氯仿—甲醇提取贝氏柯克斯体残存组分Q热疫苗的研制及其免疫保护性和安全性初步评价
发布时间：2018-09-13 14:39
【摘要】： 贝氏柯克斯体(Coxiella burnetii，又称Q热立克次体)为Q热病原体。该病原体为专性细胞内寄生菌，可通过气溶胶传播，对人和动物的感染性极强，是一种重要的生物战剂／生物恐怖剂。人、畜对贝氏柯克斯体普遍易感，畜牧相关从业人员是高危人群。不管是预防贝氏柯克斯体自然感染还是防生物武器／生物恐怖均需要有效的Q热疫苗对易感人群进行免疫接种。 本研究将国内分离的贝氏柯克斯体新桥株在鸡胚大量繁殖，然后从感染鸡胚中分离、纯化贝氏柯克斯体，用甲醛灭活纯化贝氏柯克斯体，制备全细胞疫苗(WCV)，同时采用氯仿-甲醇提取WCV，制备氯仿-甲醇提取残存组分疫苗(CMR)。采用高压气相色谱测定了两种疫苗的脂肪酸，发现CMR较WCV减少了11种脂肪酸，其中4种为不饱和脂肪酸，4种为支链脂肪酸，70％为C原子数≥18的较长链的脂肪酸。另外，两种疫苗的支链脂肪酸与直链脂肪酸、不饱和脂肪酸与饱和脂肪酸的比值也明显不同。通过蛋白单相及双向电泳分析两种疫苗蛋白，它们的蛋白电泳图谱和主要高丰度蛋白点的蛋白丰度均无明显差异。 采用CMR和WCV免疫BALB／c小鼠，，间接免疫荧光法分析免疫小鼠血清特异性抗体水平，结果显示CMR与WCV一样均能诱导高水平特异性抗体产生。CMR加Al(OH)3佐剂免疫小鼠，该免疫小鼠特异性抗体水平显著高于CMR单独免疫。用MTT法体外分析免疫小鼠脾脏淋巴细胞增殖，结果显示CMR诱导小鼠脾脏淋巴细胞增殖水平显著高于WCV，而WCV对小鼠淋巴细胞增殖有一定程度的抑制。免疫小鼠体内、外细胞因子的测定结果显示，CMR疫苗能诱导TNF-α、IFN-γ及IL-10等多种细胞因子的分泌。 用贝氏柯克斯体Ⅰ相强毒株(包括疫苗制备株和美国分离Nine Mile株和欧洲分离的Henzerling)攻击免疫小鼠，用荧光定量PCR分析小鼠脾脏贝氏柯克斯体的量。结果显示CMR与WCV一样能有效保护小鼠抵抗贝氏柯克斯体的攻击。CMR加Al(OH)3佐剂免疫小鼠的脾脏贝氏柯克斯体的量显著少于CMR单独免疫。 用大剂量WCV免疫小鼠，在接种部位出现皮下结节，小鼠的脾脏明显肿大，组织病理学检查脾脏有不同程度的网状内皮细胞增生。检查免疫小鼠肝脏功能指标，ALT和AST均明显升高。而相同大剂量CMR免疫小鼠则无上述病理学变化和肝功能损伤。 WCV疫苗是目前唯一得到美国FDA批准应用的Q热疫苗，虽然其具有良好的免疫保护效果，但其明显的副作用使得其应用受到严格限制。本研究证明采用国内分离株制备的CMR疫苗与WCV疫苗的免疫保护效果相当，但是CMR疫苗的副作用明显小于WCV，CMR疫苗的低毒性是由于氯仿-甲醇提取WCV后，清除引起机体损伤的某些脂类或显著降低某些脂类含量密切有关。CMR疫苗的良好免疫保护效果与其高效诱导机体的体液和细胞免疫应答相关。 Q热疫苗免疫小鼠能部分对抗登革病毒的感染，显示其具有非特异免疫保护作用。
[Abstract]:Coxiella burnetii (also known as Q-febrile rickettsia) is a Q-fever pathogen. The pathogen is a specific intracellular parasitic bacteria, which can be transmitted by aerosols and is highly infectious to humans and animals. It is an important biological warfare agent/bioterroric agent. People, livestock are generally susceptible to Coxiella burnetii, and livestock-related practitioners are at high risk. Population. Effective Q-fever vaccines are needed to immunize susceptible populations, whether to prevent natural Coxiella baileyi infection or biological weapons/bioterrorism.
In this study, a new bridge strain of Coxoplasma baileyi isolated in China was propagated in chicken embryos, then isolated from infected chicken embryos, purified Coxoplasma baileyi, inactivated by formaldehyde, purified Coxoplasma baileyi, prepared whole cell vaccine (WCV), and extracted WCV with chloroform-methanol to prepare residual component vaccine (CMR). The fatty acids of the two vaccines were determined by phase chromatography, and 11 kinds of fatty acids were found to be reduced by CMR compared with WCV. Among them, 4 were unsaturated fatty acids, 4 were branched chain fatty acids, and 70% were longer chain fatty acids with C atom number (>18). In addition, the ratio of branched chain fatty acids to straight chain fatty acids, unsaturated fatty acids to saturated fatty acids of the two vaccines was not significant. Similarly, there was no significant difference between the two vaccine proteins in protein electrophoresis profiles and protein abundance of major high abundance protein spots.
CMR and WCV were used to immunize BALB/c mice. Indirect immunofluorescence assay was used to analyze the level of serum specific antibodies in immunized mice. The results showed that CMR could induce high level of specific antibodies as well as WCV. The level of specific antibodies in immunized mice immunized with CMR plus Al (OH) 3 adjuvant was significantly higher than that in CMR alone. The results showed that the proliferation level of splenic lymphocytes induced by CMR was significantly higher than that induced by WCV, while WCV inhibited the proliferation of lymphocytes to a certain extent.
Immune mice were attacked with phase I virulent Coxiella baileyi strains (including vaccine preparation strains, Nine Mile strains isolated from the United States and Henzerling strains isolated from Europe). The amount of Coxiella baileyi in the spleen of mice was analyzed by fluorescence quantitative PCR. The results showed that CMR could protect mice against Coxiella baileyi as well as WCV. CMR plus Al (OH) 3 adjuvant was immune. The amount of spleen Kirk's body in spleen of epidemic mice was significantly less than that of CMR alone.
Immunized mice with high dose of WCV showed subcutaneous nodules at the site of inoculation, spleen enlargement and reticular endothelial cell hyperplasia in different degrees. The liver function indexes, ALT and AST of immunized mice were significantly increased, while the same dose of CMR immunized mice showed no pathological changes and liver function damage. Injury.
WCV vaccine is currently the only Q-fever vaccine approved by FDA in the United States. Although it has a good immune protection effect, its obvious side effects restrict its application. This study proves that the immune protection effect of CMR vaccine prepared by domestic isolates is similar to that of WCV vaccine, but the side effects of CMR vaccine are obviously less than that of WCV vaccine. The low toxicity of WCV, CMR vaccine is due to the removal of some lipids causing injury or the significant reduction of some lipids content after chloroform-methanol extraction of WCV. The good immune protection effect of CMR vaccine is related to its highly effective induction of humoral and cellular immune responses.
The mice immunized with Q-fever vaccine could partly resist the infection of dengue virus, which showed that it had non-specific immune protection.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R376
本文编号：2241462
[Abstract]:Coxiella burnetii (also known as Q-febrile rickettsia) is a Q-fever pathogen. The pathogen is a specific intracellular parasitic bacteria, which can be transmitted by aerosols and is highly infectious to humans and animals. It is an important biological warfare agent/bioterroric agent. People, livestock are generally susceptible to Coxiella burnetii, and livestock-related practitioners are at high risk. Population. Effective Q-fever vaccines are needed to immunize susceptible populations, whether to prevent natural Coxiella baileyi infection or biological weapons/bioterrorism.
In this study, a new bridge strain of Coxoplasma baileyi isolated in China was propagated in chicken embryos, then isolated from infected chicken embryos, purified Coxoplasma baileyi, inactivated by formaldehyde, purified Coxoplasma baileyi, prepared whole cell vaccine (WCV), and extracted WCV with chloroform-methanol to prepare residual component vaccine (CMR). The fatty acids of the two vaccines were determined by phase chromatography, and 11 kinds of fatty acids were found to be reduced by CMR compared with WCV. Among them, 4 were unsaturated fatty acids, 4 were branched chain fatty acids, and 70% were longer chain fatty acids with C atom number (>18). In addition, the ratio of branched chain fatty acids to straight chain fatty acids, unsaturated fatty acids to saturated fatty acids of the two vaccines was not significant. Similarly, there was no significant difference between the two vaccine proteins in protein electrophoresis profiles and protein abundance of major high abundance protein spots.
CMR and WCV were used to immunize BALB/c mice. Indirect immunofluorescence assay was used to analyze the level of serum specific antibodies in immunized mice. The results showed that CMR could induce high level of specific antibodies as well as WCV. The level of specific antibodies in immunized mice immunized with CMR plus Al (OH) 3 adjuvant was significantly higher than that in CMR alone. The results showed that the proliferation level of splenic lymphocytes induced by CMR was significantly higher than that induced by WCV, while WCV inhibited the proliferation of lymphocytes to a certain extent.
Immune mice were attacked with phase I virulent Coxiella baileyi strains (including vaccine preparation strains, Nine Mile strains isolated from the United States and Henzerling strains isolated from Europe). The amount of Coxiella baileyi in the spleen of mice was analyzed by fluorescence quantitative PCR. The results showed that CMR could protect mice against Coxiella baileyi as well as WCV. CMR plus Al (OH) 3 adjuvant was immune. The amount of spleen Kirk's body in spleen of epidemic mice was significantly less than that of CMR alone.
Immunized mice with high dose of WCV showed subcutaneous nodules at the site of inoculation, spleen enlargement and reticular endothelial cell hyperplasia in different degrees. The liver function indexes, ALT and AST of immunized mice were significantly increased, while the same dose of CMR immunized mice showed no pathological changes and liver function damage. Injury.
WCV vaccine is currently the only Q-fever vaccine approved by FDA in the United States. Although it has a good immune protection effect, its obvious side effects restrict its application. This study proves that the immune protection effect of CMR vaccine prepared by domestic isolates is similar to that of WCV vaccine, but the side effects of CMR vaccine are obviously less than that of WCV vaccine. The low toxicity of WCV, CMR vaccine is due to the removal of some lipids causing injury or the significant reduction of some lipids content after chloroform-methanol extraction of WCV. The good immune protection effect of CMR vaccine is related to its highly effective induction of humoral and cellular immune responses.
The mice immunized with Q-fever vaccine could partly resist the infection of dengue virus, which showed that it had non-specific immune protection.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R376
【参考文献】
相关期刊论文 前1条
1 孙长俭;陈梅玲;温博海;刘海洪;杨晓;牛东升;;两种Q热疫苗的脂肪酸分析[J];中国人兽共患病学报;2006年11期
本文编号：2241462
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