Persistent infection with ebola virus under conditions of partial immunity

Abstract

Ebola hemorrhagic fever in humans is associated with high mortality; however, some infected hosts clear the virus and recover. The mechanisms by which this occurs and the correlates of protective immunity are not well defined. Using a mouse model, we determined the role of the immune system in clearance of and protection against Ebola virus. All CD8 T-cell-deficient mice succumbed to subcutaneous infection and had high viral antigen titers in tissues, whereas mice deficient in B cells or CD4 T cells cleared infection and survived, suggesting that CD8 T cells, independent of CD4 T cells and antibodies, are critical to protection against subcutaneous Ebola virus infection. B-cell-deficient mice that survived the primary subcutaneous infection (vaccinated mice) transiently depleted or not depleted of CD4 T cells also survived lethal intraperitoneal rechallenge for >/==" BORDER="0">25 days. However, all vaccinated B-cell-deficient mice depleted of CD8 T cells had high viral antigen titers in tissues following intraperitoneal rechallenge and died within 6 days, suggesting that memory CD8 T cells by themselves can protect mice from early death. Surprisingly, vaccinated B-cell-deficient mice, after initially clearing the infection, were found to have viral antigens in tissues later (day 120 to 150 post-intraperitoneal infection). Furthermore, following intraperitoneal rechallenge, vaccinated B-cell-deficient mice that were transiently depleted of CD4 T cells had high levels of viral antigen in tissues earlier (days 50 to 70) than vaccinated undepleted mice. This demonstrates that under certain immunodeficiency conditions, Ebola virus can persist and that loss of primed CD4 T cells accelerates the course of persistent infections. These data show that CD8 T cells play an important role in protection against acute disease, while both CD4 T cells and antibodies are required for long-term protection, and they provide evidence of persistent infection by Ebola virus suggesting that under certain conditions of immunodeficiency a host can harbor virus for prolonged periods, potentially acting as a reservoir.

FIG. 1.

Survival and viral kinetics in wild-type (+/+), TCRβ^−/−, B-cell^−/−, β2m^−/−, and CD4^−/− mice infected with Ebola virus. (A) Viral kinetics in mice subcutaneously challenged with 100 PFU of Ebola virus. Viral antigens were assayed in the spleen (○), liver (□), kidney (▴), and serum (•) from days 3 to 16 after infection (n = 3 mice per group per time point). Antigen was assayed by capture ELISA and plotted as mean ± standard error. The detection limit of the assay is indicated by dotted lines. Wild-type, CD4^−/−, and B-cell^−/− mice efficiently controlled the viral load, whereas viral antigen levels were 2 to 3 logs higher in TCRβ^−/− and β2m^−/− groups than in controls. (B) Survival of wild-type (n = 10; ○), TCRβ^−/− (n = 5; □), B-cell^−/− (n = 10; ▪), CD4^−/− (n = 4; ▴), and β2m^−/− (n = 13; •) mice challenged subcutaneously with 100 PFU of Ebola virus. All β2m^−/− and TCRβ^−/− mice died by day 16 or 22 after infection, while all CD4^−/− and B-cell^−/− mice survived the infection.

FIG. 1.

Survival and viral kinetics in wild-type (+/+), TCRβ^−/−, B-cell^−/−, β2m^−/−, and CD4^−/− mice infected with Ebola virus. (A) Viral kinetics in mice subcutaneously challenged with 100 PFU of Ebola virus. Viral antigens were assayed in the spleen (○), liver (□), kidney (▴), and serum (•) from days 3 to 16 after infection (n = 3 mice per group per time point). Antigen was assayed by capture ELISA and plotted as mean ± standard error. The detection limit of the assay is indicated by dotted lines. Wild-type, CD4^−/−, and B-cell^−/− mice efficiently controlled the viral load, whereas viral antigen levels were 2 to 3 logs higher in TCRβ^−/− and β2m^−/− groups than in controls. (B) Survival of wild-type (n = 10; ○), TCRβ^−/− (n = 5; □), B-cell^−/− (n = 10; ▪), CD4^−/− (n = 4; ▴), and β2m^−/− (n = 13; •) mice challenged subcutaneously with 100 PFU of Ebola virus. All β2m^−/− and TCRβ^−/− mice died by day 16 or 22 after infection, while all CD4^−/− and B-cell^−/− mice survived the infection.

FIG. 2.

Protective immunity against Ebola virus challenge in B-cell^−/− mice. (A) Survival in mice challenged by intraperitoneal inoculation with Ebola virus. Naive wild-type (+/+; n = 5; □), B-cell^−/− (n = 5; ▵), or vaccinated wild-type (n = 20; •) and B-cell^−/− (n = 20; ♦) mice were infected intraperitoneally with 10⁶ PFU of Ebola virus and observed for survival. (B) Viral antigen titers in naive and vaccinated wild-type and B-cell^−/− mice infected intraperitoneally with 10⁶ PFU of Ebola virus. Naive wild-type and B-cell^−/− mice (open symbols) or vaccinated wild-type and B-cell^−/− mice (closed symbols) were sacrificed (three mice per group per time point) on days 2, 4, and 6 after infection and assayed for viral antigen in the spleen (○, •), liver (□, ▪), kidney (▵, ▴), and serum (⋄, ⧫). Mice were vaccinated subcutaneously with 100 PFU of Ebola virus 21 days prior to intraperitoneal challenge. Antigens were assayed by capture ELISA and plotted as mean ± standard error for each experimental group.

FIG. 3.

Memory CD8 T cells protect B-cell^−/− mice against lethal Ebola virus challenge. (A) Vaccinated B-cell^−/− mice undepleted (○) or depleted of CD4 T cells (•), CD8 T cells (▴), or both CD4 and CD8 cells (♦) were challenged intraperitoneally with 10⁶ PFU of Ebola virus and observed for survival (five mice per group). (B) Memory CD8 T cells are required for viral control in vaccinated B-cell^−/− mice. Vaccinated B-cell^−/− mice (undepleted or depleted of CD4, CD8, or both CD4 and CD8 T cells) were challenged with 10⁶ PFU of Ebola virus intraperitoneally 21 days after vaccination. Viral antigen levels were determined by antigen capture ELISA in the spleen (spl), liver (liv), kidney (kid), and serum (ser) on day 4 after infection (three mice per group per time point). Both undepleted and CD4 T-cell-depleted B-cell^−/− mice survived and efficiently controlled viral replication (similar to wild-type mice), whereas CD8-depleted and CD4- and CD8-depleted groups had high viral antigen titers on day 4 and died by day 8 after infection. Data are plotted as mean ± standard error.

FIG. 4.

Ebola virus persistence in mice with partial immunity. (A) The indicated groups of vaccinated mice were challenged intraperitoneally with 10⁶ PFU of Ebola virus, and animals were euthanized on days 5, 57, 70, 120, and 150 after infection. Viral antigen in the indicated tissues was quantitated by capture ELISA (bars), and infectious virus was measured by plaque assay (diamonds). Each pair of symbols (bar and diamond) represents an individual mouse. The detection limit of the antigen assay was 1:40, and for plaque assay it was 400 PFU/ml. (B) Survival of mice under different immunodeficiency conditions following Ebola virus infection. Vaccinated wild-type (+/+; n = 5; ○) or undepleted vaccinated B-cell^−/− mice challenged intraperitoneally with 10⁶ PFU of Ebola virus (n = 4; •) or vaccinated B-cell^−/− mice depleted of CD4 T cells (n = 6; ▪) 1 day prior to intraperitoneal challenge were observed for survival. Vaccinated B-cell^−/− mice (n = 3; □) not challenged intraperitoneally were also included. All mice in the three groups (i.e., vaccinated B-cell^−/−, vaccinated and intraperitoneally challenged wild-type, and B-cell^−/− mice) survived the infection, and none died during the 210-day observation period, whereas CD4 T-cell-depleted vaccinated B-cell^−/− mice died between 50 and 210 days after intraperitoneal challenge.

FIG. 5.

Pathological changes and persistence of Ebola virus in chronically infected mice. Naive B-cell^−/− and vaccinated CD4 T-cell-depleted B-cell^−/− mice were infected intraperitoneally with 10² or 10⁶ PFU, respectively, of Ebola virus. Liver sections from a naive mouse on day 5 (A and B) and a vaccinated B-cell^−/− mouse on day 70 (C and D) after infection were examined for the presence of viral antigen by immunohistochemistry (A and C; magnification, 50×) and for liver pathology by hematoxylin and eosin staining (B and D; magnification, 25×) and found to have comparable viral antigen titers (10⁵ to 10⁶) in liver homogenates. Insets show high-power (158×) views of the same sections, demonstrating infected hepatocytes (small arrow) and Kupffer's cell (large arrow) in an acutely infected mouse (A) and infected histiocytes (arrows) in a chronically infected mouse (C). Hematoxylin and eosin staining shows infiltrating neutrophils (arrows) in the day 5 liver section from a naive mouse (B) and a granuloma with histiocytes (arrows) in the day 70 liver section from a vaccinated CD4 T-cell-depleted B-cell^−/− mouse (D).