Live-attenuated foot-and-mouth disease virus vaccine engineered by codon deoptimization induces a strong protective immune response in cattle

Figures

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  Fig. 1. Clinical safety evaluation of FMDV A24-P2/P3Deopt. Six-month-old Holstein heifers of approximately 450 lb were intradermolingually inoculated with a FMDV A24 P2/P3 codon deoptimized live attenuated virus (106 or 107 pfu) or FMDV A24 wild type (2.36 × 104 pfu). Cattle were assessed for clinical score (grey bars) on days 3, 5 and 7 post-inoculation. EDTA-treated blood was assessed for signs of lymphopenia daily (orange line) and rectal body temperature was assessed daily (periods of fever shown in tan shaded boxes). n = 4 cattle/time point/treatment group.

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  Fig. 2. Viremia and virus shedding in A24-P2/P3Deopt in vivo safety study. Six-month-old Holstein heifers of approximately 450 lb were intradermolingually inoculated with a FMDV A24-P2/P3 codon deoptimized live attenuated virus (106 or 107 pfu) or FMDV A24 wild type (2.36 × 104 pfu). Serum and nasal secretion samples were collected daily for a week to assess viremia and virus shedding, respectively. Viremia analyzed by virus isolation is expressed as pfu/ml (solid red line). Viremia analyzed by real-time RT-qPCR is expressed as genome copy particles/ml of serum (GCP/ml) (dashed red line). Virus in nasal secretion analyzed by virus isolation is expressed as pfu/ml (solid blue line), while that analyzed by real-time RT-qPCR is expressed as GCP/ml (dashed blue line). Lower limits of detection are represented by a black dashed line for RT-qPCR and a solid black line for virus isolation. n = 4 cattle/time point/treatment group.

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  Fig. 3. A24-P2/P3Deopt induces strong neutralizing antibody response in cattle. Six-month-old Holstein heifers of approximately 450 lb were intradermolingually inoculated with a FMDV A24-P2/P3 codon deoptimized live attenuated virus (106 or 107 pfu) or FMDV A24 wild type (2.36 x 104 pfu). A At indicated time-points serum samples were collected to test for neutralizing antibodies (lower limit of detection represented by solid black line, while the protective titer is represented by a gray dashed line) or B antibodies against FMDV non-structural proteins (PrioCheck®ELISA). Results expressed as number of positive samples out of the total number of animals per group. n = 4 cattle/time point/treatment group. Neutralizing antibody titers assessed by a mixed-effects two-way ANOVA by treatment group and time point, followed by pairwise comparisons at each time point by treatment group with Tukey’s multiple comparison correction. Treatment group (P < 0.0001), time point (P = 0.0001) and the interaction effect (P < 0.0001) were all found to be significant. Statistically significant pairwise comparisons against FMDV A24 WT are represented in the figure by asterices and the color of the FMDV…

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  Fig. 4. A24-P2/P3Deopt vaccine efficacy study design. A Six-month-old Holstein calves of approximately 450 lb were subcutaneously inoculated with an FMDV A24-P2/P3 codon deoptimized live attenuated virus either once (105 pfu) or twice (104, 105 and 106 pfu) prior to intradermolingual challenge with 104 BID50 (2.36 × 104 pfu) FMDV A24 wild type. Samples were collected at indicated time points after vaccine inoculation (dpi) and after challenge (dpc). B Viremia and viral shedding of live modified attenuated A24-P2/P3Deopt after vaccination (days post-inoculation). Viremia analyzed by virus isolation is expressed as pfu/ml of serum (solid red line). Viremia analyzed by real-time RT-qPCR is expressed as GCP/ml (dashed red line). Virus in nasal secretion analyzed by virus isolation is expressed as pfu/ml (solid blue line), while that analyzed by real-time RT-qPCR is expressed as GCP/ml (dashed blue line). Lower limits of detection are represented by a black dashed line for RT-qPCR and a solid black line for virus isolation. n = 4 cattle/time point/treatment group.

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  Fig. 5. A24-P2/P3Deopt two dose vaccine regime protects cattle against homologous challenge. Six-month-old Holstein calves of approximately 450 lb were subcutaneously vaccinated with an FMDV A24-P2/P3 codon deoptimized live attenuated virus either once (105 pfu) or twice (106, 105 or 104 pfu) prior to intradermolingual challenge with 104 BID50 (2.36 × 104 pfu) FMDV A24 wild type. Cattle were assessed for clinical score (grey bars) on days 3, 5 and 7 post-challenge and rectal body temperature (periods of fever shown in tan shaded boxes), and percentage of lymphocytes on EDTA-treated blood (orange line) was assessed daily n = 4 cattle/time point/treatment group.

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  Fig. 6. Viremia and virus shedding in A24-P2/P3Deopt efficacy study. Six-month-old Holstein heifers of approximately 450 lb were vaccinated with indicated doses of FMDV A24-P2/P3 codon deoptimized live attenuated virus and boosted at 14 dpi for 2-dose groups. At 28 dpi all animals were challenged with 104 BID50 (2.36 × 104 pfu) of homologous A24 WT virus intradermolingually (IDL). Serum and nasal secretion samples were collected daily for a week after challenge to assess viremia and virus shedding. Viremia analyzed by virus isolation is expressed as pfu/ml (solid red line). Viremia analyzed by real-time RT-qPCR is expressed as GCP/ml (dashed red line). Virus in nasal secretion analyzed by virus isolation is expressed as pfu/ml (solid blue line), while that analyzed by real-time RT-qPCR is expressed as GCP/ml (dashed blue line). Lower limits of detection are represented by a black dashed line for RT-qPCR and a solid black line for virus isolation. n = 4 cattle/time point/treatment group.

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  Fig. 7. Adaptive immunity induced by A24-P2/P3Deopt vaccination. Six-month-old Holstein calves of approximately 450 lb were subcutaneously inoculated with an FMDV A24-P2/P3 codon deoptimized live attenuated virus either once (105 pfu) or twice (106, 105 or 104 pfu) prior to intradermolingual challenge with 104 BID50 (2.36 × 104 pfu) FMDV A24 wild type at 28 dpi. At indicated time points before and after challenge, serum samples were collected to test for A neutralizing antibodies (lower limit of detection represented by solid black line, while the protective titer is represented by a gray dashed line) or B antibodies against FMDV non-structural proteins (PrioCheck®ELISA). Number of positive samples out of the total number of animals per group. n = 4 cattle/time point/treatment group. Neutralizing antibody titers assessed by a mixed-effects two-way ANOVA by treatment group and time point, followed by pairwise comparisons at each time point by treatment group with Tukey’s multiple comparison correction. Treatment group (P < 0.0001), time point (P = 0.0016) and the interaction effect (P = 0.0047) were all found to be significant. α: Pairwise comparison at 4 dpi reveals that titers among…

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  Fig. 8. Vaccination with A24-P2P3Deopt elicits a broadly-neutralizing antibody response in cattle. Six-month-old Holstein calves of approximately 450 lb were subcutaneously inoculated with an FMDV A24-P2/P3 codon deoptimized live attenuated virus twice at doses of 106 or 105 pfu. A Serum samples were collected at specified time points after inoculation to measure neutralizing antibodies against various serotype A strains of FMDV, as well as Asia1 and O1Manisa (data not shown). The results are presented as r1 values. The dotted line at r1 = 0.3 represents the ratio generally considered to be protective. B Phylogenetic analysis of the P1 region sequence identity of the tested strains compared to A24Cruzeiro. n = 4 cattle/time point/treatment group.