Development of a Highly Effective African Swine Fever Virus Vaccine by Deletion of the I177L Gene Results in Sterile Immunity against the Current Epidemic Eurasia Strain

Abstract

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The disease is devastating the swine industry in Central Europe and East Asia, with current outbreaks caused by circulating strains of ASFV derived from the 2007 Georgia isolate (ASFV-G), a genotype II ASFV. In the absence of any available vaccines, African swine fever (ASF) outbreak containment relies on the control and culling of infected animals. Limited cross-protection studies suggest that in order to ensure a vaccine is effective, it must be derived from the current outbreak strain or at the very least from an isolate with the same genotype. Here, we report the discovery that the deletion of a previously uncharacterized gene, I177L, from the highly virulent ASFV-G produces complete virus attenuation in swine. Animals inoculated intramuscularly with the virus lacking the I177L gene, ASFV-G-ΔI177L, at a dose range of 10² to 10⁶ 50% hemadsorbing doses (HAD₅₀), remained clinically normal during the 28-day observational period. All ASFV-G-ΔI177L-infected animals had low viremia titers, showed no virus shedding, and developed a strong virus-specific antibody response; importantly, they were protected when challenged with the virulent parental strain ASFV-G. ASFV-G-ΔI177L is one of the few experimental vaccine candidate virus strains reported to be able to induce protection against the ASFV Georgia isolate, and it is the first vaccine capable of inducing sterile immunity against the current ASFV strain responsible for recent outbreaks.IMPORTANCE Currently, there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating the swine industry from Central Europe to East Asia, and they are being caused by circulating strains of African swine fever virus derived from the Georgia 2007 isolate. Here, we report the discovery of a previously uncharacterized virus gene, which when deleted completely attenuates the Georgia isolate. Importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. Interestingly, ASFV-G-ΔI177L confers protection even at low doses (10² HAD₅₀) and remains completely attenuated when inoculated at high doses (10⁶ HAD₅₀), demonstrating its potential as a safe vaccine candidate. At medium or higher doses (10⁴ HAD₅₀), sterile immunity is achieved. Therefore, ASFV-G-ΔI177L is a novel efficacious experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

Keywords: ASF; ASFV; African swine fever virus; vaccine.

FIG 1

Multiple-sequence alignment of the indicated ASFV isolates of viral protein I177L, with matching residues represented as periods and gaps in the sequence represented by dashes. The degree of conservation between the sequences is represented below the sequences.

FIG 2

Time course of I177L gene transcriptional activity. Shown are averaged microarray signal intensities (photons per pixel) of ASFV I177L, CP204L, and B646L open reading frame RNA prepared from ex vivo pig macrophages infected with ASFV at 3, 6, 9, 12, 15, and 18 h postinfection.

FIG 3

Diagram indicating the position of the I177L open reading frame in the ASFV-G genome (top). A donor plasmid with arms homologous to ASFV-G and the mCherry under the control of the p72 promoter was used to introduce the final genomic changes (bottom) to create ASFV-Georgia-ΔI177L, where the sequence of the donor plasmid mCherry reporter (red) is introduced to replace the ORF of I177L, as indicated.

FIG 4

In vitro growth characteristics of ASFV-Georgia-ΔI177L and parental ASFV-G. Primary swine macrophage cell cultures were infected (MOI, 0.01) with each of the viruses and the virus yield titrated at the indicated times postinfection. Data represent the means of the results from three independent experiments. The sensitivity of virus detection is ≥1.8 log₁₀ HAD₅₀/ml. Significant differences (*) in viral yields between the two viruses at specific times points were determined using the Holm-Sidak method (α = 0.05), without assuming a consistent standard deviation. All calculations were conducted on the software GraphPad Prism version 8. TCID₅₀, 50% tissue culture infective dose.

FIG 5

Viremia titers detected in pigs i.m. inoculated with either 10² HAD₅₀ of ASFV-Georgia-ΔI177L or 10² HAD₅₀ of ASFV-G. Each curve represents values from individual animals in each group. The sensitivity of virus detection was ≥1.8 log₁₀ HAD₅₀/ml.

FIG 6

Kinetics of body temperature values in pigs i.m. inoculated with either 10², 10⁴, or 10⁶ HAD₅₀ (top of each panel) of ASFV-Georgia-ΔI177L (filled symbols), mock inoculated (sentinels, shown in red), or 10² HAD₅₀ of ASFV-G (open symbols) (left) and after the challenge with 10² HAD₅₀ of ASFV-G (right). Each curve represents an individual animal’s values in each group. Data from sentinel animals are depicted in red.

FIG 7

Viremia titers detected in pigs i.m. inoculated with either ASFV-Georgia-ΔI177L or ASFV-G. (A to H) Animals were inoculated with either 10² HAD₅₀ of ASFV-G (A and B) or ASFV-Georgia-ΔI177L at doses of 10² (C and D), 10⁴ (E and F), or 10⁶ (G and H) HAD₅₀. (C to H) Viremias in ASFV-Georgia-ΔI177L-inoculated animals before (C, E, and G) or after (D, F, and H) the challenge with 10² HAD₅₀ of ASFV-G. Each curve represents values from individual animals in each group. The sensitivity of virus detection was ≥1.8 log₁₀ HAD₅₀/ml₅₀/ml. Data from sentinel animals are depicted in red.

FIG 8

(A to F) Anti-ASFV antibody (IgM mediated [A, C, and E] and IgG mediated [B, D, and F]) titers detected by ELISA in pigs i.m. inoculated with either 10², 10⁴, or 10⁶ HAD₅₀ of ASFV-Georgia-ΔI177L. Each curve represents values from individual animals in each group.