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

将编码VP8′或VP5′的DNA片段插入具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，构建脊髓灰质炎-rota VP4嵌合cDNA。在用pSM1 (T7) 3-VP5′、pSM1 (T7) 3-VP8′或pSM1 (T7) 2-VP8′+VP5′获得的脊髓灰质炎-轮状病毒嵌合RNA转染HeLa细胞后，可以检测到轮状病毒特异性RNA的复制。然而，轮状病毒蛋白仅在转染pSM1 (T7) 3-VP8′或pSM1 (T7) 2-VP8′+VP5′的细胞中表达。在该系统中表达的VP5′的抗原性可能较弱，这是由于VP5′与脊髓灰质炎病毒蛋白融合后的构象发生了变化。转染pSM1(T7)3-VP8′并感染感染性脊髓灰质炎病毒后，第四代细胞中检测到轮状病毒蛋白与抗血清VP4反应。通过制备高滴度脊灰-轮状病毒嵌合病毒，并利用具有口服接种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。我们测定了包括人类在内的多种哺乳动物轮状病毒VP4基因的核苷酸和氨基酸序列。VP4序列的比较分析表明，至少存在17种不同的VP4基因型，VP4基因型具有种特异性和种间亲缘性。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基因型。轮状病毒疫苗的开发应考虑到这些发现。

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.