] in the presence of 5.0 × 105 irradiated (2000 rads) BALB/c (H-2d ) splenocytes as feeders. Hybridomas that grew were expanded, characterized to
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Fig. 1. Sequence determination of the CDRs in the light (L) and heavy (H) chains expressed in the hybridomas producing the N1, N3, N4 and N6 antibodies. Primer pairs and PCR conditions used are described in Section 2.
confirm their clonality, and the properties of the monoclonal antibodies they produced. 2.4. Ascites production and purification Ascitic fluids produced by 5 × 105 of the individual hybridoma clones inoculated into individual >6-month-old pristane-treated BALB/c mice was delipidated by mixing it with 0.04 mL of 10% dextran sulphate solution (Sigma/Aldrich, St Louis, MO, USA) and 1.0 mL of 1.0 M calcium chloride (Sigma/Aldrich, St Louis, MO, USA) at 1:0.04:1 (v/v) ratio. The mixture was centrifuged 5 min later at 10,000 × g for 10 min (centrifuge model: Legend Micro 17R, Sorvall Thermo Scientific). The supernatant fraction was collected, and dialyzed in 1.0 L of 1× Tris buffered saline (TBS), pH 7.2 (137 mM NaCl and 10 mM Tris, Sigma/Aldrich, St Louis, MO, USA) in dH2 O overnight before it was loaded onto a protein G column using AKTA prime (GE Healthcare, Salt Lake City, USA). Antibodies bound to protein G were eluted with a 10.0 mM glycine–HCl (Sigma/Aldrich, St Louis, MO, USA), pH 3.0 buffer. Eluates collected in 1.0 mL fractions were dialyzed immediately against 1.0 L of 1× PBS. pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). 2.5. Analysis of the VH and VL regions of the EV71/E59-specific monoclonal antibodies RNA extracted from each of the hybridoma clones using the RNeasy kit purchased from Qiagen (Valencia, CA, USA) was reverse transcribed with the reagents supplied in the Transcriptor High Fidelity cDNA synthesis kit (Roche, IN, USA). Amplification of the CDRs of the chain expressed by the individual hybridomas was performed with polymerase chain reaction (PCR) using a forward primer, 5 -GGA TAC AGT TGG TGC AGC ATC-3 (forward), and a degenerate reverse primer 5 -GAY ATT GTG MTS ACM CAR WCT-3 using the cycling program: 94 ◦ C for 1 min, 94 ◦ C for 1.5 min, 50 ◦ C for 2.0 min, 72 ◦ C for 3.0 min, 72 ◦ C for 1.0 min for 40 cycles. The PCR products obtained were treated with BciVI restriction enzyme (Fermentas, St Leon Rot, Germany) to digest the aberrant chain DNA derived from the NSI fusion partner. Digested PCR products were electrophorized in a 2.0% agarose gel (Sigma/Aldrich, St Louis, MO, USA) to resolve the DNA fragments. DNA band of approximately
360 bp was extracted using a gel fragments extraction kit (Geneaid, Sijhih City, Taiwan), and sent to Mission Bioteck (Taiwan, Nangang, Taipei, Taiwan) for sequence determination. CDRs of the VH (hypervariable region of the heavy chain) chain were amplified using a collection of 9 degenerate forward primers and, a single reverse primer (5 -AGG GGC CAG TGG ATA GAC-3 ). The forward primers used were: OVH1 (5 -SAG GTC CAG CTG CAG CAG YYT GG-3 ), OVH2 (5 -CAG GTR CAG CTG AAG SAG TCA GG-3 ), OVH3 (5 -GAK GTG CAG CTT CAG CAG TCR GG-3 ), OVH5-1 (5 -GAV GTG AWG CTG GTG GAG TCT GA-3 ), OVH5-2 (5 -GAV GTG AWG CTG GTG GAG TCT GG-3 ), OVH11 (5 -GAA GTG CAG CTG TTG GAG ACT GG-3 ), OVH14-1 (5 -GAG GTT CAG CTG CAG CAG TCT GG-3 ), OVH14-2 (5 -GAG GTT CAG CTG CAG CAG TCT GT-3 ), and OVH15 (5 -CAG GTT CAC CTA CAA CAG TCT GG-3 ). The PCR program used to generate a DNA fragment of approximately 300 bp was set as follows: 92 ◦ C for 3 min, 92 ◦ C for 1 min, 68 ◦ C for 30 s, 72 ◦ C for 1 min, 72 ◦ C for 10 min for 25 cycles. The expected PCR product was extracted from the 2% agarose gel, and processed for sequence determination. 2.6. SDS–PAGE and Western blot Twenty microliters of the heat-inactivated (10 min in a 85 ◦ C water bath) sucrose-gradient purified EV71/E59 virus measured to contain 0.12 g of total protein determined with the BCA protein assay (Pierce, Rockford, IL, USA) was mixed with 5.0 L of 5× concentrated loading dye (Bionovas Biotech Co., Ltd., Toronto, Canada), and electrophorized in a 10% SDS–polyacrylamide gel in 1× Tris–glycine SDS-running buffer (Amersham Biosciences, Piscataway, NJ, USA). The resolved viral proteins were transferred onto a PVDF membrane (Biorad, Hercules, CA, USA) by using the Mini Trans-Blot Cell (Biorad). Protein-containing membrane was blocked in 5.0% skim milk (Anchor, New Zealand) prepared in PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA), then washed, and placed in the Mini-Protene II Multiscreen Apparatus (Biorad, Hercules, CA, USA). 2.0 g of the individual test monoclonal antibodies (N1, N3, N4, or N6) prepared in assay buffer (1.0% skim milk in PBS, pH 7.2) was added to the reaction chamber. The membrane was then treated with 0.5 mL of 1:10,000 dilution (in assay buffer) of a horse radish peroxidase (HRP)-conjugated donkey anti-mouse
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been high-performance liquid chromatography (HPLC)-purified to >90% purity. 2.8. Epitope mapping ELISA
Fig. 2. Reactivity of protein G column purified monoclonal antibodies (N1, N3, N4 and N6) against a purified preparation of HI-EV71/E59 in western blot. The assay was performed with 2.0 mg/mL of the individual monoclonal antibodies against 1.2 g/mL of the electrophorized purified EV71/E59 virus preparation.
antiserum (Jackson ImmunoResearch Lab., West Grove, PA, USA). After 0.5 h incubation, the membrane was washed, blotted dry on paper towels, and 100.0 L of Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) was then layered onto the membrane. The membrane was exposed for 0.5, 1.5 and 10 min on an X-ray film (Kodak, Rochester, NY, USA) to detect binding.
Two different formats involving the use of MaxiSorp, and CovaLink NH ELISA plates purchased from NUNC (Napperville, IL, USA) were used to immobilize the test peptides. Individual test peptide reconstituted at 2.0 mg/mL in 5.0% DMSO (Sigma/Aldrich, St Louis, MO, USA) was diluted to 10.0 g/mL in pH 9.6 carbonate–bicarbonate coating buffer (Sigma/Aldrich, St Louis, MO, USA), and coated in pools of five adjacent peptides, or individually onto each well of the MaxiSorp plate. Peptide coating onto CovaLink NH plates was done by following the procedures described by Sondergard-Andersen et al. (1990). Following overnight incubation (for MaxiSorp plate), or 30 min incubation at room temperature (for CovaLink plate), the plates were washed, and blocked in 3.0% skim milk prepared in PBS, pH 7.2 (Gibco/Invitrogen, Carlsbad, CA, USA). Varying concentrations of the individual purified monoclonal antibodies prepared in the assay buffer (1.0% skim milk in PBS pH 7.2) were then added to each of the test wells. 100.0 L of a HRP-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Lab., West Grove, PA, USA) at 1 in 10,000 dilution prepared in assay buffer was added 1 h later for detection. The wells were washed, and 50 (L of TMB peroxidase substrate (SureBlueTM , KPL, Gaithersburg, MD, USA) was added to the individual assay wells. Further colour development was stopped by adding 50.0 L of 2.0 N H2 SO4 (Sigma/Aldrich, St Louis, MO, USA) to each well 20 min later. Absorbance readings were recorded at 450 nm with an ELISA reader (Spectra Max M2 model, Sunnydale, CA, USA).
2.7. Peptide synthesis 2.9. Immuno-plaque forming inhibition assay A total of 57 15-mer peptides in overlap of 10 amino acids spanning the entire length of the VP1 capsid protein of an EV71 isolate designated, TW/2086/98, of genotype C2 were synthesized by Kelowna International Scientific Inc. (San Chung City, Taiwan) (Table 1) to characterize the fine specificities of the N1, N3, N4 and N6 monoclonal antibodies directed against the VP1 capsid protein of EV71/E59. The individual peptides used in the present study had
2.5 × 105 rhabdomyosarcoma (RD) cells (ATCC, Rockville, MD, USA) resuspended in 1.0 mL of CM were cultured in individual wells of a 24-well tissue culture plate (Corning Life Sciences, Corning, NY, USA). The cells were kept overnight in a 37 ◦ C incubator equilibrated with 5.0% CO2 to form a monolayer. The culture supernatant was removed, and 200.0 L of CM containing 100 PFUs of each test
Fig. 3. Epitope mapping studies. Maxisorp plates, and CovaLink plates were used to coate the 15-mer VP1 peptides. A concentration of 10.0 g/mL of the individual monoclonal antibodies was used in each test. Results shown represented average absorbance for each pair of the duplicate assays performed with the pooled peptides coated on MaxiSorp plate (A), CovaLink plate (B); and individual peptides coated on these plates (C and D), respectively.
H.-W. Chang et al. / Journal of Virological Methods 173 (2011) 189–195
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Fig. 4. Determination of virus neutralizing activity of the individual, and pooled EV71/E59-specific monoclonal antibodies. Results showed % neutralization of 100 PFUs of the individual purified EV71 viruses: EV71/E59 (), N0781-TW-01 (), or N2838-TW-03 (䊉) tested against varying concentrations of each of the monoclonal antibodies (A–D), and a pooled sample of the antibodies (E). An murine IgG2a subclass monoclonal antibody (clone #20102, R&D Systems, Minneapolis, MN, USA) was also assayed as control ().
virus, that had been preincubated (overnight) with varying concentrations of the individual monoclonal antibodies all belonging to the IgG2a subclass, or a mixture of the four monoclonal antibodies were added to each RD cell-containing culture wells. Cultures containing no added monoclonal antibodies, or with the addition of a monoclonal IgG2a antibody (clone #20102, R&D Systems, Minneapolis, MN, USA) were also assayed as positive, and negative controls, respectively. Following 1 h incubation at 37 ◦ C, 0.5 mL of 1.1% methanol cellulose (Merck, West Point, PA, USA) was overlaid onto the cells. The cultures were kept in a 37 ◦ C incubator equilibrated with 5.0% CO2 for 2 days. Culture supernatant was removed, and 0.5 mL of a 3.7% formalin (Merck, West Point, PA, USA) solution prepared in PBS (pH 7.2) was added to the cells. Formalin was removed the next day, and 100.0 L of 1 in 5000 dilution (prepared in assay buffer) of an anti-mouse EV71-specific monoclonal antibody, MAB 979 (Chemicon, Temecula, CA, USA) was then added to individual test wells. Plates were washed 1 h later, and 100.0 L of an anti-mouse IgG-HRP conjugate antibody (Serotec, Kidling-
ton, Oxford, UK) at 1 in 50,000 dilution (prepared in assay buffer) was added for detection. The plates were washed, and 100.0 L of TMB substrate (KPL, Gaithersburg, MD, USA) was added to each assay well. The reaction was allowed to develop for 30 min in the dark. Plagues seen as black spots under an optical microscope were counted. Virus titer was expressed as mean PFUs per mL of the two highest diluted EV71/E59 preparations tested. 3. Results 3.1. CDR sequence analysis Sequence data obtained showed that the hybridomas secreting the N1 and N4 antibodies shared the same CDR1 and CDR2, but a different CDR3, while the N3 and N6 antibody producing hybridomas expressed a different set of chains with the same CDR1 and CDR2, but a distinct CDR3 (Fig. 1). For the heavy chains expressed in the hybridomas, it was not found whether they contained different
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CDRs (data not shown). These observations support that the N1, N3, N4 and N6 antibodies are produced by distinct hybridoma clones. 3.2. Western blot analysis of purified ascitic fluids As depicted in Fig. 2, the individual monoclonal antibodies recognized a viral protein of approximately 36 kDa resolved from the sucrose gradient purified EV71/E59 virus sample. Recombinant VP1 expressed in baculovirus (Chung et al., 2006) was reported to have this molecular mass. Interestingly, the individual EV71/E59specific monoclonal antibodies also bound a protein moiety, Pr, slightly larger than VP1 (approximately 38 kDa) that was also separated in the EV71/E59 preparation. The nature of this protein is under further investigation. 3.3. Epitope mapping studies Results obtained from both ELISA formats revealed that pool 9 contained peptides recognized by each of the monoclonal antibodies (Fig. 3A and B). Fine epitope analysis performed by coating each of the peptides in pool 9 onto the MaxiSorp and CovaLink NH ELISA plates identified a peptide designated VC43, encompassing the amino acid sequence, FGEHKQEKDLEYGAC, was the target recognized by each of the monoclonal antibodies (Fig. 3C and D). 3.4. Virus neutralizing activities of monoclonal antibodies Results obtained from these studies showed that the individual monoclonal antibodies exhibited potent neutralizing activity against the EV71/E59 isolate used for immunization, as well as N0781-TW-01 also with genotype B4, and N2838-TW-03 of genotype B5 that had been found commonly in the more recent outbreaks in Taiwan. At 3log10 g/mL, N3 and N6 monoclonal antibodies completely (100.0%) blocked the infectivity of the RD cells by each of the viral isolates tested (Fig. 4B and D). At this concentration, N1 and N4 monoclonal antibodies also neutralized fully the EV71/E59 and N0781-TW-01 viruses, but neutralized N2838TW-03 infectivity by 81.0% and 88.0%, respectively (Fig. 4A and C). The monoclonal antibodies were noted to act cooperatively in neutralizing the EV71/E59 isolate. Complete inhibition of EV71/E59 virus infectivity was achieved at a markedly lower concentration (2.04log10 g/mL) of a pooled sample of the antibodies (Fig. 4E). 4. Conclusions The objective of the present study was to raise murine monoclonal antibodies that exhibit strong neutralizing activity against EV71 isolates with genotype B found commonly in the recent outbreaks in Taiwan, which could serve as targets for therapeutic development. Using live EV71/E59 for immunization, four hybridoma clones producing monoclonal antibodies found to belong to IgG2a subclass that recognize distinct antigenic sites within a common epitope (VC43) at position 211–225 of VP1 are isolated. The monoclonal antibodies exhibit modest differential virus neutralizing activity against viruses of genogroup B. At a higher concentration of 3log10 g/mL, monoclonal antibodies N3 and N6 are capable of inhibiting completely infection of 100 PFUs of the EV71/E59 virus used for immunization, N0781-TW-01 with the same genotype, as well as N2838-TW-03 of genotype B5 that has remerged in the 2008 outbreak in Taiwan (Huang et al., 2009). Although monoclonal antibodies N1 and N4 at these concentrations also neutralized completely the genotype B4 viruses, they are slightly less effective than the N3 and N6 antibodies in neutralizing the N2838-TW-03 isolate. The findings that the monoclonal antibodies act cooperatively in neutralizing the EV71/E59 virus
illustrates they recognized distinct structural sites in the VC43 epitope. Suckling ICR and BALB/c mice are susceptible to infection with high doses of live EV71 with genotype C. One-day-old mice infected with a mouse-adapted genotype C virus would develop neurological disorders similar to the symptoms seen in subjects with severe EV71 infections (Wang et al., 2004). This mouse model has been used by Chang et al. (2010) to show a monoclonal antibody against an inactivated EV71/Hn2 substrain of genotype C4 is capable of protecting newborn BALB/c mice from lethal challenge with the same isolate. To date, similar studies demonstrating that newborn mice would also develop human-like symptoms upon infection with EV71 of genogroup B have not been reported. Inoculation of high doses of EV71/E59, N0781-TW-01, or N2838-TW-03 used in the present study has not been found to cause severe infection in suckling ICR and BALB/c mice. In this setting, development of mice transgenic for the PSGL-1 and SCARB2 human EV71 receptors (Nishimura et al., 2009; Yamayoshi et al., 2009) that would acquire human-like symptoms upon infection with EV71 within genogroups B and C could provide potentially a suitable small animal model to assess the protective efficacy of the monoclonal antibodies described above. Acknowledgment This study was supported by the intramural grant #99A1VCPP02-014, from the National Health Research Institutes, Taiwan. References AbuBakar, S., Chee, H.Y., Al-Kobaisi, M.F., Xiaoshan, J., Chua, K.B., Lam, S.K., 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. 61, 1–9. Chang, G.-H., Luo, Y.-J., Wu, X.-.Y., Si, B.-Y., Lin, L., Zhu, Q.-Y., 2010. Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection. Virol. J. 7, 106–112. Chonmaitree, T., Menegus, M.A., Schervish-Swierkosz, E.M., Schwalenstocker, E., 1981. Enterovirus 71 infection: report of an outbreak with two cases of paralysis and a review of the literature. Pediatrics 67, 489–493. Chung, Y.C., Huang, J.H., Lai, C.W., Sheng, H.C., Shih, S.R., Ho, M.S., Hu, Y.C., 2006. Expression, purification and characterization of enterovirus-71 virus-like particles. World J. Gastroenterol. 12, 921–927. Foo, D.G., Alonso, S., Phoon, M.C., Ramachandran, N.P., Chow, V.T., Poh, C.L., 2007. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Res. 125, 61–68. Gilbert, G.L., Dickson, K.E., Waters, M.J., Kennett, M.L., Land, S., Sneddon, A.M., 1988. Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement. Pediatr. Infect Dis. J. 7, 484–488. Ho, M., Chen, E.R., Hsu, K.H., Twu, S.J., Chen, K.T., Tsai, S.F., Wang, J.R., Shih, S.R., 1999. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N. Engl. J. Med. 341, 929–935. Huang, S.W., Hsu, Y.W., Smith, D.J., Kiang, D., Tsai, H.P., Lin, K.H., Wang, S.M., Liu, C.C., Su, I.J., Wang, J.R., 2009. Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J. Clin. Microbiol. 47, 3653–3662. Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497. Li, X., Mao, C., Ma, S., Wang, X., Sun, Z., Yi, Y., Guo, M., Shen, X., Sun, L., Bi, S., 2009. Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides. Biochem. Biophys. Res. Commun. 390, 1126–1128. Lin, T.Y., Chang, L.Y., Hsia, S.H., Huang, Y.C., Chiu, C.H., Hsueh, C., Shih, S.R., Liu, C.C., Wu, M.H., 2002a. The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin Infect Dis 34 (Suppl. 2), S52–57. Lin, T.Y., Twu, S.J., Ho, M.S., Chang, L.Y., Lee, C.Y., 2002b. Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg. Infect. Dis. 9, 291–293. Liu, X.J., Li, W., Zhang, Y.Q., Liu, Y.M., Liu, L.Z., 2009. Clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 infection. Zhongguo Dang Dai Er Ke Za Zhi 11, 967–969. McMinn, P.C., 2003. Enterovirus 71 in the Asia-Pacific region: an emerging cause of acute neurological disease in young children. Neurol. J. Southeast Asia 8, 57–63. Nishimura, Y., Shimojima, M., Tano, Y., Miyamura, T., Wakita, T., Shimizu, H., 2009. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat. Med. 15, 794–797. Pulli, T., Koskimies, P., Hyypia, T., 1995. Molecular comparison of coxsackie A virus serotypes. Virology 212, 30–38.
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