Domestic goose as a model for West Nile virus vaccine efficacy

West Nile virus (WNV) is an emergent pathogen in the Americas, first reported in New York during 1999, and has since spread across the USA, Central and South America causing neurological disease in humans, horses and some bird species, including domestic geese. No WNV vaccines are licensed in the USA for use in geese. This study reports the development of a domestic goose vaccine efficacy model, based on utilizing multiple parameters to determine protection. To test the model, 47 geese were divided in two experiments, testing five different vaccine groups and two sham groups (challenged and unchallenged). Based on the broad range of results for individual metrics between the Challenged-Sham and Unchallenged-Sham groups, the best parameters to measure protection were Clinical Pathogenicity Index (CPI), plasma virus positive geese on days 1–4 post-inoculation and plasma virus titers, and brain histological lesion rates and severity scores. Compared to the Challenged-Sham group, the fowlpox virus vectored vaccine with inserts of WNV prM and E proteins (vFP2000) provided the best protection with significant differences in all five metrics, followed by the canarypox virus vectored vaccine with inserts of WNV prM and E proteins (vCP2018) with four metrics of protection, recombinant vCP2017 with three metrics and WNV E protein with one. These data indicate that domestic geese can be used in an efficacy model for vaccine protection studies using clinical, plasma virological and brain histopathological parameters to evaluate protection against WNV challenge.     

Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus

West Nile virus is an arthropod-borne flavivirus that has caused substantial morbidity and mortality to animals as well as humans since its introduction in to the New York area in 1999. Given that there are no antiviral drugs available for treatment of the disease, vaccines provide an efficacious alternative to control this disease. Herein we describe an attenuated WNV strain developed by the ablation of the glycosylation sites in the envelope (E) and non-structural 1 (NS1) proteins. This E_154S/NS1_{130A/175A/207A} strain showed modest reduction in multiplication kinetics in cell culture and small plaque phenotype compared to the parental NY99 strain yet displayed greater than a 200,000-fold attenuation for mouse neuroinvasiveness compared to the parental strain. Mice infected with 1000 PFU of E_154S/NS1_{130A/175A/207A} showed undectable viremia at either two or three days post infection; nonetheless, high titer neutralizing antibodies were detected in mice inoculated with low doses of this virus and protected against lethal challenge with a 50% protective dose of 50 PFU.     

Construction and characterization of a second-generation pseudoinfectious West Nile virus vaccine propagated using a new cultivation system

Safer vaccines are needed to prevent flavivirus diseases. To help develop these products we have produced a pseudoinfectious West Nile virus (WNV) lacking a functional C gene which we have named RepliVAX WN. Here we demonstrate that RepliVAX WN can be safely propagated at high titer in BHK cells and vaccine-certified Vero cells engineered to stably express the C protein needed to trans-complement RepliVAX WN growth. Using these BHK cells we selected a better growing mutant RepliVAX WN population and used this to generate a second-generation RepliVAX WN (RepliVAX WN.2). RepliVAX WN.2 grown in these C-expressing cell lines safely elicit strong protective immunity against WNV disease in mice and hamsters. Taken together, these results indicate the clinical utility of RepliVAX WN.2 as a vaccine candidate against West Nile encephalitis.     

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40–50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20–30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.     

Inflammasome signaling pathways exert antiviral effect against Chikungunya virus in human dermal fibroblasts

Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signaling pathways and leading to an increased expression of IFN-β, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1β expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV- and WNV-induced IL-1β production by these cells. Finally, inhibition of the inflammasome via caspase-1 silencing was found to enhance CHIKV replication in dermal fibroblasts. Together, these results indicate that the skin contributes to the pro-inflammatory and anti-viral microenvironment via the activation of the inflammasome in the early stages following infection with arboviruses.     

Characterization of West Nile virus live vaccine candidates attenuated by capsid deletion mutations

Protein C deletion mutants of West Nile virus (WNV) were evaluated for their potential use as live virus vaccine candidates in vivo. Double and triple mutants carrying small deletions and second-site point mutations, as well as mutants with large deletions of 36 and 37 amino acid residues were tested in a stringent mouse challenge model. The mutant viruses were found to be non-pathogenic and to induce protective immunity in a wide range of inoculation doses (10¹–10⁶ FFU). Furthermore, the effects of combining three different previously identified resuscitating point mutations, as well as the combination of a large protein C deletion with a deletion mutation in the 3′ non-coding region were studied. The data indicate that the production of safe and efficacious WNV live vaccines based on protein C deletion mutations is feasible.     

DNA vaccine for West Nile virus infection in fish crows (Corvus ossifragus)

A DNA vaccine for West Nile virus (WNV) was evaluated to determine whether its use could protect fish crows (Corvus ossifragus) from fatal WNV infection. Captured adult crows were given 0.5 mg of the DNA vaccine either orally or by intramuscular (IM) inoculation; control crows were inoculated or orally exposed to a placebo. After 6 weeks, crows were challenged subcutaneously with 105 plaque-forming units of WNV (New York 1999 strain). None of the placebo inoculated-placebo challenged birds died. While none of the 9 IM vaccine-inoculated birds died, 5 of 10 placebo-inoculated and 4 of 8 orally vaccinated birds died within 15 days after challenge. Peak viremia titers in birds with fatal WNV infection were substantially higher than those in birds that survived infection. Although oral administration of a single DNA vaccine dose failed to elicit an immune response or protect crows from WNV infection, IM administration of a single dose prevented death and was associated with reduced viremia.     

西尼罗热研究进展

西尼罗病毒属黄病毒科,为单股正链RNA病毒。它感染人类导致发展为西尼罗热。主要宿主动物为鸟类,蚊虫为传播媒介。该病在欧洲及中亚地区散发,小规模流行,于1999年在美国暴发流行及随后在北美的流行引起极大关注。另外,蚊虫叮咬以外的传播途径,如输血、器官移植、母婴传播等日益受到人们的重视,且目前还没有预防该病的疫苗。该病具有传染性强,重症病例多和病死率高的特点,对人类健康和生命安全造成较大威胁。现就西尼罗热的病原学、流行病学、临床学、实验室诊断及预防控制的研究进展做一综述。     

西尼罗病毒感染的抗体检测方法研究进展

病毒核酸编码3种结构蛋白和7种非结构蛋     

Comparison of vaccine efficacy for different antigen delivery systems for infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L.) in a cohabitation challenge model

Two strains of IPNV made by reverse genetics on the Norwegian Sp strain NVI-015 (GenBank AY379740) backbone encoding the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs were used to prepare inactivated whole virus (IWV), nanoparticle vaccines with whole virus, Escherichia coli subunit encoding truncated VP2-TA and VP2-PT, VP2-TA and VP2-PT fusion antigens with putative translocating domains of Pseudomonas aeruginosa exotoxin, and plasmid DNA encoding segment A of the TA strain. Post challenge survival percentages (PCSP) showed that IWV vaccines conferred highest protection (PCSP=42-53) while nanoparticle, sub-unit recombinant and DNA vaccines fell short of the IWV vaccines in Atlantic salmon (Salmo salar L.) postsmolts challenged with the highly virulent Sp strain NVI-015 (TA strain) of IPNV after 560 degree days post vaccination. Antibody levels induced by these vaccines did not show antigenic differences between the virulent and avirulent motifs for vaccines made with the same antigen dose and delivery system after 8 weeks post vaccination. Our findings show that fish vaccinated with less potent vaccines comprising of nanoparticle, DNA and recombinant vaccines got infected much earlier and yielded to higher infection rates than fish vaccinated with IWV vaccines that were highly potent. Ability of the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs to limit establishment of infection showed equal protection for vaccines made of the same antigen dose and delivery systems. Prevention of tissue damage linked to viral infection was eminent in the more potent vaccines than the less protective ones. Hence, there still remains the challenge of developing highly efficacious vaccines with the ability to eliminate the post challenge carrier state in IPNV vaccinology.     

In vivo efficacy and toxicity evaluation of polycaprolactone nanoparticles and aluminum based admixture formulation as vaccine delivery system

Delivery of antigen through admixture formulation containing poly caprolactone (PCL) and aluminum phosphate was studied as a promising strategy to generate antigen specific immune response. The present study demonstrates the synergistic effect of admixture formulation of PCL with reduced aluminum (PCL-Al 0.2 mg-TT and PCL-PEG-Al 0.2 mg-TT) as a potential adjuvant system using tetanus toxoid (TT) as a model antigen. On evaluation of the magnitude of efficacy for the proposed formulation by ELISA as well as challenge method, persistent and strong antibody response was obtained throughout the 180 day study period on storage at 5 ± 3 °C. In comparison to the aluminum phosphate based conventional tetanus vaccine, higher levels of IFN-γ and IL-4 were obtained with PCL-Al 0.2 mg-TT and PCL-PEG-Al 0.2 mg-TT, indicating the presence of cell mediated as well as humoral immune responses. Histopathology and serum biochemistry profile in mice further indicated the suitability of the proposed formulation. Percent adsorption/encapsulation of the antigen also increased to nearly 95% in the admixture formulation compared to 55% adsorption in the conventional tetanus vaccine. The present study established a useful baseline for designing biocompatible and effective delivery system for toxoid vaccines through judicious use of PCL based biodegradable nanoparticles in combination with aluminum phosphate.     

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn^® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.     

Pre-clinical development of a hydrogen peroxide-inactivated West Nile virus vaccine

West Nile virus (WNV) is a mosquito-transmitted pathogen with a wide geographical range that can lead to long-term disability and death in some cases. Despite the public health risk posed by WNV, including an estimated 3 million infections in the United States alone, no vaccine is available for use in humans. Here, we present a scaled manufacturing approach for production of a hydrogen peroxide-inactivated whole virion WNV vaccine, termed HydroVax-001 WNV. Vaccination resulted in robust virus-specific neutralizing antibody responses and protection against WNV-associated mortality in mice or viremia in rhesus macaques (RM). A GLP-compliant toxicology study performed in rats demonstrated an excellent safety profile with clinical findings limited to minor and transient irritation at the injection site. An in vitro relative potency (IVRP) assay was developed and shown to correlate with in vivo responses following forced degradation studies. Long-term in vivo potency comparisons between the intended storage condition (2–8 °C) and a thermally stressed condition (40 ± 2 °C) demonstrated no loss in vaccine efficacy or protective immunity over a 6-month span of time. Together, the positive pre-clinical findings regarding immunogenicity, safety, and stability indicate that HydroVax-001 WNV is a promising vaccine candidate.     

西尼罗病毒空斑形成试验方法的建立与应用

目的建立西尼罗病毒的空斑形成试验方法，应用于病毒滴度测定和实验感染蚊虫及来亨鸡血液样本中病毒的定量检测。方法将稀释的样本接种常规制备的Veto细胞单层，用琼脂糖凝胶覆盖，孵育一定时间后，加中性红染色，计数空斑数，计算空斑形成单位。结果在琼脂糖凝胶中西尼罗病毒可形成直径大约1～3mm的圆形或类圆形空斑。20％病毒鼠脑悬液的病毒滴度为10^7空斑形成单位。感染蚊虫样本和来亨鸡血液样本也出现典型的空斑。结论建立了西尼罗病毒的空斑形成试验方法，该方法可应用于病毒滴度测定和实验感染蚊虫及来亨鸡血液样本中病毒的定量检测。     

利用VSV△G＊伪型病毒建立安全、快速的西尼罗病毒抗体检测方法的尝试

目的为探讨利用水泡性口炎病毒（vesicular stomatitis virus,VSV）假病毒粒子系统包装西尼罗病毒（West Nile virus,WNV）的E蛋白可行性,构建3种E蛋白基因的真核表达质粒。以期用于WNV感染的流行病学调查。方法将表达WNN囊膜蛋白E的质粒体外转染293T细胞,然后感染VSV△G＊G,并对E蛋白表达质粒进行改造,分别在基因序列前加入信号肽,在终止密码子之前加入VSV△G蛋白的细胞质尾序列。结果包装出的VSV△G＊-WNVE滴度没有明显升高。提示WNV E蛋白与VSV的囊膜匹配性不佳,VSV△G＊-WNVE作为病毒中和抗原代替野毒进行抗体筛查还需要进一步提高转染和包装效率。结论VSV作为高效、安全的新型载体、活病毒疫苗载体和肿瘤基因治疗载体以及高危病毒的研究工具具有巨大的研究价值和应用潜力。     

新疆伊犁地区动物脑组织西尼罗病毒E基因片段的检测

目的了解新疆伊犁地区动物中西尼罗病毒（West Nile virus,WNV）感染现状,为我国WNV的防治提供资料。方法采用一步法实时荧光定量反转录-聚合酶链反应（RT-PCR）,对采自新疆伊犁地区的70头驴和100只牧羊犬的脑组织进行WNV包膜蛋白（E）基因片段检测。结果 70头驴和100只牧羊犬脑组织标本的西尼罗病毒包膜蛋白基因片段检测有2例可疑阳性,但经3%凝胶电泳法再次验证后排除。故所有待检样本WNV包膜蛋白基因检测均为阴性。结论我国新疆伊犁地区的驴和牧羊犬脑组织中未检测到WNV的感染。     

美国1999-2009年西尼罗病毒感染流行的回顾

西尼罗病毒(West Nile virus,WNV)是一种蚊传虫媒病毒,1937年从非洲乌干达西尼罗地区一名发热土著妇女血液标本中分离并得名[1].此后在非洲、欧洲和亚洲中东地区的一些国家出现WNV感染病例,表现以发热为主的临床症状,为自愈性疾病[2].但是1999年该病毒首次在美国流行,仅1个月时间报道数十例WNV感染所引发的病毒性脑炎[3].随后WNV感染在美国的分布范围逐渐扩大并迅速扩散到美国大陆全境,至今已经有数万人染病并致上千人死亡[4].     

天津市蚊虫西尼罗病毒携带调查

目的了解天津市蚊虫携带西尼罗病毒（WNV）情况。方法以灯诱法进行蚊虫采集,采用荧光定量PCR检测方法对WNV进行检测。结果天津市有候鸟栖息的湖泊、洼地周边主要蚊种为淡色库蚊（占93．68％）,同时存在凶小库蚊和三带喙库蚊。对1091只蚊虫进行西尼罗病毒检测,结果均为阴性。结论本次调查未发现蚊虫携带WNV,为预防和控制WNV传人及可能发生的流行,有必要开展对媒介蚊虫及WNV的长期监测。     

Epidemiological history and phylogeography of West Nile virus lineage 2

West Nile virus (WNV) was first isolated in Uganda. In Europe WNV was sporadically detected until 1996, since then the virus has been regularly isolated from birds and mosquitoes and caused several outbreaks in horses and humans. Phylogenetic analysis showed two main different WNV lineages. The lineage 1 is widespread and segregates into different subclades (1a–c). WNV-1a includes numerous strains from Africa, America, and Eurasia. The spatio-temporal history of WNV-1a in Europe was recently described, identifying two main routes of dispersion, one in Eastern and the second in Western Europe. The West Nile lineage 2 (WNV-2) is mainly present in sub-Saharan Africa but has been recently emerged in Eastern and Western European countries. In this study we reconstruct the phylogeny of WNV-2 on a spatio-temporal scale in order to estimate the time of origin and patterns of geographical dispersal of the different isolates, particularly in Europe. Phylogeography findings obtained from E and NS5 gene analyses suggest that there were at least two separate introductions of WNV-2 from the African continent dated back approximately to the year 1999 (Central Europe) and 2000 (Russia), respectively. The epidemiological implications and clinical consequences of lineage 1 and 2 cocirculation deserve further investigations.