Sabin株脊髓灰质炎病毒灭活疫苗中佐剂应用的初步研究

发布时间：2018-04-02 13:39

本文选题：Sabin株　切入点：脊髓灰质炎病毒灭活疫苗　出处：《中国协和医科大学》2009年硕士论文

【摘要】： 随着全球消灭脊髓灰质炎(脊灰)目标的临近,疫苗衍生病毒(VDPV)的流行、疫苗相关麻痹型病例(VAPP)的发生等对无脊灰状态成果的巩固威胁很大。在消灭脊灰的最后阶段,应尽快消灭脊灰野毒株,并使用灭活疫苗(Inactivatedpoliovirus vaccine,IPV)替代目前广泛使用的脊髓灰质炎减毒活疫苗(Oral poliovirusvaccine,OPV)来维持无脊灰状态,消除自然界的活病毒。对于新的生产厂家考虑到疫苗生产的生物安全性等因素,WHO鼓励研制Sabin株脊髓灰质炎灭活疫苗(Sabin IPV)。目前尚无成品Sabin IPV上市,于是Sabin IPV的定量检测、质量指标和免疫剂量等都还没有统一标准。前期研究显示,Sabin IPV与野毒株IPV(WT-IPV)在抗原性、稳定性等方面均有差别,于是建立在WT-IPV基础上的定量检测方法和质量控制指标并不适用于Sabin IPV。另外,Sabin IPV与WT-IPV抗原性也有差异,Sabin2型的免疫原性只是野毒株MEF-1株的1/10。为解决疫苗免疫原性不佳的问题,最常用的方法即添加疫苗佐剂。本论文旨在于建立适用于Sabin IPV的D抗原定量方法及蛋白质含量测定方法,完善Sabin IPV检定和质量控制体系;并进一步探索SabinIPV中添加佐剂对免疫效果产生的影响,探讨Sabin IPV中佐剂应用的意义和最适剂量等。本研究第一部分即建立Sabin株脊髓灰质炎病毒灭活疫苗(Sabin IPV)中D抗原含量及蛋白质含量的检测方法。首先通过摸索各步骤反应条件,优化本实验室建立的多抗ELISA法测定Sabin IPV中D抗原含量的体系,对3个批次的样品进行5次重复测定,结果的变异系数均小于10%。另外,利用蛋白质遇三氯乙酸产生沉淀的特点,对Lowry法进行改良,建立了能够准确测定Sabin IPV中微量蛋白质含量的方法,实验证明,该方法能够排除Sabin IPV中的游离氨基酸、多肽和酚红指示剂等物质的干扰,线性范围为2.5～40ug/ml,r=0.9998,最佳条件下加标平均回收率为95.32%,精密度试验结果批内和批间变异系数均小于10%。研究的第二部分通过将Al(OH)_3、DC-Chol和Al(OH)_3+DC-Chol 3种不同疫苗佐剂按不同剂量与Sabin IPV(每剂含D抗原Ⅰ型15DU、Ⅱ型16 DU和Ⅲ型22.5 DU)配伍后分别免疫Wistar大鼠,主要检测各组大鼠免疫后血清中和抗体水平、特异性IFN-γ的产生情况以及免疫后各组大鼠CD3/CD4/CD8分子表达的差异。综合上述结果评价在Sabin IPV中添加Al(OH)_3或/和DC-Chol对大鼠体液免疫和细胞免疫的影响。通过微量中和实验检测Sabin株脊髓灰质炎特异性中和抗体发现,在Sabin IPV中添加佐剂,普遍于第1针免疫后提高了大鼠血清中3个型特异性中和抗体阳转率;而且3种佐剂还在第2针免疫后提高了Ⅱ型中和抗体的阳转率;Sabin IPV中添加佐剂后,对特异性中和抗体的水平也有不同程度的影响。添加Al(OH)_3或DC-Chol在第2针免疫后就产生了高效价的Ⅰ型中和抗体,如添加0.5mgDC-Chol第2针免疫后Ⅰ型中和抗体效价几何均数(GMT)达到未添加佐剂组的3倍以上,显著提高了早期Ⅰ型中和抗体的水平;添加0.1mgAl(OH)_3组与未添加佐剂组相比,第3针免疫后Ⅱ型特异性中和抗体效价的几何均数增长2倍以上;添加Al(OH)_3或DC-Chol或Al(OH)_3+DC-Chol联合佐剂在第2针免疫后就获得了高水平的Ⅲ型特异性中和抗体,GMT比未添加佐剂组增长15倍和12倍,即大大提高了早期Ⅲ型中和抗体的水平。ELISPOT测定针对Sabin株脊髓灰质炎病毒的特异性IFN-γ的结果显示,注射不含佐剂Sabin IPV的大鼠未产生特异性IFN-γ斑点,注射添加了Al(OH)_3或DC-Chol佐剂Sabin IPV的大鼠产生了少量特异性IFN-γ斑点,其中添加0.25mgDC-Chol组效果最好,Ⅰ、Ⅱ、Ⅲ型各产生特异性IFN-γ斑点22.25±6.24、13.75±3.77和22.50±5.92 SFC/10~6cell提示添加Al(OH)_3或DC-Chol佐剂可能对大鼠针对Sabin株脊髓灰质炎病毒的特异性细胞免疫有少许增强作用;流式细胞仪检测各组大鼠CD3/CD4/CD8分子表达的差异的结果显示,Al(OH)_3或DC-Chol佐剂的应用能提高大鼠CD3~+CD4~+/CD3~+CD8~+值,提示2种佐剂的应用均能增强大鼠的免疫功能,但这种增强作用在佐剂的不同剂量组之间差别不显著。综上所述,Al(OH)_3或DC-Chol能够增强Sabin IPV的体液免疫效果,对细胞免疫也有一定增强作用,能在更短的时间内使Wistar大鼠获得更好的保护效果。
[Abstract]:With the global eradication of poliomyelitis (polio) target approaching, vaccine derived virus (VDPV) epidemic, the vaccine associated paralytic (VAPP) occurrence of polio free efforts to consolidate the threat. In the final stage of polio eradication, should as soon as possible to eradicate poliomyelitis wild strains, and the use of inactivated vaccine (Inactivatedpoliovirus vaccine IPV) to replace the widely used live attenuated poliomyelitis vaccine (Oral, poliovirusvaccine, OPV) to maintain polio free, eliminate the live virus in nature. For new products taking into account biological safety factors such as vaccine production, WHO encourages the development of Sabin inactivated poliovirus vaccine (Sabin IPV).
At present there is no finished Sabin IPV listed, so the quantitative detection of Sabin IPV, quality index and immune dose so there is no uniform standard. Previous studies have shown that Sabin IPV IPV (WT-IPV) and wild strains in antigenicity, stability and other aspects were different, and based on WT-IPV on the basis of the quantitative detection method and the quality control index is not applicable to Sabin Sabin IPV and WT-IPV IPV. in addition, there are differences in immune antigenicity, Sabin2 type of just wild strain MEF-1 1/10. to solve the problem of poor immunogenicity of vaccine, the most common method is to add adjuvant vaccine. The purpose of this paper is to establish methods for the quantitative determination method of D antigen for Sabin IPV Sabin IPV and protein content, improve the verification and quality control system; and to further explore the effect of adjuvant SabinIPV on immune effect of adjuvant Sabin, IPV should be used and the significance The optimum dosage.
The first part of this study is to establish the Sabin strain of inactivated poliovirus vaccine (Sabin IPV) method for detection of antigen content and protein content in D. First, through exploring the various steps of the reaction conditions, the content of Sabin IPV in D antigen determination optimization polyclonal antibody ELISA method established in our laboratory system of 3 batches of samples were repeated 5 times determination of the coefficient of variation of the precipitation was less than 10%. in addition, based on the characteristics of protein with three chloroacetic acid, modified Lowry method, established a method for accurate determination of trace protein content of Sabin in IPV, the experiment shows that this method can eliminate the free amino acid Sabin IPV, peptides and other substances interfere with phenol red indicator the linear range is 2.5 ~ 40ug/ml, r=0.9998, under the optimum conditions the average recovery rate was 95.32%, the results of precision test intra and interassay coefficients of variation were less than 10%.
The second part of the study by Al (OH) _3, DC-Chol and Al (OH) _3+DC-Chol 3 different adjuvants according to different dosage of Sabin and IPV (each agent containing D antigen of 15DU type I, II and III 16 DU 22.5 DU) and after Wistar rats were immunized, mainly detected in rats with immune the serum levels of neutralizing antibodies, specific IFN- gamma production and expression of rats after CD3/CD4/CD8 molecular and immunological differences. The results of evaluation in Sabin IPV add Al (OH) effect of _3 or / and DC-Chol on humoral immunity and cellular immunity in rats. By micro neutralization assay of Sabin poliovirus specific neutralizing antibody found that adding adjuvant in Sabin IPV, generally first needles after immunization increased 3 rat serum specific neutralizing antibody positive conversion rate was second; and 3 adjuvants can improve immune needle type II neutralizing antibody positive rate; Sabin IPV In addition to adjuvant, specific neutralizing antibody level has different effects. Adding Al (OH) type _3 or DC-Chol produced high titer in second needle immune neutralizing antibodies after adding 0.5mgDC-Chol second pin type 1 immune neutralizing antibody titer and geometric mean (GMT) was added to the adjuvant group has more than 3 times, significantly improve the early type of neutralizing antibody level; adding 0.1mgAl (OH) _3 group compared with the group without adjuvant, geometric neutralizing antibody titer after immunization third needle type II specific mean growth of more than 2 times; adding Al (OH) _3 (OH or DC-Chol or Al _3+DC-Chol) combined with adjuvant have high levels of type III specific neutralizing antibody in second needle immunized GMT than without adjuvant group increased 15 times and 12 times, which greatly increased the level of.ELISPOT in the early stage of type III Determination of neutralizing antibody specific for IFN- Sabin strains of polio virus Gamma results showed that injected without adjuvant Sabin IPV rats did not produce specific IFN- gamma spots, injection with Al (OH) _3 or DC-Chol Sabin IPV adjuvant in rats produced a few specific IFN- gamma spots, one of the best, adding 0.25mgDC-Chol group I, II, III type specific IFN- dot gamma 22.25 + 6.24,13.75 + 3.77 and 22.50 + 5.92 SFC/10~6cell Al (OH) suggested that adding _3 or DC-Chol adjuvant may have a little effect to enhance specific cellular immunity against poliomyelitis virus Sabin strain rats; the differential expression of /CD8 molecule CD3/CD4 in rats were determined by flow cytometry showed that Al (OH) the application of _3 or DC-Chol adjuvant can improve rat CD3~+CD4~+/CD3~+CD8~+, suggesting that the 2 Application of adjuvant can enhance the immune function of rats, but this enhancement between different adjuvant dose group was no significant difference. In summary, Al (OH) _ 3 or DC-Chol can enhance the humoral immunity effect of Sabin IPV, and enhance cell immunity. It can make Wistar rats get better protective effect in a shorter time.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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