Recombinant Ehrlichia P29 protein induces a protective immune response in a mouse model of ehrlichiosis

Abstract

Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals for which no vaccines are available. The diseases are caused by obligately intracellular bacteria belonging to the genus Ehrlichia. Several immunoreactive proteins of ehrlichiae have been identified based on their reactivity with immune sera from human patients and animals. These include the major outer membrane proteins, ankyrin repeat proteins and tandem repeat proteins (TRP). Polyclonal antibodies directed against the tandem repeats (TRs) of Ehrlichia chaffeensis TRP32, TRP47 and TRP120 have been shown to provide protection in mice. In the present study, we evaluated E. muris P29, which is the ortholog of E. chaffeensis TRP47 and E. canis TRP36, as a subunit vaccine in a mouse model of ehrlichiosis. Our study indicated that unlike E. chaffeensis TRP47 and E. canis TRP36, orthologs of E. muris (P29) and E. muris-like agent (EMLA) do not contain tandem repeats. Immunization of mice with recombinant E. muris P29 induced significant protection against a challenge infection. The protection induced by E. muris P29 was associated with induction of strong antibody responses. In contrast to development of P29-specific IgG antibodies following immunization, development of P29-specific IgG antibodies, but not IgM antibodies, was impaired during persistent E. muris infection. Furthermore, our study indicated that CD4+ T cells target P29 during E. muris infection and differentiate into IFN-γ-producing Th1 effector/memory cells. In conclusion, our study indicated that orthologs of E. muris P29 showed considerable variation in the central tandem repeat region among different species, induction of P29-specific IgG antibody response was impaired during persistent E. muris infection, and rP29 induced protective immune responses.

Keywords: Antibody; Antigen; Ehrlichia; Intracellular bacteria; Protective immunity; Vaccine.

Figure 1. Orthologs of Ehrlichia muris P29 show high variation in the central region

The N-terminal regions (A) and C-terminal regions (C) of E. muris P29 and its orthologs of E. chaffeensis Arkansas strain (TRP47) and E. canis Jake strain (TRP36) were aligned by ClustalW method. The central region of E. muris P29 and the repeat regions of orthologous E. chaffeensis TRP47 and E. canis TRP36 (B). The amino acid residues that match the consensus sequence are highlighted in grey.

Figure 2. Analysis of recombinant E. muris P29 protein, and identification of native P29 protein

(A) Purified recombinant E. muris P29 protein with N-terminal fusion tag (lane 1) or without N-terminal fusion tag (lane 2) was analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue G-250. (B) Western-blot analysis of rP29 protein. An anti-rP29 immune serum (lane 1) and an immune-serum from E. muris-infected mice containing detectable concentration of anti-rP29 IgG antibodies by ELISA (lane 2) reacted specifically with rP29. (C) Identification of native E. muris P29 protein. E. muris lysate antigen was separated on a NuPAGE Zoom gel containing an IPG well (Invitrogen) by SDS-PAGE and developed with naïve serum (lane 1), anti-rP29-serum (lane 2) or E. muris-immune serum containing detectable concentration of anti-rP29 IgG antibodies by ELISA (lane 3) using a Mini-PROTEAN II multiscreen apparatus (Bio-Rad laboratories). The anti-rP29-serum recognized a 29 kDa native E. muris protein (lane 2). The arrow head indicates the native P29 protein recognized by the E. muris-immune serum (lane 3). All images were analyzed using the MyImageAnalysis software (Thermo Scientific).

Figure 3. Immunization with recombinant E. muris P29 induces significant protection against challenge infection

C57BL/6 mice were immunized with two doses of rP29 and challenged with a low dose (A; 2 × 10³ bacteria) or a high dose (B; 1 × 10⁴ bacteria) of E. muris by the i.p. route 60 days later. Mice were sacrificed on day 10 post-challenge, and the bacterial burdens in the liver, spleen, lung, and blood were determined by quantitative real-time PCR. Unimmunized mice (saline), mice immunized with recombinant Chlamydia pneumoniae MOMP, and E. muris-immune mice served as controls. Ehrlichial copy numbers were normalized to the total DNA. Each group contained three to four mice, and data were square-root transformed and analyzed by one way ANOVA with Bonferroni post-test for comparison of multiple groups. The error bars represent the standard deviation. **, P < 0.01 and ***, P < 0.001 compared to the saline control group. ns – not significant

Figure 4. Protection induced by recombinant E. muris P29 is associated with induction of a broad IgG isotype responses

Serum IgG antibody responses specific to rP29 (A) and E. muris lysate antigen (B) in experimental groups before and on day 10 after E. muris challenge were determined by ELISA. (C) Serum immunoglobulin isotype responses specific to rP29 in mice immunized with rP29 before (rP29) and on day 10 after E. muris challenge (rP29/EM). (D) Serum immunoglobulin isotype responses specific to E. muris-lysate antigen in mice after primary (EM) and on day 10 after secondary E. muris infection (EM/EM). Data are representative of two independent experiments. (A & B) Each data point represents an individual immunized animal before E. muris challenge infection (open circles) or on day 10 after E. muris challenge (closed circles) and the horizontal solid bars represent the means. (C & D) Each bar represents the average of four immunized mice before E. muris challenge infection (open bars) or on day 10 after E. muris challenge (solid bars) and the error bars represent the standard deviation.

Figure 5. E. muris P29 induces effector/memory Th1 CD4+ T cells in infected mice

Frequencies of antigen-specific IFN-γ-producing CD4+ T cells in the spleens from separate groups of mice infected with E. muris were determined by flow cytometry. (A) Dot plots showing the percentages of P29-specific IFN-γ-producing CD4+ T cells in the spleens on day 30 after E. muris infection. (B) Frequencies of P29-specific IFN-γ-producing CD4+ T cells per million splenocytes in mice infected with E. muris on day 30 after infection. Each data point represents an individual animal, and data from two independent experiments are combined. Splenocytes from uninfected naïve mice stimulated with rP29 or E. muris lysate antigen served as controls. Background values from wells containing unstimulated splenocytes (medium only controls) were subtracted from antigen-stimulated wells for each mouse. Frequencies of IFN-γ-producing CD4+ T cells responding to E. muris lysate antigen from the same mice are presented for comparison.