Fig 1.
Study Design and comparison of viral loads in vaccinated macaques exposed to MARV-Angola.
Created with BioRender (https://biorender.com/). (A) rVSV-N4CT1-MARV-GP vector design and genome organization. The Vesiculovax vaccine encodes a truncated form (CT1) of the native VSV G (green box); the N gene was translocated from the 1st to the 4th genomic position (N4) with respect to the 3’ transcriptional promoter (blue box). The MARV GP glycoprotein is expressed from the 1st gene position from the single 3’-proximal promoter site to increase immunogen expression (orange box). (B) Survival curves of vaccinated cohorts immunized with rVSV-N4CT1-MARV-GP at -7 DPI (blue; n = 5), -5 DPI (brown; n = 5), or -3DPI (red; n = 5). A single vector control subject was vaccinated at each respective time point (black; n = 3). A log-rank test was used to determine statistical significance. Triangles on the x-axis indicate time of vaccination for each respective cohort. (C) Plasma viremia was measured by standard plaque assay at the denoted time points and reported as log10 PFU/ml. The limit of detection for this assay is 25 PFU/ml (indicated by a dotted horizontal line). (D) Viral loads were measured by RT-qPCR in whole blood and reported as log10 copies/ml at the denoted time points. The limit of detection for this assay is 1000 copies/ml (indicated by a dotted horizontal line). For (C) and (D), the average titer ± SEM is shown for each group at the denoted time points. Statistical significance was determined using a mixed-effects model with Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. Not significant (ns); p < 0.0332 (*); p < 0.0021 (**); p < 0.0002 (***). Vaccine vector created with BioRender (https://biorender.com/). Abbreviations: rVSV, recombinant Vesicular stomatitis virus; MARV, Marburg virus; GP, MARV glycoprotein; VSV, Vesicular stomatitis virus; G, VSV glycoprotein; N, VSV nucleoprotein; P, VSV phosphoprotein; M, VSV matrix protein; L, VSV polymerase; DPI, days post vaccination; PFU, plaque-forming units.
Fig 2.
Immunohistochemistry of liver and spleen tissue in MARV-infected macaques.
MARV antigen (red) IHC positivity in sinusoidal lining cells and Kupffer cells of the liver for the positive control (Control 1) (A), group day -5 vaccinated fatal subject (Fatal 1) euthanized 9 DPI (E), group day -3 vaccinated fatal subject (Fatal 4) euthanized 7 DPI (I). IHC positive mononuclear cells within the red and white pulp of the spleen for the positive control (Control 1) (B), group day -5 vaccinated fatal subject (Fatal 1) euthanized 9 DPI (F), group day -3 vaccinated fatal subject (Fatal 4) euthanized 7 DPI (J). No appreciable immunolabeling of the liver or spleen for macaques that survived from group day -7 (Survivor 1) (C, D), group day -5 (Survivor 7) (E), or group day -3 (Survivor 10) (I). All representative liver sections were taken at 20x magnification, and all spleen sections were taken at 10x magnification. IHC, immunohistochemistry; MARV, Marburg virus; DPI, days post infection.
Fig 3.
Principal component analysis and volcano plots depicting transcriptional changes in vaccinated macaques exposed to MARV-Angola.
(A) Shown are principal component (PC) analyses of all normalized transcripts on the day of challenge (0 DPI), and at early (3 DPI), mid (6 DPI), and late disease (10 DPI, or the terminal time point in fatal cases). Individual samples were filtered by disposition (fatal, survivor), disease state (0, 3, 6, 10/term DPI), group (day of vaccination; -7, -5, or -3 DPI), and group plus disposition (vector control (n = 3), -3 fatal (n = 4), -3 survivor (n = 1), -5 fatal (n = 1), -5 survivor (n = 4), -7 survivor (n = 5)). (B) Volcano plots displaying -log10(p-values) and log2 fold changes for each mRNA target on the day of challenge (0 DPI), and at early (3 DPI), mid (6 DPI), and late disease (10 DPI, or the terminal time point in fatal cases). Horizontal lines within the plot indicate adjusted p-value thresholds. Targets highlighted in blue indicate those differentially expressed in the survivor versus fatal group irrespective of time of vaccination or vector administered with a multiple hypothesis Benjamini-Hochberg false discovery rate (FDR) corrected p-value less than 0.5. Light blue dots denote mRNA targets with an adjusted p-value < 0.01; medium-light blue dots denote mRNA targets with an adjusted p-value < 0.05; medium-dark blue dots denote mRNA targets with an adjusted p-value < 0.10; dark blue dots denote mRNA targets with an adjusted p-value ≤ 0.50; gray open dots denote mRNA targets with an adjusted p-value > 0.50. Abbreviations: PC1 (principal component 1); PC2 (principal component 2); DPI, days post infection.
Fig 4.
Heatmaps depicting transcriptional changes in vaccinated macaques exposed to MARV-Angola.
The topmost upregulated (A) and downregulated (B) transcripts in vaccinated survivors immunized -7 DPI at late disease. Shown are expression changes in survivor cohorts (survivor -7 vacc (n = 5); survivor -5 vacc (n = 4)) and fatal groups (fatal vector control (n = 3); fatal -3 vacc (n = 4)) at each disease state (0, 3, 6, 10 DPI, or the terminal time point in fatal subjects) with respect to a pre-vaccination baseline. Red indicates high expression; blue indicates low expression; white indicates no change in expression. Abbreviations: Vacc, vaccinated; E, early disease (3 DPI); M, mid disease (6 DPI); L, late disease (10 DPI or terminal timepoint).
Fig 5.
Immune cell type profiling of Vesiculovax-vaccinated survivor and fatal samples.
Respective cell-type quantities for vaccinated survivor (n = 10) and fatal (n = 8) cohorts on the day of challenge (0 DPI), and early (3 DPI), mid (6 DPI), and late disease (10 DPI, or the terminal time point in fatal cases). A Benjamini-Hochberg false discovery rate (FDR) corrected p-value less than 0.05 was deemed significant for immune cell type profiling of transcripts. DPI, days post infection.
Fig 6.
Differentially expressed canonical signaling pathways in Vesiculovax-vaccinated macaques.
Heatmap of the most significantly upregulated and downregulated canonical pathways in survivor versus fatal subjects based on functional enrichment of differentially expressed transcripts (Benjamini-Hochberg false discovery rate (FDR) corrected p-value less than 0.05) in vaccinated survivor (n = 10) versus fatal (n = 8) samples. Cohorts were examined at early (3 DPI), mid (6 DPI), and late disease (10 DPI, or the terminal time point in fatal cases). DPI, days post infection. Corresponding z-scores were plotted. Red indicates high expression; blue indicates low expression; white indicates no difference in expression; gray indicates insufficient transcripts mapping to the pathway.
Fig 7.
Reciprocal endpoint dilution titers of anti-MARV GP IgG and IgM in Vesiculovax-vaccinated cynomolgus macaques.
MARV GP-specific (A) IgG and (B) IgM titers in vaccinated cohorts immunized with rVSV-N4CT1-MARV-GP at -7 DPI (blue; n = 5), -5 DPI (brown; n = 5), or -3DPI (red; n = 5), or a vector control (black; n = 3). The mean titer ± SEM is shown for each group at the denoted time points. Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; DPI, days post infection; term, terminal; MARV, Marburg virus; GP, glycoprotein; PRE, pre-vaccination baseline.
Fig 8.
Summary of potential mechanisms of Vesiculovax-mediated rapid protection.
Immunization of macaques with a rVSV-N4CT1-MARV-GP vaccine leads to early stimulation of innate immunity (e.g., transcriptional expression of ISG-, OAS-RNAse L-, and RIG-I-affiliated transcripts and HLA molecules), with resolution of the innate response coinciding with the development of a robust adaptive response and survival. Specifically vaccinated subjects exhibited increased MARV GP-specific antibodies (IgM, IgG). Moreover, protection with rVSV-N4CT1-MARV-GP correlated with higher predicted T helper 1- and cytotoxic cell-type quantities in surviving subjects as evidenced by the increased expression of transcripts encoding Tbet (TBX21), the master transcriptional regulator for T helper 1 cells, and cytoplasmic granule components (perforin and granzymes), respectively. Functional enrichment of differentially expressed transcripts (adjusted p-value < 0.05) in this study indicates potential ICOS, CD28, CD27, and lymphotoxin β signaling, suggesting the activation of effector T cells. Conversely, non-specific vaccination resulted in uncontrolled viral replication, cytokine storm, complement dysregulation, and immune checkpoint expression leading to an overall weak adaptive response, culminating in fulminant MVD and death. Created with BioRender (https://biorender.com/). Abbreviations: ISG, interferon stimulated genes; OAS-RNAse L, oligoadenylate synthetase–ribonuclease L; RIG-I, retinoic acid-inducible gene I; HLA, human leukocyte antigen; MARV, Marburg virus; GP, glycoprotein; rVSV-N4CT1-MARV-GP, Vesiculovax recombinant vesicular stomatitis virus expressing MARV GP; ICOS, inducible T-cell co-stimulator ligand; IgM, immunoglobulin M; IgG, immunoglobulin G; MVD, Marburg virus disease.