Objective:We genetically modified our non-replicating vaccinia virus NTV to improve its immunogenicity.Methods:We constructed NTV-modified strain NTV-ΔF1L-C7L by homologous recombination of vaccinia virus based on CRISPR-Cas9 technology by inserting the C7L gene while deleting the F1L gene. The recombinant virus NTV-ΔF1L-C7L was then immunized with 10 7 PFU in BALB/c mice, and the levels of humoral and cellular immunity induced by NTV-ΔF1L-C7L were detected by ELISA and ELISpot method, respectively, and the levels of neutralizing antibodies were determined by the phage-reduced neutralization assay. Results:The PCR and western- blot identification proved that the F1L gene of the constructed NTV-modified strain NTV-ΔF1L-C7L was missing, while the C7L gene was inserted back in the region, and the C7L gene could be expressed normally, indicating that the recombinant virus was constructed correctly. After immunization of mice with NTV-ΔF1L-C7L, ELISA result showed that the recombinant virus NTV-ΔF1L-C7L induced a higher level of IgG antibody than NTV; ELISpot result also showed that the recombinant virus was able to induce a higher level of IFN-γ; and the result of plaque reduction neutralization test showed that the recombinant virus was able to induce a higher level of IFN-γ antibody than that of NTV.Conclusions:We correctly constructed the NTV gene-modified strain NTV-ΔF1L-C7L, which induced stronger humoral and cellular immunity compared with NTV, and provided reference data for the research and development of replacement products for smallpox or monkeypox vaccines.

Objective:To study the cell morphological changes and related molecular mechanisms of non-replicating vaccinia virus NTV infection with human cells and to provide a scientific basis for the further optimization, transformation and application of NTV vectors.Methods:HeLa cells were infected with vaccinia virus Tiantan strain VTT and non-replicating vaccinia virus NTV, and the morphological changes of cells were observed. Then cells were harvested, and rRNA break levels were detected by electrophoresis and the molecular signals associated with apoptosis were detected by Western blotting, and the pathways and mechanisms of NTV-induced early apoptosis were preliminarily determined.Results:In this study, in terms of cell morphology, it was observed that cells infected with NTV were able to have cell rounding, wrinkles and other lesions at an earlier time compared with VTT. DAPI staining showed that NTV-infected nuclei exhibited high chromatin aggregation, marginalization, and disintegration over time. The rRNA fracture level test result indicate that rRNA has been broken and degraded after 16 hours of NTV infection. The Western blotting test result showed that the molecular signals of PARP, caspase-3 and caspase-9 that were stronger than in normal cells could be detected in NTV-infected HeLa cells, but there was no significant increase in caspase-8, while the result of VTT were the opposite of those in NTV.Conclusions:NTV can induce apoptosis in the early stage and provide a theoretical basis for the modification of vaccinia vectors.

Objective:Through the study of the cell biological characteristics and virulence in mice in vivo of three non-replicating vaccinia virus modified strains to provide reference for the development of smallpox/monkeypox vaccine replacement products.Methods:Replicating vaccinia virus Tiantan strain VTT and non-replicating Tiantan Vaccinia Strain NTV were studied in BHK-21/CEF and its modified strains NTV-C7L, NTV-△F1L-C7L, NTV-K1L were amplified, purified, and identified by Western blotting. The virulence and diffusivity of each strain in cells were evaluated by immune-plaque assay. The replication dynamics curves were used to compare the replication differences between the strains, and the weight loss was observed by intranasal route in the mouse model to compare the virulence levels of the viruses.Results:In this study, Western blotting result proved that the amplified and purified vaccinia virus strains were correct. Immunophagocytosis and replication kinetics showed that the replication capacity of the three NTV modified strains in CEF was similar to that of NTV. The diffusion ability and replication ability between Vero cells were improved, but the replication multiple was less than 100 times. The replication level of MRC-5 was significantly enhanced compared with that of NTV, and the replication ratio of NTV-C7L was more than 20000 times. The virulence in mice showed that the body weight of the three NTV modified strains had no statistical significance compared with that of NTV.Conclusions:The three NTV modified strains recovered their replication ability in human MRC-5 cells, but their virulence in mice was similar to that of NTV, which provided the preliminary conditions for being candidate strains of smallpox/monkeypox vaccine replacement products.