Deletion of B125R increases protection induced by a genotype II African swine fever vaccine candidate

Figures

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  Fig. 1. Characteristics of ASFV B125R protein. a Amino acid sequences of B125R from 19 ASFV isolates were compared using Clustal Omega, on MegAlign Pro (DNASTAR Lasergene 17). The ASFV genotypes are given beside the names of the isolates in brackets. Amino acid residues that differ are shown in bold and highlighted in a different colour. b, c Vero cells were transfected with a plasmid expressing B125R fused to an HA epitope tag at the C-terminus. The cells were fixed and permeabilised then immunostained with anti-HA antibody or pre-challenge pig sera from immunised pigs. Following secondary antibody staining with goat anti-mouse Alexa Fluor 568 or goat anti-pig Alexa Fluor 488, cells were counterstained with DAPI. b indicates which of the pre-challenge sera from pigs immunised with GeorgiaΔMGF, BeninΔMGF, OURT88/3, or BeninΔDP148R reacted with the transiently expressed B125R protein. A negative symbol – indicates no staining of B125R protein was detected, W+ indicates that low levels of B125R staining by antibodies were detected and + indicates that strong B125R staining by antibodies was detected in sera tested. An example of a vaccinated pig serum binding to transiently expressed B125R…

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  Fig. 2. The construction of recombinant ASFV virus—GeorgiaΔBDKE-CmutQ96R/K108D. a Before deleting B125R from the parental virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D (here designated GeorgiaΔBDKE-CmutQ96R/K108D), the existing fluorescent reporter gene markers flanked by loxP (TagRFP-T, mNeonGreen were removed (top panel). PBMs were infected with the parental virus and then transfected with a plasmid expressing Cre recombinase. After two days, cells not expressing any fluorescent markers were bulk sorted via FACS into complete RPMI and these cells were subjected to two rounds of limiting dilutions. The final selected virus, GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D-NoFP, which was non-haemadsorbing and non-fluorescent, but still containing the GUS marker was grown and titrated before being used as template to delete the B125R gene (bottom panel). b Using the newly produced, GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D-NoFP as parental virus, the B125R gene was deleted from nucleotides 106,585–106,886 (ASFV Georgia 2007/1: FR682468.2). The transfer plasmid, pΔB125R-tdT, contains left and right flanking regions of the B125R gene, either side of the fluorescent marker…

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  Fig. 3. Multistep growth curve of recombinant viruses compared to Georgia 2007/1. Purified PBMs were infected with the following viruses: (i) Georgia 2007/1, (ii) GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D (GΔDKE-CmutQ96R/K108D), the previous vaccine virus, (iii) GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D- NoFP (GΔDKE-CmutQ96R/K108D-NoFP), the previous vaccine virus after removing the fluorescent reporter markers, TagRFP-T and mNeonGreen, and (iv) GeorgiaΔB125RΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D (GΔBDKE-CmutQ96R/K108D), the current vaccine candidate with B125R gene deleted, at MOI 0.01 in triplicates. Viruses were harvested from both cells and supernatants at different time points and titrated on PBMs in quadruplicates. Multistep growth curves of the gene-deleted viruses and wild-type Georgia 2007/1 are shown over the course of 5 days, where Day 0 represents the inoculum (x-axis). Virus titres are shown on a log 10 scale on the y-axis as TCID50/mL. Two-way ANOVA with Dunnett’s multiple comparisons test was performed to evaluate the differences between the recombinant viruses and the wildtype virus. Significant differences are represented by asterisk where * is p < 0.05; the…

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  Fig. 4. Experimental timeline. The timeline for the 2 pig immunisation experiments is shown. Groups X, Y and D were primed and boosted with GeorgiaΔBDKE-CmutQ96R/K108D at different doses, 103, 104, and 102 TCID50 in 1 mL PBS or L2-RPMI intramuscularly, respectively. Pigs in Group C were immunised with 104 TCID50 without a booster dose. Two groups of non-immune pigs (Groups AA and F) were challenged in parallel to the immunised pigs (Groups X, Y, D, and C). In experiment 2, pigs in Group E were immunised at 104.5 TCID50 and were culled at 23 dpi, where † denotes the culling of Group E.

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  Fig. 5. Daily temperatures of pigs. Rectal temperatures were recorded daily for pigs immunised and boosted with GΔBDKE-CmutQ96R/K108D at different doses a 103 (Group X). b 104 (Group Y). c 102 (Group D). d Pigs immunised with 104 without a booster dose (Group C). The temperatures for the non-immune, challenge control groups were recorded following challenge with Georgia 2007/1 isolate, for (e) experiment 1 and f experiment 2. g pigs immunised with 104.5 TCID50 dose (Group E).

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  Fig. 6. Daily clinical scores of pigs. Cumulative clinical scores based on clinical signs observed daily for pigs immunised and boosted with GΔBDKE-CmutQ96R/K108D at different doses a 103 (Group X), b 104 (Group Y), and c 102 (Group D). d Pigs immunised (104) without a booster dose (Group C) and The clinical scores for the non-immune, challenge control groups were recorded following challenge with Georgia 2007/1 isolate, for (e) experiment 1 and (f) experiment 2. g Pigs immunised with 104.5 TCID50 dose (Group E).

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  Fig. 7. Post-mortem macroscopic lesion scoring. Lesions observed were scored and displayed as a cumulative score observed in different organs of the pig (different coloured bars). Lesions in the thoracic cavity include the presence of thoracic exudates as well as lesions affecting cardio-respiratory system (pink bar). Lesions in the abdominal cavity include the presence of ascites along with the presence of lesions affecting the gastrointestinal system including the stomach, intestines, liver and gallbladder (blue bar). Lesions in lymphoid tissues including tonsils, thymus, spleen and several lymph nodes are depicted by the black bar. Results for individual pigs in different groups as labelled on the x-axis are shown in (a) for experiment 1; and (b) for experiment 2. † above the individual bars or groups denotes the days on which the individual pigs or groups of pigs were culled. Days post-immunisation are indicated as dpi and days post-challenge as DPC.

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  Fig. 8. Levels of ASFV genome in whole blood from pigs. Viral genome copies per mL blood were determined by qPCR for pigs immunised and boosted with GΔBDKE-CmutQ96R/K108D at different doses a 103 (Group X), b 104 (Group Y), and c 102 (Group D), d pigs immunised (104) without a booster dose (Group C). Viremia for the non-immune, challenge control groups were measured following challenge with Georgia 2007/1 isolate, for e experiment 1 and f experiment 2. The cut-off for accurate detection is 102 ASFV genome copies/mL; below this, the exact genome copy cannot be determined. Any value below this indicates the presence of ASFV genome but the exact titres cannot be determined. g Pigs immunised with 104.5 TCID50 dose (Group E).

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  Fig. 9. Levels of infectious ASFV in whole blood from pigs. Levels of viremia were determined by titrating virus in PBMs from the whole blood of pigs immunised and boosted with GΔBDKE-CmutQ96R/K108D at different doses a 103 (Group X), b 104 (Group Y), and c 102 (Group D), d pigs immunised (104)without a booster dose (Group C) and g pigs immunised with 104.5 TCID50 dose (Group E). Viremia for the non-immune, challenge control groups was measured following challenge with Georgia 2007/1 isolate. Results for the control Group AA in experiment 1 are shown in (e) and Group F for experiment 2 in (f). The dashed line on the y-axis represents the cut-off below which accurate measurement of infectious virus per mL blood was not obtained. Any value below this indicates the isolation of infectious ASFV but the exact titres cannot be determined. Results for Group E were below this level (g).

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  Fig. 10. ASFV-specific immune responses following immunisation of pigs with GeorgiaΔBDKE-CmutQ96R/K108D. a The numbers of IFN-γ producing cells in PBMCs collected pre-immunisation, pre-boost or pre-challenge (x-axis) and stimulated with Georgia 2007/1 isolate were measured by ELISpot assays. Pigs in groups D, X, and Y were immunised and boosted with 102, 103, and 104 TCID50 GΔBDKE-CmutQ96R/K108D, while Group C was only primed with 104 TCID50 GΔBDKE-CmutQ96R/K108D. Each dot/symbol represents the mean frequencies of IFN-γ producing cells per million PBMCs from an individual pig in each group. The standard error mean (SEM) shown is for the average mean frequencies per group. Two-way repeated measure ANOVA with Tukey’s multiple comparison test was performed to find differences between the different groups, where * denotes p < 0.05. b Antibody responses of pigs were measured on different days pre and post-immunisation and challenge using a blocking ELISA against ASFV VP72 protein. Results are presented as percentage of blocking where values above 50% blocking were considered as positive antibody responses, while anything below 40% was considered as negative. Samples with blocking between…

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  Fig. 11. Shedding of ASFV in swabs from pigs. Viral DNA was extracted from swabs from a nasal, b oral and, c rectal cavities of pigs in Group E on different days until 23 dpi. These pigs were immunised with 104.5 TCID50 GΔBDKE-CmutQ96R/K108D. As a comparison, d nasal, e oral, and f rectal swabs show results from swabs collected from the non-immune, challenge control pigs (Group F) from experiment 2. Genome copies per/ml log 10 are shown on the y-axis. The x-axis shows days post-immunisation or days post-challenge when samples were collected.