Enhanced Immune Responses Against Japanese Encephalitis Virus Infection Using Japanese Encephalitis Live-Attenuated Virus Adjuvanted with Montanide GEL 01 ST in Mice

Abstract

Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in human and animal. To prevent JEV infection, an effective live-attenuated vaccine is needed. In the article, JEV attenuated strain, SCYA201201 of GI genotype, which was mixed with 10% concentrate GEL 01 ST adjuvant (Montanide™ GEL 01 ST), was selected for a vaccine candidate and its immunogenicity was evaluated in mice. Our results showed that JEV mixed with GEL 01 ST elicited production of both IgG1 and IgG2a antibodies, and enhanced virus-specific crossprotective intergenotypic response in mice. Proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was detected in JEV mixed with GEL 01 ST group (p < 0.05). In the JEV + 10% GEL 01 ST vaccinated group, the proportion of CD4⁺ T cells in spleen was significantly higher than that of control group (p < 0.05), and the yields of interleukin (IL)-2, IL-4, and interferon-γ in the splenocyte supernatant were also significantly higher than that of control group (p < 0.05). Moreover, complete protection was provided after JEV challenge in mice in JEV mixed with GEL 01 ST group, and early immunity was detected in those mice immunized with JEV mixed with GEL 01 ST. These findings confirm that GEL 01 ST can enhance JEV live-attenuated immunogenicity, and JEV +10% GEL 01 ST used as vaccine candidates provide protection against JEV infection in a mouse model, which could be used as potential vaccine candidates in pig.

Introduction

Japanese encephalitis virus (JEV) is one of the most common agents of viral encephalitis in humans and animals (Ashraf et al. 2016), with most cases occurring in southern and eastern Asia (Solomon 2006, Liang and Huanyu 2015, ). It is believed that nearly three billion people suffer from JEV infection, with estimated 50,000 cases annually, of which 15,000 are fatal, and up to 50% of survivors have severe residual neurological complications (Ghosh et al. 2009, Impoinvil et al. 2013, Campbell et al. 2015).

Vaccination is the most cost-effective and the best preventive strategy for the treatment of encephalitis, and is widely used to improve both human and animal health (Wang et al. 2015). Adjuvant is often required in subunit, inactivated, and DNA vaccines, to enhance the activation of dendritic cells resulting in the generation of strong antigen-specific immune responses, but due to the adverse effects on live viruses, adjuvants are generally not added to these formulations (Hilton et al. 2002, Singh and O'Hagan 2003). However, Montanide™ has developed a range of adjuvants that have been shown to be safe and efficient in a wide variety of vaccine types (Coffman et al. 2010, Mohan et al. 2013). The Montanide GEL 01 ST, a new adjuvant based on the dispersion of highly stable gel microparticles of sodium polyacrylate in water, is a robust, nontoxic, and effective adjuvant that can induce balanced immune responses (both cellular and humoral) with minimal or no side effects (Tabynov et al. 2016).

Both genotype I (GI) JEV and the Taiwan clade are newly introduced and have evolved relatively rapidly and genotype III (GIII) has been replaced by genotype I in South Korea, Thailand, and China (Zhou et al. 2018). However, all approved vaccine products are derived from GIII strains (Ye et al. 2015, Chu et al. 2017, Hegde and Gore 2017). As the current live-attenuated JEV vaccine used in China is derived from GIII strain SA14–14–2, the efficacy of the vaccine to protect against GI has been evaluated, with results suggesting that immunization with GIII JEV vaccine does not provide complete protection against GI JEV. Thus, a more efficacious GI JEV vaccine is needed (Fan et al. 2012, 2013, Han et al. 2014, Do et al. 2015).

The objectives of the current study were to evaluate the immunogenicity of JEV GI strain SCYA201201 mixed with adjuvant Montanide GEL 01 ST. The first phase aimed to investigate the effect of Montanide GEL 01 ST on JEV viral survival, and determine the optimum concentrate of GEL 01 ST adjuvant, whereas the second phase aimed to determine immune responses against JEV using Japanese encephalitis live-attenuated virus adjuvanted with 10% concentrate Montanide GEL 01 ST in mice.

Materials and Methods

Animals and ethics statement

Three-week-old female KM mice were purchased from Chengdu Dossy Experimental Animal Co., Ltd. (Chengdu, China). The animal experiments were conducted in compliance with the animal protocol approved by the College of Veterinary Medicine at Sichuan Agricultural University. All procedures performed in the present study involving animals had been approved by the Institutional Animal Care and Use Committee of Sichuan Agriculture University (Approval Number RW2016-088), and followed the National Institutes of Health guidelines.

Cells, virus, and adjuvant preparation

BHK-21 cells were maintained in Double modified Eagle's medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) containing penicillin/streptomycin (Life Technologies, Inc., Gibco/Brl Div, Grand Island, NY), and 2 mM l-glutamine in a humidified atmosphere at 37°C, 5% CO₂. The JEV of GI live-attenuated strain SCYA201201-F126, which was identical to SCYA201201-86 (GenBank:ANV27916.1), was maintained in our laboratory. The SCYA201201-F126 strain was derived from the SCYA201201-86 strain, which was attenuated by passage on the BHK-21 cell from 86 generation to 126 generation (Zhou et al. 2018). The JEV strain SA14-14-2 (GIII) (GenBank:AF315119), JEV SC2013 strain (GIII), and the JEV SCYA201201 strain (GI) (GenBank:KM658163), which isolated from pig cerebrospinal fluid in 2012 (Yuan et al. 2016) (Sichuan, China). Montanide GEL 01 ST (abbreviated as GEL 01 ST) polymeric adjuvant was a kind gift from SEPPIC (France). A final 10%, 15%, and 20% of GEL 01 ST, respectively, was added into diluted JEV live vaccine and mixed by manual shaking.

Effect of different concentrations of GEL 01 ST on JEV in vitro and in mice

Viral survival test in vitro

Three experimental groups were designated based on differing concentrations of adjuvant GEL 01 ST mixed with the JEV (10^7.0 PFU/mL) live-attenuated strain (JEV +10% GEL 01 ST, JEV +15% GEL 01 ST, and JEV +20% GEL 01 ST), and JEV alone was used as control group. All groups were allowed to mix for 1 h at 37°C, diluted 10-fold (starting at 0.1:0.9 mL) in DMEM, and added to six-well tissue culture plates containing a 90% confluent monolayer of BHK-21 cells. For the plaque assay, cells cultured in the various groups were incubated for 1.5 h at 37°C with gentle shaking every 15 min in 5% CO₂, after which the plates were incubated for 3–4 days at 37°C in 5% CO₂, fixed with formalin, and stained with 1% Crystal Violet. The plaque-forming units (PFU) of a virus solution (virus/mL) were counted by plaque numbers, and the logarithm for the PFU of the virus was calculated.

Virulent challenge in mice

Eighty mice were randomly divided into eight groups (10/each group). Mice were intraperitoneally immunized with 0.3 mL/mice in all groups. Mice in group 1–3 were immunized with different concentrations of adjuvant GEL 01 ST mixed with the JEV (10^7.0 PFU/mL) live-attenuated strain (JEV +10% GEL 01 ST, JEV +15% GEL 01 ST, and JEV +20% GEL 01 ST), and remaining mice in groups 4–8 were immunized with JEV alone, three various concentrations of adjuvant (10%, 15%, and 20%), and PBS, respectively. Two weeks after immunization, a challenge was carried out using the 75LD₅₀ JEV virulent strain, and mice were monitored daily for 21 days. The survival rate was determined using the formula, 100 × (no. of survivors)/(nos. of challenged).

Effect of 10% concentrate GEL 01 ST on JEV immunogenicity

Mice group and vaccination

Sixty-three-week-old female KM mice (15/each group) were randomly divided into four groups, and were intraperitoneally immunized (0.3 mL/mice) with JEV (10^7.0 PFU/mL) +10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST, or PBS, respectively.

Antibody determination of IgG1 and IgG2a in mice

Two weeks after immunization, sera isolated from 10 mice/each group were examined by enzyme-linked immunosorbent assay (ELISA) for IgG1 and IgG2a titers according to the manufacturer's instructions (ICL). Antibody titers were calculated from the serum dilution that gave an OD₆₃₀ of 0.3 above that of the preimmune sera.

Lymphocyte proliferation assay

Five mice from each group were sacrificed on the 14th day after immunization, and spleens were removed. Briefly, spleens were harvested aseptically and processed by gentle dissociation with a sterile stainless steel sieve and glass pestle and a suspension of splenocytes was then cultured in incomplete RPMI medium (Gibco). Cell suspensions were centrifuged at 300 × g for 5 min to isolate erythrocytes which were subsequently lysed by placing the suspension on ice for 5 min. Cells were washed three times with Hank's Balanced Salt Solution (HyClone) and resuspended in complete RPMI medium. Two hundred microliters of cells at a concentration of 2 × 10⁵ cells/mL were cultured in 96-well culture plates. Cells were stimulated with inactivated JEV antigen and concanavalin A (5 μg/mL) (Sigma) and incubated for 72 h at 37°C in a 5% CO₂ incubator. Lymphocyte proliferation assays were performed using the MTS reagent with a Cell Proliferation Kit (Promega), according to the manufacturer's instructions. Briefly, lymphocytes were incubated in 96-well culture plates with MTS reagent for 4 h, and the absorbance was measured at 490 nm using an ELISA reader.

Analysis of the spleen T cell subsets

Aliquots of the splenocytes prepared as mentioned above were subjected to T cell subset analysis by flow cytometry. Briefly, cell counting was performed and the concentration of the spleen cells was adjusted to 10^6.0 cells/mL. Hundred microliters of cell suspension was transferred to another centrifuge tube, and stained with rat anti-mouse CD3-fluorescein (FITC), R-phycoerythrin/cyanine 5 (SPRD) (Southern Biotech), rat anti-mouse CD4-R-phycoerythrin (PE) (Southern Biotech), and rat anti-mouse CD8a-PerCP (Southern Biotech) at 4°C for 15 min in the dark. Stained cells were washed in PBS and were resuspended in 0.45 mL PBS. Resuspended cells were analyzed for T cell subsets by BD fluorescence-activated cell sorting (FACS) using Calibur Flow Cytometer (BD) and the results of analysis were collected (Fu et al. 2013, Zhang et al. 2016).

Analysis of IL-2, IL-4, and IFN-γ cytokine response

Supernatants from cell cultures were harvested after 72 h and stored at −80°C. The level of interleukin (IL)-2, IL-4, and interferon (IFN)-γ were determined in culture supernatants using an ELISA Kit (NEO, China), according to the manufacturer's instructions.

Effect of 10% concentrate GEL 01 ST on JEV crossprotection of different genotypes

Mice group and vaccination

Eighty-three-week-old female KM mice (20/each group) were randomly divided into four groups, and were intraperitoneally immunized (0.3 mL/mice) with JEV (10^7.0 PFU/mL) +10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST, or PBS, respectively.

Plaque reduction neutralization test

Two weeks after immunization, the sera (10 mice/each group) were collected by orbital phlebotomy. To determine the serum-neutralizing antibody titers of immunized mice, sera were diluted twofold (starting at 1:10) in DMEM containing 2% FBS. After heat inactivation of complements, 100 μL of each serial dilution was incubated with an equal volume of JEV solution containing ∼1000 PFU/mL. The mixture was then added to BHK-21 cells in duplicate wells (∼100 PFU/well). After absorption, the infected BHK-21 cells were overlaid with DMEM containing 5% FBS and 1% methylcellulose. After incubation for 4 days, the plaques were stained and counted. The neutralizing antibody titer was defined as the reciprocal of the maximum dilution of serum that yielded a 50% plaque reduction (PRNT₅₀) when compared with that of the virus controls without anti-JEV serum.

Challenge of genotype 1 and 3 JEV in mice

Mice were challenged with an intraperitoneal injection of a lethal dose of 75LD₅₀ JEV of the SCYA201201 strain (GI) and the SC2013 strain (GIII) on the 14th day after immunization and then monitored daily for 21 days. The survival rate was determined using the formula, 100 × (no. of survivors)/(nos. of challenged).

Effect of 10% concentrate GEL 01 ST on JEV early immunity

Mice group and vaccination

Three-week-old female KM mice were randomly divided into four groups (10 mice/each group), and were intraperitoneally immunized (0.3 mL/mice) with JEV (10^7.0 PFU/mL) +10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST, or PBS, respectively.

Anti-JEV IgG detection using ELISA

The blood samples from four immunized mice groups (n = 10) were collected every other day after the second day postvaccination, until the 14th day. Anti-JEV IgG titers in mice against different immunogens were determined by ELISA, according to the manufacturer's instructions (Combined, China) (Liu et al. 2016). The results are presented as mean ± standard deviation (SD) of A450 nm (absorbance 450 nm).

Virulent challenge in mice

Mice were injected intraperitoneally with JEV virus. On the third day after immunization and every subsequent odd number day until day 15 and then monitored daily for 21 days. The survival rate was determined using the formula, 100 × (no. of survivors)/(nos. of challenged).

Effect of 10% concentrate GEL 01 ST on protection rate in mice immunized with low-level JEV virus titer

JEV was serially diluted 10-fold (starting at 1:10) in DMEM (GIBCO) and mixed with 10% GEL 01. The live attenuated JEV stock solution was ∼10^7.0 PFU/mL, whereas the 10- and 100-fold dilutions were ∼10^6.0 and 10^5.0 PFU/mL, respectively. Mixtures were intraperitoneally injected (0.3 mL/mice), and mice were challenged using a dose of 75LD₅₀ JEV 2 weeks after the first immunization, and then monitored daily for 21 days. The survival rate was determined using the formula, 100 × (no. of survivors)/(nos. of challenged).

Statistical analysis

Experiments were conducted in triplicate, and repeated three times. A univariate analysis of variance using SPSS software (SPSS, Chicago, IL) was used to analyze the significance among treatments. Error bars represent the SD or standard error of the mean.

Results

Different concentrations of adjuvant GEL 01 ST had no significant impact on JEV viral survival in vitro

Results are shown in Fig. 1 and demonstrate that adjuvant GEL 01 ST had a marginal effect on JEV live-attenuated strain. Specifically, with increasing concentrations of GEL 01 ST, there were decreasing numbers of plaques. Results showed that plaque formations were approximately, 10^7.2, 10^7.1, 10^7.0, and 10^7.6 PFU/mL for JEV + 10% adjuvant, JEV + 15% adjuvant, and JEV + 20% adjuvant and control groups, respectively. Each PFU of JEV (log₁₀/mL) from the experimental groups was directly compared with the control group, and overall percentages of plaque formation compared with the control group (100%) were calculated and found to be 94.7%, 93.4%, and 92.1%, for the 10%, 15%, and 20% adjuvant concentrations, respectively.

FIG. 1.

Effect of different concentrations of GEL 01 ST on JEV viral survival in vitro. JEV live-attenuated strain was mixed with three concentrations (10%, 15%, and 20%) of adjuvant GEL 01 ST, and JEV + PBS was used as control group. Then all groups were allowed to mix for 1 h at 37°C, diluted 10-fold (starting at 1:10) in DMEM, and added to six-well tissue culture plates containing a 90% confluent monolayer of BHK-21 cells. The PFU of a virus solution (virus/mL) were counted by plaque numbers, and the logarithm for the PFU of the virus was calculated. DMEM, Double modified Eagle's medium; JEV, Japanese encephalitis virus; PFU, plaque-forming units.

JEV live-attenuated vaccines with different concentrations of GEL 01 ST provide 100% protection in mice

Figure 2 depicts the protective efficacy of JEV live-attenuated strain mixed with various concentrations of adjuvant GEL 01 ST in mice challenged with lethal dose of 75LD₅₀ JEV. The results showed that the survival rate of the JEV + 10% GEL 01 ST group, the JEV + 15% GEL 01 ST group, and the JEV + 20% GEL 01 ST group were both 100%, the survival rate of the JEV group was 70%, and there was no survival mice in the remaining four groups. Overall, results indicate the JEV plus adjuvant groups provided complete clinical protection, and suggest that GEL 01 ST adjuvant may be able to resist the destabilizing antigenic conditions in media.

FIG. 2.

Clinical protection of JEV live-attenuated strain adjuvanted with different concentrations of GEL 01 ST. Three-week female mice were intraperitoneally immunized with 0.3 mL/mice, and JEV titer is 10^7.0 PFU/mL in all JEV groups with or without GEL 01 ST. Two weeks after immunization, a challenge was carried out using the 75LD₅₀ JEV virulent strain, and mice were monitored daily for 21 days. The survival rate was determined.

JEV + 10% GEL 01 ST enhance Th1 phenotype response in mice

To characterize the type of immune responses elicited from the vaccine/adjuvant mixture, the level of IgG1 and IgG2a subclasses were determined with results demonstrating that the level of both antibody subclasses were significantly higher in the JEV + 10% GEL 01 ST immunized groups compared with the JEV alone and negative control groups (IgG1, p < 0.0001; IgG2a, p < 0.0001; Fig. 3). In addition, IgG2a responses predominated over IgG1 responses (p < 0.01; Fig. 3), as these have been shown to mediate Th2 and Th1 immune responses, respectively (Fig. 3). The results indicated that immunization of mice with JEV + 10% GEL 01 ST, and JEV alone triggered a Th1-type immune, in the JEV +10% GEL 01 ST and JEV groups, due to the level of IgG1 increased notably when compared with IgG2a, which was biased toward a Th1 phenotype response.

FIG. 3.

Identification of IgG1 and IgG2a subclasses in serum in mice immunized with JEV live-attenuated strain adjuvanted with 10% GEL 01 ST. Three-week female mice were intraperitoneally immunized with JEV (10^7.0 PFU/mL) +10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST or PBS, respectively. Two weeks after immunization, sera isolated from four groups of mice were examined by ELISA for IgG1 and IgG2a. The results are expressed as the mean specific absorbance ± SD. The asterisk “***” indicates significance at p < 0.001. “Δ” versus the blank control and negative control. ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

JEV + 10% GEL 01 ST enhance cell-mediated immune response in mice

As shown in Fig. 4, a significant proliferative T cell immune response was noted in the four groups of mice immunized. Similarly, in the JEV +10% GEL 01 ST group, a strong T cell proliferative response was found in the concanavalin A-positive control (p < 0.001), whereas a medium T cell proliferative response was detected in JEV alone group. However, neither the 10% GEL 01 ST group or PBS group elicited a proliferative T cell response (Fig. 4).

FIG. 4.

Lymphocyte proliferation. Three-week female mice were intraperitoneally immunized with JEV (10^7.0 PFU/mL) + 10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST or PBS, respectively. Mice were sacrificed on the 14th day after immunization, and spleens were removed. The splenocytes was cultured in incomplete RPMI medium, and lymphocyte proliferation assays were performed using the MTS reagent with a Cell Proliferation Kit. The results are expressed as the mean specific absorbance ± SD. The asterisk “**” indicates significance at p < 0.01 and “***” indicates significance at p < 0.001. “Δ” versus the blank control and negative control.

Distribution of CD3⁺, CD4⁺, and CD8⁺ T cells in the spleen cells were evaluated by FACS. Overall trends in the proportion of CD3⁺ and CD4C⁺ T cells were higher in immunized mice than the control group animals (Fig. 5). In JEV +10% GEL 01 ST group, all of CD3⁺, CD4⁺, and CD8⁺ showed the highest level, which were significantly higher than the control group (p < 0.05).

FIG. 5.

Analysis of the spleen T cell subsets by FACS. The levels of CD3⁺ (A), CD3⁺ CD4⁺ (B), and CD4⁺ CD8⁺ (C) T cell subsets in splenocytes from immunized groups and the negative control group were analyzed by FACS. The average measured percentages of CD3⁺, CD3⁺ CD4⁺, and CD4⁺ CD8⁺ T cells with representative scatter diagrams of the T cell subsets detected by flow cytometry method. The mean with SDs were represented as bar graph, and are representatives of the percentages of CD3⁺, CD3⁺ CD4⁺, and CD4⁺ CD8⁺ T cells of spleens. The asterisk “*” indicates significance at p < 0.05, “**” indicates significance at p < 0.01. “Δ” versus the blank control and negative control.

The levels of IL-2, IL-4, and IFN-γ in the supernatant of splenocytes isolated from mice vaccinated with JEV +10% GEL 01 ST were significantly higher than the level in the supernatant of splenocytes isolated from mice vaccinated with JEV alone and also those of the 10%GEL 01 ST or PBS groups (Fig. 6A, B, p < 0.001; Fig. 6C, p < 0.01).

FIG. 6.

Analysis of IL-2, IL-4, and IFN-γ cytokine response. The levels of IL-2 (A), IL-4 (B), and IFN-γ (C) in the supernatant in splenocytes from immunized groups and the negative control group were analyzed by ELISA. Results are expressed in picograms per milliliter and are the mean ± SD. The asterisk “**” indicates significance at p < 0.01 and “***” indicates significance at p < 0.001. “Δ” versus the negative control. IFN, interferon; IL, interleukin.

JEV + 10% GEL 01 ST enhance virus-specific crossprotective intergenotypic response in mice

The ability of JEV to produce neutralization antibodies in sera from the various immunized groups were also examined in vitro using PRNT₅₀. Mice were injected with JEV +10% GEL 01 ST, JEV alone, 10% GEL 01 ST, or PBS. Sera were collected on day 14 after immunization and checked for JEV-neutralizing antibodies. As shown in Table 1, no antibodies were detected (<1/5) in the 10% GEL 01 ST and PBS group, whereas a medium titer of antibodies (>1/20) was detected in the sera from mice immunized with JEV alone. As expected, a high-titer (>1/40 complete protection in mice) concentration of neutralizing antibodies was found in the sera of mice immunized with JEV +10% GEL 01 ST.

Table 1.

JEV + 10% GEL 01 ST Enhance Virus-Specific Crossprotective Intergenotypic Response in Mice

Mode ^a	Neutralizing antibody ^b		Virulent challenge ^c
Mode ^a	JEV GI strain	JEV GIII strain	JEV GI strain	JEV GIII strain
JEV +10% GEL 01 ST	>1/40	>1/40	9/10	10/10
JEV	>1/20	>1/20	7/10	7/10
10% GEL 01 ST	<1/5	<1/5	0/10	0/10
PBS	<1/5	<1/5	0/10	0/10

Three-week female mice were intraperitoneally immunized with 0.3 mL/mice in all groups, and JEV titer is 10^7.0 PFU/mL in JEV + 10% GEL 01 ST and JEV alone groups.

Sera were collected on day 14 after immunization and checked for JEV-neutralizing antibodies by PRNT₅₀. They are expressed as the reciprocals of serum dilution, yielding a 50% reduction in plaque numbers for the viral genotype.

Two weeks after the first immunization, mice were challenged intraperitoneally with a dose of 75LD₅₀ JEV of GI strain or GIII strain. The survival rate was determined using the formula, 100 × (no. of survivors)/(nos. of challenged).

JEV, Japanese encephalitis virus.

Moreover, our data indicated an induction of crossprotection improved by the effects of JEV + 10% GEL 01 ST, and more importantly, demonstrated that JEV + 10% GEL 01 ST could also enhance a crossprotection response across the viral GI strain and GIII strain (Table 1).

JEV + 10% GEL 01 ST elicit increased anti-JEV IgG and provide an early protection after challenge in mice

The results presented in Fig. 7A and B are as follows. High antibody titers were observed in mice immunized with JEV +10% GEL 01 ST. Medium antibody titers were observed in mice immunized with JEV alone, whereas immunization with 10% GEL 01 ST or PBS alone did not elicit an immune response (p < 0.01; Fig. 7A).

FIG. 7.

Anti-JEV IgG increased and protective efficiency enhanced by using JEV + 10% Gel 01 ST in mice model of early immunity. Three-week female mice were intraperitoneally immunized with JEV (10^7.0 PFU/mL) + 10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST or PBS, respectively. (A) On the 2nd, 4th, 6th, 8th, 10th, 12th, and 14th day after immunization, 5 mice were randomly selected from the four groups (n = 10) to collect blood samples. Anti-JEV IgG titers in mice against different immunogens were determined by ELISA. The asterisk “**” indicates significance at p < 0.01 and “***” indicates significance at p < 0.001. “Δ” versus the blank control and negative control. (B) On the 3rd, 5th, 7th, 9th, 11th, 13th, and 15th day after immunization, 10 mice were challenged intraperitoneally with a dose of 75 LD₅₀ JEV and then monitored daily for 21 days. The survival rate was determined. The asterisk “**” indicates significance at p < 0.01 and “***” indicates significance at p < 0.001.

Three-week-old female KM mice were randomly divided into three groups, and were intraperitoneally immunized with JEV (10^7.0 PFU/mL) +10% GEL 01 ST, JEV (10^7.0 PFU/mL), 10% GEL 01 ST, or PBS, respectively. Mice were challenged with a lethal dose of 75LD₅₀ JEV on the day 3 after immunization and every subsequent odd numbered day until day 15 and then monitored daily for 21 days. The survival rate in mice preimmunized with JEV +10% GEL 01 ST and given the lethal doses at the specified days were found to be 60%, 80%, 100%, 100%, 100%, 100%, and 100%, respectively. The survival rate of mice preimmunized with JEV alone and given lethal doses at the specified days were 20%, 40%, 40%, 60%, 40%, 70%, and 70%, respectively. Mice preimmunized with 10% GEL 01 ST or PBS alone had a 100% mortality rate (Fig. 7B).

JEV + 10% GEL 01 ST enhances the immunogenicity of JEV live-attenuated vaccines with low-level virus titer

JEV live-attenuated vaccines (LAV) (10^7.0 PFU/mL) were diluted 10-fold (starting at 1:10) in DMEM and after serial dilutions, were mixed with 10% GEL 01 ST. Results indicated that JEV live-attenuated viral stock solutions mixed with GEL 01 ST afforded higher survival rate than the JEV alone group. Specifically there were survival rates of 0%, 0%, 20%, 50%, 50%, 60%, 70%, and 100%, for PBS alone, 10% GEL 01 ST, JEV (10^5.0 PFU/mL), JEV + GEL 01 ST (10^5.0 PFU/mL), JEV alone (10^6.0 PFU/mL), JEV +10% GEL 01 ST (10^6.0 PFU/mL), JEV alone (10^7.0 PFU/mL), and JEV (10^7.0 PFU/mL) + 10% GEL 01 ST, respectively (Table 2).

Table 2.

JEV + 10% GEL 01 ST Enhances the Immunogenicity of Japanese Encephalitis Virus Live-Attenuated Vaccines with Lower Levels of Virus Titer

Mode ^a	No. of survivors/10 in group	% Survival ^b
JEV (10^7.0 PFU/mL) + 10% GEL 01 ST	10/10	100
JEV (10^7.0 PFU/mL)	7/10	70
JEV (10^6.0 PFU/mL) + 10% GEL 01 ST	6/10	60
JEV (10^6.0 PFU/mL)	5/10	50
JEV (10^5.0 PFU/mL) + 10% GEL 01 ST	5/10	50
JEV (10^5.0 PFU/mL)	2/10	20
10% GEL 01 ST	0/10	0
PBS	0/10	0

Three-week female mice were intraperitoneally immunized with 0.3 mL/mice in all groups. The JEV titer is 10^7.0, 10^6.0, and 10^5.0 PFU/mL, respectively.

Two weeks after the first immunization, mice were intraperitoneally challenged with a dose of 75LD₅₀ JEV, then monitored daily for 21 days and the survival rate was determined.

Discussion

JEV is a major cause of acute encephalitis in human and animal. Vaccination is the only viable strategy practiced to control the clinical JEV disease and transmission in swine (Mansfield et al. 2017). Commercially “killed” vaccines are usually coadministered with adjuvant to enhance the immunogenicity of the vaccine (Hervé et al. 2016), but this is not common practice when using LAV. This is the result of several factors that include the possibility that the adjuvant may affect the efficiency of the LAV, and as LAVs proliferate in the body, there is little need to coadminister adjuvants. Results from the current study suggest that although viral strain safety parameters were enhanced using GEL 01 ST, the immunogenicity of virus standard was not reached. Regardless, we still consider JEV live-attenuated strain coadministered with the adjuvant GEL 01 ST to be a valid vaccine for several reasons. First, GEL 01 ST is a new adjuvant based on the dispersion of highly stable calibrated spheric micronic gel particles of sodium polyacrylate in water, which is both convenient and effective. Second, several studies have indicated that an inactivated PRRSV vaccine mixed with GEL 01 ST induced crossprotective responses across the viral genotype (Tabynov et al. 2016). This is of specific importance as the major from JEV has switched from III to I in China. Third, traditional adjuvants needed to be emulsified (Galliherbeckley et al. 2015). This slowed vaccination/adjuvant mixing and also made injection inconvenient, occasionally causing adverse effects at the injection site. The manufacturers of GEL 01 ST recognized these drawbacks and developed this adjuvant to not only reduce thickness of the mixture, but also produce an adjuvant that was able to overcome destabilizing antigenic media and conditions, and at the same time keep safety parameters and efficacy at the appropriate level (Campbell et al. 2015, Arous et al. 2013).

Modified live vaccines are widely used in veterinary medicine, as well as in human medicine, to control many infectious diseases in a wide variety of hosts (Impoinvil et al. 2013, Li et al. 2013). Our study describes four findings concerning JEV live-attenuated strain mixed with adjuvant, and may have a significant impact on the overall understanding how specific adjuvants can enhance the effects of these types of vaccines. First, 10% concentrate GEL 01 ST mixing JEV of 10^7.0 PFU/mL produced higher protect in mice compared with only JEV. Second, the finding that JEV + 10% GEL 01 ST provided protection against a lethal dose of infection during the early immune phase is important for vaccine to prevent the breakout of JEV in the farm. Three, JEV + 10% GEL 01 ST group conveyed increased cellular immunity and survival rates in mice compared with immunization with JEV alone group. Lastly, JEV + 10% GEL 01 ST also elicits virus-specific crossprotective intergenotypic response in mice.

We observed that the GEL 01 ST adjuvant enhanced its effectiveness. Preimmunization of mice with both JEV +10% GEL 01 ST or JEV alone triggered a significantly increased IgG antibody titers on day 5 of the injection compared with JEV alone. All of CD3⁺, CD4⁺, and CD8⁺ showed the highest level, which was significantly higher than the JEV group.

The relevant IgG antibody subclass and Th type are critical for protection against a particular disease. The IgG1 and IgG2a subclasses have been determined to be markers of T helper 2 (Th2) and Th1 immune responses, respectively. In our study, we found that the JEV + 10% GEL 01 ST group induced significant humoral immune responses in which IgG2a was higher than IgG1. These findings suggest that antibody-mediated immune responses are Th1 phenotype responsive against JEV infection.

There are several immunomodulatory cytokines that are believed to be responsible for the clearance of JEV virus. Interestingly, in our study, mice receiving JEV mixed with Gel 01 ST were better protected against virulent JEV-infected than JEV-vaccinated mice, and protection rate are 100% and 70%, respectively, whereas upregulation of proinflammatory cytokine IFN-γ has been observed in JEV +10% GEL 01 ST group and upregulation of IL-2 and IL-4 in JEV group. These results suggested that IFN-γ may be playing a key role in protecting mice to combat against virulent JEV infection.

In addition, the results of GI and GIII JEV strain challenge demonstrated that a JEV + 10% GEL 01 ST adjuvant vaccine could induce crossprotective response across the viral genotype, and GI and GIII were 90% and 100%, respectively, compared with JEV groups. These results are consistent with the PRNT that the GEL 01 ST boost with JEV live-attenuated strain was able to generate higher NA titers (>1:40), a concept which has already been shown to occur in PRRSV virus vaccination (Campbell et al. 2015). In summary, our results show that the addition of GEL 01 ST adjuvant to JEV modified live vaccine can confer increased protection to combat JEV of GI strain and GIII strain, but not to test other genotype of JEV challenge, the higher titers of ELISA and neutralizing antibodies, and reduced IFN-γ and cytokine production in JEV + 10% GEL 01 ST group. Therefore, the commercially available Gel 01 ST adjuvant may be a useful tool in improving the efficacy of live vaccines.

Conclusion

In conclusion, different concentrations of GEL 01 ST adjuvant alone had a marginal impact on virus survival, and can provide enhanced protection after challenge in mice. JEV + 10% GEL 01 ST produced higher level of antibodies and conveyed the increase of cellular immunity and survival rates in mice compared with immunization with JEV alone. JEV + 10% GEL 01 ST provided protection against a lethal dose of infection during the early immune phase, and enhanced the immunogenicity of JEV LAVs with low-level virus titer. Moreover, JEV + 10% GEL 01 ST also induced crossprotective responses across the viral genotype. Our results demonstrated that GEL 01 ST adjuvant could be used in JEV live-attenuated virus vaccine, and JEV + 10% GEL 01 ST could be used as a potential vaccine candidate.

Footnotes

Acknowledgments

This work was supported by Sichuan Science and Technology Program in China (2018HH0127), Education Department of Sichuan in China (18ZB0471), and National Key R & D projects in China (no. 2016YFD0500403). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.

Author Disclosure Statement

No conflicting financial interests exist.

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