Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice

Microbes and Infection 14 (2012) 169e176 www.elsevier.com/locate/micinf

Original article

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice Senji Tafuku a,b,1, Takeshi Miyata a,1, Masayuki Tadano c, Ryotarou Mitsumata a,b,2, Hirochika Kawakami a,b, Tetsuya Harakuni a, Tomomitsu Sewaki b, Takeshi Arakawa a,d,* a

Molecular Microbiology Group, Department of Tropical Infectious Diseases, COMB, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan b GenoLac BL Corporation, 1831-1 Oroku, Naha, Okinawa 901-0152, Japan c Department of Molecular Virology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan d Division of Host Defence and Vaccinology, Department of Microbiology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan Received 7 March 2011; accepted 25 September 2011 Available online 4 October 2011

Abstract Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1e3), domains I and II (D1e2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1e3, D1e2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1e3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-g T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Japanese encephalitis virus; E protein; NS1; Recombinant proteins; Mucosal vaccine; Adjuvant

1. Introduction Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), an arbovirus that belongs to the family Flaviviridae. The family also includes several medically important viruses such as dengue virus, West Nile virus, and yellow fever virus. JEV is the most important cause of epidemic

* Corresponding author. Molecular Microbiology Group, Department of Tropical Infectious Diseases, COMB, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan. Tel./fax: þ81 98 895 8974. E-mail address: [email protected] (T. Arakawa). 1 These two authors contributed equally to this work. 2 Present address: Denka Seiken Co., Ltd., 3-4-2 Nihonbashi, Kayaba-cho, Chuo-ku, Tokyo 103-0025, Japan.

viral encephalitis in Southeast Asia, China, India and parts of Oceania. Disturbingly, JEV has recently spread to newer geographic locations such as Australia and Pakistan and is, therefore, an emerging infectious disease of significance in these areas. Nearly half of the world’s population live in JE endemic countries, and approximately 50,000 cases and 12,500e17,500 deaths are reported annually [1]. JE is no longer endemic in Japan with less than 10 cases reported annually; however, transmission of the virus by mosquitoes such as Culex tritaeniorhynchus and Culex vishnui still continues. Despite such low endemicity, the existence of JEV in Japan can be clearly demonstrated by annual surveillance of hemagglutinin inhibition antibody-positive domestic pigs; these surveys reveal that a large proportion (up to 100%), of the pigs become seropositive for the virus [2]. Although JE is no longer endemic in Japan (due mainly to mosquito control

1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.09.004

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measures and immunization programs), the disease has the potential to re-emerge if herd immunity thresholds decrease. Circumstances where this could happen include, for example, increases in the proportion of unvaccinated individuals within a population, and/or increases in the frequency of infectious mosquito bites, perhaps due to environmental factors such as global warming or destruction of infrastructures. Since there are no effective drugs against JEV, nor any alternate specific treatment for the disease, a routine vaccination program for disease endemic areas is currently the only control strategy available. The infected adult mouse brain-derived formalin-inactivated whole virus vaccine, based on the Nakayama strain (JEVaxÒ manufactured by BIKEN, Japan) was the only internationally available JE vaccine approved by the World Health Organization (WHO); however, production of this vaccine ceased in 2005. Consequently, the WHO has placed high priority on the development of new JE vaccines [3e8]. Development of safe, effective and affordable alternate JE vaccines are urgently needed, and progress toward such vaccines are starting to be made. For example, a primary hamster kidney cell-derived live attenuated vaccine based on the SA14-14-2 strain (which has been used in China for the past 20 years), has recently been licensed for use in India and South Korea [6,9]. In February 2009, a Vero cell-derived inactivated JE vaccine based on the Beijing-1 strain (JEBIK V, BIKEN) was licensed in Japan. Similarly, a Vero cellderived formalin-inactivated whole virus vaccine based on SA14-14-2 strain adjuvanted with aluminum hydroxide (IC51, Intercell, Australia) was licensed in Europe, USA and Australia in 2009 [10,11]. Another promising JE vaccine candidate that is now at an advanced stage of development is a Vero cell-derived genetically engineered chimeric live attenuated vaccine (ChimeriVaxÔ-JE, Acambis, UK/USA), which employs the yellow fever vaccine virus 17D (YF-17D) as a genetic backbone for expression of the prM and the envelope (E) protein of JEV (SA14-14-2 strain) [12]. In addition to the aforementioned next-generation JE vaccines, a number of studies have demonstrated the immunogenicity and protective efficacy of recombinantly expressed JEV proteins (e.g., those expressed in Escherichia coli, baculovirus, or mammalian cell expression systems), recombinant live virus-based vaccines (e.g., poxvirus, adenovirus, and yellow fever virus), and plasmid DNA-based vaccines [8]. Among these vaccine candidates, purified recombinant proteins are generally considered promising for the development of vaccines, primarily because of their safety record. E. coli expression systems are particularly attractive because of their ease of manipulation and low costs. In addition, recombinant protein-based subunit vaccines, when combined with suitable adjuvants, could be as highly protective as the existing inactivated vaccines. By exploiting suitable adjuvants or vaccine delivery systems, long lasting immunity could also be induced by an immunization regimen with acceptable numbers of doses [13e16]. Based on such premise a number of laboratories have expressed JEV E protein domains, epitopes derived from the E protein, or nonstructural proteins (NS1), and have demonstrated

their ability to induce virus-neutralizing antibodies and/or protective efficacy against lethal virus challenge [17e26]. We have recently demonstrated that a mouse brain-derived formalin-inactivated JE vaccine was effective in eliciting virus-neutralizing antibodies when administered intranasally (i.n.) to mice [18]. Here, we extend our previous study to evaluate whether i.n. immunization with E. coli-expressed structural or nonstructural JEV protein provides protective immunity against a lethal JEV challenge in mice. 2. Materials and methods 2.1. Expression and purification of recombinant JEV E and NS1 proteins The sequences of the PCR primers used to amplify the ectodomain of the E protein gene or NS1 gene derived from the JEV Beijing-1 strain are as follows: ED1F978, 50 -cgc gga tcc g ttc aac tgt ctg gga atg ggc-30 ; ED2R1850, 50 -gcg aag ctt cat ttt cag cct gca ttt tag-30 ; ED3F1851, 50 -cgc gga tcc g gac aaa ctg gct ctg aaa ggc aca-30 ; ED3R2183, 50 -gcg aag ctt cgt gct tcc agc ctt gta cca-30 ; NS1F2478, 50 -cgc gga tcc g gac act gga tgt gcc att gac-30 ; NS1R3523, 50 -gcg aag ctt agc atc aac ccg tga tct gac-30 . Underlined sequences indicate BamHI or HindIII restriction enzyme recognition sites. A plasmid containing the full-length JEV E or NS1 gene was used as a template. The following combinations of the forward and the reverse primer sets were used: domain IeIII (D1e3), ED1F978/ED3R2183; domain IeII (D1e2), ED1F978/ ED2R1850; domain III (D3), ED3F1851/ED3R2183; NS1, NS1F2478/NS1R3533. The amplified fragments were digested with BamHI and HindIII, and inserted into the corresponding sites of the pET-22b vector (MERCK KGaA, Darmstadt, Germany). E. coli BL21(DE3) (MERCK KGaA) was transformed with the engineered plasmids. Transformed cells were cultured in LB broth containing ampicillin (100 mg/ml), and expression was induced over a 4 h period using 0.5 mM IPTG, when cell density reached 0.6 OD600. Cells were harvested, resuspended in sonication buffer (50 mM sodium phosphate, 50 mM NaCl, pH 7.6), lysed on ice by sonication (3 min  3, Output 2, Duty 30, TOMY UD-201), and centrifuged (10,000  g, 20 min) to separate the supernatant from inclusion bodies. The inclusion bodies were suspended in the sonication buffer with 1% Triton-X and centrifuged again as described above. This procedure was repeated an additional two times to purify the inclusion bodies, prior to solubilizing them in a solution containing 8 M urea, 20 mM sodium phosphate, 300 mM NaCl, pH 7.6. The preparation was centrifuged (as described above) to remove insoluble debris. The protein solution was subjected to nickel-affinity chromatography (HisTrap FF, GE Healthcare, USA) according to the manufacturer’s instructions. The concentration of the eluted protein samples was adjusted to 0.2 mg/ml and dialyzed (Spectra/Por CE Dialysis Membrane 3.5e5 kD, Spectrum Laboratories, Inc., CA, USA) stepwise against phosphate-buffered saline (PBS) for D3, or against a refolding buffer (100 mM Tris, 1 mM EDTA, 0.2 M

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NaCl, pH 8.0) for D1e3, D1e2 and NS1, containing decreasing amounts of urea (4, 2, 1, 0.5, 0.25, and 0 M  2, 6 h for each) at 4  C. After dialysis, samples were centrifuged again and the proteins were concentrated by using sizeexclusion membrane filter (Amicon Ultra15, MWCO 10,000; Millipore, Billerica, MA, USA). Concentrations of 10 mg/ml for D3 and 1e3 mg/ml for the other three recombinant proteins were achieved. Endotoxin was removed from each purified protein using polymyxin B (Detoxi-Gel Endotoxin Removing Gel, Thermo Scientific, Inc., Rockford, IL, USA), and we confirmed that endotoxin levels were less than 500 pg/mg of protein (Pyrogent Single Test Vials, Cambrex, East Rutherford, NJ, USA). 2.2. Mouse immunization Seven-week-old female BALB/c mice (Japan SLC, Shizuoka, Japan) (12e18 per group) were immunized with 50 mg of the E. coli-expressed recombinant protein (i.e., D1e3, D1e2, D3, or NS1) by the i.n. or intraperitoneal (i.p.) route, using 20 ml volumes containing 1 mg of cholera toxin (CT; List Biological Laboratories, Campbell, CA, USA) or 200 ml volumes containing aluminum hydroxide (Thermo Scientific, Inc.), for the i.n. or i.p. immunization regimen, respectively. Mice were immunized 7 (at weeks 0, 1, 3, 5, 7, 9, and 11) or 5 times (at weeks 0, 1, 3, 5, and 11) for the i.n. or i.p. immunization regimen, respectively. As a positive control a mouse brain-derived formalin-inactivated JE vaccine of Beijing-1 strain (ChemoSero-Therapeutic Research Institute, Kumamoto, Japan) was i.p. administered to 18-week-old female BALB/c mice (10 per group) twice in a volume of 100 ml at 3-day intervals at week 11, according to the manufacturer’s immunization protocol. Blood samples were collected at week 12 from the tail vein for serum IgG titer determination and virus-neutralization analysis. Animal experimental protocols were approved by the institutional Animal Care and Use Committee, and animal experiments were conducted according to the institutional ethical guidelines for animal experiments. 2.3. Serum IgG titer determination Indirect ELISA was conducted to determine serum IgG titers by using antisera induced and collected at week 12 as described in the section of Mouse immunization. Briefly, 96-well microtiter plates (Nalge Nunc International, Rochester, NY, USA) were coated with 100 ml/well of the E. coli-expressed recombinant protein (10 ng/ml of D1e3, D1e2, D3, or NS1) or 100 ml/well of the mouse brain-derived formalin-inactivated JE vaccine (Beijing-1 strain, Chemo-Sero-Therapeutic Research Institute). The plates were blocked with 10% skimmed milk in PBS-T (PBS with 0.05% Tween-20) for the recombinant proteins or 1% bovine serum albumin (BSA) in PBS-T for the JE vaccine, respectively. Serially diluted antisera and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:2500, Promega, Madison, WI, USA), followed by its substrate were added to wells. After 20 min of incubation at room temperature, the reaction was stopped by adding 1 N sulfuric acid, and the

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absorbance was measured at 450 nm (OD450) using a microplate reader (Bio-Rad Laboratories Inc., Redmond, WA, USA). Serum IgG titers were defined as the highest serum dilution at which the OD450 reached the baseline. The baseline was defined as the sum of the OD450 value and the standard deviation (SD) multiplied by 2, for 100-fold diluted pre-immune sera (i.e., baseline ¼ [OD450 þ (SD  2)]). 2.4. Virus neutralization and virus challenge BHK-21 cells were grown at 37  C in Eagle’s MEM containing 2% fetal calf serum (2F-EMEM). The C6/36 clone of Aedes albopictus cells [27] was cultured at 28  C in 10FEMEM supplemented with 0.1 mM of non-essential amino acids. The JaGAr01 strain of JEV was propagated in C6/36 cell culture. Viral solutions were stored at 80  C prior to use. Mice were immunized as described in the section of Mouse immunization, and virus-neutralizing antibody titers were determined for the antisera collected at week 12 by using the 50% focus reduction neutralization test (FRNT50) [28]. Briefly, viral solutions at 100 focus forming units were mixed with an equal volume of heat-inactivated test immune sera diluted in 2F-EMEM. The virus/serum mixture was incubated at room temperature for 1.5 h, and applied to a BHK-21 cell monolayer in 96-well plates. The plate was incubated at room temperature for 1.5 h. After incubation, additional 2F-EMEM was added and the plate incubated in a CO2 incubator at 37  C for 24 h. Cells were fixed with methanol at room temperature for 5 min, and incubated with rabbit anti-JEV antiserum and goat anti-rabbit HRP-conjugated IgG (American Qualex International, Inc., San Clemente, CA, USA). The peroxidase reaction was developed using 0.01% H2O2 and 3-30 -diaminobenzidine tetrahydrochloride (DAB, Wako Chemical, Osaka, Japan) in PBS. To evaluate the vaccine efficacy, mice immunized as described in the section of Mouse immunization with the E. coli-expressed recombinant proteins (i.e., D1e3, D1e2, D3, or NS1; 12e18 per group) or with the mouse brainderived formalin-inactivated JE vaccine (Beijing-1 strain, Chemo-Sero-Therapeutic Research Institute; 10 mice per group) were challenged at week 12 with a lethal amount of the JEV JaGAr01 strain (5LD50) by i.p. injection followed by intracerebral inoculation with 50 ml of PBS. The animals were monitored daily to detect signs of illness/distress such as ruffled fur or paralysis and the survival rates were recorded. 2.5. Cytokine analysis Lymphocytes were isolated from the cervical lymph nodes or spleens from nonimmune mice or mice immunized with D1e3 or NS1 by the i.n. route one week after the last immunization (at week 12). To obtain antigen presenting cells (APCs), the spleens of nonimmune mice were mechanically disrupted, and the disrupted tissues were suspended in Hank’s Balanced Salt Solutions (HBSS; Gibco Inc., Grand Island, NY, USA) and centrifuged (420  g, 10 min). The collected cells were incubated in ACTB hemolytic buffer (0.017 M Tris

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buffer, pH 7.65, 0.75% NH4Cl) for 5 min at room temperature, washed and centrifuged again (as described above). The cell concentration was adjusted to 1  108 cells/ml with RPMI1640 medium (Wako Chemical) supplemented with 10% FBS, 50 mM 2-ME, 10 ml/ml Antibiotic Antimycotic Solution (SigmaeAldrich, St. Louis, MO, USA), and treated with 75 mg/ml of mitomycin C solution (Nacalai Tesque Inc., Kyoto, Japan) at 37  C for 30 min to obtain the APCs. Isolated lymph node cells (1  105 cells/50 ml) and APCs (5  104 cells/50 ml) were incubated together with or without each recombinant protein (final concentration; 1 mg/ml) for 24 h in 96-well plates. Culture supernatants were analyzed by using a Cytokine ELISA Kit according to the manufacturer’s instruction (R&D Systems, Minneapolis, MN, USA). 2.6. Statistical analysis Statistical significance was determined using the WilcoxoneManneWhitney test for serum IgG and virus-neutralizing antibody titers. Differences were considered significant when P < 0.05 or P < 0.01. 3. Results

A

E protein HWYKAGST

FNCLGMGN 1

402

D1-3 FNCLGM

KCRLKM D1-2

1

291 DKL

GST

D3

292

402

NS1 protein DTGCAID

VRSRVDA

1

NS1

352 His

6

B (kDa)

Anti-JEV

Anti-NS1

50 37

3.1. Expression of E and NS1 proteins in E. coli 25 20

NS1 N

D3

D1-2 D

15 D1-3 D

The JEV E protein domains [D1e3 (amino acids 1e402), D1e2 (amino acids 1e291), and D3 (amino acids 292e402)], or the NS1 protein (amino acids 1e352) were expressed in E. coli as hexahistidine-tag fusion proteins (Fig. 1A). All constructs expressed as inclusion bodies were solubilized by 8 M urea, buffer exchanged by dialysis against PBS or refolding buffer, and analyzed by the Western blot (Fig. 1B). The calculated molecular masses based on their primary structures were 43.4 kDa, 31.6 kDa, 11.8 kDa, and 40.1 kDa for D1e3, D1e2, D3, and NS1, respectively. The apparent molecular masses of the detected protein bands closely matched with those calculated values. The nickel-affinity purified proteins were soluble in PBS, but more than 100-fold differences in the final protein yields were observed among the different protein constructs. Although all constructs contained a similar level of protein prior to purification process, their final yields were vastly different. This was primarily because their solubility in PBS was significantly different; D3 was the most soluble and thus exhibited the highest yield, reaching 60 mg/l culture, followed by D1e3 and NS1 with a level of 3 mg/l culture, and D1e2 with a level of 0.5 mg/l culture had the lowest yield. Proteins with low yields showed a tendency to make aggregation during purification process. We also noted that all proteins, with the exception of D3, were difficult to solubilize in PBS at high concentrations.

Fig. 1. Analysis of JEV E and NS1 proteins expressed in Escherichia coli. (A) Schematic drawing of three recombinant E protein constructs (D1e3, D1e2 and D3) and NS1 protein with the flanking amino acid sequences. The amino acid numbers indicate the first and the last amino acid of each recombinant protein designed to be expressed in E. coli. All protein constructs were engineered as fusion proteins with a hexahistidine (His  6) tag. (B) Western blot analysis of the four recombinant proteins expressed and purified from E. coli. E protein constructs were detected by rabbit anti-JEV antisera (1:1000) and NS1 protein by rabbit anti-NS1 antisera (1:1000).

the antisera were tested against the formalin-inactivated JE vaccine (Fig. 2B), the reactivity of the D1e3 and D1e2 proteins were reduced by 10e100 folds, compared with the reactivity against the corresponding recombinant proteins (compare Fig. 2A and B). However, the reactivity of the D3 remained at similar level for both routes (Fig. 2B). As expected anti-NS1 antisera did not react with the inactivated virus, because the NS1 protein is not present on the surface of the virus (Fig. 2B).

3.2. Immunogenicity of E and NS1 proteins in mice BALB/c mice were immunized with each purified protein by the i.n. or i.p. route as described in the Materials and methods. All recombinant proteins were immunogenic when administered by both immunization routes (Fig. 2A). When

3.3. Virus-neutralizing antibody and protective immunity induction Virus-neutralizing antibodies were analyzed using the FRNT50 as described in the Materials and methods. All

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Anti-recombinant protein serum IgG titer (log10)

A

7

**

3.4. Antigen-specific cytokine induction in the cervical lymph nodes of mice i.n. immunized with D1e3 or NS1 protein

**

6 5

**

**

** **

**

We analyzed various cytokines expressed in mice immunized with the JEV E protein antigen through i.n. or i.p. route by using a mouse cytokine array kit (data not shown), and then we selected several cytokines whose expressions were appeared to be augmented (Fig. 4). We found that both IL-2 and IL-17, but not IFN-g, were significantly augmented in the cervical lymph nodes of the i.n. immunized mice with D1e3 or NS1 (Fig. 4).

*

4 3

i.n.

NS1

D3

i.p.

7 6 **

i.n.

D1-2

NS1

D3

NS1

D3

**

D1-2

2

**

**

4 3

**

D1-3

**

5

D1-3

Anti-JE Vac serum IgG titer (log10)

D1-2

D1-3

NS1

D3

D1-2

D1-3

2

B

173

i.p.

Fig. 2. Serum IgG induced in female BALB/c mice (12e18 per group) by immunization with recombinant D1e3, D1e2, D3 or NS1. IgG titers for (A) each recombinant protein or (B) the formalin-inactivated JE vaccine (JE Vac, Beijing - 1 strain). Mice were immunized as described in the Materials and methods section, and the antisera collected at week 12 were used to determine the IgG titers. Titers were defined as the highest serum dilution at which the OD450 reached the baseline. The baseline was defined as the sum of the OD450 value and the standard deviation (SD) multiplied by 2 for 100-fold diluted pre-immune sera (i.e., baseline ¼ [OD450 þ (SD  2)]). * or ** denotes values that are significantly different from the nonimmune serum using the WilcoxoneManneWhitney test (P < 0.05 or 0.01, respectively).

recombinant proteins except NS1 induced neutralizing antibodies with average titers ranging from 50 to 100 (Fig. 3A). As expected, anti-NS1 antisera did not show any virusneutralization capacity. Next, immunized mice were challenged with the JEV JaGAr01 strain. This challenge experimental protocol was found to consistently kill at least 75% of the unimmunized female BALB/c mice within seven days post-infection. Both i.n. (Fig. 3B) and i.p. (Fig. 3C) immunizations with the E protein constructs conferred 90%e100% protection, while NS1 conferred 40% and 80% protection by i.p. and i.n. routes, respectively.

4. Discussion WHO policy now discourages the use of mouse brainderived vaccines, and Japanese legislation has suspended the routine use of the formalin-inactive JE vaccine; this has resulted in vaccine production being discontinued after 2005. A new Vero cell-derived vaccine (Beijing-1 strain), licensed in 2009, was designed to replace such vaccines. Various other types of JE vaccine are now at advanced stages of development or already available on the international markets. Although still at the preliminary stage of preclinical studies, research on the development of recombinant JE vaccines using E or NS1 protein is ongoing in several laboratories; the results of such studies are briefly summarized here [17e25]. Chia et al. have shown that a sub-fragment of the E protein produced in E. coli was protective to mice when 25 mg of the antigen was used in combination with Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IFA) [24]. In this study, the vaccines were administered by two subcutaneous injections and the N-terminal domain (designated as EA) provided 66.6% protection, whereas the C-terminal domain (designated as EB) provided only 20% protection; this compared with a 4% survival rate in the adjuvant only control group [24]. Wu et al. reported that i.p. immunization with 100 mg of thioredoxineDIII fusion protein with CFA or cationic liposome, followed by three boosters using IFA or various liposome preparations, conferred 60% or 80% protection in mice, respectively (the PBS control group did not survive the challenge infections) [23]. In a different study Alka et al. reported that 5.5 mg of DIII administered three times to mice via the intramuscular route with Freund’s adjuvant or Alhydrogel conferred 78.6% protection (the control group did not survive their challenge infections) [21]; Lin et al. first demonstrated the protective efficacy of an E. coli-synthesized recombinant NS1 protein against a lethal JEV challenge (survival rate, immunized:unimmunized; 87.5%:25%) when three 100 mg doses of antigen were administered with CFA or IFA by the i.p. route [25]. Unlike D1e2, the D3 was successfully expressed in recombinant systems as a soluble antigen and independently fold as a single entity [21,23]. Consistent with those previous findings, we demonstrated that only the D3 achieved high level expression in E. coli and was readily refolded following purification from the inclusion bodies. Considering the fact

S. Tafuku et al. / Microbes and Infection 14 (2012) 169e176

C

3

Survival rate (%)

* * *

* 2

*

*

JE Vac, D1-2, D3 * D1-3 *

100

50 NS1 Nonimmune

NS1

D3

D1-2

D1-3

D3

NS1

D1-2 i.n.

i.p.

Nonimmune

0

1 D1-3

A

Virus-neutralizing antibody titer (log10)

174

0

5

10

15

20

Days after challenge

B JE Vac, D1-3, D1-2, D3 *

100

Survival rate (%)

NS1 *

50

Nonimmune

0 0

5

10

15

20

Days after challenge

Fig. 3. Anti-viral immunity induced in female BALB/c mice by immunization with recombinant D1e3, D1e2, D3 or NS1. (A) Virus-neutralizing antibody titers against the JEV JaGAr01 strain for the antisera collected at week 12. Titers less than 10 were considered negative in the virus-neutralization assays [28]. Protection against virus challenge with the JaGAr01 strain by (B) i.n. or (C) i.p. immunization regimens. Mice were immunized as described in the Materials and methods section with the recombinant protein constructs (12e18 mice per group) or the Beijing-1 strain of the formalin-inactivated JE vaccine (JE Vac, 10 mice per group). Immunized mice were challenged with the JEV (5LD50), and their survivals were monitored for three weeks. * denotes values that are significantly different from the nonimmune serum using the WilcoxoneManneWhitney test (P < 0.01). The data for the nonimmune mouse survival curves in graphs B and C are identical.

1500 IFNIL-2

pg/ml

1000

IL-17 Granzyme B

500

0 Antigen stimulation

+

+ D1-3

NS1

Fig. 4. A cytokine profile analysis for mice i.n. immunized with D1e3 or NS1. Cervical lymph nodes were isolated from the immunized mice and stimulated by the corresponding antigen to determine the antigen-specific cytokine secretion from isolated lymphocytes. Levels of cytokine secretion were compared between cells incubated with and without the addition of each antigen.

that D3 contains several virus-neutralizing epitopes [29e31], we drew a conclusion that the D3 should be placed at the top priority for consideration in the development of JE subunit vaccine. Further, the existence of the only one JEV serotype which does not necessitate the production of a multivalent vaccine is a tremendous advantage when subunit vaccine is ought to be manufactured. Furthermore, the D3 of JEV E protein is known to be highly conserved among many strains of JEV isolates, and therefore, a recombinant protein-based JEV vaccine developmental strategy may reliably be able to focus on the D3. The majority of pathogens initiate infection through mucosal tissues. Hence mucosal vaccines, which can induce both local and systemic immunity, are generally considered advantageous over parenteral vaccines against mucosal pathogens. However, mucosal vaccine design is no longer confined to the control of mucosal infections, making the application of such vaccines to non-mucosal infections theoretically possible [18,32,33]. We demonstrated for the first time that the recombinant E protein domains as well as NS1 protein, when administered through the nasal route, are protective against a lethal virus challenge. We also found that antigen-specific IL-17 was induced in the draining lymph nodes of the i.n.

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immunized mice. This result is consistent with a previous finding that Th17 response was induced by nasal immunization with an antigen co-administered with CT [32]. Th17 cells and their effector cytokines have been reported as important mediators for host defense against various infections, especially extracellular bacterial and fungal infections, in large part through neutrophil recruitment and production of antimicrobial peptides [34,35]. Furthermore, a protection against inhalation anthrax was demonstrated to be mediated by CTinduced Th17 cells, and IL-17 was found to be involved in serum antibody response [36]. Taken these reports together, it is plausible to speculate that IL-17 induced in our i.n. immunization regimen was an important mediator of CT adjuvant activity and that may have mediated protection against lethal JEV challenge infection. Protection against JEV is primarily, if not exclusively, neutralizing antibody-dependent, thus any immunization regimens, including mucosal vaccines, that induce neutralizing antibodies with sufficiently high magnitude and duration will have a potential to be effective. Rauthan et al. previously reported an experimental result on the first trial of oral immunization using the ectodomain of JEV E protein [19]. The immunization induced virus-specific antibody response, but it failed to neutralize JEV in vitro and did not provide any protection against lethal virus challenge [19]. In a clear contrast to their oral immunization experimental results, our present study demonstrated the protective efficacy of the i.n. immunization regimen against lethal virus infection using a series of recombinant E protein domains as well as the NS1 protein. Furthermore, our study raised the possibility that i.n. immunization regimen could be applicable to other flavivirus including West Nile virus and other neurotropic viruses. Domestic pigs are the important amplifier of the virus in JE endemic and even in nonendemic countries; for example, almost all newborn pigs become seroconverted once they spend a one season of summer in Japan where JE is no longer endemic [2]. Thus, besides economic benefits to use an antiabortive pregnancy veterinary vaccine for female pigs, it is believed to be desirable in a public health point of view that veterinary JE vaccine that are economically feasible to mass vaccinate all newborn pigs should, in theory, significantly interfere virus amplification process in the environment, and would consequently reduce the chance of human infection. A strategy for needle-less i.n. vaccine may be within the scope of the development of such mass vaccination regimen. The most challenging and significant obstacle to the development of an effective subunit protein-based vaccine is identification of suitable adjuvants and/or delivery systems [13,14,16]. In this study, we used CT because no superior i.n. adjuvant currently exists. However, because CT with GM1ganglioside binding affinity is redirected to the brain when administered by the i.n. route, raising a serious concern for the use of CT-related adjuvants in i.n. immunization regimens. Consequently, the design of strong but safe mucosal adjuvants is eagerly awaited. Crucially, the future success of many subunit protein-based mucosal vaccines is dependent on the development of effective adjuvants and delivery systems.

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Acknowledgment This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences from the Bio-oriented Technology Research Advancement Institution, and the Okinawa Industry Promotion Public Corporation (Naha, Okinawa, Japan).

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