Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue,West Nile and Japanese encephalitis viruses

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax™ technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax™-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.     

Flavivirus vaccines

Diseases caused by flavivirus infection have been a scourge of mankind for over three centuries; with yellow fever, dengue fever and Russian spring-summer encephalitis causing epidemics resulting in thousands of fatalities. Due to the development of a safe and efficacious live-attenuated vaccine against yellow fever, this disease is no longer such a threat in countries where adequate vaccination is practised. A similarly safe and efficacious inactivated vaccine against central-European tick-borne encephalitis has also been developed and this has drastically reduced the incidence of this disease in many countries where it is endemic. In spite of these successes, the development of vaccines against other pathogenic flaviviruses, causing diseases such as dengue fever and Russian spring-summer encephalitis, have not been successful. This review attempts to summarize the development of flavivirus vaccines to date and identify areas for future improvements. Problems associated with designing flavivirus vaccines are discussed and the advantages and disadvantages of future strategies for vaccine development are considered.     

A review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available

Genus flavivirus comprises many important human pathogens causing public health problems worldwide. Some flavivirus infections are characterized by a relatively high mortality rate and/or high sequelae rate in survivors. Because most flavivirus life cycles are maintained between arthropod vectors and amplifying/reservoir hosts in the absence of humans, eradication of flaviviruses might be extremely difficult. Flavivirus vaccine development is considered a reasonable method to prevent flavivirus infections. Some vaccines have been successfully developed, but others have not, regardless of much effort. This review article describes currently available flavivirus vaccines against yellow fever, Japanese encephalitis, and tick-borne encephalitis. In addition, the current status of dengue and West Nile virus vaccine development is reviewed and problems regarding their development are discussed.     

Live virus vaccines based on a yellow fever vaccine backbone: Standardized template with key considerations for a risk/benefit assessment

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called “chimeric virus vaccines”). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for marketing (to date in Australia, Thailand, Malaysia, and the Philippines) is a vaccine against the flavivirus, Japanese encephalitis (JE), which employs a licensed vaccine (yellow fever 17D) as a vector. In this vaccine, two envelope proteins (prM-E) of YF 17D virus were exchanged for the corresponding genes of JE virus, with additional attenuating mutations incorporated into the JE gene inserts. Similar vaccines have been constructed by inserting prM-E genes of dengue and West Nile into YF 17D virus and are in late stage clinical studies. The dengue vaccine is, however, more complex in that it requires a mixture of four live vectors each expressing one of the four dengue serotypes. This vaccine has been evaluated in multiple clinical trials. No significant safety concerns have been found. The Phase 3 trials met their endpoints in terms of overall reduction of confirmed dengue fever, and, most importantly a significant reduction in severe dengue and hospitalization due to dengue. However, based on results that have been published so far, efficacy in preventing serotype 2 infection is less than that for the other three serotypes. In the development of these chimeric vaccines, an important series of comparative studies of safety and efficacy were made using the parental YF 17D vaccine virus as a benchmark. In this paper, we use a standardized template describing the key characteristics of the novel flavivirus vaccine vectors, in comparison to the parental YF 17D vaccine. The template facilitates scientific discourse among key stakeholders by increasing the transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.     

Development of immunizing agents against dengue]

The four serotypes of dengue flaviviruses are transmitted mainly by the Aedes aegypti mosquito, and some epidemics have been attributed to Ae. albopictus, Ae. polynesiensis, and various species of the Ae. scutellaris complex. The risk factors involved in dengue mortality and morbidity are related to the human host (genetic characteristics of infected persons; lifestyles, immune status, and health conditions of people; basic sanitation of dwellings; and water supply) and to the virus (genetic variability between and among serotypes, different pathogenicities, and geographic distribution). Notwithstanding the lack of knowledge of the immunopathobiology of dengue fever, important advances have been made in terms of a protective immune response, using attenuated dengue viruses or antigens produced by means of recombinant technologies. Efforts have been made since the 1940s to develop dengue vaccines. Immunity acquired from natural infection is specific for each serotype, and as many as three different serotype infections have been reported in one individual. For this reason, a tetravalent vaccine may likely be needed. Candidate vaccines against the four serotypes have been tested in volunteers and have proven to be immunogenic and safe. Although attenuated live virus vaccines are promising, more study is needed regarding their effectiveness and safety. Currently, several studies are ongoing to develop dengue vaccines using antigens from structural proteins (particularly E glycoprotein) and nonstructural proteins, with recombinant DNA technology and other biomolecular technologies. With the same goal, various expression vectors are being used, including Escherichia coli, baculovirus, vaccinia virus, and yellow fever virus. Unfortunately, no satisfactory results have been obtained in humans. The need for effective dengue vaccines is great, given the serious worldwide problem of the transmission of the four serotypes. Effective immunization against dengue would contribute to its prevention, with a positive cost-benefit relationship. Endemic dengue affects young children, and they should be immunized through the Expanded Program on Immunization.     

Immune evasion strategies of flaviviruses

Flavivirus is a genus of the family Flaviviridae. It includes West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and several other viruses which lead to extensive morbidity and mortality in humans. To establish infection and replication in the hosts, flaviviruses have evolved a variety of strategies to modulate the host's immune responses. In this review, the strategies employed by flaviviruses to evade the innate and adaptive immunity of host are summarized based on current studies, with a major focus on the inhibition of interferon, complement, natural killer (NK) cell, B cell, and T cell responses. This review aims to provide an overview of the current understanding for the mechanisms used by flaviviruses to escape the host's immune response, which will facilitate the future studies on flavivirus pathogenesis and the development of anti-flavivirus therapeutics.     

Live attenuated tetravalent dengue vaccine

The development of a live attenuated tetravalent dengue vaccine is currently the best strategy to obtain a vaccine against dengue viruses. The Mahidol University group developed candidate live attenuated vaccines by attenuation through serial passages in certified primary cell cultures. Dengue serotype 1, 2 and 4 viruses were developed in primary dog kidney cells, whereas dengue serotype 3 was serially passaged in primary African green monkey kidney cells. Tissue culture passaged strain viruses were subjected to biological marker studies. Candidate vaccines have been tested as monovalent (single virus), bivalent (two viruses), trivalent (three viruses) and tetravalent (all four serotype viruses) vaccines in Thai volunteers. They were found to be safe and immunogenic in both adults and children. The Mahidol live attenuated dengue 2 virus was also tested in American volunteers and resulted in good immune response indistinguishable from those induced in Thai volunteers. The master seeds from the four live attenuated virus strains developed were provided to Pasteur Merieux Connaught of France for production on an industrial scale following good manufacturing practice guidelines.     

Is it time for a new yellow fever vaccine?

An inexpensive live attenuated vaccine (the 17D vaccine) against yellow fever has been effectively used to prevent yellow fever for more than 70 years. Interest in developing new inactivated vaccines has been spurred by recognition of rare but serious, sometimes fatal adverse events following live virus vaccination. A safer inactivated yellow fever vaccine could be useful for vaccinating people at higher risk of adverse events from the live vaccine, but could also have broader global health utility by lowering the risk-benefit threshold for assuring high levels of yellow fever vaccine coverage. If ongoing trials demonstrate favorable immunogenicity and safety compared to the current vaccine, the practical global health utility of an inactivated vaccine is likely to be determined mostly by cost.     

Flavivirus vaccines: Virus-like particles and single-round infectious particles as promising alternatives

The genus flavivirus of the Flaviridae family includes several human pathogens, like dengue, Zika, Japanese encephalitis, and yellow fever virus. These viruses continue to be a significant threat to human health. Vaccination remains the most useful approach to reduce the impact of flavivirus fever. However, currently available vaccines can induce severe side effects or have low effectiveness. An alternative is the use of recombinant vaccines, of which virus-like particles (VLP) and single-round infectious particles (SRIP) are of especial interest. VLP consist of the virus structural proteins produced in a heterologous system that self-assemble in a structure almost identical to the native virus. They are highly immunogenic and have been effective vaccines for other viruses for over 30 years. SRIP are promising vaccine candidates, as they induce both cellular and humoral responses, as viral proteins are expressed. Here, the state of the art to produce both types of particles and their use as vaccines against flaviviruses are discussed. We summarize the different approaches used for the design and production of flavivirus VLP and SRIP, the evidence for their safety and efficacy, and the main challenges for their use as commercial vaccines.     

主要黄病毒E蛋白抗原表位分析与初步鉴定

目的 对主要黄病毒结构蛋白的抗原表位情况进行系统分析,鉴别共同及特异性抗原表位并对可能的特异性抗原表位进行原核表达.方法 分别利用DNAStar及ANTHEPROT软件对登革病毒1～4型、流行性乙型脑炎(乙脑)病毒及黄热病毒E蛋白的亲水性、抗原性、可塑性、表面可及性和Garnier-Robson的二级结构进行分析,预测可能的抗原表位.在此基础上,根据表位位置和氨基酸序列的相似性,分析6种病毒的共有及特异性抗原表位,并参考GenBank中的序列信息,对预测的抗原表位进行比对,进一步分析抗原表位在不同病毒株中的保守性.然后利用pET32及pMAL-c2x系统对可能的特异性抗原表位进行高效原核表达、Western blot验证其抗原性.结果 经系统的生物信息学分析,共预测获得登革病毒1～4型、乙脑病毒及黄热病毒共有抗原表位15个,病毒特异性抗原表位47个.利用pET32及pMAL-c2x系统对6种病毒部分可能的特异性抗原表位进行了高效表达.Western blot结果显示登革病毒1型及2型表达抗原片段表现良好的抗原反应原性,并与试验中所用其他黄病毒及甲病毒多克隆抗体无明显的交叉反应.而所表达的登革病毒3型和4型、乙脑病毒及黄热病毒片段无抗原反应原性.结论 6条原核表达抗原片段经Western blot验证,获得登革病毒1型和2型特异性抗原片段.     

林芝地区4种虫媒传染病血清学调查

目的了解西藏林芝地区健康人群乙脑、登革热、森林脑炎和肾综合征出血热抗体水平,为林芝地区的虫媒传染病的诊断和防控提供参考依据。方法2010年7—8月,根据既往已证实存在疟疾病本地流行等经验,选择海拔2000m以下的墨脱县达木乡、背崩乡、墨脱镇和察隅县下察隅镇为调查区域,调查对象为在调查时段在家中的当地常住居民。采集不同年龄段健康人群血清样本。用间接ELISA法对上述调查对象血清样本进行乙脑、登革热、森林脑炎和肾综合征出血热IgG抗体的检测。结果共采集并完成了350份（其中墨脱县249份,察隅县101份）血清标本的检测,乙脑、登革热、森林脑炎和肾综合征出血热的阳性标本数分别为27、6、12和2份,阳性率分别为7．71％（27／350）、1．71％（6／350）、3．43％（12／350）和0．57％（2／350）。察隅县森林脑炎阳性率高于墨脱县,分别为6．93％（7／101）和2．00％（5／249）（P〈0．05）;海拔≤1500m与〉1500m地区的人群森林脑炎病毒阳性率分别为1．97％（5／254）和7．29％（7／96）,两者差别有统计学意义（P〈0．05）,且随着海拔增高,阳性率升高;森林脑炎病毒抗体阳性率在不同年龄组间差别有统计学意义（P〈0．05）,其中11—50岁年龄组的阳性率最高,达5．88％（11／187）,≤10、〉50岁年龄组的阳性率均为O。结论林芝地区存在乙脑和森林脑炎病毒的自然疫源地的可能性极大,尚不确定存在登革热和肾综合征出血热传染病的可能。     

Characterization of immune responses induced by inactivated,live attenuated and DNA vaccines against Japanese encephalitis virus in mice

Vaccination is the most effective countermeasure for protecting individuals from Japanese encephalitis virus (JEV) infection. There are two types of JEV vaccines currently used in China: the Vero cell-derived inactivated vaccine and the live attenuated vaccine. In this study, we characterized the immune response and protective efficacy induced in mice by the inactivated vaccine, live attenuated vaccine and the DNA vaccine candidate pCAG-JME, which expresses JEV prM-E proteins. We found that the live attenuated vaccine conferred 100% protection and resulted in the generation of high levels of specific anti-JEV antibodies and cytokines. The pCAG-JME vaccine induced protective immunity as well as the live attenuated vaccine. Unexpectedly, immunization with the inactivated vaccine only induced a limited immune response and partial protection, which may be due to the decreased activity of dendritic cells and the expansion of CD4+CD25+Foxp3+ regulatory T cells observed in these mice. Altogether, our results suggest that the live attenuated vaccine is more effective in providing protection against JEV infection than the inactivated vaccine and that pCAG-JME will be a potential JEV vaccine candidate.     

Japanese encephalitis virus/yellow fever virus chimera is safe and confers full protection against yellow fever virus in intracerebrally challenged mice

Yellow fever (YF) is an acute viral haemorrhagic disease caused by the yellow fever virus (YFV), which remains a potential threat to public health. The live-attenuated YF vaccine (17D strain) is a safe and highly effective measure against YF. However, increasing adverse events have been associated with YF vaccinations in recent years; thus, safer, alternative vaccines are needed. In this study, using the Japanese encephalitis live vaccine strain SA14-14–2 as a backbone, a novel chimeric virus was constructed by replacing the pre-membrane (prM) and envelope (E) genes with their YFV 17D counterparts.The chimeric virus exhibited a reduced growth rate and a much smaller plaque morphology than did either parental virus. Furthermore, the chimera was much less neurovirulent than was YF17D and protected mice that were challenged with a lethal dose of the YF virus. These results suggest that this chimera has potential as a novel attenuated YF vaccine.     

Onset and duration of protective immunity to IA/IB and IE strains of Venezuelan equine encephalitis virus in vaccinated mice.

Three vaccines developed for protection against IA/IB subtypes of Venezuelan equine encephalitis (VEE) virus were evaluated in mice for the ability to protect against systemic and mucosal challenges with a virulent virus of the IE subtype. The vaccines were the formaldehyde-inactivated C-84 and live attenuated TC-83 vaccines currently administered to people under investigational new drug (IND) status, and a new live attenuated vaccine candidate, V3526. V3526 was superior for inducing protection to VEE IA/IB within a week of vaccination, and protection persisted for at least a year. All three vaccines induced long-term clinical protection against peripheral or mucosal challenge with IE virus, with the mucosal immunity induced by attenuated vaccines lasting longer than that induced by the inactivated vaccine. These data show that the molecularly cloned V3526 vaccine induces equivalent or improved immunity to homologous and heterologous VEE viruses than the existing vaccines.     

两种流行性乙型脑炎疫苗基础免疫的血清抗体观察

为考察流行性乙型脑炎 (乙脑 )减毒活疫苗和灭活疫苗的免疫效果和安全性 ,在新沂市和邳州市分别对 14 1名和 99名无乙脑疫苗接种史的 12～ 2 4月龄儿童 ,分别用乙脑减毒活疫苗和灭活疫苗进行基础免疫 ,在免疫前和免疫后 1个月采血用反向被动凝集抑制试验检测血清抗体。结果 :14 1名经减毒活疫苗免疫的儿童 ,抗体阳性率由免疫前的 2 2 70 %上升至免疫后的 87 2 3 % ,GMT由 1∶12 60上升为 1∶46 13 ;99名用灭活疫苗初免 2针的儿童 ,抗体阳性率由 13 13 %上升至 67 68% ,GMT由 1∶11 83上升至 1∶2 2 5 3。对免疫前抗体阴性的儿童 ,分别注射乙脑减毒活疫苗和灭活疫苗后 ,抗体阳转率分别为 83 49%和 62 79%。两种疫苗的安全性均好 ,接种后仅 17人 ( 0 0 7% )有轻度发热 ,11人 ( 0 0 4% )出现局部红肿。而减毒活疫苗出现反应的人数又少于灭活疫苗。本次观察表明 ,乙脑减毒活疫苗的免疫效果明显高于灭活疫苗 ,安全性好 ,接种次数少 ,适宜于推广使用。     

登革-2型病毒一步法TaqMan荧光定量RT-PCR检测方法建立

目的 建立一种针对登革2型病毒（DENV-2）的快速、灵敏、特异的检测方法。方法 从GenBank下载105株DENV-2病毒毒株全基因序列,应用生物软件Bioedit进行比对分析筛选出保守序列,在保守区域设计特异引物和探针,建立检测DENV-2的TaqMan荧光定量逆转录聚合酶链反应（RT-PCR）方法。结果 该方法检测灵敏度达到10² copies/μL;特异性验证中除登革2型病毒有明显扩增外,与墨累谷脑炎病毒、西尼罗病毒、蜱传脑炎病毒、登革病毒1、3、4型、黄热病毒和科萨努尔森林病毒、乙型脑炎病毒均无交叉反应。重复性实验结果表明该方法的组间和组内的变异系数（CV）均<2%。对人工感染DENV-2的87只蚊虫标本进行比对实验,结果荧光定量RT-PCR检测出56份阳性,传统RT-PCR只检测出16份阳性,差异有统计学意义（χ²=37.908,P=0.000）。结论 TaqMan荧光定量RT-PCR方法检测DENV-2,特异性强、敏感性高。     

Trends in flavivirus infections in Japan

Although Japanese encephalitis has declined as an important cause of illness and death in Japan, infection with other flaviviruses has become a public health concern. Recently, reports of imported dengue cases, as well as isolations of tick-borne encephalitis virus, have increased.     

Comparison of purified psoralen-inactivated and formalin-inactivated dengue vaccines in mice and nonhuman primates

Dengue fever, caused by dengue viruses (DENV 1–4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1–4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV.     

Aedes aegypti in Brazil: genetically differentiated populations with high susceptibility to dengue and yellow fever viruses

Aedes aegypti was eliminated from Brazil in 1955, but re-infested the country in the 1970s. Dengue outbreaks have occurred since 1981 and became endemic in several cities in Brazil after 1986. Urban yellow fever has not occurred since 1942, and only jungle yellow fever cases have been reported. A population genetic analysis using isoenzyme variation combined with an evaluation of susceptibility to both yellow fever and dengue 2 viruses was conducted among 23 A. aegypti samples from 13 Brazilian states. We demonstrated that experimental infection rates of A. aegypti for both dengue and yellow fever viruses (YFV) are high and heterogeneous, and samples collected in the endemic and transition areas of sylvatic yellow fever were highly susceptible to yellow fever virus. Boa Vista, a border city between Brazil and Venezuela, and Rio de Janeiro in the Southeast region are considered as the most important entry points for dengue dissemination. Considering the high densities of A. aegypti, and its high susceptibility to dengue and yellow fever viruses, the risk of dengue epidemics and yellow fever urbanization in Brazil is more real than ever.     

Alpha-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-γ secreting CD8⁺ T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.