Japanese Encephalitis Virus Vaccine Research Articles

A Monoclonal Antibody Specific for Japanese Encephalitis Virus with High Neutralizing Capability for Inclusion as a Positive Control in Diagnostic Neutralization Tests.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 μg/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV.

Antibody Persistence up to 3 Years After Primary Immunization With Inactivated Japanese Encephalitis Vaccine IXIARO in Philippine Children and Effect of a Booster Dose.

An inactivated Vero cell culture derived Japanese encephalitis virus vaccine (IXIARO) requires a booster dose 1 year after primary schedule for long-term antibody persistence in adults. The aim of this study is to evaluate immunogenicity and safety of a booster dose in children 2 months to <18 years of age. This is a randomized, controlled open-label study in the Philippines. Three hundred children vaccinated with IXIARO in a previous trial were randomized 1:1 to receive either no booster or a booster 12 months after initiation of the primary series. Neutralizing antibody titers were assessed before and after the booster and up to 3 years after primary series. Safety endpoints included the rate of subjects with solicited adverse events (AEs), unsolicited AEs and serious AEs within 1 month after the booster. Geometric mean titer declined by 1 year after the primary series, but titers remained above the established protective threshold in 85%-100% of children depending on age group. The booster led to a pronounced increase in geometric mean titer and 100% seroprotection rate in all age groups. The booster was well tolerated, with AE rates lower compared with the primary series. Most AEs were mild. A booster dose of IXIARO administered 12 months after the primary immunization was well tolerated and highly immunogenic.

Development of a Japanese encephalitis virus genotype V virus-like particle vaccine in silkworms.

To counter the spread of multiple Japanese encephalitis virus (JEV) variants harboured in alternative host species and highly neurotoxic variants with new antigenicity, such as genotype V (Muar), methods for developing more effective and low-cost vaccines against a variety of epidemic JEV strains are required. Here, we successfully synthesized large amounts of a Muar virus-like particle (MVLP) vaccine for JEV in silkworm pupae by using a Bombyx mori nuclear polyhedrosis virus recombinant consisting of JEV codon-optimized envelope (E) DNA. In particular, histopathological examination suggested that MVLP was efficiently synthesized in body fat tissues as well as epithelial cells. Quantitative analysis indicated that one silkworm pupa produced 724.8 µg of E protein in the MVLP vaccine. Electron microscopic examination of purified MVLP vaccine defined a typical MVLP morphological structure. Detailed MVLP antigen assessment by immune-electron microscopy revealed that the majority of MVLPs were covered with approximately 10 nm projections. Boosted immunization with MVLP antigens in mice and rabbits tended to show improved plaque inhibition potency against homologous Muar and heterologous Nakayama, but less potency to Beijing-1 strains. Notably, mixed immune rabbit antisera against Nakayama and Muar VLP antigens led to an increase in the low antibody reaction to Beijing-1. Additionally, a stopgap divalent JEV vaccine consisting of MVLP and Nakayama VLP and its immune mouse serum significantly increased plaque inhibition titre against Muar, Nakayama and Beijing-1 strains. These findings suggested that low-cost MVLP vaccines prepared in silkworm pupae are suitable for providing simultaneous protection of individuals in developing countries against various JEV strains.

The Functions of Bursal Hexapeptide (BHP) on Immune Response and the Molecular Mechanism on Immature B Cell.

The bursa of Fabricius (BF) is an acknowledged central immune organ, and is important to B cell differentiation. Bursal hexapeptide (BHP) is the recently reported bursalderived peptide, while its inducing function on immune response is uncertain. The main objective of this study was to analyze the immune responses to JEV vaccine in mice induced by BHP plus JEV vaccine, and to detect the signal and biological functions of BHP on immature B cells. Mice were immunized with Japanese encephalitis virus (JEV) vaccine and BHP from 0.01 mg/mL to 0.25 mg/mL to detect antibody response and cellular immune response, respectively. The production of IgG, IgG1 and IgG2a specific to JEV in serum from immunized mice were measured by ELISA, and T cell subpopulation from immunized mice were detected with using fluorochrome conjugated mAbs of the corresponding PE-Cys/FITC/PE by flow cytometry. Spleen cells from all immunized mice were harvested after one week of second immunization for lymphocyte proliferation assay. Mouse immature B cell WEHI-231 cell was treated with 0.01μg/mL BHP for 4h, and analyzed the involved biological function and pathway of differentially expressed genes with gene microarray. BHP co-immunization with JEV vaccine generated significant increased antibody levels, neutralizing antibody titers and spleen lymphocyte viability, compared to that of vaccine control. The subpopulations of T cells in spleen lymphocytes were significantly modified in the mice coimmunized with JEV vaccine and BHP. The analysis results of gene expression profiles of WEHI- 231 mouse immature B cells with BHP treatment showed that the regulated genes with BHP treatment were involved various immune related biological functions, including proliferation and activation of lymphocyte and T cell, T cell mediated immunity and regulation of adaptive immune response. Furthermore, BHP stimulated three significant enriched pathways, including amphetamine addiction, long-term potentiation, and RIG-I-like receptor signaling pathway. Our results indicated BHP induced significant humoral and cellular immunity to JEV vaccine, and regulated various biological processes and signalling related to immune activation in immature B cells. These results proposed the immunomodulatory function and mechanism of BHP on immune induction, which provided the novel insight on the candidate reagent for immune improvement.

Full-Length Genome Sequence of Japanese Encephalitis Virus Strain FC792, Isolated from Guangxi, China.

ABSTRACTWe report here the complete genomic sequence of Japanese encephalitis virus (JEV) strain FC792, isolated from aborted fetuses of sows which were unimmunized with JEV vaccines in Guangxi Province, southern China. The complete JEV genome of strain FC792 had the highest nucleotide homology (99.7%) and amino acid identity (99.4%) with the sequence of JEV strain SA14-14-2 (GenBank accession number AF315119). Phylogenetic analysis showed that strain FC792 had the closest phylogenetic relationship to the sequence of strain YUNNAN0901 (GenBank accession number JQ086762). This study will help us understand the molecular pathogenesis and genetic diversity of genotype III Japanese encephalitis virus in pigs.

Epidemiological Profile of Acute Viral Encephalitis

To study the etiology and clinico-epidemiological profile of acute viral encephalitis in children with acute encephalitis syndrome (AES). An observational study including 100 patients fulfilling the criteria for AES was conducted in children of age group 1mo - 16y. Viral isolation was done on RD cells, HEp-2 cells and Vero cells from the cerebrospinal fluid samples of suspected viral encephalitis (VE) cases. An enzyme immunoassay for IgM antibodies was performed for measles, mumps, Varicella zoster virus (VZV), Herpes simplex virus 1 (HSV1) and Japanese encephalitis virus (JEV). Multiplex polymerase chain reaction (PCR) was done for Cytomegalovirus, Epstein Barr virus (EBV), HSV1 & 2, VZV, Enterovirus, Parecho virus, Human Herpes virus (HHV 6, 7) and Parvovirus B19. A micro neutralization test was performed for Enterovirus 71. Out of enrolled 100 patients, 73 were of probable viral encephalitis. HSV1 (31.50%) was the commonest virus followed by Adenovirus (10.95%), Parvovirus (2.73%), JE virus (1.36%), Enterovirus (1.36%), EBV (1.36%), and mixed infection with HSV & EBV (1.36%). HSV 1 caused significant morbidity in children. The common computed tomography (CT) findings were hypodensities in the fronto- parietal lobe followed by cerebral edema. The landscape of AES in India has changed in the previous decade, and both outbreak investigations and surveillance studies have increasingly reported non-JEV etiologies; because of these findings there is a need to explore additional strategies to prevent AES beyond vector control and JEV vaccination.

Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice

We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 − 5μg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.

Secreted expression of Japanese encephalitis virus prME in Pichia pastoris and immunogenicity evaluation of the virus-like particles in mice

The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×10⁶ Da to 20×10⁶ Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.

A Japanese Encephalitis Virus Vaccine Inducing Antibodies Strongly Enhancing In Vitro Infection Is Protective in Pigs.

The Japanese encephalitis virus (JEV) is responsible for zoonotic severe viral encephalitis transmitted by Culex mosquitoes. Although birds are reservoirs, pigs play a role as amplifying hosts, and are affected in particular through reproductive failure. Here, we show that a lentiviral JEV vector, expressing JEV prM and E proteins (TRIP/JEV.prME), but not JEV infection induces strong antibody-dependent enhancement (ADE) activities for infection of macrophages. Such antibodies strongly promoted infection via Fc receptors. ADE was found at both neutralizing and non-neutralizing serum dilutions. Nevertheless, in vivo JEV challenge of pigs demonstrated comparable protection induced by the TRIP/JEV.prME vaccine or heterologous JEV infection. Thus, either ADE antibodies cause no harm in the presence of neutralizing antibodies or may even have protective effects in vivo in pigs. Additionally, we found that both pre-infected and vaccinated pigs were not fully protected as low levels of viral RNA were found in lymphoid and nervous system tissue in some animals. Strikingly, the virus from the pre-infection persisted in the tonsils throughout the experiment. Finally, despite the vaccination challenge, viral RNA was detected in the oronasal swabs in all vaccinated pigs. These latter data are relevant when JEV vaccination is employed in pigs.

Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector.

Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice.

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-γ+ CD4+ and CD8+ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus-specific IL-17+ CD4+ 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.

Post-licensure, phase IV, safety study of a live attenuated Japanese encephalitis recombinant vaccine in children in Thailand

BackgroundJapanese encephalitis is a mosquito-borne viral disease endemic in most countries in Asia. A recombinant live, attenuated Japanese encephalitis virus vaccine, JE-CV, is licensed in 14 countries, including Thailand, for the prevention of Japanese encephalitis in adults and children. MethodsThis was a prospective, phase IV, open-label, multicentre, safety study of JE-CV conducted from November 2013 to April 2015, to evaluate rare serious adverse events (AEs). JE-CV was administered to 10,000 healthy children aged 9months to <5years in Thailand as a primary (Group 1) or booster (Group 2) vaccination. Serious AEs (SAEs), including AEs of special interest, up to 60days after administration were evaluated. Immediate Grade 3 systemic AEs up to 30min after JE-CV administration were also described. ResultsThe median age of participants was 1.1years in Group 1 and 3.8years in Group 2. SAEs were reported in 204 (3.0%) participants in Group 1 and 59 (1.9%) participants in Group 2. Among a total of 294 SAEs in 263 participants, only three events occurring in two participants were considered related to vaccination. All three cases were moderate urticaria, none of which met the definition of AEs of special interest for hypersensitivity. AEs of special interest were reported in 28 (0.4%) participants in Group 1 and 4 (0.1%) participants in Group 2; none were considered related to vaccination. Febrile convulsion was the most frequently reported AE of special interest: 25 (0.4%) participants in Group 1; and 2 (<0.1%) in Group 2. There were no cases of Japanese encephalitis reported. No Grade 3 immediate systemic AEs were reported after any JE-CV vaccination. ConclusionsOur study did not identify any new safety concerns with JE-CV and confirms its good safety profile.This study was registered on www.clinicaltrials.gov (NCT01981967; Universal Trial Number: U1111-1127-7052).

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

Cross-protection induced by Japanese encephalitis vaccines against different genotypes of Dengue viruses in mice.

Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) are closely related mosquito-borne flaviviruses that cause very high global disease burdens. Although cross-reactivity and cross-protection within flaviviruses have been demonstrated, the effect of JEV vaccination on susceptibility to DENV infection has not been well elucidated. In this study, we found that vaccination with the JEV inactivated vaccine (INV) and live attenuated vaccine (LAV) could induce cross-immune responses and cross-protection against DENV1-4 in mice. Despite the theoretical risk of immune enhancement, no increased mortality was observed in our mouse model. Additionally, low but consistently detectable cross-neutralizing antibodies against DENV2 and DENV3 were also observed in the sera of JEV vaccine-immunized human donors. The results suggested that both JEV-LAV and JEV-INV could elicit strong cross-immunity and protection against DENVs, indicating that inoculation with JEV vaccines may influence the distribution of DENVs in co-circulated areas and that the cross-protection induced by JEV vaccines against DENVs might provide important information in terms of DENV prevention.

Change in Dengue and Japanese Encephalitis Seroprevalence Rates in Sri Lanka

BackgroundSri Lanka has been affected by epidemics of dengue infections for many decades and the incidence and severity of dengue infections have been rising each year. Therefore, we investigated the age stratified seroprevalence of dengue infections in order to facilitate future dengue vaccine strategies. In addition, since the symptomatic dengue infections have increased during the past few decades, we also investigated the possible association with Japanese Encephalitis Virus (JEV) antibody seropositivity with symptomatic dengue in a community cohort in Sri Lanka.Methods1689 healthy individuals who were attending a primary health care facility were recruited. Dengue and JEV antibody status was determined in all individuals and JEV vaccination status was recorded.Results1152/1689 (68.2%) individuals were seropositive for dengue and only 133/1152 (11.5%) of them had been hospitalized to due to dengue. A significant and positive correlation was observed for dengue antibody seropositivity and age in children (Spearmans R = 0.84, p = 0.002) and in adults (Spearmans R = 0.96, p = 0.004). We observed a significant rise in the age stratified seroprevalence rates in children over a period of 12 years. For instance, in year 2003 the annual seroconversion rate was 1.5% per annum, which had risen to 3.79% per annum by 2014. We also found that both adults (p<0.001) and in children (p = 0.03) who were hospitalized due to dengue were more likely to be seropositive for JEV antibodies. However, 244 (91.4%) of adults who were seropositive for JEV had not had the JEV vaccine.ConclusionsDengue seroprevalence rates have risen significantly over the last 12 years in Sri Lanka, possibly due to increased transmission. As individuals who were hospitalized due to dengue were more likely to be seropositive for JEV, the possibility of cross-reactive assays and/or of JEV infection on immunity to the DENV and clinical disease severity should be further investigated.

Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design

Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzed effects of E138K and K279M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use.

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III.

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.

Longitudinal study of CD4 T cell responses following Japanese encephalitis virus vaccination (VIR2P.1165)

Abstract Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. There are currently a number of vaccines shown to effectively prevent disease. In the United States, an alum adsorbed vaccine, which consists of a formaldehyde-inactivated, attenuated JEV strain is available. Vaccine protection has been shown to be mediated by neutralizing antibody responses. However, the CD4 T cell response associated with vaccine protection has not been fully characterized. In particular, the JEV model also presents an opportunity to study the kinetics of T cell responses elicited by a multi-dose vaccination in human subjects. In this study, we characterized the longitudinal response of JEV-specific CD4 T cells following vaccination using tetramer guided epitope mapping (TGEM), direct ex vivo MHC class II tetramer staining, and intracellular cytokine staining. A total of 67 epitopes restricted by 7 common HLA-DRB1 alleles and one DRB5 allele were identified. Vaccination elicited a robust CD4 T cell response, which was substantially boosted after the second dose of vaccine was administered. JEV-specific CD4 T cells exhibited a memory phenotype and expressed CCR4 and CXCR3. Consistent with this surface phenotype, JEV specific responses were polyfunctional, secreting IFN-g, IL-4 and sometimes IL-17a. These results indicate that JEV vaccination induces a robust, polyfunctional CD4 T cell response that is similar in magnitude to other highly effective vaccines.