Single dose VSV-based vaccine protects mice against lethal heterologous Crimean-Congo hemorrhagic fever virus challenge

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe, sometimes fatal hemorrhagic fever (CCHF) in humans. Currently, there are no approved therapies against CCHF. In this study we used the recombinant vesicular stomatitis virus (VSV) platform to generate live-attenuated recombinant CCHF vaccine candidates expressing the CCHFV nucleoprotein (NP) and glycoprotein precursor (GPC). As one approach, we utilized the established VSV expressing the full-length Ebola virus glycoprotein (VSV-EBOV) or a truncated version of the EBOV glycoprotein and added the CCHFV-NP (VSV-CCHFnp1 or VSV-CCHFnp2, respectively). Additionally, we prepared a vaccine candidate, VSV-CCHFgpc, in which the VSV glycoprotein was replaced with the CCHFV-GPC. Vaccine constructs induced CCHFV-specific IgG antibodies comprising largely IgG2c subclass. Only, the VSV-CCHFgpc vaccine candidate induced significant T cell immune responses directed against epitopes in the CCHFV-NSm and Gc proteins. Efficacy of the vaccine candidates was evaluated using a prime-only approach in a transiently immune-suppressed mouse model. Animals vaccinated with VSV-CCHFnp2 succumbed to lethal CCHFV challenge, while the VSV-CCHFgpc vaccine candidate afforded partial protection. In contrast, vaccination with VSV-CCHFnp1 uniformly protected animals against death. Our results demonstrate the promise of VSV-CCHFnp1 as a vaccine candidate for CCHFV and warrant continued development.

Fig. 1. VSV vector design and characterization.

a Schematic representation depicting the genomic organization of the VSV vectors. N nucleoprotein, P phosphoprotein, M matrix protein, G glycoprotein, L polymerase, EBOV-GP Ebola virus full-length glycoprotein, EBOV GP∆GC∆MLD Ebola virus glycoprotein lacking the glycan cap and mucin-like domain, CCHFV-NP CCHFV full-length nucleocapsid protein, CCHFV-GPC_del53 CCHFV glycoprotein precursor lacking 53 amino acid sequences of the carboxy terminal (see also Supplementary Fig. 1A). b Intracellular protein expression. Vero E6 cells were infected with each virus at an MOI of 0.01. CCHFV-NP, Gc, and EBOV-GP expression was detected by immunofluorescence following permeabilization. VSV-M served as a control (magnification, 10x). c In vitro growth kinetics of the VSV vectors. Vero E6 cells were infected with VSV vectors at an MOI of 0.01. Cell culture supernatants were collected at the indicated time points. The infectious virus was titrated using a TCID₅₀ assay. One representative experiment in triplicate is shown. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test in Prism 10 (GraphPad), and results are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Comparisons with p values >0.05 were not displayed. Data shown as geometric mean plus standard deviation. Graphical illustrations were prepared with Adobe Illustrator version 28.7 (public domain).

Fig. 2. VSV-CCHFgpc vaccination induces significant B cell and T cell responses.

Six-week-old C57BL/6J mice were vaccinated intraperitoneally with 1 × 10⁴ PFU VSV-CCHFnp1 or VSV-CCHFnp2 or VSV-CCHFgpc or VSV-EBOV on day -28. On day 0, groups of six mice were euthanized and blood and spleen samples were collected for evaluation of immune responses. Whole virion ELISA (a) or specific isotypes (b) was used to detect IgG responses elicited by the CCHFV-NP vaccines. c CCHFV-Gc specific IgG antibodies were evaluated by recombinant ELISA or d specific isotypes. CCHFV-GP38-specific IgG antibodies were evaluated by recombinant ELISA (e) or specific isotypes (f). Dashed line represents the cut-off for seropositivity, which was set at 3 standard deviations above the mean absorbance of wells that received no serum. g, h CCHFV-specific T cell responses were measured using IFN-γ ELISpot. The number of spot-forming cells against individual CCHFV-NP (g) or GPC peptide pools (h), the mitogen concanavalin A or DMSO vehicle alone are shown. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test (a, c, e) or two-way ANOVA with Sidak’s multiple comparisons test (b, d, f, g, h) and results are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Comparisons with p values >0.05 were not displayed. Data shown as geometric mean plus standard deviation.

Fig. 3. VSV-CCHFnp1 vaccine protects mice from death against heterologous CCHFV challenge.

Six-week-old C57BL/6J mice were vaccinated intraperitoneally (IP) with 1 × 10⁴ PFU VSV-CCHFnp1 or VSV-CCHFnp2 or VSV-CCHFgpc or VSV-EBOV on day -28 relative to challenge. On day 0, mice were treated IP with MAR1-5A3 to block type I IFN receptor signaling and challenged IP with 100 TCID₅₀ CCHFV strain UG3010. Mice were weighed daily (a), and monitored for survival (b). N = 8 mice per group. c–e Viral RNA in indicated tissues at day 5 post challenge was quantified by RT-qPCR. f–h Replicating virus in indicated tissues at day 5 post challenge was quantified by a TCID₅₀ assay. N = 6 mice per group. One animal in the VSV-EBOV group succumbed during viral challenge at day 0 (cause unknown), leaving five animals in the group. The dashed line indicates the limit of detection. Statistical comparisons were calculated using log-rank test with Bonferroni’s correction for multiple comparisons test (b) one-way ANOVA with Tukey’s multiple comparisons test (c–h) and results are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Comparisons with p values >0.05 were not displayed. Data shown as geometric mean plus standard deviation.

Fig. 4. VSV-CCHFnp1 vaccine reduces liver and spleen pathology.

Six-week-old C57BL/6J mice were vaccinated intraperitoneally (IP) with 1 × 10⁴ PFU VSV-CCHFnp1, VSV-CCHFnp2, VSV-CCHFgpc, or VSV-EBOV on day -28 relative to challenge. On day 0, mice were treated IP with MAR1-5A3 to block type I IFN receptor signaling and challenged IP with 100 TCID₅₀ CCHFV strain UG3010. Liver tissue (a–h) and spleen tissue (i–p) were collected from euthanized animals for Hematoxylin and Eosin staining and CCHFV anti-NP immunohistochemistry at day 5 post challenge. All photomicrographs taken at 200x; scale bar = 100 µm.

Fig. 5. VSV-CCHFgpc vaccines prevents weight loss and reduces viral RNA load following challenge with the CCHFV strain Hoti.

Six-week-old C57BL/6J mice were vaccinated intraperitoneally (IP) with 1 × 10⁴ PFU VSV-CCHFnp1, VSV-CCHFgpc, or VSV-EBOV on day -28 relative to challenge. On day 0 mice were treated IP with MAR1-5A3 to block type I IFN receptor signaling and challenged IP with 100 TCID₅₀ CCHFV strain Hoti. a Mice were weighed daily. N = 8 mice per group. Statistical significance compared to VSV-EBOV vaccinated animals is shown. b–d Viral RNA in indicated tissues at day 5 post challenge was quantified by RT-qPCR. N = 6 mice per group. Statistical comparisons calculated using two-way ANOVA with Sidak’s multiple comparisons test (a) or one-way ANOVA with Tukey’s multiple comparisons test (b–d) and results are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Comparisons with p values >0.05 were not displayed. Data shown as geometric mean plus standard deviation.

Fig. 6. VSV vaccines reduce liver pathology.

Six-week-old C57BL/6J mice were vaccinated intraperitoneally (IP) with 1 × 10⁴ PFU VSV-CCHFnp1, VSV-CCHFnp2, VSV-CCHFgpc, or VSV-EBOV on day -28 relative to challenge. On day 0 mice were treated IP with MAR1-5A3 to block type I IFN receptor signaling and challenged IP with 100 TCID₅₀ CCHFV strain Hoti. Day 5 post challenge Liver tissue (a–f) and spleen tissue (g–l) were collected on day 5 post challenge for Hematoxylin and Eosin staining and CCHFV anti-NP immunohistochemistry. All photomicrographs taken at 200x; scale bar = 100 µm.