【摘要】：目的: 建立SARS相關冠狀病毒感染恒河猴動物模型,并用該模型評價一種實驗性SARS冠狀病毒滅活疫苗免疫原性、保護性與安全性,同時也為該疫苗進一步完善及投入臨床試驗提供理論和實驗依據(jù)。 方法: 動物免疫分3組,分別為肌肉注射0.5μg、5μg、50μg疫苗組及磷酸鹽緩沖液(PBS)對照組,同時設超大劑量(5000μg)觀察疫苗安全性。于初次免疫后7天再次以同等劑量加強免疫,0.5μg、5μg、50μg劑量組和陽性對照組于免疫后22天接受SARS相關冠狀病毒的攻擊,并在攻毒后15天處死所有動物。觀察免疫和攻毒前后動物臨床癥狀和生化指標的改變。ELISA檢測IgG和IgA抗體,觀察不同動物組IgG的變化和鼻咽部IgA分泌情況,了解動物體液免疫和粘膜免疫的情況。雙抗體夾心ELISA試劑盒定量分析恒河猴血清IL-4和IFN-γ水平。采用病毒分離、免疫熒光、光鏡及RT-PCR方法對實驗動物不同時間、不同組織或分泌物進行檢測。病理切片了解免疫和攻毒前后動物臟器的改變。 結果: 1.血清IgG抗體反應:大部分免疫猴在初次免疫7天后產生較高IgG滴度(100),高劑量組呈現(xiàn)高水平抗體滴度(≥1600),隨疫苗劑量的升高,免疫應答亦增強。 2.粘膜免疫:肌肉接種引起的粘膜抗體反應,12只免疫動物中有5只(41.7%)表達SARS-CoV特異性分泌型IgA抗體。 3.細胞因子評價:初次免疫后,5μg,50μg疫苗組動物血清中IFN-γ顯著增加,明顯高于免疫前水平(P0.05和0.01),再次免疫后繼續(xù)增加。0.5μg劑量組動物僅在再次免疫后IFN-γ顯著升高(P0.05),而PBS組無明顯變化。三個實驗組及對照組IL-4水平在免疫前后均無顯著變化。 4.血清中和抗體效價:在初次免疫后14天,三個免疫組出現(xiàn)不同水平針對SARS-CoV的中和抗體,同時隨著疫苗劑量的增加,中和抗體滴度也升高。 5.組織病理學檢查:各免疫組及陰性對照動物及肺部、肝臟、腎臟病理檢查均未發(fā)現(xiàn)異常,而陽性對照動物肺部及肝臟病理學檢查均發(fā)現(xiàn)異常變化。
[Abstract]:Objective: to establish an animal model of SARS associated coronavirus infection in rhesus monkeys and to evaluate the immunogenicity of an experimental SARS coronavirus inactivated vaccine. Protection and safety also provide theoretical and experimental basis for further improvement and application of the vaccine in clinical trials. Methods: animal immunizations were divided into 3 groups, which were intramuscularly injected with 0.5 渭 g of 5 渭 g or 50 渭 g of (PBS) phosphate buffer, respectively. The safety of the vaccine was observed at a high dose (5000 渭 g). On the 7th day after the first immunization, the same dose of 0.5 渭 g ~ 5 渭 g ~ (5 渭 g) ~ (50 渭 g) and the positive control group were given SARS related coronavirus attack again, and all the animals were killed 15 days after the initial immunization. To observe the changes of clinical symptoms and biochemical indexes before and after immunization. Elisa was used to detect IgG and IgA antibodies, to observe the changes of IgG and the secretion of nasopharyngeal IgA in different groups, and to understand the humoral immunity and mucosal immunity of animals. Serum levels of IL-4 and IFN- 緯 in rhesus monkeys were determined by double antibody sandwich ELISA kit. Virus isolation, immunofluorescence, light microscopy and RT-PCR were used to detect different tissues and secretions of experimental animals at different time. Pathological sections were used to understand the changes of animal organs before and after immunization. Results: 1. Serum IgG antibody response: most of the immunized monkeys produced high IgG titer (100) 7 days after the first immunization, and the high level antibody titer (鈮，

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