A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine

Figures

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  Figure 1. Clinical Response and Serology Following Inoculation with ASFV-G-ΔI177L and Challenge with ASF-G. At 0 dpi, swine were intramuscularly inoculated with 106 median hemadsorbing dose (HAD50) of ASFV-G-ΔI177L or vehicle control. At 28 days post-inoculation (dpi) all animals were challenged by intramuscular injection of 102 HAD50 of ASFV-G and monitored for up to 20 days post-challenge (dpc). (A) Kaplan–Meier survival curve following challenge. ** p-value = 0.0011 (B) Serum viral titers were measured by HAD50 at various post-inoculation and post-challenge time points, and titers are displayed in Log10. Difference in slope of average titer change over time was assessed by linear regression. **** p-value < 0.0001 (C) Rectal temperatures were measured at various post-inoculation and post-challenge time points and are displayed in degrees Celsius. Difference in slope of average temperature change over time was assessed by linear regression. **** p-value < 0.0001 (D) Serum anti-ASFV antibody titers were measured by ELISA at various time points during the post-inoculation and post-challenge period among ASFV-G-ΔI177L vaccinated group and are expressed as the reciprocal Log10 titer.…

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  Figure 2. Serum Cytokine Concentrations. At various time points following inoculation (A–G) and challenge (H–N), blood was collected, allowed to clot, centrifuged and the serum collected and assayed for a variety of cytokines. n = 2–12 swine/treatment/time point. Mock-vaccinated swine shown in blue and ASFV-G-ΔI177L-vaccinated swine shown in purple. ** p-value < 0.01, *** p-value < 0.001 when compared with time-matched control. † p-value < 0.05 †† p-value < 0.01 when compared against treatment-matched baseline at 0 dpi.

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  Figure 3. Memory T Cell and NK Cell Panel Following Inoculation. At various time points, heparinized blood was collected, PBMCs isolated and stained for flow cytometric analysis. (A) Gating strategy for this panel showing ultimate isolation of NK cells (CD3−CD4−CD8+), CD4+, CD8+ and CD4+CD8+ Memory T cells and IFNγ expression within those subsets. In the case of CD4+, CD8+ and CD4+CD8+ T cells, these were each subjected to CD44+CD25−CD27− identification of the memory subset and then plotted for their expression of CD62L vs. CCR7 in order to determine central memory T cells (CD62L+CCR7+) as well as effector memory T cell (CD62L−CCR7−) before analyzing the IFNγ expression for each memory population. Briefly, a generous gate was drawn around the lymphocyte population in FSC-H vs. SSC-H, then singlets were selected from a SSC-A vs. SSC-H plot, live cells were selected by gating around Viability negative cells. Expression of CD4 vs. CD8 was then assessed among CD3− as well as CD3+ cells. CD4−CD8+ were then selected from the CD3− population and assessed for IFNγ expression as NK cells. Each of three groups of T cells—CD4+CD8−, CD4−CD8+ and CD4+CD8+—were then each subjected to the same gating…

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  Figure 4. Memory T Cell and NK Cell Populations Following Inoculation. At various time points following inoculation, heparinized blood was collected, PBMCs isolated and stained for flow cytometric analysis. Upon ex vivo stimulation with ASFV-G, the change (ASFV-G-stimulated well minus unstimulated well) in CD8+ (A) Central (Tcm) and (B) Effector memory T (Tem) cells were calculated as a percentage of the CD4−CD8+ population, and IFNγ+ CD8+ (C) Tcm and (D) Tem cells were calculated as a percentage of the memory cell parent population. Additionally, the change (ASFV-G-stimulated well minus unstimulated well) in CD4+ (E) Tcm and (F) Tem cells were calculated as a percentage of the CD4+CD8− population, while IFNγ+ CD4+ (G) Tcm and (H) Tem cells were calculated as a percentage of the memory parent population. Finally, the change (ASFV-G-stimulated well minus unstimulated well) in CD4+CD8+ double positive (I) Tcm and (J) Tem cells were calculated as a percentage of the CD4+CD8+ population, while IFNγ+ CD4+CD8+ (K) Tcm and (L) Tem cells were calculated as a percentage of the memory parent population. (M) Upon ex vivo stimulation with ASFV-G, the change (ASFV-G-stimulated well minus unstimulated…

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  Figure 5. γδT cell and Regulatory T cell Panel Following Inoculation. At various time points isolated PBMCs were stained for flow cytometric analysis. (A) Gating strategy for this panel showing ultimate isolation of γδT cells and Treg, along with IFNγ expression within those subsets. Gates were determined by fluorescence minus one (FMO) analysis. (B) γδT cells (unstimulated well) as a percentage of their parent population were assessed among ex vivo unstimulated cells. (C) Change (ASFV-G-stimulated well minus unstimulated well) in IFNγ+ γδT cells as a percentage of the parent population following ex vivo stimulation with ASFV-G. Change (ASFV-G-stimulated well minus unstimulated well) in (D) Treg cells and (E) IFNγ+ Treg cells as a percentage of their parent population were assessed following ex vivo stimulation with ASFV-G. Mock-vaccinated swine shown in blue and ASFV-G-ΔI177L-vaccinated swine shown in purple. n = 2–10 swine/treatment/time point. ** p-value < 0.01 **** p-value < 0.0001 when compared with time-matched control. † p-value < 0.05 ††† p-value < 0.001 †††† p-value < 0.0001 when compared against treatment-matched baseline at 0 dpi.

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  Figure 6. Memory T Cell and NK Cell Populations Following Challenge. At various time points following challenge, heparinized blood was collected, PBMCs isolated and stained for flow cytometric analysis. Upon ex vivo stimulation with ASF-G, the change (ASFV-G-stimulated well minus unstimulated well) in CD8+ (A) Central (Tcm) and (B) Effector memory T (Tem) cells, along with the IFNγ+ CD8+ (C) Tcm and (D) Tem cells were calculated as a percentage of the parent population. Additionally, the change (ASFV-G-stimulated well minus unstimulated well) in CD4+ (E) Central (Tcm) and (F) Effector memory T (Tem) cells, along with the IFNγ+ CD4+ (G) Tcm and (H) Tem cells were calculated as a percentage of the parent population. Finally, the change (ASFV-G-stimulated well minus unstimulated well) in CD4+CD8+ (I) Central (Tcm) and (J) Effector memory T (Tem) cells, along with the IFNγ+ CD4+CD8+ (K) Tcm and (L) Tem cells were calculated as a percentage of the parent population (M) Upon ex vivo stimulation with ASF-G, the change (ASFV-G-stimulated well minus unstimulated well) in IFNγ+ NK cells as a proportion of NK cells was also calculated. Mock-vaccinated swine shown in blue and ASFV-G-ΔI177L-vaccinated…

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  Figure 7. γδT cell and Regulatory T cell Panel Following Challenge. At various time points, heparinized blood was collected, PBMCs isolated and stained for flow cytometric analysis. (A) γδT cells (unstimulated well) as a percentage of their parent population were assessed among cells ex vivo unstimulated. (B) Change (ASFV-G-stimulated well minus unstimulated well) in IFNγ+ γδT cells as a percentage of their parent population following ex vivo stimulation with ASFV-G. Change (ASFV-G-stimulated well minus unstimulated well) in (C) Treg cells and (D) IFNγ+ Treg cells as a percentage of their parent population were also assessed following ex vivo stimulation with ASFV-G. Mock-vaccinated swine shown in blue and ASFV-G-ΔI177L-vaccinated swine shown in purple. n = 2–6 swine/treatment/time point. ** p-value < 0.01 when compared with time-matched control. † p-value < 0.05 †† p-value < 0.01 when compared against treatment-matched baseline at 0 dpi.