Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment

Abstract

The study of Ebola virus (EBOV) pathogenesis in vivo has been limited to nonhuman primate models or use of an adapted virus to cause disease in rodent models. Herein we describe wild-type EBOV (Makona variant) infection of mice engrafted with human hematopoietic CD34⁺ stem cells (Hu-NSG™-SGM3 mice; hereafter referred to as SGM3 HuMice). SGM3 HuMice support increased development of myeloid immune cells, which are primary EBOV targets. In SGM3 HuMice, EBOV replicated to high levels, and disease was observed following either intraperitoneal or intramuscular inoculation. Despite the high levels of viral antigen and inflammatory cell infiltration in the liver, the characteristic histopathology of Ebola virus disease was not observed, and this absence of severe immunopathology may have contributed to the recovery and survival of some of the animals. Future investigations into the underlying mechanisms of the atypical disease presentation in SGM3 HuMice will provide additional insights into the immunopathogenesis of severe EBOV disease.

Keywords: Ebola; flow cytometry; hemorrhagic fever; humanized NSG-SGM3 mice; immune response; immunopathology; myeloid cell; virus.

Figure 1.

Weight loss and survival of SGM3 HuMice inoculated with Ebola virus (EBOV) Makona. A and B, Weight change in humanized mice with CD34⁺ cells from donor 1 (DR1) and donor 2 (DR2) inoculated intraperitoneally (A) or intramuscularly (B) with Dulbecco's modified Eagle's medium (DMEM; mock) or EBOV Makona at indicated doses. Data are presented on indicated days after infection. C, Survival in HuMice inoculated intraperitoneally or intramuscularly with DMEM (DR1 and DR2; n = 8) or with 10³ focus-forming units (FFU) intraperitoneally (DR1 and DR2; n = 6), 10³ FFU intramuscularly (DR1 and DR2; n = 6), 10 FFU intraperitoneally (DR1; n = 3), or 10 FFU intramuscularly (DR1; n = 3) of EBOV Makona.

Figure 2.

Ebola virus (EBOV) RNA loads in the blood and tissues of SGM3 HuMice. RNA quantitation in blood (A), spleen (B), and liver (C) samples collected at the indicated time points from HuMice inoculated with the indicated doses of EBOV Makona. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was specific for the EBOV nucleoprotein (NP) gene, and focus-forming unit (FFU) equivalents were based on standards generated from stock virus. Samples collected 1 and 3 days after infection are from DR1; samples collected 5 and 8 days after infection are from intramuscularly infected DR2 animals only; no intraperitoneally infected animals were analyzed at these time points. Terminal and survivor samples are from both donors and are from animals in Figure 1 that were euthanized because of disease (terminal) or at 28 days after infection (survivor).

Figure 3.

Histopathologic changes and antigen staining of spleen and liver serial samples in SGM3 HuMice inoculated with Ebola virus (EBOV). Original magnification ×100 of spleen and liver samples stained with hematoxylin-eosin (HE) or an EBOV-specific anti-VP40 antibody to assess pathologic changes and antigen distribution in animals inoculated intramuscularly (10³ focus-forming units). Control animals had a baseline low level of macrophage infiltration (top row). By 5 days after infection, antigen was apparent in both spleen and liver, and by 8 days after infection, extensive macrophage infiltration appears in both the liver and spleen of infected animals. Antigen was present in the red pulp of the spleen and in most hepatocytes of the liver. Terminal samples were similar to samples collected 8 days after infection. In survivors (28 days after infection; intramuscularly or intraperitoneally inoculated), antigen was less abundant and infiltrating macrophages less apparent in both tissue types (bottom row). Abbreviation: IHC, immunohistochemistry analysis.

Figure 4.

High magnification of histopathologic changes and antigen staining in the livers of SGM3 HuMice inoculated with Ebola virus (EBOV). Tissues are stained as described in Figure 3; panels are representative of SGM3 HuMouse samples obtained 8 days after infection, and images are 400× original magnification. The hepatic architecture was disrupted by infiltrating macrophages, many of which contained a brown granular pigment (by hematoxylin-eosin [HE] staining; black arrows), and by lymphocytes and multinucleate giant cells (by HE staining; white arrows). Numerous hepatocytes contained eosinophilic intracytoplasmic inclusions (by HE staining; blue arrows). Viral antigen was detected in hepatocytes (by immunohistochemistry [IHC] analysis; white arrows) and macrophages (by IHC analysis; blue arrows) and was lining the space of Disse (by IHC analysis; black arrows). In the spleen, the red pulp contained numerous macrophages, many with a brown granular pigment (by HE staining; black arrows). Viral antigen was primarily found within macrophages (by IHC analysis; blue arrow) and extracellularly in the red pulp. Small numbers of cells morphologically consistent with dendritic cells (by IHC analysis; black arrows) were positive for viral antigen within the lymphoid follicular center.

Figure 5.

Flow cytometric assessment of the human immune system development in naive SGM3 HuMice. A, Splenocytes from naive animals (20 weeks after engraftment) stained with mouse- or human-specific CD45 to determine reconstitution and residual mouse leukocyte levels. B and C, Blood cell (B) and splenocyte (C) frequencies in naive animals were determined following staining with the indicated immune cell markers. Panels are representative of 3 animals. Abbreviations: HLA-DR, human leukocyte antigen–D related, major histocompatibility complex class II antigen; mDC, myeloid dendritic cells; NK, natural killer; pDC, plasmacytoid dendritic cells.

Figure 6.

Flow cytometric analysis of blood leukocytes of SGM3 HuMice. Red blood cells were lysed from whole blood, and remaining cells were stained with multicolor panels of antibodies specific for myeloid and lymphoid cell types and for overall leukocytes (CD45). Data for each panel are from 3 mock-inoculated HuMice (closed circles) and 3 HuMice intramuscularly inoculated with Ebola virus (10³ focus-forming units; open circles) from each time point (in days). *P < .5, **P < .1, and ***P < .01, by 1-way analysis of variance, with a Tukey post hoc test. Abbreviations: gMFI, geometric mean fluorescence intensity; mDC, myeloid dendritic cells; NK, natural killer; ns, nonsignificant.

Figure 7.

Flow cytometric analysis of splenocytes of SGM3 HuMice. Single-cell suspensions from spleen samples were lysed, and splenocytes were stained with multicolor panels of antibodies specific for myeloid and lymphoid cell types and overall leukocytes (CD45). Data for each panel are from 3 mock-inoculated HuMice (closed circles) and 3 HuMice intramuscularly inoculated with Ebola virus (10³ focus-forming units; open circles) from each time point (in days). *P < .5, **P < .1, and ***P < .01, by 1-way analysis of variance, with a Tukey post hoc test. Abbreviations: gMFI, geometric mean fluorescence intensity; mDC, myeloid dendritic cells; ns, nonsignificant.