埃博拉疫苗和药物研究进展

埃博拉病毒(Ebola virus)是丝状病毒科的一员,可导致埃博拉出血热,致死率达25%~90%不等。2014年暴发的埃博拉疫情席卷了西非各地,极高的致死率引起了全世界的恐慌。鉴于埃博拉病毒严重威胁人类公共健康,科学研究毫无懈怠,多种疫苗如rVSV-ZEBOV和ChAd3-ZEBOV等已经进入临床试验阶段,多种药物如TKMEbola和ZMapp等已用于埃博拉患者的紧急治疗。本文总结了近期埃博拉疫苗和药物的最新研究进展。     

埃博拉病毒疫苗研究进展及相关专利浅析

通过非专利数据库的文献检索,总结了埃博拉病毒（EBOV）研究的最新进展,通过专利文献数据库文献检索分析EBOV疫苗的专利申请基本情况、发展趋势和技术点。结果发现专利申请量与疫情的爆发有一定的相关性;专利申请人多为政府主导的机构或非营利性机构;最新的专利申请均关注GP蛋白对病毒侵染的重要性,针对GP蛋白的抗体和疫苗正在开发中,其中Mapp生物制药公司的产品“ZMapp”作为试验性生物药物目前效果最佳。     

埃博拉病毒疫苗rVSV-ZEBOV的研究进展

埃博拉病毒被列为A类病原体,感染后可引起埃博拉出血热,具有高传染率和高致死率。研发安全有效的抗病毒疫苗迫在眉睫。目前正在研发的埃博拉病毒疫苗包括病毒载体疫苗、蛋白疫苗、DNA疫苗等,其中最有希望的是重组水疱性口炎病毒载体疫苗rVSV-ZEBOV。该疫苗在预防和治疗埃博拉出血热方面具有较高的安全性和有效性,有望在2018年上市。为了深入了解rVSV-ZEBOV疫苗,现主要从制备方法、药理学研究和作用机制等方面对该疫苗进行介绍。     

重组水疱性口炎病毒疫苗的研制现状

水疱性口炎病毒引起的水疱性口炎是一种人兽共患病.该病毒可刺激感染的宿主产生细胞、体液和黏膜免疫应答,是一种有希望的疫苗载体,可表达病毒、细菌和寄生虫的多种蛋白.以水疱性口炎病毒为载体的病原体疫苗包括埃博拉病毒(rVSV-GP)、马尔堡病毒(rVSV-G)、Ⅰ型人类免疫缺陷病毒(rVSV-env/gag)、猴免疫缺陷病毒...     

埃博拉病毒疫苗和药物的研发进展及启示

2014年空前的西非埃博拉疫情,使得埃博拉病毒受到了全世界的广泛关注。针对埃博拉病毒病,目前还没有获批上市的疫苗和药物,但有几种疫苗和药物已在非人灵长类动物中证实了保护效果,其中少数品种已进入临床试验阶段。本文集中介绍几种最具前景的疫苗和药物,并对其发展前景进行了分析和预测。     

表达埃博拉病毒表面糖蛋白的rVSV载体疫苗的效力与效果

正以表达扎伊尔埃博拉病毒表面糖蛋白的重组、可复制水泡性口炎病毒为基础的疫苗(rV SV-ZEBOV)是有希望的埃博拉病毒候选疫苗。本文主要描述了在西非的几内亚地区所做临床试验临时分析的结果。此次标签开放、群随机的环状疫苗接种实验,Basse-Guinée(几内亚,西非)的埃博拉病毒疑似病例由国家检测系统的埃博拉应急小组独立进行确诊。     

The structure of vesicular stomatitis virus membrane A phosphorus nuclear magnetic resonance approach

The proton decoupled 40.48 M Hz ³¹P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (serotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The ³¹P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups.The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the ³¹P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the ³¹P phospholipid spectrum were also demonstrated.     

Association of polyadenylic acid with messenger RNA of vesicular stomatitis virus

Polyadenylate (poly(A)) sequences are associated with the 28 S and 13–15 S messenger RNA species of vesicular stomatitis virus. These sequences contain approximately 125 to 150 nucleotides. Virion RNA contains little or no poly(A) sequences. The association of poly(A) with viral messenger RNA species and the gross distribution of poly(A) among these species remain unaltered even when the RNA is synthesized in the presence of cordycepin or cycloheximide and whether viral messenger RNA is polyribosome-bound or free. Also, when viral translation is completely inhibited by superinfection with poliovirus, there is no effect on poly(A) association with the messenger RNA of vesicular stomatitis virus.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

SARS冠状病毒病毒样颗粒疫苗的研究进展

2003年严重急性呼吸综合征(SARS)大流行之后,研究人员为防控SARS冠状病毒(SARS-CoV)的传播研制了多种不同形式的候选疫苗,大多是利用SARS-CoV表面的一种或多种结构蛋白制备的。SARS-CoV病毒样颗粒是具有较好的应用前景的一种候选疫苗,现将其制备方法及免疫原性做简要综述。     

Expression of the vesicular stomatitis virus nucleocapsid protein controlled by the lambda PR and PL promoters in Escherichia coli

Expression vectors were constructed in which a cDNA specifying the vesicular stomatitis virus nucleocapsid (VSV N) protein was inserted near the translational initiation region downstream from the thermoinducible P_R or P_L promoter of bacteriophage λ. Expression of the VSV N-protein was determined by a radioimmunoassay with monoclonal antibody prepared against the VSV N-protein. The expression of the VSV N-protein in Escherichia coli was low with either system. However, the deletion of a part of leader sequence from the translational initiation signal to the VSV N-gene resulted in at least 30-fold increase in production of the VSV N-protein. The VSV N-protein in E. coli was also analyzed by radioimmune blot after separation of proteins by gel electrophoresis. Degraded proteins reacted with the antibody were also observed in the cell extracts.     

具备定点偶联功能的HBc-VLPs的制备与性质鉴定

乙型肝炎病毒核心蛋白HBc,可在体外自组装形成二十面体对称结构的病毒样微粒VLPs。VLPs可将外源序列重复且高密度地展示在表面,VLPs进入机体后能够快速诱导机体产生针对外源性抗原的特异性体液免疫及细胞免疫应答,具有极强的免疫原性与生物活性。因此,HBc-VLPs可以作为一种安全、有效的疫苗载体。文中设计了一种能够实现与抗原定点偶联的HBc-VLPs,并开发了一套高效制备HBc-VLPs的方法。通过定点突变技术,使翻译后的多肽序列第80位氨基酸由Ala变为Cys,在HBc-VLPs的主要免疫显性区域引入一个定点交联位点,构建了原核表达载体pET28a(+)-hbc,表达、纯化获得了高纯度的HBc(A80C) 单体蛋白;在PB缓冲体系中,HBc(A80C) 蛋白自组装形成HBc-VLPs纳米粒子。粒度仪的测定结果表明,HBc-VLPs纳米微粒的平均粒径为29.8 nm,透射电子显微镜观察到HBc-VLPs形成粒径约为30 nm的球形微粒,其形态与天然的HBV微粒相似。以流感病毒M2e抗原肽为模式抗原,通过Sulfo-SMCC氨基-巯基双功能交联剂,将M2e定点连接于HBc-VLPs通过突变引入的Cys残基处,制备了M2e-HBc-VLPs模式疫苗,通过细胞荧光示踪,验证了HBc-VLPs结构的完整性与M2e的正确交联。动物免疫实验表明该疫苗能够有效刺激小鼠产生抗原特异性的IgG抗体,验证了疫苗载体HBc-VLPs的有效性。研究结果为HBc-VLPs作为疫苗载体的研究奠定了基础,能够促进HBc-VLPs载体疫苗的研发以及HBc-VLPs在其他领域的应用。     

成人T细胞白血病细胞靶向型水泡性口炎病毒的构建及应用

目前肿瘤治疗主要使用放疗、药物化疗,具有很大的毒、副作用,研发肿瘤靶向性药物是未来发展趋势.成人T细胞白血病(adult T cell leukemia,ATL)是一种由HTLV-1病毒引起的人恶性CD4 T淋巴细胞白血病,目前尚无有效治疗方法.溶瘤性水泡性口炎病毒(vesicular stomatitis virus,VSV)是一种肿瘤治疗病毒载体,利用HIV-1囊膜蛋白gp160对野生型VSV病毒进行假型化改造,研制了具备人CD4受体靶向性的重组VSV病毒(VSV-△G-gp160G),在对ATL病人肿瘤细胞进行的体外杀伤实验(ex vivo)中,显示出良好的应用前景.