Vaccination with the Crimean-Congo hemorrhagic fever virus viral replicon vaccine induces NP-based T-cell activation and antibodies possessing Fc-mediated effector functions

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.

Keywords: CCHFV; Fc-mediated effector function; antibody; nucleoprotein; vaccine.

Figure 1

Humoral and cellular immune responses to CCHFV VRP vaccination. (A) Schematic study overview. Male and female C57BL6/J mice were divided into 8 experimental groups containing 10 vaccinated animals and 4 controls, sexes distributed evenly. Animals were given a single dose of 1 x 10⁵ TCID₅₀ CCHFV VRP particles. At the indicated timepoints after vaccination (days post vaccination; DPV), 14 animals were euthanized (10 vaccinated and 4 control animals) and samples were collected. (B) VRP-derived CCHFV S segment RNA levels detected by RT-qPCR in various tissues at the indicated timepoints post vaccination. (C) IgG and IgM antibodies against CCHFV NP, Gn, Gc, and GP38 were quantified by ELISA using the corresponding purified proteins. (D) Splenocytes were isolated from vaccinated and control C57BL6/J mice and IFN-ɣ production was measured by ELIspot. Splenocytes were incubated with peptides spanning NP or NSm-Gc (GPC). Spot counts were normalized to PMA-induced spots. (E) Data represented in panel D was reformatted to show male (M) v. female (F) mice. Dots represent individual animals. Means and standard deviations are indicated. DPV, days post vaccination; Per. LN, Peripheral lymph nodes; AAU, arbitrary antibody units; SFU, spot-forming units. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. If not indicated, no significance was found.

Figure 2

Cellular immune responses and Fc-mediated antibody functionality induced by VRP vaccination. (A, B) Immune cells were isolated from spleen, peripheral lymph nodes, and liver of vaccinated and control C57BL6/J mice at the indicated timepoints post vaccination. Quantity (A) and activation status (B) of B cells, CD4⁺ and CD8⁺ T cells was determined by flow cytometry. (C) Isotypes of anti-NP IgG antibodies were determined by ELISA isotype-specific secondary antibodies. (D) Avidity of IgG antibodies against CCHFV NP was determined by treating plasma samples with 1 M NH₄SCN prior to ELISA analysis. IgG avidity index was calculated by dividing the mean OD of NH₄SCN-treated plasma by the mean OD of PBS-treated plasma. (E) Antibody-dependent cellular phagocytosis (ADCP) activity of NP-specific antibodies induced by VRP vaccination was quantified by incubating plasma of vaccinated or control mice with CCHFV NP-coated green fluorescent beads. After formation of immune complexes, THP-1 cells were added and incubated overnight. The next day, cells were washed and analyzed on a Guava cytometer. Phagocytic score was calculated by multiplying the percentage of bead-positive cells with the overall median fluorescence intensity. (F) Antibody-dependent complement deposition (ADCD) activity of NP-specific antibodies induced by VRP vaccination was determined by incubating plasma of vaccinated or control mice with CCHFV NP-coated red fluorescent beads. After formation of immune complexes, guinea pig complement was added, followed by the addition of a C3-specific FITC-labeled antibody. ADCD activity was calculated as fold induction over mock-vaccinated animals. Dots represent individual animals. Means and standard deviations are indicated. Statistical significance: *p<0.05, **p<0.01. If not indicated, no significance was found.