Experiments involving CCHFV were conducted in the BSL-4 laboratory at the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA). Experiments involving cDNA-encoding viral sequences were performed in accordance with approved Institutional Biosafety Committee protocols. Animal studies were conducted in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the CDC Institutional Animal Care and Use Committee (IACUC; #3342). The CDC is fully accredited by AAALAC International.

Vero-E6 cells stably expressing the codon-optimized Oman-98 GPC were described previously (Scholte et al., 2023). BSR-T7/5 cells were a kind gift of K.K. Conzelmann (Ludwig-Maximilians-Universität, Munich, Germany). Vero-E6, BSR-T7/5, and Huh7 cells were cultured in DMEM supplemented with fetal calf serum, non-essential amino acids, sodium pyruvate, L-glutamine, penicillin/streptomycin, and puromycin (Vero-E6 Oman-98 GPC cells only). THP-1 cells (ATCC TIB-202) were cultured in RPMI supplemented with fetal calf serum and antibiotics, as above.

CCHF VRP stocks were generated as previously described (Scholte et al., 2019). Briefly, CCHF VRPs were generated by transfecting HuH-7 cells with plasmids encoding CCHFV strain IbAr10200 S and L genomic segments (pT7-S and pT7-L; GenBank KJ648914 and KJ648913) and strain Oman-98 glycoprotein (pCAGGS-GPC-Oman; GenBank ALT31693.1) along with plasmids encoding the codon-optimized polymerase (pCAGGS-L), nucleoprotein (pCAGGS-NP), and T7 polymerase (pCAGGS-T7). Supernatants were propagated and quantified via immunofluorescent TCID₅₀ assay using Vero-E6 cells stably expressing the codon-optimized Oman-98 GPC; immunostaining was performed using a rabbit polyclonal anti-NP CCHFV antibody (1:2500; IBT, 04–0011) followed by a goat anti-rabbit secondary antibody (1:2500; Alexa Fluor 488, Invitrogen, A-11008). Wells were scored visually for the presence or absence of fluorescent cells, and TCID₅₀ values were calculated using the Reed-Muench method (Reed and Muench, 1938). All vaccine stocks were verified to be mycoplasma free (MycoAlert PLUS detection kit, Lonza LT07), and genomic sequences were confirmed via next-generation sequencing prior to in vivo studies. Backtiter quantification of vaccine inoculum used on day of vaccination (day 0) was performed as described above.

For these studies, a healthy heterogeneous cohort of 12 adult rhesus macaques (6 males [5.8–11.1 kg] and 6 females [4.3–6.8 kg]; all 4–5 years of age) was selected based on previous clinical history and records and on current health parameters/status (i.e., normal values on recent health assessments, negative for simian immune deficiency virus, and no chronic conditions or health concerns). For α = 0.05 and a difference of 1.25 standard deviations, 12 animals provide a power of 80%. Animals were social/pair housed in a climate-controlled laboratory with a 12 h day/night cycle in standard NHP cages, and given NHP chow (High Protein Monkey Diet, Labdiet 5045) and water ad libitum. Per standard institution practices, animals were also provided daily produce enrichment items including fruits and/or vegetables and received destructible enrichment/treats twice per week. Animals were kindly provided for use in this non-terminal study by the Laboratory Branch of the CDC Division of HIV Prevention and transferred back to originating protocols upon study completion. Animals had previously been used in hepatitis B vaccine studies, and to assess doxycycline vaginal inserts in a vaginal chlamydia challenge. Vaginal swabs from all 6 chlamydia-challenged females tested negative for chlamydia over 3 months prior to study initiation. Female and male rhesus monkeys had not been actively on study for approximately 4 and 10 months, respectively, before CCHF VRP vaccination.

For vaccination, blood collection, and mucosal swabbing, animals were anesthetized with ketamine (2–10 mg/kg) intramuscularly (IM) in the cranial quadriceps using a 22-gauge needle. After anesthesia, animals were monitored every 15 min until they were ambulatory and alert, indicating full recovery. On vaccination day, the fur on the left or right lateral thigh was clipped, and vaccine was administered IM with a 23-gauge, 1-inch needle in the clipped area (target dose: 1 × 10⁶ TCID₅₀; backtiter dose: 7 × 10⁵ TCID₅₀; 1 mL total volume).

Prior to vaccine inoculation, animals were sedated, and 5 mL blood was collected from the femoral vein using a 5 mL syringe with a 23-gauge, 1-inch needle. The needle was removed, and the blood was divided into 3 different collection tubes: ~500 μL each into an EDTA tube (BD Microtainer with K2E; 365974) and a lithium/heparin (LiH) tube (Sarstedt Micro tube 1.3 mL LH; 41.1393.105), and ~4 mL into a sodium citrate CPT Vacutainer (BD Biosciences; 362760). Blood draw volumes were in compliance with IACUC policy (single sampling ≤10% of total body weight [TBV]; ≤7.5% TBV taken weekly; repeated serial sampling ≤7.5% TBV within a 7-day period).

Daily mentation/activity levels were recorded as bright alert reactive (BAR) or quiet alert responsive (QAR), along with any other descriptors of activity noticed during assessment. Hydration status was recorded as adequate (no sunken eye appearance, skin laxity, or droopiness) or inadequate (mild, moderate, or severe based on sunken eye appearance or skin looseness/tenting as observed cage-side). Heart rate and electrocardiogram (ECG) monitoring were recorded using an Avante DRE ASM 5000 monitor. Respiration was assessed via auscultating the lungs, and SpO₂ was recorded with a NONIN 8600V pulse oximeter. Body temperatures were recorded with a rectal thermometer probe (Welch Allyn SureTemp Plus 690 handheld electronic thermometer). Clinical chemistry was analyzed on whole-blood samples collected in LiH using Piccolo Xpress chemistry analyzers with the Comprehensive Metabolic Panel (Abaxis). Complete blood counts (CBC) were performed on whole-blood samples collected in EDTA using the Vetscan HM5 hematology analyzer (Abaxis).

RNA was extracted from whole blood collected in EDTA (50 μL), and from oral, nasal, and rectal mucosal swabs collected using sterile polyester-tipped applicators (Puritan; 25–800 1PD 50) placed in 1 mL of MagMAX lysis buffer. MagMAX Pathogen RNA/DNA kits (Thermo Fisher Scientific 4462359) were used in conjunction with the 96-well ABI MagMAX extraction platform; RNA was eluted into 75 μL of elution buffer. Viral RNA was quantified using a primer/probe set targeting the NP open reading frame of the S genomic segment of CCHFV strain IbAr10200 (forward: 5′-CAG GAC ATG GAC ATA GTG GC-3′; reverse: 5′-ATT GCC CTT GAC GTT GTA GG-3′; probe: 5′-CCC TTG TTG GCA AGC AAT CCC-3′ [all IDT]) using the SuperScript III Platinum One-Step RT-qPCR kit (Thermo Fisher 11732088). Tissue RNA levels were normalized using the validated reference gene Ppia (Davies et al., 2023). Viral RNA copy numbers were quantified via standard curves generated from an RNA standard of known concentration (IDT).

EDTA plasma samples were analyzed from each animal 0, 3, 7, 14, and 21 days post vaccination. Plasma was heat-inactivated at 56 °C for 30 min and analyzed (25 μL) using the ProcartaPlex NHP Cytokine and Chemokine 30-plex panel (Thermo Fisher EPX300–40044-901) according to the manufacturer’s instructions. Data were generated using the Luminex xMAP INTELLIFLEX system and analyzed using the ProcartaPlex Analysis App software (Thermo Fisher). All analytes were calculated based on standard curves and reported as mean fold change in mean fluorescence intensity compared to baseline (day 0) values (Fig. 2C), or as absolute values (pg/mL, Supplemental Fig. 1).

CCHFV anti-glycoprotein-specific (Gn, Gc, and GP38), and anti-NP-specific IgG antibodies were detected as described previously (Scholte et al., 2023). Briefly, Immulon 2HB 96-well microtiter plates were coated with 100 μL of 500 ng/mL antigens prepared in PBS and incubated overnight at 4 °C. Wells were washed 4 × with 300 μL PBS-T (0.1% Tween-20 in PBS) and blocked (5% w/v non-fat dry milk in PBS-T) for 1 h at room temperature. Following blocking, heat-inactivated plasma samples prepared in blocking buffer with 2-fold serial dilutions (range 1:200 to 1:102,400) were added to the wells in duplicate. After 1 h incubation, wells were washed, and anti-rhesus IgG linked to horseradish peroxidase (HRP; 1:3000; Southern Biotech 4700–05) was added to the wells. Following 1 h incubation, wells were washed, TMB Ultra ELISA substrate (Thermo Fisher 34028) was added, and samples were incubated for 20 min. The reaction was stopped by adding ELISA stop solution (Thermo Fisher SS040), and optical density was read at 450 nm on a Synergy Neo2 instrument (BioTek) microplate reader. A cut-off value was determined for each plate based on the average absorbance value of 12 negative control wells per plate plus 5 standard deviations. The highest dilutions with a signal above the determined cut-off value were assigned as the endpoint titers. For the detection of antigen-specific IgG subclasses, mouse anti-rhesus IgG1-specific (Absolute Antibody Ab01623–4.0), IgG2-specific (Creative Biolabs NHP-AB055), IgG1/3-specific (Creative Biolabs NHP-AB052), and IgG4-specific (Absolute Antibody Ab01624–1.1) antibodies were used in 1:3000 dilution along with anti-mouse IgG-HRP (Invitrogen 61–6520). For the detection of antibody avidity, ELISAs were performed as described above with an additional step. Following 1 h incubation of plasma samples and washing, replicates of samples were incubated with PBS (untreated) or with 6 M urea (treated) for 10 min. The assay was completed as described above, and the area under the curves (AUCs) was calculated for untreated and treated wells. The avidity index was determined by dividing the AUC of the urea-treated wells by that of the PBS-treated wells and then multiplying by 100 (Almanzar et al., 2013).

Antibody-dependent complement deposition (ADCD) and antibody-dependent cellular phagocytosis (ADCP) assays were adapted from previously reported methods (Butler et al., 2019; Fischinger et al., 2019). Recombinant CCHFV NP generated based on strain Hoti was biotinylated (EZ-Link^™ Sulfo-NHS-LC-Biotinylation Kit, Thermo Fisher 21435) and coupled to fluorescent red or green neutravidin microspheres (Thermo Fisher F8775/F8776). Antigen-coated beads were incubated 2 h at 37 °C with heat-inactivated (30 min at 56 °C) NHP plasma in sodium citrate. 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 for 15 min at room temperature with FITC-conjugated anti-guinea pig complement C3 (MP Biomedicals 0855385). For ADCP assays, beads were incubated overnight at 37 °C with 2 × 10⁴ THP-1 cells per well. Beads and cells were analyzed on a Guava cytometer. Phagocytic score was calculated by multiplying the percentage of bead-positive cells by the overall median fluorescence intensity.

Blood was collected in sodium citrate CPT Vacutainers (BD Biosciences 362760), and peripheral blood mononuclear cells (PBMCs) were isolated per the manufacturer’s recommendations. Briefly, tubes were centrifuged for 15–20 min at 1500–1800×g. The top plasma layer was removed and stored for other downstream applications/analyses. The remaining mononuclear cells were washed twice with PBS before dilution in RPMI supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (2 million cells/mL). PBMCs (200,000 cells/well) were stimulated with peptides spanning the CCHFV strain IbAr10200 NP protein (Abclonal; 15-mers with 9 amino acids overlap; 1 μg/mL; 90% purity). PBMCs were mixed with stimuli and seeded in IFN-gamma ELIspot plates (MabTech 3421M-4HPW). DMSO was used as a negative control and phorbol myristate acetate (PMA)/ionomycin (BioLegend, 423302) was used as a positive control. PBMCs to be stimulated with PMA/ionomycin were diluted 1:20 (10,000 cells/well) to prevent confluence and ensure countable spots. PMA/ionomycin was used at the concentration recommended by the manufacturer (1:500; 0.05 μg/mL PMA and 1 μg/mL ionomycin final concentration). IFN-gamma spots were counted with a CTL ELIspot reader and normalized to PMA-induced spots. Antigen-specific spots were determined by subtracting spots in the DMSO-treated wells.

Sodium citrate plasma samples were heat-inactivated (30 min at 56 °C) and 2-fold serially diluted in DMEM (starting dilution 1:10). Diluted plasma samples were mixed with an equal volume of DMEM containing 200 TCID₅₀ of recombinant CCHFV reporter virus expressing ZsGreen1 fluorescent protein (Oman-ZsG; total volume: 100 μL). Mixtures were incubated 1 h at 37 °C and subsequently overlaid onto BSR-T7 cells seeded in 96-well plates. Cells were incubated 5–7 days, and ZsG-positive wells were scored to determine neutralizing activity. Mouse monoclonal antibodies (mAbs; 11E7 and 30F7) were used as a positive control. 11E7 and 30F7 mouse mAbs were obtained from the Joel M. Dalrymple-Clarence J. Peters USAMRIID Antibody Collection through BEI Resources, NIAID, NIH: Pre-Gc Glycoprotein, Clone 30F7 (NR-40288) and Clone 11E7 (NR-40277).

All graphs were generated using GraphPad Prism version 10.2.3. Fig. 1A was created with BioRender.com.

Twelve rhesus macaques (adult; 6 males, 6 females) were vaccinated IM with CCHF VRP. Daily cage-side evaluations were performed until study completion (28 days post vaccination) to assess mentation, activity level, hydration status, and the injection site. Fur at the injection site was clipped prior to vaccination to facilitate evaluation for papules, redness, bruising, and other signs of injection site reactions. The injection site was examined cage side 2–3 h post vaccination, when acute injection site reactions were most likely to occur, and then daily afterwards.

A total of 7 sedation events were performed over the course of the study, on days −4, 0, 3, 7, 14, 21, and 28 post vaccination (Fig. 1A). During sedation events, blood and non-invasive clinical data (weight, body temperature, respiration, heart rate, SpO₂, and ECG) were collected. Two baseline measurements were obtained for CBC and chemistry, along with non-invasive clinical data 4 days prior to vaccination (day −4), and immediately prior to vaccination on day 0. All clinical data were evaluated independently by two licensed and ACLAM-boarded clinical veterinarians experienced with NHPs. Post-vaccination clinical findings were largely unremarkable (Fig. 1B). Throughout the 28-day study period, mentation/activity level were recorded as within normal limits, hydration status was adequate, and no injection site-associated erythema or swelling were observed. Four of the twelve animals lost weight (max weight loss <10% from baseline). While weight loss due to vaccination alone cannot be ruled out, animals under daily study observations and sedated ≥ once weekly often exhibit weight loss; all changes were within expected limits for the frequency of handling and sedation. No abnormalities were observed cage side, except for a single observation of vomit in the cage of one animal on day 10. Investigation revealed that the animal received an uncommon enrichment food item less than 24 h prior to the vomiting episode. No other signs of GI upset or toxicity were noted. Overall, no notable deviations from baseline were observed by non-invasive sampling post vaccination, and no clinical alterations noted were considered vaccine related.

To assess safety of the vaccine, VRP dissemination (blood) and mucosal shedding (oral, nasal, and rectal swabs) were evaluated on days 0, 3, 7, 14, 21, and 28 post vaccination. All samples were CCHFV-negative by RT-qPCR, supporting the non-spreading nature of the vaccine (Fig. 1C). To assess changes in blood parameters post vaccination, complete blood counts and clinical chemistry were performed. No notable changes were seen from baseline following vaccination (Fig. 2A). At both baseline timepoints, elevated alkaline phosphatase (ALP) and decreased glucose and K⁺ were observed in a subset of animals (Fig. 2B). These values are believed to be within normal limits for the colony or associated with fasting prior to anesthesia. Increased ALP is also associated with amyloidosis deposits in the liver, which are common age-related changes resulting in liver enzyme elevation (Rice et al., 2013) and have been reported in NHPs. Additionally, inflammation was evaluated by cytokine/chemokine analysis using Luminex up to 21 days post vaccination (Fig. 2C). No clinically relevant differences from baseline (day 0) were observed in plasma cytokine/chemokine levels post vaccination.

Previous work on the VRP characterized responses to vaccination in mice following vaccination alone (Scholte et al., 2023) and in vaccinated mice after CCHFV challenge (Sorvillo et al., 2024). Our study is the first to characterize the immune responses to VRP vaccination in NHPs. IgG responses targeting a spectrum of viral antigens were evaluated, including the glycoproteins (Gn, Gc, GP38) and nucleocapsid protein (NP). Overall, the primary antibody responses detected were against NP: anti-NP antibodies were detected in half of the animals on day 7, in all animals on day 14, and were comparable in magnitude in all animals on days 21 and 28 (Fig. 3A). A subset of animals (9 of 12) developed anti-Gc antibodies, but no antibodies were detected against Gn or GP38.

To better characterize the IgG response, we evaluated the development of IgG1, IgG2, IgG1/3, and IgG4 subclasses of anti-NP antibodies (Fig. 3B). No IgG2 nor IgG4 were detected, but both IgG1 and IgG3 subclasses were present in 11 of 12 (91.6%), and 12 of 12 animals (100%), respectively. Additionally, anti-NP antibody avidity and function were evaluated (Fig. 3C). The overall quality of antibody binding (avidity) began to increase on day 14 and continued through day 28. NP-specific antibodies were able to bind complement and induce ADCD activity starting on day 14, with similar levels on days 21 and 28. NP-specific antibodies were also able to mediate phagocytosis, with activity detected starting on day 14. Consistent with previous VRP vaccine studies indicating low or absent neutralizing responses (Kainulainen et al., 2019; Sorvillo et al., 2024; Welch et al., 2023), very low-level neutralizing activity was observed in a small subset of animals on days 7, 14, and 21 (1:10 dilution).

Our previous CCHF VRP studies (Scholte et al., 2023; Sorvillo et al., 2024), as well as studies of VRP vaccines against Lassa (Kainulainen et al., 2018) and Nipah viruses (Welch et al., 2023), indicate a predilection for and importance of NP-targeted antibodies. Herein, we found a similar predominance of NP-mediated antibody responses in rhesus macaques. To further characterize NP responses, we assessed cell-mediated responses by ELISpot in PBMCs collected on days 0, 3, 7, 14, 21, and 28 post vaccination using NP peptides (Fig. 3D). As previously observed in mice (Scholte et al., 2023), NP-specific T cell activation (IFN-gamma expression) was detected, peaking on day 7 and waning afterwards.