Antibodies targeting the Crimean-Congo Hemorrhagic Fever Virus nucleoprotein protect via TRIM21

Abstract

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a negative-sense RNA virus spread by Hyalomma genus ticks across Europe, Asia, and Africa. CCHF disease begins as a non-specific febrile illness which may progress into a severe hemorrhagic disease with no widely approved or highly efficacious interventions currently available. Recently, we reported a self-replicating, alphavirus-based RNA vaccine that expresses the CCHFV nucleoprotein and is protective against lethal CCHFV disease in mice. This vaccine induces high titers of non-neutralizing anti-NP antibodies and we show here that protection does not require Fc-gamma receptors or complement. Instead, vaccinated mice deficient in the intracellular Fc-receptor TRIM21 were unable to control the infection despite mounting robust CCHFV-specific immunity. We also show that passive transfer of NP-immune sera confers significant TRIM21-dependent protection against lethal CCHFV challenge. Together our data identifies TRIM21-mediated mechanisms as the Fc effector function of protective antibodies against the CCHFV NP and provides mechanistic insight into how vaccines against the CCHFV NP confer protection.

Fig. 1. Adoptive transfer of repNP vaccinated mouse sera increases survival in naïve CCHFV infected mice.

Sera stocks for adoptive transfer were confirmed to have CCHFV-specific antibodies via (a) whole virion IgG ELISA and isotype/subtype whole virion IgG ELISA. Naïve WT C57BL6/J mice were treated with sera from repNP or sham vaccinated mice on day −1 (1tx) or days 0 and +3 (2tx) relative to lethal challenge with 100 TCID₅₀ CCHFV strain UG3010. Mice (N = 8) were (b) weighed daily and monitored for (c) survival until day 14 p.i. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P > 0.05, ****P < 0.0001. Data shown as mean plus standard deviation.

Fig. 2. repNP vaccination is efficacious in the absence of Fcγ Receptors, Complement, and NK cells.

WT C57BL6/J or B6NTac mice, FcγR^−/−, C3^−/− and WT mice depleted of NK cells were (a, b) vaccinated with 1ug of Sham or repNP RNA on day −28 relative to lethal CCHFV challenge. b WT mice were depleted of NK cells on Day −2, 1, 4, 7, and 10 relative to CCHFV challenge by IP treatment with NK1.1 antibody. On D0, groups of mice were evaluated for immunological response to vaccine or treated with MAR1-5A3 antibody and infected with a lethal dose of 100 TCID₅₀ CCHFV strain UG3010. Mice (N = 8) were (c) weighed daily and monitored for (d) survival until day 14 post-infection (p.i.). On D5 p.i., groups of mice (N = 6) were euthanized and evaluated for (e) viral genome copies via qRT-PCR and (f) infectious virus via TCID₅₀ in the blood, liver, and spleen. WT repNP mice are pooled C57BL/6 and B6NTac mice vaccinated with repNP RNA. Sham mice are pooled C57BL/6, B6NTac, FcγR^−/−, and C3^−/− mice vaccinated with Sham RNA. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data shown as mean plus standard deviation.

Fig. 3. repNP vaccination fails to protect TRIM21^−/− mice.

WT C57BL6/J or TRIM21^−/− mice were (a) vaccinated with 1ug of Sham or repNP RNA on day −28 relative to lethal CCHFV challenge. On D0, groups of mice were evaluated for immunological response to vaccine or treated with MAR1−5A3 antibody and infected with a lethal dose of 100 TCID₅₀ CCHFV strain UG3010. Mice (N = 8) were (b) weighed daily and monitored for (c) survival until day 14 p.i. On D5 p.i., groups of mice (N = 6) were euthanized and evaluated for (d) viral genome copies via qRT-PCR, and (e) infectious virus via TCID₅₀ in the blood, liver, and spleen. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data shown as mean plus standard deviation. Exact p values: (c) P = 0.0008.

Fig. 4. repNP vaccination protects WT but not TRIM21^−/− mice from liver pathology.

200X magnification of liver pathology from (a, b) WT sham vaccinated mice, (c, d) TRIM21^−/− sham vaccinated mice, (e, f) WT repNP vaccinated mice, and (g, h) TRIM21^−/− repNP vaccinated mice with (left) HE and (right) anti-CCHF IHC reactivity staining. Images are representative of groups of N = 6 mice. Liver samples from sham vaccinated and TRIM21^−/− repNP vaccinated mice show clusters of necrotic cellular debris multifocally dispersed throughout the hepatic plates. Sinusoidal mononuclear cells (Kupffer cells) and necrotic hepatocytes are immunoreactive. The liver samples from WT repNP vaccinated mice are normal.

Fig. 5. NP-immune sera protects WT but not TRIM21^−/− mice from lethal CCHFV infection.

Sera stocks for adoptive transfer were the same as described in Fig. 1. Naïve WT C57BL6/J and TRIM21^−/− mice were treated with 400 ul of sham or NP-immune sera (a) 6 h prior to lethal challenge with 100 TCID₅₀ CCHFV strain UG3010. Mice (N = 8) were (b) weighed daily and monitored for (c) survival until day 14 p.i. Data shown as mean plus standard deviation. Significance was calculated using one-way ANOVA; ns P > 0.05, ***P < 0.001. Exact p-values: (c) P = 0.0001.

Fig. 6. WT mice depleted of CD4+ and CD8 + T-cells and CD8^−/− mice are protected by repNP vaccination.

On D5 p.i., groups of mice (N = 6) from Fig. 3 were euthanized and evaluated for cellular immune responses to infection via (a) IFNу ELISpot shown as cumulative responses against peptides spanning the entire CCHFV NP (SFC: spot forming cells). For T-cell depletion study, WT C57BL6/J mice were (b) vaccinated with Sham or repNP RNA on day −28 relative to lethal CCHFV challenge. On days −5, −2, and +5 relative to CCHFV challenge, mice were treated with isotype or αCD4 and αCD8 antibody to deplete mice of T-cell populations. On D0, groups of mice were evaluated for immunological response to vaccine or treated with MAR1−5A3 antibody and infected with a lethal dose of 100 TCID₅₀ CCHFV strain UG3010. Mice (N = 8) were (c) weighed daily and monitored for (d) survival until day 14 p.i., On D5 p.i., groups of mice (N = 6) were evaluated for (e) depletion of CD4+ and CD8 + T-cell populations via Flow Cytometry. Antibody treatment achieved a 96.3% depletion of CD4 + T-cells and 98.5% depletion of CD8 + T-cells. In the second study, groups of WT C57BL6/J and CD8^−/− mice were vaccinated and infected as above. Mice (N = 8) were (f) weighed daily and monitored for (g) survival until day 14 p.i. Dashed lines indicate limit of detection. Data shown as mean plus standard deviation. Significance was calculated using one-way ANOVA; ns P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact p-values: (d) both P = 0.0002, (g) both P = 0.0002.

Fig. 7. NP-immune sera blocks CCHFV infection in vitro.

To investigate antibody-dependent intracellular neutralization (ADIN), L929 cells were electroporated with antibody and efficiency of electroporation was verified via FLOW cytometry measuring internalization of control anti-mouse AF488 antibody with and without electroporation (EP) andcells were gated by AF488- or AF488+ (Supplementary Fig. 5b). Next, L929 cells were electroporated with (a) mouse sham or NP-immune sera, as used in adoptive transfer studies, and infected with MA-CCHFV. Viral growth was monitored via TCID₅₀ 72 h p.i., Study was performed with four technical replicates. Pre-immune and NP-immune sera from cynomolgus macaques vaccinated with our repNP vaccine was pooled and evaluated for CCHFV-specific antibody via (b) ELISA and then electroporated into L929 cells to assess (c) ADIN capacity in MA-CCHFV infected cells as above. Human sera was confirmed positive or negative for CCHFV specific antibodies via ELISA and assessed for (d) ADIN capacity as above. Data graphed to show inhibitory concentrations in mean plus standard deviation. Lower dashed line indicates limit of detection (LOD) of assay and upper dashed lines indicate the average TCID₅₀ (Log₁₀) of sham, pre-immune, or negative sera samples. Significance was calculated using one-way ANOVA; ns P > 0.05, ****P < 0.0001. Exact p-values: (d) *P = 0.0113; **P = 0.0049; ***P = 0.0004.