Adenoviral vectored vaccination protects against Crimean-Congo Haemorrhagic Fever disease in a lethal challenge model

Abstract

Background: The tick-borne bunyavirus, Crimean-Congo Haemorrhagic Fever virus (CCHFV), can cause severe febrile illness in humans and has a wide geographic range that continues to expand due to tick migration. Currently, there are no licensed vaccines against CCHFV for widespread usage.

Methods: In this study, we describe the preclinical assessment of a chimpanzee adenoviral vectored vaccine (ChAdOx2 CCHF) which encodes the glycoprotein precursor (GPC) from CCHFV.

Findings: We demonstrate here that vaccination with ChAdOx2 CCHF induces both a humoral and cellular immune response in mice and 100% protection in a lethal CCHF challenge model. Delivery of the adenoviral vaccine in a heterologous vaccine regimen with a Modified Vaccinia Ankara vaccine (MVA CCHF) induces the highest levels of CCHFV-specific cell-mediated and antibody responses in mice. Histopathological examination and viral load analysis of the tissues of ChAdOx2 CCHF immunised mice reveals an absence of both microscopic changes and viral antigen associated with CCHF infection, further demonstrating protection against disease.

Interpretation: There is the continued need for an effective vaccine against CCHFV to protect humans from lethal haemorrhagic disease. Our findings support further development of the ChAd platform expressing the CCHFV GPC to seek an effective vaccine against CCHFV.

Funding: This research was supported by funding from the Biotechnology and Biological Sciences Research Council (UKRI-BBSRC) [BB/R019991/1 and BB/T008784/1].

Keywords: Antibodies; CCHFV; ChAdOx2; Heterologous immunisation; Prime and boost; T cells; Vaccine.

Fig. 1

CCHFV-specific IgG responses following ChAd and MVA immunisation. Immunisation schedules of the two mouse strains (a). Prime-boost regimens received prime vaccination on day 0 of experiment, and prime only regimens and prime-boost regimens received prime and boost vaccination respectively on day 14. Antibody responses were measured in the serum of BALB/c (n = 8) (b) and A129 (n = 4) mice (c) collected 3 weeks after the final immunization. CCHFV Gc-specific (left panel) and Gn-specific (right panel) IgG responses were quantified by standardised ELISA. Individual data points expressed as logarithmic ELISA units (EU log10) are shown here as a scatter dot plot with boxes showing the median and interquartile range and whiskers showing minimum and maximum. For (b, left panel) significant differences were determined by a one-way ANOVA with Tukey post-hoc analysis and data in graphs (b, right panel) and (c) analysed with Kruskal–Wallis test with Dunn's correction for multiple comparisons between vaccination groups. Dotted lines represent the quantified level of response from control immunised mice.

Fig. 2

Measurement of CCHFV-specific antibody-mediated neutralisation and avidity. Antibody neutralisation responses and avidity were measured in the serum of (a) BALB/c (n = 8) and (b) A129 (n = 4) mice collected 3 weeks after the final immunisation. Neutralisation was assessed by measuring inhibition of CCHFV tecVLPs entry into A549 cells, shown by individual data points expressed as logarithmic IC50 values (left panels). Avidity of CCHFV Gc specific IgG responses was measured using a NaSCN chemical displacement ELISA (right panels). Individual data points are shown here as a scatter dot plot with boxes showing the median and interquartile range and whiskers showing minimum and maximum. Significant differences were determined by one-way ANOVA with Tukey post-hoc analysis. Dotted lines represent the quantified level of response from control immunised mice.

Fig. 3

Detection of IgG subclasses in BALB/c and A129 mice immunised with ChAd and MVA regimens. Samples with detectable CCHFV Gc-specific responses were normalised and diluted to 1 EU. IgG subclasses were quantified by optical density and data displayed as scattered dot plots with bars showing the median and IQR, and as heatmap with median OD values of each group. Individual data points represent OD of a single mouse. BALB/c mice data (n = 8) (a) in each graph were analysed with a one-way ANOVA with Tukey post-hoc analysis, and A129 (n = 4) (b) data analysed with Kruskal–Wallis test followed by a post hoc Dunn's multiple comparison test to compare differences between vaccination groups. Dotted lines represent the assay limit of quantification.

Fig. 4

CCHFV-specific cellular responses in mice immunised with ChAd and MVA regimens. CCHFV antigen-specific IFN-γ responses in mouse splenocytes were assayed by IFN-γ ELISPOT assays. The summed IFN-γ ELISPOT responses in BALB/c (a) and A129 mice (b) are displayed as individual data points as a scatter dot plot with boxes showing the median and interquartile range and whiskers showing minimum and maximum. Responses to individual peptide pools are displayed by stacked bars of IFN-γ responses in BALB/c (c) and A129 mice (d), with lines showing the median with IQR. Significant differences were determined by one-way ANOVA with Tukey post-hoc analysis. Dotted lines represent the quantified level of response from control immunised mice.

Fig. 5

Assessing the protective effect of ChAd and MVA regimens against challenge with CCHFV in A129 mice. Challenge timeline overview (a) for A129 mice (n = 6 mice per group). Mice in prime-boost regimens received prime vaccination on day 0 of experiment. Prime only regimens and prime-boost regimens received prime and boost vaccination respectively on day 14. Mice were challenged on day 36 with a 100 μl volume of 200 ffu CCHFV that was intradermally administered. All surviving mice were euthanised on day 56. Following challenge, all mice were monitored for changes in rectal temperature (b) and bodyweight (c) that are displayed as the recorded median of each regimen group with error bars representing IQR, as well as Kaplan–Meier survival plot (d) displaying percentage survival up to 20 days post challenge.

Fig. 6

Viral load and histopathological analysis following lethal CCHFV challenge of A129 mice immunised with ChAd or MVA. A129 mice (n = 6 per group) vaccinated with ChAd or MVA CCHF were challenged with CCHFV, and twenty days post–challenge (day 56) all surviving animals were killed humanely and tissue samples taken for analysis. (a) Graphs show RNA levels measured in blood, spleen and liver by RT-PCR analysis for CCHFV gene expression, with control vaccinated mice analysed for viral RNA 5 days post challenge after reaching humane endpoints. Each point represents the mean value of triplicate measurements in an individual animal. Lines show mean ± standard deviation. Sections of spleen and liver were fixed, stained, and examined by pathology; graphs show combined subjective scores of all animals in all groups for (b) microscopic changes from histopathology on the liver and spleen using a scoring system [normal (0), minimal (1), mild (2), moderate (3), marked (4)], and (c) total score for viral antigen staining of the liver and spleen, with the system [occasional single cell staining (1); scattered, positive staining (2); frequent, scattered staining (3); and marked, patchy to diffuse staining throughout the tissue (4)]. Horizontal bars represent median value.

Fig. 7

Representative images of the histopathological changes in the spleen and liver of A129 mice after challenge with CCHFV. A129 mice were challenged with a lethal dose of CCHFV 21 days after prime or prime-boost immunisation with ChAd or MVA. Haematoxylin and eosin-stained sections and IHC- stained sections for viral antigen were examined from mice that were euthanised after meeting either the study or humane endpoints. In the spleen of the control mice (a), there was mild to marked lymphocytolysis in the white pulp, with prominent, tingeable body macrophages; in the red pulp, a variable increase in macrophages and some neutrophils, were noted. Inset, higher power image showing lymphocyte apoptosis and tingeable body macrophages in the white pulp. HE. Prominent, diffuse, staining of viral antigen was noted within the parenchyma (b), most prominent in cells in the red pulp. Inset, higher power image of stained cells. IHC. In the liver of the control mice (c), there was a moderate to marked, multifocal hepatocyte necrosis scattered randomly throughout the parenchyma (arrows) and accompanied by variable numbers of inflammatory cells, primarily neutrophils. Inset, higher power image of necrotic hepatocytes. HE. Prominent, diffuse, staining of viral antigen (d) was noted within the parenchyma, most prominent in intra-lesional hepatocytes. Inset, higher power image of cells staining positive for viral antigen. IHC. In the spleen (e, f) and liver (g, h) of all mice in the treated groups, microscopic lesions associated with CCHF infection and staining for viral antigen, were absent. HE. IHC. Scale bars are shown at 100 μM for large low power images, and 50 μM scale bars for magnified square higher power images.

Fig. S1 correl

Figure S1. Detailed serology following immunisation of BALB/c mice with various ChAd and MVA regimens. (a and b) Relationship between levels of CCHFV-Gc specific IgG and tecVLP neutralisation IC50 correlation displayed by scattered dot plot (left panel) and correlation heatmap matrix (right panel). (c) Relationship between levels of tecVLP neutralisation IC50 and avidity IC50, displayed as scattered dot plot. Statistical analyses were performed using two-tailed Pearson correlation test.