Characterization of a novel inactivated Salmonella enterica serovar Enteritidis vaccine candidate generated using a modified cI857/λ PR/gene E expression system

Abstract

A new strategy to develop an effective vaccine is essential to control food-borne Salmonella enterica serovar Enteritidis infections. Bacterial ghosts (BGs), which are nonliving, Gram-negative bacterial cell envelopes, are generated by expulsion of the cytoplasmic contents from bacterial cells through controlled expression using the modified cI857/λ P(R)/gene E expression system. In the present study, the pJHL99 lysis plasmid carrying the mutated lambda pR37-cI857 repressor and PhiX174 lysis gene E was constructed and transformed in S. Enteritidis to produce a BG. Temperature induction of the lysis gene cassette at 42°C revealed quantitative killing of S. Enteritidis. The S. Enteritidis ghost was characterized using scanning and transmission electron microscopy to visualize the transmembrane tunnel structure and loss of cytoplasmic materials, respectively. The efficacy of the BG as a vaccine candidate was evaluated in a chicken model using 60 10-day-old chickens, which were divided into four groups (n = 15), A, B, C, and D. Group A was designated as the nonimmunized control group, whereas the birds in groups B, C, and D were immunized via the intramuscular, subcutaneous, and oral routes, respectively. The chickens from all immunized groups showed significant increases in plasma IgG and intestinal secretory IgA levels. The lymphocyte proliferation response and CD3(+) CD4(+) and CD3(+) CD8(+) T cell subpopulations were also significantly increased in all immunized groups. The data indicate that both humoral and cell-mediated immune responses are robustly stimulated. Based on an examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate vaccine can provide efficient protection against virulent challenge.

Fig 1

Growth and lysis of JOL1313 containing pJHL99 and of JOL860, naïve wild-type S. Enteritidis. At time zero, the cultures were shifted from 37°C to 42°C. Gene E-mediated lysis of JOL1313 resulted in a dramatic decrease in the OD₆₀₀ (I) and CFU/ml (II).

Fig 2

Electron microscopic analyses of JOL1313 by SEM (I and II) and TEM (III and IV). (I and III) Presence of transmembrane tunnels, indicated by arrowheads, and loss of cytoplasmic material after lysis. (II and IV) Intact bacteria before lysis.

Fig 3

Antigen-specific humoral immune responses in nonimmunized control (A), intramuscularly immunized (B), subcutaneously immunized (C), and orally immunized (D) groups were determined at each week postimmunization. (Left) Plasma IgG concentrations (ng/ml). (Right) Secretory IgA concentrations (ng/ml). Antibody levels are expressed as means ± standard errors of the mean. The asterisks indicate significant differences between the antibody titers of the immunized and nonimmunized groups (P < 0.05).

Fig 4

Lymphocyte proliferative responses against S. Enteritidis-specific antigen in immunized and nonimmunized chickens at 3 weeks postvaccination. The antigen-specific lymphocyte proliferative response is expressed as the SI. The asterisks indicate significant differences between the SIs of the immunized and nonimmunized groups (P < 0.05). Group A, nonimmunized control; group B, intramuscularly immunized; group C, subcutaneously immunized; group D, orally immunized.

Fig 5

Flow cytometric analysis of T lymphocyte subpopulations. (I) Two-dimensional density plots for CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cell populations. The plots represent events for one representative chicken from each group. The population is represented as a percentage of gated cells. (II) CD3⁺ CD4⁺ T lymphocyte populations in immunized and nonimmunized chickens. (III) CD3⁺ CD8⁺ T lymphocyte populations in immunized and nonimmunized chickens. The values are shown as means ± standard errors of the mean of 5 chickens per group. The asterisks indicate significant differences between the T cell subpopulations of the immunized and nonimmunized groups (P < 0.05). Group A, nonimmunized control; group B, intramuscularly immunized; group C, subcutaneously immunized; group D, orally immunized.