重组卡介苗的研究与应用

重组卡介苗可同时表达多种病原体抗原，已成为新的疫苗载体。对重组卡介苗的研究方法和进展，包括重组卡介苗疫苗载体、启动子、电转化方法、绿色荧光蛋白的作用、重组耻垢分枝杆菌疫苗及重组卡介苗的作用机制等进行综述，并介绍了重组卡介苗在表达病毒、细菌及寄生虫抗原和表达细胞因子及抗肿瘤等方面的应用。     

DNA疫苗的作用机制及影响因素

DNA疫苗(DNA vaccine)是1990年从基因治疗(genetic thera-py)研究领域发展起来的一种全新疫苗,也称核酸疫苗(nucleicacid vaccine)。DNA疫苗是把含有编码某种抗原蛋白的外源基因克隆到真核质粒表达载体上,然后将重组质粒直接导入动物细胞内,并通过宿主细胞的转录系统使外源基因在动物体内表达抗原蛋白,从而诱导宿主产生对该抗原蛋白的免疫应答,以达到预防和治疗疾病的目的。     

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.     

纳米递送系统在mRNA肿瘤疫苗中的研究进展

肿瘤疫苗是肿瘤免疫治疗中极具发展前景的治疗策略之一,其通过递送肿瘤抗原促进抗原递呈过程,进而激活抗肿瘤免疫反应。信使RNA (messenger RNA, mRNA)疫苗是一种新型疫苗,通过向体内递送特定抗原的mRNA序列并表达相应抗原蛋白,从而激活机体免疫系统达到免疫治疗的目的。mRNA疫苗与传统疫苗相比具有生产周期短、有效性高和免疫原性强等优势,近年来mRNA疫苗在肿瘤免疫治疗中的应用引起广泛关注,但mRNA的不稳定性和低递送效率限制了其应用。纳米递送系统能有效解决mRNA疫苗递送的难题,极大地促进mRNA肿瘤疫苗的研究进程和临床应用,已成为mRNA疫苗研究的热点。本文对mRNA肿瘤疫苗进行介绍,重点对纳米递送系统在mRNA肿瘤疫苗中的应用进行综述,以期为mRNA肿瘤疫苗高效递送及肿瘤免疫治疗提供新思路和新方法。     

肿瘤疫苗Astuprotimut-R

黑色素瘤相关抗原-3（MAGE-3）为肿瘤特异性免疫治疗的理想靶分子。葛兰素史克公司研发的重组抗肿瘤融合蛋白疫苗Astuprotimut—R（GSK-249553），系将MAGE-3表位融合到流感嗜血杆菌蛋白D抗原的部分序列中所制得，即MAGE-3重组蛋白。     

新型基因治疗疫苗可避免危险的副作用

肿瘤蛋白为肿瘤生长所必需,因而是肿瘤疫苗的目标。然而,编码功能蛋白的基因疫苗可把癌基因引入正常细胞,导致注射部位癌细胞发生。 荷兰莱登大学研究人员研制的疫苗仅释放来源于肿瘤相关抗原的、必需的T细胞表位。他们证明,这种疫苗能保护小鼠防御致死性肿瘤攻击。此外,应用复制缺陷性重组腺病毒(rAd)较易大量生产疫苗。     

植物制剂防治自身免疫性疾病研究进展

植物转基因技术是一种新兴的技术手段,它给低成本、大规模生产重组蛋白带来了广阔的前景.此文综述了目前该领域中应用的各种蛋白表达技术,并对植物疫苗在预防和治疗自身免疫病方面的研究进展,尤其对口服自身抗原诱导机体免疫耐受予以重点介绍.     

基因疫苗黏膜免疫及其机制的研究进展

基因疫苗是将编码外源性抗原的基因插入到含真核表达系统的载体质粒上,然后将重组质粒直接导人人或动物体内,让其在宿主细胞中表达抗原蛋白,诱导机体产生免疫应答.以达到预防疾病的目的.现已证实基因疫苗的有效性及安全性,但存在人体不能对基因疫苗进行有效的吸收以及机体对抗原的免疫耐受等不足,使其在人体中的免疫效果不佳.近年来,对基因疫苗的免疫策略、免疫效果及作用机制已进行了大量的研究.肌肉注射法因外源基因在体内的表达水平较低,免疫效果不够理想.基因枪法、电穿孔等递送技术的发展均显著提高了基因疫苗的活体递送效率,使基因疫苗免疫具有较强的诱导免疫应答的能力[1-2].     

人乳头瘤病毒重组蛋白疫苗酶联免疫检测法的建立及其应用

目的建立人乳头瘤病毒重组蛋白疫苗(HPV16 L2E6E7)酶联免疫的检测方法,用于疫苗发酵过程中的重组蛋白表达的监控。方法用常规方法制备兔抗HPV16 L2E6E7多克隆抗体作为包被抗体,商品化小鼠抗HPV16 E7单克隆抗体为检测抗体,夹心ELISA方法检测HPV16 L2E6E7抗原参考品,建立抗原标准曲线,确定线性范围及检测限,同时验证该方法的特异性。结果建立了检测HPV16 L2E6E7抗原含量的夹心ELISA方法,抗原参考品系列浓度在19.53~1 250 ng·m L-1内有很好的线性(R2>0.99)和回收率(90%~110%);特异性好,不受发酵液中宿主菌蛋白的干扰。结论建立了人乳头瘤病毒重组蛋白疫苗重组抗原含量的测定方法,为该疫苗的发酵工艺的质控提供了有效技术手段。     

新一代HIV疫苗将首次进行临床试验

据推测,由九种主要病毒抗原制成的人类免疫缺陷症病毒(HIV)疫苗(如美国Therion生物制品公司的TBC-3B疫苗)将取代由一种HIV抗原制成的疫苗。这种多抗原疫苗可能首先进入市场。 TBC-3B疫苗的Ⅰ期临床试验由美国国立变态反应和传染病研究所(NIAID)进行。这种疫苗是首次在人体中试验的重组疫苗,它能表达几种主要的HIV蛋白。 公司总裁Panicali说,“公司研究方法是将尽可能多的抗原插入疫苗中以模拟HIV”。在活疫苗病毒的天然结构中表达主要的HIV抗原。将三种HIV基因env、gag和pol插入疫苗病毒中,后者又能产生9种HIV蛋白,包括基质、核心、包膜、核壳体、逆转录酶、整合酶和蛋白酶抗原。仅表达包膜蛋白的疫苗似不能产生病毒株交叉性保护作用。 HIV包膜糖蛋白能诱生结合游离病毒的抗体,因此能进一步预防感染。虽然包     

重组4-1BB配体对治疗性重组人乳头瘤病毒16型蛋白疫苗的佐剂作用

目的 研究重组4-1BB配体(recombinant 4-1BB ligand,r4-1BBL)作为佐剂对治疗性重组人乳头瘤病毒16型(human papillomavirus-16,HPV16)蛋白疫苗(HPV16蛋白疫苗)免疫效果的影响.方法 对4-1BBL胞外功能区进行密码子优化,并通过NdeⅠ和XhoⅠ酶切位点定向插入pET28a表达载体.将获得的重组表达载体转化至大肠埃希菌BL21 (DE3),并用异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达.用蛋白质印迹法对重组蛋白进行鉴定,并用非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电子显微镜分析重组蛋白结构.用CCK-8试剂盒检测纯化r4-1BBL对小鼠T细胞体外增殖的影响.同时将r4-1BBL与HPV16蛋白疫苗联合免疫C57BL/6小鼠,用酶联免疫斑点试验检测小鼠的细胞免疫应答水平,观察r4-1BBL对HPV16蛋白疫苗抑制肿瘤生长的影响.结果 密码子优化的4-1BBL胞外功能区能在重组表达载体中表达,且表达产物同时以包涵体和可溶性形式存在.可溶性产物的结构主要为二聚体,电子显微镜下可以观察到类似亚单位的结构.纯化r4-1BBL可促进小鼠脾淋巴细胞的体外增殖.与单纯HPV16蛋白疫苗相比,r4-1BBL联合HPV16蛋白疫苗能诱导更强的特异性细胞免疫应答(t=3.525,P=0.024)和产生更明显的肿瘤生长抑制作用(t=2.534,P=0.021).结论 r4-1BBL能在小鼠中提高HPV16蛋白疫苗的免疫效果,具有作为HPV16蛋白疫苗佐剂的潜力.     

含融合自杀基因CDglyTK复制型重组腺病毒的构建与鉴定

目的:构建含融合自杀基因CDglyTK复制型重组腺病毒,并在肺腺癌细胞A549中验证其复制能力和溶瘤特性。方法:建立重叠延伸PCR方法,用该方法制备的E1A-IRES片段插入AdEasy载体构建含融合自杀基因CDglyTK复制型重组腺病毒rAdE1A-CDglyTK。并用该病毒与复制缺陷型重组腺病毒rAd-CDglyTK分别感染肿瘤细胞A549,从荧光蛋白表达、细胞病变及病毒增殖等方面进行比较,以确定其复制能力及溶瘤特性。结果:成功构建了含融合自杀基因CDglyTK的复制型重组腺病毒rAdE1A-CDglyTK,该病毒感染肿瘤细胞A549后报告基因绿色荧光蛋白(GFP)的表达,能大量增殖并出现明显的细胞病变效应(CPE)。结论:构建的复制型重组腺病毒在肿瘤细胞内的复制能力、溶瘤特性和外源基因的表达明显优于目前广泛应用的复制缺陷型重组腺病毒。     

重组四价人乳头瘤病毒16/18/52/58型病毒样颗粒疫苗制剂工艺的优化

目的 考察不同制剂工艺流程下制备的重组四价人乳头瘤病毒（human papillomavirus,HPV）16/18/52/58型病毒样颗粒疫苗的抗原吸附效果、稳定性及免疫原性,根据实验结果筛选出最合适的制剂配方及工艺。方法  通过改变重组四价HPV疫苗的缓冲体系,以及氢氧化铝佐剂的含量、混合工艺、吸附方式与吸附条件,配制不同的实验疫苗,观察其外观,并分别进行蛋白质稳定性、抗原吸附率和小鼠体内半数有效剂量的检测。 结果 重组四价HPV疫苗原液与450 μg/ml氢氧化铝佐剂在pH6.5的组氨酸缓冲体系下混合后的实验疫苗,外观均一性良好,抗原蛋白稳定性最高,Tm=87.4 ℃,小鼠体内免疫原性较好;搅动吸附条件下的4型别HPV抗原吸附率明显高于静止吸附;铝佐剂和抗原蛋白的吸附与混合先后顺序,不同吸附比例、温度和时间对疫苗外观无显著影响,抗原吸附率均大于99.00%。结论 确定了重组四价HPV16/18/52/58型病毒样颗粒疫苗合适的制剂工艺条件。     

Old and new vaccine approaches

The conventional, currently available vaccines, though quite successful, suffer from a few shortcomings which hamper future vaccine development. We present herewith some of the new approaches that are presently being pursued, including (1) the development of recombinant, or genetically engineered, vaccines which are based either on the expression of the relevant protective antigen and its formulation into vaccine, or the production of live vaccines, where an appropriate live vector (virus or bacterium) presents the foreign antigen. (2) The development of naked DNA vaccines that include the gene(s) coding for the relevant protective antigen(s). (3) Peptide vaccines that include defined B cell and T cell epitopes, either in a chemically synthesized molecule or in a synthetic recombinant construct. The efficacy of such vaccines is usually dependent on adequate presentation and delivery, namely, carrier/adjuvant technology. (4) Therapeutic vaccines, based on all of the above approaches, may be applied for chronic or long-term infections, or for noninfectious diseases including autoimmune diseases, various neurological disorders, allergy and cancer.     

An efficient,chemically-defined semisynthetic lipid-adjuvanted nanoparticulate vaccine development system

A novel vaccine development platform that enables the site-specific conjugation of synthetic lipid adjuvants to recombinant proteins was produced. This technology facilitates the simple and efficient production of homogeneous, chemically-defined, semisynthetic lipoprotein vaccines. Using a polytope ‘string-of-beads’ approach, a synthetic gene incorporating seven Streptococcus pyogenes M protein strain-specific antigens, and a conserved M protein antigen (J14) was produced, expressed, and attached to a lipoamino acid based adjuvant (lipid core peptide; LCP). Nanoparticles (40 nm diameter) of an optimal size for stimulating antibody-mediated immunity were formed upon the addition of these lipoproteins to aqueous buffer (PBS). Systemic antigen-specific IgG antibodies were raised against all eight antigens in C57BL/6 J mice, without the need to formulate with additional adjuvant. These antibodies bound cell surface M proteins of S. pyogenes strains represented within the polytope sequence, with higher antibody levels observed where a dendritic cell targeting peptide (DCpep) was incorporated within the LCP adjuvant.From the Clinical EditorIn this study, a novel vaccine development system is presented, combining adjuvants with recombinant protein antigens, and presenting the antigen in a nanoparticle system optimized for antibody production. They demonstrate efficient vaccination in a murine model system without the need for additional adjuvants.     

Development and application of PROVAX adjuvant formulation for subunit cancer vaccines

A major challenge facing the development of subunit vaccines comprised of well-defined recombinant antigens is their weak immunogenicity and inability to induce effective cytotoxic T cell (CTL) responses. Adjuvants aimed at increasing the immunogenicity of recombinant antigens remain a focus in vaccine development. The potency of an adjuvant is linked to specific stimulation of T cell responses, involving TH1 and TH2 subsets of CD4(+) T helper cells and CD8(+) CTL and B cell-mediated antibody responses. As a result of the existence of two distinct intra-cellular pathways for antigen processing, immunization with exogenous antigens often shows a greater propensity for T helper and antibody responses, but not CD8(+) CTL responses. However, existing experimental evidence suggests that CD8(+) CTLs, which are critical in the elimination of viral-infected and neoplastic cells, can be elicited with soluble antigens when delivered in appropriate formulations or adjuvants. This review focuses on the properties of PROVAX adjuvant in inducing antigen-specific CTL responses, antibody responses and tumor regression in experimental models and its potential application for the development of recombinant cancer vaccines.     

Baculovirus-expressed influenza vaccine. A novel technology for safe and expeditious vaccine production for human use

Effectiveness of the influenza vaccine in persons with high-risk conditions needs to be improved. In this paper, the authors review various vaccination strategies, including repeated doses of the vaccine or the use of higher hemagglutinin (HA) content vaccines that have been shown to result in improved immunogenicity. A recombinant HA vaccine produced in insect cells using the baculovirus vectors system presents the possibility for safe and expeditious vaccine production. The high purity of the antigen enables administration at much higher doses without a significant increase in side effects in human subjects. An overview of the use of this production system for the development of alternative influenza vaccine targets is also provided, such as neuraminidase and possibly M2. However, the role of M2 may be more appropriate as an adjuvant vaccine in combination with standard HA vaccine supplement and needs further evaluation. The conclusion that the insect cell-baculovirus production technology is a modern solution for rapid viral or parasitic antigen production is made and that this technology is particularly suitable for influenza where annual adjustment of the vaccine is required. In addition, a highly purified recombinant protein vaccine results in an improved influenza vaccine response in those with high-risk medical conditions.     

Microtiter Plate-Based Differential Scanning Fluorimetry: A High-Throughput Method for Efficient Formulation Development

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ～ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.     

Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants

Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products.     

Carcinoembryonic antigen (CEA)-based cancer vaccines: recent patents and antitumor effects from experimental models to clinical trials

Carcinoembryonic antigen (CEA), a glycosylated protein of MW 180 kDa, is overexpressed in a wide range of human carcinomas, including colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Accordingly, CEA is one of several oncofetal antigens that may serve as a target for active anti-cancer specific immunotherapy. Experimental results obtained by employing animal models have supported the design of clinical trials using a CEA-based vaccine for the treatment of different types of human cancers. This review reports findings from experimental models and clinical evidence on the use of a CEA-based vaccine for the treatment of cancer patients. Among the diverse CEA-based cancer vaccines, DCs- and recombinant viruses-based vaccines seem the most valid. However, although vaccination was shown to induce a strong immune response to CEA, resulting in a delay in tumor progression and prolonged survival in some cancer patients, it failed to eradicate the tumor in most cases, owing partly to the negative effect exerted by the tumor microenvironment on immune response. Thus, in order to develop more efficient and effective cancer vaccines, it is necessary to design new clinical trials combining cancer vaccines with chemotherapy, radiotherapy and drugs which target those factors responsible for immunosuppression of immune cells. This review also discusses relevant patents relating to the use of CEA as a cancer vaccine.