Nucleocapsid protein-based vaccine provides protection in mice against lethal Crimean-Congo hemorrhagic fever virus challenge

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is an acute, often fatal viral disease characterized by rapid onset of febrile symptoms followed by hemorrhagic manifestations. The etiologic agent, CCHF orthonairovirus (CCHFV), can infect several mammals in nature but only seems to cause clinical disease in humans. Over the past two decades there has been an increase in total number of CCHF case reports, including imported CCHF patients, and an expansion of CCHF endemic areas. Despite its increased public health burden there are currently no licensed vaccines or treatments to prevent CCHF. We here report the development and assessment of the protective efficacy of an adenovirus (Ad)-based vaccine expressing the nucleocapsid protein (N) of CCHFV (Ad-N) in a lethal immunocompromised mouse model of CCHF. The results show that Ad-N can protect mice from CCHF mortality and that this platform should be considered for future CCHFV vaccine strategies.

Fig 1. Efficacy of single-dose and prime-boost vaccination.

(A,B) Single-dose vaccination. IFNAR^-/- mice (n = 9 for Ad-N; n = 6 for Ad-wt) were vaccinated with recombinant adenoviruses (1.25×10⁷ ifu) 28 days prior to lethal CCHFV infection (1000 LD₅₀). The animals were monitored for weight as a group (A) and disease progression/survival (B) over 30 days. (C,D) Prime-boost vaccination. IFNAR^-/- mice (n = 9 for Ad-N; n = 3 for Ad-wt) were vaccinated with recombinant adenoviruses 56 (1.25×10⁷ IFU; intramuscular) and 28 (10⁸ IFU; intranasal) days prior to lethal CCHFV infection (1000 LD₅₀). The animals were monitored for weight (C) and disease progression/survival (D) over 30 days. Data is reported as the means. *** p<0.001, ****p<0.0001.

Fig 2. CCHFV loads in single-dose and prime-boost vaccinated and challenged mice.

Groups of IFNAR^-/- mice were either single-dose (1.25×10⁷ IFU; intramuscular) or prime-boost (1.25×10⁷ IFU; intramuscular / 10⁸ IFU; intranasal) vaccinated with Ad-N or Ad-wt and challenged with 1000 LD₅₀ of CCHFV 28 days following final vaccination. Mice (n = 9 per group) were anesthetized, bleed and euthanized to harvest organ samples on day 3 post CCHFV challenge. Viral loads were analyzed by quantitative RT-PCR or infectivity assay. (A) Viremia analyzed by quantitative RT-PCR; (B) Viremia analyzed by TCID₅₀ assay; (C) liver virus load analyzed by TCID₅₀ assay; (D) spleen viral loads analyzed by TCID₅₀ assay. Data is shown as individual organ data points, the mean and the standard error of the mean. The dotted line illustrates the limit of detection of the TCID₅₀ assay. * p<0.05, ** p<0.01, **** p<0.0001.

Fig 3. Liver histopathology and CCHFV antigen distribution in single-dose and prime-boost vaccinated and challenged mice.

Groups of IFNAR^-/- mice were either single, (1.25×10⁷ IFU; intramuscular) or prime-boost (1.25×10⁷ IFU; intramuscular / 10⁸ IFU; intranasal) vaccinated with Ad-N or Ad-wt and challenged with 1000 LD₅₀ of CCHFV 28 days following final vaccination. Mice (n = 9 per group) were anesthetized, bleed and euthanized to harvest organ samples on day 3 post CCHFV challenge. Thin-sections of liver material were stained with hematoxylin and eosin (H&E) or with N₁₀₂₈ rabbit polyclonal serum (anti-CCHFV N serum) (IHC). (A) Liver H&E of control-vaccinated mice (Ad-wt), (B) Liver H&E of prime-vaccinated mice (Ad-N); (C) Liver H&E of prime-boost-vaccinated mice (Ad-N); (D) Liver IHC of control-vaccinated mice (Ad-wt); (E) Liver IHC of prime-vaccinated mice (Ad-N); (F) Liver IHC of prime-boost-vaccinated mice (Ad-N). Images are at a magnification of 10x with 500x insets.