# Protective efficacy of a genetically modified attenuated vaccinia virus Tiantan strain against monkeypox virus challenge in a small animal model

## TL;DR

A genetically modified version of the vaccinia virus Tiantan strain shows reduced virulence and provides protection against monkeypox in a mouse model.

## Contribution

A novel attenuated vaccinia virus strain is developed with deletions in virulence and immune regulatory genes for safer MPXV protection.

## Key findings

- The attenuated virus replicates in CEF cells but is impaired in human-derived cells.
- Vaccinated mice showed significantly lower MPXV viral loads in lungs and genital organs.
- The virus exhibits reduced virulence in small animals and induces cross-neutralizing antibodies.

## Abstract

Vaccinia virus (VACV) confers cross-protective immunity against monkeypox virus (MPXV), the causative agent of mpox, and has therefore been extensively exploited as a preventive vaccine. VACV Tiantan strain (VTT) is a second-generation smallpox vaccine used in China in the last century, and there are consistent efforts to minimize its virulence and ensure its best safety for potential clinical applications. In this study, an attenuated VACV rVTT△C12K2△A45 was constructed by deletion of gene segments related to virulence genes, host range genes, immune regulatory genes, and other functional genes from the VTT genome by genetic engineering. Attenuation characteristics of rVTT△C12K2△A45 were confirmed by smaller plaque size, lower replication capacity in various mammalian cell lines along with tests for neurotoxicity in mice, and lesion formation on rabbit skin. Immunization in BALB/c mice with rVTT△C12K2△A45 induced both anti-MPXV and anti-VACV neutralizing antibodies. Animals vaccinated with rVTT△C12K2△A45 showed lower MPXV viral loads in the lungs and genital organs compared to the non-immunized mice.

The World Health Organization declared monkeypox a “Public Health Emergency of International Concern” twice, in 2022 and 2024, respectively. Smallpox vaccines have shown efficacy in protecting against monkeypox because of the cross-protective immunity among orthopoxviruses. The vaccinia virus Tiantan strain (VTT) played a critical role in China’s smallpox eradication campaign. Here, we construct an attenuated vaccinia virus by deletion of different ranges of genes in the VTT genome. This attenuated vaccinia virus replicates like its parental VTT strain in production CEF cells but is severely impaired in human-derived cells like 2BS, MRC-5, and WI-38 cells. Meanwhile, this virus shows significantly reduced virulence in small animals. Animals vaccinated with this attenuated vaccinia virus showed lower monkeypox virus (MPXV) viral loads in the lungs and genital organs compared to the non-immunized mice after MPXV challenge. Our data suggest the potential of this genetically engineered VTT strain as a MPXV vaccine candidate.