Protective efficacy of the recombinant lysozyme-PMAP36 fusion protein-inactivated Salmonella Typhimurium vaccine candidate via oral immunization in a murine model

Abstract

The aim of this study was to evaluate protective efficacy of S. Typhimurium ghost vaccine candidate lysed by the recombinant lysozyme-PMAP36 fusion protein via oral immunization in a murine model. Sixty BALB/c mice were equally divided into 4 groups. Group A mice were inoculated with 20 μL of sterile phosphate-buffered saline (PBS). Groups B-D mice were immunized with approximately 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ cells of the vaccine candidate, respectively, in 20 μL of PBS. Salmonella-outermembrane-proteins-specific serum IgG was considerably higher in groups B–D than in group A. The interleukin-10 and interferon-γ levels in groups B–D were significantly higher than in group A. Following challenge with wild-type S. Typhimurium, all immunized groups showed a significant level of protection compared with group A. The highest protection was shown in group D. Overall, these results show that oral immunization with the candidate vaccine can effectively protect mice from S. Typhimurium infection.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in domestic animals, as well as in humans (1,2). S. Typhimurium in a mouse infection model can cause symptoms similar to those observed after S. Typhi infection in humans (1). An effective method of preventing this disease is vaccination against salmonellosis (2). For effective protection against Salmonella, the cellular immune response is vital (3), and the humoral immune response is also known to contribute to the clearance of Salmonella infection through serum IgG and secretory IgA (3). The use of live attenuated vaccine is a routine protocol to prevent salmonellosis, but it may pose a great risk due to its potential to revert to a virulent strain, shedding in feces and thus infecting animals and humans coming in contact with the infected animals. Several attenuated Salmonella vaccine candidates have been generated by mutating or deleting virulence-associated pathogenicity islands or metabolism-associated genes (4–6). Hence, many different approaches, such as recombinant proteins, vector vaccines, subunit vaccines, and killed vaccines, have been tried for protection of salmonellosis, with varying degrees of success (3,4,7,8).

The nonliving bacterial envelopes without cytoplasmic contents (bacterial ghost) share the antigenic and functional determinants of the membranes with their living bacteria (9,10). The action mechanism of antimicrobial peptides (AMPs) is disruption of barrier function of its membrane by formation of a transmembrane tunnel or alteration of bacterial membrane permeabilization, without disturbing the integrity of the membrane (9–11). Porcine myeloid antimicrobial peptide-36 (PMAP36) has the highest positive charge among AMPs of pigs reported until now (12). The enzyme attacks the membranes of both Gram-negative and Gram-positive bacteria, resulting in weakening of the mechanical strength of the membranes and finally lysing of the membranes (13).

The objective of the present study was to estimate the protective efficacy of the S. Typhimurium ghost vaccine candidate lysed by the recombinant lysozyme-PMAP36 fusion protein via oral immunization in a murine model. Oral immunization with the S. Typhimurium ghost vaccine candidate can powerfully induce serum IgG and cytokines associated with Th-1 type immune response. In addition, the ghost vaccine candidate can effectively protect mice from systemic infections by S. Typhimurium through oral immunization.

Salmonella Typhimurium isolate, HJL 456, was used for construction of the inactivated vaccine by the recombinant lysozyme-PMAP36 fusion protein and as the malignant challenge strain. Escherichia coli BL21 (DE3), HJL 505, was used for overexpression of recombinant lysozyme-PMAP36 fusion proteins (14). The recombinant fusion protein was expressed in HJL505 and purified according to a previously reported method (14). All purified proteins were mixed with 50% glycerol and stored at −70°C until further use.

The fusion protein-inactivated S. Typhimurium vaccine candidate was prepared according to the method described in a previous study (14).

Sixty female BALB/c mice were divided into 4 groups of 15 mice each. The mice were orally primed at 6 wk of age [0-week post-prime inoculation (WPPI)] and were orally boosted at 8 wk of age (2 WPPI). The mice in group A were inoculated with sterile PBS as the control. Groups B, C, and D were inoculated with approximately 1 × 10⁷ cells, 1 × 10⁸ cells, and 1 × 10⁹ cells of the Salmonella vaccine candidate, respectively, in 20 μL of PBS. Blood and fecal samples were collected at 0, 2, and 4 WPPI for evaluation of the immune response.

Salmonella Typhimurium OMPs-specific IgG and IgA titers in serum and fecal samples, respectively, were examined according to the enzyme-linked immunosorbent assay (ELISA) methods described by Moon et al (14).

Five mice from each group were sacrificed, and their spleens were removed aseptically at 4 WPPI. Splenocytes were prepared according to the modified methods described previously (11). Briefly, the splenocytes were stimulated in vitro with the Salmonella vaccine candidate (10⁸ cells/well), concanavalin A (0.5 μg/well) as a positive control, or medium as an unstimulated control, and incubated at 37°C, 5% CO₂, and 95% humidity. After 48 h of re-stimulation, the supernatants were collected and used for the measurement of cytokine concentration. The cytokine concentration in the stimulated supernatants such as IL-10 and IFN-γ, was evaluated using the mouse cytokine ELISA Ready-SET-GO reagent set according to the manufacturer’s instructions (eBioscience, San Diego, California, USA).

For challenge experiments, the challenge strain (HJL456) was prepared according to the method described by Hur et al (4). All mice were orally challenged with 2 × 10⁸ colony-forming units (CFU) of the challenge strain in 20 μL of PBS at 4 WPPI. The mice were subsequently monitored up to 14 d after challenge for mortality.

In order to observe differences among the different vaccinated groups, absorbance data values of ELISA were used for analysis of variance with the post-hoc Tukey test for pairwise comparisons using SPSS version 16.0 (SPSS, Chicago, Illinois, USA). A P-value < 0.05 was considered to indicate statistical significance.

The mean serum immunoglobulin (Ig) G titers against the outer membrane proteins (OMPs) of S. Typhimurium in groups A–D mice were about same at 0 WPPI. The mean serum IgG titers in group A mice were maintained by the end of this study. However, the mean serum IgG titers in groups B–D mice were significantly increased gradually from 2 WPPI (P < 0.05) until 4 WPPI (P < 0.05) (Figure 1). In addition, the mean fecal IgA titers against the OMPs in groups A–D mice were about the same at 0 WPPI as serum IgG titers. The fecal IgA titers in group A mice were sustained by 4 WPPI. However, the fecal IgA titers in groups B–D mice were increased gradually from 2 WPPI and were significantly induced at 4 WPPI (P < 0.05) (Figure 1).

Figure 1.

Serum IgG and fecal IgA titers against OMPs of Salmonella Typhimurium in mice orally immunized with the recombinant lysozyme-PMAP36-inactivated S. Typhimurium vaccine candidate. Group A mice (□) were immunized with sterile PBS; group B mice ( ) were immunized with 1.0 × 10⁷ cells of the vaccine candidate; group C mice ( ) were immunized with 1.0 × 10⁸ cells of the candidate; group D mice ( ) were immunized with 1.0 × 10⁹ cells of the candidate. Data shown are the means for all mice in each group, and error bars show the standard deviations (SD). Asterisks indicate a significant difference between the values of the groups immunized with the vaccine candidate (*P < 0.05) and those of the control group.

As shown in Figure 2, the mean concentrations of interleukin (IL)-10 against the S. Typhimurium ghost vaccine from splenocytes of groups B–D mice were approximately 2.6, 2.9, and 3.7 times higher than that in group A mice (P < 0.05), respectively. The mean interferon (IFN)-γ concentrations of groups B–D mice were also significantly increased by approximately 2.7, 3.0, and 4.2 times respectively, compared with that in group A mice (P < 0.05).

Figure 2.

IL-10 (pg/mL) and IFN-γ (pg/mL) concentrations in the supernatants of splenocytes stimulated with the vaccine candidate at 4 WPPI. Group A mice (□) were immunized with sterile PBS; group B mice ( ) were immunized with 1.0 × 10⁷ cells of the vaccine candidate; group C mice ( ) were immunized with 1.0 × 10⁸ cells of the vaccine candidate; group D mice ( ) were immunized with 1.0 × 10⁹ cells of the vaccine candidate. Data are the means for all mice in each group; error bars show SDs. Asterisks indicate a significant difference between the values of groups B, C, and D mice (*P < 0.05) and those of the control group mice.

All mice in group A died between day 7 and day 11 after challenge. However, 6 mice in groups B and C survived until the end of the study. A total of 8 out of 10 mice in group D survived until the end of the study (Figure 3).

Figure 3.

Survival rates of mice challenged with S. Typhimurium strain in mice immunized with the vaccine candidate. Group A mice were immunized with sterile PBS; group B mice were immunized with 1.0 × 10⁷ cells of the vaccine candidate; group C mice were immunized with 1.0 × 10⁸ cells of the vaccine candidate; group D mice were immunized with 1.0 × 10⁹ cells of the vaccine candidate.

The recombinant lysozyme-PMAP36 fusion protein-S. Typhimurium ghost cells were used as a vaccine candidate herein similar to the previous study (14). The membrane integrity and intracellular alteration of the S. Typhimurium lysed cells before and after interaction with the recombinant fusion protein was demonstrated by transmission electron microscopy (TEM). These results indicate that the fusion protein may be maintained in storage conditions and may function effectively.

In this study, we first investigated the immune responses induced after oral immunization with the recombinant fusion protein-inactivated S. Typhimurium ghost vaccine candidate and the protective efficacy of it against a virulent S. Typhimurium infection in a murine model. A vaccine that produced a protective immune response such as IgA at mucosal surfaces is an ideal vaccine to inhibit the entry and colonization of pathogens and to provide protection from disease at the same area (8). In addition, the optimal vaccine should be efficiently producing IgG, because humoral immune responses are generally important to prevent bacterial infections (8). In this study, humoral immune responses such as serum IgG and fecal IgA were significantly increased in mice immunized orally by the vaccine candidate compared with control mice. These results show that oral immunization with the S. Typhimurium ghost vaccine can efficiently induce protective humoral immune responses.

IFN-γ is secreted by dendritic cells, natural killer cells, and Th1-type cells (15). Induction of IFN-γ, associated with CMI, during infection with intracellular bacterial pathogens such as Salmonella, is crucial for the clearance of the intracellular bacterial infection (16,17). The Th1-type immune response starts within few hours after infection resulting in elimination of Salmonella and protection against re-infection (5). In the present study, high levels of IFN-γ concentrations were significantly induced in the immunized mice compared with control group mice as in previous studies (4,14). This result shows that oral immunization with the S. Typhimurium ghost vaccine candidate produces sufficient cytokines, which are associated with Th1-type immune response. Generally, mucosal IgA is strongly induced by Th2-type immunity. IL-10 is one of the major cytokines that enhance IgA production (18). Furthermore, the IL-10 levels of all mice immunized with the vaccine candidate were significantly higher than those in control mice. This result demonstrates that secretion of IL-10, which is associated with an enhanced IgA response, was also powerfully enhanced by our ghost vaccine.

High protection of attenuated Salmonella vaccine candidates against virulent S. Typhimurium infection in a murine model was reported in previous studies (4,8). In addition, intramuscular immunization with the ghost vaccine candidate showed perfect protection against salmonellosis in mice (14). Similarly, 80% of group D mice in this study were protected against Salmonella infection after challenge with a virulent wild-type Salmonella Typhimurium isolate, and only 40% of groups B and C mice died after challenge. However, all control mice died after challenge. These results suggest that oral immunization with the S. Typhimurium ghost vaccine candidate can elicit antibodies and can be effective in protecting against salmonellosis in mouse models, even though this vaccine candidate is inactivated.

The results of this study show that the fusion protein-inactivated Salmonella vaccine candidate can induce a robust serum antibody and Th-1 type immune response through oral immunization in mice. Furthermore, oral immunization with the vaccine candidate, irrespective of the inoculation dose, confers effective protection against virulent S. Typhimurium infection. Mice immunized with 1 × 10⁹ cells of the vaccine candidate had the best protection against virulent S. Typhimurium infection. These results suggest that the fusion protein-inactivated Salmonella vaccine candidate can be a good basis for the development of a novel vaccine candidate against salmonellosis. In addition, these results imply that the vaccine candidate can be used through various inoculation routes.