A Novel Vaccine against Crimean-Congo Haemorrhagic Fever Protects 100% of Animals against Lethal Challenge in a Mouse Model

<div><p>Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15–70% of reported cases are fatal. There is no approved vaccine available, and preclinical protection <i>in vivo</i> by an experimental vaccine has not been demonstrated previously. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus glycoproteins. Cellular and humoral immunogenicity was confirmed in two mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. This vaccine protected all recipient animals from lethal disease in a challenge model adapted to represent infection via a tick bite. Histopathology and viral load analysis of protected animals confirmed that they had been exposed to challenge virus, even though they did not exhibit clinical signs. This is the first demonstration of efficacy of a CCHF vaccine.</p></div

Immunohistochemistry of tissues from A129 mice, 4 days after challenge with CCHFv.

<p>A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A–C) or MVA-GP (D–E). Four days after challenge, sections of spleen (A, D) and liver (B–C, E) were fixed, immunohistochemically stained with CCHFv-specific antibody, and examined microscopically. Tissues in panels A, B, D and E were from the same individuals as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091516#pone-0091516-g007" target="_blank">Figures 7A, 7B, 7C and 7D</a>, respectively. A diffuse staining pattern of viral proteins was found in tissues from animals that received the MVA 1974 negative control. However, in MVA-GP vaccinated animals, the only staining found was of a minimal degree, in liver from one individual. (A) A few, scattered cells with cytoplasmic staining within the parenchyma. (B) Frequent, diffuse, positively stained hepatocytes. (C) Scattered, small, elongated cells consistent with Kupffer cells, with cytoplasmic staining. (D) Normal parenchyma. (E) A few, positively stained cells within an inflammatory cell focus.</p

Severity of microscopic lesions in HE stained tissues from vaccinated A129 mice, challenged with CCHFv.

<p>Numbers of animals in each group, according to severity rating of histological lesions.</p

IFN-γ ELISpot responses from vaccinated A129 and 129Sv/Ev mice, 7 days after booster vaccination.

<p>Splenocytes from animals vaccinated with MVA-GP (orange), MVA 1974 or saline (black) were restimulated with peptides derived from the CCHFv glycoprotein. A129 mice data (solid bars) were pooled from 2 independent experiments (n = 8). 129Sv/Ev mice n = 5 (hatched bars). Mean ± SEM is plotted. (A) Summed antigen responses from all peptide pools. Splenocytes from MVA-GP vaccinated mice, but not control mice, responded to GP-specific peptides, indicating similar T-cell responses between mouse strains. (B) Antigen responses from MVA-GP vaccinated mice, according to peptide pool. Immunogenicity was not evenly distributed across the antigen, but some peptide pools were more immunogenic than others. Responses were specific to the glycoprotein, and similar between mouse strains.</p

Antibody responses from vaccinated A129 and 129Sv/Ev mice.

<p>Sera from vaccinated mice were tested for reactivity with CCHFv-infected (lane 3) or uninfected (lane 2) SW13 cells by Western blotting. Lane 1 shows a molecular weight marker. Blots show proteins reactive with serum from representative individual animals 7 days after booster vaccination (A–E) or representative pooled serum 14 days after booster vaccination (F). Secondary antibody used was specific for mouse IgG (A–C, F), or mouse IgG, IgA and IgM (D–E). Arrows highlight CCHFv-specific proteins, indicating specific antibody responses in both mouse strains. (A) 129Sv/Ev mouse vaccinated with MVA-GP. (B, D–E) A129 mouse vaccinated with MVA-GP. (C) A129 mouse vaccinated with MVA 1974. (F) Pooled sera from A129 mice vaccinated with MVA-GP.</p

Frequency of immunohistochemically stained cells in tissues from selected vaccinated A129 mice, challenged with CCHFv.

<p>Numbers of animals in each group, according to frequency of cells stained by immunohistochemistry.</p

Schematic representation of the vaccine vector.

<p>Nucleotides 34 to 5082 of the CCHFv M segment (orange) were fused at the amino-terminus to the 36 amino acid residue tPA leader sequence, via 11 residues derived from the Gateway recombination process. The carboxy-terminus was fused to the 14 amino acid residue V5 epitope, via 11 residues of Gateway sequence, and 8 residues of a linker peptide. This product was under the control of the mH5 promoter, and between MVA flanks for insertion into the Deletion III site of the MVA genome. The insertion cassette also contained the enhanced GFP gene downstream of the p11 promoter, for identification of recombinant virus. The mucin-like domain is represented by ‘mu’.</p

Tissue histology of A129 mice, 4 days after challenge with CCHFv.

<p>A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A–B) or MVA-GP (C–D). Four days after challenge, sections of spleen (A, C) and liver (B, D) were fixed, HE stained, and examined for pathology. More severe pathology was found in mice that received MVA 1974, compared to those that received MVA-GP. (A) Marked lymphocyte loss with prominent apoptotic bodies, and infiltration by macrophages. (B) Marked, multifocally extensive hepatocyte necrosis (arrows). (C) A single infiltration of macrophages in the white pulp (asterisk) (scored minimal). (D) Scattered, multifocal areas of hepatocellular necrosis with a mixed inflammatory cell infiltrate (arrows) (scored moderate).</p

Efficacy of MVA-GP in A129 mice challenged with CCHFv.

<p>A129 mice (n = 6) were challenged with double the minimum lethal dose of CCHFv 14 days after booster vaccination with MVA-GP (orange triangles), MVA 1974 (grey squares) or saline (black circles) and monitored for the following 14 days. All mice vaccinated with MVA-GP survived lethal challenge, and did not show any signs of illness, become febrile, or exhibit weight loss. (A) Percentage of surviving animals. (B) Signs of illness. Mean ± standard deviation is plotted. (C) Body temperature. Mean ± standard deviation is plotted. (D) Weight change as a percentage of body weight at challenge. Mean ± standard deviation is plotted.</p