Chimeric Mice with Competent Hematopoietic Immunity Reproduce Key Features of Severe Lassa Fever

<div><p>Lassa fever (LASF) is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR^-/-) was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology.</p></div

Depletion of myeloid cells has no impact on the disease.

<p>(A) Mice were depleted for CD11b or CD11c cells by treating IFNAR^{-/- CD11b} or IFNAR^{-/- CD11c} mice with diphtheria toxin prior to infection. The mice were inoculated i. p. with 1,000 FFU LASV Ba366. Mice were monitored for survival, weight loss and temperature. AST activity, virus titer in blood and virus titer in organs were measured at the indicated time points. The normal range for AST and the limit of detection for the virus titer in blood are shaded in gray. Mean and standard deviation are shown. Statistical analysis was done as indicated in Material and Methods. (B) Bone marrow derived macrophages (upper graph) and dendritic cells (lower graph) were inoculated with different pathogenic and non-pathogenic arenaviruses with a MOI of 0.01. Aliquots of the cell culture supernatant were taken daily and analyzed for infectious virus particles (n = 3).</p

CD8 T cells drive vascular leakage during LASV infection.

<p>(A) IFNAR^{-/- Bl6} mice were depleted of CD8 T cells with anti-CD8 antibody or remained untreated as indicated in the figure. Each group consisted of n = 5 mice. Mice were inoculated i. p. with 1,000 FFU MORV, LASV Ba366 or were Mock infected with PBS. Mice were injected intravenously with 100 μl Evans Blue on day 7 p. i., euthanized and perfused with NaCl. Lung and Liver were analyzed for vasculear leakage. Mean and standard deviation are shown. Differences in vasculear leakage were analyzed using Mann-Whitney non-parametric test. (B) Plasma concentrations of FAS, FAS ligand and TNFα were determined via Luminex in samples obtained at days 1, 4 and 7 p. i. from CD8 depleted or undepleted IFNAR^{-/- Bl6} mice, infected with MORV (in red) or LASV Ba366 (in black). Mean and standard deviation are shown. Differences in the curves were analyzed using Kruskal-Wallis test and Dunn’s post test.</p

Depletion of rescues LASV-infected mice.

<p>IFNAR^{-/- Bl6} mice were depleted for CD4 T cells (B), CD8 T cells (C) or both (D) with anti-CD4 or anti-CD8 antibodies. Control mice (A) remained untreated (Not depleted) or received an isotype control antibody (isotype control). All groups were inoculated i. p. with 1,000 FFU of LASV Ba366. Mice were monitored for survival, weight loss and temperature. AST activity, virus titer in blood and virus titer in organs were measured at the indicated timepoints. The normal range for AST and the limit of detection for the virus titer in blood are shaded in gray. Mean and standard deviation are shown. The T cell depleted groups were compared to the undepleted group. Statistical analysis was done as indicated in Material and Methods.</p

Inflammation in response to LASV vs MORV infection.

<p>(A) Chimeric IFNAR^{-/- Bl6} mice were inoculated with 1,000 FFU LASV Ba366, MORV or mock infected. 7 days p. i. liver sections were processed for immunohistochemical staining using the indicated antibodies. Mice were inoculated i. p. with 1,000 FFU LASV or MORV and euthanized at day 0, 4 and 7 p. i. (n = 3). Changes in cellularity of CD4 and CD8 T cells (B) and granulocytes (C) were analyzed using flow cytometry. Mean and standard deviation are shown. Differences in the curves were analyzed via Two-Way ANOVA. (D). Mice were inoculated i. p. with 1,000 FFU LASV Ba366 or MORV and euthanized at day 7 p. i. The virus titers in spleen and liver were determined. Mean and standard devation are shown. Statistical analysis was done using Mann-Whitney non-parametric test.</p

Chimeric mice show signs of infection-associated immunopathology.

<p>(A) Chimeric IFNAR^{-/- Bl6} mice, Bl6^IFNAR-/- mice, IFNAR^{-/- IFNAR-/-} mice or Bl6^Bl6 mice were inoculated i.p with 1,000 FFU with LASV Ba366. Plasma concentration of FAS, FAS ligand, TNFα, Granzyme B, IFNγ and MIP-1β were analyzed at the indicated time points post infection via Luminex assay. Mean and standard deviation are shown. Differences between the indicated groups were analysed via non-parametric Kruskal-Wallis test followed by Dunn’s post-test. (B) Chimeric IFNAR^{-/- Bl6} mice, Bl6 ^IFNAR-/- mice, IFNAR^{-/- IFNAR-/-} mice or Bl6 ^Bl6 mice were inoculated i.p with 1,000 FFU with LASV Ba366. Seven days post infection liver tissue sections were processed for H&A staining. The arrows indicate areas of lymphocytic infiltration.</p

Chimeric mice are susceptible to infection with LASV.

<p>Scheme of the generation of chimeric mice (A). Chimeric IFNAR^{-/- Bl6} mice (B), Bl6 ^IFNAR-/- mice (C), IFNAR^{-/- IFNAR-/-} mice (D) or Bl6 ^Bl6 mice (E) were inoculated i.p with 1,000 FFU with LASV Ba366. Survival, relative weight loss, changes in temperature, AST activity and viremia in blood and in organs were measured. For the determination of virus titers in organs, representative mice of each group were sacrificed on day 7 p.i. The normal range for AST and the limit of detection for the virus titer in blood are shaded in gray. Mean and standard deviation are shown.</p

Chimeric mice are less susceptible to non-pathogenic arenaviruses.

<p>Chimeric IFNAR^{-/- Bl6} mice (A), Bl6 ^IFNAR-/- mice (B), IFNAR^{-/- IFNAR-/-} mice (C) or Bl6 ^Bl6 mice (D) were inoculated i.p. with 1,000 FFU LASV Ba366 (in black, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005656#ppat.1005656.g001" target="_blank">Fig 1</a>) or MORV (in red). Survival, relative weight loss, changes in temperature, AST activity and viremia in blood were measured. The normal range for AST and the limit of detection for the virus titer in blood are shaded in gray. Mean and standard deviation are shown. Statistical analysis was done as indicated in Material and Methods.</p

NK cells in survivors and in fatalities.

<p>The frequency of NK (<b>A</b>), and of NK subsets (<b>B:</b> CD56^bright, CD56^dim, and CD56^neg) was analyzed in HD (n = 14), EBOV-survivors (n = 7) and EBOV-fatalities (n = 6). Statistical analysis was performed by using Mann Whitney test and differences were considered significant with a p<0.05, and highlighted with an asterisk. *: p<0.05; **: p<0.01; ***: p<0.001.</p

Frequency of Vδ2 T-cells in survivors and in fatalities.

<p>The frequency of Vδ2 (<b>A</b>), Vδ2^posCD95^pos (<b>B</b>), of Vδ2^posCCR7^neg (<b>C</b>) and Vδ2^posCTLA-4^pos (<b>D</b>) was analyzed in HD (n = 14), EBOV-survivors (n = 10) and EBOV-fatalities (n = 6). Statistical analysis was performed by using Mann Whitney test and differences were considered significant with a p<0.05 and highlighted with an asterisk.*: p<0.05; **: p<0.01; ***: p<0.001.</p