A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

Protective Efficacy of Centralized and Polyvalent Envelope Immunogens in an Attenuated Equine Lentivirus Vaccine

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.     

中国株EIAV S2基因逆向突变感染性克隆的构建及体外感染性评价

以中国株EIAV的驴强毒株EIAVDV115与疫苗株EIAVFDDV15的S2基因变化规律为依据,利用反向遗传技术对EIAV弱毒疫苗株全基因组感染性克隆pFDDV3-8的S2基因区进行逆向突变,构建了含有强毒株EIAVDV115S2基因区内4个稳定点突变的克隆质粒pFDDVS2r1-3-4-5,将该质粒转染FDD后进行体外继代盲传,经RT-PCR、逆转录酶活性、间接免疫荧光等检测后,显示经EIAV克隆质粒pFDDVS2r1-3-4-5转染的FDD盲传3代后,可在转染的FDD细胞培养物的上清液中检测到EIAV逆转录酶活性;该细胞培养物经RT-PCR和间接免疫荧光检测均呈EIAV阳性;在电镜下可观察到典型EIAV粒子。证明已成功拯救经EIAVS2基因逆向突变后的衍生病毒vpFDDVS2r1-3-4-5。比较vpFDDVS2r1-3-4-5衍生病毒与亲本克隆衍生病毒的复制动力学,表明前者的复制比后者略滞后。以上结果提示,对疫苗株S2基因的突变未明显影响EIAV的体外复制。     

马传染性贫血强/弱毒嵌合病毒的体外构建

马传染性贫血病毒(equine infectious anemia virus,EIAV)引起马传染性贫血(简称马传贫),导致马持续性感染和反复病毒血症[1].EIAV与人免疫缺陷病毒Ⅰ型(HIV-1)同属反转录病毒科慢病毒属,二者有很多相似的特性[2].在反转录病毒前病毒基因组两端含有长末端重复序列(long terminal repeat,LTR).LTR含有真核启动子,其中含有病毒转录调控顺式作用位点,病毒编码的反式作用因子与其结合后可以反式激活基因的表达,对病毒基因的表达和其它生命活动起重要调控作用[3,4].因此,LTR序列的变异可能会引起病毒转录和复制方式的改变,进而引起其细胞嗜性和致病性的改变[5,6].为了探讨LTR在EIAV病毒复制和转录过程中的作用,并进一步研究EIAV的致病和免疫机制,用EIAV强毒L株LTR置换了以前构建的EIAV DLA(弱毒)感染性分子克隆中的LTR,构建了马传贫强/弱毒嵌合分子克隆,并获得了具有感染性的强/弱毒嵌合病毒.     

EIAV弱毒疫苗免疫马外周血单核细胞IL-2表达水平的研究

目的:通过定量监测马传染性贫血病毒(EIAV)弱毒疫苗免疫马外周血单核细胞(PBMC)IL-2表达水平的变化特征,探讨EIAV弱毒疫苗的免疫保护机制。方法:用实时定量RT-PCR技术建立了马外周血PBMCIL-2表达水平的定量检测方法。在不同的时间点定期对4组(疫苗免疫组、健康对照组、强毒攻毒组和EIAV自然感染组)12匹马的外周血PBMCIL-2表达水平进行了检测,同时观察了临床症状及体温变化等指标。疫苗株免疫动物8个月后用强毒攻毒,观察了攻毒前后IL-2表达水平的变化。结果:(1)疫苗免疫马外周血PBMCIL-2的表达量显著高于健康对照组及自然感染组(P<0.01),且免疫后攻毒IL2继续升高,4匹疫苗免疫马均获得完全保护;(2)强毒攻毒对照组IL2表达量随疾病进展波动,发热期明显下降。结论:首次证明EIAV弱毒疫苗可诱导马外周血PBMC表达高水平的IL-2,提示IL-2在疫苗的免疫保护应答中发挥了重要作用;IL-2表达水平还与EIAV感染后的疾病进展密切相关。     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.     

T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

[目的]马传染性贫血病毒(EIAV)弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型.为探讨IFN-γ表达水平与疫苗保护性免疫的关系,本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-γ水平的方法.[方法]我们将分离的马传贫弱毒疫苗免疫马(FDDV)、强毒感染马(LV)和健康马的外周血单核细胞(PBMC),体外分别经病毒(FDDV)和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-γ抗体等过程后,进行流式检测.[结果]疫苗免疫马产生的特异性IFN-γ水平为CD4 1.7(0.9%/CD8 6.1(1.2%,而强毒组则为CD4 0.6(0.1%/CD8 2.4(0.9%.[结论]本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-γ染色和淋巴细胞亚型的方法,具有良好的特异性,稳定性和重复性.为研究EIAV弱毒疫苗免疫保护机制奠定了基础.     

Enhanced safety and efficacy of live attenuated SIV vaccines by prevaccination with recombinant vaccines

The only vaccines shown to be protective against intravenous challenge with virulent virus in the simian immunodeficiency virus (SIV)/macaque model are attenuated live SIVs. However, these vaccines have several disadvantages: 1) they persist indefinitely in vaccinated macaques; 2) they are pathogenic to neonatal macaques; and 3) they are lethal in some adult macaques. To enhance the safety and efficacy of these vaccines, we immunized macaques first with recombinant vaccines and then inoculated the animals with SIV(delta(nef)). In the first experiment, preimmunized macaques advanced to disease slower than controls after challenge with virulent SIV; five animals survived for 3 years without disease and only the vaccine virus (SIV(delta(nef)) could be isolated at this time. In the second experiment, preimmunized animals had lower virus loads and no disease compared to controls.     

马传染性贫血病毒减毒疫苗诱导马外周血单个核细胞白细胞介素12的转录

目的 探讨马传染性贫血病毒(EIAV)驴白细胞减毒疫苗(DLV)免疫马后,外周血单个核细胞(PBMC)中白细胞介素12(IL-12)mRNA转录水平与免疫保护应答的关系,揭示DLV的免疫保护机制。方法 应用分子克隆及实时定量反转录-聚合酶链反应(RT-PCR)技术,建立马PBMC中IL-12 mRNA转录水平的定量检测方法,在不同时间点定期观察4组(疫苗免疫组、阴性对照组、强毒株阳性对照组和EIAV自然感染组)12匹马PBMC中IL-12 mRNA转录水平及分布特征,同时监测体温变化等指标。疫苗株免疫动物8个月后,用EIAV强毒株攻击,观察攻击前、后IL-12 mRNA转录水平的变化。结果 DLV免疫马在免疫期内,PBMC中IL-12 mRNA转录的量略高于阴性对照组及自然感染组,但免疫后8个月用EIAV强毒株攻击,IL-12 mRNA转录量显著升高,4匹免疫马获得完全保护;强毒株阳性对照组IL-12转录量随疾病进展波动,发热期下降。结论 本研究首次证明EIAV减毒疫苗可诱导马外周血PBMC中IL-12基因高效转录,其转录水平与DLV的免疫保护密切相关。此结果在分子水平为阐明DLV的免疫保护机制提供了新的实验依据。     

马传染性贫血病毒非编码区LTR嵌合克隆的生物学特性

将已构建的马传染性贫血病毒LTR强弱毒嵌合克隆衍生毒vLGFD9-12体内接种健康试验马,在150d观察期内,各组试验动物体症均未见异常。血液学分析发现,vLGFD9-12嵌合克隆衍生病毒与亲本弱毒疫苗株的白细胞与血红蛋白含量总体上没有明显的规律性的变化。在动物外周血中均检测到一定的病毒RNA拷贝数,但拷贝数较低。二者在诱导EIAV特异性淋巴细胞增殖功能和特异性细胞毒性杀伤反应中,亦具有相似的变化趋势和效应。本项研究为进一步确定我国马传贫弱毒疫苗株毒力致弱及免疫保护的分子机制奠定了重要的分子生物学基础。     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

马传染性贫血病毒白细胞弱毒疫苗株及其亲本毒驴强毒株前病毒基因组比较分析

为阐明马传染性贫血白细胞弱毒疫苗株(EIAVDLV)的致弱和免疫保护机理,对EIAVDLV121及其亲本驴强毒株(EIAVDV117)前病毒全基因组序列进行了测定,并结合准种理论,分析了EIAV疫苗致弱过程中基因组进化特点。利用LA-PCR技术对EIAVDV117和EIAVDLV121的前病毒基因组分两段进行扩增,分别获得4个和10个前病毒全基因组序列。EIAVDV117前病毒基因组平均为8236bp,G C含量38.0。EIAVDLV121前病毒基因组平均8249bp,G C含量37.3。两者的前病毒基因组平均差异率为2.8。其中S2、LTR和env基因差异较大,分别为4.1、3.9和3.1。此外,S2、S3和env推导的氨基酸的差异明显,分别为10.4、5.6和4.8(gp90为6.8)。EIAVDLV121各基因的异质性均显著高于EIAVDV117。研究发现体外培养的EIAVDLV121至少有5种类型的LTR混合存在。在gp90推导的氨基酸序列上,EIAVDV117比EIAVDLV121平均多2个N-糖基化位点,总数为19,其中3个为EIAVDV117特有。EIAVDLV121有1个疫苗株特有N-糖基化位点。研究结果为进一步探讨马传染性贫血弱毒疫苗生物学特性提供信息。     

马传染性贫血弱毒疫苗株核衣壳蛋白基因的表达与鉴定

从感染驴白细胞的马传染性贫血弱毒疫苗株前病毒DNA中克隆了编码核衣壳蛋白 (pll)的基因 ,在大肠杆菌中得到了表达 ,而表达的蛋白是一种可溶性的融合蛋白 ,其氨基端带有 6个组氨酸的标签 ,因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。经间接ELISA和免疫印迹试验检测 ,这种表达的融合蛋白可与马传贫阳性血清样品发生反应 ,而与健康马血清无任何反应 ,显示其具有良好的抗原性和特异性 ,可用于马传贫弱毒疫苗株在体内外复制及在接种马体内免疫应答的研究。     

Vaccination with inactivated virus but not viral DNA reduces virus load following challenge with a heterologous and virulent isolate of feline immunodeficiency virus

It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIV(PET)) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIV(AM6) and FIV(GL8). This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIV(GL8)-infected cats. In contrast, DNA vaccines based on either FIV(PET) or FIV(GL8), which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIV(PET) but had no measurable effect on virus load when the infecting virus was FIV(GL8). These results indicate that the more virulent FIV(GL8) is intrinsically more resistant to vaccinal immunity than the FIV(PET) strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.     

Host Defense Mechanism-Based Rational Design of Live Vaccine

Live attenuated vaccine (LAV), mimicking natural infection, provides an excellent protection against microbial infection. The development of LAV, however, still remains highly empirical and the rational design of clinically useful LAV is scarcely available. Apoptosis and caspase activation are general host antiviral responses in virus-infected cells. Utilizing these tightly regulated host defense mechanisms, we present a novel apoptosis-triggered attenuation of viral virulence as a rational design of live attenuated vaccine with desired levels of safety, efficacy, and productivity. Mutant influenza viruses carrying caspase recognition motifs in viral NP and the interferon-antagonist NS1 proteins were highly attenuated both in vitro and in vivo by caspase-mediated cleavage of those proteins in infected cells. Both viral replication and interferon-resistance were substantially reduced, resulting in a marked attenuation of virulence of the virus. Despite pronounced attenuation, the viruses demonstrated high growth phenotype in embryonated eggs at lower temperature, ensuring its productivity. A single dose vaccination with the mutant virus elicited high levels of systemic and mucosal antibody responses and provided complete protection against both homologous and heterologous lethal challenges in mouse model. While providing a practical means to generate seasonal or pandemic influenza live vaccines, the sensitization of viral proteins to pathogen-triggered apoptotic signals presents a potentially universal, mechanism-based rational design of live vaccines against many viral infections.     

Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.     

马传染性贫血病毒N-连接糖基化回复突变的感染性克隆构建

根据马传贫强毒株EIAV-L和疫苗株EIAV-FDD表面蛋白gp90的N-连接糖基化的变化规律,采用PCR定点突变的方法,对全长感染性克隆pLGFD3-8上的N-连接糖基化的差异区域进行改造后,构建成含有3个N-连接糖基化位点突变的感染性克隆pLGNl91N236N246.将其转染驴胎皮肤细胞(FDD),通过用逆转录酶活性、间接免疫荧光和RT-PCR方法检测而确定其感染性.结果表明,在FDD细胞中盲传三代后,在细胞培养物中可检测到逆转录酶活性,RT-PCR和间接免疫荧光检测均呈阳性,电镜下见到典型的EIAV颗粒.这一结果可能对N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究而奠定良好的基础.     

Preexisting vaccinia virus immunity decreases SIV-specific cellular immunity but does not diminish humoral immunity and efficacy of a DNA/MVA vaccine

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.     

A modified vaccinia Ankara virus (MVA) vaccine expressing African horse sickness virus (AHSV) VP2 protects against AHSV challenge in an IFNAR -/- mouse model

African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2).