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. 2023 Nov 28;15(12):2335.
doi: 10.3390/v15122335.

Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing

Elizabeth E Zumbrun  1 , Carly B Garvey  1   2 , Jay B Wells  1   2 , Ginger C Lynn  1   2 , Sean Van Tongeren  1   2 , Jesse T Steffens  1   2 , Kelly S Wetzel  1   2 , Laura M Gomba  1   2 , Kristan A O'Brien  1   2 , Franco D Rossi  1 , Xiankun Zeng  1 , Eric D Lee  1 , Jo Lynne W Raymond  1 , Diana A Hoffman  1 , Alexandra N Jay  1 , Elizabeth S Brown  1   2 , Paul A Kallgren  1 , Sarah L Norris  1 , Jean Cantey-Kiser  3 , Humza Kudiya  4 , Chris Arthur  4 , Christiana Blair  4 , Darius Babusis  4 , Victor C Chu  4 , Bali Singh  4 , Roy Bannister  4 , Danielle P Porter  4 , Tomas Cihlar  4 , John M Dye  1
Affiliations

Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing

Elizabeth E Zumbrun et al. Viruses. .

Abstract

Marburg virus (MARV) causes severe disease and high mortality in humans. The objective of this study was to characterize disease manifestations and pathogenesis in cynomolgus macaques exposed to MARV. The results of this natural history study may be used to identify features of MARV disease useful in defining the ideal treatment initiation time for subsequent evaluations of investigational therapeutics using this model. Twelve cynomolgus macaques were exposed to a target dose of 1000 plaque-forming units MARV by the intramuscular route, and six control animals were mock-exposed. The primary endpoint of this study was survival to Day 28 post-inoculation (PI). Anesthesia events were minimized with the use of central venous catheters for periodic blood collection, and temperature and activity were continuously monitored by telemetry. All mock-exposed animals remained healthy for the duration of the study. All 12 MARV-exposed animals (100%) became infected, developed illness, and succumbed on Days 8-10 PI. On Day 4 PI, 11 of the 12 MARV-exposed animals had statistically significant temperature elevations over baseline. Clinically observable signs of MARV disease first appeared on Day 5 PI, when 6 of the 12 animals exhibited reduced responsiveness. Ultimately, systemic inflammation, coagulopathy, and direct cytopathic effects of MARV all contributed to multiorgan dysfunction, organ failure, and death or euthanasia of all MARV-exposed animals. Manifestations of MARV disease, including fever, systemic viremia, lymphocytolysis, coagulopathy, and hepatocellular damage, could be used as triggers for initiation of treatment in future therapeutic efficacy studies.

Keywords: Marburg virus; animal model; animal rule; cynomolgus macaque; filovirus; intramuscular; nonhuman primate; pathogenesis; telemetry.

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Conflict of interest statement

H.K., C.A., C.B., D.B., V.C.C., B.S., R.B., D.P.P. and T.C. are current or former employees and may be shareholders of Gilead Sciences, Inc. Other authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or Department of Defense. The use of trade or manufacturers’ names in this publication does not constitute an official endorsement of any commercial products. This report may not be cited for purposes of advertisement.

Figures

Figure 14
Figure 14
Immune-Privileged Tissues from MARV-Exposed Animals. Representative images of tissues from MARV-exposed animals are shown with IHC and ISH staining from adjacent sections. Brain (A): brain parenchymal IHC, (B): adjacent brain parenchymal ISH, (C): brain ventricles/choroid plexus IHC, (D): adjacent brain ventricles/choroid plexus ISH; eye (E): IHC at low magnification, (F): IHC at high magnification, (G): ISH at low magnification, (H): ISH at high magnification; testis (I): IHC, (J): ISH; and epididymis (K): IHC, (L): ISH. Positive staining from IHC tissue appears as a brown precipitate, and positive ISH staining is red. The scale bar is 50 μm.
Figure 1
Figure 1
Kaplan–Meier Plot of Survival of MARV-Exposed and Mock-Exposed Cynomolgus Macaques. The survival of MARV-exposed (blue) and Mock-exposed (red) cynomolgus macaques is plotted on a Kaplan–Meier plot. All (n = 12) MARV-exposed animals succumbed between Days 8 and 10 PI. All (n = 6) mock-exposed animals survived until the end of the study.
Figure 2
Figure 2
Daily Maximum Responsiveness Scores. The maximum responsiveness score for each animal on each day is displayed. At each awake observation, animals were assigned a responsiveness score using a five-point scale as described in the Methods. Animals were euthanized when they reached a responsiveness score of four. FD = Found Deceased.
Figure 3
Figure 3
Plasma MARV RNA (RT-PCR). Top panel shows group means; error bars represent standard deviation. Bottom panel shows values for individual animals. LLOQ = 5.90 log10 ge/mL; LOD = 4.20 log10 ge/mL; ULOQ = 11.90 log10 ge/mL.
Figure 4
Figure 4
Serum Infectious Virus. MARV plaque assays were performed on serum samples collected from mock-exposed and MARV-exposed animals at intervals throughout the study. LOD = 1.70 log10 pfu/mL; ULOQ = 9.18 log10 pfu/mL. Horizontal bars represent group means.
Figure 5
Figure 5
Body Temperature Measured by Telemetry—Representative Example. Temperature readings assessed by telemetry for a representative MARV-exposed animal are shown above. Analysis of body temperature (°C). Body temperature values displaying normal (♦), fever (♦), hyperpyrexia (♦), and hypothermia (♦); baseline average values are in gray (▬). Anesthesia use (▲). Days post inoculation = calendar days.
Figure 6
Figure 6
Body Temperature Measured by Telemetry. Results of the individual MARV-exposed (blue open circles) and mock-exposed (red open triangles) animals for the maximum daily temperature elevation value (∆TMax; top panel), daily percentage of significant temperature elevation values (TESig; middle panel), and daily fever-hours (fever-h; bottom panel). Days post-inoculation = calendar days. Horizontal bars in all panels represent group means.
Figure 7
Figure 7
Interval between Onset of Decreased Temperature or Hypothermia and Time Deceased in MARV-Exposed Animals. Horizontal bars represent means. Significantly decreased temperature: temperature greater than 3 standard deviations below mean values calculated from the baseline period for longer than 2 h. Hypothermia: greater than 2.0 °C below baseline for longer than 2 h.
Figure 8
Figure 8
Activity Measured by Telemetry—Representative Example. Activity readings assessed by telemetry for a representative MARV-exposed animal are shown above. Values + 3 SD (♦) or −3 SD (♦) from baseline are statistically significant; values < 3 SD (♦) are not significant. Daytime (0600–1800 h) values are shown with solid blue lines (──♦──); nighttime (1800–0600 h) values are shown with dotted blue lines (- - -♦- - -). Baseline average values for daytime and nighttime are shown as black dotted lines (- - - -).
Figure 9
Figure 9
Timing of Statistically Significant Clinicopathologic Changes. The p-values shown represent the comparison of MARV-exposed change from baseline to mock-exposed change from baseline. Alb, albumin; Bili, bilirubin; Ca, calcium; Creat, creatinine; Fbg, fibrinogen; Glu, glucose; Lymph, lymphocytes; MCH, mean corpuscular hemoglobin; MCHC, MCH concentration; MCV, mean corpuscular volume; Mono, monocytes; MPV, mean platelet volume; Neut, neutrophils; Plt, platelets; RDW, RBC distribution width; WBC, white blood cells. In the schematic, white cells denote a lack of statistical significance. The pink shading is a heat map with deeper pink denoting greater significance. p-Values denoted as 0.001 are either p = 0.001 or p < 0.001.
Figure 10
Figure 10
Inflammation and Coagulation Parameters. Measures of inflammation (neutrophils, albumin, and lymphocytes) and coagulation (fibrinogen, APTT, PT, and D-dimers) are depicted for individual MARV-exposed animals (blue circles) and mock-exposed animals (red triangles). Horizontal bars represent means.
Figure 11
Figure 11
Hepatobiliary Parameters. Measures of hepatobiliary function (ALT, AST, ALP, GGT, and bilirubin) are depicted for individual MARV-exposed animals (blue circles) and mock-exposed animals (red triangles). Horizontal bars represent means.
Figure 12
Figure 12
Renal Parameters. Measures of renal function (BUN and creatinine) are depicted for individual MARV-exposed animals (blue circles) and mock-exposed animals (red triangles). Horizontal bars represent means.
Figure 13
Figure 13
Target Organs of MARV Infection. Representative images of liver (A–C), kidney (D–F), spleen (G–I), and inguinal lymph node (J–L) tissues from MARV-exposed animals are shown with IHC (left panel), ISH (middle panel) and H&E (right panel) staining from adjacent sections shown. Positive staining from IHC tissue appears as a brown precipitate and the positive ISH staining is red. The scale bar is 50 µm.
Figure 15
Figure 15
Clinical Progression of Acute MVD in the IM/MARV Cynomolgus Macaque Natural History Study.
Figure 16
Figure 16
Potential Triggers for Treatment on Days 4–6 PI. Horizontal bars represent means.
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