Co-Deletion of A238L and EP402R Genes from a Genotype IX African Swine Fever Virus Results in Partial Attenuation and Protection in Swine

Abstract

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), resulting in up to 100% mortality in pigs. Although endemic in most sub-Saharan African countries, where all known ASFV genotypes have been reported, the disease has caused pandemics of significant economic impact in Eurasia, and no vaccines or therapeutics are available to date. In endeavors to develop live-attenuated vaccines against ASF, deletions of several of the ~170 ASFV genes have shown contrasting results depending on the genotype of the investigated ASFV. Here, we report the in vivo outcome of a single deletion of the A238L (5EL) gene and double deletions of A238L (5EL) and EP402R (CD2v) genes from the genome of a highly virulent genotype IX ASFV isolate. Domestic pigs were intramuscularly inoculated with (i) ASFV-Ke-ΔA238L to assess the safety of A238L deletion and (ii) ASFV-Ke-ΔEP402RΔA238L to investigate protection against challenge with the virulent wildtype ASFV-Ke virus. While A238L (5EL) gene deletion did not yield complete attenuation, co-deletion of A238L (5EL) and EP402R (CD2v) improved the safety profile of the single deletions, eliciting both humoral and cellular immune responses and conferred partial protection against challenge with the virulent wildtype ASFV-Ke virus.

Keywords: ASFV; attenuation; gene deletion; protection; vaccine; virulence.

Figure 1

Body temperature and clinical scores: Rectal temperatures in individual pigs inoculated with 10² HAD₅₀ of ASFV-Ke (A) or 10² TCID₅₀ ASFV-Ke-∆A238L (B). Mean +/− SEM of rectal temperatures in ASFV-Ke (black) and ASFV-Ke-∆A238L (maroon) groups (C). Cumulative clinical scores in pigs inoculated with ASFV-Ke (D) or ASFV-Ke-∆A238L (E). Mean +/− SEM of cumulative clinical scores in ASFV-Ke (black) and ASFV-Ke-∆A238L (maroon) groups (F).

Figure 2

Virus replication in the blood (viral genome copies) after inoculation with 10² TCID₅₀ of ASFV-Ke (black) or ASFV-Ke-∆A238L (maroon).

Figure 3

ASFV-specific antibody response after inoculation with ASFV-Ke-∆A238L. The percentage of blocking by p72-specific serum antibodies at indicated times was tested using a commercial competitive ELISA. The threshold range is indicated by a grey bar.

Figure 4

Body temperatures and clinical scores: Rectal temperatures in individual pigs mock-immunised with PBS (A) or immunised with 10² TCID₅₀ of ASFV-Ke-∆EP402R∆A238L (B) before and after challenge with ASFV-Ke. Mean +/− SEM of rectal temperatures in PBS (blue) and ASFV-Ke-∆EP402R∆A238L (red) (C) and ASFV-Ke challenged groups. Cumulative clinical scores in pigs mock-immunised with PBS (D) or ASFV-Ke-∆EP402R∆A238L-immunised (E) and ASFV-Ke challenged groups. Mean +/− SEM of cumulative clinical scores in mock-immunised (blue) and ASFV-Ke-∆EP402R∆A238L-immunised (red) (F) and ASFV-Ke challenged groups.

Figure 5

Virus replication in the blood (viral genome copies) after immunisation with PBS (blue) or ASFV-Ke-∆EP402R∆A238L (red) and subsequent challenge with ASFV-Ke (A). Detection of virus DNA in tissues prepared post-mortem from animals immunised with PBS (blue) or ASFV-Ke-∆EP402R∆A238L (red) after challenge with ASFV-Ke (B).

Figure 6

ASFV-specific antibody and cellular immune responses: percentage of blocking by p72-specific serum antibodies after mock-immunisation with PBS (A) or immunisation with ASFV-Ke-∆EP402R∆A238L (B) determined using a commercial blocking ELISA. Mean+/−SEM of percentage blocking in PBS- or ASFV-Ke-∆EP402R∆A238L-immunised groups (C). IFNγ ELISPOT using PBMC isolated from animals at day 28 after immunisation with PBS (blue) or ASFV-Ke-∆EP402R∆A238L (red) and stimulation with either medium or ASFV-Ke wild type (wt) infection at an MOI of 0.1 (D).