Lethal Japanese Encephalitis Virus Challenge Research Articles

Japanese Encephalitis DNA Vaccines with Epitope Modification Reduce the Induction of Cross-Reactive Antibodies against Dengue Virus and Antibody-Dependent Enhancement of Dengue Virus Infection

Infection with viruses belonging to the genus Flavivirus, such as Japanese encephalitis virus (JEV) and dengue virus (DENV), is a worldwide health problem. Vaccines against JEV and DENV are currently available. However, the dengue vaccine possibly increases the risk of severe dengue due to antibody-dependent enhancement (ADE). Moreover, the Japanese encephalitis (JE) vaccine reportedly induces cross-reactive ADE-prone antibodies against DENV, potentially leading to symptomatic dengue. Therefore, it is necessary to eliminate the risk of ADE through vaccination. In this study, we attempted to develop a JE vaccine that does not induce ADE of DENV infection using an epitope modification strategy. We found that an ADE-prone monoclonal antibody cross-reactive to DENV and JEV recognizes the 106th amino acid residue of the E protein of JEV (E-106). The JE DNA vaccine with a mutation at E-106 (E-106 vaccine) induced comparable neutralizing antibody titers against JEV to those induced by the wild-type JE DNA vaccine. Meanwhile, the E-106 vaccine induced 64-fold less cross-reactive ADE-prone antibodies against DENV. The mutation did not compromise the protective efficacy of the vaccine in the lethal JEV challenge experiment. Altogether, the modification of a single amino acid residue identified in this study helped in the development of an ADE-free JE vaccine.

Re-burying Artificially Exposed Surface of Viral Subunit Vaccines Through Oligomerization Enhances Vaccine Efficacy.

Viral subunit vaccines often suffer low efficacy. We recently showed that when taken out of the context of whole virus particles, recombinant subunit vaccines contain artificially exposed surface regions that are non-neutralizing and reduce their efficacy, and thus these regions need to be re-buried in vaccine design. Here we used the envelope protein domain III (EDIII) of Japanese encephalitis virus (JEV), a subunit vaccine candidate, to further validate this important concept for subunit vaccine designs. We constructed monomeric EDIII, dimeric EDIII via a linear space, dimeric EDIII via an Fc tag, and trimeric EDIII via a foldon tag. Compared to monomeric EDIII or linearly linked dimeric EDIII, tightly packed EDIII oligomers via the Fc or foldon tag induce higher neutralizing antibody titers in mice and also protect mice more effectively from lethal JEV challenge. Structural analyses demonstrate that part of the artificially exposed surface areas on recombinant EDIII becomes re-buried in Fc or foldon-mediated oligomers. This study further establishes the artificially exposed surfaces as an intrinsic limitation of subunit vaccines, and suggests that re-burying these surfaces through tightly packed oligomerization is a convenient and effective approach to overcome this limitation.

A Novel Recombinant Virus-Like Particles Displaying B and T Cell Epitopes of Japanese Encephalitis Virus Offers Protective Immunity in Mice and Guinea Pigs.

Virus-like particles (VLPs) are non-replicative vectors for the delivery of heterologous epitopes and are considered one of the most potent inducers of cellular and humoral immune responses in mice and guinea pigs. In the present study, VLP-JEVe was constructed by the insertion of six Japanese encephalitis virus (JEV) envelope protein epitopes into different surface loop regions of PPV VP2 by the substitution of specific amino acid sequences without altering the assembly of the virus; subsequently, the protective efficacy of this VLP-JEVe was evaluated against JEV challenge in mice and guinea pigs. Mice immunized with the VLP-JEVe antigen developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. The neutralizing and hemagglutination inhibition (HI) antibody responses were also induced in guinea pigs vaccinated with VLP-JEVe. In addition, immunization with VLP-JEVe in mice induced effective neutralizing antibodies and protective immunity against PPV (porcine parvovirus) challenge in guinea pigs. These studies suggest that VLP-JEVe produced as described here could be a potential candidate for vaccine development.

Novel Japanese encephalitis virus NS1-based vaccine: Truncated NS1 fused with E. coli heat labile enterotoxin B subunit.

BackgroundCurrent vaccines against Japanese encephalitis virus (JEV) of flaviviruses have some disadvantages, such as the risk of virulent reversion. Non-structural protein NS1 is conserved among flaviviruses and confers immune protection without the risk of antibody-dependent enhancement (ADE). Therefore, NS1 has become a promising vaccine candidate against flaviviruses.MethodsA NS1-based vaccine (LTB-NS1∆63) with a truncated NS1 protein (NS1∆63) fused to E. coli heat-labile enterotoxin B subunit (LTB) was expressed in E.coli and explored for its ability to induce immune responses. Safety of LTB-NS1∆63 was assessed by determining its toxicity in vitro and in vivo. Protective capability of LTB-NS1∆63 and its-induced antisera was evaluated in the mice challenged with JEV by analyzing mortality and morbidity.FindingsLTB-NS1∆63 induced immune responses to a similar level as LTB-NS1, but more robust than NS1∆63 alone, particularly in the context of oral immunization of mice. Oral vaccination of LTB-NS1∆63 led to a higher survival rate than that of NS1∆63 or live-attenuated JEV vaccine SA14–14–2 in the mice receiving lethal JEV challenge. LTB-NS1∆63 protein also significantly decreases the morbidity of JEV-infected mice. In addition, passive transfer of LTB-NS1∆63-induced antisera provides a protection against JEV infection in mice.InterpretationNS1∆63 bears JEV NS1 antigenicity. Besides, LTB-NS1∆63 could serve as a novel protein-based mucosa vaccine targeting JEV and other flaviviruses.FundingThis work was supported by the National Natural Science Foundation, Jiangxi Province Science and Technology Committee, Education Department of Jiangxi Province.

Immune responses and protective effects against Japanese encephalitis induced by a DNA vaccine encoding the prM/E proteins of the attenuated SA14-14-2 strain

Japanese encephalitis virus (JEV) is the causal pathogen of Japanese encephalitis (JE), which has become a severe public health problem and is one of the most rapidly spreading mosquito-borne diseases worldwide. Currently, there is no specific treatment for JEV. A vaccine would be an effective measure for reducing morbidity and mortality. Although the live attenuated vaccine SA14-14-2 has been approved in some countries, it is still necessary to develop safer, more effective, and less costly vaccines. In this study, a DNA vaccine candidate, pV-SA14ME, expressing the prM/E proteins of SA14-14-2 was inoculated into BALB/c mice via intramuscular electroporation, and the immunogenicity and degree of protection were evaluated. We found that administration of 50 μg pV-SA14ME via electroporation via three immunizations could induce persistent humoral and cellular immune responses and effectively protect mice against lethal JEV challenge. This study provides a basis for the subsequent promotion and use of the vaccine and lays the foundation for its further use in swine and humans.

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BackgroundJapanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine.ResultsWe generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15–20 μg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4–6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge.ConclusionThese results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Antibodies generated by immunization with the NS1 protein of West Nile virus confer partial protection against lethal Japanese encephalitis virus challenge

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are two medically important flaviviruses that can cause severe hemorrhagic and encephalitic diseases in humans. Immune responses directed against the NS1 protein of flaviviruses can confer protection against lethal viral challenge. Previous studies have shown that the WNV NS1 protein harbors epitopes that elicit antibodies that cross react with JEV. Here we demonstrate that the WNV NS1 protein not only contains cross-reactive epitopes, but that the antibodies elicited by these cross-reactive epitopes provide partial protection against lethal JEV challenge in a mouse model. Mice immunized with WNV NS1 protein showed reduced morbidity and mortality following both intracerebral and intraperitoneal JEV challenge. WNV NS1 immunization attenuated the extent of lung pathology generated following JEV challenge, and delayed the appearance of other pathological findings including vascular cuffing. By screening and identifying the specific WNV NS1 protein-derived peptides recognized by serum antibodies elicited by immunization with WNV NS1 protein and by JEV challenge, we found after JEV challenge will induce several new epitopes, but which epitope primarily contribute to antibody-mediated cross protection need further evaluation. The knowledge and reagents generated in this study have potential applications in vaccine and subunit vaccine development for WNV and JEV.

Inhibition of japanese encephalitis virus infection by flavivirus recombinant e protein domain III

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.

Multiple linear epitopes (B-cell, CTL and Th) of JEV expressed in recombinant MVA as multiple epitope vaccine induces a protective immune response

Epitope-based vaccination might play an important role in the protective immunity against Japanese encephalitis virus (JEV) infection. The purpose of the study is to evaluate the immune characteristics of recombinant MVA carrying multi-epitope gene of JEV (rMVA-mep). The synthetic gene containing critical epitopes (B-cell, CTL and Th) of JEV was cloned into the eukaryotic expression vector pGEM-K1L, and the rMVA-mep was prepared. BALB/c mice were immunized with different dosages of purified rMVA-mep and the immune responses were determined in the form of protective response against JEV, antibodies titers (IgG1 and IgG2a), spleen cell lymphocyte proliferation, and the levels of interferon-γ and interleukin-4 cytokines. The results showed that live rMVA-mep elicited strongly immune responses in dose-dependent manner, and the highest level of immune responses was observed from the groups immunized with 107 TCID50 rMVA-mep among the experimental three concentrations. There were almost no difference of cytokines and neutralizing antibody titers among 107 TCID50 rMVA-mep, recombinant ED3 and inactivated JEV vaccine. It was noteworthy that rMVA-mep vaccination potentiates the Th1 and Th2-type immune responses in dose-dependent manner, and was sufficient to protect the mice survival against lethal JEV challenge. These findings demonstrated that rMVA-mep can produce adequate humoral and cellular immune responses, and protection in mice, which suggested that rMVA-mep might be an attractive candidate vaccine for preventing JEV infection.

Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections

A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292–402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

Antiviral Activity ofIsatis indigoticaExtract and Its Derived Indirubin against Japanese Encephalitis Virus

Isatis indigotica is widely used in Chinese Traditional Medicine for clinical treatment of virus infection, tumor, and inflammation, yet its antiviral activities remain unclear. This study probed antiviral activity of I. indigotica extract and its marker compounds against Japanese encephalitis virus (JEV). I. indigotica methanol extract, indigo, and indirubin proved less cytotoxic than other components, showing inhibitory effect (concentration-dependent) on JEV replication in vitro. Time-of-addition experiments proved the extract, indigo, and indirubin with potent antiviral effect by pretreatment (before infection) or simultaneous treatment (during infection), but not posttreatment (after entry). Antiviral action of these agents showed correlation with blocking virus attachment and exhibited potent virucidal activity. In particular, indirubin had strong protective ability in a mouse model with lethal JEV challenge. The study could yield anti-JEV agents.

SiRNA-mediated suppression of Japanese encephalitis virus replication in cultured cells and mice

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South-East Asia and there is a pressing need to develop novel therapeutic options against it. Gene silencing by RNA interference has therapeutic potential by way of degrading the RNA genome of JEV. Four small hairpin RNAs (shRNAs) targeting different locations in the JEV genome were evaluated for antiviral activity against JEV in different cell lines and the mouse model of disease. shN8010, an shRNA targeting the NS5-coding sequence of JEV, had significant antiviral activity in cultured cells. JEV titres were suppressed by 99% in human embryonic kidney cells at 24 h post-infection (p.i.) when shN8010 was delivered using a plasmid. Further, shN8010 delivered using recombinant adenovirus caused 99% and 95% suppression of JEV titres at 24 h p.i. in porcine stable kidney and Vero cells, respectively. In Neuro-2a cells, JEV titres at 24 h p.i. were suppressed by 90% when shN8010 was delivered using a recombinant adenovirus or retrovirus. Four-week-old FvB/J mice treated intracerebrally with recombinant adenovirus-delivered shN8010 1 week prior to lethal intraperitoneal JEV challenge showed no protection, although the mean survival time was prolonged. In a similar experiment, retrovirus-delivered shN8010 provided 100% protection to mice following the lethal JEV challenge. NS5-targeting shRNA (shN8010) had very significant antiviral activity in both cultured cells and the mouse model of JEV infection.

Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO ®) induced neutralizing antibody titers

Immune sera from volunteers vaccinated in a blinded Phase 3 clinical trial with JE-VAX ® and a new Japanese encephalitis virus (JEV) vaccine (IC51 or IXIARO), were tested for the ability to protect mice against lethal JEV challenge. Sera from IXIARO vaccinated subjects were pooled into four batches based on neutralizing antibody measured by plaque reduction neutralization test (PRNT 50 titer): high (∼200), medium (∼40–50), low (∼20) and negative (<10). Pooled sera from JE-VAX ® vaccinated subjects (PRNT 50 titer ∼ 55) and pooled JEV antibody negative pre-vaccination sera were used as controls. Groups of ten 6- to 7-week-old female ICR mice were injected intraperitoneally with 0.5 ml of each serum pool diluted 1:2 or 1:10, challenged approximately 18 h later with a lethal dose of either JEV strain SA14 (genotype III) or strain KE-093 (genotype I) and observed for 21 days. All mice in the non-immune serum groups developed clinical signs consistent with JEV infection or died, whereas high titer sera from both IXIARO and JE-VAX ® sera protected 90–100% of the animals. Statistical tests showed similar protection against both JEV strains SA14 and KE-093 and protection correlated with the anti-JEV antibody titer of IXIARO sera as measured by PRNT 50. Ex vivo neutralizing antibody titers showed that almost all mice with a titer of 10 or greater were fully protected. In a separate study, analysis of geometric mean titers (GMTs) of the groups of mice vaccinated with different doses of IXIARO and challenged with JEV SA14 provided additional evidence that titers ≥ 10 were protective.

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.

Immunogenicity and protective efficacy of the E. coli-expressed domain III of Japanese encephalitis virus envelope protein in mice

Domain III of Japanese encephalitis virus (JEV) envelope protein (E-DIII) was synthesized in E. coli as a fusion protein containing maltose-binding protein (MBP-E-DIII) or six contiguous histidine residues (His-E-DIII) at its N-terminus. MBP-E-DIII was found both in the soluble as well as the insoluble fraction of the bacterial lysate, while His-E-DIII was found exclusively in the inclusion bodies. These purified proteins were examined in mice for their immunogenicity in presence of an aluminium hydroxide based-adjuvant Alhydrogel and Freund's adjuvant. While both proteins generated anti-JEV antibodies that neutralized JEV activity in vitro, His-E-DIII generated higher antibody titers than MBP-E-DIII. Mice immunized with His-E-DIII in presence of Alhydrogel generated antibody titers similar to those induced by the commercial vaccine and protected mice against lethal JEV challenge.

Detection of enteric adenovirus in childhood gastroenteritis with real-time PCR

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-γ and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.

Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-γ and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.

Effect of Cytokine‐Encoding Plasmid Delivery on Immune Response to Japanese Encephalitis Virus DNA Vaccine in Mice

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti-JEV humoral and cellular immune responses. We now show that intra-muscular co-administration of mice with pMEa and pGM-CSF, encoding murine granulocyte-macrophage colony-stimulating factor or pIL-2, encoding murine interleukin-2 given 4 days after pMEa, augmented anti-JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra-dermal co-administration of pMEa and pGM-CSF in mice using the gene gun, enhanced anti-JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-γ and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.

Immunogenicity in mice of a cationic microparticle-adsorbed plasmid DNA encoding Japanese encephalitis virus envelope protein

Previously, we described a plasmid pMEa, synthesizing the anchored form of Japanese encephalitis virus (JEV) envelope protein that generated virus-neutralizing antibodies in mice upon intra-muscular injection. The immunized mice showed significant protection against lethal JEV challenge. In order to improve the efficacy of plasmid DNA immunization against JEV, methods need to be employed that would enhance neutralizing antibody titers. Plasmid DNA adsorbed to cationic microparticles has recently been shown to significantly improve its immunogenicity. In the present study, we have adsorbed the plasmid pMEa on cationic microparticles and have compared its immunogenicity with the naked plasmid DNA. As seen in ELISA, the microparticle-adsorbed DNA induced higher titers of anti-JEV antibodies when compared to those induced by the naked DNA. No difference, however, was seen in JEV neutralization titers. The microparticle-adsorbed DNA induced a mixed Th1–Th2 kind of immune responses as opposed to Th1 type of immune responses elicited by the naked DNA.