Nan Gao ¹ Tianhan Yang ¹ Lanlan Dong ² Wanda Tang ^3* Kangli Cao ⁴ Longfei Ding ¹ Cuisong Zhu ¹ Shimeng Bai ^5* Ai Xia ^2* Chen Zhao ^1* Haoran Peng ^3* Jianqing Xu ^6* Xiaoyan Zhang ^6*

• ¹ Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
• ² Institutes of Biomedical Sciences, Fudan University, Shanghai, China
• ³ Department of Microbiology, Second Military Medical University, Shanghai, China
• ⁴ Clinical Center of Biotherapy, Zhongshan Hospital, Fudan University, Shanghai, China
• ⁵ The Second Hospital Affiliated with the School of Medicine, Southern University of Science and Technology, Bio-therapeutic Center, National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, Shenzhen, China
• ⁶ Clinical Center of Biotherapy, Zhongshan Hospital, Fudan University, Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences, Fudan University, Shanghai, China

Coronaviruses and influenza viruses are significant respiratory pathogens that cause severe disease burdens and economic losses for society. Due to their diversity and evolution, vaccines typically require periodic updating to remain effective. An additional challenge is imposed by the possible coinfection of SARS-CoV-2 and influenza, which could increase disease severity. Here, we developed a vaccinia vaccine, named rTTV-RBD-HA2, broadly targeting coronavirus and influenza virus. This vaccine expresses three fusion proteins, each comprising the receptor-binding domain (RBD) from one of the three highly pathogenic coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV) and the conserved HA stalk region from two influenza viruses (pdmH1N1 and nH7N9) belonging to groups 1 and 2, respectively. The multi-targeting nature of this vaccine was validated by its success in inducing antibody responses to the three RBDs and both group 1 and 2 HAs in mice. Importantly, it also generated robust T cell responses to all the immunogens, which could be mobilized to the lung through intranasal vaccination. Consistent with this broad immunogenicity profile, when administered via intramuscular priming and two intranasal boosts, rTTV-RBD-HA2 effectively protected vaccinated mice against challenges of the wild-type SARS-CoV-2 virus, the Omicron XBB variant, and the influenza A H1N1 and H3N2 viruses. Our results collectively support the candidacy of recombinant rTTV-RBD-HA2 as a novel respiratory virus vaccine that provides cross-protection against coronaviruses and influenza viruses, surpassing the breadth of previous vaccines. Additionally, they underscore the importance of establishing a strong mucosal T cell response in the development of a universal respiratory virus vaccine.