Rapid generation of replication-deficient monovalent and multivalent vaccines for bluetongue virus: protection against virulent virus challenge in cattle and sheep

Abstract

Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Fig 1

Characterization of reassortant disabled viruses generated by reverse genetics. (A) Left panel: the genomic profile of purified dsRNA from cells infected with the reassortant BTV-2D was analyzed by nondenaturing PAGE. BTV-1 and BTV-2 profiles were included as a control. Right panel: details of the genomic profile of dsRNA from each of the reassortant disabled viruses. Arrowheads indicate differences in the mobility of segments S2 and S6. (B) Replication in normal (upper panel) or complementing (lower panel) cells of the reassortant disabled BTV strains. Monolayers of normal BSR or complementing BSR9 cell lines were infected at a multiplicity of infection (MOI) of 0.1, and samples were collected as indicated. Cells and supernatant were harvested, and the total titer was determined by TCID₅₀. Total titers at the indicated time points are expressed as TCID₅₀/ml.

Fig 2

Detection of group-specific antibody response in sera of vaccinated cattle by ELISA. (A) Antibodies directed against the VP7 protein of BTV were detected using a competition ELISA kit. The data points represent the mean antibody responses of the animals in each group (vaccinated with the BTV2D, -4D, or -8D strain or the control) with standard deviations. Results are expressed as percentages of negativity (% Negativity) compared to the negative control included in the kit and were classified into positive (≤65%), uncertain (>65% but ≤75%), and negative (>75%) groups. Gray arrows indicate the first and second vaccination times (V1 and V2), and the black arrow indicates the time of challenge with homologous virulent virus serotypes (C). The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○). (B) Early detection of IgG and IgM antibodies after the first vaccination. The data represent the mean antibody responses of the animals in each group with standard deviations. The samples were evaluated by the sample-to-positive ratio expressed as a percentage (% S/P) and were classified into positive (% S/P > 30), uncertain (% S/P ≤ 30 but > 25), and negative (% S/P ≤ 25) groups. The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○).

Fig 3

Virus replication in vaccinated cattle challenged with a virulent strain. Detection of genomic BTV RNA was performed using a real-time RT-PCR assay in postchallenged samples from cattle vaccinated with the monoserotype vaccines. The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○). The data points represent the means and standard deviations of RNA copy numbers of the animals in each group.

Fig 4

Detection of group-specific antibody response in sera of sheep vaccinated with multivalent vaccines by ELISA. (A) Antibodies directed against the VP7 protein of BTV were detected using a competition ELISA kit. The data points represent the mean antibody responses of the animals in each group (vaccinated with the vaccine cocktail and control) with standard deviations. Results are expressed as percentages of negativity (% Negativity) compared to the negative control included in the kit and were classified into positive (≤65), uncertain (>65 but ≤75), and negative (>75) groups. The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○). Arrows indicate first and second vaccination times (V1 and V2), and the black arrow indicates the time of challenge with homologous virulent virus serotypes (C). (B) Early detection of IgG and IgM antibodies after the first vaccination. The data represent the mean antibody responses of the animals in each group (vaccinated with the vaccine cocktail and control) with standard deviations. The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○). The samples were evaluated by the sample-to-positive ratio expressed as a percentage (% S/P) and were classified into positive (% S/P > 30), uncertain (% S/P ≤ 30 but > 25), and negative (% S/P ≤ 25) groups.

Fig 5

Neutralizing antibody response of vaccinated sheep. Samples of sera from vaccinated (gray columns) and control (open columns) animals were taken 21 days after the second vaccination and tested for neutralizing activity against all BTV serotypes included in the vaccine (BTV-1, -2, -4, -8, -13, and -21) as indicated. The neutralizing titer in each animal was determined as the maximum dilution of serum that showed complete protection.

Fig 6

Clinical signs and viremia in sheep vaccinated with the multivalent cocktail vaccine. (A) Body temperature as a sign of BT disease was recorded at different time points for all animals in each group. (B) The presence of genomic RNA in blood from all animals was determined by real-time RT-PCR at the indicated times during the experiment. V1, V2, and C represent the times of the first and second vaccinations and challenge, respectively. The samples were as follows (symbol): 2D plus BTV-2 (■), 4D plus BTV-4 (⧫), 8D plus BTV-8 (●), control plus BTV-2 (☐), control plus BTV-4 (◊), and control plus BTV-8 (○).