(D-E) Percentages of IFN- and/or TNF-producing CD4+CD8+, CD8+ and CD4+ T cells (D) or myeloid cells (CD3-CD172a+) (E) in PBMC from unvaccinated (n = 7C8) and vaccinated (n = 10) pigs after mock or BA71CD2 stimulation. BGLAP and unique DE genes recognized in the unvaccinated and vaccinated groups. (C) List of representative GO terms enriched in DE genes from BA71CD2-stimulated PBMC from unvaccinated and vaccinated pigs. The size of the dots represents the number of DE genes associated with the GO term, and the color indicates the unfavorable log10 value of the false discovery rate (FDR). (D) Heatmap depicting normalized RNA-seq-derived log2CPM values of representative DE genes (extension of Fig 2C).(DOCX) ppat.1010931.s004.docx (342K) GUID:?1ACDA31C-AB4E-4B9D-82C5-EFC2F7EFF497 S5 Fig: Pigs vaccinated with 106 pfu of BA71CD2 from which submandibular lymph node cells were used do not show major clinical signs. (A) Rectal temperatures from individual animals in each group. (B) Clinical scores measured throughout the experiment. Each row represents an animal within the group.(DOCX) ppat.1010931.s005.docx (250K) GUID:?BED60069-93E4-4715-847C-42CAEA55702A S6 Fig: IFN ELISpot and analysis of scRNA-seq data in submandibular LN cells. (A) Pigs were vaccinated with Zibotentan (ZD4054) 106 pfu of BA71CD2 (n = 6) and three weeks later levels of ASFV-specific cells in submandibular LN were measured by IFN ELISpot using BA71CD2 as stimulus. Unvaccinated pigs (n = 6) were used as unfavorable control. (B) Quantity of genes differentially expressed between the unvaccinated and the vaccinated pig in each cluster. (C) Quantity of cells in each cluster recognized by scRNA-seq. (D) Pearsons correlation between the quantity of DE genes and the number Zibotentan (ZD4054) of cells in each cluster. Each dot represents a cluster, and the value of the total quantity of cells is the result of the addition of cells from each sample.(DOCX) ppat.1010931.s006.docx (151K) GUID:?CAD439E1-2106-48BC-9CD1-50EB8E09D272 S7 Fig: Gene ontology enrichment analysis of scRNA-seq-derived clusters shows the activation of innate immunity in cells from your vaccinated pig. List of representative GO terms enriched for each cluster in DE genes from BA71CD2-stimulated submandibular LN cells from your vaccinated pig. The size of the dots represents the number of DE genes associated with the GO term, and the color indicates the unfavorable log10 value of the false discovery rate (FDR).(DOCX) ppat.1010931.s007.docx (237K) GUID:?C09EEF43-5687-479A-929C-4FD5D7CB45F8 S8 Fig: Violin plots depicting scRNA-seq-derived expression levels of representative ISG in each cluster. Asterisks denote differential expression: ** p value adjusted 0.01, *** p value adjusted 0.001.(DOCX) ppat.1010931.s008.docx (411K) GUID:?E89D5F01-247F-400E-B76A-0F193282CD2D S9 Fig: A cytotoxic recall response in BA71CD2-vaccinated animals is usually revealed by scRNA-seq analysis of LN cells but not by RNA-seq analysis of PBMC stimulated for any shorter period. (A) Violin plots showing expression levels from scRNA-seq-derived data of and profilin 1 (ASFV-specific activation. (B) RNA-seq-derived expression levels as log2CPM values of representative cytotoxic markers in PBMC after 10 hours of ASFV-specific activation.(DOCX) ppat.1010931.s009.docx (148K) GUID:?9DE02E48-A500-4D55-8CA0-3D1F7D5F9C71 S1 Table: Pigs vaccinated with BA71CD2 do not show viral DNA neither in serum nor in whole blood. The table shows genomic comparative copies (GEC)/ml assessed by qPCR assay targeting the ASFV PK gene. Samples obtained at day 0, 14 and 21 postvaccination from pigs receiving 106 pfu of BA71CD2 were used. Undet: Undetectable.(XLSX) ppat.1010931.s010.xlsx (12K) GUID:?649F5053-5E6F-4B9D-9608-8AE6674892DC S2 Table: BA71CD2-vaccinated animals do not show BA71CD2 replication in sera after Georgia2007/1 challenge. The table shows Ct values obtained using Taqman primer/probe units targeting LacI, only present in Zibotentan (ZD4054) the BA71CD2 genome. Serum samples from BA71CD2-vaccinated pigs at days 10 and 13 after Georgia2007/1 challenge were tested. Positive controls of the assay were: 1) nasal swabs samples from BA71CD2-vaccinated pigs at days 7 and 14 postvaccination, and 2) serum from a na?ve pig where BA71CD2 was added.(XLSX) ppat.1010931.s011.xlsx (14K) GUID:?85982E19-B002-4EB8-AD43-A4E937FADA8F S3 Table: GO terms enriched in RNA-seq-derived DE genes from PBMC from unvaccinated animals stimulated with BA71CD2. (XLSX) ppat.1010931.s012.xlsx (27K) GUID:?5C690963-2D2C-4558-800A-802357DDBCD9 S4 Table: GO terms enriched in RNA-seq-derived DE genes from PBMC from vaccinated animals stimulated with BA71CD2. (XLSX) ppat.1010931.s013.xlsx (64K) GUID:?EDDFCE54-DD24-40D0-AFC6-5AC40B44770E S5 Table: Statistical analysis results from microfluidic quantitative PCR assay data. (XLSX) ppat.1010931.s014.xlsx (28K) GUID:?EB3BA79A-AAAA-45B5-BC20-20DEC952A4A7 S6 Table: BA71CD2-vaccinated animals show low or undetectable levels of BA71CD2 in submandibular lymph node cells. Submandibular.