病毒样颗粒用作轮状病毒亚单位疫苗(英)

目前正在人体中评价轮状病毒亚单位疫苗的效果，已在杆状病毒中克隆出编码各种轮状病毒结构蛋白的基因，并在杆状病毒表达系统中共表达出重组蛋白。这些表达的蛋白能自发装配成病毒样颗粒（VLP），各种不同组合结构蛋白共表达后即形成由各种不同蛋白构成的VLP。     

基因工程表达的颗粒状病毒样结构及其用作疫苗释放系统

作者用新型的杆状病毒多重基因表达载体同时表达蓝舌病病毒(BTV)的三、四或五种结构基因,表达产物可成功地组装为病毒样颗粒(VLP)及核心样颗粒(CLP).初步临床试验证明VLP是安全有效的疫苗,具有多方面的优越性.多组分的VLP还曾用作多种免疫原(包括B和T细胞表位)的疫苗释放系统.此表达系统有可能广泛用于疫苗、杀虫剂、诊断和蛋白质试剂的生产行业.     

病毒样颗粒疫苗的免疫机制研究及应用进展

病毒样颗粒（virus-like particle，VLP）因具有良好的免疫原性以及较高的安全性，逐渐成为了当今药物及疫苗研究的热点。鉴于VLP具有高度重复表面结构的特性，其可通过多种途径引起有效的体液和细胞免疫应答。目前可用多种表达系统表达VLP，且随着对VLP研究的深化，已有多种VLP疫苗进入临床试验阶段且部分VLP疫苗已经商业化。此文对VLP免疫机制及VLP疫苗研究进展做一综述。     

由H9N2流感病毒HA、NA和M1蛋白构成的流感病毒样颗粒诱导BALB/c小鼠产生保护性免疫应答

禽类与人类的流感病毒之间发生基因重配,可能导致流感病毒在人群间迅速大规模流行。美国Novavax公司Pushko等研制成一种由禽流感病毒A/香港/1073/99(H9N2)的HA、NA和M1蛋白构成的病毒样颗料(VLP)疫苗,该疫苗能诱导BALB/c小鼠产生保护性免疫应答。将编码流感病毒A/香港/1073/99株HA、NA和M1蛋白的基因克隆至杆状病毒表达载体,形成重组杆状病毒,转染Sf9昆虫细胞,扩大培养,蔗糖密度梯度离心收获VLP。进行凝胶过滤层析、电镜检查鉴定VLP。以雌性BALB/c小鼠为模型进行两次试验。第一次分别于0,28天皮下接种10μgVLP,采集初免前和…     

16型人乳头瘤病毒疫苗研究进展

16型人乳头瘤病毒(HPV16)与宫颈癌的发生关系密切,由于该病毒尚不能在体外有效培养,而限制了疫苗的研究进展.目前通过分子生物学方法在体外表达的HPV16病毒样颗粒(VLP)成为疫苗研究的热点.研究表明VLP可诱导机体产生抗病毒攻击的细胞免疫和体液免疫应答,从而为基因工程亚单位疫苗的研制提供了科学依据.     

16型人乳头瘤病毒疫苗研究进展

16型人乳头瘤病毒（HPV16）与宫颈癌的发生关系密切,由于该病毒尚不能在体外有效培养,而限制了疫苗的研究进展。目前通过分子生物学方法在体外表达的HPV16病毒样颗粒（VLP）成为疫苗研究的热点。研究表明VLP可诱导机体产生抗病毒攻击的细胞免疫和体液免疫应答,从而为基础工程亚单位疫苗的研制提供了科学依据。     

新一代重组病毒亚单位疫苗

发展重组病毒亚单位疫苗有两大热点:其一,候选抗原的表达策略日趋注重于产生更近天然的抗原结构,如病毒样颗粒(VLP);其二,为提高免疫原性而使用新型佐剂.本文从抗原设计、佐剂及其在动物和人体中的免疫原性等方面对12种重要的人类病毒亚单位疫苗的研究进展作了评述.     

制备轮状病毒亚单位疫苗的病毒样颗粒的生化及免疫学特性比较

本文比较了组成轮状病毒亚单位疫苗的两种病毒样颗粒 (VLP)的生化及免疫学特性 ,以及 VL P加铝或 QS2 1佐剂的免疫原性 ,评介了这些非肠道接种疫苗用于人体的可能性。　　用于试验的重组杆状病毒分别表达牛轮状病毒核壳蛋白 VP2、VP6和猿猴轮状病毒VP7T(片段 )或 VP7F(全段 ) ,共感染或三次感染 sf9昆虫细胞 ,自行装配成两种杂交体6 / 7- VLP和 2 / 6 / 7- VLP。离心纯化后测定其纯度和组成 ,结果表明 VL P的纯度约为90 % ,2 / 6 / 7T- VLP中 VP2、VP6、VP7分别为 1 5 %、5 1 %、30 %左右 ;重复离心后 ,2 / 6 /7T- VLP较 6…     

昆虫细胞表达的重组流感血凝素疫苗的免疫效果评价

应用DNA重组技术可以快速克隆流感病毒血凝素(HA)基因并使其在真核系统中有效地表达.应用重组杆状病毒在昆虫细胞中表达的HA蛋白,对青年人接种后,可诱生抗体并预防感染.本文作者对这一重组蛋白在老年人中的免疫效果进行了评价.用表达流感病毒A/北京/32/92(H3N2)HA基因cDNA全序列的重组杆状病毒感染昆虫细胞产生的HA抗原纯度在99%以上,该重组抗原称为rHAO.将rHAO疫苗溶于PBS使接种量分别为15、45或135μg/0.5ml,同时设立三价亚病毒疫苗和安慰剂对照.     

流感病毒血凝素在多种系统中表达的研究进展

流感疫苗是预防流感病毒感染最有效的方法。传统灭活流感疫苗通过在鸡胚中培养病毒后经纯化获得。流感每年都会发生季节性流行,流感病毒高度多变的特性使生产有效疫苗成为一项挑战。为了克服流感疫苗生产对鸡胚的依赖,需要开发新的流感疫苗生产策略。由于血凝素是流感病毒主要表面抗原之一,重组血凝素亚单位疫苗为流感疫苗的生产提供了一个方案。本文将对流感病毒血凝素在大肠埃希菌、毕赤酵母、昆虫细胞、哺乳动物细胞多种系统中表达的研究进行综述。     

Application of baculovirus-insect cell expression system for human therapy

A major advantage of recombinant DNA technology is its flexibility allowing for "on demand" production of specific proteins with theurapeutic value in heterologous expression systems. Gene expression vectors based on baculovirus, insect virus attacking mostly lepidopteran species, are frequently used for relatively inexpensive and fast production of such proteins. This expression system is recognized as one of the most powerful technologies for commercial synthesis of glycoproteins originating from vertebrate themselves or from vertebrate viruses. Glycosylation pathways utilized by insects are not identical, though they are similar to vertebrate glycosylation pathways. In the review special attention is given to the development of new virus-like particles (VLPs) potential vaccines which represent a novel class of subunit vaccines that are able to stimulate efficiently cellular and humoral immune responses against viral agents. Apart from production of vertebrate proteins or VLPs "on demand " in insect cells, a new exciting field of using baculovirus as gene delivery system to vertebrate cells was recently open which has a great potential for future uses of baculovirus as effective gene therapy vector.     

流感疫苗研究的新策略

疫苗接种是预防流感最有效的方法,但流感病毒的变异给疫苗研制带来了极大的困难,而减毒活疫苗能使机体获得持久免疫力,因此成为预防流感的重要疫苗.然而,目前研发的减毒活疫苗仍存在制备条件苛刻、安全性较差等问题.鉴于miRNA是调控宿主和病毒相互作用的关键因子,miRNA介导的减毒活疫苗给流感疫苗研发提供了新的思路.此文对近年来基于miRNA的流感疫苗研究进展做一综述.     

Immunization by influenza virus-like particles protects aged mice against lethal influenza virus challenge

Influenza virus-like particles (VLPs) were produced in Sf9 insect cells by co-expressing the matrix protein M1 and the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) using the recombinant baculovirus expression system. The VLPs were morphologically similar to influenza virions. Both HA and NA proteins were incorporated into VLPs and these proteins retained their functional activities. Further, influenza VLPs but not inactivated influenza viruses (IIV) stimulated secretion of inflammatory cytokines from mouse bone marrow-derived dendritic cells (BMDC). Immunogenicity of influenza VLPs and their protective efficacies against lethal influenza virus challenge were evaluated in young and aged mice. Immunization with influenza VLPs induced strong antibody responses against HA that inhibited hemagglutination by influenza virus, similar to IIV vaccines. Compared to young mice, antibody responses in aged mice immunized with a low dose of either influenza VLPs or IIV vaccines exhibited markedly reduced avidity for HA. However, immunization of aged mice with a high dose of influenza VLPs induced antibody responses with high avidity similar to those in young mice. Furthermore, all vaccinated animals survived a lethal challenge by a mouse-adapted influenza virus (A/PR/8/34), indicating that influenza VLPs are highly efficacious for protection against influenza virus infection in both young and aged mice.     

Virus-like particles as a vaccine delivery system: myths and facts

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this review we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus-like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the marketplace we argue that this technology has now 'come-of-age' and must be considered a viable vaccine strategy.     

Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier

We report the preparation and the immunogenicity of a conjugate vaccine obtained by chemically conjugating a variant of the extracellular peptide fragment of influenza type A M2 protein to the human papillomavirus (HPV) viruslike particle (VLP). Conjugates comprised of approximately 4,000 copies of the antigenic peptide per VLP are obtained as the result of the reaction between a C-terminal cysteine residue on the peptide and the maleimide-activated HPV VLP. The resulting conjugates have an average particle size slightly larger than the carrier and present enhanced overall stability against chemical and thermal-induced denaturation. The M2-HPV VLP conjugates lost the binding affinity for anti-HPV conformational antibodies but retained reactivity to a M2-specific monoclonal antibody. The conjugate vaccine formulated with aluminum adjuvant and delivered in two doses of 30-ng peptide was found to be highly immunogenic and conferred good protection against lethal challenge of influenza virus in mice. These results suggest that HPV VLP can be used as a carrier for synthetic or small antigens for the development of subunit vaccines.     

Recombinant influenza B virus HA and NA antigens administered in equivalent amounts are immunogenically equivalent and induce equivalent homotypic and broader heterovariant protection in mice than conventional and live influenza vaccines

Influenza B virus is an important cause of acute upper respiratory disease in humans. Vaccination is the primary method of control of influenza related disease, yet vaccine methodology and production technology have not changed in over 40 years. In this study, we compare the efficacy of recombinant baculovirus produced protein based neuraminidase containing influenza B vaccines with conventional inactivated influenza vaccine (CIV) and live-attenuated influenza vaccine (LAIV) in a murine model. All HA containing vaccines stimulated antibody and protected against an infectious challenge with homotypic virus (B/Harbin/7/94), only recombinant protein based (rHA + rNA and rNA) vaccines containing immunogenic amounts of influenza neuraminidase (NA) protected against challenge with a significantly antigenically different heterovariant virus (B/Beijing/243/1997), as measured by a reduction in mean pulmonary virus titers. This report demonstrates with influenza B virus, in a side-by-side comparison with CIV and LAIV in a murine model system the superiority of vaccines containing immunogenic NA over currently approved CIV and LAIV vaccines.     

基于神经氨酸酶的流感疫苗研究进展

当前广泛使用的季节性流感疫苗的主要抗原成分包括2个重要的流感病毒表面糖蛋白,血凝素和神经氨酸酶(neuraminidase,NA).研究表明NA抑制性抗体虽然不能防止流感病毒感染,但能有效抑制病毒扩散,因此基于NA设计的疫苗开始受到重视.NA的相对保守性也使此类疫苗有潜力成为广谱流感疫苗.目前基于NA的多种形式流感疫苗,包括DNA疫苗、病毒样颗粒疫苗、重组载体疫苗、重组亚单位疫苗以及联合其他流感病毒蛋白的疫苗等,在动物模型中均表现出诱导交叉保护的能力.此文综述了近年来发表的有关基于NA的流感病毒疫苗的研究.     

Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities

Influenza vaccination represents the cornerstone of influenza prevention. However, today all influenza vaccines are formulated as liquids that are unstable at ambient temperatures and have to be stored and distributed under refrigeration. In order to stabilize influenza vaccines, they can be brought into the dry state using suitable excipients, stabilizers and drying processes. The resulting stable influenza vaccine powder is independent of cold-chain facilities. This can be attractive for the integration of the vaccine logistics with general drug distribution in Western as well as developing countries. In addition, a stockpile of stable vaccine formulations of potential vaccines against pandemic viruses can provide an immediate availability and simple distribution of vaccine in a pandemic outbreak. Finally, in the development of new needle-free dosage forms, dry and stable influenza vaccine powder formulations can facilitate new or improved targeting strategies for the vaccine compound. This review represents the current status of dry stable inactivated influenza vaccine development. Attention is given to the different influenza vaccine types (i.e. whole inactivated virus, split, subunit or virosomal vaccine), the rationale and need for stabilized influenza vaccines, drying methods by which influenza vaccines can be stabilized (i.e. lyophilization, spray drying, spray-freeze drying, vacuum drying or supercritical fluid drying), the current status of dry influenza vaccine development and the challenges for ultimate market introduction of a stable and effective dry-powder influenza vaccine.     

Formulation and stabilization of recombinant protein based virus-like particle vaccines

Vaccine formulation development has traditionally focused on improving antigen storage stability and compatibility with conventional adjuvants. More recently, it has also provided an opportunity to modify the interaction and presentation of an antigen/adjuvant to the immune system to better stimulate the desired immune responses for maximal efficacy. In the last decade, there has been a paradigm shift in vaccine antigen and formulation design involving an improved physical understanding of antigens and a better understanding of the immune system. In addition, the discovery of novel adjuvants and delivery systems promises to further improve the design of new, more effective vaccines. Here we describe some of the fundamental aspects of formulation design applicable to virus-like-particle based vaccine antigens (VLPs). Case studies are presented for commercially approved VLP vaccines as well as some investigational VLP vaccine candidates. An emphasis is placed on the biophysical analysis of vaccines to facilitate formulation and stabilization of these particulate antigens.