Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.

Fig. 1. MAb-9D5 protects mice from CCHFV infection.

A IFNAR^−/− mice (N = 10/group) were treated with mAb-9D5 (blue circles) or isotype control antibody (red squares) IP on days −1 and +3. Mice were challenged SC with CCHFV strain IbAr 10200 on day 0 and survival and group weight loss were monitored and plotted using Prism software. ****P < 0.0001. Source data is provided. B Representative H&E ISH staining of livers of CCHFV strain IbAr 10200-infected mice treated with mAb-9D5 (days 4, 8, and 12) or isotype control antibody (day 4). ISH-stained tissue was counterstained with hematoxylin. C Liver sections from CCHFV strain IbAr 10200-infected mice treated with anti-NP or isotype control were stained with anti-CLEC4F (green) and anti-CCHFV NP antibodies (red). Cell nuclei were stained with DAPI (blue). D IFA straining for CD68+ macrophages (green) and CD45+ leukocytes (red) or Ki67+ proliferating cells (green) and MPO+ neutrophil granulocytes (green) in livers of anti-NP or isotype control-treated and -infected mice. Nuclei are stained with DAPI (blue). µM micrometer. B–D N = 5 mice/group for days 4 and 8, and N = 2 mice for day 12. Samples with the most severe pathology are shown.

Fig. 2. MAb-9D5 does not provide postexposure protection.

A IFNAR^−/− mice (N = 3) (circles, squares and right side up triangles) were euthanized four days post infection with strain IbAr 10200 or uninfected (N = 1) (upside down triangles), and the presence of NP, G_C, and GP38 proteins in the sera was determined by MAGPIX assay. The line shows the mean of the two displayed replicates. B IFNAR^−/− mice (N = 10 per group) were treated with two doses of the mAb-9D5 (1 mg/dose) on days −1/ + 3 (black circle) or +1/ + 4 (red square) or isotype control antibody days +1/ + 3 (aqua triangle) and survival and group weight monitored. Mice were SC-infected with CCHFV strain IbAr 10200 on day 0 and survival and weight monitored. Log-rank test comparing each treatment group to the isotype control; ***P = 0.0004. Source data is provided.

Fig. 3. Surface localization of NP in CCHFV-infected cells.

Non-permeabilized A549 cells infected with CCHFV strain IbAr 10200 were stained with the indicated antibodies against CCHFV viral proteins (NP; mAb-9D5 or Gc; mAb-8A1; green) and cell mask (red). Cell nuclei were stained with DAPI (blue). Representative images are shown from two experiments, each with six replicates. µM micrometer.

Fig. 4. Fc-domains do not impact the protective efficacy of mAb-9D5 in IFN-I antibody-blockaded mice.

FcR^−/−, C3^−/−, or B6:129 (wild-type) mice (N = 10/group) were injected SC with two doses of the mAb-9D5 (black squares) or an isotype control antibody (red circle) (1 mg/dose) onD-1/ + 3. IFN-I was blocked on day +1 using mAb-5A3 (2.5 mg) injected IP. Mice were challenged with CCHFV strain IbAr 10200 by the IP route. Survival and group weights were plotted. Log-rank test; ****P < 0.0001, ***P = 0.0004, **P = 0.0012. Source data is provided.

Fig. 5. Binding domain of mAb-9D5 on the NP.

A Structural representation of the NP monomer. The dashed box indicates the magnified NP region depicting the mAb-9D5 binding region identified by LC-MS. B Epitope amino acid comparisons between diverse strains of CCHFV and related nairoviruses IbAr 10200 (accession #MH483987.1), Afg09 (accession #HM452305.1), Kosova Hoti (accession #JN173797.1), Oman (accession #DQ211645.1), Semunya (accession #DQ076413), Senegal (accession #DQ211640), Aigai virus (accession #NC_078226), Hazara virus (accession #NC_038711) and Erve virus (accession #JF911699). C BLI binding kinetics of mAb-9D5 hybridoma with nairovirus NPs. BLI SA biosensors were loaded with NP (antigen) at concentration of 500 nM. Anti-NP mAb was evaluated in triplicate at concentrations of 700, 350, and 100 nM. NB not binding. Source data is provided.

Fig. 6. Heterologous protection of mice by mAb-9D5.

IFNAR^−/− (N = 10) mice were treated with two doses of the mAb-9D5 (blue circles; green triangles) or an isotype control antibody (red squares; purple triangles) on day −1/ + 3, and challenged SC with either IbAr 10200 (blue circles, red squares) or Afg09 (green triangles, purple triangles). Survival and percent group weight change were monitored. Log-rank test; **P = 0.0055, *P = 0.011. Source data is provided.