Fig 1.
Immunoblot showing PrPSc levels in brain and spinal cord of cervid KI mice.
(A) Schematic diagram showing the study design for vaccination and (B), the time points of feces and urine collection. Ddi vaccinated group (n = 8), CPG control group (n = 8) and Mmo vaccinated group (n = 8) were inoculated intraperitoneally with 1% brain homogenate (BH) of mouse-adapted reindeer CWD. Samples were digested with 50 µg/ml PK and loaded onto SDS-PAGE as labelled at the top of the blot, with (C) showing PrPSc levels in brain and (E) in spinal cord. Membranes were probed with the anti-PrP mAb 4H1 (1:500). Naïve depicts brain homogenate of non-inoculated KI mice. The PrPSc levels in brain (C) and spinal cord (E) were densitometrically quantified for the three vaccination groups ((D) brain, (E) spinal cord), the graphs in (G), (H) and (I) compare brain signals to spinal cord for the three vaccination groups (Ddi, CpG and Mmo). SB stands for sample buffer and denotes a free lane. Graphs were generated by GraphPad Prism (version 10). Statistical analysis was done using paired-t test. ns = not significant, * p-value = 0.0440 and ** p-value = 0.0038.
Fig 2.
Humoral immune response of Ddi and Mmo vaccinated KI mice to immunogens.
ELISA plates were coated with Ddi immunogen (A, B) or Mmo immunogen (C, D). Sera were isolated after the 4th dose of vaccination (n = 8 per vaccination group.) All sera were diluted at 1:100, CpG mice sera were used as a negative control and GAM was used as secondary antibody at 1:4,000. One-way ANOVA was done followed by Tukey’s test. Statistical significance = **** p-value <0.0001, *** p-value = 0.0007. (E) End-point ELISA antibody dilutions are shown for the two vaccinated groups. Each mouse antibody titer was determined by endpoint dilution and is represented by a data point. The y-axis represents the serum fold dilution, and the x-axis represents the treatment groups. The dashed horizontal line represents the cut-off, which is the average OD of CpG sera + 5 x SD.
Table 1.
Difference in the CWD shedding in feces between KI vaccinated and control groups at different time points.
Fig 3.
RT-QuIC data for the PMCA products showing the difference in the CWD seeding activity between KI vaccinated and control groups at 350 dpi.
Ten % pooled fecal homogenate from all groups (two cages per group) was extracted using IOME followed by three rounds of PMCA reactions using KI wildtype cervid PrPC substrate seeded with 1:10 dilution of the fecal homogenate, in duplicates for all vaccinated and control groups. Positive control for the PMCA was naïve feces spiked with mouse-adapted CWD reindeer; negative control was naïve feces. Samples and controls were subjected to 3 rounds of PMCA. The PMCA products were analyzed using RT-QuIC assay at 10-4 to 10-7 dilution. (A) Representative RT-QuIC graphs showing the seeding activity in feces from KI mice vaccinated subcutaneous with Ddi or Mmo, or injected with CPG as control, followed by intraperitoneally inoculation with mouse-adapted CWD. Samples were considered positive when 2 out of 4 wells crossed the threshold, defined as the average relative fluorescent unit (RFU) of the negative control group plus five times its standard deviation. The y-axis represents the RFU, and the x-axis represents the time in hours (hr). (B) Summary of the RT-QuIC analysis of prion seeding in the 350 dpi pooled feces of KI mice. The heat map indicates the percentage of positive RT-QuIC replicates out of the total of four replicates analyzed. The scale ranges from 0 (all replicates were negative) to 100 (all replicates were positive). Graphs were generated using GraphPad Prism (version 10).
Fig 4.
Seeding activity in individual fecal samples of KI vaccinated and control group at 200 dpi.
Three rounds of PMCA reactions were done using KI substrate seeded with individual 10-1 dilution of 200 dpi 10% fecal homogenate samples after IOME, in duplicates for all vaccinated and control groups. Positive control for the PMCA was naïve feces spiked 1:100 with mouse-adapted CWD and negative control was naïve feces subjected to three cycles of PMCA reaction. The PMCA products were analyzed using RT-QuIC assay at 10-1 dilution. (A-C), representative RT-QuIC graphs showing the seeding activity in feces from KI mice vaccinated with Ddi or Mmo and CPG control group. Samples were considered positive when 2 out of 4 wells crossed the threshold, which defined as the average RFU of the negative control group plus five times its standard deviation. The y-axis represents the RFU, and the x-axis represents the time in hours (hr). (D), Chi square test, (E), time to threshold, (F), maximum of range and (G), area under curve. Graphs were generated using GraphPad Prism (version 10). Statistical analysis was done using Chi-square test (D) and **** p-value < 0.0001, or One-way ANOVA followed by a Tukey’s multiple comparison. For maximum of range (F) ** p-value = 0.0046, for area under curve (G) ** p-value = 0.0034. Data in (E) were not significant. ns: not significant.
Fig 5.
Representative Western blots showing PrPSc levels in third round of PMCA reactions seeded with urine of Ddi, Mmo vaccinated or CPG control KI mice in duplicates at different time points.
(A), 150 and 250 dpi and (B), 450 dpi samples were subjected to IOME followed by three rounds of PMCA. Negative control for the PMCA is naïve urine and positive control for the PMCA was naïve urine spiked (1:100) with brain homogenate of mouse-adapted CWD subjected to PMCA reaction. PMCA products were digested for 1 hour with 37 µg/ml PK at 37°C and were probed with anti-PrP mAb 4H11 (1:500). NBH: normal brain homogenate, used as negative PMCA control. (C-E), summary of the RT-QuIC analysis of prion seeding in the pooled urine of KI mice at (C) 150 dpi, (D) 250 dpi, and (E) 450 dpi. The heat map indicates the percentage of positive RT-QuIC replicates out of the total of four replicates analyzed. The scale ranges from 0 (all replicates were negative) to 100 (all replicates were positive). Graphs were generated using GraphPad Prism (version 10).
Fig 6.
Anti-PrP antibodies do not interfere with the IPR readout method.
Ten % fecal homogenates from naïve KI mice were treated with pre-immune mouse sera, post-immune mouse sera, mAb 4H11, or pAb 142 antibodies for 90 minutes followed by spiking 1:100 with brain homogenate of mouse adapted CWD, or without spiking (naïve), and all the samples were subjected to full IPR method. (A) Immunoblot showing PrPSc of PMCA reactions (in duplicate) pre-incubated with pre-immune sera (mouse Mmo4 and CpG7) or post-immune sera (mouse Ddi1 and Ddi2), either CWD spiked or not (naïve). Right shows spiked only reactions and spiked reactions pre-treated with mAb 4H11 or pAb 142 antibodies. SB, sample buffer. (B) RT-QuIC data showing the seeding activity in pre-treated and spiked samples from A. In both A and B, there was no detectable difference between anti-body treated and not treated samples.