A Recombinant 47-kDa Outer Membrane Protein Induces an Immune Response against Orientia tsutsugamushi Strain Boryong - PubMed (original) (raw)

A Recombinant 47-kDa Outer Membrane Protein Induces an Immune Response against Orientia tsutsugamushi Strain Boryong

Sangho Choi et al. Am J Trop Med Hyg. 2017 Jul.

Abstract

We investigated the 47-kDa outer membrane protein (OMP), which is a periplasmic serine protease and an antigenic major surface protein of Orientia tsutsugamushi, as a vaccine candidate. We developed a conventional subunit vaccine expressing recombinant 47-kDa OMP (rec47) and a DNA vaccine (p47). In mouse immunization experiments, intranasal immunization with rec47 alone or with rec47 plus heat-labile enterotoxin B subunit from Escherichia coli or plus cholera toxin (CT) as adjuvants induced a higher amount of rec47-specific antibodies than intramuscular immunization with p47 alone or with p47 plus pBOOST2-samIRF7/3 (pB) as adjuvant. Moreover, the combination of rec47 and CT induced a strong cellular immune response to 47-kDa OMP, as demonstrated by a spleen cell proliferation assay, and also induced Th1- and Th2-type cytokine production, as demonstrated by a cytokine enzyme-linked immunosorbent assay. Intranasal immunization with rec47 plus CT was the most effective method for the induction of humoral and cell-mediated immune responses. Furthermore, relatively strong protection against homologous O. tsutsugamushi strain Boryong challenge was observed in mice immunized with rec47 plus CT. Therefore, 47-kDa OMP is an attractive candidate for developing a prophylactic vaccine against scrub typhus by O. tsutsugamushi infection.

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Figures

Figure 1.

Figure 1.

Humoral immune response upon the different treatments, as measured in sera from immunized mice. 47-kDa outer membrane protein–specific immunoglobulins including total (A) IgG, (B) IgG1, (C) IgG2a, and (D) IgA were measured by indirect enzyme-linked immunosorbent assay after every immunization. Statistically significant differences between the PBS control group and each recombinant protein immunization group are indicated with * (P < 0.05) or ** (P < 0.01). Statistically significant differences between the pVAX1 control group and each DNA vaccination group are indicated with † (P < 0.05) or †† (P < 0.01).

Figure 2.

Figure 2.

Spleen cell proliferation assay to measure cell-mediated immunization. The isolated spleen cells were restimulated with 1 μg of rec47. Cell viability was measured as the percentage of viable cells at 24, 48, and 72 hours after restimulation. Statistically significant differences between the PBS control group and each recombinant protein immunization group are indicated with * (P < 0.05) or ** (P < 0.01). Statistically significant differences between the pVAX1 control group and each DNA vaccination group are indicated with † (P < 0.05) or †† (P < 0.01).

Figure 3.

Figure 3.

Cytokine release in immunized mice sera. After the third immunization, the concentration of distinct Th1- and Th2-type cytokines in mouse serum was measured by using a multiplex assay. (A) interferon gamma (IFN-γ), (B) interleukin (IL)-2, (C) IL-4, (D) IL-5, (E) IL-6, (F) IL-10, and (G) IL-12 (p70). Statistically significant differences between the PBS control group and each recombinant protein immunization group are indicated with * (P < 0.05) or ** (P < 0.01). Statistically significant differences between the pVAX1 control group and each DNA vaccination group are indicated with † (P < 0.05) or †† (P < 0.01).

Figure 4.

Figure 4.

Cytokine release from spleen cells isolated from immunized mice. The isolated spleen cells were restimulated with 1 μg of rec47. Cytokines were measured at 24 hours after restimulation using an enzyme-linked immunosorbent assay. (A) interferon gamma (IFN-γ), (B) tumor necrosis factor (TNF)-α, (C) interleukin (IL)-2, (D) IL-5, (E) IL-6, and (F) IL-10. Statistically significant differences between the PBS control group and each recombinant protein immunization group are indicated with * (P < 0.05) or ** (P < 0.01). Statistically significant differences between the pVAX1 control group and each DNA vaccination group are indicated with † (P < 0.05) or †† (P < 0.01).

Figure 5.

Figure 5.

Evaluation of the protection against homologous challenge in immunized mice. Orientia tsutsugamushi strain Boryong was injected intraperitoneally into mice immunized with rec47 alone or rec47 plus LTB/CT and into mice immunized with p47 alone or p47 and pB, and their respective control treatments. The mortality was monitored daily for 2 weeks. The survival rate was calculated as the ratio of the number of living mice to the total number of challenged mice in a group. Statistically significant differences between the PBS control group and each recombinant protein immunization group are indicated with * (P < 0.05). Statistically significant differences between the pVAX1 control group and each DNA vaccination group are indicated with † (P < 0.05).

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