Capsid-Specific Antibody Responses of Domestic Pigs Immunized with Low-Virulent African Swine Fever Virus

Figures

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  Figure 1. Schematic representation of experiment involving large, white inbred Babraham pigs that were immunized with low-virulent OURT88/3 (OT3) before challenge with virulent OURT88/1 (OT1), as published previously [50]. Partial protection from challenge with related ASFV was observed. (†) indicates that the animal was euthanized before the conclusion of the experiment because it had reached its humane endpoint.

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  Figure 2. Schematic representation of experiment involving outbred domestic pigs that were immunized with low-virulent OURT88/3 and challenged with the related virulent OURT88/1. Survivors were then challenged further with the heterologous Georgia 2007/1 [50]. Partial protection from challenge with homologous and heterologous virulent ASFV was observed. (†) indicates that the animal was euthanized before the conclusion of the experiment because it had reached its humane endpoint.

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  Figure 3. Schematic of modified LACA developed to study ASFV antigen-specific antibody responses of immunized and challenged animals. (a) Sera samples are incubated with cell lysates containing a mixture of specific Nluc-tagged ASFV proteins of interest and irrelevant proteins. (b) Plates are coated with protein A and blocked to prevent unspecific binding. (c) Nluc-tagged antigens bound to their respective antibodies are then captured on protein A, and (d) the presence of antigen-binding antibodies is indirectly measured through the Nluc activity. Irrelevant antibodies bound to protein A are not detected since these do not bind the Nluc-tagged antigens.

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  Figure 4. Longitudinal antibody responses of inbred Babraham animals to ASFV recombinant proteins (a,b) CP204L/p30, (c,d) B646L/p72 co-expressed with chaperone B602L, (e,f) B646L/p72, and (g,h) B602L chaperone detected with antigen-specific LACAs at selected days post-immunization (dpi). (a,c,e,g) Antigen-specific antibody kinetics of each animal are plotted. (b,d,f,h) Antibody responses consolidated as a group at each relevant time point are plotted. Only time points where there were four data sets or more were included in this analysis. Lines indicate the mean. Each data point corresponds to a single animal. The point of challenge with virulent OURT88/1 (OT1, 18 dpi) is denoted by the arrowhead in each graph. P/N Ratio: ratio of luciferase activity of each sample to the luciferase activity of the negative control. Dashed line indicates the cutoff determined from the mean and 3x standard deviation of all negative sera samples in each experiment. Blue, Mock: mock control animals immunized with PBS, Pink, NP: OURT88/3 immunized animals that were not protected from OURT88/1, Green, R: OURT88/3 immunized animals that recovered from OURT88/1. * p < 0.05, ** p < 0.01, *** p < 0.005, mixed…

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  Figure 5. Longitudinal antibody responses of inbred Babraham pigs to ASFV recombinant capsid proteins (a,b) D117L/p17, (c,d) B438L/p49, (e,f) M1249L, and (g,h) E120R/p14.5 detected with antigen-specific LACAs at selected dpi. (a,c,e,g) Antigen-specific antibody kinetics of each animal are plotted. (b,d,f,h) Antibody responses consolidated as a group at each relevant time point are plotted. Only time points where there were four data points or more were included in this analysis. Lines indicate the mean. Each data point corresponds to a single animal. The point of challenge with virulent OURT88/1 (OT1, 18 dpi) is denoted by the arrowhead in each graph. P/N Ratio: ratio of luciferase activity of each sample to the luciferase activity of the negative control. Dashed line indicates the cutoff determined from the mean and 3 × standard deviation of all negative sera samples in each experiment. Blue, Mock: mock control animals immunized with PBS, Pink, NP: OURT88/3 immunized animals that were not protected from OURT88/1, Green, R: OURT88/3 immunized animals that recovered from OURT88/1. * p < 0.05, ** p < 0.01, mixed effects model.

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  Figure 6. Heatmaps of the virological and immunological parameters of inbred Babraham animals. (a,b) Data reported previously [50], (a) viremia, (b) anti-ASFV antibody titer as determined by fixed-cell ELISA on BA71V-infected Vero cells [50], and (c–i) recombinant ASFV protein-specific LACAs targeting (c) CP204L/p30, (d) B646L/p72 co-expressed with B602L chaperone, (e) B646L/p72, (f) B602L chaperone, (g) D117L/p17, (h) M1249L, and (i) E120R/p14.5. Data plotted as (a) Log10 genome copy numbers/mL, (b) Log2 antibody titer, and (c–i) Log10 of P/N ratio. Each row denotes the responses of a single animal. The negative cutoff for each protein-specific LACA was determined from the mean and 3x standard deviation of all negative sera samples in each experiment. Animal numbers are indicated on the y-axis and the time post-immunization is denoted on the x-axis. Crosses indicate samples that were not available for analysis. Arrowheads denote the immunization and ASFV challenge time points. Blue, Mock: mock control animals immunized with PBS, Pink, NP: OURT88/3 immunized animals that were not protected from OURT88/1, Green, R: OURT88/3 immunized animals that recovered from OURT88/1.

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  Figure 7. Longitudinal antibody responses of outbred domestic pigs to ASFV recombinant proteins (a) CP204L/p30, (b) B646L/p72 co-expressed with chaperone B602L, (c) B646L/p72, (d) B602L chaperone, (e) D117L/p17, (f) B438L/p49, (g) M1249L, and (h) E120R/p14.5 detected with protein-specific LACAs at selected dpi. Antigen-specific antibody kinetics of each animal are plotted. Each data point corresponds to a single animal. The point of challenge with virulent OURT88/1 (OT1, 21 dpi) and virulent Georgia 2007/1 (G7, 42 dpi) is denoted by the black and orange arrowheads, respectively. P/N Ratio: ratio of luciferase activity of each sample to the luciferase activity of the negative control. Dashed line indicates the cutoff determined from the mean and 3x standard deviation of all negative sera samples in each experiment. Purple: OURT88/3 immunized animal that suffered from chronic ASF, Pink: OURT88/3 immunized animal that was not protected from OURT88/1, Green: OURT88/3 immunized animals that recovered from OURT88/1, but were not protected from Georgia 2007/1, Black: OURT88/3 immunized animals that recovered from OURT88/1 and Georgia 2007/1.

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  Figure 8. Heatmaps of the virological and immunological parameters of outbred domestic animals. (a,b) Data collected in previous study [50], (a) viremia, (b) anti-ASFV antibody titer as determined by fixed-cell ELISA on BA71V infected Vero cells [50], and (c–i) recombinant ASFV protein-specific LACAs targeting (c) CP204L/p30, (d) B646L/p72 co-expressed with B602L chaperone, (e) B646L/p72, (f) B602L chaperone, (g) D117L/p17, (h) M1249L, and (i) E120R/p14.5. Data plotted as (a) Log10 genome copy numbers/mL, (b) Log2 antibody titer, and (c–i) Log10 of P/N ratio. Each row denotes the responses of a single animal. The negative cutoff for each protein-specific LACA was determined from the mean and 3x standard deviation of all negative sera samples in each experiment. Animal numbers are indicated on the y-axis and the time post-immunization is denoted on the x-axis. Crosses indicate samples that were not available for analysis. Arrowheads denote the immunization and ASFV challenge time points. Purple: OURT88/3 immunized animals that suffered from chronic ASF; Pink: OURT88/3 immunized animals that were not protected from OURT88/1; Green: OURT88/3 immunized animals that recovered from OURT88/1 but…