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, 28 (12), 3032-3046.e6

A VP35 Mutant Ebola Virus Lacks Virulence but Can Elicit Protective Immunity to Wild-Type Virus Challenge

Affiliations

A VP35 Mutant Ebola Virus Lacks Virulence but Can Elicit Protective Immunity to Wild-Type Virus Challenge

Courtney Woolsey et al. Cell Rep.

Abstract

Zaire ebolavirus (EBOV) VP35 protein is a suppressor of type I interferon (IFN) production, an inhibitor of dendritic cell maturation, and a putative virulence determinant. Here, a recombinant EBOV encoding a mutant VP35 virus (VP35m) is demonstrated to activate RIG-I-like receptor signaling and innate antiviral pathways. When inoculated into cynomolgus macaques, VP35m exhibits dramatic attenuation as compared to wild-type EBOV (wtEBOV), with 20 or 300 times the standard 100% lethal challenge dose not causing EBOV disease (EVD). Further, VP35m infection, despite limited replication in vivo, activates antigen presentation and innate immunity pathways and elicits increased frequencies of proliferating memory T cells and B cells and production of anti-EBOV antibodies. Upon wtEBOV challenge, VP35m-immunized animals survive, exhibiting host responses consistent with an orderly immune response and the absence of excessive inflammation. These data demonstrate that VP35 is a critical EBOV immune evasion factor and provide insights into immune mechanisms of EBOV control.

Keywords: Ebola; RIG-I; RLR signaling; VP35; filovirus; innate immunity; interferon; pathogenesis; primate.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. VP35 Regulates MAVS-Dependent Host Responses to wtEBOV Infection
(A) IFN-β promoter reporter assay to assess VP35 mutant IFN-inhibitory function. HEK293T cells were transfected with plasmids expressing wt VP35 or VP35 mutants (F239A, K319A, R322A, KRA-K319A/R322A, FKRA, and F239A/K319A/R322A at 10 or 100 ng) along with an IFN-β promoter-firefly reporter plasmid and Renilla reporter as a transfection control. SeV infection was used as an inducer of RIG-I activation and 20 h later luciferase activities were determined. Fold increase indicates Firefly luciferase normalized to Renilla luciferase values, with the empty vector (E) value set to 1. The error bars represent SD of three independent replicates. The p value was determined by a one-way ANOVA test followed by multiple comparisons using Tukey’s test. *p < 0.05, **p < 0.001, ***p <0.0005, ****p < 0.0001. (B) Average viral titers of wtEBOV or VP35m in A549 wt or MAVS KO cells. Supernatants from infected cells were plated in duplicate and viral titers were determined at the indicated hours post-infection by plaque assay. PFU, plaque-forming units. The error bars represent SD of two independent replicates. (C) Venn diagram comparing DEGs between VP35m- (n = 2) and wtEBOV-infected (n = 2) A549 cells at 72 h post-infection. Heatmap representing functional enrichment of DEGs common to either infection or exclusively only following VP35m or wtEBOV infection; color intensity represents the statistical significance (shown as −log10 of the q-value); range of colors is based on the lowest and highest −log10(q-value) for the entire set of GO processes. The number of DEGs enriching to each GO term is listed within each box; blank boxes represent lack of significant enrichment to a given GO term. (D and E) Heatmap representing gene expression (shown as absolute normalized reads per kilobase per million mapped reads [RPKM] values) of DEGs that enriched to “defense response to virus” in A549 cells (D) or A549 MAVS KO cells (E) infected with wtEBOV or VP35m at 1 and 72 h. The range of colors is based on scaled and centered RPKM values of the entire set of genes (red represents increased expression, while blue represents decreased expression); each column represents one biological replicate. (F) Venn diagram comparing DEGs between VP35m- (n = 2) and wtEBOV-infected (n = 2) A549 MAVS KO cells at 72 h post-infection. Heatmap representing functional enrichment of DEGs common to either infection or exclusively only following VP35m or wtEBOV infection. (G) Heatmap representing gene expression of DEGs common to either infection that enriched to “cell division”; each column represents one biological replicate.
Figure 2.
Figure 2.. VP35m Infection Results in Mild Clinical Signs and Protects Animals against Subsequent wtEBOV Challenge
(A) Study timeline. (B) Plasma levels of C-reactive protein (CRP). (C) Viral loads determined by qRT-PCR using probe targeting VP30. (D) Infectious virus was quantified by plaque assay on Vero cells. (E) Clinical score. (F) Aspartate transaminase (AST) and blood urea nitrogen (BUN) levels. (G) Platelet counts. (H) White blood cells (WBCs) and lymphocyte (LY) counts. Data in (B)–(H) are presented as mean ± SEM. *p % 0.05 and **p % 0.01 at the indicated time point compared to 0 DPI. The VP35m underlined time points correspond to the challenge with the VP35m portion of the study. The wtEBOV underlined time points correspond to the challenge with wtEBOV of animals previously infected with VP35m.
Figure 3.
Figure 3.. VP35m Induced Antigen Presentation and Regulated Innate Immunity
(A and C) Bar graph depicting statistically significant GO processes to which cluster 3 (A) or cluster 4 (C) genes enriched; the line graph represents −log10(false discovery rate [FDR]) of the enriched term. (B and D) Heatmaps representing gene expression (shown as absolute normalized RPKM values) of the DEGs in cluster 3 that enriched to “inflammatory response” and “response to cytokine” (B) and DEGs in cluster 4 that enriched to response to cytokine (D). The range of colors is based on scaled and centered RPKM values of the entire set of genes (red represents increased expression, while blue represents decreased expression); each column represents the median RPKM values for each DPI. For gene expression data shown in (A)–(D), the number of biological replicates at each time point are as follows: day 0 (n = 5), day 3 (n = 5), day 6 (n = 4), day 10 (n = 5). (E) Monocyte (CD14+HLA-DR+/−) and dendritic cell (DC; CD14−HLA-DR+) levels measured by flow cytometry (n = 4). (F) Frequency of classical (CD16) and CD16+ intermediate or nonclassical monocyte subsets (n = 4). (G) Frequency of classical and CD16+ intermediate or nonclassical monocyte subsets expressing CD86 (n = 4). (H) Frequencies of myeloid DCs (mDCs; CD123CD11c+), plasmacytoid DCs (pDCs; CD123+CD11c), and other DCs (CD123CD11c+) (n = 4). (I) Frequency of DC subsets expressing CD86 (n = 4). Data are presented as mean ± SEM in (C)–(F). For changes in the frequencies within innate immune subsets, a nonparametric trend where each time point is modeled by its own mean was assumed for statistical analysis; *p % 0.05 compared to 0 DPI; *p % 0.05 for CD16monocytes and mDCs and #p ≤ 0.05 for other DCs.
Figure 4.
Figure 4.. Disruption of VP35 Function Resulted in Development of Adaptive Immunity
(A) Bar graph depicting statistically significant GO processes to which cluster 5 genes enriched; the line graph represents −log10(FDR) of the enriched term. (B) Heatmap representing gene expression (shown as absolute normalized RPKM values) of the DEGs in cluster 5 that enriched to “immune effector process”; range of colors is based on scaled and centered RPKM values of the entire set of genes (red represents increased expression while blue represents decreased expression); each column represents the median RPKM values for each DPI. For gene expression data in (A) and (B), the number of biological replicates at each time point are as follows: day 0 (n = 5), day 3 (n = 5), day 6 (n = 4), and day 10 (n = 5). (C) Frequency of CD4+ and CD8+ T cells and CD20+ B cells (n = 4). (D and E) The magnitude of CD4 T cell (n = 4) (D) and CD8 T cell (n = 4) (E) proliferation was determined by measuring changes in the frequency of Ki67+ cells within naive, central (CM), and effector (EM) memory subsets. (F) B cell proliferation was determined by measuring the frequency of Ki67+ within naive and memory subsets (n = 4). (G) Average IgG antibody titers directed against EBOV GP as measured in duplicate by ELISA (n = 4). (H and I) Frequency of naive, CM, and EM T cells within CD4 (H) and CD8 (I) subsets. (J) Frequency of naive and memory B cells (n = 4). Data are presented as mean ± SEM in (C)–(J). For changes in proliferation frequencies within lymphocyte subsets, a nonparametric trend where each time point is modeled by its own mean was assumed for statistical analysis; *p ≤ 0.05 and #p ≤ 0.05 at the indicated time point compared to 0 DPI (*p ≤ 0.05 for Ki67+ CD4 CM T cells, Ki67+ CD8 CM T cells, CD8 EM T cells and memory B cells and #p % 0.05 for Ki67+ CD8 EM T cells).
Figure 5.
Figure 5.. Animals Exhibit Sustained Gene Expression Changes Associated with Cellular Metabolism and Cell Cycle following VP35m Infection and Limited Gene Expression Changes following Subsequent wtEBOV Challenge
(A) Bar graph depicting GO processes to which cluster 1 genes (see Figure S3B) enriched; the line graph represents −log10(FDR) of the enriched term. (B) Heatmap representing gene expression (shown as absolute normalized RPKM values) of the DEGs in cluster 1 that enriched to “response to stress.” The range of colors is based on scaled and centered RPKM values of the entire set of genes (red represents increased expression, while blue represents decreased expression); each column represents the median RPKM values for each DPI. (C) Bar graph depicting statistically significant GO processes to which cluster 2 genes (see Figure S3C) enriched; the line graph represents −log10(FDR) of the enriched term. (D) Network depicting direct interactions of DEGs in cluster 2 with an RPKM R25 that map to the GO process “cellular metabolic process.” For gene expression data shown in (A)–(D), the number of biological replicates at each time point are as follows: day 0 (n = 5), day 3 (n = 5), day 6 (n = 4), and day 10 (n = 5). (E) Bar graph depicts number of protein-coding differentially expressed genes (DEGs; defined as those R2-fold change compared to 0 DPI and FDR-corrected p value ≥ 0.05) that have human homologs in VP35m-infected animals following challenge with wtEBOV (VP35m-wtEBOV); day 0 (n = 3), day 3 (n = 4), day 6 (n = 4), day 10 (n = 4), and day 28 (n = 4). (F) Bar graph depicting statistically significant GO processes to which DEGs at 10 and 28 days after back-challenge enriched; the line graph represents −log10(FDR) of the enriched term.

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