Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model

Abstract

The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

Keywords: CME; Ehrlichia canis; GP19; mice; recombinant protein; vaccine prototype.

Figure 1

Recombinant protein of Ehrlichia canis (rGP19) expression was affirmed with the HRP conjugated IgG antibody against rGP19 in Western blot analysis. Lane M: protein marker; rGP19: recombinant protein GP19 induced with IPTG; non-induced: non-induced rGP19 with IPTG.

Figure 2

The association between antibody responses induced by the rGP19 vaccine and the development of antigen-specific antibody responses, pre- and post-E. canis infection in mice. rGP19 vaccine prototypes provide the protection against the pathogen measured by an ELISA using a purified rGP19 as the antigen on day 0 (pre-Ehrlichia canis challenging), and days 7 and 14 (post-E. canis challenging).

Figure 3

Immunization with rGP19 provided protection against Ehrlichia canis infection in mice. The protection against E. canis infection in the immunized mice with rGP19 was determined by the ehrlichial load in the blood (A), liver (B), and spleen (C) samples measured by quantitative PCR (qPCR) and showed lower ehrlichial loads on days 7 and 14 post-challenge (** p < 0.01) compared with mice immunized with adjuvant.

Figure 3

Immunization with rGP19 provided protection against Ehrlichia canis infection in mice. The protection against E. canis infection in the immunized mice with rGP19 was determined by the ehrlichial load in the blood (A), liver (B), and spleen (C) samples measured by quantitative PCR (qPCR) and showed lower ehrlichial loads on days 7 and 14 post-challenge (** p < 0.01) compared with mice immunized with adjuvant.

Figure 4

Analyses of cytokine mRNA expression in mice using qPCR. Bar graphs represent relative IFNG, IL-1a, IL-4, IL-6, and TNF mRNA expression levels after normalization to the GAPDH gene in naive and immunized mice with later Ehrlichia canis challenge group (50 µg rGP19, 100 µg rGP19, and adjuvant). Data are represented as mean ± SE (n = 5 each treatment), one-way ANOVA, * p < 0.05, ** p < 0.01.

Figure 5

Ehrlichia canis GP19-specific IFN-γ-producing CD4+ T cells develop during E. canis infection in mouse splenocyte determined by flow cytometry. Naive and immunized mice infected with E. canis over a period of 7 days (A) and 14 days (B) post-infection. The data plot represents the populations of lymphocytes (black dot), CD3+ T lymphocytes (blue dot), CD4+ T lymphocytes (green dot), and IFN-γ molecules in CD4+ T lymphocytes (pink dot).

Figure 6

Mean of the frequencies of IFN-γ-producing CD4+ T lymphocytes in the splenocytes of mice on days 14 post-Ehrlichia canis infection when stimulated with different treatments. Data are represented as mean ± SE, one-way ANOVA, * p < 0.05, ** p < 0.01.

Figure 7

Cytokine network involved macrophage, T cell (CD4+ and CD8+) and B cell activation in the immune response against Ehrlichia canis. Abbreviations: 4-1BBL: 4-1BB ligand; APRIL: a proliferation-inducing ligand; CD: cluster of differentiation; IFN: interferon; IL-: interleukin; MIF: macrophage migration inhibitory factor; TNF: tumor necrosis factor; TGF: transforming growth factor. Additionally, * represents the cytokine expression at the molecular level in this study and previous study [13].