Sublingual immunization with Japanese ... - Wiley Online Library

Microbiol Immunol 2016; 60: 846–853 doi: 10.1111/1348-0421.12458

ORIGINAL ARTICLE

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice Eun-Young Lee1,#, Joo-Young Kim2,#, Deuk-Ki Lee1, Il-Sub Yoon1, Hae Li Ko1, Ji-Woo Chung1, Jun Chang2 and Jae-Hwan Nam1 1

Department of Biotechnology, Catholic University of Korea, Bucheon, 420-743 and 2Division of Life & Pharmaceutical Sciences, Ewha Women's University, Seoul 120-750, Korea

ABSTRACT The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live-attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live-attenuated vaccine, whereas the titers of IgG2a induced by the live-attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus-specific IFN-gþ CD4þ and CD8þ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus-specific IL-17þ CD4þ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL-17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live-attenuated and recombinant MVA vaccines. Key words

intramuscular, Japanese encephalitis virus, SA14-14-2, sublingual.

The Japanese encephalitis virus (JEV), which is transmitted by Culex mosquitoes, is the leading cause of viral encephalitis in Asia. JEV belongs to the Flavivirus genus of the Flaviviridae family. The Flaviviridae family contains more than 70 viruses with positive-sense, single-strand RNA genomes (11 kb) that encode a polypeptide comprising the capsid protein C (core protein), the matrix protein (envelope protein M), the major envelope

protein E, and a number of small nonstructural proteins (NS1, NS2A, NS2B, NS4A and NS4B). This single polyprotein is cleaved co- and post-translationally by viral and host proteases to form both structural and nonstructural viral proteins. This is an essential step in producing infectious progeny viruses (1, 2). Commercially available JEV vaccines include a formalin-inactivated vaccine produced in mouse brain

Correspondence Jae-Hwan Nam, Department of Biotechnology, Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, 420-743, Korea. Tel: þ82 2 2164 4852; fax: þ82 2 2164 4865; email: [email protected] #

These authors contributed equally to this work.

Received 22 July 2016; revised 1 November 2016; accepted 9 December 2016. List of Abbreviations: CT, Cholera toxin; i.p., intraperitoneal; JEV, Japanese encephalitis virus; MVA, modified vaccinia virus Ankara; PRNT, Plaque reduction neutralization test; RANTES, regulated upon activation normal T cells expressed and secreted; s.l., sublingual.

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Sublingual immunization with JEV vaccine

and a live-attenuated vaccine, SA14-14-2, produced in primary hamster kidney cell cultures, both of which perform efficiently (3). A JEV vaccine very recently approved by the Food and Drug Administration is produced in Vero cells, purified from virus-infected cell supernatants and inactivated by formalin (3). JEV vaccines fall into four classes: inactivated mouse brainderived, inactivated cell culture-derived, live-attenuated and live recombinant (chimeric) vaccines (4, 5). For all vaccines developed to date, the immunization routes have been s.c. or i.m. injections (4). However, the s.l. route for vaccine delivery has recently been introduced because it can induce both mucosal and systemic immune responses similar to those induced by the conventional routes of vaccine administration. In addition, in the presence of CT adjuvant, s.l. administration can also induce a cytotoxic T lymphocyte response (6). Previous studies have shown that injection via i.m. or i.p. routes of two types of recombinant MVA expressing JEV K94P05 prM/E under a strong synthetic poxvirus promoter (vJH6) or modified H5 vaccinia virus promoters (vJH9) can induce JEV-specific neutralizing antibodies in mouse and swine and completely protect mice against JEV challenge (7, 8). SA14-14-2, the only commercially available live-attenuated JEV vaccine, has some advantages for JEV vaccination programs, including its efficiency, strong immunogenicity, and low cost to developing countries. It is administered by s.c. injection (9). In this study, we compared the effectiveness of s.l. and i.m. administration of a JEV live-attenuated vaccine (SA-14-14-2) and a recombinant MVA vaccine including JEV prM/E (vJH6).

MATERIALS AND METHODS Animals Four-week-old C57BL/6 male mice (Dae Han Bio Link, Chungbuk, Korea) were housed in a controlled environment (inverted 12-week daylight cycle) with free access to food and water. The animal studies were conducted in accordance with the Institutional Animal Care and Use Committee of the Sungsim Campus of the Catholic University of Korea. Immunization and challenge The mice were allocated to the following seven groups (n ¼ 6/group): (i) control group (i.m.), negative control mice treated with PBS alone; (ii) CT group, mice were given 0.5 mg/kg of CT (Sigma, St. Louis, MO, USA) s.l.; (iii) PBS þ JEV group, mice were given PBS i.m. injection at 0, 2 and 11 weeks, then challenged with © 2016 The Societies and John Wiley & Sons Australia, Ltd

JEV one week later; (iv) SA14-14-2/CTþJEV (s.l.) group, mice were given SA14-14-2/CT s.l. at injection at 0, 2 and 11 weeks, then challenged with JEV one week later; (v) SA14-14-2þJEV (i.m.) group, mice were given SA14-142 i.m injection at 0, 2 and 11 weeks, then challenged with JEV one week later; (vi) MVA-vJH6/CTþJEV group (s.l.), mice were given MVA-vJH6/CT s.l injection at 0, 2 and 11 weeks, then challenged with JEV one week later; and (vii) MVA-vJH6þJEV group (i.m.), mice were given MVA-vJH6 i.m. injection at 0, 2 and 11 weeks, then challenged with JEV one week later (Fig. 1). For s.l. injection, 2.55 105 pfu/100 mL of SA14-14-2 and 1.0 106 pfu/100 mL of MVA-vJH6 were used (Table 1). Mice were challenged intraperitoneally with 1 107 pfu/ mouse of JE-Beijing-1 virus. ELISA The national reference standard for JEV vaccines, which originates from inactivated infected mouse brain, was applied in a coating buffer (0.05 M carbonate–bicarbonate buffer) to ELISA microplates (high binding; Greiner, Frickenhausen, Germany). The microplates were then incubated overnight at 4°C. To minimize nonspecific binding, the microplates were then blocked with 1% BSA in 0.1% Tween 20 in PBS-T at room temperature for 1 hr. The serum samples were then diluted 1:100 in PBS-T and the microplates incubated with these samples at 37°C for 1 hr. After incubation, the microplates were washed three times with PBS-T and incubated with HRPconjugated goat anti-mouse in the dark for 1 hr at 37°C followed by incubation with 3,30 ,5,50 -tetramethylbenzidine substrate (BD Biosciences, San Diego, CA, USA). After 15 min, the reaction was stopped with stop solution (2N sulfuric acid). Absorbance was measured at 450 nm with a microtiter plate reader within 30 min of addition of the stop solution. The IgG subclasses of JEV-specific antibodies were quantitated using the same protocol but replacing the detection antibody with either biotinylated rat anti-mouse IgG1 (Santa Cruz Biotechnology, Bergheim, Germany) or rat anti-mouse IgG2a (Santa Cruz Biotechnology) followed by avidin-conjugated HRP (BD Pharmingen, San Diego, CA, USA). Flow cytometry analysis Splenocytes were harvested from the immunized mice 1 week after the last immunization and incubated with JEV E peptides (a.a. 60–68 and 436–445) and recombinant human IL-2 (BioLegend, San Diego, CA, USA) in Iscove's Modified Dulbecco's Media containing 10% FBS and Brefeldin A (eBioscience, San Diego, CA, USA). Following incubation, the cells were blocked with anti-mouse CD16/32 (BD Pharmingen), surface stained 847

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Fig. 1. Schedule of vaccination and challenge. Six 4-week-old female Balb/c mice per group were used to evaluate immunogenicity and protective efficacy of the vaccines. Mice were s.l. or i.m. inoculated with 2.55 105 pfu/mouse of vaccine three times on weeks 0, 2 and 11 under anesthesia induced by by i.p. injection of Zoletil 30 mg/kg and Rompun 10 mg/kg. One week after the final immunization, sera were harvested from the facial veins of the mice for analysis of antibody responses by ELISA and neutralization assays, after which the mice were challenged with JE-Beijing-1 virus (1 107 pfu/mouse).

with anti-mouse CD4 PE/Cy7 and anti-mouse CD8 APC/Cy7 (BioLegend), and fixed in BD FACS lysing solution (BD Pharmingen). For intracellular staining, the fixed cell suspensions were permeabilized in FACS buffer (0.5% FBS, 0.09% NaN3 in PBS) containing 0.5% saponin and stained with anti-mouse IFN-g PE, antimouse IL-17 PerCP/Cy5.5 and anti-mouse granzyme B Alexa Fluor 647 (BioLegend). The cells were assessed using a BD LSRFortessa (BD Biosciences, San Diego, CA, USA) and the data analyzed using FlowJo software (TreeStar, Ashland, OR, USA). RT–qPCR analysis RNA was extracted from mouse spleen tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and used as the template for cDNA synthesis using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). RT–qPCR analysis was performed using an iCycler MyiQ Single Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex Taq II (Takara, Shiga, Japan) and normalized to 18S mRNA. The strength of

Table 1. Neutralizing antibody titers assessed using PRNT 1 week

expression of the proinflammatory cytokines TNF-a and RANTES was assessed by RT-PCR. The real-time PCR primers used were: TNF-a forward primer, 50 -CTG TAG CCC ACG TCG TAG C-30 and reverse primer, 50 -TTG AGA TCC ATG CCG TTG-30 ; RANTES forward primer, 50 -AGC AGC AAG TGC TCC AAT C-30 and reverse primer, 50 -GGG AAG CGT ATA CAG GGT C-30 . Plaque reduction neutralization test Sera from immunized mice were collected and equal volumes from each mouse (100 mL/mouse) pooled, aliquoted into sterile microtubes, and stored at 70°C until analysis. Neutralizing antibody titers were measured with a 50% plaque-reduction end-point assay, as reported previously (15). Potency was determined by using PRNT in a monolayer of BHK-21 cells to quantitate JEV neutralizing antibody responses in a group of immunized mice. Various dilutions of immune serum ranging from 1:10 to 1:320 were made in Eagle's Minimum Essential Medium containing 5% FBS. The diluted sera were mixed with the challenge virus (100 pfu) and incubated for 90 min at 36 1°C. The incubated samples were added to the BHK-21 cells and the cells covered twice with agarose medium. After 1–2 days, the plaque numbers were counted and the 50% reduction in potency calculated.

after the third immunization prior to JEV challenge Immunizing virus

Route of immunization

Immunization

Neutralizing antibody

PBS Cholera toxin SA-14-14-2

Intramuscular Sublingual Sublingual Intramuscular Sublingual Intramuscular

NA NA 2.55 105 pfu 2.55 105 pfu 1.0 106 pfu 1.0 106 pfu

NA NA 160 320 160 320

MVA

Mice were inoculated with the same dose on each occasion. Sera were pooled from six mice per group. Virus-specific neutralizing antibody titers were determined by 50% endpoint PRNT (PRNT50) NA, not applicable.

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Histology Mouse spleen tissues were fixed in 10% neutral formalin. After fixation, the samples were embedded in paraffin and stained with hematoxylin and eosin or periodic acid Schiff. Statistical analysis All analyses were performed using GraphPad Prism (version 5.01; GraphPad Software, La Jolla, CA, USA). All values were calculated as mean SD. The values were compared using a t-test or one-way anova followed by a © 2016 The Societies and John Wiley & Sons Australia, Ltd


post-hoc Tukey test for multiple comparisons. Differences were considered significant at P < 0.05.

RESULTS Effect of immunization with MVA-vJH6 and SA14-14-2 through by s.l. and i.m. routes The humoral immune responses of the mice immunized with MVA-vJH6 and SA14-14-2 were quantitated by ELISA. Four-week-old Balb/c mice were immunized by s.l. or i.m. and boosted twice with the same vaccine. Sera collected before challenge and 1 week after the third vaccination were assayed. The immunization schedule is shown in Figure 1. After the third immunization, serum samples were collected and pooled to measure JEV-specific IgG and neutralizing antibody titers (Fig. 2, Table 1). All immunized mice had significant increases in total IgG, IgG1 and IgG2a after the third immunization. SA14-14-2 vaccination via both the s.l. and i.m. routes induced both IgG1 and IgG2a JEV-specific antibody responses, IgG2a titers being 1.5-fold higher than IgG1 titers (Fig. 2), indicating good induction of a Th1 cell response (16). By contrast,

MVA-vJH6 immunization s.l. and i.m. generated titers of IgG1 that were approximately twice those of IgG2a, indicating that the Th2 cell response was stronger (Fig. 2) (16). However, both vaccine types administered via s.l. and i.m. routes induced neutralizing antibodies (1:160–1:320) (Table 1). These findings suggest that both s.l. and i.m. routes induce equivalent titers of JEV-specific IgG isotypes and neutralizing antibodies. Protective immunity after JEV challenge Induction of proinflammatory cytokines by JEV challenge was investigated by quantifying the mRNAs for cytokines using RT-qPCR. There were significantly greater amounts of mRNA for RANTES and TNF-a in unvaccinated mice challenged with JEV than in the vaccine groups (Fig. 3). As shown in Figure 3, MVA-vJH6 and SA14-14-2 immunization by both i.m. and s.l. routes significantly decreased induction of expression of both TNF-a and RANTES after JEV challenge. The spleen is responsible for storage and production of red blood cells and is composed of red pulp and white pulp. The white pulp produces and develops both immune and blood cells, whereas the red pulp is responsible for

Fig. 2. IgG, IgG1 and IgG2a titers in mouse serum 1 week after the third immunization. JEV-specific IgGs were measured by ELISA. Sera from six mice per group were pooled and measured in triplicate. PBS represents the negative control for the i.m. route, and CT the negative control for the s.l. route. P values were obtained using anova. Different letters (a, b, c, and d) on the bars indicated statistically significant differences.


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Fig. 3. RT-qPCR analysis of proinflammatory cytokines in mouse spleens 1 week after challenge with JEV. Amounts of TNF-a and RANTES mRNA were measured. The value of the PBS group was normalized to 1 and relative values for the other groups determined accordingly. The spleens from six mice per group were measured individually. P values were obtained using anova. Different letters (a, b, c, and d) on the bars indicated statistically significant differences.

purifying the blood and removing dead or old blood cells. Because the spleen serves a critical role in immune function by purifying the blood and helping the immune system to recognize and attack foreign pathogens and allergens (10), the effects of vaccination on the histology of mouse spleen

after JEV challenge were investigated. Both vaccines, administered by either s.l. or i.m. routes, protected against focal hyperplasia of the white pulp induced by JEV challenge (Fig. 4). All the above data indicate that the s.l. route is as effective as the i.m. route for vaccine efficacy.

Fig. 4. (a) Histopathologic findings in the spleens of mice 1 week after challenge with JE-Beijing-1 virus. All images are at 20 magnification. (b) Comparison of the size of the white pulp (WP), which comprises lymphoid tissue and represents sites of inflammation. Spleens from six mice per group were analyzed separately. P values were obtained using anova. Different letters (a, b, ac, and d) on the bars indicated statistically significant differences.

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Fig. 5. JEV-specific CD8+ T cell responses. Splenocytes were harvested from the immunized mice 1 week after the third immunization prior to JEV challenge and stimulated with JEV E peptide (a.a. 60–68). JEV-specific CD8þ T cells (JEV E peptide 60–68þ) were measured by flow cytometry. (a) Representative and (b) quantitative flow cytometric data for JEV-specific CD8þ IFN-gþ and granzyme Bþ T cells. The splenocytes from six mice per group were measured individually. P values were obtained using anova. Different letters (a, b, and ab) on the bars indicated statistically significant differences.

Assessment of CD4þ and CD8þ T cell responses to MVA-vJH6 and SA14-14-2 administered s.l. and i.m. To assess the efficiency of both vaccines in inducing immune responses against JEV, the activities of immune cells, in particular CD4þ and CD8þ T cells, were tested. JEV-specific CD8þ T cells were assessed using intracellular cytokine staining and flow cytometric analysis. Immunization with SA14-14-2 and MVA-vJH6 s.l. and i.m. appeared to increase numbers of JEV-specific CD8þ IFN-gþ T cells, but not of CD8þ granzyme Bþ T cells (Fig. 5). In addition, IFN-gþ CD4þ T cell numbers appeared to increase for both immunization routes and vaccine types; however, this increase was only significant after vaccination with recombinant MVA-vJH6 via the i.m. route (Fig. 6). It is notable that the increase in IFN-gþ T cell numbers © 2016 The Societies and John Wiley & Sons Australia, Ltd

after MVA-vJH6 vaccination (Figs. 5b and 6b) was not matched by increases in the appropriate IgG isotype in serum (Fig. 2d). This apparent discrepancy may be attributable to differences in the detection methods used. However, these data do demonstrate that both SA-14-14-2 and recombinant MVA can induce immune responses that include both Th1 and Th2 cells. Interestingly, IL-17þ CD4þ T cell numbers, which were measured using JEV E peptide (a.a. 436–445), were increased in the mice immunized with both vaccines via both s.l. and i.m. routes, but not in the CT-only immunized mice (Fig. 6).

DISCUSSION Vaccination is the most powerful tool for fighting virus-induced diseases. However, some obstacles have 851

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Fig. 6. JEV-specific CD4+ T cell response. Splenocytes were harvested from the immunized mice 1 week after the third immunization prior to JEV challenge and stimulated with JEV E peptide (a.a. 436–445). JEV-specific CD4þ T cells (JEV E peptide 436–445þ) were measured by flow cytometry. (a) Representative and (b) quantitative flow cytometric data for JEV-specific CD4þ IFN-gþ and IL-17þ T cells. The splenocytes from six mice per group were measured individually. P values were obtained using anova. Different letters (a, b, and ab) on the bars indicated statistically significant differences.

prevented the wide use of vaccines in both developed and underdeveloped countries; namely, various adverse immunological reactions, the high cost of production and fear of needles (11). S.l. administration is a simple means of bypassing “needle phobia” or “fear of shots” (12). In this study, we compared the efficacy of s.l. versus i.m. routes for administering two types of JEV vaccine: a live-attenuated vaccine (SA14-14-2) that is already commercially available and recombinant MVA (vJH6), a chimeric virus vector vaccine. We found that immunization of mice with MVAvJH6 and SA14-14-2 via s.l. versus i.m. routes induced similar titers of JEV-specific IgG. Interestingly, we found that mice that received MVA-vJH6 via either i.m. or s.l. routes achieved higher titers of IgG1 (Th2) than of IgG2a (Th1), which suggests a Th2-driven (humoral) immune response. In contrast, SA14-14-2 immunization via either i.m. or s.l. routes induced higher tiers of IgG2a 852

(Th1) than of IgG1 (Th2), which suggests a Th1-driven (cellular) immune response. However, the differences between Th1 and Th2 responses for both vaccine types were not significant. Moreover, total JEV-specific IgG titers were similar regardless of whether i.m. and s.l. routes were used, whereas titers of neutralizing antibodies in mice immunized via the i.m. route were twice those of mice immunized via the s.l. route for both SA14-14-2 and MVA-vJH6 vaccines. However, a twofold increase is an insignificant difference in neutralizing antibody titer and a titer of 1:160 was sufficient to protect against JEV challenge. Thus, immunization via both s.l. and i.m. routes induced similar titers of neutralizing antibody. In addition, immunization through both routes completely protected against JEV challenge, this being confirmed by reduced induction of proinflammatory cytokines and protection against focal white pulp hyperplasia. All our data indicate that s.l. administration © 2016 The Societies and John Wiley & Sons Australia, Ltd


is as effective as i.m. injection, at least for the two vaccines used in this study. Both the SA14-14-2 and MVA-vJH6 vaccines given i.m. or s.l. tended to increase numbers of virus-specific IFN-gþ CD4þ and CD8þ T cells. This indicates that vaccination induced and maintained memory T cells, because IFN-g, the signature cytokine of Th1 type immune responses, is produced by stimulated T cells and has important immunomodulatory effects (13, 17). Interestingly, IL17þ CD8þ T cell numbers were increased only by s.l. administration. The cytokine IL-17 has been implicated in regulation of inflammation and autoimmune diseases (14). It is notable that our recent data shows that mice immunized with ovalbumin in the absence of CT adjuvant produce lower antibody titers than those immunized in the presence of CT adjuvant (data not shown). Thus, s.l. immunization requires mixing of CT with the vaccine to trigger an appropriate immune response. The mucosal adjuvant activity of CT involves induction of IL-1 and other proinflammatory cytokines (6, 14). Thus, we suspect that CT may nonspecifically trigger the induction of IL-17; however, administration of CT alone did not induce IL-17. These results suggested that both MVA-vJH6 and SA14-14-2 can induce equivalent humoral and cellular immune responses via s.l. and i.m. routes, indicating that s.l. administration vaccine is a very strong candidate for clinical use in the field.

ACKNOWLEDGEMENTS This research was supported by the Korean Healthcare Technology Research and Development Project of the Ministry of Health & Welfare (HI13C0826), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, Information and Communications Technology and Future Planning (NRF-2015M3A9 B5030157), and by a grant from the KHIDI funded by the Ministry of Health & Welfare, Korea (HI15C2955) and KFDA (16172INFECTION268).

DISCLOSURE The authors declare that they have no conflicting interests associated with this study.


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