Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates

Containment of highly lethal Ebola virus outbreaks poses a serious public health challenge. Although an experimental vaccine has successfully protected non-human primates against disease, more than six months was required to complete the immunizations, making it impractical to limit an acute epidemic. Here, we report the development of accelerated vaccination against Ebola virus in non-human primates. The antibody response to immunization with an adenoviral (ADV) vector encoding the Ebola glycoprotein (GP) was induced more rapidly than with DNA priming and ADV boosting, but it was of lower magnitude. To determine whether this earlier immune response could nonetheless protect against disease, cynomolgus macaques were challenged with Ebola virus after vaccination with ADV-GP and nucleoprotein (NP) vectors. Protection was highly effective and correlated with the generation of Ebola-specific CD8(+) T-cell and antibody responses. Even when animals were immunized once with ADV-GP/NP and challenged 28 days later, they remained resistant to challenge with either low or high doses of virus. This accelerated vaccine provides an intervention that may help to limit the epidemic spread of Ebola, and is applicable to other viruses.     

埃博拉病毒感染数量的一个数学模型

埃博拉病毒病(EVD)是严重的、往往致命的人类疾病,病死率高达90%.埃博拉病毒病疫情主要发生在中非和西非靠近热带雨林的边远村庄.该病毒通过野生动物传到人,并且通过人际间传播在人群中蔓延.病情严重的患者需要获得重症支持治疗,无论对人还是对动物都无可用的已获正式许可的特异性治疗办法或者疫苗.由于缺乏有效的治疗手段和人用疫苗,提高对感染埃博拉危险因素的认识以及个人可以采取一些保护措施,这是减少人类感染和死亡的唯一方法.本文建立一个埃博拉病毒的数学模型,对疫情进行实证分析;并且对疫情的发展也做了一个预测.     

埃博拉病毒入侵宿主细胞的分子机制

 埃博拉病毒是一类能够感染并引起人和灵长类动物发生埃博拉出血热的烈性囊膜病毒。发现近40年中,埃博拉病毒给人类生命带来了极大威胁。然而,目前人们对于埃博拉病毒的了解非常有限,尤其是病毒与其宿主细胞受体的结合机制和膜融合机制相关信息的缺失,使得针对埃博拉病毒的特效药物的设计和研发工作阻碍重重。本文综述了埃博拉病毒分类、形态、病毒蛋白和病毒生命周期,着重介绍了高福院士团队在埃博拉病毒入侵宿主细胞的分子机制研究中的成果。通过结构学手段解析了埃博拉病毒激活态囊膜糖蛋白GPcl与宿主细胞受体NPC1分子的复合物结构,从原子水平上阐明了埃博拉病毒与宿主细胞相互识别的机制,并在结构基础上对病毒的膜融合促发机制做出推测,提出以埃博拉病毒为代表的新的（第5种）囊膜病毒膜融合激发机制,为抗埃博拉病毒药物和疫苗的设计提供了结构基础。     

Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys.

Infection of macaques with simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) are useful models for studies of immunotherapy and vaccination against HIV as well as for testing of antiviral drugs. Vaccine research showing protective immunity in immunized monkeys has indicated that it will be possible to develop a vaccine for prevention of human HIV infection, although many hurdles remain. The design of an HIV vaccine would be helped if the basis of the protective immunity could be elucidated. Passive immune prophylaxis offers a means to determine the relative role of antibodies in protection against infection. We have studied whether a transfer of antibodies can prevent HIV-2 and SIVsm (SIV of sooty mangabey origin) infection in cynomolgus monkeys. Sera with high antibody titres were collected, heat-treated and injected into naive animals 6 h before challenge with 10-100 monkey-infectious doses of live homologous virus. All control animals treated with normal monkey serum (n = 6) or no serum (n = 39) became infected by the challenge virus, whereas five out of seven animals pretreated with antibody-containing serum at a dose of 9 ml kg-1 resisted infection. Thus passively transferred antibodies can protect against a low-dose lentivirus challenge in a nonhuman primate.     

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.     

埃博拉病毒感染的实验室诊断方法研究进展

 埃博拉病毒病是由埃博拉病毒引起的一种急性出血性传染病,具有极高的传染性,病死率高达90%,世界卫生组织已将埃博拉病毒列为对人类危害最严重的病毒之一,目前虽有一个被美国食品和药物管理局(FDA)批准用于紧急治疗的实验药物TKM-Ebola,但尚无批准上市的用于预防埃博拉病毒的疫苗.建立快速、准确、简便的实验室检测方法对及时进行临床诊断救治、开展流行病学调查并最终控制其传播流行具有重要意义.本文综述埃博拉病毒实验室检测方法的原理、应用及进展,介绍病毒分离、电子显微镜观察、逆转录聚合酶链反应法、抗原检测试验、抗体酶联免疫吸附试验、血清中和试验及其他检测方法.     

Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.     

Fruit bats as reservoirs of Ebola virus

The first recorded human outbreak of Ebola virus was in 1976, but the wild reservoir of this virus is still unknown. Here we test for Ebola in more than a thousand small vertebrates that were collected during Ebola outbreaks in humans and great apes between 2001 and 2003 in Gabon and the Republic of the Congo. We find evidence of asymptomatic infection by Ebola virus in three species of fruit bat, indicating that these animals may be acting as a reservoir for this deadly virus.     

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1.     

埃博拉病毒病：流行病学、生态学、诊断、治疗及控制

 埃博拉病毒（Ebolavirus）是埃博拉病毒病（Ebola Virus Disease, EVD）的病原体,1976 年首次在非洲发现,目前确认该病毒包括5 个种,其中苏丹型（Sudan ebolavirus）、扎伊尔型（Zaire ebolavirus）、塔伊森林型（Taï Forest ebolavirus）和本迪布焦型（Bundibugyo ebolavirus）均有感染人发病的记录;莱斯顿型（Reston ebolavirus）可致人隐性感染,并与多起猕猴暴发疫情有关,曾在菲律宾的猪中检出。人类埃博拉病毒病的病死率为25%~90%,疫情均发生在非洲,主要集中在10°N-10°S 的非洲地区,本次西非疫情是规模最大的暴发流行,截至2014 年8 月20 日已报告2615 例病例。该病是动物源性传染病,目前证据支持果蝠可能为病毒的自然储存宿主。该病在人群中主要通过接触传播,有症状的病人才具有传染性。未采取正确防护措施的医护人员、家庭护理人员及接触病人血液、体液,或接触病人血液、体液等污染的物品,或接触病例尸体的人是高风险感染人群。本病起病急,早期表现为发热、厌食、虚弱无力等非特异性症状,可通过检测病毒核酸、抗原、抗体等方法确诊。目前尚无批准上市的特效药和疫苗,以对症和支持治疗为主。预防控制策略主要包括早期发现病例、及时调查处置、追踪和密切观察接触者,以及有效的医院内和社区的感染控制。     

Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen

A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant.     

Chemical basis of antigenic variation in foot-and-mouth disease virus

One of the difficulties in controlling foot and mouth disease by vaccination is the occurrence of the virus as seven distinct serotypes because immunity conferred by vaccination against one serotype leaves the animals susceptible to infection by the other six. Moreover, the antigenic variation, even within a serotype, can be so great that immunity against the homologous strain of virus need not necessarily ensure protection against infection by other viruses within that serotype. Here we report the separation of three natural antigenic variants, distinguishable in cross-neutralization tests from an isolate of foot-and-mouth disease virus (FMDV). The serological differences could also be demonstrated by antisera elicited by synthetic peptides corresponding to residues 141-160 of the capsid polypeptide VP1, showing that this region contains a major immunogenic site of the virus. The results have practical implications for the choice of viruses for vaccine production.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (T(EM)) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIV(MAC239) infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4(+) memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8(+) T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8(+) or CD4(+) lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated T(EM) responses might significantly contribute to an efficacious HIV/AIDS vaccine.     

Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine.

Rabies infection of domestic and wild animals is a serious problem throughout the world. The major disease vector in Europe is the red fox (Vulpes vulpes) and rabies control has focused on vaccinating and/or culling foxes. Culling has not been effective, and the distribution of five vaccine baits is the only appropriate method for the vaccination of wild foxes. Although some European countries have conducted field vaccination campaigns using attenuated rabies virus strains, their use has not been extensively approved because they retain pathogenicity for rodents and can revert to virulence. These strains cannot be used in North America because they are pathogenic for the striped skunk (Mephitis mephitis) and are ineffective in the raccoon (Procyon lotor). We have constructed a recombinant vaccinia virus, VVTGgRAB, expressing the surface glycoprotein (G) of rabies virus (ERA strain). The recombinant was a highly effective vaccine in experimental animals, in captive foxes and in raccoons. We report here the results of a large-scale campaign of fox vaccination in a 2,200 km2 region of southern Belgium, an area in which rabies is prevalent. After distribution, 81% of foxes inspected were positive for tetracycline, a biomarker included in the vaccine bait and, other than one rabid fox detected close to the periphery of the treated area, no case of rabies, either in foxes or in domestic livestock, has been reported in the area.     

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.     

HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus

Early treatment of acute HIV-1 infection followed by treatment interruptions has shown promise for enhancing immune control of infection. A subsequent loss of control, however, allows the correlates of protective immunity to be assessed. Here we show that sudden breakthrough of plasma viraemia occurred after prolonged immune containment in an individual infected with HIV-1 at a time when 25 distinct CD8+ T-cell epitopes in the viral proteins Gag, RT, Integrase, Env, Nef, Vpr, Vif and Rev were being targeted. Sequencing of the virus in plasma and cells showed that superinfection with a second clade-B virus was coincident with the loss of immune control. This sudden increase in viraemia was associated with a decline in half of the CD8+ T-cell responses. The declining CD8+ T-cell responses were coupled with sequence changes relative to the initial virus that resulted in impaired recognition. Our data show that HIV-1 superinfection can occur in the setting of a strong and broadly directed virus-specific CD8+ T-cell response. The lack of cross-protective immunity for closely related HIV-1 strains, despite persistent recognition of multiple CD8 epitopes, has important implications for public health and vaccine development.     

Effect of immunization with a vaccinia-HIV env recombinant on HIV infection of chimpanzees

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV) and is now recognized as a worldwide epidemic for which there is no cure or vaccine. Chimpanzees are the only other animals that can be infected by HIV, and therefore the chimpanzee-HIV model system is useful for testing potential HIV vaccines. However, with one exception, there have been no reports of clinical manifestations of AIDS in chimpanzees. We report here results of an HIV vaccine trial in which nine chimpanzees were first immunized with either a recombinant vaccinia virus expressing the envelope glycoproteins of HIV strain LAV-1 (v-env5) or a control recombinant vaccinia virus and were then challenged with a high or low dose of LAV-1. Although HIV-specific antibody and T-cell responses were elicited by immunization, virus was isolated from lymphocytes of all challenged chimpanzees, indicating that immunization did not prevent infection by HIV. Among the animals that received a higher dose of LAV-1, one of two control chimpanzees, but none of the four v-env5-immunized chimpanzees developed substantial and persistent lymphadenopathy.     

Characterization of the Antigenicity and Immunogenicity of the Escherichia Coli Produced RNase Domain of the Classical Swine Fever Virus Glycoprotein E

E^rns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. E^rns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the E^rns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the E^rns retained its antigenicity and induced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The results suggest that other essential factors rather than simply enhancing the immunogenicity of E^rns should be taken into consideration when E^rns is enrolled as one of the components of a candidate vaccine.     

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.