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

ABSTRACT African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83–100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.


Supplementary Figures
expressing TagRFP-T + and mNeonGreen + , were isolated by single cell sorting into PBMs using FACS.Following subsequent rounds of single cell isolation and limiting dilutions, three recombinant ASFV: (i) GeorgiaΔK145RΔEP153R-CD2v_mutQ96R (G∆KE_CmutQ96R), (ii) GeorgiaΔK145RΔEP153R-CD2v_mutQ96R/K108D (G∆KE_CmutQ96R/K108D) and (iii) GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D (G∆DKE_CmutQ96R/K108D) were obtained.(B) Viral DNA was extracted from 3 recombinant viruses and whole genomes were sequenced.Sequences obtained were assembled using the NGS-based whole genome variant analysis module on the SeqMan NGen.The table summarizes the total number of reads, total and percentages of assembled reads and median coverage depth for each of the three recombinant viruses.on the surface.

Production of ASFV recombinant Georgia07/1 with deletions of K145R, EP153R only or with DP148R and expressing modified CD2v proteins
To delete the EP153R gene combined with replacing wildtype EP402R with mutant versions by homologous recombination, sequences from the ASFV Georgia 2007/1 (FR682468.Recombinant viruses expressing both TagRFP-T and mNeonGreen were isolated via a combination of single cell FACS sorting and rounds of limiting dilutions [1].Full genome sequencing of recombinant ASFV using an Illumina MiSeq instrument using a 600 cycle v3 cartridge.Sequences obtained were assembled using the NGS-based whole genome variant analysis module on the SeqMan NGen (DNASTAR Lasergene).

Multistep growth curves
Purified PBMs from 2 different pigs were infected at a multiplicity (MOI) of 0.01 in triplicates.Total infectious virus titres were determined and repeated measure two-way ANOVA, with Dunnett's multiple comparisons test was performed to compare replication of recombinant and wildtype ASFV.

Western blots
Lysates from Vero cells transiently expressing wildtype or mutant CD2v or purified PBMs infected with recombinant ASFV were separated by SDS/PAGE and transferred onto Amersham Hybond P 0.45 PVDF blotting membranes (Cytiva).Blots were probed with rat anti-HA monoclonal antibody (Roche) and secondary goat anti-rat IgG HRP conjugated antibody (Invitrogen).Bound antibodies were detected by ECL Select (Amersham) and imaged on Syngene G: Box.

In vivo immunization and challenge
Animal experiments were carried out at the Pirbright Institute under UK Home Office License 7088520.Groups of 6 female Landrace-Large White cross pigs with initial weights of 17 -30kg were inoculated and challenged via the intramuscular route (IM).Group of 3 non-immune pigs were challenged in parallel to act as controls.Clinical signs were recorded daily and blood samples were collected on selected days [2].
Figure 1 shows the schedule of 4 experiments conducted.In experiment 1, one group (K) was immunised with GeorgiaΔKE-CmutQ96R.In experiment 2, one group was immunised with GeorgiaΔKE-CmutQ96R/K108D (O) and a second group with GeorgiaΔDKE-CmutQ96R/K108D (N).In experiment 3 two groups of pigs were immunised with different doses of GeorgiaΔDKE-CmutQ96R/K108D.In experiment 4, 2 groups of pigs were immunized with two higher doses GeorgiaΔDKE-CmutQ96R/K108D and culled between 7 -17 days to investigate the tissue dissemination of the virus.Viruses were back titrated to verify dosages used.
Figure S1.Scheme for construction of recombinant ASFV viruses (A) To delete ASFV EP153R gene in combination with replacing wildtype EP402R with mutant versions, sequences from ASFV Georgia 2007/1 (FR682468.2) genome were commercially synthesised and cloned in plasmids -p∆EP153R-PP30mNG-CD2v_mutQ96R (single amino acid mutation) and p∆EP153R-PP30mNG-CD2v_mutQ96R/K108D (double amino acid mutation).These plasmids contain 720bp from the left flanking region of EP153R (position 73086 -73805), the first LoxP site, a fluorescent reporter gene (mNeonGreen), controlled by ASFV VP30 promoter, the second LoxP site, followed by mutant versions of the EP402R gene expressing CD2v protein with an HA epitope tag fused at the 3' end.The CD2v mutations included either a single amino acid mutation at position 96 (Q → R) (position 74639 -74641, nucleotides CAA to CgA) or double amino acid mutations at position 96 (Q → R) (position 74639 -74641, nucleotides CAA to Cgc) and at position 108 (K → D) (position 74675 -74677, nucleotides AAA to gAt).The CD2v protein was under control of CD2v promoter (position 74281 -74353).Finally, a 609bp sequence flanked the stop codon at the right end of EP402R (position 75417 -76025).The LoxP sites is to facilitate removal of the reporter gene in the future.Purified PBMs were first infected with GeorgiaΔK145R or GeorgiaΔDP148RΔK145R, recombinant ASFV lacking gene(s) K145R and/or DP148R, then transfected with the transfer plasmid from.The parental viruses both contained a red fluorescent protein, TagRFP-T.After 36-48 post infection-transfection, recombinants

Figure S2 .
Figure S2.Surface representations of the CD2v and hCD2 immunoglobulin superfamily V-set domains.In Panel (A), mutations of CD2v that produced strong effects in the mutational screen are coloured magenta, and weak grey.In panel (B) the putative V-set domain of CD2v and that of human CD2, with the GFCC'C" β-sheets to the front, are coloured according to amino acid properties: negatively charged (D, E)red, positively charged (R, H, K)blue, polar (S, T, N, Q)yellow, and hydrophobic (A, V, I, L, M, F, Y, W)green.

Figure S4 .
Figure S4.Daily rectal temperatures of pigs.Rectal temperatures were recorded daily for pigs immunized with (A) G∆KE_CmutQ96R (Group K), (C) G∆KE_CmutQ96R/K108D (Group O), and (D, F, G, I and J) G∆DKE_CmutQ96R/K108D (Groups N, S, T, V and W).(B, E, H) The temperatures for the naïve, challenge control groups for each experiment were recorded following challenge with Georgia 2007/1 isolate.

Figure S5 .
Figure S5.Post-mortem macroscopic lesion scoring.Lesions observed during postmortems were scored and displayed as a cumulative score observed in different sections of the pig.Results for individual pigs in experiments 1 to 4 are shown in panels A to D. Lesions in the thoracic cavity (pink) include the presence of thoracic exudates as well as lesionsaffecting cardio-respiratory system.Lesions in the abdominal cavity (blue) include the presence of ascites along with the presence of lesions affecting the gastrointestinal system including the stomach, intestines, liver, and gallbladder.Lesions in the lymphoid tissues (black) include the tonsils, thymus, spleen, and various lymph nodes.The † above the individual or grouped bars denotes pig(s) that were culled before the end of the experiments and the day of cull for experiments 1 -3.In experiment 4, 3 Group V pigs were culled at 7dpi and the other 3, at 14dpi, while Group W pigs were all culled 17dpi at the end of the experiment.

Table S1 :
The mean ASFV genome copies per gram tissue (in log10) post-challenge.