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1.
Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic stability and growth characteristics of MVA-MERS-S make it a suitable candidate vaccine for clinical testing. Vaccinated mice produced high levels of serum antibodies neutralizing MERS-CoV. Thus, MVA-MERS-S may serve for further development of an emergency vaccine against MERS-CoV.  相似文献   

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Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH4Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.  相似文献   

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Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.  相似文献   

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将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)修饰的ORF5(MORF5)与ORF6基因分别克隆入笔者构建的改良型痘苗病毒安卡拉株(Modified vaccinia virus ankara, MVA) 转移载体pLR-gpt的两个多克隆位点中, 构建重组转移载体pLR-MORF5/ORF6。经脂质体介导, 将构建好的pLR-MORF5/ORF6 转染已感染亲本MVA 2h的BHK-21细胞单层, 用药物选择性培养基(MXHAT)在24孔板上进行连续筛选纯化, 得到带有筛选标记的重组病毒rMVAgpt-MGP5/M。将rMVAgpt-MGP5/M 感染表达Cre重组酶的BHK-21细胞(BHK-Cre), 获得删除筛选标记的重组病毒rMVA-MGP5/M。PCR证明MORF5与ORF6成功插入MVA基因组中; Western blotting与间接免疫荧光证明重组病毒能表达MGP5与M蛋白; 重组病毒的生长特性表明与亲本MVA出现病变的时间及病毒滴度基本一致。结果表明, 本研究成功构建了共表达PRRSV MGP5与M蛋白的重组MVA, 并且表达产物具有免疫原性; 外源基因的插入不影响MVA复制, 具较好的稳定性, 从而为PRRSV新型基因工程疫苗的研制奠定了良好的基础。  相似文献   

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将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)修饰的ORF5(MORF5)与ORF6基因分别克隆入笔者构建的改良型痘苗病毒安卡拉株(Modified vaccinia virus ankara, MVA) 转移载体pLR-gpt的两个多克隆位点中, 构建重组转移载体pLR-MORF5/ORF6。经脂质体介导, 将构建好的pLR-MORF5/ORF6 转染已感染亲本MVA 2h的BHK-21细胞单层, 用药物选择性培养基(MXHAT)在24孔板上进行连续筛选纯化, 得到带有筛选标记的重组病毒rMVAgpt-MGP5/M。将rMVAgpt-MGP5/M 感染表达Cre重组酶的BHK-21细胞(BHK-Cre), 获得删除筛选标记的重组病毒rMVA-MGP5/M。PCR证明MORF5与ORF6成功插入MVA基因组中; Western blotting与间接免疫荧光证明重组病毒能表达MGP5与M蛋白; 重组病毒的生长特性表明与亲本MVA出现病变的时间及病毒滴度基本一致。结果表明, 本研究成功构建了共表达PRRSV MGP5与M蛋白的重组MVA, 并且表达产物具有免疫原性; 外源基因的插入不影响MVA复制, 具较好的稳定性, 从而为PRRSV新型基因工程疫苗的研制奠定了良好的基础。  相似文献   

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The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.  相似文献   

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Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA401 or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA401 induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.Close to 2.3 million of children globally are infected with human immunodeficiency virus type 1 (HIV-1). The majority of neonatal infections occur in utero or intrapartum and, in the absence of preventative interventions, up to 29% of infants breast-fed by infected mothers acquire HIV-1 (6). Furthermore, HIV-1-infected children face a worse prognosis than adults in that, without antiretroviral treatment (ART), 25% of perinatally infected children progress to AIDS within 1 year (10), and the median time to AIDS for the remaining children is less than 7 years (2). It is now clearly established that maternal and extended infant ART can substantially reduce transmission of HIV-1 through breast-feeding (23). However, in a resource-poor setting, many logistical barriers to implementation of the ART-based prevention of mother-to-child-transmission (PMTCT) remain (23). Because nutrition and hygiene makes breast milk an important determinant of infant survival (22, 28), formula feeding as a protective measure against HIV-1 acquisition is recommended only if it is AFASS (acceptable, feasible, affordable, sustainable, and safe). Unfortunately, AFASS it is still not for majority of infected mothers in sub-Saharan Africa. Also, mixed bottle and breast feeding is associated with a 10-fold increase in HIV-1 transmission relative to exclusive breast-feeding (4). Thus, an effective infant vaccine against HIV-1 infection is the best and safest solution for PMTCT of HIV-1 with the added practical option of prolonging breast-feeding.Neonatal immunity is immature compared to the adult immune system (25). The differences include naivety of the immune cells, a tendency to develop Th2 responses (5) and antigen-presenting cells with inefficient cytokine production (35). For example, human cord blood T cells proliferated poorly and produced low levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) when endogenous antigen-presenting cells presented the antigen (35, 44). Also, infant myeloid dendritic cells are less efficient in priming Th1 responses because of their decreased responsiveness to Toll-like receptor stimulation, lower levels of surface costimulatory molecules, and lower production of IL-12 (8, 27). In several infections, qualitative and quantitative differences between human newborn and adult responses were detected (1, 9, 26, 37). In contrast, other studies of infants reported proliferation as well as IL-2 and IFN-γ production by T cells equal to that of adults following T-cell receptor-independent activation (21, 46). These latter observations indicate that neonate T cells are not intrinsically “locked” into an immature phenotype but, given the correct stimuli, they can develop mature immune responses (25). The requirement for specific stimuli will likely differ for different pathogens and vaccine vectors.Mycobacterium bovis bacillus Calmette-Guérin (BCG) is commonly delivered at birth as an antituberculosis vaccine as a part of the WHO Expanded Programme on Immunization (EPI). It has been reported by several studies to promote an adultlike Th1 response in newborns (16, 24, 34, 43), although it was also suggested that delaying the BCG delivery to 10 weeks of age benefits the quantity and quality of BCG-induced CD4 T-cell responses (20). BCG and related mycobacterial vectors have been explored as vaccines against other infectious agents, including human and simian immunodeficiency viruses (19), and in adult animals showed immunogenicity and protection (3, 36, 39, 47, 48). The only clinical study of recombinant BCG (rBCG) in adults failed to provide consistent efficacy (7). We have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a heterologous vaccine platform for PMTCT of HIV-1 through infected breast milk (18), where it is critical to prime HIV-1-specific responses as soon as possible after birth. These responses could be boosted a few weeks later or shortly after the already busy EPI by heterologous vaccines delivering the same HIV-1-derived immunogen. To this extent, we constructed the novel candidate vaccine BCG.HIVA401 (36) by inserting a gene coding for the HIV-1 clade A-derived immunogen HIVA (14) into recombinant BCG strain AREAS-401 (40). AERAS-401 is a newly developed strain that displayed enhanced safety (40) and immunogenicity (11, 15) in murine models relative to its parental BCG vaccine strain Danish SSI-1331. Increased safety represents an important feature should the BCG.HIVA401 vaccine be deployed in babies born to HIV-1-infected mothers. We showed that BCG.HIVA401 in a heterologous combination with recombinant modified vaccinia virus Ankara MVA.HIVA and recombinant ovine atadenovirus OAdV.HIVA induced robust polyfunctional HIV-1-specific T-cell responses in adult macaques (36). Here, we assess the safety and immunogenicity of the BCG.HIVA prime-MVA.HIVA boost regimen in newborn rhesus macaques.  相似文献   

13.
To understand how Middle East respiratory syndrome coronavirus (MERS-CoV) transmitted from bats to humans, we compared the virus surface spikes of MERS-CoV and a related bat coronavirus, HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases. These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells. These mutations therefore played critical roles in the bat-to-human transmission of MERS-CoV, either directly or through intermediate hosts.  相似文献   

14.
We identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also significantly inhibited infection. These findings indicate that both 2F9 and YS110 are potential therapeutic agents for MERS-CoV infection. YS110, in particular, is a good candidate for immediate testing as a therapeutic modality for MERS.  相似文献   

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对表达狂犬病毒糖蛋白的重组痘苗病毒VVM11KRG株生物学性质进行了研究,该重组病毒的特点是:(1)糖蛋白基因插入到痘苗病毒天坛株基因组HindⅢM片段中;(2)启动子为痘苗病毒天坛株P11晚期启动子;(3)不含外源lac基因。该重组病毒在CV-1细胞和鸡胚细胞上繁殖滴渡略高于另一株重组痘苗病毒VVTK11KRG,在鸡胚细胞中繁殖滴度第三天达到最高,在CV-1细胞中第二天达到最高。温度稳定性与天坛株相比没有明显改变。重组病毒在家兔皮内的毒力比亲本株(天坛株)低。间接免疫荧光和Westernblot都证明了狂犬病毒糖蛋白有良好的表达。通过Southernblot证实糖蛋白基因准确地插入到HindⅢM片段中。重组痘苗病毒启动子与部分糖蛋白基因克隆到pGEM3zf(-)质粒上,对该段DNA序列分析表明不会产生移码和融合蛋白,从而为该重组病毒的使用提供了明确的基因背景资料。  相似文献   

17.
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.  相似文献   

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目的:为了避免中东呼吸综合征冠状病毒(MERS-CoV)感染与中和试验中操作活病毒带来的生物安全隐患,构建只具有一次感染能力而无复制能力的MERS假病毒,建立MERS假病毒系统,并应用于中和抗体检测。方法:构建含有MERS-CoV S基因的重组质粒pcDNA3.1-MERS-S,与缺失Env基因、含有萤光素酶报告基因的HIV-1骨架质粒pNL4-3.Luc.RE共转染293T细胞,收获含有假病毒的上清;通过Western印迹、细胞感染实验和血清中和试验,确定是否包装出MERS假病毒,及是否能有效应用于细胞感染与中和试验。结果:MERS假病毒pMERS-S培养上清经Western印迹鉴定出相对分子质量为25×103的HIV-1 P24蛋白和相对分子质量为180×103的MERS-CoV S蛋白;与阴性对照假病毒pEnv-相比,pMERS-S能有效感染MERS-CoV敏感细胞系Huh-7,在感染细胞中产生荧光信号,感染细胞的假病毒量与产生的荧光信号呈明显的量效关系;在MERS假病毒中和试验中,pMERS-S能被MERS-CoV中和抗体中和而失去感染力,反映抗体对MERS-CoV的中和活性。结论:建立了不依赖于BSL-3高等级生物安全条件的MERS假病毒系统,并有效应用于中和抗体检测,为MERS-CoV疫苗、药物评价及病毒致病机制研究提供了良好的技术支撑手段。  相似文献   

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Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater than 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.  相似文献   

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