第三代百日咳疫苗毒性国家标准品的制备和标定

目的:制备和标定第三代百日咳疫苗毒性国家标准品,用于百日咳疫苗毒性检测.方法:按照《中国药典》三部(2015版)的相关要求,选用百日咳疫苗生产菌株CS株进行发酵罐培养,将收获的百日咳菌液灭活后分装冻干,作为第三代百日咳疫苗毒性国家标准品的候选品.按照《中国药典》三部要求对候选品进行检定,以第二代百日咳疫苗毒性国家标准品...     

百日咳杆菌黏着素的纯化及生物学特性研究

目的  建立从天然百日咳杆菌中提纯百日咳杆菌黏着素（pertactin,PRN）的方法,研究PRN的生物学特性并确定PRN在无细胞百日咳疫苗中的作用。方法  百日咳杆菌CS株于发酵罐培养后,经热浸提、硫酸铵分级沉淀及离子交换层析得到纯化PRN,根据《中华人民共和国药典》2005年版三部的要求,对纯化PRN进行纯度检测及生物学特性研究。结果  各项检测显示,纯化PRN的纯度在95%以上,其相对分子质量和等电点与标准PRN一致。纯化PRN对小鼠具有保护效力,且其保护效力随PRN免疫浓度的增加而增强。未检到纯化PRN的特异性毒性和异常毒性。结论  建立了从天然百日咳杆菌中提纯PRN的方法,而且纯化PRN具有免疫原性和保护效力。     

第一代无细胞百日咳疫苗毒性国家参考品制备工艺的研究

目的筛选无细胞百日咳疫苗毒性国家参考品的最佳生产工艺。方法选用百日咳CS菌株(编号58003),对不同的培养方式进行比较研究,选择最佳毒性国家参考品的生产工艺。结果该参考品经过不同培养方式比较后,确定了最终生产工艺,将培养的百日咳菌加入保护剂后分装制备成冻干品。结论该生产工艺制备的样品含有较强的毒性,同时能进行规模生产,满足了毒性国家参考品各项检测指标的要求。     

百日咳患儿及密切接触者的细菌培养和药物敏感性检测

目的:调查通过聚合酶链式反应(PCR)确诊百日咳住院患儿及其密切接触者百日咳鲍特菌培养分离情况,并检测患儿分离株对9种抗生素的最低抑菌浓度(MIC),为临床合理用药提供参考.方法:对2015年8月至2017年6月深圳市儿童医院通过PCR确诊的百日咳住院患儿及其密切接触者,采集鼻咽拭子进行百日咳鲍特菌培养,并对患儿分离株...     

痉挛性咳嗽患儿百日咳鲍特菌感染状况及临床特征分析

目的:了解痉挛性咳嗽患儿百日咳鲍特菌感染状况并分析其临床特征.方法:回顾性分析2019年1～12月于本院进行咽拭子聚合酶链式反应(PCR)及血清人百日咳毒素IgG抗体(PT-IgG)检测的疑似百日咳鲍特菌感染的痉挛性咳嗽患儿病历资料,根据检测结果分为百日咳组与类百日咳组,并比较两组患儿的临床特征.结果:共纳入147例痉...     

新一代百日咳无细胞菌苗和重组菌苗

４０年代研制的百日咳全细胞菌苗成功地控制了百日咳流行。对这种菌苗的反应原性的担心导致无细胞菌苗的研制，日本应用无细胞菌苗作常规免疫，证明它安全，有效而且副作用较小。具有不同抗原组分和浓度的新型菌苗正在进行临床试验，以进一步提高无细胞菌苗的效力。用某些化学方法脱毒的百日咳类毒素的毒性仍可恢复，为了解决这个问题，研制了含有遗传学方法脱毒的百日咳毒素（ＰＴ）的重组百日咳菌苗。Ｓ１基因的定位诱变能消除ＰＴ     

应用中国仓鼠卵巢细胞法检测百日咳毒素毒性方法的建立及初步验证

目的  建立用中国仓鼠卵巢（Chinese hamster ovary,CHO）细胞检测百日咳毒素（PT）毒性的方法,并对其适用性进行初步验证。方法  根据PT能特异地使CHO细胞簇集的特性,以国家PT标准品为参考品,取多批脱毒后PT进行检测,摸索适宜的孵育时间、细胞悬液浓度等实验条件,建立PT毒性的体外检测方法。对PT参考品进行倍比系列稀释,确定本法的检测限。对5批脱毒后PT进行重复检测,验证本法的重复性。结果  利用不同浓度CHO细胞对脱毒PT进行测定,确定最佳细胞浓度为5×104/ml,并通过最佳浓度细胞确定簇集结果的最佳判定时间为PT接种后48 h。本法对PT标准品的检测限定为125~500 pg/ml。用本法重复测定5批脱毒PT的残余毒性,变异系数为0.0%~34.6%,说明该法重复性良好。结论  建立的CHO细胞检测法快捷、简便,能灵敏、准确地检测脱毒PT的残余毒性,可用于相关疫苗产品的质量控制。     

组分百日咳疫苗安全性检测体外替代方法的应用和初步评价

目的：用一种组分百日咳疫苗残留毒性的体外替代检测方法，更稳定和客观地评价疫苗成品的生产一致性。方法：验证直接定性法和间接定量法2种中华仓鼠卵巢细胞簇集试验方法，分别对6个国内外厂家的10种组分百日咳疫苗产品毒性进行定量和定性检测，并使用百日咳毒素参考品将体外法与小鼠组胺致敏试验进行桥接和初步评价。结果：体外定量试验的灵敏度为0.0026±0.0003 IU·mL^-1，几何变异系数为13%，体外定性试验的灵敏度为0.0067±0.0016 IU·mL^-1，几何变异系数为24%，组胺致敏试验的灵敏度为3.3 IU·mL^-1；所有样品的体外定性结果均为阴性，小鼠组胺致敏试验结果均合格，体外定量结果与小鼠组胺致敏结果具有良好的相关性（r=0.737，P＜0.05）。结论：本研究在国内首次使用 CHO细胞簇集试验方法检测百日咳疫苗成品，并进行了初步评价。该方法灵敏度高于小鼠组胺致敏试验，可应用于百日咳疫苗毒性及毒性逆转检测。该方法需要更广泛的推广应用和数据积累，以达到完全替代动物实验的目标。     

检测百日咳毒素的双抗体夹心ELISA法的建立

目的  建立白喉-破伤风-无细胞百日咳疫苗生产中百日咳毒素（pertussis toxin, PT）含量的测定方法。方法  免疫家兔制备高效价抗PT血清,纯化抗PT多克隆抗体并进行酶标记,建立双抗体夹心ELISA法并对其进行验证。结果  建立的方法在0~20 ng/ml PT测量区间呈现最佳线性,相关系数＞0.99,且重复性好。检测百日咳丝状血凝素和黏着素,结果均为阴性,说明该法特异性良好。试验内10次、不同试验间3次测定16.0、8.0、4.0、3.0 ng/ml PT,变异系数为2.8%~9.7%,回收率为95.7%~106.0%,精密度和准确度验证均符合常规质量控制要求,定量限度为3.0 ng/ml。结论  该方法能有效检测百日咳杆菌大罐发酵过程中分泌的PT,为PT质量控制奠定了重要基础。     

百日咳黏着素研究进展

百日咳黏着素(pertactin,PRN)是所有有毒力的百日咳杆菌产生的一种非纤毛性凝集原,存在于菌体外膜上,属于自主转运蛋白.它能产生保护水平的抗体,已经成为百日咳组分疫苗的重要抗原成分.近年来许多疫苗接种率较高的国家均有百日咳发病率增高的报告,其中一个因素就是百日咳杆菌的一些抗原(百日咳毒素、PRN、凝集原)型别的转换导致疫苗保护效果的下降.此文就PRN的分子结构、生物学特征、流行病学及纯化工艺方面作一综述.     

Transferability of dermal temperature histamine sensitization test for estimation of pertussis toxin activity in vaccines

All current acellular pertussis vaccines (ACVs) contain detoxified pertussis toxin (PT) as a major component. An essential part of the safety evaluation of these vaccines, required by regulatory authorities, is to monitor their active PT content and to check for reversion to toxicity of the detoxified PT. Although various in vitro tests are under investigation, the only practicable means for detecting active PT at present is the histamine sensitization test. The methods given in the European Pharmacopoeia and in the US Pharmacopoeia are based on recording a binary response to histamine challenge (using a lethal end point). A more sensitive method based on measurement of rectal temperature is given in the Japanese Minimum Requirements for Biological Products. More recently, a refinement of this method based on dermal temperature measurement has been developed for ACVs in combination with diphtheria and tetanus vaccines (DTaP). We show that this method also can be used for more complex combination vaccines and is readily transferable. Furthermore use of dermal temperature provides a more precise quantitative estimate of toxin activity than the binary response, leading to an increase in information from a specified number of animals, or allowing a reduction in the number of animals required. We suggest that, pending the development of an alternative in vitro replacement method, the temperature based method may serve as an intermediate solution to the estimation of PT activity giving a precise estimate with reduction in animal numbers.     

检测百日咳毒素活性的中国仓鼠卵巢细胞簇集法的建立和初步应用

目的  建立检测百日咳毒素（pertussis toxin,PT）活性的中国仓鼠卵巢（Chinese hamster ovary,CHO）细胞簇集法,并将建立的方法应用于无细胞百日咳疫苗生产的质量控制。方法  将PT标准品系列稀释后与培养的CHO细胞混合,观察PT引起CHO细胞簇集的敏感度,并研究反应时间、反应溶液的pH值、缓冲液浓度、加样顺序等因素对PT引起CHO细胞簇集的影响。应用建立的方法检测百日咳疫苗生产过程中脱毒前后的PT活性及丝状血凝素和百日咳黏着素组分中残留的PT活性。    结果  PT使CHO细胞发生最佳簇集的理想反应时间是24 h,最适pH值是7~8,适宜氯化钠缓冲液浓度为0.15 mol/L,最优加样顺序是先加CHO细胞再加PT。建立的CHO细胞簇集法的检测敏感度为10 pg/ml PT,且重复性良好。结论  CHO细胞簇集法可用于PT活性检测。     

Investigation in a model system of the effects of combinations of anthrax and pertussis vaccines administered to service personnel in the 1991 Gulf War

The toxicity and immunogenicity of the anthrax and pertussis vaccine combinations used in the 1991 Gulf War was assessed in NIH, A/J and Balb/c mice. Inoculation of pertussis vaccines, vaccine combinations, or aluminium salt caused illness, splenomegaly and significant weight loss. Although some animals recovered eventually, a lethal form of ascites developed in some NIH mice and body weights of A/J and Balb/c mice remained below normal levels. Inoculation of anthrax vaccine produced little effect. Exposure to diluted vaccine combinations produced less serious side effects of shorter duration. Single vaccinations induced specific IgG1 antibodies whereas a mixture of IgG1 and IgG2a was produced after multiple injections. Antigen stimulation of spleen cells from mice exposed to pertussis vaccines induced high levels of NO and IL-6, whereas stimulated spleen cells from mice exposed to anthrax vaccine produced only low levels of IL-6. In mice, pertussis vaccines act as an adjuvant for anthrax vaccine, but these vaccines are also the major cause of toxicity of the vaccine combination. The relatively high vaccine dose used, together with the low sensitivity of mice to anthrax toxin, emphasises that caution should be exercised in applying these results to human recipients of these vaccines.     

Reduced-antigen combined diphtheria-tetanus-acellular pertussis vaccine (Boostrix)

The reduced-antigen combined diphtheria-tetanus-acellular pertussis vaccine (dTpa) is intended for use as a booster dose in individuals aged > or =4 years. A single dose of dTpa elicited generally similar levels of antibodies against pertussis antigens (pertussis toxoid [PT], filamentous haemagglutinin [FHA] and pertactin [PRN]) as a similar monovalent pertussis booster vaccine (ap) in adolescents or adults, irrespective of their prevaccination serological status or vaccination history. Levels of antibodies directed against diphtheria toxoid were similar in recipients of dTpa or a licensed reduced-antigen combined diphtheria-tetanus booster vaccine (Td). However, levels of antitetanus antibodies were significantly higher in recipients of Td vaccines compared with those receiving dTpa. Similar serological response rates were observed for anti-PT, -FHA and -PRN between those receiving dTpa or ap and a similar high percentage of recipients of dTpa and the Td vaccines had seroprotective levels of antibodies against diphtheria and tetanus toxoid. The most frequently reported local adverse reactions following immunisation with dTpa included pain, redness and swelling; general symptoms included fatigue, headache and fever.     

On technological and immunological benefits of multivalent single-injection microsphere vaccines

Purpose. With the aim of developing multivalent vaccines for single-injection, we examined the feasibility of combining antigens in biodegradable microspheres. Such vaccines are expected to improve vaccination coverage by reducing the number of vaccination sessions required to generate immunity. Methods. Mono- and multivalent vaccines of Haemophilus influenzae type b (Hib) conjugate, diphtheria toxoid (DT), tetanus toxoid (TT), and pertussis toxin (PT) in poly (lactic acid) and poly(lactic-coglycolic acid) microspheres were prepared by spray drying, and the influence of coencapsulated antigens and excipients on antigen loading, release, and stability was examined. Two tetravalent formulations were tested in guinea pigs. Results. Monovalent Hib and PT vaccines showed loading efficiencies of 10% (Hib) and 30% (PT) in both polymers. The loading efficiencies increased upon addition of trehalose and, even more, when the antigens were coencapsulated in di- and trivalent combinations. Highest loading efficiencies (>80%) were achieved with trivalent formulations (DT + PT + Hib) that also contained coencapsulated albumin. The percentage of antigen released during 24 h of incubation was typically 10-40% and decreased as loading efficiency increased. Enzyme-linked immunosorbent assay (ELISA) data revealed that TT, DT, and PT remained antigenic throughout the encapsulation and subsequent release processes. Finally, all antigens maintained their immunogenicity, since strong and sustained antibody responses were elicited after a single injection of tetravalent microsphere vaccines (DT + TT + PT + Hib) in guinea pigs. Conclusions. This study reveals technologic benefit as well as an immunological potential of multivalent single-injection microsphere vaccines. The results support our hypothesis that coencapsulation of several antigens may intrinsically improve entrapment of antigenic and immunogenic antigen probably by virtue of increased protein concentration during microencapsulation leading to mutual stabilization of the components.     

Acellular pertussis vaccines for use among infants and young children

Pertussis is still one of the most common vaccine-preventable childhood diseases in developed countries. Infants, particularly those < 6 months, are the most susceptible and those who suffer the greatest disease burden and mortality. In the 1970s, concerns about the reactogenicity of whole-cell vaccines led to a decrease in vaccine coverage and later the re-emergence of the disease in many countries. The advent of acellular vaccines in recent years has constituted an important advance in the acceptance of this immunisation and consequently the control of the disease. The efficacy of acellular pertussis vaccines is ～ 59 – 93%, similar to whole-cell vaccines, but all available data confirm the substantial improvement in safety of the new vaccines. With the licensure of acellular pertussis vaccines and combined vaccines containing them, pertussis immunisation has become significantly developed. Furthermore, the possibility of continuing to vaccinate adolescents and adults with new diphtheria, tetanus, and pertussis (dTap) vaccines is an important step in achieving control and elimination of the disease.     

Acellular pertussis vaccines for use among infants and young children

Pertussis is still one of the most common vaccine-preventable childhood diseases in developed countries. Infants, particularly those < 6 months, are the most susceptible and those who suffer the greatest disease burden and mortality. In the 1970s, concerns about the reactogenicity of whole-cell vaccines led to a decrease in vaccine coverage and later the re-emergence of the disease in many countries. The advent of acellular vaccines in recent years has constituted an important advance in the acceptance of this immunisation and consequently the control of the disease. The efficacy of acellular pertussis vaccines is approximately 59 - 93%, similar to whole-cell vaccines, but all available data confirm the substantial improvement in safety of the new vaccines. With the licensure of acellular pertussis vaccines and combined vaccines containing them, pertussis immunisation has become significantly developed. Furthermore, the possibility of continuing to vaccinate adolescents and adults with new diphtheria, tetanus, and pertussis (dTap) vaccines is an important step in achieving control and elimination of the disease.     

Acellular pertussis vaccines: new preparation. Better tolerated but less active?

(1) In France SmithKline Beecham market a three-component acellular pertussis vaccine in the form of a tetravalent vaccine and a pentavalent vaccine. Pasteur M/erieux MSD also market in France a two-component acellular pertussis vaccine in the form of a tetravalent vaccine and a pentavalent vaccine. (2) The two pentavalent vaccines are indicated for an early booster injection at age 16-18 months; the two tetravalent vaccines are indicated for a late booster injection at age 11-13 years. (3) The published assessment file on these vaccines is limited. (4) Immunological studies show that the acellular pertussis valency does not reduce the antigenicity of the valencies with which it is combined (diphtheria, tetanus and poliomyelitis), although there is some doubt regarding the Haemophilus influenzae tybe b valency. (5) In the absence of direct comparisons with the cellular pertussis vaccine available in France in tetravalent or pentavalent vaccines, indirect evidence and data from a trial comparing the acellular two-component vaccine with the cellular vaccine (Vaxicoq degrees ) marketed in France (combined with diphtheria and tetanus valencies) suggest that clinical protection is slightly lower after primary vaccination with the two acellular vaccines. (6) In children, acellular pertussis vaccines are globally better tolerated than the cellular pertussis vaccine. (7) In adolescents, a small study has shown that the three-antigen acellular pertussis vaccine is relatively well tolerated. But the acellular vaccine was released too recently onto the international market to know the precise incidence of rare and severe adverse effects. (8) The new French vaccination schedule for pertussis now includes a booster between 11 and 13 years. Only long-term epidemiological follow-up can show if routine vaccination of adolescents against pertussis will have an impact on the incidence of pertussis in infants and adults. (9) The incidence of Haemophilus b meningitis must continue to be monitored.     

检测百日咳抗原的系列酶免疫测定试剂盒的稳定性及其在百日咳疫苗生产中的应用

 目的   评估检测百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamentous haemagglutinin,FHA)和黏着素(pertactin,Prn)的3种酶免疫测定试剂盒的稳定性及其在无细胞百日咳疫苗（acellular pertussis vaccine,aPV）生产质量控制中的应用。 方法   对3种酶免疫测定试剂盒进行热加速试验（37 ℃放置3 d）和长期稳定性试验（4 ℃放置6月）,根据试剂盒的标准曲线相关系数、最高浓度标准品与阴性对照的吸光度值之比（P/N）值、质控品回收率评估试剂盒的稳定性。将3种试剂盒用于aPV生产的发酵、纯化、吸附阶段的PT、FHA、Prn定量检测,以确定3种试剂盒对aPV生产质量控制的可行性。 结果   3种酶免疫测定试剂盒的37 ℃热加速试验和4 ℃长期稳定性试验的各项质量参数均符合相关要求。采用试剂盒定量各生产阶段的百日咳抗原含量显示：百日咳杆菌的发酵终点在第42~46 h;各3批PT、FHA和Prn组分液的纯化回收率分别为49.24%、56.12%和63.65%、68.75%、55.60%和49.76%、75.73%、60.63%和50.10%。采用单独吸附的铝佐剂抗原吸附率高于采用混合吸附,但单独和混合吸附方式制备的疫苗的效力无差异。 结论   检测PT、FHA和Prn的3种酶免疫测定试剂盒具有良好的稳定性,可用于aPV生产的质量控制。