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However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR\/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-\u0394F1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-\u0394F1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (10\n                      <jats:sup>3<\/jats:sup>\n                      PFU) or \n\na single dose (10\u2075 PFU) of NTV-\u0394F1L-C7L, comparable to that conferred by VTT. These findings demonstrate that NTV-\u0394F1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        A highly attenuated \nNTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-\u0394F1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-\u0394F1L-C7L, demonstrates several significant advantages. Its robust\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-\u0394F1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00943-26","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T13:00:51Z","timestamp":1785502851000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Enhanced replication and immunogenicity of a genome-modified non-replicating vaccinia virus Tiantan strain for robust low-dose protection in mice"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7355-5287","authenticated-orcid":false,"given":"Jiao","family":"Ren","sequence":"first","affiliation":[{"name":"National Key Laboratory of Intelligent Tracking and Forecasting for Infectious 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