Vero-E6 cells stably expressing codon-optimized CCHFV Oman strain glycoprotein were generated by cloning the ORF into an episomal vector (System Biosciences) with an additional puromycin resistance cassette, transfecting cells with this construct, and selecting stable clones under puromycin selection. Uniform expression of the glycoprotein was verified by immunostaining with monoclonal antibodies 11E7 and 13G8 (BEI Resources). Sequence of the transgene carried by the cell line was verified by PCR with Phusion Human Specimen Direct PCR Kit and next-generation sequencing (Illumina). Cells were cultured in DMEM supplemented with fetal calf serum, non-essential amino acids, sodium pyruvate, L-glutamine, antibiotics, and puromycin. THP-1 cells (ATCC TIB-202) were cultured in RPMI supplemented with fetal calf serum, and antibiotics.

The CCHFV VRP vaccine contains the S and L genome segments of CCHFV strain IbAr10200 combined with ectopically expressed Oman strain GPC (Scholte et al., 2019; Spengler et al., 2019; Spengler et al., 2021). The stock used in this study also expresses the ZsGreen reporter gene from the S segment ORF, using a similar strategy as described for infectious recombinant CCHFV (Welch et al., 2017; Welch et al., 2019). The original VRP stock was generated by transfecting Huh7 cells with plasmids expressing the IbAr10200 S and L genome segments under control of a T7 promoter, as well as plasmids encoding T7, NP, and human codon-optimized GPC and L proteins. Stocks were amplified and quantified by TCID₅₀ determination in Vero-E6 cells stably expressing the codon-optimized Oman GPC. The VRP sequence was verified by Illumina next-generation sequencing and confirmed to be mycoplasma free.

Six-week-old male and female C57BL6/J mice (Jackson Laboratory #000664) were kept in a climate-controlled laboratory with a 12 h day/night cycle; provided commercially available mouse chow (Teklad Global 18% Protein Rodent Diet) and water ad libitum; and group-housed on autoclaved corn cob bedding (Bed-o’Cobs^® ¼”, Anderson Lab Bedding) with cotton nestlets in an isolator-caging system (Tecniplast GM500) with a HEPA-filtered inlet and exhaust air supply. Mice were vaccinated subcutaneously with a target dose of 1 x 10⁵ TCID₅₀ VRP in the dorsum/interscapular region (back-titer dose: 8 x 10⁴ TCID₅₀). Control animals were mock-vaccinated with DMEM only. Equal numbers of male and female mice were divided between 8 groups representing the pre-determined terminal sampling timepoints. At each predetermined timepoint, 14 mice were euthanized (5 male and 5 female vaccinated animals; 2 male and 2 female mock-vaccinated mice). At the time of euthanasia, terminal blood was collected by intracardiac puncture under deep isoflurane anesthesia in lithium heparin microtubes (Sarstedt). Animals were subsequently euthanized and tissues were harvested at necropsy. All animal experiments were approved by the CDC Institutional Animal Care and Use Committee and performed in an AAALAC-International approved facility.

RNA was extracted from lithium-heparinized whole blood and homogenized tissue samples using the MagMAX-96 Total RNA Isolation Kit (ThermoFisher) on a 96-well ABI MagMAX extraction platform with a DNaseI treatment step. RNA was quantified using a one-step, real-time reverse-transcription polymerase chain reaction (RT-PCR) assay targeting the NP gene sequence (Spengler et al., 2019) and normalized to 18S with a SuperScript III Platinum One-Step qRT-PCR Kit. Relative copy numbers were determined based on a dilution series of an in vitro RNA transcript of known copy number run in parallel. Relative RNA levels of selected immune genes in blood and lymph node tissues were quantified using murine Taqman assays (ThermoFisher) and normalized to β-actin mRNA levels.

Plasma samples were analyzed using a magnetic Th1/Th2 cytokine 11-plex mouse ProcartaPlex panel (Luminex EPX110-20820-901) per manufacturer’s instructions.

Kosovo Hoti strain sequences were used for the expression of all proteins. The NP sequence was optimized for bacterial expression and cloned into pET28a (Twist Bioscience). After transformation into E. coli BL21 (DE3) strain, a bacterial culture was grown, and induced with 1 mM IPTG when OD was between 0.4-0.6, and transferred to 16°C for overnight incubation. Cells were harvested by centrifugation, resuspended in lysis buffer (500 mM NaCl, 20 mM Tris-Cl [pH 7], 0.1% Triton-X, 5% glycerol, 1 mM MgCl₂, 25 U/ml benzonase), and sonicated. The cleared lysates were filtered through 0.2-micron PES membranes and loaded onto HisTrap Excel columns (Cytiva) for immobilized metal affinity chromatography. Following His purification, N-terminal His-GST was cleaved with HRV-3C protease cleavage enzyme (3CC-N3133, Acro Biosystems).

The CCHFV Gn, Gc, and GP38 sequences were cloned into pTWIST (for Gn) or pEEV (Kainulainen et al., 2021) (for Gc and GP38) plasmids by Twist Bioscience. Proteins were expressed in Expi293F cells after transfection using FectoPro transfection reagent (Polypus). For the constructs with furin cleavage sites, plasmids were co-transfected with furin plasmid at a 4:1 ratio. Supernatants were harvested 4-6 days post transfection, filtered through 0.2-micron PES membranes and purified by IMAC using HisTrap Excel columns. Proteins were further purified using size exclusion chromatography, quantified, and stored at -80°C. All expression and purification steps were confirmed by polyacrylamide gel electrophoresis. For additional details, see supplemental Methods .

Plates were coated with antigen in PBS and incubated overnight at 4°C. Wells were washed with PBS-T (0.1% Tween-20 in PBS) and blocked with blocking buffer (5% [w/v] non-fat dry milk in PBS-T). Plasma samples diluted in blocking buffer were added to the wells at 1:1000 (IgG) or 1:500 dilutions (IgM). After 1h incubation at RT, wells were washed and anti-mouse IgG or IgM HRP was added to the wells, and incubated 1h at RT. After washing, TMB Ultra ELISA substrate was added and incubated 10 min at RT. The reaction was stopped using ELISA stop solution and OD was read at 450 nm. Antibody activity units were determined based on calibrator curves generated using mouse IgG and IgM standards. Antibody concentrations in samples were determined by interpolating the concentrations of the standards that corresponded to the absorbance value of the sample. For additional details, see Supplemental Methods .

IgG avidity of NP-specific antibodies was determined by treating plasma samples with 1M NH₄SCN prior to ELISA analysis as described above. IgG avidity index was calculated by dividing the mean OD of NH₄SCN-treated plasma by the mean OD of PBS-treated plasma, and multiplying this number by 100 to obtain a percentage.

Cells were incubated with mouse Fc block and surface stains ( Supplementary Table S1 ) for 30 min on ice. Cell viability was determined using Tonbo Ghost Dye Violet 510. Cells were washed and resuspended in Cytofix/Cytoperm (BD) for 20 min on ice. Stained cells were analyzed on a Stratedigm S1000EXi. For additional details, see Supplemental Methods .

Splenocytes were diluted to 2 x 10⁶ cells/ml in RPMI containing 10% heat-inactivated FBS and antibiotics, and seeded onto ELIspot plates (MabTech 3321-4HPW) containing peptides spanning the CCHFV IbAr10200 NP protein (AbClonal, 15-mers with 9 AA overlap). Alternatively, peptides spanning the NSm-Gc domain of the Oman GPC were used (Genscript; 15-mers with 4 AA overlap). PMA and DMSO were used as positive and negative controls, respectively. Spots were counted using a CTL ELIspot reader and normalized to PMA-induced spots.

These assays were adapted from (Butler et al., 2019; Fischinger et al., 2019). Recombinant NP was biotinylated (ThermoFisher 21435) and coupled to fluorescent red or green neutravidin microspheres (ThermoFisher F8775/F8776). Antigen-coated beads were incubated with mouse plasma (2 h at 37°C). For ADCD assays, guinea pig complement (Cedarlane CL4051) diluted in gelatin veronal buffer (CompTech B102) was added and incubated 15 min at 37°C. Beads were washed and incubated 15 min at RT with FITC-conjugated anti-guinea pig complement C3 (MP Biomedicals 0855385). For ADCP assays, beads were incubated overnight at 37°C with 1 x 10⁴ THP-1 cells per well. Beads or cells were analyzed on a Guava cytometer. Fold ADCD activation was calculated using naïve mouse plasma. Phagocytic score was calculated by multiplying the percentage of bead-positive cells with the overall median fluorescence intensity. For additional details, see Supplemental Methods .

Data were visualized and analyzed for statistical relevance using GraphPad Prism v.9. Figure 1A was created with Biorender.com.

To characterize the kinetics of cellular and humoral immune responses that potentially correlate with protection induced by the CCHFV VRP vaccine in immunocompetent mice, a cohort of C57BL6/J mice was vaccinated with a single dose (1 x 10⁵/mouse) of the VRP vaccine. Animals were divided into 8 experimental groups containing 10 vaccinated animals and 4 mock-vaccinated controls, with equal numbers of males and females ( Figure 1A ). A subset of animals was euthanized at pre-determined timepoints, ranging 1-56 days post vaccination. Blood, liver, spleen, peripheral lymph nodes, and a selection of other tissues were collected.

To investigate if RNA derived from the VRP vaccine could be detected after vaccination, RNA was isolated from various tissues, including blood, lymph nodes, and spleen, and analyzed for the presence of CCHFV S segment RNA by RT-qPCR. One day after vaccination, low levels of VRP-derived RNA could be detected in the liver, spleen, gonads, heart and lungs of one animal, and in the liver and spleen of a second animal. No VRP-derived RNA could be detected in any of the tested tissues of the other 8 vaccinated animals 1 day after vaccination. In addition, low levels of VRP-derived RNA could be detected in the peripheral lymph nodes of 1 animal 3 days post vaccination, and in the peripheral lymph nodes of 2 animals 7 days post vaccination ( Figure 1B ).

Additionally, we looked at vaccine-derived protein expression by flow cytometry. In this study, the CCHF VRP vaccine expressed a reporter gene (ZsGreen1; ZsG) from the S segment. This allows direct visualization of cells containing VRP RNA, as ZsG is expressed alongside NP. To assess the presence of ZsG-positive cells as an indicator of VRP-derived RNA, liver, spleen and peripheral lymph node samples were processed for flow analysis 1 and 3 days post vaccination. No ZsG signal was detected in any of these tissues harvested at either timepoint (data not shown).

Next, we examined the induction of localized or systemic innate immune responses after vaccination. It was previously observed that the VRP vaccine protects mice from lethal CCHFV challenge given 3 days after vaccination (Spengler et al., 2021). This could potentially be due to innate immune responses induced by VRP vaccination. The replicating VRP RNA may be recognized by pattern recognition receptors and result in the production of IFNs and proinflammatory cytokines. The presence of VRP-derived antigens, such as the glycoproteins studding the particles and newly synthesized NP and L, can also induce innate immune responses (Kang and Compans, 2009). Generally, immune cells, including dendritic cells and monocytes/macrophages at the vaccination site or draining lymph nodes take up the vaccine and express cytokines upon activation, such as IFN-ɣ, IFN-α, and IL-6. This activation ultimately results in efficient induction of the adaptive immune response (Matsumura et al., 2022). To assess if the VRP vaccine induces innate immune responses, we quantified levels of cytokines involved in the T helper cell response in plasma samples obtained during the first 5 days after vaccination. We did not observe any significant changes in cytokine levels ( Supplementary Figure S1A ).

In addition, we examined the expression levels of a limited selection of immune genes (ISG15, CCL-2, IFN-β, and IFN-ɣ) by RT-qPCR using RNA extracted from blood and lymph node samples obtained during the first week after vaccination. No upregulation of these genes was observed ( Supplementary Figure S1B ).

We previously found that CCHFV VRP vaccination protects mice from lethal infection. In these mice, NP-specific antibodies were found in samples collected pre-challenge at 28 days post vaccination (Scholte et al., 2019; Spengler et al., 2019). Additionally, we found that mice vaccinated 3 days before lethal CCHFV challenge are protected from death (Spengler et al., 2021). However, the longitudinal kinetics of vaccine-induced humoral and cellular immunity associated with these protective vaccination timepoints have not been examined. To fully characterize the kinetics of CCHFV-specific antibody development following VRP vaccination in immune-competent mice, we analyzed plasma samples obtained at the indicated timepoints (1-56 days post vaccination). IgM and IgG binding antibodies against CCHFV NP, Gn, Gc and GP38 were analyzed by ELISA ( Figure 1C ). Similar to previous reports, we detected robust antibody levels against NP at 28 days post vaccination. We detected NP-specific IgM and IgG as early as 7 days post vaccination, with IgG levels reaching a plateau on day 21, and IgM levels waning after day 7. Antibodies against any of the GPC components (Gc, Gn, GP38) could not be detected. This study expands our understanding of the humoral response to CCHFV VRP vaccination with additional timepoints providing insight into the antibody kinetics, as well as 2 novel antigens (Gn and GP38). No differences in antibody responses were observed between male and female animals (data not shown).

In previous studies, the cellular immune response to VRP vaccination was not assessed. Recent studies suggest that both cellular and humoral immune responses are required for vaccine-mediated protection against viral infection. To assess the cellular immune response to CCHFV VRP vaccination, we used ELIspot assays to examine antigen-specific T cell activation. Splenocytes were collected from vaccinated and mock-vaccinated control animals at the indicated timepoints and restimulated with CCHFV NP peptides. A peak in NP-specific T cell activation was observed 7 days post vaccination ( Figure 1D ). Male animals had a slightly earlier response (5 days after vaccination), whereas female mice overall showed a stronger response to NP restimulation ( Figure 1E ). Restimulation of splenocytes with CCHFV GPC (NSm-Gc) peptides yielded a modest response 5 days post vaccination, which waned quickly after. Female vaccinated animals had a slightly stronger response to the GPC peptides than male animals ( Figure 1E ). Overall, the antigen-specific T-cell activation after VRP vaccination was predominantly NP-based, and only weak GPC-specific activation could be detected.

To further examine effects of vaccination on immune cells, we assessed B and T cells in liver, spleen, and peripheral lymph nodes at the indicated timepoints after vaccination by flow analysis ( Figures 2A, B ; Supplementary Figure S2 ). Liver and spleen are primary targets of CCHFV, and peripheral lymph nodes were selected as potential initial sites of VRP replication, VRP-induced antigen presentation, and immune cell activation. We determined total numbers of B cells, and CD4⁺ and CD8⁺ T cells, as well as T cell activation status (CD69⁺, CD44⁺). Overall, few vaccine-specific changes were observed at any of the tested timepoints. We saw a modest [but statistically significant (p<0.01)] increase in total B cells, and CD4⁺ and CD8⁺ T cells in the spleens of vaccinated animals 21 days post vaccination, compared to mock-vaccinated animals euthanized on the same day ( Figure 2A ). In the liver, we found elevated levels of CD4⁺ CD44⁺ and CD8⁺ CD44⁺ T cells at various timepoints (14, 21, 28 days) after vaccination ( Figure 2B ). No differences were seen between vaccinated and control animals in the lymph node samples ( Supplementary Figure S2 ).

Both neutralizing and non-neutralizing antibodies can contribute to protection against CCHFV infection. Antibodies targeting NP have been reported to lack neutralizing activity (Zivcec et al., 2017; Leventhal et al., 2022; Hu et al., 2023). Non-neutralizing antibodies can be protective through other mechanisms, including Fc-mediated effector functions like antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement deposition (ADCD). We set out to characterize the vaccine-induced NP-specific antibodies and elucidate how their non-neutralizing activity could contribute to protection. First, we determined the IgG subclass profiles of NP-specific antibodies, as this can provide some insight into antibody functionality. C57BL6/J mice can produce IgG1, IgG2b, IgG2c, and IgG3. Murine IgG1 antibodies poorly facilitate Fc-mediated effector functions, whereas IgG2b and IgG2c have been described to facilitate Fc-mediated effector functions (Collins, 2016). We detected NP-specific IgG1, IgG2b, and IgG2c, but not IgG3 ( Figure 2C ), indicating that these NP-specific antibodies have the potential for Fc-mediated effector functionality. Next, we determined avidity of IgG NP antibodies produced in response to vaccination. Avidity is a measure of the strength of binding between IgG and its epitope. Avidity of NP IgG increased gradually over time, indicating antibody maturation, enhancing their protective activity ( Figure 2D ).

To investigate the potential mechanisms underlying the expected contribution of the NP-based humoral response to vaccine-mediated protection, we measured Fc-mediated antibody functionality ( Figures 2E, F ). Vaccine-induced NP-specific antibodies possessed both ADCD and ADCD activities. ADCD activity could first be detected 7 days after vaccination and plateaued 28 days after vaccination, largely following NP IgG kinetics. ADCP activity associated with NP-specific plasma antibodies followed similar kinetics. These results support a protective role for VRP-induced NP antibodies through Fc-mediated effector functions.