Single dose, dual antigen RNA vaccines protect against lethal Crimean-Congo haemorrhagic fever virus infection in mice

Background Crimean-Congo Haemorrhagic Fever Virus is a tick -borne bunyavirus prevalent across Asia, Africa, the Middle East, and Europe. The virus causes a non-specific febrile illness which may develop into severe haemorrhagic disease. To date, there are no widely approved therapeutics. Recently, we reported an alphavirus-based replicon RNA vaccine which expresses the CCHFV nucleoprotein (repNP) or glycoprotein precursor (repGPC) and is protective against lethal disease in mice. Methods Here, we evaluated engineered GPC constructs to find the minimal enhancing epitope of repGPC and test two RNA vaccine approaches to express multiple antigens in vivo to optimize protective efficacy of our repRNA. Findings Vaccination with repNP and a construct expressing just the Gc antigen (repGc-FL) resulted in equivalent immunogenicity and protective efficacy compared to original repNP + repGPC vaccination. This vaccine was protective when prepared in either of two vaccine approaches, a mixed synthesis reaction producing two RNAs in a single tube and a single RNA expressing two antigens. Interpretation Overall, our data illustrate two vaccine approaches to deliver two antigens in a single immunization. Both approaches induced protective immune responses against CCHFV in this model. These approaches support their continued development for this and future vaccine candidates for CCHFV and other vaccines where inclusion of multiple antigens would be optimal. Funding This work was supported by the Intramural Research Program, NIAID/NIH, HDT Bio and MCDC Grant #MCDC2204-011. Copyright Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 2024;101: 105017 Published Online February 2024 https://doi.org/10. 1016/j.ebiom.2024. 105017