Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge

Abstract

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (10⁶ HAD₅₀). Importantly, animals infected with 10⁴ 50% hemadsorbing doses (HAD₅₀) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination.

Importance: Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

Keywords: 9GL; ASFV; African swine fever virus; UK; vaccine.

FIG 1

Amino acid sequence alignment of protein products of 9GL (B119L) and UK (DP96R) from different ASFV isolates. Virus isolates of various temporal and geographic origins, including those obtained from ticks and pig sources, were compared. The partial deletions introduced into ASFV-G that yielded ASFV-G-Δ9GL/ΔUK are shown between brackets. (.), identical amino acid residues; (*), stop codon.

FIG 2

In vitro growth characteristics of ASFV-G-Δ9GL/ΔUK and parental ASFV-G-Δ9GL and ASFV-G. Primary swine macrophage cultures were infected (MOI = 0.01) with each of the viruses, and the virus yields were titrated at the indicated times postinfection. The data represent the means and standard deviations from three independent experiments. The sensitivity of virus detection was ≥1.8 log₁₀ HAD₅₀/ml.

FIG 3

Viremia titers detected in pigs i.m. inoculated with either 10² (A), 10⁴ (B), or 10⁶ (C) HAD₅₀ of ASFV-G-Δ9GL/ΔUK and i.m. challenged (indicated by arrows) 28 days later with 10³ HAD₅₀ of ASFV-G. Each curve represents data from an individual animal. The gray circles show the viremia of control animals infected with 10² HAD₅₀. The sensitivity of virus detection was ≥1.8 log₁₀ HAD₅₀/ml.

FIG 4

Viremia titers detected in pigs i.m. challenged (indicated by arrows) with 10³ HAD₅₀ of ASFV-G at 7 (A), 14 (B), or 21 (C) days post-i.m. infection with 10⁴ HAD₅₀ of ASFV-G-Δ9GL/ΔUK. Each curve represents data from an individual animal. The circles show the viremias of mock-vaccinated and challenged animals. The sensitivity of virus detection was ≥1.8 log₁₀ HAD₅₀/ml.

FIG 5

Serological ASFV-specific antibodies detected by ELISA and immunoperoxidase staining (IPA), along with the number of circulating ASFV-specific IFN-γ-producing cells for each individual pig at the time of challenge in animals infected with either 10² (A), 10⁴ (B), or 10⁶ (C) HAD₅₀ of ASFV-G-Δ9GL/ΔUK. The survival status of the swine is indicated as survival (open symbols) or no survival (solid symbols). neg, negative; dotted lines, threshold for positivity.

FIG 6

Serological ASFV-specific antibodies detected by ELISA and IPA, along with the number of circulating ASFV-specific IFN-γ-producing cells, for each individual pig at the time of challenge at 7 (A), 14 (B), or 21 (C) days postinfection with 10⁴ HAD₅₀ of ASFV-G-Δ9GL/ΔUK. The survival status of the swine is indicated as survival (open symbols) or no survival (solid symbols). Dotted lines, threshold for positivity.

FIG 7

Evaluation of systemic levels of different host cytokines at the time of challenge, either 7 or 14 days postinfection with ASFV-G-Δ9GL/ΔUK. The values were determined for individual animals and are expressed as the concentration per milliliter of serum, as described in Materials and Methods. The survival status of the swine is indicated as survival (open symbols) or no survival (solid symbols).