Clin Exp Vaccine Res.  2016 Jul;5(2):148-158. 10.7774/cevr.2016.5.2.148.

Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate

• Won G
• Chaudhari
• Lee JH

• KMID: 2421562
• DOI: http://doi.org/10.7774/cevr.2016.5.2.148

Abstract

PURPOSE
Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid.
MATERIALS AND METHODS
The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization.
RESULTS
Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4+ and CD8+ T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4+ and CD8+ T-cell population and IL-6 cytokine in mRNA levels.
CONCLUSION
Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.

Keyword

Salmonella; Ghost vaccine; Immunization scheme; Protection; Fowl typhoid

MeSH Terms

Birds
Chickens
Cytokines
Immunization*
Immunoglobulin A, Secretory
Immunoglobulin G
Interferons
Interleukin-6
Lymphocyte Activation
Plasma
RNA, Messenger
Salmonella enterica
Salmonella*
Serogroup
Spleen
T-Lymphocytes
Typhoid Fever
Vaccination
Cytokines
Immunoglobulin A, Secretory
Immunoglobulin G
Interferons
Interleukin-6
RNA, Messenger

Figure

• 

  Fig. 1 The plasma IgG levels in chickens against the outer membrane protein were determined for 7 weeks PPI. Heparinized blood (1 mL) was collected from both vaccinated and unvaccinated groups (n=5). Antibody levels were expressed as mean±standard deviation values for each week post-immunization. Statistical significance was defined at p-values ≤0.05 or 0.01. Arrow indicates that the booster was performed at fourth week PPI. PPI, post-prime immunization; group A, non-immunized control; group B, orally primed and boosted; group C, primed orally and boosted intramuscularly; group D, primed and boosted intramuscularly; group E, primed intramuscularly and boosted orally. *p < 0.05 vs. unvaccinated control.

• 

  Fig. 2 The intestinal secretory IgA (sIgA) antibody response was measured in chickens against the outer membrane protein for 7 weeks PPI. Antibody levels were expressed as mean±standard deviation values for each week post-immunization. Statistical significance was defined at p-values ≤0.05 or 0.01. Arrow indicates that the booster was performed at fourth week PPI. PPI, post-prime immunization; group A, non-immunized control; group B, orally primed and boosted; group C, primed orally and boosted intramuscularly; group D, primed and boosted intramuscularly; group E, primed intramuscularly and boosted orally. *p < 0.05 vs. unvaccinated control.

• 

  Fig. 3 The lymphocyte stimulation responses determined at 3-week post-booster immunization against the sonicated bacterial cell protein suspension antigen. The stimulation index of lymphocyte sample from the chickens was determined by the peripheral lymphocyte proliferation assay. Statistical significance was defined at p-values ≤0.05 or 0.01. Group A, non-immunized control; group B, orally primed and boosted; group C, primed orally and boosted intramuscularly; group D, primed and boosted intramuscularly; group E, primed intramuscularly and boosted orally. *p<0.05 vs. unvaccinated control.

• 

  Fig. 4 Evaluation of cellular immune responses from spleens of chickens post-prime immunization by intramuscular route. (A) Flowcytometry scatter dot plots for CD45+, macrophage cell populations. The plots represent events for one representative chicken from each group. The gating and the quadrants are set according to the standard procedures of the BD Biosciences flowcytometer. (B) Bar graphs represent CD45+ macrophage population in immunized and non-immunized chickens. Values are shown as mean±standard deviation of 5 chickens per group. (C) The mRNA production of IFN-γ cytokine in spleens from chickens after prime vaccination. The mRNA amount of IFN-γ was measured by SYBER green–based real-time reverse transcription polymerase chain reaction. The total RNA was isolated from the spleens of the chickens at 3-week post-prime immunization. Data is expressed as geometric mean with standard deviation. APC, allophycocyanin; IFN-γ, interferon γ; PE, phycoerythrin; group I, non-immunized control; group II, primed and boosted intramuscularly. *p≤0.05 vs. unvaccinated control.

• 

  Fig. 5 Evaluation of cellular immune responses from spleens of chickens post-booster vaccination by intramuscular route. (A) Flowcytometry scatter dot plots for CD3+, CD4+, CD8+ T-cell populations. The plots represent events for one representative chicken from each group. The gating and the quadrants are set according to the standard procedures of the BD Biosciences flowcytometer. (B) Bar graphs represent CD3+ CD4+ T-lymphocytes population in immunized and non-immunized chickens. (C) Bar graphs represent CD3+ CD8+ T-lymphocytes population in immunized and non-immunized chickens. Values are shown as mean±standard deviation of 5 chickens per group. (D) The mRNA production of IL-6 cytokine in spleens from chickens after booster vaccination. The mRNA amount of IL-6 was measured by SYBER green-based real-time reverse transcription polymerase chain reaction. The total RNA was isolated from the spleens of the chickens at 3-week post-booster immunization. Data is expressed as geometric mean with standard deviation. APC, allophycocyanin; IL-6, interleukin 6; PE, phycoerythrin; group I, non-immunized control; group II, primed and boosted intramuscularly. *p≤0.05 vs. unvaccinated control.

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