Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle

Abstract

Background: Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain.

Methods: Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle.

Results: PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia.

Conclusion: Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

Keywords: Pasteurella multocida; gram-negative; hemorrhagic septicemia; surface lipoprotein; vaccine.

Figure 1

Immunogenicity and antibody subclasses present in sera from C57Bl/6 mice immunized with different PmSLP-1 formulations. Antisera from individual mice (n = 2 mice for naïve and n = 9 mice for each vaccinated groups) were tested in technical duplicate for (A) total IgG, (B) IgG1, (C) IgG2b, and (D) IgG2c after one dose (gray circles) and two doses (black circles) of vaccine. Titres were compared with a two-way ANOVA with multiple comparisons with Uncorrected Fisher’s least significant difference (LSD) at each time point. Line represents median. Comparisons to naïve were ignored due to the naïve group being under powered (n = 2) for analysis. Only significant differences are indicated, other comparisons are not significant, * represents p ≤ 0.05, ** represents p ≤ 0.01, and **** represents p ≤ 0.0001.

Figure 2

Duration of immunity in mice. Persistence of vaccine elicited antibodies for 26 weeks after booster for all groups (A) and separated by individual mice for each formulation including PmSLP-1 formulated with Montanide Gel 02 + Poly(I:C) (B), Montanide Gel 02 (C), Alum (D), Emulsigen-D (E), and Montanide ISA 61 (F). Titres at 26 weeks after booster are compared to titres at 2 weeks after booster (G). Titres were compared with a two-way ANOVA with multiple comparisons with uncorrected Fisher’s LSD comparing titres from each formulation at 2 weeks and 26 weeks. Line represents median for panel (G). * represents p < 0.05, ns represents non-significant difference.

Figure 3

Immunogenicity and antibody subclasses present in plasma from Holstein cattle immunized with different PmSLP-3 adjuvant formulations. Plasma from individual calves was tested by ELISA for PmSLP-3–specific (A) IgG, (B) IgG1, and (C) IgG2. Samples from each calf at each time point were tested in triplicate on three separate plates for a total of nine technical data points per sample, with the line representing the median. Performed as part of cattle trial 1 at the University of Calgary. Titres were compared with a two-way ANOVA with multiple comparisons with uncorrected Fisher’s LSD at each time point. Only significant differences are indicated, other comparisons are not significant, ** represents p ≤ 0.01, and *** represents p ≤ 0.001.

Figure 4

Immunization of cattle with PmSLP-3 antigen for adjuvant selection. Groups of five 6-month-old Holstein calves were immunized with 200 µg of PmSLP-3 formulated with AlOH, Montanide Gel 02 + Poly(I:C), or Montanide ISA 61 adjuvant according to the manufacturer’s instructions. Blood samples were collected for 12 months, and anti–PmSLP-3 plasma IgG was measured by ELISA. (A) Average of all animals per group shown, as well as titres of each calf from animals that received PmSLP-3 vaccine formulated with (B) aluminum hydroxide gel (alum), (C) Montanide Gel 02 + Poly(I:C), or (D) Montanide ISA 61. Plasma samples were also obtained at 3 years (36 months) after booster and compared to the titres obtained at 12 months after booster (E). Performed as part of cattle trial 1 at the University of Calgary. Titres were compared with a two-way ANOVA with multiple comparisons with uncorrected Fisher’s LSD comparison. Line represents median for panel (E). Only significant differences are indicated, other comparisons are not significant, * represents p ≤ 0.05, *** represents p ≤ 0.001, and **** represents p ≤ 0.0001.

Figure 5

Mucosal IgG endpoint titres for each Holstein calf immunized s.c. with PmSLP-3 formulated with aluminum hydroxide gel (blue), Montanide Gel 02 + Poly(I:C) (red), or Montanide ISA 61 (purple). Data are shown at weeks after first immunization, with doses given at day 0 and day 21. Performed as part of cattle trial 1 at the University of Calgary. Titres at each time point were compared with a repeated-measures two-way ANOVA with multiple comparisons with Tukey’s multiple comparisons test. Line represents median. Only significant differences are indicated, other comparisons are not significant, * represents p ≤ 0.05, and ** represents p ≤ 0.01.

Figure 6

Evaluation of different adjuvants on immunogenicity and protection by PmSLP-3 vaccine formulations. (A) Immunogenicity of sera from Zebu cattle that received either aluminum potassium sulfate only (Adjuvant only, “AlkSO4,” white), the local Bacterin (red), or PmSLP-3 formulated with aluminum potassium sulfate (light purple), aluminum hydroxide gel (AlOH, dark purple), or Montanide ISA 61 (teal) against purified PmSLP-3 protein. Serum was evaluated at baseline (day 0), after one dose of vaccine (day 21), or after two doses of vaccine (day 35). Each dot represents the mean of technical triplicates with line representing median. (B) Survival curves of all cattle after challenge with a lethal dose of P. multocida serogroup B bacteria. Performed as cattle trial 3 at NVI. Serum immunogenicity compared by two-way ANOVA with Dunnett’s multiple comparisons test after each dose against cattle that received adjuvant only. Survival curves compared the PmSLP-3–immunized animals individually against adjuvant only controls with Log-rank (Mantel–Cox) test. Only significant differences are indicated, other comparisons are not significant, * represents p ≤ 0.05, ** represents p ≤ 0.01, and **** represents p ≤ 0.0001.

Figure 7

Evaluation of needed immunogen dosing and number of immunizations. (A) Immunogenicity of sera from Zebu cattle that received either Montanide ISA 61 only (Adjuvant only, white), or PmSLP-3 formulated with Montanide ISA 61 against purified PmSLP-3 protein. Cattle received either a single dose of 50 µg of PmSLP-3 vaccine (light purple) or 100 µg of PmSLP-3 vaccine (light blue) on day 21, or received two doses of 50 µg of PmSLP-3 vaccine (dark purple) or 100 µg of PmSLP-3 vaccine (dark blue) on day 0 and day 21. Each dot represents the mean of technical triplicates with line representing median. (B) Survival curves of all cattle after challenge with a lethal dose of P. multocida serogroup B bacteria. Performed as cattle trial 4 at NVI. Serum immunogenicity compared by two-way ANOVA with Dunnett’s multiple comparisons test. Survival curves compared individually against adjuvant only with Log-rank (Mantel–Cox) test. Only significant differences are indicated, other comparisons are not significant, ** represents p ≤ 0.01, and **** represents p ≤ 0.0001.

Figure 8

Duration of immunity after immunization with PmSLP-3 antigen. (A) Groups of three 6-month-old female Holstein calves were vaccinated i.m. with PmSLP-3 antigen formulated with Montanide ISA 61 either once (day 0) or twice (day 0 and day 21) with either 50 µg or 100 µg of protein antigen. PmSLP-3–specific plasma IgG was evaluated at 3 weeks (gray) and 8 weeks (black) after day 0. (B) PmSLP-3–specific plasma IgG was evaluated at 12 months (gray) and 18 months (black) after immunization to evaluate for the duration of immunity. Performed as cattle trial 2 at the University of Calgary. Serology was run on animals in separate experiments due to the long time span between sampling and, therefore, is plotted separately. Plasma immunogenicity compared by two-way repeated-measures ANOVA with Uncorrected Fisher’s LSD multiple comparisons test. Line represents median. Only significant differences are indicated, other comparisons are not significant, * represents p ≤ 0.05, and ** represents p ≤ 0.01.

Figure 9

Evaluation of duration of immunity by PmSLP-3 formulated with Montanide ISA 61. (A) Immunogenicity of Zebu cattle immunized with PmSLP-3 (teal), the local bacterin (red), or control (naïve) animals (black). PmSLP-3–specific serum IgG was evaluated prior to immunization as well as at 1 month, 3 months, 4 months, 6 months, and 7 months after vaccination. Data represent median and range of 8 to 10 animals per group. (B) PmSLP-3–specific serum IgG from individual cattle immunized with PmSLP-3. (C) Survival curves of all cattle after challenge with a lethal dose of P. multocida serogroup B bacteria 7 months after immunization. Performed as cattle trial 5 at NVI. Serum samples were tested in technical triplicate, and endpoint titres were compared by two-way repeated-measures ANOVA with post-hoc analysis by Tukey’s multiple comparisons test at each time point. Survival curves compared individually with Log-rank (Mantel–Cox) test. ns represents non-significant, * represents p ≤ 0.05, ** represents p ≤ 0.01, and *** represents p ≤ 0.001.

Figure 10

Evaluation of protection against a serogroup E strain of P. multocida by a single dose of PmSLP-3 vaccine formulated with Montanide ISA 61. (A) Immunogenicity of sera from Zebu cattle that received either PmSLP-3 formulated with Montanide ISA 61 (teal), the local bacterin (red), or control (naïve) animals (white) tested against purified PmSLP-3 protein. Each dot represents the mean of technical triplicates and line represents median. (B) Survival curves of all cattle after challenge with a lethal dose of P. multocida serogroup E bacteria. Performed as cattle trial 6 at NVI. PmSLP-3 titres tested by two-way repeated-measures ANOVA with Tukey’s multiple comparisons test with titres compared to baseline. Survival curves compared individually against adjuvant only with Log-rank (Mantel–Cox) test. Only significant differences are noted forl (A), non-significant differences are not shown. ns represents non significant, * represents p ≤ 0.05, ** represents p ≤ 0.01, and *** represents p ≤ 0.001.