Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host

Abstract

Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

Fig 1. E. chaffeensis-specific IgG response following Ech_0660 vaccination and secondary challenge with wild-type E. chaffeensis.

Total E. chaffeensis-specific IgG was measured in the plasma at multiple time points by ELISA in dogs vaccinated with the Ech_0660 mutant and challenged with wild-type E. chaffeensis via needle inoculation (group 1), or vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis via tick-transmission (group 2). Unvaccinated control dogs were infected with wild-type E. chaffeensis or the non-attenuated Ech_0480 mutant via tick-transmission (group 3). Each line is representative of a single animal.

Fig 2. CD4⁺ T cells from Ech_0660 mutant vaccinated and wild-type E. chaffeensis infected animals proliferate in response to E. chaffeensis antigen.

PBMC from dogs vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis via needle inoculation (group 1, left panels), vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis via tick inoculation (group 2, middle panels), or unvaccinated and infected with wild-type E. chaffeensis or Ech_0480 via tick inoculation (group 3, right panels) were labeled with Cell Trace Violet, then cultured for 5 days at 4x10⁶ cells/mL in the presence or absence of 10 ug/mL E. chaffeensis host-cell free lysate grown in the tick ISE6 cell line. On day 5, CD4⁺ T cells were analyzed by flow cytometry for Cell Trace Violet dilution as a measure of proliferation. (A) Representative Cell Trace Violet dilution profiles, gated on total live cells and total CD3⁺CD4⁺ T cells. (B) The percentage of CD4⁺ T cells that have proliferated in response to E. chaffeensis antigens as measured over the course of the experiment. The background (mock stimulated) proliferation was subtracted, and results represent change over mock. Each line is representative of a single animal.

Fig 3. CD4⁺ T cells from Ech_0660 mutant vaccinated and wild-type E. chaffeensis infected animals produce IFNγ in response to E. chaffeensis antigen.

PBMC from dogs vaccinated with the Ech_0660 mutant and challenged with wild-type E. chaffeensis (groups 1–3, as in Fig 2) were cultured for 5 days at 4x10⁶ cells/mL in the presence or absence of 10 ug/mL E. chaffeensis host-cell free lysate grown in the tick ISE6 cell line. On day 5, brefeldin A was added for the last 6 hours of culture. CD4⁺ T cells were stained for intracellular expression of IFNγ and analyzed by flow cytometry. (A) Representative flow plots from animals in groups 1, 2 and 3, gated on total live cells and total CD3⁺CD4⁺ T cells. (B) The percentage of IFNγ⁺ cells of total CD4⁺ T cells in the blood measured over the course of the experiment. Background (mock stimulated) IFNγ production was subtracted, and results represent change over mock.

Fig 4. CD8⁺ T cells from Ech_0660 vaccinated and wild-type E. chaffeensis infected animals proliferate and produce IFNγ in response to E. chaffeensis antigen.

CD8⁺ T cell proliferation and IFNγ production were measured using similar approaches as in Figs 2 and 3. PBMC from dogs in groups 1–3 were cultured for 5 days at 4x10⁶ cells/mL in the presence or absence of 10 ug/mL E. chaffeensis host-cell free lysate. On day 5 of culture, CD8⁺ T cells were analyzed by flow cytometry for (A) proliferation as measured by Cell Trace Violet dilution; and (B) intracellular production of IFNγ. The frequencies of responding CD8⁺ T cells were measured over the course of the experiment. Results were gated on total live cells and total CD3⁺CD8⁺ T cells. Background (mock stimulated) proliferation or IFNγ production was subtracted and results represent change over mock.

Fig 5. PBMC from Ech_0660 vaccinated and wild-type E. chaffeensis infected animals secrete IFNγ and IL-17 in response to E. chaffeensis antigen.

PBMC from dogs vaccinated with Ech_0660 and challenged with wild-type E. chaffeensis (groups 1–3, as in Fig 2) were collected on day 7 post-secondary challenge with wild-type E. chaffeensis. PBMC were cultured for 5 days at 4x10⁶ cells/mL in the presence or absence of 10 ug/mL E. chaffeensis host-cell free lysate grown in the tick ISE6 cell line. On day 5, cell culture supernatants were collected and later analyzed by ELISA for secretion of (A) IFNγ, (B) IL-17, and IL-4 (not shown). Each bar is representative of a single animal.