African Swine Fever Vaccine Candidate ASFV-G-ΔI177L Produced in the Swine Macrophage-Derived Cell Line IPKM Remains Genetically Stable and Protective against Homologous Virulent Challenge

Abstract

ASFV vaccine candidate ASFV-G-ΔI177L has been shown to be highly efficacious in inducing protection against challenges with the parental virus, the Georgia 2010 isolate, as well as against field strains isolated from Vietnam. ASFV-G-ΔI177L has been shown to produce protection even when used at low doses (10² HAD₅₀) and shows no residual virulence even when administered at high doses (10⁶ HAD₅₀) or evaluated for a relatively long period of time (6 months). ASFV-G-ΔI177L stocks can only be massively produced in primary cell macrophages. Alternatively, its modified version (ASFV-G-ΔI177L/ΔLVR) grows in a swine-derived cell line (PIPEC), acquiring significant genomic modifications. We present here the development of ASFV-G-ΔI177L stocks in a swine macrophage cell line, IPKM, and its protective efficacy when evaluated in domestic pigs. Successive passing of ASFV-G-ΔI177L in IPKM cells produces minimal genomic changes. Interestingly, a stock of ASFV-G-ΔI177L obtained after 10 passages (ASFV-G-ΔI177Lp10) in IPKM cells showed very small genomic changes when compared with the original virus stock. ASFV-G-ΔI177Lp10 conserves similar growth kinetics in primary swine macrophage cultures than the original parental virus ASFV-G-ΔI177L. Pigs infected with 10³ HAD₅₀ of ASFV-G-ΔI177Lp10 developed a strong virus-specific antibody response and were completely protected against the challenge with the parental virulent field isolate Georgia 2010. Therefore, IPKM cells could be an effective alternative for the production of ASFV vaccine stocks for those vaccine candidates exclusively growing in swine macrophages.

Keywords: ASF; ASFV; ASFV vaccine; ASFV-G-ΔI177L.

Figure 1

Replication of ASFV-G-ΔI177L in successive passages in IPKM cells. ASFV-G-ΔI177L and ASFV-G were passed 10 consecutive times (MOI = 1) in IPKM cell cultures. Viral yield in each passage was quantified in primary cultures of swine macrophages and titers expressed as HAD₅₀/_mL. Titrations were conducted by duplicated (presented data represent one set of them).

Figure 2

In vitro growth kinetics of ASFV-G-ΔI177Lp10 and parental ASFV-G-ΔI177Lp0 in primary swine macrophage cell cultures (MOI = 0.01). As control, ASFV-Gp0 and ASFV-Gp10 were tested under similar conditions. Samples were taken from two independent experiments at the indicated time points and titrated in swine macrophages. Data represent means and standard deviations. Sensitivity using this methodology for detecting virus is ≥log10 1.8 HAD₅₀/mL. (*) Indicates significant differences. Statistically significant differences at specific time points between groups were evaluated by ANOVA analysis and confirmed by Tukey’s honest significance test (≤0.05). Analyses were conducted using the software JMP Pro version 16.0.0.

Figure 3

Evolution of body temperature in animals (5 animals/group) IM inoculated with 10³ HAD₅₀ of ASFV-G-ΔI177Lp10 or mock-inoculated and challenged 28 days later with 10² HAD₅₀ of parental virulent ASFV-G. Data represent individual animals.

Figure 4

Viremia titers detected in animals (5 animals/group) IM inoculated with 10³ HAD₅₀ of ASFV-G-ΔI177Lp10 or mock-inoculated and challenged 28 days later with 10² HAD₅₀ of ASFV-G. Data represent individual animals. Sensitivity of virus detection: ≥10^1.8 TCID₅₀/_mL.

Figure 5

Anti-ASFV antibody titers detected by ELISA in pigs IM inoculated with 10³ HAD₅₀ of ASFV-G-ΔI177Lp10. Each point represents values from individual animals. Titers are expressed as the log₁₀ of inverse of the highest serum dilution that still duplicates OD of the pre-inoculation serum.