Development of poliovirus-rotavirus chimeric vaccine by using poliovirus artificial interfering particle RNA

我们将编码VP8‘或VP5’的DNA片段插入到含有脊髓灰质炎病毒人工DI基因组的pSM1(T7)3或pSM1(T7)2载体中，构建了脊髓灰质炎病毒VP4嵌合基因。在从pSM1(T7)3-VP5‘、pSM1(T7)3-VP8’或pSM1(T7)2-VP8‘+VP5’获得的脊髓灰质炎病毒嵌合RNA的HeLa细胞中，可检测到轮状病毒特异性RNA的复制。而轮状病毒蛋白只有在pSM1(T7)3-VP8‘或pSM1(T7)2-VP8’+VP5‘细胞中才有表达。在该系统中表达的VP5‘的抗原性可能较弱，这是因为VP5’与脊髓灰质炎病毒蛋白融合后的构象发生了变化。PSM1(T7)3-VP8‘转染组和传染性细小病毒感染组在第4代细胞中均可检测到与VP4抗血清反应的轮状病毒蛋白。通过制备高效价脊髓灰质炎病毒嵌合病毒，并利用具有口服免疫效力的脊髓灰质炎病毒受体转基因小鼠，研究脊髓灰质炎嵌合病毒的免疫原性。对VP4基因序列的比较分析表明，至少有17种VP4基因型别，且VP4基因型间存在种属特异性和种间亲缘关系。研究还表明，一些人轮状病毒具有与动物轮状病毒相关的VP4基因类型。这些发现应该被考虑到轮状病毒疫苗的开发中。

We constructed polio-rota VP4 chimeric cDNA by inserting the DNA fragments encoding VP8' or VP5' into pSM1 (T7) 3 or pSM1 (T7) 2 vector having poliovirus artificial DI genome. In the HeLa cells transfected with polio-rota chimeric RNA obtained from pSM1 (T7) 3-VP5', pSM1 (T7) 3-VP8', or pSM1 (T7) 2-VP8'+VP5', replication of rotavirus-specific RNA could be detected. However, the expression of rotaviral protein was observed only in the cells transfected with pSM1 (T7) 3-VP8' or pSM1 (T7) 2-VP8'+VP5'. The antigenicity of the expressed VP5' might be weak in this system because of the conformational change in the from of the VP5' fused with polioviral protein. Rotaviral protein reactive with antiserum to VP4 could be detected in the cells at the fourth generation after transfection with pSM1(T7)3-VP8' and infection with infectious poiliovirus. By preparing of polio-rota chimeric virus with high titer and by using the transgenic mice with poliovirus receptor which have the potency of oral inoculation, the immunogenicity of the poliorota chimeric virus would be investigated.We determined the nucleotide and deduced amino acid sequences of the VP4 genes of the rotaviruses from various mammalian species including humans. Comparative analysis of the VP4 sequences showed that there are at least 17 kinds of VP4 genotypes and that species specificity and interspecies relatedness in VP4 geneotypes are present. It was also shown that some human rotaviruses have VP4 geneotypes related to those of animal rotaviruses. These findings should be taken into considerataion for rotavirus vaccine development.