Replicon particle vaccination induces non-neutralizing anti-nucleoprotein antibody-mediated control of Crimean-Congo hemorrhagic fever virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar^-/- mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar^-/- mice with CCHF VRP 28, 14, 7, or 3 days prior to challenge, all known to confer complete protection, significantly reduced CCHFV viral load, mucosal shedding, and markers of clinical disease, with greater reductions associated with longer vaccination periods. Interestingly, there were no significant differences in innate immune responses, T cell activation, or antibody titers after challenge between groups of mice vaccinated a week or more before challenge, but higher anti-NP antibody avidity and effector function (ADCD) were positively associated with longer vaccination periods. These findings support the importance of antibody-mediated responses in VRP vaccine-mediated protection against CCHFV infection.

Fig. 1. CCHF VRP platform requires single-round replication and antigen specificity for efficacy.

a Study timeline: Ifnar^-/- mice were vaccinated with a UV-inactivated CCHF VRP inoculum (n = 5) 28 days prior to challenge or with a non-specific LASV VRP inoculum (n = 5) 3 days prior to challenge. Vaccines were administered subcutaneously (SC) with a dose of 1.00 × 10⁵ TCID₅₀/animal. All mice were challenged SC with 100 TCID₅₀ CCHFV strain Turkey04 and followed to their terminal endpoint (endpoint criteria described in Methods). After challenge, b weight loss (% change from baseline at -1 dpi), clinical score (described in Methods), and survival were recorded daily for each animal. Weight and survival data from historical controls are represented by dashed gray lines. Tissues (liver, spleen, ovary/testis, kidney, lung, heart, eye, brain, whole blood) and oropharyngeal and rectal swabs were collected from terminal animals and evaluated by RT-qPCR to quantify levels of CCHFV RNA using a primer/probe set specific for the NP ORF of the CCHFV S gene segment. Individual animals are represented. Bars and error bars indicate mean ± standard error of the mean (SEM).

Fig. 2. CCHF VRP vaccination reduces potential for virus transmission after infection.

a Study timeline for vaccination, challenge, and serial euthanasia. Cohorts of Ifnar^-/- mice (n = 8–10) were vaccinated SC with CCHF VRP or LASV VRP (both at 1.00 × 10⁵ TCID₅₀), or left unvaccinated (no VRP, given DMEM alone) 28, 14, 7, or 3 days (-28D, -14D, -7D, -3D) prior to challenge with lethal CCHFV Turkey04 (100 TCID₅₀, SC). Cohorts from each group (n = 4–5) were serially euthanized 3 or 6 dpi. b After challenge, weight loss (% change from baseline at -1 dpi) and clinical score (described in Methods) were recorded daily for each animal. Paired oropharyngeal (OP) and rectal (R) swabs were collected from all animals at the time of euthanasia and used for c viral RNA quantification (individual animals are represented, bars and error bars indicate mean ± SEM) or d CCHFV isolation (individual animals are represented). vRNA was quantified by RT-qPCR using a primer/probe set specific for the NP ORF of the CCHFV S gene segment. CCHFV was isolated through inoculation, fixation, and immunostaining of BSR-T7/5 cells. Statistics for each vaccine group were calculated as significant change compared to unvaccinated control animals (No VRP, given DMEM alone) at equivalent timepoint (3 or 6 dpi) using multiple two-tailed t-tests (Mann-Whitney). Only statistically significant results are reported; *p < 0.5; **p < 0.01 (Supplementary Table 1).

Fig. 3. CCHF VRP reduces clinical disease by controlling viral load and improving liver function.

a Tissues including liver, spleen, ovary/testis (gonad), kidney, lung, heart, eye, brain, and whole blood were collected from mice in all vaccine cohorts at the time of euthanasia 3 or 6 days post infection (dpi). Viral RNA was quantified via RT-qPCR using primers/probe specific for the NP ORF of the CCHFV S gene segment. b Clinical chemistry analytes from each animal were assessed using whole blood collected peri-mortem (intracardiac bleed) in lithium heparin and analyzed via the General Chemistry 13 Panel on the Piccolo Xpress analyzer. GLU glucose, BUN blood urea nitrogen, ALB albumin, ALT alanine aminotransferase, AST aspartate aminotransferase, TP total protein, CRE creatinine. Viral load and clinical chemistry statistics for each vaccine group were calculated as significant change compared to unvaccinated control animals (No VRP, given DMEM alone) at equivalent timepoint (3 or 6 dpi) using multiple two-tailed I-tests (Mann-Whitney). Only statistically significant results are reported; *p < 0.5; **p < 0.01 (Supplementary Tables 1-2). Individual animals are represented. Bars and error bars indicate mean ± SEM.

Fig. 4. Vaccination reduces pathology and CCHFV antigen in liver and spleen.

a Mean hepatic inflammation and necrosis and splenic lymphoid reactivity scores (0–4) (error bars represent standard deviation). Mean liver inflammation and necrosis scores were reduced with vaccination 1 week or more prior to inoculation, compared to short-course (-3D) or unvaccinated controls (no VRP, given DMEM alone). Mean liver inflammation scores were similar for short-course and unvaccinated animals at both 3 and 6 days post infection (dpi), but mean liver necrosis score decreased from 3 to 6 dpi in -3D animals and increased from 3 to 6 dpi in unvaccinated animals. Consistent lymphoid reactivity was present only in -3D and unvaccinated animals and increased from 3 to 6 dpi. Individual animals are represented. Bars and error bars indicate mean ± SEM. b Liver and spleen pathology (top two rows) and CCHFV antigen detection by immunohistochemistry (bottom two rows) at 6 dpi. No or rare small foci of inflammation and hepatocyte necrosis (arrows) were present with vaccine administration 7 or more days prior to inoculation. Livers in -3D and unvaccinated animals both have prominent inflammation and necrotic hepatocytes (arrowheads). Spleens from -28D- and -14D-vaccinated animals showed non-reactive follicles, while -7D-vaccinated animals showed mild reactivity, and spleens from -3D-vaccinated and unvaccinated mice showed similar, marked lymphoid reactivity characterized by follicular expansion by lymphoblasts and prominent plasma cells (*). Immunohistochemistry for CCHFV shows immunostaining (red) of necrotic hepatocytes in -3D and unvaccinated livers; more numerous, confluent clusters of hepatocytes were stained in livers of unvaccinated animals than in short-course vaccinees. Scattered staining of histiocytes was present in a -3D spleen and more prevalent in the spleen of an unvaccinated animal. No immunostaining was seen in livers or spleens from animals vaccinated 1 week or more prior to inoculation. Original magnifications 20 ×, scale bars are 50 μM. Top two rows: hematoxylin-eosin (H&E) stain; bottom two rows: CCHF immunohistochemistry (IHC) with Fast Red chromogen.

Fig. 5. Disseminated CCHFV antigen is present in tissues with minimal or no pathology in short-course-vaccinated (-3D) and unvaccinated animals at 6 dpi.

Top row: -28D vaccination; middle row: short-course (-3D) vaccination; bottom row: unvaccinated. In unvaccinated (no VRP, given DMEM alone) and short-course-vaccinated animals, CCHFV antigen (red; arrows) was present in scattered cells within the leptomeninges of the brain, ciliary body of the eye, valvular stroma of the heart, adrenal cortex, renal interstitium, pancreatic islets, ovarian follicles and stroma, uterine endometrial stroma, and gastric lamina propria and submucosa/serosa. No immunostaining was seen in the same tissue types from -28D vaccinated animals. Original magnifications: 20 × (heart, kidney, ovary, uterus, stomach); 40 × (brain, eye, pancreas, adrenal); scale bars are 50 μM. CCHFV immunohistochemistry with Fast Red chromogen.

Fig. 6. Anti-NP CCHFV antibodies are present early after challenge in short-course-vaccinated animals.

a Cytokine/chemokine responses in mice vaccinated with CCHF VRP, LASV VRP, or left unvaccinated (no VRP, given DMEM alone) 3 days prior to challenge and euthanized 3 or 6 days post infection (dpi) were analyzed using the ProcartaPlex Mouse Th1/Th2 Cytokine and Chemokine panel and 25 µL mouse plasma. b CCHFV-specific T-cell responses were evaluated using IFN-gamma ELISpot assay. Peptides covering the CCHFV IbAr10200 NP or Oman-98 GPC NSm-Gc domain (numbers represent aa positions) were used to stimulate splenocytes harvested from vaccinated animals. Data are reported as the number of spot-forming cells (SPC)/1.00 x 10⁶ cells. c CCHFV-specific NP, Gn, Gc, and GP38 antibody responses (IgM and IgG) were evaluated via ELISA and are reported as endpoint dilution titers. Antibody titers, T-cell responses, and cytokine/chemokine levels were compared statistically between vaccine groups by timepoint (3 or 6 dpi) using multiple two-tailed t-tests (Mann-Whitney) and only statistically significant results are reported; *p < 0.5, **p < 0.01. Individual animals are represented. Bars and error bars indicate mean ± SEM.

Fig. 7. Early control of virus replication is associated with presence of anti-NP antibodies rather than innate or cellular immune responses.

a Cytokine/chemokine responses in mice vaccinated with CCHF VRP 28, 14, or 7 days prior to challenge or left unvaccinated (no VRP, given DMEM alone) and euthanized 3 or 6 days post infection (dpi) were analyzed using the ProcartaPlex Mouse Th1/Th2 Cytokine and Chemokine panel and 25 µL mouse plasma. b CCHFV-specific T-cell responses were evaluated using IFN-gamma ELISpot assay. Peptides covering the CCHFV IbAr10200 NP or Oman-98 GPC NSm-Gc domain (numbers represent aa positions) were used to stimulate splenocytes harvested from vaccinated animals. Data are reported as the number of spot-forming cells (SPC)/1 × 10⁶ cells. c CCHFV-specific NP, Gn, Gc, and GP38 antibody responses (IgM and IgG) were evaluated via ELISA using mouse plasma and reported as endpoint dilution titers. Antibody titers, T-cell responses, and cytokine/chemokine levels were compared statistically between vaccine groups by timepoint (3 or 6 dpi) using multiple two-tailed t-tests (Mann-Whitney) and only statistically significant results are reported; *p < 0.5, **p < 0.01. Individual animals are represented. Bars and error bars indicate mean ± SEM.

Fig. 8. Anti-NP antibody quality and complement-mediated effector function are highest in animals vaccinated 28 days prior to challenge.

a Anti-NP antibody quality was assessed using an avidity ELISA in which mouse plasma was exposed to varying concentrations of a chaotropic agent (ammonium thiocyanate). Antibody quality was evaluated in plasma from mice vaccinated 28, 14, or 7 days prior to challenge and euthanized 3 dpi. b Antibody-dependent complement deposition (ADCD) assay and (c) antibody-dependent cellular phagocytosis (ADCP) assays were performed using plasma from mice in all vaccine cohorts at 3 and 6 dpi. Fold ADCD activation was calculated using naïve mouse plasma. ADCP phagocytic score was calculated by multiplying the percentage of bead-positive cells by the overall median fluorescence intensity. Data were compared statistically between vaccine groups by timepoint (3 or 6 dpi) using multiple two-tailed t-tests (Mann-Whitney) and only statistically significant results are reported; *p < 0.5, **p < 0.01. Individual animals are represented. Bars and error bars indicate the mean ± standard deviation (SD).