A Pool of Eight Virally Vectored African Swine Fever Antigens Protect Pigs against Fatal Disease

Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease.

between all stages.

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Protein lysates were resolved with 10% bis-tris gels, transferred to PVDF membranes, blocked 24 with 5% milk powder and then probed overnight with primary antibody diluted in 5% BSA.

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Secondary antibodies were diluted in 5% milk powder, all solutions were based on TBS containing 26 0.2% Tween 20. Bands were detected by enhanced chemiluminescence (Pierce) with either X-ray 27 film or with a Syngene G-box.

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IgG specific sandwich ELISA 30 MaxiSorp 96 well plates were coated overnight at 2 -8°C with recombinant CP204L and B602L 31 (Reis et al., 2007) diluted in sodium carbonate buffer at 2 μg/ml. Plates were washed five times with 32 PBS containing 0.2% tween 20 (PBS-T) and then blocked for one hour at 37°C with 5% milk powder  The following humane end-points were used in both experiments.

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1. Pyrexia a) The animal will be euthanized if it has a fever above 40.5°C and shows other clinical signs at the beginning of the third consecutive day. b) If the animal has no other clinical signs, apart from a raised temperature, it will be euthanized at the beginning of the fourth consecutive day.

Behavior
The animal will be euthanized if it has a tendency to stay isolated, shows a delayed response to stimuli (gets up slowly when touched), and/or presents an abnormal posture e.g. head hung or back arched for two consecutive days.

Anorexia
The animal will be euthanized if it is not eating on a second consecutive day.

Digestive system
The animal will be euthanized if it has hemorrhagic diarrhea.

Respiratory system
Any pig showing an increase in breathing rate at the beginning of the second day without improvement will be euthanized at the beginning of the second day.
6. Lameness All animals showing lameness will be treated with antibiotics and/or suitable analgesia at first signs. a) Those animals showing non-weight bearing lameness will be euthanized 48 hours after treatment if it does not show improvement. b) Those animals showing weight bearing lameness will be euthanized beginning of the fifth consecutive day after treatment if lameness has not improved significantly. c) If there is a recurrence of the lameness, due to a regulated procedure, within 21 days from initial onset of lameness the animal will be euthanized on the same day. d) If the animal becomes lame, due to a regulated procedure, more than 21 days from the first signs of initial lameness it will be treated as if it was the first instance as point one above. e) If the animal becomes lame due to a regulated procedure a third time, it will be euthanized on the same day.

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Any animal showing three or more of the above clinical signs combined on a single day will be euthanized on the same day even if the duration of individual endpoints has not been reached.

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Endpoint 7 was the most common reason for euthanasia (9 animals), followed by endpoints 1b and 3

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(six animals each) and endpoint 2 (1 animal). No animals were euthanized for reaching endpoints 4, 44 5 or 6.    Figure S7: Comparison of antigen specific immune responses between Experiments 1 and 2. Sera from taken from animals pre-challenge (day 59) were analysed using B602L and CP204L ELISAs. Individual data points represent the mean of duplicate wells and error bars indicated the standard deviation. This data is taken from Figure 1C and 5B (B602L) and Figure 1E, 5C (CP204L). Differences in the means of the two experiments were analysed using Welch's t-test, **** p ≤ 0.0001, ** p = 0.0015. Macroscopic lesions were evaluated as described by Galindo-Cardiel et al., 2013 [44]. The tissues evaluated and the gross lesions observed are indicated on the graph by different patterns and/or colours. All of the animals in the control group and those immunised with Antigen Pool C were euthanized six days post challenge. The animals immunised with Antigen Pool A were euthanized twenty days post challenge.