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Efficient production of Tymovirus like particles displaying immunodominant epitopes of Japanese Encephalitis Virus envelope protein

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Pallichera Vijayan Shahana a,b, Dipankar Das a, Abhinay Gontu a, Dev Chandran a, Kapil Maithal a,⇑

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a b

Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Telangana, India Department of Biotechnology, Acharya Nagarjuna University, Nagarjunanagar, Guntur 522510, Andhra Pradesh, India

a r t i c l e

i n f o

Article history: Received 10 January 2015 and in revised form 26 March 2015 Available online xxxx Keywords: Japanese Encephalitis Virus Virus like particle Physalis Mottle Virus Immunodominant epitopes In vitro refolding

a b s t r a c t Japanese Encephalitis (JE) is a mosquito borne arboviral infection caused by Japanese Encephalitis Virus (JEV). It is a major cause of viral encephalitis in Asian countries including India. In the present study, we have used a Tymovirus [i.e. Physalis Mottle Virus (PhMV) coat protein (CP)], which forms virus like particles (VLPs) as a template to display immunodominant epitopes of JEV envelope (E) protein. The immunodominant epitopes of JEV were inserted at the N-terminus of the wild type PhMV CP, and these constructs were cloned and expressed in Escherichia coli. The chimeric proteins were purified from the inclusion bodies and evaluated for VLP formation. The purified protein was identified by Western blotting and VLP formation was studied and confirmed by transmission electron microscopy and dynamic light scattering. Finally, the immunogenicity was studied in mice. Our results indicate that the chimeric protein with JEV epitopes assembled efficiently to form VLPs generating neutralizing antibodies. Hence, we report the purified chimeric VLP would be a potent vaccine candidate which needs to be evaluated in a mouse challenge model. Ó 2015 Published by Elsevier Inc.

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Introduction

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Japanese encephalitis (JE)1, caused by the Japanese Encephalitis Virus (JEV), is a mosquito-borne arboviral infection and a leading cause of viral encephalitis in Asia [1]. Approximately 50,000 cases are reported annually from JE endemic countries like Japan, China, Korea and Taiwan, as well as other Asian countries [2]. The hallmark of JE infection is an acute inflammation of the brain, resulting in a fatal outcome in 25% of cases and residual neuropsychiatric sequelae in 30% of cases. The first clinically recorded case of JE in India was reported in Tamil Nadu in 1955 [3] and has been followed by several reports of outbreaks including the outbreak in 2005 that killed 1700 people mostly children and left thousands more disabled [4]. The global impact of this disease is calculated as a measure of the overall

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⇑ Corresponding author at: Research and Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Telangana, India. Tel.: +91 40 64545389; fax: +91 40 23005958. E-mail address: [email protected] (K. Maithal). 1 Abbreviation used: JE, Japanese Encephalitis; JEV, Japanese Encephalitis Virus; PhMV, Physalis Mottle Virus; CP, coat protein; VLPs, virus like particles; DALYs, disability-adjusted life years; JGMV, Johnsongrass mosaic virus; FBS, fetal bovine serum; bME, b-mercaptoethanol; HRP, horseradish peroxidase; TMB, tetramethylbenzidine; TB, terrific broth.

disease burden and is expressed as the number of years lost due to ill-health, disability or early death and was estimated in 2008 as 709,000 disability-adjusted life years (DALYs) [5]. Different approaches are used for treatment of the disease at various stages of the disease condition, like antiviral drugs against the virus and the use of vaccines in preventing the disease [6]. The first generation JE vaccines were associated with problems such as unwanted adverse neurological reactions caused by the nature of mouse brain-derived vaccine and a lack of compliance for follow-up vaccination [7]. The development of second-generation vaccines like the present cell culture JE vaccine is an important step toward development of a consistent and safer vaccine against JEV. Recombinant sub-unit vaccines provide new options of benefit to endemic countries because of their improved safety profile and lower dosage requirements. Advances in the field of molecular biology and the availability of bio-informatics tools have paved the way for the development of new vaccines for many viral diseases. These tools have made way for transfer of many pools of genes of interest and their efficient expression in a host cell of our choice. The genome and molecular structure of JEV is well characterized and the complete genome of more than 50 strains of JEV is available in the GenBank. This enables the gene pool of JEV to be used in different platforms for

http://dx.doi.org/10.1016/j.pep.2015.03.017 1046-5928/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: P.V. Shahana et al., Efficient production of Tymovirus like particles displaying immunodominant epitopes of Japanese Encephalitis Virus envelope protein, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.03.017

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cloning and expression. JEV is a small spherical virion approximately 50 nm in diameter, with a non segmented positive sense RNA genome [8]. The single stranded positive-sense RNA is wrapped in a nucleocapsid and surrounded by an envelope glycoprotein. The RNA consists of a single open reading frame capped by a 95 bp long 50 UTR and a 580 bp long 30 UTR. [9]. The viral RNA encodes a single polyprotein, which is cleaved by viral and cellular proteases into ten functionally distinct proteins, three structural (capsid, C; membrane, M; and envelope, E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The glycoprotein E is a structural protein forming projections on the outer surface, and its C-terminal domain traverses through the membrane. It is responsible for a number of important processes that include virion assembly, receptor binding and membrane fusion [10,11]. The E protein is the principal target for neutralization in vitro and in vivo by specific antibodies and is associated with the induction of a protective neutralizing antibody response in hosts. It is a homodimer with 3 domains. Domain I is the central b-barrel, domain II is an elongated dimerization region and domain III is a C-terminal immunoglobulin like module [12]. Neutralizing epitopes on the lateral surface of domain III have been identified, including residues 333 [13], 373–399 [14] 306, 331, and 387 [15] and stretches from 356 to 445 [16]. Virus like particles (VLPs) are composed of non infectious viral coat proteins [17]; the use of VLPs formed from self assembling coat proteins from a number of icosahedral viruses has been widely explored for clinical applications like vaccine production and gene therapy [18]. Literature data support this with the use of hepatitis B virus [19], polio virus [20] blue tongue virus [21], rotavirus [22], cauliflower mosaic virus [23], and Johnsongrass mosaic virus (JGMV) [24]. Various platforms have been used for the development of such VLP vaccines in the past. In the present study, we have developed a VLP based vaccine for JEV by displaying the immunodominant epitopes of JEV E protein on Physalis Mottle Virus (PhMV) coat protein (CP). The PhMV-based VLP display system is used in the present study for JEV antigen display as it has been shown to be a promising system for expression [25]. PhMV is a spherical plant virus consisting of a single-stranded, plus-sense RNA genome with 6670 base pairs [26,27]. The RNA genome is encapsidated in a protein shell comprising 180 chemically identical CP subunits arranged in an icosahedral symmetry. The bacterially expressed recombinant CP (rCP) could self-assemble into stable VLPs and the deletion of 30 amino acid residues or the addition of 41 amino acid residues at the N-terminus of rCP did not affect its assembly into Triangulation number-3 (T-3) capsids [28]. In this context, we have used PhMV CP to display immunodominant epitopes of JEV. A 42 amino acid stretch of the epitopes from JEV E protein was cloned at the N terminus of PhMV CP. The protein expressed as inclusion bodies in Escherichia coli, and, on refolding, the protein formed VLPs which were characterized by light scattering in a sucrose gradient, Western blotting and electron microscopy. The immunogenicity of the VLPs was evaluated in mice and found to be highly immunogenic, generating neutralizing antibodies.

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Materials and methods

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Bacterial strain and chemicals

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The synthetic genes encoding JEV envelope protein and PhMV CP were codon optimized for the E. coli expression system and were synthesized at GENEART, Regensburg, Germany. The pRSETA expression vectors were from Life Technologies, California, USA. The bacterial competent cells XL10-GoldÒ and BL21 (DE3) pLysS were purchased from Stratagene, California,

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USA, whereas Rosetta-gami and Tuner (DE3) pLysS were from Novagen, Wisconsin, USA. Gel extraction kit and plasmid isolation kits were procured from Qiagen, Limburg, Netherlands. The restriction and ligation enzymes were purchased from New England Biolabs (NEB), Hertforshire, UK. Minimum essential medium eagle (modified) with Hanks’ salts (HMEM), fetal bovine serum (FBS) and penicillin–streptomycin–glutamine mix were purchased from GIBCO-Life Technologies, California, USA. Fine chemicals like isopropyl b-D-1-thiogalactopyranoside (IPTG), b-mercaptoethanol (bME), guanidine hydrochloride, Tris buffer, ethylenediaminetetraacetic acid (EDTA), glycerol, diaminobenzidine (DAB), horseradish peroxidase (HRP)-conjugate and tetramethylbenzidine (TMB) substrate were purchased from Sigma–Aldrich Chemical Company, St. Louis, MO, USA. General laboratory chemicals like citric acid, trisodium citrate, sucrose, urea, sodium chloride, potassium chloride, magnesium sulfate and magnesium chloride were purchased from Merck, Mumbai, India while tryptone and yeast extract were from HiMedia, Mumbai, India. Plasticware like six well tissue culture plates and Maxisorb ELISA plates were purchased from Nunc, New York, USA. Adju-phos alum for blending the vaccine was procured from Brenntag, Mulheim an der Ruhr, Germany.

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Construction of the recombinant plasmid and protein expression

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The synthetic gene with immunodominant epitopes of the JEV envelope gene (Accession Number: AF080251, 126 bp) and PhMV CP (Accession Number: 20177482, 564 bp) were codon optimized for expression in E. coli. PhMV CP was synthesized with XhoI and HindIII sites at 50 and 30 ends respectively with a stop codon at the end of the reading frame. A KpnI site was introduced as a silent mutation between the 44th and 45th amino acid residues of the CP for the insertion of heterologous sequences. This PhMV CP of 564 bp was cloned at the XhoI and HindIII sites of the pRSETA vector. The JEV envelope synthetic genes were synthesized with an NdeI site at the 50 end and KpnI site at the 30 end. These heterologous sequences were swapped in frame into the NdeI and KpnI sites of the PhMV CP. The plasmids were transformed into XL10-GoldÒ ultracompetent cells and plated on Luria Bertani (LB) agar plates containing 100 lg/mL ampicillin. After overnight incubation of the plates at 37 °C, single colonies were picked and the plasmid DNA isolation was done using a Qiagen Plasmid DNA isolation kit. The inserts in the chimeric constructs were screened by restriction enzyme digestion analysis and confirmed by DNA sequencing. The chimera was named J1, whereas the coat protein construct alone was called PhMVwt. The plasmid was transformed into different bacterial hosts for expression and different parameters were optimized. The expression of the protein was assessed using varied media, temperatures, IPTG concentrations, induction periods and hosts. The optimal conditions for protein expression were evaluated by SDS–PAGE and Western blot analysis using a 15% gel.

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Isolation of the inclusion bodies

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The protein was expressed using standard procedure in a shake flask under IPTG induction. Briefly, an overnight grown culture was inoculated (1% v/v) with the clone into a 2 L flask containing 500 ml terrific broth (TB). The flask was incubated at 37 °C with constant shaking at 170 rpm. Once the optical density (600 nm) of the culture reached 0.6 A.U., it was induced with 0.5 mM IPTG for 4 h at 37 °C. Subsequently, the cells were harvested by centrifugation at 4000g for 20 min. The supernatant was discarded and the pellet was resuspended in 10 ml of lysis buffer (50 mM Tris buffer, 5 mM EDTA, pH 8.5) per 1 g of wet cell pellet. The resuspended cell lysate was subjected to sonication (Sonics Vibracell,

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USA) at 35% amplitude with a 5.5/5.5 pulse rate. The lysed culture was centrifuged at 10,000g for 30 min, and supernatant and pellet samples were analyzed on SDS–PAGE for the presence of the protein of interest.

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Solubilization of the inclusion bodies, refolding and purification of VLPs

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b-Mercaptoethanol (bME), urea and guanidine hydrochloride were used to solubilize the inclusion bodies. The solubilizing efficiency of each reagent was evaluated by incrementally increasing the concentration of each reagent. Briefly, the inclusion body pellet was washed with 1% sodium deoxycholate followed by two washes with distilled water. The pellet was then divided equally into multiple aliquots and solubilized with varying concentrations of bME (1–8 M), urea (1–8 M) and guanidine hydrochloride (1–8 M) dissolved in resuspension buffer (50 mM Tris, pH 8.5) respectively. The samples were centrifuged at 12,000g for 15 min and the supernatants obtained were analyzed for extraction efficiency of J1 protein by SDS–PAGE and Western blot. Once the optimal conditions for solubilization of the inclusion bodies were identified, the soluble fractions were pooled and the solution was added drop wise in ice cold refolding buffer (50 mM Tris, 0.5 mM EDTA, 5% sucrose, 10% glycerol, pH 8.5) at 1:2 dilution. Subsequently, the refolded sample was subjected to dialysis in dialysis buffer (50 mM Tris, pH 8.5) at 4 °C (Table 2).

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Characterization of the J1 protein

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(1) Western blot analysis The purified VLPs were electrophoretically resolved on a SDS–PAGE and transferred to nitrocellulose membrane. The blot was developed using antibodies raised in mice against Vero cell cultured inactivated JEV antigen. (2) Formation of light scattering zone The purified protein was subjected to ultracentrifugation at 140,000g on a linear sucrose gradient (40–10%) dissolved in citrate buffer (pH 5.4) to check for the specific light scattering zone, a characteristic feature of PhMV VLPs [26]. The zone was collected and subjected to another round of ultracentrifugation at 140,000g for 3 h. The final pellet was resuspended in 50 mM Tris buffer (pH 8.5). The protein was further analyzed by SDS–PAGE and Western blotting with antibodies raised in mice against inactivated JEV antigen. (3) Electron microscopy The VLPs were coated onto carbon-shadowed, formvar coated grids and visualized by negative staining with 1% uranyl acetate. These grids were examined with a transmission electron microscope (Hitachi H-7500) at a magnification range of 60,000–80,000. (4) Particle size analysis Particle size distribution of the purified PhMVwt and J1 VLPs were analyzed on a 90-Plus particle size distribution

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analyzer (Brookhaven Instruments, New York, USA) by dynamic light scattering (DLS) at 660 nm, keeping the scattering angle of 90°.

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Immunogenicity studies

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3–4 week old, BALB/c mice, weighing 20–25 g were procured from the National Institute of Nutrition (NIN), Hyderabad, for the study. Animal treatment and care was carried out according to Institutional Animal Ethics Guidelines. Mice were immunized intramuscularly with 50 ll of J1 or PhMVwt consisting of 20 lg VLPs, 0.05% Adju-phos adjuvant in phosphate buffered saline (PBS) respectively. JEEVÒ vaccine was used as a positive control (1/10th recommended dosage for the adults), whereas placebo (containing only adjuvant and PBS) and unvaccinated groups were used as negative controls. A booster dose was given 3 weeks post primary immunization. The animals were bled on the 3rd, 4th, 5th, 6th and 8th week and the serum was stored at 20 °C until further use. Each group consisted of ten mice as described in Table 3.

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(1) Indirect ELISA The reactivity of J1 VLPs to the polyclonal antibodies raised against JEEVÒ vaccine in mice was tested by indirect ELISA (I-ELISA). Briefly, the ELISA microtiter plates were coated with 500 ng of J1 or PhMVwt antigen per well in 0.5 M carbonate buffer, pH 9.6, overnight at 4 °C. Plates were washed thrice with washing buffer (0.1% PBST) and blocked with 3% (w/v) skimmed milk in PBST for 1 h. Subsequently, plates were washed thrice with washing buffer and polyclonal antibodies raised against JEEVÒ in mice were added at a dilution of 1:500. The plate was then incubated at 37 °C for 1 h followed by three washes with washing buffer. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was added to each well at 1:5000 dilution and incubated for another 1 h. Following three washes with 0.1% PBST, TMB (3,30 ,5,50 -tertamethylbenzidine) peroxidase substrate (1 mg tablet in 10 ml of citrate buffer pH 5.4 and 10 ll H2O2) was added to each well and incubated in the dark for 10 min. The peroxidase reaction was quenched by 1.25 M H2SO4 and absorbance was measured at 450 nm using an ELISA plate reader (Spectra Max 190, Molecular Devices, USA). PhMVwt antigen was used as a negative control. (2) Plaque Reduction Neutralization Test (PRNT) The serum samples obtained from mice immunized with J1, PhMVwt and other controls were heat inactivated at 56 °C for 30 min. 6-well cell culture plates were plated with Vero cells at a cell density of 5  105/well and incubated at 37 °C, 5% CO2 overnight. JEV was maintained in virus maintenance medium (HMEM with 1% serum and 1% penicillin–streptomycin–glutamine) and diluted accordingly to get 50 plaque forming units (pfu) per well.

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Table 1 Protein sequences of PhMVwt, JEV1 and J1. S. No

Construct

Amino acid sequence

1

PhMVwt

2 3

JEV1 J1

LEDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSLQTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVYGGQ SFVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTASIQISGRIRLSKPMLIAN HMKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSGGSGGGT HMKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSGGSGGGTAETAAQVSLQTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVYGGQS FVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTASIQISGRIRLSKPMLIAN

(1) Aminoacid sequence of PhMVwt. (2) Aminoacid sequence of JEV1 epitope. (3) Aminoacid sequence of the final construct of PhMV with JEV1 epitope labeled as J1.

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Table 2 Buffers used for inclusion bodies purification. Lysis buffer Resuspension buffer Refolding buffer Dialysis buffer

Tris Tris Tris Tris Tris 8.5 Tris

50 mM, 50 mM, 50 mM, 50 mM, 50 mM,

EDTA 5 mM, pH 8.5 b-mercaptoethanol (1–8) M, pH 8.5 urea (1–8) M, pH 8.5 guanidine hydrochloride (1–8) M, pH 8.5 EDTA 0.5 mM, sucrose 5%, glycerol 10%, pH

50 mM, pH 8.5

Table 3 Animal experimental groups for immunogenicity studies. Group

Antigen

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J1 PhMVwt JEEV (commercial vaccine) Placebo Unvaccinated Control

The heat inactivated serum samples were diluted two fold in the virus maintenance medium and the virus and serum samples were mixed in equal volumes and incubated for 90 min at 37 °C with intermittent shaking. The spent medium was discarded from the confluent Vero cells, the antigen antibody mix was added to the corresponding wells, the plates were incubated at 37 °C for 90 min, and 2 ml of 1% methyl cellulose was overlaid. The plates were again incubated for 96 h at 37 °C in a CO2 incubator. Subsequently, the methyl cellulose overlay was carefully discarded and the plates were stained with crystal violet. The wells were observed for plaque reduction (50%) in comparison to the virus inoculated wells. The neutralizing antibody titers were expressed as the reciprocal of the highest initial serum dilution that inhibited 50% or greater of the plaque formation compared with the virus control titration (PRNT50).

Fig. 1. Expression optimization samples were analyzed by SDS–PAGE using a 15% gel. (A) Optimization in different medium. Lane 1 – uninduced LB medium, Lane 2 – induced LB medium, Lane 3 – uninduced TB medium, Lane 4 – induced TB medium, Lane 5 – uninduced SOB medium, Lane 6 – induced SOB medium, Lane 7 – uninduced 2XYT medium, Lane 8 – induced 2XYT medium, Lane 9 – standard protein molecular weight markers. (B) Optimization of different temperatures. Lane 1 – uninduced 16 °C, Lane 2 – induced 16 °C, Lane 3 – uninduced 25 °C, Lane 4 – induced 25 °C, Lane 5 – uninduced 30 °C, Lane 6 – induced 30 °C, Lane 7 – uninduced 37 °C, Lane 8 – induced 37 °C, Lane 9 – standard protein molecular weight markers. (C) Optimization in different IPTG concentrations. Lane 1 – uninduced 0.5 mM, Lane 2 – induced 0.5 mM, Lane 3 – uninduced 1 mM, Lane 4 – induced 1 mM, Lane 5 – uninduced 2 mM, Lane 6 – induced 2 mM, Lane 7 – uninduced 5 mM, Lane 8 – induced 5 mM, Lane 9 – standard protein molecular weight markers. (D) Optimization of different time periods of induction. Lane 1 – uninduced 4 h, Lane 2 – induced 4 h, Lane 3 – uninduced 6 h, Lane 4 – induced 6 h, Lane 5 – uninduced 12 h, Lane 6 – induced 12 h, Lane 7 – uninduced 24 h, Lane 8 – induced 24 h, Lane 9 – standard protein molecular weight markers. (E) Optimization in different hosts. Lane 1 – uninduced BL21, Lane 2 – induced BL21, Lane 3 – uninduced Tuner, Lane 4 – induced Tuner, Lane 5 – uninduced Rosetta gami, Lane 6 – induced Rosetta gami, Lane 7 – uninduced BL21pLysS, Lane 8 – induced BL21pLysS, Lane 9 – standard protein molecular weight markers.

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Results

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Cloning, expression and purification of J1 VLPs

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The codon optimized JEV1 epitope sequence [24] encoding the polypeptide sequence given in Table 1 was synthesized and cloned into the pRSETA vector and restriction enzyme digestion analysis confirmed the integration of the 126 bp JEV immunodominant epitope gene into the chimeric construct. This was further confirmed by DNA sequencing analysis. The sequenced plasmid construct was transformed into different bacterial hosts for expression. The expression conditions were optimized as follows (Fig. 1): (a) Medium: Luria Bertani (LB), Terrific Broth (TB), Super Optimal Broth (SOB) and 2XYT medium, (Fig. 1A) (b) induction temperatures: 16 °C, 25 °C, 30 °C, 37 °C, (Fig. 1B) (c) IPTG concentrations: 0.5 mM, 1 mM, 2 mM, 5 mM, (Fig. 1C) (d) induction periods: 4 h, 6 h, 12 h, 24 h (Fig. 1D) and (e) expression hosts: BL21 (DE3), BL21 (DE3) pLysS, Rosetta-gami, Tuner (DE3) pLysS (Fig. 1E). The expression of the clone was analyzed on SDS–PAGE and Western blot. The best expression was seen in Tuner (DE3) pLysS expression host cells cultured in TB medium grown at 37 °C and induced with 0.5 mM IPTG for a period of 4 h (Fig. 2A and B). The bacterial cultures were subjected to lysis, and it was found that under all expression conditions the J1 protein expressed as inclusion bodies. The inclusion bodies from the finalized expression conditions were isolated and were sequentially solubilized in varying concentrations of bME (1–8 M), urea (1–8 M) and guanidine hydrochloride (1–8 M). The solubilization efficiency of each reagent was analyzed by SDS–PAGE and Western blot (Fig. 3A–F). It was evident that solubilization buffer with 1–8 M bME was most efficient (Fig. 3A and B) as compared to urea (Fig. 3C and D) and guanidine hydrochloride (Fig. 3E and F) for solubilizing the J1 protein. Further, it is also evident that 8 M bME was most suitable and showed maximal extraction of the J1 protein. The extracted protein was refolded in vitro using the refolding buffer and the protein was dialyzed against the dialysis buffer for three days with repeated buffer changes to remove bME from the protein. SDS–PAGE and Western blot analysis of the dialyzed protein showed a band of 20 kDa corresponding to the expected size of the J1 protein (Fig. 4A and B).

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Characterization of the J1 protein

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The final purified protein yield of 20 mg of VLPs per 1 L of bacterial culture was obtained with a concentration of 2 mg/ml. The dialyzed protein was subjected to sucrose density gradient ultracentrifugation and formation of a light scattering zone, characteristic feature of PhMV VLPs was obtained [28]. The identity of this purified protein was confirmed by Western blot (data not shown). The VLPs formed were analyzed using transmission electron microscopy (TEM) and it revealed the presence of spherical particles of diameter in the range of 28 ± 3 nm indicating that the chimeric proteins are indeed capable of self-assembly in vitro into VLPs (Fig. 5A–C). In accordance with the electron microscopy results, the particle size analysis by DLS at 660 nm also showed a mean size of the PhMVwt and J1 as 32.0 ± 6.4 nm and 32.1 ± 6.5 nm, respectively (Fig. 6A and B). The particle size data from EM and particle size analysis are in good agreement with earlier published literature [28].

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Immunogenicity studies

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Indirect ELISA results for the serum samples obtained with purified J1 VLPs showed the specificity of the purified antigen with a commercially available JEEVÒ vaccine. The seroconversion criteria

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Fig. 2. Expression of J1 protein E. coli was analyzed by SDS–PAGE and Western blot using a 15% gel. SDS–PAGE (A) and Western blot (B) probed with antibodies raised against inactivated JEV virus in mice. Lane 1 – uninduced culture, Lane 2 – induced culture and Lane 3 – standard protein molecular weight markers.

was the increase in the absorbance value equal to or greater than three times that of the pre-immune serum. The strong ELISA titers produced by J1 antigen in comparison to the control PhMVwt antigen revealed that the display of the epitopes was successful in generating an immune response and indicate the immunogenic potential of the J1 antigen (Fig. 7). Virus neutralization antibody titer in JEV is important to determine the potency of the antigen [29]. A titer above 10 in PRNT50 is indicative of a seroprotective titer [30]. The PRNT50 was done with the challenge virus prepared by passaging the Beijing-1 virus strain in the brain of suckling mice. The seroconversion titers of PRNT50 for J1 and the commercial vaccine JEEV were above 50. PhMVwt, placebo and unvaccinated control did not generate any neutralizing antibodies. In the present study J1 VLPs generated antibody titers on par with the commercial inactivated vaccine indicating the effective display of JEV epitopes on the capsid (Fig. 8).

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Discussion

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There are several advantages of using VLPs: they eliminate the need to maintain virus stocks or viral reservoirs, preventing the accidental release of the virus; maintenance of containment facilities is easier, and, above all, they are easier to handle than the cell culture systems. VLP-vaccines for human papillomavirus (HPV) [31] and Hepatitis B virus [32] have already received regulatory approval and are commercially available. More VLP-based vaccines are in the pipeline for viruses such as H5N1 influenza [33] and chikungunya virus [34]. VLPs serve as an excellent vehicle for expression of foreign epitopes as they are non-pathogenic, non-replicating and often self-assembling molecules [35]. High level expression of foreign genes in the host cells, especially E. coli, often results in the formation of inclusion bodies (IBs). In the present study, the immunodominant epitopes of the E protein of JEV were expressed in PhMV CP as IBs. The sequenced clone was optimized for expression with different parameters to increase the expression level and solubility of the protein. But neither altering the media, competent cells for expression, induction temperature, period of induction nor IPTG concentration helped in expressing the protein in soluble form. A comparative better expression was seen in Tuner E. coli cells in terrific broth (TB) medium. Varying IPTG concentrations did not show much effect on the expression levels of the recombinant protein. Lower temperature

397

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Fig. 3. SDS–PAGE (A, C, E) and Western blot (B, D, F) analysis using a 15% gel. IBs were extracted by bME and analyzed by SDS–PAGE (A) and Western blot (B). IBs were extracted by urea and analyzed by SDS–PAGE (C) and Western blot (D). IBs were extracted by guanidine hydrochloride and analyzed by SDS–PAGE (E) and Western blot (F). Lane 1–8 is 1–8 M bME in A and B, urea in C and D, guanidine hydrochloride in E and F respectively, Lane 9 – standard protein molecular weight markers.

Fig. 4. Analysis of final purified J1 VLPs by SDS–PAGE (A) and Western blot (B) using a 15% gel. Lane 1 – purified J1 VLPs, Lane 2 – standard protein molecular weight markers. 420 421 422

did not show any effect on the solubility of the protein. Under all of the above optimized conditions the protein expressed as IBs. Purifying the intact VLPs displaying JEV epitopes from IBs was

the main goal. Hence the IBs were isolated, solubilized and refolded in vitro. Isolation of IBs to homogeneity before solubilization is a prerequisite to improve the recovery of intact VLPs. It was done using standard procedures of sonication for cell lysis and sodium deoxycholate washes to remove contaminants, especially proteases that may have absorbed onto the hydrophobic IBs. Efficient isolation is important as the presence of contaminating proteins reduces the refolding yield of denatured proteins [36]. In general inclusion bodies are solubilized by chaotropic agents like urea, guanidine hydrochloride [37] and thiocyanate salts; detergents like sodium dodecyl sulfate (SDS) [38], sodium N-lauroylsarcosine (NLS) [39] and reducing agents like bME or dithiothreitol [40]. In the present study, solubilizing the IBs with urea and guanidine hydrochloride yielded less protein. High concentrations of chaotropic agents when used to solubilize protein aggregates generates random coil structure and also loss of secondary structure of the protein [41]. In the present study, solubilizing the IBs with the reducing agent bME gave the most promising result. An ideal solubilizing agent should have the ability to disrupt both hydrophobic interaction and disulfide bond formation yet protect the native-like secondary structure of the protein. It is hypothesized that bME would have aided in this role. bME is used to reduce disulfide bonds, and it acts as a biological antioxidant by scavenging hydroxyl radicals, by which the tertiary structure and the quaternary structure of some proteins can be disrupted [42]. This ensures that a protein solution contains

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Fig. 5. TEM images at a magnification range of 60,000–80,000. (A) PhMVwt VLPs, (B) J1 VLPs before refolding and (C) J1 VLPs after refolding.

Fig. 6. Particle size analysis of PhMVwt VLPs and J1 VLPs using HORIBA scientific nano partica, Nano particle size analyzer (Scattering angle 90°). (A) PhMV wt VLPs, (B) J1 VLPs.

Fig. 7. Indirect ELISA. ELISA was done by coating the plate with J1 protein or PhMVwt protein and probing it with JEEVÒ serum to assess the reactivity of J1 and PhMVwt with JEEVÒ vaccine serum, placebo serum and unvaccinated mice serum.

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monomeric protein molecules, instead of disulfide linked dimers or higher order oligomers. This unique feature helps in solubilization of inclusion body protein aggregates without completely unfolding the proteins, and, thus, helping in high recovery of the desired protein in bioactive form [41]. It is demonstrated in this study that a high concentration of bME is required to completely solubilize the inclusion bodies. The gradual addition of 8 M bME efficiently and completely extracted the proteins from the inclusion bodies.

Refolding was done by pulse renaturation of the solubilized protein in the refolding buffer followed by slow and continuous dialysis. The refolding buffer contained sucrose and glycerol as additives, which were used to decrease the degree of aggregates and misfolding of protein [43]. Pulse renaturation involves the gradual addition of solubilized protein to the renaturation or refolding buffer to allow the protein to refold into proper conformation [44].

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Fig. 8. PRNT study in mice. J1 VLPs, PhMVwt VLPs, JEEVÒ vaccine were used to raise polyclonal antibodies in mice to check for the neutralizing antibody titer. Group1 – J1, Group 2 – PhMVwt, Group 3 – JEEVÒ vaccine, Group 4 – Placebo and Group 5 – Unvaccinated.

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The removal of the solubilizing agent was done by slow dilution or dialysis. Generally, precipitation at the stage of folding intermediates formation is seen with the removal of denaturant by dialysis. In the present study, the pulse renaturation was followed by dialysis with 50 mM Tris buffer, pH 8.5. The dialysis was done for 3–4 days with continuous buffer changes. The amount of VLPs obtained by this method is higher quantitatively than that obtained with the use of traditional chaotropic agents. The final purified protein was characterized by various methods. The primary test was the formation of a light scattering zone in a sucrose gradient, which is a characteristic feature of PhMV VLPs. This was followed by a Western blot developed with a specific serum raised in mice against inactivated JEV virus. This conformed the efficient display of the JEV epitopes on the purified VLPs from the IBs. The VLP sizes of J1 as seen in TEM and particle size analysis were correlative and in agreement with the literature data for PhMV VLPs. ELISA titers showed the seroconversion and PRNT titers confirmed the generation of neutralizing antibodies against JEV. Here we report, for the first time, the display of JEV immunodominant epitopes on PhMV CP, their purification from bacterial inclusion bodies, and their successful display characterized by TEM, particle size analysis, ELISA and PRNT. The simple purification methodology, cost effective process and good yields of the purified protein makes this an efficient process for the generation of a subunit vaccine (J1 VLP). The protein requires evaluation in a mouse challenge model to confirm its efficacy.

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Acknowledgement

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We would like to thank Dr. Shailendra Sahu for the help provided for the mice inoculations, Dr. Y. Naga Malleshwari (Acharya N.G. Ranga University, Hyderabad) for her help with the electron microscopy and the Spectroscopy Department of Centre for Cellular and Molecular Biology, Hyderabad, India for their help with the particle size analysis.

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