A protective multiple gene-deleted African swine fever virus genotype II, Georgia 2007/1, expressing a modified non-haemadsorbing CD2v protein

Figures

• 

  Figure 1.. Experimental timeline. The timeline for four pig immunization experiments is shown. In experiment 4 shown in panel D † denotes the pigs and shows days that they were culled. In experiments 1, 2, and 3, groups of 3 non-immune pigs were challenged in parallel to the immunized pigs.

• 

  Figure 2.. Sequences and transient expression of wildtype and mutant CD2v. Panel (A) shows the structure-based alignment of the putative CD2v and human CD2 Ig1 domains. Residues with 100% identity are boxed, and numbering is according to the CD2v sequence. Mutations that showed strong effects in the mutation screen are coloured magenta and weak grey. β-strands are labelled according to the human CD2 structure (PDB: 1HNF). Asterisks denote human CD2-CD58 interface residues (PDB: 1QA9). The accession numbers for these sequences are: CD2v Georgia, YP_009927182 (NCBI), and human CD2, P06729 (UniProt). For Panel (B), Vero cells were transfected with pcDNA3.1 plasmids expressing Georgia CD2v, wildtype (WT), or mutants with indicated single or double amino acid substitutions. After 48 h, RBC were added, or the cells were fixed and stained for expression of HA tag fused downstream of the signal peptide at the N-terminus of CD2v. Representative fields demonstrating the presence or absence of RBC rosettes (HAD), as indicated, are shown. Panel (C) summarizes results from repeat experiments and different fields representing HAD relative to wildtype CD2v: +++ similar levels; ++ reduced levels; +/−…

• 

  Figure 3.. In vitro characterization of ASFV recombinant viruses expressing mutant CD2v. Panel (A) shows brightfield images of PBM cells infected with the recombinant or wildtype Georgia07/1 viruses, mNeonGreen expression in infected cells (green), and a merged image overlaying brightfield and green channels. The red arrows in GΔKE_CmutQ96R-infected cells show reduced numbers of rosettes compared to the wildtype virus. The scale bars represent 25 µm. Panel (B) shows Western blots of lysates of PBMs from two pigs infected at 0.1 MOI with GΔKE_CmutQ96R (Lane 1) and GΔDKE_CmutQ96R/K108D (Lane 3) at 4 dpi and probed to detect the C-terminal HA epitope tag. HA-specific bands were not detected in lanes with mock-infected (lane 4) or GΔKE_CmutQ96R/K108D-infected cell lysates (lane 2). This virus lacks the C-terminal HA tag. Arrows show bands of expected molecular weight for full-length glycosylated (∼80 kDa) CD2v or the C-terminal cleavage fragment. The blue arrow shows a higher MW (∼120 kDa) CD2v detected at higher levels lysates from infected PBMs from pig 2. Panel (C) shows titres of virus harvested from combined cell extracts and supernatants at different days post-infection of PBMs infected…

• 

  Figure 4.. Clinical scores of pigs. Cumulative clinical scores per day for pigs immunized with (A) GΔKE_CmutQ96R (Group K), (C) GΔKE_CmutQ96R/K108D (Group O), and (D, F, G, I, and J) GΔDKE_CmutQ96R/K108D (Groups N, S, T, V, and W). (B, E, H) The cumulative scores per day for the naïve challenge control groups for each experiment were recorded following challenge with Georgia 2007/1 isolate.

• 

  Figure 5.. Level of viremia in pigs. Virus titres present in whole blood collected at different days post-immunization or challenge are shown for individual pigs as indicated. Pigs were immunized with (A) GΔKE_CmutQ96R (Group K), (C) GΔKE_CmutQ96R/K108D (Group O), and (D, F, G, I, and J) GΔDKE_CmutQ96R/K108D (Groups N, S, T, V, and W). Panels B, E, H show virus titres detected in blood from control non-immune pigs challenged with Georgia 2007/1 isolate. The dashed line on the y-axis represents the cut-off below which accurate measurement of infectious virus per mL blood was not obtained.

• 

  Figure 6.. Immune responses of immunized pigs. (A) Antibody responses are shown against ASFV VP72 protein using a blocking ELISA, of pigs immunized with GΔKE_CmutQ96R (Group K), GΔKE_CmutQ96R/K108D (Group O), and GΔDKE_CmutQ96R/K108D (Groups N, S, and T) on different days after immunization and challenge (x-axis). Results are presented as % blocking, where values >50% blocking are positive. (B) Levels of CD2v antibody in the serum of immunized pigs, pre-challenge (Groups K, O, and N) detected by ELISA are shown. (C) Shows antibody levels against ASFV P30 protein measured by ELISA in the same pigs as in Panel B. The numbers of IFN-γ producing cells detected in PBMCs of immunized pigs (Groups K, O, N, and S) were measured via ELISpot assays. PBMCs were stimulated with either (D) Georgia 2007/1 isolate or (E) a genotype I, Benin 97/1 isolate. Results are presented as mean number of IFN-γ producing cells per million PBMC of individual pigs.