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The current Japanese encephalitis (JE) vaccine derived from G3 JE virus (JEV) can induce protective immunity against G1-G4 JEV genotypes. However, protective efficacy against the emerging G5 genotype has not been reported. Using in vitro and in vivo tests, biological phenotype and cross-immunoreactions were compared between G3 JEV and G5 JEV (wild strains). The PRNT90 method was used to detect neutralizing antibodies against different genotypes of JEV in JE vaccine-immunized subjects and JE patients. In JE vaccine-immunized mice, the lethal challenge protection rates against G3 and G5 JEV wild strains were 100% and 50%, respectively. The seroconversion rates (SCRs) of virus antibodies against G3 and G5 JEV among vaccinated healthy subjects were 100% and 35%, respectively. All clinically identified JE patients showed high levels of G3 JEV neutralizing antibodies (≥1:10-1280) with positive serum geometric mean titers (GMTs) of 43.2, while for G5 JEV, neutralizing antibody conversion rates were only 64% with positive serum GMTs of 11.14. Moreover, the positive rate of JEV neutralizing antibodies against G5 JEV in pediatric patients was lower than in adults. Low levels of neutralizing/protective antibodies induced by the current JE vaccine, based on the G3 genotype, were observed against the emerging G5 JEV genotype. Our results demonstrate the need for more detailed studies to reevaluate whether or not the apparent emergence of G5 JEV can be attributed to failure of the current vaccine to induce appropriate immune protectivity against this genotype of JEV.

Japanese encephalitis virus (JEV), a neuroinvasive and neurovirulent orthoflavivirus, can be prevented in humans with the SA 14 -14-2 vaccine, a live-attenuated version derived from the wild-type SA 14 strain. To determine the viral factors responsible for the differences in pathogenicity between SA 14 and SA 14 -14-2, we initially established a reverse genetics system that includes a pair of full-length infectious cDNAs for both strains. Using this cDNA pair, we then systematically exchanged genomic regions between SA 14 and SA 14 -14-2 to generate 20 chimeric viruses and evaluated their replication capability in cell culture and their pathogenic potential in mice. Our findings revealed the following: ( i ) The single envelope (E) protein of SA 14 -14-2, which contains nine mutations (eight in the ectodomain and one in the stem region), is both necessary and sufficient to render SA 14 non-neuroinvasive and non-neurovirulent. ( ii ) Conversely, the E protein of SA 14 alone is necessary for SA 14 -14-2 to become highly neurovirulent, but it is not sufficient to make it highly neuroinvasive. ( iii ) The limited neuroinvasiveness of an SA 14 -14-2 derivative that contains the E gene of SA 14 significantly increases (approaching that of the wild-type strain) when two viral nonstructural proteins are replaced by their counterparts from SA 14 : ( a ) NS1/1’, which has four mutations on the external surface of the core β-ladder domain; and ( b ) NS2A, which has two mutations in the N-terminal region, including two non-transmembrane α-helices. In line with their roles in viral pathogenicity, the E, NS1/1’, and NS2A genes all contribute to the enhanced spread of the virus in cell culture. Collectively, our data reveal for the first time that the E protein of JEV has a dual function: It is the master regulator of viral neurovirulence and also the primary initiator of viral neuroinvasion. After the initial E-mediated neuroinvasion, the NS1/1’ and NS2A proteins act as secondary promoters, further amplifying viral neuroinvasiveness.

Small viral protein domains expressed in Escherichia coli could serve as a cost-effective alternative for the development of subunit vaccines. However, due to their inherently low immunogenicity, such E. coli-expressed protein domains are rarely used in practice. In this study, we evaluated the ability of five divalent metal ions—Co2+, Cu2+, Mn2+, Ni2+, and Zn2+—to enhance the immunogenicity of an E. coli-expressed Japanese encephalitis virus envelope protein domain III (JEV-ED3; ∼12 kDa). Biophysical analyses using DLS, SLS, CD, and tryptophan fluorescence spectroscopy revealed that trace amounts of metal ions did not alter the conformational integrity of JEV-ED3, which remained natively folded and monomeric in the presence or absence of the tested ions, except for Zn2+. We then assessed the immunogenicity of JEV-ED3 in the presence and absence of these metal ions in a mouse model. Among them, 5 μM Co2+ produced the strongest enhancement, increasing anti-JEV-ED3 IgG titers by more than 15-fold compared to the no‑cobalt control. Furthermore, Co2+ also significantly boosted the anti-JEV antibody response—by over 7-fold—when co-administered with an inactivated JEV vaccine. The cobalt-induced immune response was Th2-biased (elevated IgG1 and IL-4 levels) and long-lasting, as evidenced by increased CD44+CD62L+ memory T cell populations measured by flow cytometry. Importantly, Co2+-induced antibodies exhibited potent live virus-neutralizing activity in the FRNT₅₀ assay. MTT assays confirmed that the metal concentrations used were well below cytotoxic levels. Collectively, these findings suggest that trace amounts of cobalt can markedly enhance the immunogenicity of otherwise weakly immunogenic protein domains, offering a promising strategy for subunit vaccine development. •LOw concnetration of divalent metal ions did not have any effects on the protein structure.•A trace amount of cobalt ions enhanced the generation of antibodies against JEV-ED3.•It generated memory T-cells upon injection of JEV-ED3.•Cobalt-mediated anti-JEV ED3 antibodies neutraized live JE viruses.•Cobalt ions below the cytotoxic threshold level have high potential for immune modulation.

Flavivirus infections pose a significant global health burden, highlighting the need for safe and effective vaccination strategies. Co-administration of different vaccines, including licensed flavivirus vaccines, is commonly practiced providing protection against multiple pathogens while also saving time and reducing visits to healthcare units. However, how co-administration of different flavivirus vaccines de facto affects immunogenicity, particularly with respect to T cell responses, is only partially understood. Antigen-specific T cell responses were assessed in study participants enrolled in a previously conducted open-label, non-randomized clinical trial. In the trial, vaccines against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), or yellow fever virus (YFV) were administered either individually or concomitantly in different combinations in healthy study participants. Peripheral blood samples were collected before vaccination and at multiple time points afterward. To analyze antigen-specific CD4+ and CD8+ T cell responses, PBMCs were stimulated with overlapping peptide pools from TBEV, JEV, YFV, and Zika virus (ZIKV) envelope (E), capsid (C), and non-structural protein 5 (NS5) viral antigens. A flow cytometry-based activation-induced marker (AIM) assay was used to quantify antigen-specific T cell responses. The results revealed remarkably similar frequencies of CD4+ and CD8+ T cell responses, regardless of whether vaccines were administered individually or concomitantly. In addition, administering the vaccines in the same or different upper arms did not markedly affect T cell responses. Finally, limited cross-reactivity was observed between the TBEV, JEV, and YFV vaccines, and related ZIKV-specific antigens. TBEV or JEV vaccines can be co-administered with the live attenuated YFV vaccine without any markedly altered antigen-specific CD4+ and CD8+ T cell responses to the respective flaviviruses. Additionally, the vaccines can be delivered in the same or different upper arms without any significant altered influence on the T cell response. From a broader perspective, these results provide valuable insights into the outcome of immune responses following simultaneous administration of different vaccines for different but related pathogens.

Escherichia coli is a powerful and cost-effective platform for producing recombinant proteins. However, E. coli- produced proteins lack side-chain glycosylation and may be misfolded due to non-native disulfide bonds, often leading to poor immunogenicity. As a result, they are commonly perceived as unsuitable for use as antiviral vaccine antigens. This study addresses this challenge using the small 12 kDa envelope protein domain III of the Japanese encephalitis virus (JEV-EDIII) as a model. We demonstrate that the low immunogenicity of E. coli- produced proteins can be effectively overcome by employing a solubility-controlling peptide tag (SCP-tag) composed of five isoleucines (C5I). E. coli-produced JEV-EDIII oligomerized into 100 nm (Rh) soluble oligomers upon attachment of the C5I-tag, whereas the untagged JEV-EDIII remained monomeric (Rh ∼ 1.9 nm). The C5I-tag significantly enhanced anti-JEV EDIII IgG titers, as evidenced by ELISA, and increased the population of memory T cells in the spleen, as assessed by flow cytometry. Most notably, the C5I-tagged JEV-EDIII elicited neutralizing antibodies, confirmed by the FRNT50 neutralization assay using live JEV. These findings suggest that oligomerization via SCP-tagging offers a promising, adjuvant-free approach for producing neutralizing antibodies with long-term T cell memory, paving the way for developing E. coli- produced, protein domain-based vaccines. •Five-residue isoleucine tag produces subvisible aggregates of the JEV-ED3 protein.•The C5I-tag retains the native secondary and tertiary structure of the JEV-ED3.•The C5I-tag strongly enhances the immunogenicity of the JEV-ED3.•The IgG produced against the C5I-tagged proteins is specific to the native JEV-ED3 epitope.•The C5I-tagged JEV-ED3 induces a long-term memory and production of neutralizing IgG.

Defining the immune dynamics of Japanese encephalitis (JE) in healthy individuals is crucial for assessing population susceptibility and evaluating the effectiveness of vaccinations. We conducted a cross-sectional serological survey of anti-JEV IgG antibodies and anti-JEV neutralizing antibodies (nAbs) in Guangzhou City, Zhanjiang City and Heyuan City of Guangdong Province, China. A total of 691 participants were included from 2018-2022, among whom 50 were dengue IgG antibody positive and 641 were dengue IgG antibody negative. In the total population, the anti-JEV IgG antibody positivity rate detected by enzyme-linked immunosorbent assay (ELISA) was 51.37% (95% CI: 47.64-55.11%), and the neutralizing antibody positivity rate detected using the microneutralization test (MNT) was 73.22% (95% CI: 69.92-76.54%). Among the 641 dengue IgG antibody-negative subjects, the anti-JEV IgG antibody positivity rate by ELISA and the neutralizing antibody positivity rate by MNT were 48.05% (95% CI: 44.17-51.93%) and 72.07% (95% CI: 68.59-74.56%), respectively. Comparable geographical seroprevalences of either anti-JEV IgG or neutralizing antibody were observed in Guangzhou City, Heyuan City and Zhanjiang City, respectively (49.52% vs. 48.04% vs. 47.45%, 65.71% vs. 70.46% vs. 76.47%, respectively). Antibody positivity rates in all age groups exhibited a U-shaped curve, with the lowest rate occurring in the 7-18-year-old age group. With respect to the vaccine dose, the anti-JEV nAb positivity rate and geometric mean titer (GMT) detected by MNT were higher in those who received two doses of live attenuated vaccine than in those who received one dose or 0 doses (80.57% vs. 55.81% vs. 55.09% and 25.92 vs. 12.19 vs. 16.47, respectively). In the 641 dengue IgG antibody-negative subjects, moderate consistency between the MNT and ELISA results was observed (Kappa = 0.47, rs = 0.76). The high seroprevalence in participants indicated a neglected transmission of JE, which highlights the importance of strengthening the surveillance of JEV in this area. The vaccination program against JEV is highly needed because of immune gaps in adults, e.g., boosters for adults aged 7-39 years.

IntroductionVaccination remains the most effective strategy for preventing and controlling Japanese encephalitis (JE). The Japanese encephalitis virus (JEV) seroconversion has been documented in sheep and goats across various countries, with occasional fatal cases occurring among sheep on farms in China. Despite the widespread use of attenuated live vaccines, the efficacy of these vaccines in protecting sheep against JE remains uncertain. This study aimed to assess the protective efficacy of currently available attenuated vaccines against genotype I (GI) JEV strains isolated from sheep using a mouse challenge model.MethodsIn this study, vaccination-challenge experiments were conducted using a mouse challenge model to assess the efficacy of attenuated vaccines. The specific vaccines tested were the SA14-14-2 (GI) and SD12-F120 (GI) attenuated live vaccines. The neutralizing antibodies generated by these vaccines were titrated to evaluate their levels of protection. Mice were immunized with high, medium, or low doses of the vaccines and then challenged with either homologous or heterologous JEV strains. The challenge strains included the SH2201 (GI) and N28 (GIII) strains. Viremia levels and the development of encephalitis lesions were monitored as indicators of protection.ResultsThe neutralizing antibody titers against the sheep-derived SH2201 (GI) strain were significantly lower in mice immunized with the SA14-14-2 (GIII) vaccine compared to those receiving the SD12-F120 (GI) vaccine. Immunization with high and medium doses of SA14-14-2 (GIII) vaccine provided complete protection against challenge with the homologous N28 (GIII) strain but only partial protection against the heterologous SH2201 (GI) strain. Mice immunized with medium and low doses of SA14-14-2 (GIII) vaccine showed varying levels of viremia and developed characteristic encephalitis lesions after being challenged with the heterologous SH2201 (GI) strain. Conversely, mice immunized with high and medium doses of the SD12-F120 (GI) vaccine exhibited 100% protection against the challenge with the homologous SH2201 (GI) strain.DiscussionThe results of this study suggest that while the SA14-14-2 (GIII) attenuated live vaccine offers partial protection against sheep-derived GI strains, it is not fully effective against heterologous strains like SH2201 (GI). This highlights a significant gap in the ability of the current vaccines to protect across different JEV genotypes and host species. In contrast, the SD12-F120 (GI) vaccine demonstrated stronger protection against the homologous SH2201 (GI) strain. These findings indicate a pressing need for the development of new vaccination strategies that can provide broader and more effective protection against JE, particularly in diverse host species and against a wide range of JEV genotypes.

Japanese encephalitis virus (JEV) is a mosquito-borne pathogen that causes severe neurologic disease. Its endemicity in Asia has prompted its inclusion in nationwide immunization programs. However, the Philippines, which is also endemic for related viruses like dengue (DENV), has not yet adopted this practice. Vaccine hesitancy is a major challenge, exacerbated by concerns over cross-reactive antibodies that may enhance viral infection. This study aimed to determine whether IMOJEV vaccination would induce cross-neutralizing or enhancing antibodies against DENV. Pre- and one-month post-vaccination samples from IMOJEV-vaccinated Filipino children (9-24 months old) were analyzed. A reporter virus particle (RVP)-based neutralization assay against JEV showed neutralization in 28/29 subjects post-vaccination. Presence of DENV2-reactive antibodies was measured via DENV2 VLP ELISA, which revealed increased DENV2 binding reactivity post-vaccination. Pre-vaccination DENV2 binding reactivity also had no significant correlation with the JEV vaccine response. RVP-based neutralization and enhancement assays against DENV2 showed that there was no significant change in neutralizing or enhancing antibody activity against DENV2 after JEV vaccination. This study shows that IMOJEV vaccination elicited a JEV neutralizing response in 97% of vaccinees and that the magnitude of JEV neutralizing titers post-vaccination was not associated with pre-existing binding antibodies to DENV2. Further, while live JEV vaccination increases DENV2-binding antibodies, this cross-reactivity does not lead to DENV2 enhancement. These findings contribute to a better understanding of the orthoflavivirus antibody response following immunization and the influence of pre-existing heterologous orthoflavivirus antibodies. This could guide vaccination strategies, especially in orthoflavivirus-endemic regions.

Current Japanese encephalitis vaccines are derived from strains of genotype III, yet heterologous genotypes are emerging in endemic areas. Inactivated vaccines given to European travelers were found to elicit protective levels of neutralizing anti-bodies against heterologous strains of genotypes I–IV.

Background Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus , and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination. Methods Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells. Results A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %). Conclusion In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.