Complete Protection Against Lethal Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Challenge in Mice Afforded by Immunization with Modified mRNA Encoding the Nucleocapsid Protein (NP) Alone

Keskin, S.; Pavel, S. T. I.; Sak, R.; Bahadori, F.; Aslan, A. F.; Aktas, M.; Karakecili, F.; Kalkan, A.; Özdarendeli, A.; Doymaz, M. Z.

doi:10.1101/2025.04.21.649815

bioRxiv · 10.1101/2025.04.21.649815

Complete Protection Against Lethal Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Challenge in Mice Afforded by Immunization with Modified mRNA Encoding the Nucleocapsid Protein (NP) Alone

Keskin, S.·Pavel, S. T. I.·Sak, R.·Bahadori, F.·Aslan, A. F.·Aktas, M.·Karakecili, F.·Kalkan, A.·Özdarendeli, A.·Doymaz, M. Z.

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Abstract

BackgroundThe Crimean-Congo Hemorrhagic Fever Virus (Orthonairovirus haemorrhagiae) causes a hemorrhagic fever with mortality rates reaching up to 40%. For years, this virus has maintained its position among the top priority pathogens identified by the World Health Organization (WHO). This is due to its endemic presence across a vast region--from Africa and Spain to the Balkans, the Middle East, and throughout Asia--its potential for human-to-human transmission, and the lack of an effective and approved vaccine or treatment. Therefore, the development of an effective vaccine against CCHFV is of critical importance. Building on the success of mRNA-based vaccines during the Coronavirus Disease 2019 (COVID-19) pandemic, this study reports the development of a messenger ribonucleic acid (mRNA) vaccine candidate expressing the nucleocapsid protein (NP) of CCHFV. The CCHFV NP in vitro transcript (IVT) was designed with pseudouridine ({Psi}) nucleoside modification. MethodsAs part of the preclinical characterization of the IVT vaccine candidate, the biochemical and immunological properties of NP were confirmed in Huh-7 cells transfected with IVT NP-{Psi}mRNA. Afterwards, the efficacy of IVT NP-{Psi}mRNA immunization was evaluated in immunocompetent BALB/c and transiently immunosuppressed (IS) C57BL/6 mice. In CCHFV challenge studies, IS C57BL/6 mice were used. IS C57BL/6 mice were immunized intramuscularly with 2 doses of NP-{Psi}mRNA, either naked or encapsulated in Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, administered 14 days apart. FindingsHigh levels of CCHFV NP-specific humoral (IgM and IgG) and cellular (cytokine and lymphoproliferative) responses were demonstrated in BALB/c mice immunized with IVT NP-{Psi}mRNA. In challenge experiments, 100% protection was achieved by both the naked and PLGA-encapsulated IVT NP-{Psi}mRNA immunizations. Additionally, full protection was observed in mice immunized with inactivated CCHFV, whereas only 20% protection was detected in the unmodified IVT NP-mRNA vaccinated animals. In the protected mice, viral clearance was observed in the spleen, liver tissues, and blood on day 14 post-challenge. InterpretationThis study demonstrates that NP, the most abundant protein of the virus, is capable of providing full protection as a standalone vaccine candidate. Furthermore, our report represents a crucial milestone in identifying a future vaccine candidate and paves the way for subsequent clinical studies. FundingsThis study was supported by the Bezmialem Vakif University Scientific Research Project BAP#20220402. Additionally, this study received support from TUBITAK 2211/C. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/649815v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@6e5aa4org.highwire.dtl.DTLVardef@100f150org.highwire.dtl.DTLVardef@e2f004org.highwire.dtl.DTLVardef@fbcf7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Keskin, S., Pavel, S. T. I., Sak, R., Bahadori, F., Aslan, A. F., Aktas, M., Karakecili, F., Kalkan, A., Özdarendeli, A., Doymaz, M. Z.. 2025-04-23. Complete Protection Against Lethal Crimean-Congo Hemorrhagic Fever Virus (CCHFV) Challenge in Mice Afforded by Immunization with Modified mRNA Encoding the Nucleocapsid Protein (NP) Alone. https://doi.org/10.1101/2025.04.21.649815

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