伤寒Vi多糖、白喉、破伤风联合疫苗初探

目的 制备成人及青少年用吸附伤寒Vi多糖、白喉、破伤风联合疫苗，观察伤寒Vi多糖含量、白喉及破伤风效价。方法 按设计的配方,将白喉、破伤风类毒素加入佐剂中吸附后，再加入伤寒Vi多糖，要求配制的吸附伤寒Vi多糖、白喉、破伤风联合疫苗每人用剂量0．5ml伤寒Vi多糖含量≥30μg．白喉类毒素效价≥2IU，破伤风类毒素效价≥40IU。结果 三组配方试验样品每人用剂量0．5ml伤寒Vi多糖含量均低于30μg，白喉类毒素效价3．127IU～5．911IU，破伤风类毒素效价7．784IU～23．976IU。除白喉类毒素效价达到要求外，伤寒Vi多糖含量及破伤风类毒素效价未达到要求。结论 初探试验表明，试验的联合疫苗存在抗原干扰．伤寒Vi多糖含量及破伤风类毒素效价下降。     

家庭接触百日咳后百日咳类毒素菌苗的效果

作者就百日咳类毒素菌苗对家庭接触百日咳的儿童的效果进行了随机双盲安慰剂对照试验。 3450名健康儿童于3、5和12月龄随机接种白喉-破伤风类毒素(DT)或白喉-破伤风-百日咳类毒素(DTP)。每种菌苗剂量0.5ml,含25Lf白喉类毒素、7Lf破伤风类毒     

间接血凝试验与毒素中和试验测定小鼠血清白喉抗毒素的比较

作者用不同剂量的吸附白喉、破伤风类毒素、吸附DTP和纯化白喉类毒素免疫42组小鼠,然后用间接血凝试验(IHA)和毒素中和试验(TN)测定小鼠血清中的白喉抗毒素含量。实验证明,TN和IHA测定的白喉抗毒素滴度的相关系数(r)是0.91,回归方程是:y=0.7935x+0.0647。TN滴度和IHA滴度在统计学上无明显差异。IHA的灵敏度比TN高,可测出白喉抗毒素的最低水平为0.00039IU/ml。IHA在不同时间测定同一血清的滴度几乎相等,证实了这一试验的可重复性。结果表明,IHA可代替TN用于测定白喉抗毒素,     

婴儿接种乙型肝炎和DTP联合疫苗的免疫应答

作者对179名婴儿分别于2、4、6月龄于大腿前外侧肌肉接种3剂乙型肝炎和DTP联合疫苗(DTP-HBV),并分别在接种前、第2次接种后2个月及第3次接种后1个月采血,检测抗体水平。由父母在日记卡上记录症状。本研究使用的疫苗由SmithKline公司生产,每剂0.5ml,含10μg HBsAg、≥30IU白喉类毒素、≥60IU破伤风类毒素和15OU百日咳杆菌。采用放射免疫法测定抗-HBs;用间接固相酶免疫法测定抗破伤风IgG和抗白喉IgG抗体滴度;用ELISA测定抗百日咳抗体滴度。     

应用Stainer培养基与TFF系统制备白喉类毒素

目的  验证WHO推荐的Stainer培养基与切向流过滤(TFF)系统能否制备符合我国现行规程要求的精制白喉类毒素.方法   用PW8菌株、Stainer培养基进行了三批白喉杆菌产毒培养,用TFF微孔过滤和超滤系统分别进行了澄清与浓缩.毒素经甲醛脱毒后,用TFF超滤系统对类毒素进行了超滤精制,得到三批精制白喉类毒素,按我国现行规程主要技术指标进行评价  .结果   产毒效价分别为180Lf/ml、210Lf/ml和155Lf/ml.精制类毒素纯度分别为1900Lf/mgPN、1860Lf/mgPN、1760Lf/mgPN.毒性试验和毒性逆转试验全部合格.结论   应用Stainer培养基与TFF系统可获得符合我国现行规程要求的精制白喉类毒素.     

铝吸附和液体白喉-破伤风类毒素加强免疫后的IgE应答

本文旨在研究铝吸附和非吸附白喉-破伤风类毒素(DT)加强免疫后的IgE应答情况及IgE与副反应之间的关系。 作者用双盲法随机将104名(男68,女36)10岁学龄儿童分为两组:一组51人,接种铝吸附DT;另一组53人,接种非吸附液体DT。104名儿童均于婴儿期接种过3×0.5ml的吸附DT。吸附DT含30Lf/ml白喉类毒素和7.5Lf/ml破伤风类毒素。于上臂外侧三角肌处深皮下接种。分别于接种前和接种4周后采血,用改良Phadebas RAST检测抗白喉和破伤风类毒素IgE,>0.1 Phadebas RAST单位(PRU)/ml者判为阳性。     

瑞典儿童对破伤风的免疫力和免疫接种

本文分析了瑞典457名儿童接种破伤风菌苗后血清抗毒素水平及其保护作用。作者将457名儿童分为四组:5,6,10,16岁年龄组。其中,1963～1979年出生的儿童接种非吸附白-百-破三联菌苗,每毫升含20×10~9灭活的百日咳杆菌、30Lf白喉类毒素和5Lf破伤风类毒素。1979年及其后出生的儿童接种0.5毫升磷酸铝吸附白喉-破伤风二联菌苗(DT),含7.5Lf/ml破伤风类毒素和1.0Lf/ml白喉类毒素。于生后第2或第3个月进行首次基础免疫,间隔4～6周进行第2     

试验表明戊二醛灭活的百日咳菌苗是一种安全菌苗

作者对含有不同方法灭活的百日咳菌苗的吸附DTP作了安全试验。将百日咳杆菌134和509株的悬液等量混合,混合后每ml含有8×10~9菌体。将40ml磷酸铝吸附白喉破伤风类毒素(其中每ml含有白喉类毒素110Lf、破伤风类毒素22Lf)分别和40ml不同的百日咳菌苗混合,制成两批菌苗。这些百日咳菌苗采用五种方法灭活:加     

吸附DTP特异毒性检测

DTP的特异毒性的检测是不可缺少的,作者对DTP接种小鼠的剂量及途径进行了研究。由六家生产厂提供的112份吸附DTP样品,采用三种试验方法:(1)腹腔接种1人用剂量;(2)皮下接种1人用剂量;(3)0.5人用剂量作腹腔接种。每份样品接种5只体重17～20g的LACA小鼠。每ml样品中含白喉类毒素≤50Lf;破伤风类毒素10Lf;百日咳杆菌     

按两种程序接种DTaP-IPV-Hib联合疫苗的安全性和免疫原性

作者将液体白喉-破伤风-无细胞百日咳菌苗-脊髓灰质炎灭活疫苗（DTaP-IPV）和冻干b型流感杆菌（Hib）结合菌苗混合制成五价DTaP-IPV/Act-HIB联合疫苗。每0.5ml疫苗含白喉类毒素（DT）≥30IU、破伤风类毒素（TT）≥40IU、戊二醛灭活的百日咳类毒素（PT）25μg、天然丝状血凝素（FHA）25μg以及1、2、3型脊髓灰质炎病毒40、8、32D抗原单位、与24μg纯化TT结合的Hib荚膜多糖10μg。按2、4、6月龄初免、13月加强免疫或3、5月龄初免、12月龄加强免疫的程序,对236名婴儿进行免疫,于每次接种后3日内记录各类不良反应,并于初免后、加强免疫前后采血。用ELISA检测抗DT、TT抗体以及抗PT、FHA抗体;用中和试验（NT）检测抗各型脊髓灰质炎病毒抗体;用放射免疫法检测抗Hib荚膜多糖抗体。同时用NT检测抗DT抗体和抗PT抗体。     

破伤风类毒素和白喉类毒素原液的质量分析

目的  分析破伤风类毒素（tetanus toxoid,TT）和白喉类毒素（diphtheria toxoid,DT）原液的细菌内毒素含量、单体与多聚体之比以及游离氨基数。方法  取TT和DT原液各10批,用凝胶法、高效液相色谱法及三硝基苯磺酸法分别检测原液的细菌内毒素、单体和多聚体以及游离氨基。 结果  各10批TT、DT原液的细菌内毒素含量均值分别为1.14和0.35 EU/Lf。TT原液的单体平均含量为72.1%,是多聚体的3倍;DT原液的单体平均含量为88.9%,是多聚体的8倍。脱毒后,每个TT蛋白分子含游离氨基数均值为43.5,比破伤风毒素减少约60%;每个DT蛋白分子含游离氨基数均值为9.9,比白喉毒素减少约75%。10批TT、DT的单体与多聚体含量之比以及游离氨基数的批间一致性较好（变异系数：1.59% ~16.26%）。 结论   细菌内毒素含量、单体与多聚体之比以及游离氨基数检测指标可用于对TT、DT生产过程的控制以及批间一致性的监控。     

Modified Heat-Stable Toxins (hSTa) of Enterotoxigenic Escherichia coli Lose Toxicity but Display Antigenicity after Being Genetically Fused to Heat-Labile Toxoid LT(R192G)

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea in humans and animals. Heat-stable (STa) and heat-labile (LT) enterotoxins produced by ETEC disrupt fluid homeostasis in host small intestinal epithelial cells and cause fluid and electrolyte hyper-secretion that leads to diarrhea. ETEC strains producing STa or LT are sufficiently virulent to cause diarrhea, therefore STa and LT antigens must be included in ETEC vaccines. However, potent toxicity and poor immunogenicity (of STa) prevent them from being directly applied as vaccine components. While LT toxoids, especially LT(R192G), being used in vaccine development, STa toxoids have not been included. A recent study (IAI, 78:316-325) demonstrated porcine-type STa toxoids [pSTa(P12F) and pSTa(A13Q)] elicited protective anti-STa antibodies after being fused to a porcine-type LT toxoid [pLT(R192G)]. In this study, we substituted the 8th, 9th, 16th, or the 17th amino acid of a human-type STa (hSTa) and generated 28 modified STa peptides. We tested each STa peptide for toxicity and structure integrity, and found nearly all modified STa proteins showed structure alteration and toxicity reduction. Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT(192)-STa(-toxoid) fusions. Constructed fusions were used to immunize mice, and immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea.     

脉冲式破伤风类毒素聚乳酸微球动物免疫效果研究

目的　研究破伤风类毒素聚乳酸微球在动物体内的免疫效果及影响因素。方法　观察破伤风类毒素聚乳酸微球免疫动物后1年中,动物血清抗体反应及免疫记忆反应的规律。结果　微球中抗原释放为脉冲式,释放时间与聚乳酸分子量、微球载药量及粒径有关,微球引起的抗体反应显著高于破伤风类毒素溶液一次免疫,与溶液3次免疫效果相似。结论　创制了一种新型脉冲式释药系统,一次注射完成全程免疫,有巨大的应用价值     

检测破伤风类毒素的酶联免疫吸附法的建立及应用

目的  建立定量检测白喉-破伤风-无细胞百日咳疫苗生产过程中破伤风类毒素（tetanus toxoid,TT）的方法。方法  TT免疫家兔以制备高效价血清抗TT抗体。辛酸-硫酸铵沉淀法纯化抗TT多克隆抗体并进行辣根过氧化物酶标记,建立双抗体夹心ELISA法。结果  建立的ELISA法与丝状血凝素、百日咳毒素及白喉类毒素无交叉反应,特异性较好。该ELISA法在0.5~16.00 Lf/L TT测量区间有最佳线性,决定系数＞0.99。实验内和实验间检测14.0、12.0、6.0、3.0和1.5 Lf/L TT,变异系数为4.7%~9.8%,回收率为92.7%~109.0%,精密度和准确度均符合常规质控要求。该法的定量下限为1.5 Lf/L TT。结论  建立的ELISA法可有效检测破伤风疫苗纯化过程中的TT含量,为破伤风疫苗质量控制奠定了基础。     

凝胶过滤层析纯化白喉类毒素工艺的优化

目的  通过定制设计优化白喉类毒素（diphtheria toxoid,DT）层析纯化工艺。方法  将经硫酸铵沉淀的精制DT原液浓缩后分别用Sepharose 4FF （4FF）和Sephacrayl S-300（S-300）凝胶过滤层析进行进一步纯化。根据定制设计,将上样体积、样品浓度、洗脱流速、层析介质设为实验因子,将层析载量、纯度、收率设为响应值,建立数学模型,确定凝胶过滤层析的最优层析参数,并对实验结果进行工艺验证。结果  定制设计的实验结果显示,S-300凝胶过滤层析纯化的DT纯度和收率均高于4FF凝胶过滤层析。进一步分析确定,S-300凝胶过滤层析的最佳参数为：上样体积2.5%,样品浓度45 mg/ml,洗脱流速45 cm/h。当以最佳参数进行S-300凝胶过滤层析纯化时,层析工艺的验证结果符合预期。结论  S-300比4FF更适宜作为DT纯化工艺的凝胶层析介质。     

Preparation and characterization of diphtheria toxoid-loaded elastic vesicles for transcutaneous immunization

The objective was to develop surfactant-based vesicle formulations containing diphtheria toxoid (DT) for transcutaneous immunization. Formulation variables were molar ratio of the surfactants (sucrose-laurate ester, octaoxyethylene-laurate ester, and sodium bistridecyl sulfosuccinate), DT concentration, pH, and ionic strength. The formulations were characterized by visual inspection, dynamic light scattering and zeta potential measurements. DT loading efficiency and capacity of the vesicle formulations were studied by protein assay and enzyme-linked immunosorbent assay. DT-containing vesicle formulations were optimized for colloidal stability. The pH had a dramatic effect on DT-vesicle association: at pH 4.5 > 70% of the protein was associated with the vesicles, vs. < 20% at pH 5.0. By varying the ionic strength of the buffer, we revealed that hydrophobic interactions play an important role in the association. DT-vesicles formulated at pH 4.5 were stable and showed high association of DT with preserved antigenicity, and are therefore excellent candidates for future in vivo studies.     

Haemophilus influenzae Type b Vaccine: Reconstitution of Lyophilised PRP-T Vaccine with a Pertussis-containing Paediatric Combination Vaccine,or a Change in the Primary Series Immunisation Schedule,May Modify the Serum Anti-PRP Antibody Responses

Summary

Immunogenicity data obtained after primary series immunisations against Haemophilus influenzae type b (Hib), using a vaccine prepared by conjugating the capsular polysaccharide of Hib to tetanus toxoid (ActHIB?), were compiled from 146 study groups comprising 85 clinical trials or vaccination programs conducted between 1987 and 1999. ActHIB? was administered as a monovalent lyophilised vaccine, injected either in association with another paediatric vaccine (at separate administration sites) or in combination (where the different vaccines are mixed together in the same syringe before injection). Review of these data reveals two trends. First, PRP-T vaccine, given either alone or in combination with DTwcP, resulted in a stronger anti-PRP serum antibody response than when PRP-T was combined with DTacP vaccine. Second, an accelerated (i.e. one-month interval) immunisation schedule tended to induce a poorer anti-PRP antibody response than did the more widely spaced, standard inoculation schedules. Although an in-depth analysis of these over 11 000 study subjects on an individual basis with multivariate analysis or multifactorial statistical methods might eventually provide working hypotheses to fully understand these phenomenon, the various licensed, PRP-T-containing paediatric combination vaccines have proved to be clinically effective.     

Alginate Nanoparticles as a Promising Adjuvant and Vaccine Delivery System

During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system.     

破伤风类毒素对甲型副伤寒脱毒脂多糖的免疫增强作用

目的 增强多糖抗原在婴幼儿中的免疫原性,提供有效的免疫保护.方法 采用液体培养,收集菌体,脂多糖(LPS)提取,纯化脂多糖,活化脱毒多糖(O-SP)与破伤风类毒素(TT)共价结合.ELISA法检测O-SP及结合物的免疫效果.结果 单纯甲型副伤寒脱毒脂多糖未能诱导小鼠产生抗体,而与TT结合后,具有良好的免疫原性,能产生体液免疫反应,诱导小鼠产生较高水平的抗脂多糖抗体和抗破伤风抗体.结论 破伤风类毒类对甲型副伤寒脱毒脂多糖具有免疫增强作用.     

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits (n=4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1+/-25.8 microm. Mean encapsulation efficiency was 20.3+/-3.2% (n=3). Antigenicity of encapsulated TT was 90.5+/-1.8% (n=3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM+TT (P<0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P<0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different (P>0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM+TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM+TT was 175 times higher than the protective levels.