Construction Of Sabin 1 Poliovirus As Vaccine Vectors And Application In Development Of Universal Influenza Vaccine

The primary means of controlling influenza virus at the present time is vaccination. The compositions of vaccine are changed yearly according to WHO recommendation. This is because RNA polymerase of influenza virus are less faithful than are DNA polymerases and are not able to correct errors by a proof- reading mechanism, thus mutations occur enough to provide ample source for the selection of naturally accumulated variants. The antibodies exist in human body induced by vaccination or exposure to previously circulating virus could not efficiently prevent the infection of mutated strains. A universal vaccine,which protects effectively against all influenza A strains and provides long- lasting immunity, will definitely be an attractive solution to overcome the threat of this promiscuous virus. It involves a vaccine based on the extra- cellular domain of the influenza M2 protein (M2e), which has remained nearly invariable since the virus was isolated in 1933. However, M2e is less immuno- genic since it is a small peptide of 24 amino acids. In this study, we aim to modify the Sabin 1 poliovirus as a vaccine vector and rescue the recombinant virus carrying the fusion gene of Cholera Toxin B subunit and M2e. Our strategy is to use the live attenuated virus to boost the immunogenicity of this peptide.Three fragments of Sabin1 poliovirus were amplified by RT-PCR and cloned into pGEM vectors. The overlapping sequences between neighboring fragments contain unique restriction enzyme sites within the genome. Thus, the 3 fragments could be joined together to form the whole genome by enzyme digestion and ligation. The sequencing results show that three nucleotides are different from the genome sequence in GenBank. Only one mutation, 6735 A→G, results in the change from 250Lysine to 250Arginine on polymerase 3D.In order to construct the gonome as a vector, a cassette containing EcoRI, XmaI, XhoI sites and artificial protease site was introduced adjacent before the initial codon of PV ORF. A similar cassette was also inserted into the P1/P2 joint site. Thus, two kinds of basic PV vectors could be derived based on the different insertion sites of extrageneous genes. The modification were carried out on different plasmids and accomplished by overlapping PCR methods.To improve the transfection efficiency of vector-derived RNA, further modifications were made on the vectors. A hammer-headed Ribozyme (Rz) motif was added just before the 5'-terminal of genome, thus the transcribed RNA could cleavage itself in vitro, releasing the additional nucleotides derived from plasmid and leaving PV RNA with the authentic 5'-end. A poly(A)40 stretch was patched to the 3'-terminal of genome to stabilize the transcribed RNA. Two modifications were carried out on target plasmids by primer extension PCR and fragment replacement, respectively. The original and derived plasmids were assembled into 8 kinds of vaccine vectors with different combination of various cassettes and motifs.The plasmid carrying vectors were linearized by enzyme digestion and were used as templates to synthesize RNAs by T7 RNA polymerase in vitro. Agrose electrophoresis result demonstrated that the dedived RNA are homogenous and intact in size, thus are suitable for transfection. The RNAs carrying Rz motif were subject to self-cleavage in reaction buffer and small Rz released were visulized by PAGE. Rz fragments accumulate as time progress and reach to platform at 1.5 h.To optimize the transfection condition, RNAs extracted from Sabin1 virus were transfected into Vero cells through different methods and reagents. The plaques achieved by 1μg RNA at different methods were calculated. The results suggested that liposome Tfx-20 has the highest efficiency with 1.1~1.5×103 infectious clones/μg RNA and exhibits the minimum toxicity on cells. RNAs transcribed from 8 kinds of vectors were transfected into Vero cell using establish- ed method. Only vectors containing both the Rz and poly(A)40 motifs can transfect cells with high efficiency, with 500~800 clones/μg RNA. When 10μg RNAs were transfected, CPE could appear as early as 60 h post- transfection. The RNAs carrying only poly(A)40 can also produce infectious viruses with lower efficiency, while RNA from vectors bearing only Rz or neither of the motifs failed to rescue live viruses on Vero cells.Cholera Toxin B subunit (CTB) gene was amplified by PCR and fused to M2e by primer extension PCR. EcoRI and XhoI sites were designed to 5'-and 3'- end of fusion gene, respectively. The gene was inserted into the polylinker site of pPV1RzA by double digestion and re-ligation. Rescued recombinant virus carrying CTBM2e gene, rPV-CTBM2e, was obtained by the established mthod. Similarly, a recombinant virus carrying the receptor binding domain gene from SARS-CoV Spike protein, rPV-S-RBD, was also prepared using the same vector. The foreign inserts were confirmed by RT-PCR and sequencing. rPV-CTBM2e was very stable during the 12 round of passage in Vero cell, while rPV-S-RBD lost the foreign insert quickly in serial passages.Monoclonal antibodies against M2e were prepapred by immunizing Balb/c mice with M2e peptide coupling to KLH carrier proteins. 7 clones of McAb were screened by ELISA, and 5 of them can bind to natural M2e displayed on Vero cells, which were infected with influenza virus.Vero cells infected with rPV-CTBM2e and rPV-S-RBD were treated with paraformaldehyde and permeated with Triton X-100. The expressed proteins were detected by McAb agsinst M2e and polyclonal antibodies against SARS-CoV and visualized by FITC-labeled secondary antibodies.The fluorescence signals appear mainly in the cytoplasm, thus confirmed the expression of foreign proteins. Our study involves in construction of Sabin1 poliovirus as a vaccine vector with high efficiency and provides a powerful tool for development of universal influenza vaccine and SARS-CoV vaccine.