人轮状病毒ZTR-5株灭活疫苗的制备及小鼠血清免疫学评价

目的: 研究人轮状病毒ZTR-5株灭活疫苗的制备及在实验小鼠中的免疫原性评价。方法: 轮状病毒ZTR-5株在MA104细胞上经蚀斑筛选纯化后,获得单一克隆接种至Vero细胞上适应性培养,免疫荧光定量检测病毒的感染性滴度,对收获的病毒液进行离心、超滤、分子筛纯化,甲醛灭活,抗原定量检测Al(OH)₃吸附制备的实验性疫苗。使用不同剂量(8EU、32EU、128EU、256EU)经肌内注射免疫小鼠,共免疫三次,免疫间隔2周。采用间接ELISA法检测血清特异性抗体效价。 结果: 通过蚀斑纯化,筛选得到一株纯化的病毒株ZTR-5纯-1,在Vero细胞上适应性后感染性滴度达7.35logCCID₅₀/ml;大量培养收获的病毒原液滴度为7.57logCCID₅₀/ml,制备获得轮状病毒样品抗原含量为2 560EU/ml;经肌内注射,初次免疫后,所有剂量组动物均获得抗体阳转,阳转率为100%;第一次加强免疫后,各组血清特异性抗体水平均明显增高,免疫剂量为128EU和256EU的两组小鼠血清抗体效价均达1∶10 240;第二次加强免疫后,各剂量组(8EU、32EU、128EU、256EU)血清抗体效价依次达1∶5 120,1∶7 456,1∶14 481.54,1∶14 481.54。 结论:人轮状病毒ZTR-5株可在Vero细胞上稳定增殖,所制备的疫苗具良好免疫原性,用128EU/2次免疫即可获得良好的免疫效果。

Preparation and Immunogenicity of a Scalable Inactivated Vaccine, Strain ZTR-5 in Mice

Objective: To study preparation and immunogenicity of a scalable inactivated human rotavirus vaccine, strain ZTR-5 in mice. Method: The clones of rotavirus ZTR-5 strain cultured in MA104 cells were purified by plaque screening and inoculated to Vero cell for adaptive cultivation, then determined for infectious titer by FFA, the harvest virus solution was purified by Centrifugation, ultrafiltration and molecular sieve, then inactivated by formaldehyde, quantitatively detected for content of antigen with Al(OH)₃ adsorption which was experimental vaccine. Using different doses (8EU, 32EU, 128EU, 256EU) immunized mice by intramuscular injection, a total of three immune and immune interval of 2 weeks. Detected for the serum antibody titers by indirect ELISA. Results: By plaque purification, screened a purified strain that was the virus ZTR-5 pure-1,and infectious titer reached 7.35logCCID₅₀/ml after adaptive cultivation in Vero cells. The infectious titer of virus original solution acquired after large-scale cultivation was 7.57logCCID₅₀/ml, and the antigenic content of the manufactured and obtained rotavirus samples was 2 560EU/ml. All animals of dose group had received positive seroconversion after the initial immunization by intramuscular injection and seroconversion rate was 100%. After the first booster immunization, the serum antibody levels were significantly higher, and the mice serum antibody titers of the two dose groups (256EU and 128EU) reached 1∶10 240. After the second booster immunization, each dose group (8EU, 32EU, 128EU, 256EU) serum antibody titers followed up 1∶5 120, 1∶7 456, 1∶14 481.54, 1∶14 481.54. Conclusion: Human rotavirus ZTR-5 strain was stably proliferated in Vero cell, and the prepared vaccine had good immunogenicity and got a good immune effects with 128EU/2 times.