Immunogenicity and protective efficacy of the current inactivated Japanese encephalitis vaccine against different Japanese encephalitis virus strains

Mouse brain-derived, inactivated Japanese encephalitis (JE) vaccine has been internationally used for many years. It is believed that this vaccine made a great contribution to the reduction of JE patients in several countries. Mouse brain-derived, Beijing-1 and Nakayama JE vaccines induce high levels of neutralizing antibodies. High levels of induced antibodies are maintained at least for 3-4 yr. The induced antibodies are cross-reactive to heterologous strains; however, the neutralizing antibody titers against heterologous strains are usually lower than those against homologous strains. Considering that both Nakayama and Beijing-1 JE vaccines showed high levels of protective efficacy in Taiwan and Thailand where strains other than Nakayama and Beijing-1 were circulating, we conclude that the current inactivated JE vaccine can induce high levels of protective immunity against heterologous JE virus strains.     

Effect of current Japanese encephalitis vaccine on different strains of Japanese encephalitis virus

Inactivated Japanese encephalitis (JE) vaccine was given to healthy children and their neutralizing antibody titres against nine JE virus strains were measured. After two shots of the vaccine in the first year, most of the children showed seroconversion, but their neutralizing antibody titres were not high, and one year after vaccination their titres had decreased to low levels. However, a booster dose of vaccine in the second year had a marked effect on the antibody response. The neutralizing antibodies induced by the vaccine reacted to almost the same extent with nine different JE virus strains, though significantly higher titres against the Nakayama strain, the vaccine strain, were observed after vaccination in the second year.     

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [Macaca mulatta

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys (Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1mg plasmid DNA given IM, or 5 microg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5mg DNA given IM or 5 microg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.     

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.     

Dengue tetravalent DNA vaccine increases its immunogenicity in mice when mixed with a dengue type 2 subunit vaccine or an inactivated Japanese encephalitis vaccine

We previously developed a dengue tetravalent DNA vaccine that can induce neutralizing antibodies against four dengue viruses in mice. Here, we demonstrated that immunogenicity of our tetravalent vaccine is synergistically increased in mice by co-immunization with dengue type 2 virus (DENV2) subviral extracellular particles (D2EPs) or inactivated Japanese encephalitis vaccine (JEVAX). A single immunization with a mixture of 100 microg of the tetravalent vaccine and 150 ng of D2EPs or a 1/10 dose of JEVAX induced moderate levels of neutralizing antibodies in a 90% plaque reduction assay. Immunized mice were protected from "artificial" viremia created by intravenous injection with DENV2.     

Boosting with an adenovirus-based vaccine improves protective efficacy against Venezuelan equine encephalitis virus following DNA vaccination

There is a requirement for a vaccine that protects against the alphavirus, Venezuelan equine encephalitis virus (VEEV). Previous work has shown that DNA vaccines encoding structural proteins of VEEV can elicit immune responses and protection against VEEV though this protection is incomplete against airborne VEEV. In this study, we demonstrate that particle-mediated epidermal delivery of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein. This results in an improved Th2-type IgG response, an increase in neutralising antibody and a significant increase in protection against airborne VEEV. This indicates that prime-boost may be a suitable immunisation regimen for providing protection against airborne VEEV.     

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 microg of pNJEME by intramuscular (i.m.) injection, and 3 microg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 microg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 microg of pNJEME or by gene gun administration with 3 microg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.     

Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2.     

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.     

流行性乙型脑炎减毒活疫苗一针次强化免疫较长期效果评价

目的 探讨一针次流行性乙型脑炎(乙脑)减毒活疫苗强化免疫措施在乙脑自然疫源地环境1～3年较长期防控效果,为制定下一步防控措施提供依据.方法 以贵州省2004-2006年仅经历过一次乙脑减毒活疫苗强化免疫活动且自然暴露时间相同的儿童作为观察对象,动态分析其强化免疫后1～3年发病专率变化,对该人群所有乙脑确诊病例开展免疫史回顾性调查.结果 强化免疫县当年乙脑发病率较前一年均大幅度下降,下降幅度平均为62.36%.但个别县强化免疫1～2年后疫情出现反弹,病例中强化免疫目标儿童占73.28%,随机调查27例,均未接种过乙脑疫苗.免疫后1～3年观察对象发病专率分别为6.25/10万、5.77/10万、4.54/10万,差异无统计学意义(X2=0.73,P＞0.05).调查观察对象中所有实验室诊断病例52例,乙脑疫苗接种率为1.92%,低于对照组(29.41%),二者差异有统计学意义(X2=52.74,P＜0.01).结论 在贵州省乙脑自然疫源地环境中一针次乙脑减毒活疫苗强化免疫措施1～3年较长期疫情控制效果良好,提高目标儿童接种率是落实措施效果的有效手段.个别县强化免疫后1～2年疫情出现反弹与当地强化免疫活动实际接种率低有关.     

流行性乙型脑炎DNA疫苗研究新进展

流行性乙型脑炎(Japanese encephalitis,JE)疫苗防治问题是黄病毒领域主要研究内容之一。自1997年WHO提出以流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)为研究目标,集中进行黄病毒多价疫苗研制与开发以来,JE DNA疫苗研究相继报道。虽然已证明了JE DNA疫苗在防治JEV感染方面的实验可行性,但是该类疫苗还不能像常规灭活或减毒活疫苗那样诱导强烈免疫应答。本文着重近3年来有关JE DNA疫苗免疫原性与增强效应等方面研究进展综述如下。     

In vivo electroporation improves immune responses to DNA vaccination in sheep

In vivo electroporation was utilised to enhance plasmid DNA expression in sheep muscle to improve the immune response to DNA vaccination. DNA encoding enhanced green fluorescence protein expressed at higher levels in sheep muscle following in vivo electroporation which caused minimal muscle damage. Groups of seven sheep were then given three intramuscular injections of plasmids encoding two Haemonchus contortus Ag, with and without electroporation at 0, 3 and 7 weeks. Humoral responses were enhanced in electroporated sheep. Four weeks after vaccination, all groups were injected subcutaneously with recombinant Ag formulated in Quil A. Induction of vaccine-specific immune memory was demonstrated in DNA-vaccinated sheep.     

Induction of cross-protection against two wild-type Taiwanese isolates of Japanese encephalitis virus using Beijing-1 strain DNA vaccine

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is an important human pathogen. Mouse brain-derived, inactivated JEV vaccines have contributed greatly to the reduction in numbers of JE patients in several countries, including Taiwan. However, mice immunized with the Nakayama strain inactivated vaccine show lower protection against a lethal strain of Beijing-1 JEV than those immunized with the homologous vaccine. DNA vaccine encoding the envelope (E) protein gene appears to provide protection against JEV in the mouse model, but it is not known whether such vaccines would confer cross-protection for mice against different strains of JEV. In this study, we evaluated the ability of pE, a plasmid DNA vaccine encoding the Beijing-1 envelope protein to elicit cross-protective immunity against infection with the homologous Beijing-1 strain and two lethal Taiwanese isolates of JEV, CH2195 and CEN. Our results showed that mice immunized with pE were protected against lethal challenge with Beijing-1 JEV as well as two Taiwanese isolates. In addition, nai;ve mice were cross-protected by passive transfer of sera from immunized animals, indicating the crucial role of humoral immunity in protection. These results demonstrate that JEV DNA vaccine can provide effective protection against infection not only with homologous virus, but also with heterologous virus.     

流行性乙型脑炎减毒活疫苗加强免疫的Meta分析

目的 系统评价乙脑减毒活疫苗加强免疫效果并探讨其必要性。方法 计算机检索中国知网、万方数据知识服务平台、Cochrane Database 、PubMed、Web of Science数据库,收集乙脑减毒活疫苗加强免疫的研究,检索时限均为从建库至2015年3月。由2名研究者按照纳入与排除标准筛选文献、提取资料和评价纳入研究的方法学质量,使用Stata 12.0 软件进行统计计算。结果 总共纳入9篇文献。Meta分析结果显示：基础免疫后一个月合并阳性率为79%（95% CI：69%～90%）,基础免疫一年后,接种者的抗体水平下降（合并阳性率51%,95% CI：17%～86%）,加强免疫后抗体水平明显升高（合并阳性率98%,95% CI：97%～99%）。结论 乙脑减毒活疫苗加强免疫能提高人群乙脑抗体阳性率和抗体平均几何滴度,有利于达到对人群长期保护的目的。     

Immunogenicity and protective efficacy of a DNA vaccine against Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge.     

Construction and evaluation of a chimeric pseudoinfectious virus vaccine to prevent Japanese encephalitis

Multiple vaccines exist to control Japanese encephalitis (JE), but all suffer from problems. We have developed a new type of flavivirus vaccine, a pseudoinfectious virus (RepliVAX WN) that prevents West Nile virus (WNV)-induced disease. Here, we describe production of a chimeric RepliVAX (RepliVAX JE) that expresses the JE virus (JEV) prM and E proteins. Our prototype RepliVAX JE replicated poorly in cells, but blind passage produced a better-growing derivative, and analyses of this derivative allowed us to engineer a second-generation RepliVAX (RepliVAX JE.2) that grew to high titers. RepliVAX JE.2 elicited neutralizing antibodies in both mice and hamsters and provided 100% protection from a lethal challenge with JEV or WNV, respectively. These results demonstrate the utility our RepliVAX platform for producing a JE vaccine.     

Single dose of inactivated Japanese encephalitis vaccine with poly(gamma-glutamic acid) nanoparticles provides effective protection from Japanese encephalitis virus

Japanese encephalitis (JE) is a serious disease caused by the JE virus (JEV), and vaccination is the only way to prevent the diseases. In Japan, the only JE vaccine currently available is an inactivated vaccine that requires multiple doses for effective protection; therefore, an effective single-dose vaccine is needed. However, no report of an effective protocol for a single dose of JE vaccine in animals has been published. Here, we evaluated the efficacy of a single-dose vaccination in mice to which the JE vaccine was given with or without adjuvant. Biodegradable poly(gamma-glutamic acid) nanoparticles (gamma-PGA-NPs) were used as a test adjuvant. Remarkably, a single dose of JE vaccine with gamma-PGA-NPs enhanced the neutralizing antibody titer, and all of the immunized mice survived a normally lethal JEV infection, while only 50% of the mice that received a single dose of JE vaccine without gamma-PGA-NPs survived. The use of aluminum as the adjuvant showed similar levels of enhanced efficacy. These results show that gamma-PGA-NPs are a novel and safe adjuvant for JE vaccine, and that a single dose of JE vaccine with gamma-PGA-NPs provides effective protection from lethal JEV in mice. A similar protocol, in which a single dose of JE vaccine is mixed with gamma-PGA-NPs, may be useful for the immunization of humans.     

Characterization of live-attenuated Japanese encephalitis vaccine virus SA14-14-2

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 was licensed decades ago and now approved for clinical use in most JE endemic countries. Large-scale clinical trials demonstrate ideal safety and efficacy profile of this Chinese vaccine. The SA14-14-2 vaccine was derived from a virulent strain SA14 after hundreds of serial passaging in cells and animals, concern about virulence reversion remains exist. In the present study, to study the in vitro and in vivo genetic and attenuation stability of the vaccine virus, SA14-14-2 was serially passaged in Vero cells and mouse brain followed by sequence comparison and attenuation phenotype analysis. The results showed that no significant mutation was acquired after serial passaging in Vero cells except a single Ser66Leu mutation within capsid protein, which had no effect on viral virulence in mice. Importantly, serial passaging of SA14-14-2 in suckling mouse brain resulted in emergence of adaptive mutations and increased virulence in mice. Population and plaque-purified clone consensus sequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E, T226R and I270T, sequentially occurred and become predominant during serial passaging in suckling mouse brain. Especially, these adaptive mutations were close related with the enhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimental evidence of highly genetic and attenuation stability of SA14-14-2 following passaging in Vero cells, and reveal the potential virulence reversion during passaging in mouse brain in association with critical adaptive mutations in E protein. These findings are important for quality control and evaluation of live JE vaccines and will help understand the attenuation mechanism of flavivirus vaccine.     

Long-term follow-up of Japanese encephalitis chimeric virus vaccine: Immune responses in children

BackgroundA single dose of live attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) was shown to be immunogenic and well tolerated when given either as a booster to formalin-inactivated Japanese encephalitis (JE)-vaccine (mouse brain-derived vaccine [MBDV])-primed 2–5-year-olds, or as a primary vaccination to JE-vaccine-naïve 12–24-month-old toddlers in Thailand. A 5-year follow-up assessment of immune response persistence over time was conducted.MethodsFour additional visits (at 2, 3, 4, and 5 years) for immunologic assessments were added to the original 12-month open-label crossover study, in which 100 healthy children aged 2–5 years with a history of two-dose primary vaccination with MBDV (according to the Thai Expanded Program for Immunization schedule), and 200 healthy JE-vaccine-naïve 12–24-month-old toddlers, were randomized 1:1 to receive JE-CV, containing ⩾4 log₁₀ plaque forming units, 1 month before or after hepatitis A control vaccine.ResultsIn MBDV-primed 2–5-year-olds (n = 78), the immune response to the JE-CV vaccine persisted up to at least 5 years after vaccination with a single dose of JE-CV, with all (n = 78) children seroprotected at the year 5 visit (geometric mean titers [GMT]: 252 1/dil). There was no decrease of seroprotection rate over time (100% at 6 months post-vaccination and 96.8% (90.3–98.9) at 5 years post-vaccination). In JE-vaccine-naïve toddlers, a protective immune response persisted up to at least 5 years in 58.8% (50.9–66.4) after a single-dose administration of JE-CV (GMT 26.7 1/dil; sensitivity analysis).ConclusionsA single-dose of JE-CV as a booster following MBDV administration provided long-lasting immunity. In JE-vaccine-naïve toddlers, despite relatively high seroprotection rates persisting over time, a subsequent booster dose is recommended following a JE-CV primary vaccination for long-term protection.This study was registered on www.clinicaltrials.gov (NCT00621764).     

Potentiation of an anthrax DNA vaccine with electroporation

DNA vaccines are a promising method of immunization against biothreats and emerging infections because they are relatively easy to design, manufacture, store and distribute. However, immunization with DNA vaccines using conventional delivery methods often fails to induce consistent, robust immune responses, especially in species larger than the mouse. Intramuscular (i.m.) delivery of a plasmid encoding anthrax toxin protective antigen (PA) using electroporation (EP), a potent DNA delivery method, rapidly induced anti-PA IgG and toxin neutralizing antibodies within 2 weeks following a single immunization in multiple experimental species. The delivery procedure is particularly dose efficient and thus favorable for achieving target levels of response following vaccine administration in humans. These results suggest that EP may be a valuable platform technology for the delivery of DNA vaccines against anthrax and other biothreat agents.