JGV Papers in Press. Published June 19, 2014 as doi:10.1099/vir.0.062562-0

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Production of a neutralizing antibody against envelope protein of dengue virus type 2 using linear array epitope (LAE) technique

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Peng-Yeh Laia, Chia-Tse Hsub, Shao-Hung Wangc, Jin-Ching Leed, Min-Jen Tsenga, Jaulang

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Hwanga, e, Wen-Tsai Jia and Hau-Ren Chena,*

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a

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Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102,

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Taiwan.

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b

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Biochemical Engineering, Kao Yuan University, Luzhu District, Kaohsiung City 82151,

Department of Life Science, Institute of Molecular Biology and Institute of Biomedical

Department of Chemical and Biochemical Engineering and Institute of Chemical and

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Taiwan

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c

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University, Chiayi City 60004, Taiwan.

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d

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80708,Taiwan

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e

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11031, Taiwan.

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*Correspondence to Dr. Hau-Ren Chen, Department of Life Science, National Chung Cheng University.

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168, Sec. 1, University Road, Min-Hsiung, Chia-Yi 62102, Taiwan.

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Tel, 886-5-2720411 ext 66503; Fax, 886-5-2722871; e-mail, [email protected]

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Running title: Novel antibody generated by LAE technique

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Abstract: 245

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Text including figure legends: 4832

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Total 5 figures and 0 table

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Category: Animal virus-Positive-strand RNA

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Department of Microbiology, Immunology and Biopharmaceuticals, National Chiayi

Department

of

Biotechnology,

Kaohsiung

Medical

University,

Kaohsiung

city

Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei City

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Abstract

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Dengue virus belongs to Flavivirus and contains a positive-stranded RNA genome

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Binding of dengue virus to host cells was mediated through domain III of the viral

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envelope protein. Many therapeutic monoclonal antibodies (mAbs) against domain III

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have been generated and characterized because of its high antigenicity. We have

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previously established a novel PCR method named linear array epitope (LAE)

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technique for producing monoclone-like polyclonal antibodies. To prove this method

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could be utilized to produce antibody against epitopes with low antigenicity, a region

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of 10 amino acids (V365NIEAEPPFG374) from domain III of the envelope protein in

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dengue virus serotype 2 (DENV2) was selected to design the primers for LAE

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technique. A DNA fragment encoding 10 directed repeats of these 10 amino acids for

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producing the tandem repeated peptides was obtained and fused it with

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GST-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from

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E. coli and used as antigen for immunizing rabbits to obtain polyclonal antibody.

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Furthermore, this EIII antibody could recognize envelope proteins either ectopically

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overexpressed or synthesized by DENV2 infection using immunoblot and

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immunofluorescence assays. Most importantly, this antibody was also capable of

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detecting DENV2 virions by ELISA assay and could block viral entry into BHK-21

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cells as shown by immunofluorescence and qRT-PCR assays. Taken together, LAE

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technique could be applied for production of antibody against antigen with low

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antigenicity successfully and carries a high potential to produce antibodies with good

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quality for academic research, diagnosis and even therapeutic application in the

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future.

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Abbreviations: LAE: linear array epitope, TR-PCR: template-repeated polymerase

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chain reaction, PEIa: exotoxin receptor-binding domain A of Pseudomonas

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531. Introduction 54

Dengue virus is a considerable threat to public health all worldwide. The outbreaks

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and epidemics caused by virus infection become a serious public health issue in many

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tropical and subtropical countries. Dengue viruses are classified into four serotypes

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(DENV1 to DENV4) and are transmitted to human by mosquito vectors such as Aedes

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aegypti and Aedes albopictus. Virus infection may cause different kinds of diseases,

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including dengue fever (DF) with febrile illness, dengue haemorrhagic fever (DHF)

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with plasma leakage syndrome and dengue shock syndrome (DSS). Approximately

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100 million cases of DF and 500,000 cases of DHF and DSS occur every year, with a

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mortality rate of 5% (Gubler, 2002; Wilder-Smith & Gubler, 2008). Therefore, it is

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important and urgent to develop proper vaccines for prevention of the disease. In the

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past decades, there are several approaches attempting to develop dengue virus

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vaccines, including live attenuated viruses, chimeric viruses, recombinant subunit

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antigens, expression vector-based vaccines and DNA vaccines (Chokephaibulkit &

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Perng, 2013; Heinz & Stiasny, 2012; Lee et al., 2012; Leng et al., 2009; Schmitz et al.,

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2011). However, there is currently no commercially available vaccine against dengue

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virus.

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Dengue virus belongs to the family of Flaviviridae, genus Flavivirus, with a

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positive-sense single-stranded RNA genome. The RNA genome of 10.7 kb in length

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has a 5’ cap structure but lacks 3’ poly A tail, and encodes a polyprotein which is

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cleaved into three structural proteins (core, premembrane and envelope) and seven

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non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by host

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and/or viral proteases. Among the three structural proteins, the premembrane and

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envelope proteins are the primary antigenic targets of humoral immune response in

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human (Wan et al., 2013). The neutralizing antibodies are usually induced by the

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envelope protein (Murphy & Whitehead, 2011). The envelope protein is consisted of

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495 amino acids with a molecular weight of about 60 kDa. It has been suggested that

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the major role of the envelope protein is host cell attachment and entry (Bhardwaj et

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al., 2001). The ectodomain of envelope protein is composed of three domains,

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including domain I, II and III (Modis et al., 2003; 2004; Rey et al., 1995). Domain I

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contains mainly type-specific non-neutralizing epitopes and is thought to be the

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molecular hinge region involved in low-pH-triggered conformational changes

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(Roehrig et al., 1998). Domain II is the dimerization domain, and is involved in

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virus-mediated membrane fusion. It contains many cross-reactive epitopes eliciting

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neutralizing and non-neutralizing monoclonal antibodies (Rey et al., 1995; Roehrig et

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al., 1998). Domain III is an immunoglobulin-like structure containing the most distal

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projecting loops from the virion surface. According to X-ray crystal structure, domain

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III contains 10 β-strands (A to G and a small extra sheet, AxCxDx). In addition,

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domain III carries multiple type- and subcomplex-specific epitopes eliciting

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neutralizing monoclonal antibodies that have been hypothesized to block virus

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binding to the host cell receptor (Bhardwaj et al., 2001; Chiu & Yang, 2003; Modis et

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al., 2003; 2004; Rey et al., 1995; Sukupolvi-Petty et al., 2007; Volk et al., 2004; Yu et

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al., 2004). Since envelope domain III plays important roles in viral entry, it is a good

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target for generating neutralizing antibody.

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Linear array epitope technique (LAE) is a method for preparation of an immunogen

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containing multiple copies of a defined peptide in a linear alignment. This method

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was designed to overcome poor immune response induced by traditional

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immunogenic peptide/protein antigens. The tandem repeated epitopes are able to

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efficiently induce a strong immune response in vivo (Yi et al., 2006) and prime CD4+

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T cells in vitro (Barratt-Boyes et al., 1999). Immunization of female rabbits with the

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LAE-produced immunogen that contained the exotoxin receptor-binding domain A of

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Pseudomonas (PEIa) and 12 copies of gonadotropin-releasing hormone (GnRH)

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resulted in the generation of high-titer antibodies specific for GnRH. The anti-GnRH

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antibodies effectively neutralized GnRH activity in vivo, as demonstrated by the

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degeneration of the ovaries in the injected rabbits (Hsu et al., 2000). The

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LAE-produced gastrin-releasing peptide (GRP) immunogen also induced antibody

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potentially inhibiting breast cancer cell proliferation in vitro and in vivo (Guojun et al.,

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2008). A rabbit anti-mouse EKLF (erythroid Krűppel-like factor) antibody induced by

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LAE-produced immunogen could be used to detect EKLF in mouse embryonic

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samples by Western blot and immunostaining, and was applicable for chromatin

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immunoprecipitation (ChIP) assay (Shyu et al., 2007).

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In this study, we used LAE technique to produce a tandem-repeated domain III region

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(V365NIEAEPPFG374)10 of DENV2 envelope protein as the immunogen (referred as

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EIII epitope hereafter). We demonstrated that anti-EIII antibody induced by this novel

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immunogen could be used to detect the envelope protein either ectopically

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overexpressed or synthesized by DENV2 infection in cells. Importantly, the antibody

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could be used to detect dengue virions and to block dengue viral entry into BHK-21

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cells successfully.

1212. Results 122

Epitope selection and the principle of linear array epitope (LAE) technique

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To test whether LAE technique could be used to induce antibodies against DENV2

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envelope domain III, we chose an epitope (V365NIEAEPPFG374) consisting of

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β-strand and random coil secondary structures, and was not co-localized with previous

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neutralizing epitopes (Crill & Chang, 2004; Gromowski & Barrett, 2007; Lin et al.,

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1994; Thullier et al., 2001; Wan et al., 2013). According to the three-dimensional

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structures

129

(www.rcsb.org/pdb), this epitope seemed to be localized on the surface of the protein

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(Fig. S1). To synthesize repeated EIII epitope fused with GST, two oligonucleotides

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(oligo Den E-A and Den E-B) were designed for template-repeated PCR (TR-PCR).

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Oligo Den E-A encodes VNIEAEPPFG peptide, and its 5’ and 3’ halves are

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complementary to those of oligo Den E-B, respectively (Figure 1). The TR-PCR

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products containing repeated EIII epitope were used as the templates for the second

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PCR (adapter-PCR) to introduce the restriction enzyme sites and stop codon. The

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products of both TR-PCR and adapter-PCR showed the ladder pattern in 8% PAGE

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(Figure 1d). The lowest band (the major one in lane 3) among the ladders was the

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monomer, and the higher bands were multimers. The DNA products were eluted and

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cloned into T vectors, followed by screening for positive clones. Finally, a clone with

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10 tandem repeated epitopes was obtained.

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Preparation of tandem repeated antigen for generating rabbit antiserum against

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domain III of the envelope protein

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The DNA fragment with 10 repeats of EIII epitope was subcloned into two expression

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plasmids for producing tandem repeated antigens, generating GST-Den EIII10-His6

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fusion protein and PEIa-Den EIII10-His6 fusion protein (data not shown) in E. coli

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BL21(DE3). The induced proteins were purified by glutathione Sepharose and nickel

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column, respectively. The Coomassie blue staining for purification of GST-Den

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EIII10-His6 protein is shown in Figure 2a. Elution fractions 3 to 7 were collected as the

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antigen to immunize rabbits. The harvested antiserum against GST-Den EIII10-His6

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protein was named EIII antibody, and the antibody titer against recombinant

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full-length domain III fused with His6 tag (referred as EIII-His6 hereafter) was

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monitored by ELISA every two weeks. Even as low as 10 ng coated EIII-His6 could

of

the

envelope

protein

obtained

from

protein

data

bank

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be significantly detected by this antibody; the OD410 values of 102- and 103-fold

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diluted sera were much higher than those of control (Fig. 2b). The titer of the

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antiserum achieved plateau after 6 weeks (data not shown).

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Evaluation of the specificity of EIII antibody by various assays

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To further confirm the specificity of the harvested EIII antibody, ELISA and

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immunoblot assay was performed. Recombinant EIII-His6 proteins from four

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serotypes of DENV were used to test whether EIII Ab could recognize EIII protein of

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all serotypes. In ELISA assay, Fig. 2c showed that EIII Ab mainly detected EIII-His6

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of DENV2 with 104-fold dilution, but cross-reacted with EIII-His6 of all four

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serotypes with 102-fold dilution. In immunoblot assay, EIII antibody could recognize

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purified EIII-His6, PEIa-Den EIII10-His6 and GST-Den EIII10-His6, but not the

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unrelated proteins such as PEIa-His6 (the protein product of empty pPEIa-His6 vector),

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TAF7-His6 (a transcriptional factor) and NS3-C-His6 (the N-terminal truncated NS3

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protein of dengue virus), suggesting the high specificity of LAE-induced antibody

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(Fig. 3a). We next examined if EIII antibody could detect full-length DENV2

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envelope protein in cells. Figure 3b and 3c showed that EIII antibody could

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specifically detect the DENV2 envelope proteins ectopically overexpressed in

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BHK-21 or HEK293T cells by Western blot analysis and immunofluorescence assay,

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respectively. Furthermore, we tested if the antibody could be used in

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immunoprecipitation assay. The cell lysates from HEK293T cells transfected with

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vector only or with pCMV-3FLAG-DENV2 envelope were incubated with EIII

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antibody, resulting in immunoprecipitation of ectopically expressed FLAG-tagged

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DENV2 envelope proteins (Fig. 3d).

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Application of EIII antibody for detecting virions and blocking viral entry

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Since EIII antibody could recognize recombinant envelope proteins in BHK-21 and

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HEK293T cells, we examined if it could recognize the envelope protein of dengue

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virus virions. C6/36 cells were infected with DENV2 for 10 to 12 days and the cell

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lysates or the media were collected for Western blot analysis. EIII antibody could

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clearly detect dengue virus premembrane/envelope heterodimer protein in

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virus-infected cell lysates as indicated in the filled arrow (Sukupolvi-Petty et al., 2010;

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Zhang et al., 2003). EIII antibody also detected envelope protein in the medium

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fraction of virus-infected culture weakly as indicated in the open arrow (Fig. 4a). To

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further confirm this result, EIII antibody was used to detect virus-infected BHK-21

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cells at different time points after infection. As shown in Figure 4b, EIII antibody

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could detect virus envelope proteins in the infected cells and the signal increased

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commensurate to the infection time, which correlated with the replication cycle of

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dengue virus. Since the envelope protein is located on the surface of the virus, the

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antibody was tested for virions recognition. Different amounts of dengue viruses were

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coated on the ELISA plate and serially diluted EIII antibody was used to detect the

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virions. The results showed that EIII antibody with 10- to 103-fold dilutions could

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detect virions in a dosage-dependent manner (Fig. 4c).

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Finally we tested if EIII antibody could be applied for blocking dengue virus infection.

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Dengue viruses were pre-incubated with serially diluted EIII antibody and the

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virus-antibody mixture was used to infect BHK-21 cells. Viruses were detected for

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NS3 by immunofluorescence and qRT-PCR assays. The immunofluorescence signals

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were quantified by counting green fluorescence-positive cells in different fields, and

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the results showed that EIII antibody with 10-fold dilution significantly reduced the

200

fluorescence signals (Fig. 5a and 5b). The levels of NS3 RNA were also decreased by

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EIII antibody (Fig 5c), suggesting that viral entry might be blocked effectively. In

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order to further verify the process, we treated EIII Ab either before or after dengue

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virus infection and found that EIII Ab affected viral infection significantly only in

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pretreated condition, indicating that EIII Ab blocked virus infection through blocking

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of viral attachment (Fig. 5d). Since EIII Ab could block DENV2 infection, we further

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test if this Ab would block viral entry of other serotypes. All fours serotypes of

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DENV were used to evaluate the blocking ability as done in Fig. 5c. The result

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suggested EIII Ab specifically blocked DENV2 infection although DENV4 could not

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be determined (Fig. S3)

2103. Discussion 211

Infection by dengue virus is a global concern. However, there is still no approved

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antiviral treatment available so far. Several candidate vaccines remain in preclinical or

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clinical evaluation (Durbin & Whitehead, 2010). The main strategy of these candidate

214

vaccines is to induce neutralizing antibodies to block virus infection. The best target

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for blocking virus infection is the structural proteins on the virion’s surface.

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According to previous studies, many neutralizing antibodies were induced by the

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envelope proteins. Based on epitope mapping data, many epitopes of the type-specific

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neutralizing antibodies against individual flaviviruses are localized to domain III,

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whereas neutralizing monoclonal antibodies that cross-react with other flaviviruses

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are localized primarily to domain II (Beasley & Barrett, 2002; Cecilia & Gould, 1991;

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Chavez et al., 2010; Crill & Chang, 2004; Crill & Roehrig, 2001; Fonseca et al., 1994;

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Goncalvez et al., 2004a; Goncalvez et al., 2004b; Lin et al., 1994; Oliphant et al.,

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2005; Oliphant et al., 2006; Stiasny et al., 2006). Some studies further defined contact

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residues for serotype-specific, subcomplex-specific and cross-reactive monoclonal

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antibodies that recognize domain III of the envelope protein in dengue virus; the

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epitopes of serotype-specific neutralizing monoclonal antibodies are localized to the

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BC, DE and FG loop on the lateral ridge of domain III, whereas subcomplex-specific

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monoclonal antibodies recognize A strand of domain III (Gromowski & Barrett, 2007;

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Gromowski et al., 2008; Lok et al., 2008; Rajamanonmani et al., 2009;

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Sukupolvi-Petty et al., 2007; Thullier et al., 2001). To prove that LAE technique

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could improve epitopes of poor antigenicity, we chose the epitope (V365 N I E A E P P

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F G374) localized between E-strand and F-strand of domain III, which has not been

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reported to induce any neutralizing antibody. During the antigen purification process,

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there were some ladder-like minor bands located under the major bands (Fig. 2a). We

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speculate that the tandem repeated sequences might affect antigen translation or

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degradation. This tandem repeated antigen indeed induces EIII antibody production,

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which could be utilized for basic biochemical assays, such as Western blot analysis,

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immunofluorescence and co-immunoprecipitation. In addition, this antibody could

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specifically detect EIII-His6 (Fig. 3a), ruling out the possibility that it recognized the

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junction sequences between two repeated epitopes. More importantly, this antibody

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could be also applied for detecting dengue virus virions and blocking viral infection

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into BHK-21 cells. According to literature search, this is the first report of application

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of LAE technique in virus-related research.

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From epidemiological studies, it is generally believed that heterologous antibodies

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that fail to neutralize the infecting serotype contribute primarily to the development of

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DHF/DSS in both infants and adults (Halstead, 2003). Those sub-neutralizing

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antibodies enhance viral uptake by Fcγ receptor (FcγR)-dependent or Fcγ-independent

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mechanisms called antibody-dependent enhancement (ADE) (Halstead & O'Rourke,

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1977; Huang et al., 2006). To reduce the risk of ADE, the neutralizing antibodies

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must be protective against each of the four dengue virus serotypes. This might be

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achieved by choosing a conserved epitope in different serotypes to produce tetravalent

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neutralizing antibodies by LAE technique. Therefore, this is the second reason why

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we chose a region of 10 amino acids (V365NIEAEPPFG374) from domain III of the

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envelope protein in DENV2 as the target of epitope. This sequence is 100% conserved

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in DENV1, DENV2 and DENV3 but is only with two amino acids difference in

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DENV4 (Fig. S2a). Interestingly, even this minor divergence might reflect in the

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generated EIII Ab: 2000-fold diluted EIII Ab recognizes recombinant EIII-His6 of

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DENV1 to DENV3, but not DENV4 (Fig. S2b). However, 100-fold diluted EIII Ab

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could detect all the recombinant EIII-His6 of four serotypes (data not shown).

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Similarly, EIII Ab majorly recognizes recombinant EIII-His6 of DENV2 under low

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concentration (104-fold dilution), but cross-reacts with EIII-His6 of all four serotypes

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under high concentration (102-fold dilution) in ELISA (Fig. 2c). Surprisingly, Fig. 5

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and Fig. S3 also show that EIII Ab specifically blocks DENV2 infection to BHK-21

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cells although the epitope are identical in DENV1, DENV2 and DENV3. We

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speculate this specific blockage might derive from higher avidity of EIII Ab with

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envelope protein of DENV2, suggesting that the the antibody generated by LAE not

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only recognizes the sequence but also discriminate structural arrange of corresponding

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antigen.

269

To further delineate the process of blockage, we further examined whether that EIII

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Ab blocked virus infection through the blockage of viral attachment to host receptor

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(Fig. 5d). Our result is consistent with previous studies, which suggests that domain

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III also play important roles in binding to the host cell receptors (Bhardwaj et al.,

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2001; Chiu & Yang, 2003; Crill & Roehrig, 2001; Hung et al., 2004).

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Taken together, LAE technique can be used to develop antigens with higher

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antigenicity or antibodies with higher specificity through simple PCR-based method

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even without any DNA template. Moreover, the LAE-produced polyclonal antibody

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recognized the same epitope, displaying the property of monoclonal antibody and may

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be considered as monoclone-like polyclonal antibody. Such antigens and antibodies

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have high potential for uses in academic research, diagnosis and therapeutic

280

application in the future.

2814. Methods 282

Template-repeated PCR (TR-PCR) and adapter-PCR

283

KOD Hot Start DNA Polymerase (Novagen) was used in TR-PCR. Two

284

oligonucleotides were used in this reaction: Den E-A: 5’-GTT AAC ATC GAA GCC

285

GAA CCT CCT TTT GGT-3’ and Den E-B: 5’-GGC TTC GAT GTT AAC ACC

286

AAA AGG AGG TTC-3’. In TR-PCR, the two primers were also functional as

287

templates. The PCR programs were 30 cycles of denaturation at 94°C for 1 minute,

288

annealing at 44°C for 1 minute, and polymerization at 72°C for 1 minute, followed by

289

a final polymerization step at 72°C for 10 minutes. After TR-PCR, the PCR product

290

was then used as template for adapter-PCR reaction catalyzed by Taq polymerase

291

(Fermentas). Two oligonucleotides were used in this reaction: Den E-5b: 5’-GGA

292

TCC GTT AAC ATC GAA GCC-3’ and Den E-3he: 5’-GAA TTC ATT AAA GCT

293

TAC CAA AAG GAG GTT C-3’. The PCR condition were 5 cycles of denaturation at

294

94°C for 1 minute, annealing at 44°C for 1 minute, and polymerization at 72°C for 1

295

minute, followed by 10 cycles of denaturation at 94°C for 1 minute, annealing at 48°C

296

for 1 minute, and polymerization at 72°C for 1 minute. Finally the reaction was

297

terminated with polymerization step at 72°C for 10 minutes. The product of adaptor

298

PCR was cloned into T vector for sequencing. The correct construct was then

299

subcloned into expression vectors pGEX-His6 or pPEIa-His6.

300

Antigen and antibody preparation

301

Recombinant GST-Den EIII10-His6 protein was expressed in BL21 (DE3) with 1 mM

℃ for 3hrs followed by breaking cells with sonication. After centrifugation,

302

IPTG at 37

303

the soluble protein was purified with Glutathione-Sepharose 4B (Amersham

304

Pharmacia). One hundred or five hundred micrograms of purified GST-Den

305

EIII10-His6 proteins were mixed with Freund’s complete adjuvant (Chemicon) and

306

then subcutaneously injected into female New Zealand white rabbits four times with

307

two weeks intervals. Sera were collected from rabbits’ ears biweekly and titer was

308

tested by ELISA. The sera were purified by Millipore montage® antibody purification

309

kit.

310

Enzyme-linked immunosorbent assay (ELISA)

311

The recombinant EIII-His6 proteins are the courtesy of Dr. Chih-Hsiang Leng in

312

National Health Research Institute, Taiwan (Leng et al., 2009). The consensus

313

sequence for EIII from DENV1 was obtained by alignment of the amino acid

314

sequences from five different isolates of DENV1, including Brazil/97-11/1997,

315

Jamaica/CV1636/1977,

316

Thailand/AHF 82-80/1980. The consensus sequences for EIII from DENV2, DENV3

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and DENV4 were obtained by alignment of the amino acid sequences from different

318

isolates, respectively. For DENV2, 16681-PDK53, China/D2-04, Jamaica/1409/1983,

319

Malaysia M2, Malaysia M3, Peru/IQT2913/1996, Thailand/0168/1979, Puerto

320

Rico/PR159-S1/1969,

321

Thailand/16681/84,

322

referenced. Strains for DENV3 included Philippines/H87/1956, China/80-2/1980,

323

Singapore/8120/1995, Martinique/1243/1999, and Sri Lanka/1266/2000. Strains for

324

DENV4

325

Thailand/0348/1991 Singapore/8976/1995, and Thailand/0476/1997. 10 ng, 100 ng

Nauru/West

Pac/1974,

Tonga/EKB194/1974 Thailand/NGS-C/1944,

included

Singapore/S275/1990

P27914, and

Philippines/H241/1956,

and

Thailand/PUO-218/1980,

Thailand/TH-36/1958

were

Dominica/814669/1981,

326

or 1000 ng of EIII-His6 proteins or dengue virus virion were coated on 96-well plates

327

with coating buffer (10 mM Na2CO3, 35 mM NaHCO3, pH 9.6) at 37

328

4

℃ for 2 hrs or

330

℃ overnight. After coating, samples were blocked by blocking solution (1% BSA and 0.0375% saponin in PBS) at 37℃ for 2 hrs or at 4℃ for overnight. The samples were incubated with EIII antibody at 37℃ for 2 hrs and washed with 120 μl PBS

331

three times. Then the samples were incubated with goat anti-rabbit IgG-HRP second

332

antibody at 37

333

substrate solution [2,2-azino-diethylbenzthiazoline sulphonic acid (ABTS) 0.54mg/ml,

334

0.1M citric acid pH 4.2, 3μl 30% H2O2/ml] was incubated with samples at room

335

temperature for 15 minutes. The signals were detected by multiscan ELISA reader

336

with A410 nm (Dynatech).

337

Cell cultures, transfection, virus strain and infection:

338

Baby hamster kidney (BHK-21) cells and human embryonic kidney (HEK293T) cells

339

were cultured at 37

340

medium (Gibco) supplemented with 10% fetal bovine serum (Biowest). C6/36, a

341

mosquito cell line established from Aedes albopictus was grown at 28

342

in RPMI 1640 medium (Gibco) supplemented with 10% FBS. Cells were transfected

343

with pCMV-3FLAG or pCMV-3FLAG-envelope using TurbofectTM reagent

344

(Fermentas). DENV1 (Myanmar 3886201 strain), DENV2 (PL046, NGC strain),

345

DENV3 (98TW503 strain) and DENV4 (H241 strain) stock was prepared in C6/36

346

cells and titrated by plaque assay. In infection experiments, appropriated cells were

347

seeded on culture plates and incubated at 37

329

℃ for 1 hour and washed three times with 120 ul PBS. Finally,

℃ with 5% CO in Dulbecco's modified Eagle's medium (DMEM) 2

℃ with 5% CO

2

℃ overnight. The next day, cells were

℃ for 2 hrs by adding dengue virus-containing culture supernatant of a

348

infected at 37

349

MOI of 0.02 or 12. After infection, cells were washed with PBS and cultured in fresh

350

medium.

351

Western blot and Co-immunoprecipitation (Co-IP):

352

Samples and pre-stained marker were subjected to a 10% or 12.5% polyacrylamide

353

gel for electrophoresis. The proteins were then electrophoretically transferred from gel

354

onto PVDF membrane by a semi-dry transfer system (Biometra). After transferring

355

onto the PVDF membranes and subsequent blocking, the blots were incubated with

356

the respective primary antibodies, followed by the secondary antibody conjugated

357

with horseradish peroxidase (HRP). Finally the signal was detected by adding

358

chemiluminescence reagent (PerkinElmer) and visualized using Chemi Genius2

359

CG2/D2A

360

immunoprecipitation, 150 micrograms cell lysates from HEK293T cells transfected

361

with empty vector or pCMV-3FLAG-envelope plasmid were immunoprecipitated with

362

EIII antibody using Dynabeads protein A system (Invitrogen), followed by Western

363

blot with FLAG antibody.

364

Immunofluorescence assay (IFA):

365

In dengue virus blocking assay, BHK-21 cells were seeded on cover glasses in

366

six-well plates (105 or 3X 105 cells per well) and incubated at 37°C for overnight. EIII

367

antibody at different dilutions was incubated with dengue virus with a MOI of 0.02 at

368

4°C for 1 hour. The virus-antibody mixture was then added to BHK-21 and incubated

369

for an additional hour at 4°C. Negative controls received PBS instead of antibody.

370

After infection, BHK-21 cells were washed three times with 1 ml cold PBS and

machine

(Syngene)

or

using

traditional

X-ray

film.

For

371

cultured in DMEM medium supplemented with 10% FBS. At different time points

372

after infection, the cells on cover glasses were then permeabilized with 0.25% Triton

373

X-100 followed by 100% methanol fixation at -20°C for 5 minutes. After fixation,

374

cells were incubated with rabbit-anti-Den2 NS3 antibody followed by incubation with

375

anti-rabbit-IgG-Alexa488 (Molecular Probes) secondary antibody. Finally, cover

376

glasses were mounted on slide with Vectashield mounting medium (Vector Labs) with

377

4, 6-diamidino-2-phenylindole (DAPI). The slides were observed by fluorescence

378

microscope or confocal microscope. In transfection experiment, pCMV-3FLAG or

379

pCMV-3FLAG-envelope plasmid was transfected to HEK293T cells by using

380

TurbofectTM transfection reagent (Fermentas). The primary antibody was EIII or

381

FLAG and the secondary antibody was anti-rabbit-IgG-Alexa488 or anti-mouse

382

IgG-Alexa555 (Molecular Probes) in this experiment.

383

Quantitative reverse transcription polymerase chain reaction (qRT-PCR):

384

Total RNA was isolated from mock infected or dengue virus infected cells by REzol

385

C&T reagent (PROTECH). 100 ng or 1μg RNA was used as template for qRT-PCR

386

using KAPA SYBR® FAST One-Step qRT-PCR kit (KAPA Biosystem) or ABI

387

qRT-PCR kit and ABI StepOne detection system and analysis software (Applied

388

Biosystems). Levels of NS3 were normalized to β-actin mRNA levels using a

389

comparative threshold cycle (2–[delta] Ct) method which converts differences of cycle

390

numbers to test gene/β-actin ratios.

391

Statistical Analysis

392

The data were analyzed using either t-test or Mann–Whitney U test and the results

393

with p value less than 0.05 were considered significant.

394 3955. Acknowledgements 396

We thank Dr. Wen Chang (Institute of Molecular Biology, Academia Sinica, Taiwan)

397

for providing plasmid pET-21C-EIII for expression of domain III protein and Dr.

398

Huey-Nan Wu (Institute of Molecular Biology, Academia Sinica, Taiwan) for

399

providing plasmid pMR-SpeI-prME-HA1-RzD for construction of full-length

400

envelope protein and NS3 antibody for Western blot. We highly appreciate

401

Chih-Hsiang Leng and Hsin-Wei Chen (National Institute of Infectious Diseases and

402

Vaccinolog, National Health Research Institute) for providing purified recombinant

403

EIII-His6 proteins of four serotypes DENV. We also thank Dr. Ying-Ray Lee, (Chia-Yi

404

Christian Hospital) for providing Dengue virus type 2 (PL046 strain) for infection

405

experiments. This study is based in part on sequence data from the Taiwan Pathogenic

406

Microorganism Genome Database provided by Centers for Disease Control, Ministry

407

of Health and Welfare, Taiwan (supported by National Research Program for Genome

408

Medicine 99-0324-01-F-12). The interpretation and conclusions contained herein do

409

not represent those of Centers for Disease Control, Ministry of Health and Welfare,

410

Taiwan. This work was supported by National Science Council grants

411

NSC93-2311-B-194-002 for Hau-Ren Chen.

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561 562 563

564

Figure legends

565 566

Fig. 1. The linear array epitope (LAE) technique includes TR-PCR and adapter-PCR steps

567

(a) Template-repeated PCR (TR-PCR): Oligo Den E-A and oligo Den E-B are used as

568

primers and templates for TR-PCR. (b) Primers designed for TR-PCR. The peptide

569

sequences of epitope chosen are VNIEAEPPFG. Oligo Den E-A encodes this peptide

570

and oligo Den E-B is partially complementary to oligo Den E-A as indicated. The 5’

571

half of oligo Den E-A (shown as a) is complementary to the 5’ half of oligo Den E-B

572

(shown as a’) and the 3’ half of oligo Den E-A (shown as b) is complementary to the

573

3’ half of oligo Den E-B (shown as b’). (c) Adapter-PCR: The products of TR-PCR

574

(3μl) are subjected to adapter-PCR as templates. Adapter primer Den E-5b and Den

575

E-3he introduced proper restriction enzyme sites, leading to have a Bam HI site at 5’

576

end, a Hind III/Eco RI site at 3’ end, and two stop codons at the end of coding region.

577

The PCR products are then subcloned into a T vector. (d) The products of TR-PCR

578

(lane 2) and adapter-PCR (lane 3) are analyzed on 8% PAGE with ethidium bromide

579

stain. Lane 1 and 4 indicate 100 bp DNA markers.

580 581

Fig. 2. Novel tandem-repeated antigen was used to generate anti-EIII antiserum from rabbit

582

(a) Recombinant GST-Den EIII10-His6 protein was expressed in E. coli BL21 (DE3)

583

and was purified through Glutathione-Sepharose affinity column. Elution fractions

584

were analyzed on 10% SDS-PAGE and stained with Coomassie blue. Lane N, total

585

lysates before induction. Lane I, total lysates after 3 hrs IPTG induction. Lane Wash,

586

wash fraction during column purification. Elution fractions 1 to 8 indicated fractions

587

eluted by 10 mM glutathione from affinity column. (b) The titer of anti-EIII antiserum

588

from rabbit was measured by ELISA. 10 ng, 100 ng and 1000 ng of EIII-His 6 proteins

589

from DENV2 PL046 were coated onto ELISA plate and serial dilutions of EIII Ab

590

were added into the plate to measure OD410. No antigen (Ag) coated, no primary (1’)

591

antibody and no secondary (2’) antibody are all negative control sets. (c) The titer of

592

anti-EIII antiserum from rabbit was measured by ELISA. 10 ng, 100 ng and 1000 ng

593

of recombinant EIII-His6 proteins from four serotypes were coated onto ELISA plate

594

and serial dilutions of EIII Ab were added into the plate to measure OD410. No antigen

595

(Ag) coated, no primary (1’) antibody and no secondary (2’) antibody are all negative

596

control sets.

597 598

Fig. 3. The specificity of EIII antibody was evaluated in various assays

599

(a) The specificity of EIII antibody was first examined by Western blot. Lane 1:

600

purified EIII-His6, lane 2: PEIa-His6, lane 3: PEIa-Den EIII10-His6, lane 4: TAF7-His6,

601

lane 5: NS3-C-His6, lane 6: GST-Den EIII10-His6. Arrow indicated PEIa-Den

602

EIII10-His6 and arrow head indicated GST-Den EIII10-His6, respectively. (b) BHK-21

603

cells were transfected with empty vector or FLAG-envelope plasmid. Then, cell

604

lysates were analyzed by Western blot using either anti-EIII or anti-FLAG antibodies.

605

(c) Overexpression of FLAG-envelope in HEK293T cells were analyzed by

606

immunofluorescence assay using either anti-EIII or anti-FLAG antibodies. (d)

607

HEK293T cells were transfected with empty vector or pCMV-3FLAG-envelope

608

plasmid. Then, 150 μg total protein of cell lysates were immunoprecipitated with 10,

609

20 or 50 μl EIII antibody, followed by Western blot using anti-FLAG antibody. Input

610

was loaded with 15 μg of total protein.

611

Fig. 4. EIII antibody could recognize viral envelope proteins and virions

612

(a) C6/36 cells were infected by dengue virus and the medium was collected post 10

613

to 12 days infection. The cells were lysed by 2X sample buffer and the proteins either

614

in medium fraction (med) or in the cell fraction were analyzed by Western blot. The

615

first two lanes indicate mock infection. The other six lanes indicate three independent

616

experiments (Exp.1, 2 and 3) for dengue virus infection. Filled Arrow, open arrow and

617

arrow head indicated the band of premembrane/envelope heterodimer, envelope and

618

NS3 proteins, respectively. (b) BHK-21 cells were infected by dengue virus (MOI: 12)

619

and cells were harvested after 12 or 24 hrs infection, followed by Western blot

620

analysis with EIII or NS3 antibody. (c) Virions with different dilution folds were

621

coated in ELISA plate and were detected by anti-EIII antibody.

622

Fig. 5. EIII antibody could block dengue viral entry

623

(a) Purified EIII antibodies with serial dilution were pre-mixed with DENV2 PL046

624

strain, and then the antibody-virus mixtures were used to infect BHK-21 cells. After

625

infection for 48 hrs, cells were fixed with methanol and then dengue viruses were

626

detected by anti-NS3 (1:200) antibody. The signals were quantified in (b) by counting

627

the green fluorescence positive cells/DAPI positive cells in different fields. The

628

percentage of green fluorescence positive cells were measured based on over 180

629

DAPI-positive cells examined). The data was analyzed by Mann–Whitney U test (*

630

p