Fig 1.
Phylogenetic diversity of PmSLP mature protein sequences from published isolates of P. multocida including the associate capsule type, geographic location, associated disease, and animal host.
PmSLP sequences separate into four distinct clusters, two of which are further separated into two sub-clusters each.
Fig 2.
Evaluation of PmSLP vaccines using a mouse sepsis model.
A. Determination of the lethal dose for a PmSLP group 1 (H246) and B. a group 3 (H229) isolate. N = 3–6 mice per group (3 mice per group for infectious dose of 106 CFU; 4 mice per group use for infectious doses between 500 CFU to 105 CFU; 6 mice per group for doses lower than 500 CFU). C. Survival of vaccinated and control animals after challenge with the BRD-PmSLP antigen-matched strain H246. N = 4 BRD-PmSLP, N = 6 HS-PmSLP, N = 7 Adjuvant). D. Survival of vaccinated and control animals after challenge with the HS-PmSLP antigen-matched strain H229. N = 8 per group. Log-rank (Mantel Cox) tests performed for survival curve comparisons; ****, p<0.0001; **, p<0.01; ns, not significant. E,F. Cumulative clinical scores depicted for individual animals over the course of 72 hours. Dotted lines at 10 indicate the clinical cut-off used for determining humane endpoint. G,H. Bacterial burden in tail bleeds depicted for individual animals over the duration of the challenge trial. I. Serum IgG titre in vaccinated or adjuvant control animals measured using purified BRD-PmSLP or HS-PmSLP proteins as capture. J. Serum IgG titre in vaccinated or adjuvant control animals measured using heat inactivated whole bacteria as capture. For I and J, boxplot with individual animals is shown. 2-way ANOVA with Tukey’s multiple comparisons test performed on log2 transformed data to calculate p-value. ***, p<0.005; ****, p<0.0001; ns, not significant.
Table 1.
Challenge strains of P. multocida utilized in these studies.
Fig 3.
Evaluation of bivalent versus single component PmSLP vaccines using a murine infection model.
Survival of vaccinated and control animals after challenge with the A. BRD-PmSLP antigen-matched strain H246 or B. HS-matched H229 strain. N = 4 per group. Log-rank (Mantel Cox) tests performed for survival curve comparisons; **, p<0.01. C, D. Cumulative clinical scores depicted for individual animals over the course of 48 hours. Dotted line at 10 indicate the clinical cut-off used for determining humane endpoint. E, F. Bacterial burden in tail bleeds depicted for individual animals over time. G, H. Serum IgG titre in vaccinated or adjuvant control animals measured using purified BRD-PmSLP or HS-PmSLP proteins as capture. Boxplot with individual animals is shown. Ordinary 1-way ANOVA with Tukey’s multiple comparisons test performed on log2 transformed data to calculate p-value. ***, p<0.005; ****, p<0.0001; ns, not significant.
Fig 4.
Short term stability of PmSLP antigens.
A. Comparison of (i) thermal profile and (ii) first derivative of BRD-PmSLP protein lyophilized and stored at 4°C for 7 days. B. Comparison of (i) thermal profile and (ii) first derivative of HS-PmSLP protein lyophilized and stored at 4°C, room temperature (RT), and 37°C for 14 days. C. Survival of mice that received different HS-PmSLP vaccine preparations after challenge with matched strain H229. Vaccine 1, HS-PmSLP antigen stored at -80°C after purification and formulated prior to each dose administration; Vaccine 2, HS-PmSLP antigen stored at -80°C after purification, formulated prior to first dose and stored at 4°C until the booster; Vaccine 3, lyophilized HS-PmSLP antigen reconstituted and formulated immediately prior to each dose administration. N = 6 mice for adjuvant, N = 5 mice for each vaccinated group. Log-rank (Mantel Cox) tests performed for survival curve comparisons; **, p<0.01. D. Clinical scores of individual mice over 36 hours post intraperitoneal challenge. E. Bacteremia in tail vein sample at 18h post infection. Line at median, error bars depict interquartile range. Ordinary 1-way ANOVA with Dunnett’s multiple comparison test performed to compare vaccinated groups to adjuvant control. *, p<0.05.
Fig 5.
Long term stability of PmSLP antigens.
A. Comparison of (i) thermal profile and (ii) first derivative of HS-PmSLP protein lyophilized and stored at 4°C or room temperature for 1 year. B. Survival of mice that received different HS-PmSLP vaccine preparations after challenge with the matched strain H229. Vaccine 1, HS-PmSLP antigen stored at -80°C for 1 year after purification. Vaccine 2, lyophilized HS-PmSLP stored for 1 year at 4°C. Both vaccines were formulated immediately prior to each dose administration. N = 4 mice for adjuvant control, N = 4 mice for each vaccinated group. Log-rank (Mantel Cox) tests performed for survival curve comparisons; **, p<0.01. C. Clinical scores of individual mice over 48 hours post intraperitoneal challenge. D. Bacteremia in tail vein sample at 15h post infection. Line at median, error bars depict interquartile range. Ordinary 1-way ANOVA with Dunnett’s multiple comparison test performed to compare vaccinated groups to adjuvant control. **, p<0.01. E. Serum IgG titre in vaccinated or control animals against the purified protein antigen. Boxplot with individual animals is shown. 1-way ANOVA with Tukey’s multiple comparisons test performed to compare each group to all other groups using log2 transformed data. ****, p<0.0001; ns, not significant.
Table 2.
Experimental layout of PmSLP cattle trials.
Fig 6.
Cattle immunogenicity studies using the BRD-PmSLP vaccine.
Ai, B: BRD-PmSLP antigen specific IgG detected in serum of cattle from Trials 1 and 2 respectively. Aii: Cross-reactivity of serum from Trial 1 against HS-PmSLP protein. (Left) Background subtracted absorbance readings obtained for each animal using serially diluted serum. Dotted line represents the cut-off used for calculating IgG titre. (Right) Boxplot displaying individual animals. 2-way ANOVA with Dunnett’s multiple comparisons test performed on log2 transformed data to compare post vaccination titres to baseline. * p<0.05, *** p<0.005, ****p<0.0001, ns = not significant.
Fig 7.
Cattle immunogenicity studies using the HS-PmSLP vaccine.
Ai, B: HS-PmSLP antigen specific IgG detected in serum of cattle from Trials 3 and 4 respectively. Aii: Cross-reactivity of serum from Trial 3 against BRD-PmSLP protein. (Left) Background subtracted absorbance readings obtained for each animal using serially diluted serum. Dotted line represents the cut-off used for calculating IgG titre. (Right) Boxplot displaying individual animals. 2-way ANOVA with Dunnett’s multiple comparisons test performed on log2 transformed data to compare post vaccination titres to baseline. * p<0.05, ** p<0.01, ****p<0.0001, ns = not significant. Vaccine 1 formulated with Montanide Gel02 + Poly(I:C), Vaccine 2 formulated with Alhydrogel. Adjuvant controls received Montanide Gel02 + Poly (I:C) only.
Fig 8.
Survival of Zebu cattle immunized with two different HS-PmSLP vaccine formulations or the matched bacterin following a lethal challenge with a Serogroup B HS isolate.
N = 8 animals per group. Log-rank (Mantel Cox) tests performed for survival curve comparisons; ***, p<0.005; **, p<0.01.