Pathogenesis of Viral Hemorrhagic Fever
Transcript of Pathogenesis of Viral Hemorrhagic Fever
Dr. Tom Geisbert
Pathogenesis of Viral Hemorrhagic Fever
28 June 2008
National Emerging Infectious Diseases LaboratoriesBoston University Medical Center
Viral Hemorrhagic Fevers of HumansVirus FamilyGenus Virus Disease Nat.Distrib. Source (Days)Arenaviridae
Arenavirus Lassa Lassa Fever West Africa Rodent 5-16New World† New World HF Americas Rodent 5-16
BunyaviridaeNairovirus CCHF CCHF Africa, C.Asia, E.EU. Tick 3-12Phlebovirus Rift Valley fever Rift Valley fever Africa, SaudiArabia Mosquito 2-6Hantavirus Agents of HFRS HFRS Asia, Balkans, EU. Rodent 9-35
FiloviridaeEbolavirus Ebola Ebola HF Africa Unknown 2-21Marburgvirus Marburg Marburg HF Africa Unknown 2-21
FlaviviridaeFlavivirus Dengue Dengue HF Asia,Africa,Americas Mosquito Unknown
Yellow fever Yellow fever Africa, Americas Mosquito 3-6Omsk HF Omsk HF Central Asia Tick 2-9Kyasanur Forest Kyasanur Forest India Tick 2-9disease disease
* Indicates HF viruses that pose serious threat as biological weapons† New World Arenaviridae include: Junin (Argentine HF); Machupo (Bolivian HF); Sabia (Brazilian HF);
Guanarito (Venezuelan HF); Whitewater Arroyo (Unnamed)
Virus Person-to-Person Mortality,% Vaccine Treatment Transmission
Lassa Yes 15-20 None Ribavirin, supportiveJunin Yes 15-30 IND-Junin Ribavirin, supportiveMachupo Yes 25-35 IND-Junin Ribavirin, supportiveGuanarito ? 25-35 None Ribavirin, supportiveSabia ? 33 (1/3) None Ribavirin, supportive
Rift Valley fever No < 1 IND (x2) Ribavirin, supportive
Ebola Yes 50-90 None SupportiveMarburg Yes 23-90 None Supportive
Yellow fever No 20 Live 17D SupportiveOmsk HF No 0.5-10 None SupportiveKyasanur Forest No 3-10 FIA, India Supportivedisease
Viral Hemorrhagic Fevers of Humans
Most HF viruses require BSL-4 containment
An acute febrile illness characterized by malaise, myalgia, and prostration dominated by general abnormalities of vascular permeability, and regulation. Bleeding manifestations often occur, particularly in severe cases; they are usually diffuse and reflect widespread vascular damage rather than life-threatening volume loss.
“The doctors at Kisumu decide that he should go to Nairobi Hospital in East Africa so he boards an aircraft and ends up bleeding and puking blood and black stuff the whole way there. The skin on his face is starting to droop more as it separates from his face. When the aircraft arrives he stumbles off and gets in a taxi headed for the Nairobi hospital. After he arrives he is left in the waiting room and this is when he "crashes", or "bleeds out". He loses consciousness, vomits blood all over the floor, his bowels release and parts of his intestines flood out in a mess of blood and black particles.”
Hemorrhagic Fever:
Filoviruses: Taxonomy
Mononegavirales
Rhabdoviridae Filoviridae Paramyxoviridae
Marburgvirus Ebolavirus
Reston
SudanZaire
Ivory Coast
Nucleotide and amino acid differences between EBOV and MARV are ~ 55%
EBOV species show 37-41% difference in nucleotide and amino acid sequences Uganda???
Ebola and Marburg Virus Outbreaks
~ 1300 Fatal Cases of Ebola HF ~ 360 Fatal Cases
of Marburg HF
Animal Models of Ebola and Marburg Viruses
MiceMice- ZEBOV (i.p. route only)
- MARV???
Guinea pigs Guinea pigs (Strain 13, Outbred)(Strain 13, Outbred)
- ZEBOV- SEBOV?- MARV-Musoke- MARV-Ravn- MARV –’67
Nonhuman Primates (NHP)Nonhuman Primates (NHP)
- Rhesus monkeys (ZEBOV, MARV’s)- Cynomolgus monkeys (ZEBOV, SEBOV, REBOV, MARV’s)- African green monkeys (ZEBOV, MARV’s)- Hamadryas baboons (ZEBOV)
Cynomolgus macaque Guinea pig Mouse
Fibrin Deposition in Spleen of Ebola-Infected Animals
Fibrin Deposition in Human Tissues – Marburg Ravn
Rhesus
Pathogenesis: 22
Vaccine: 2
Treatment: 8
Cynomolgus
Pathogenesis: 10
Vaccine: 8
Treatment: 2
African Green Monkey
Pathogenesis: 11
Vaccine: 0
Treatment: 0
NHP Species Used for Filovirus Studies
Feature Mouse Guinea Pig African Green
Cynomolgus Macaque
Rhesus Macaque
Human
Fever No Moderate Yes Yes Yes Yes
Peak viremia 10^8.0-9.0 10^5.0 10^5.5-6.5 10^6.0-7.0 10^6.0-7.0 10^6.5
↑ liver enzymes Yes Yes Yes Yes Yes Yes
Lymphopenia(process of lymphocyte death)
Yes(PCD-like apoptosis)
Yes(?)
Yes (Classical apoptosis)
Yes (Classical apoptosis)
Yes(Classical apoptosis)
Yes(Classical apoptosis)
Neutrophilia Yes Yes Yes Yes Yes Yes
Thrombocytopenia Modest Yes Yes Yes Yes Yes
Macular rash No No No Yes Yes Yes
↑ blood clotting times No Yes Yes Yes Yes Yes
↑ levels of D-dimers NT NT NT Yes Yes Yes
Fibrin deposition No Minimal Moderate Yes Yes Yes
↑ Nitrate levels NT NT NT Yes Yes Yes
In vivo target cells Mono/Mac,
DC?, hepat
Mono/Mac,
DC?, hepat
Mono/Mac,
DC?, hepat
Mono/Mac,
DC, hepat
Mono/Mac, DC, hepat
Mono/Mac, DC, hepat
Increased levels of circulating cytokines
IL-6, TNF-a NT NT IL-6, TNF-a, IFN-a
IL-6, IL-10, TNF-a, IFN-a
IL-6, IL-10, TNF-a, IFN-a
Summary of Clinical Features Filovirus Infection
Filovirus Species Number Mortality Mean Day of Death
Ebola-Zaire (’95) Cynomolgus 36 100% 6.6 (Mode = 6) (range 5-9)
Ebola-Zaire (’95) Rhesus 25 100% 8.4 (Mode = 8) (range 7-10)
Ebola-Sudan (Gulu) Cynomolgus 4 50% 8.5 (7, 10)
Ebola-Sudan (’76) Cynomolgus 5 100% 7.6 (Mode = 8)
Ebola-Sudan (’76) Rhesus 1 100% 17
Ebola-Ivory Coast Cynomolgus 5 60% 13
Marburg (Angola) Cynomolgus 1 100% 8
Marburg (Angola) Rhesus 6 100% 7.3 (Mode = 7)
Marburg (Ci67) Cynomolgus 4 100% 7.8 (Mode = 8)
Marburg (Musoke) Cynomolgus 4 100% 9 (Mode = 9)
Marburg (Musoke) Rhesus 5 100% 11.4 (Mode = 11)
Marburg (Ravn) Cynomolgus 1 100% 8
Marburg (Ravn) Rhesus 3 66% 9.5 (8, 11)
Disease Course in Filovirus-Infected Macaques
1000 pfu, i.m. injection
Route of Exposure vs. Disease Course
1976 Outbreak of Ebola-Zaire
InjectionMean incubation period: 6.3 days
Mortality: 100% (85/85)
Contact ExposureMean incubation period: 9.5 days
Mortality: 80% (119/149)
0 1 2 3 4 5 6 7 8 9 10 11 12
Days PI
Terminal
0 1 2 3 4 5 6 7 8
Days PI
Terminal
EBOV-Zaire Cynomolgus disease course –10 pfu, i.m.
EBOV-Zaire Cynomolgus disease course –1000 pfu, i.m.
Challenge Dose vs. Disease Course
Fever, Viremia, Rash, Anorexia
0 1 2 3 4 5 6 7 8 9 10 11
Days PI
Terminal
0 1 2 3 4 5 6 7 8
Days PI
Terminal
MARV-Angola Rhesis disease course –40 pfu, i.m.
MARV-Angola Rhesus disease course –1000 pfu, i.m.
Challenge Dose vs. Disease Course
Fever, Viremia, Rash, Anorexia
Can a better understanding of pathogenesis facilitate the development of effective interventions?
Ebola Pathogenesis Study Design
21 Cynomolgusmacaques
d 1
d 2
d 3
d 4
d 5
d 6
4
4
4
3
3
3
1000 PFU
i.m. injection
Marburg Pathogenesis Study Design
d 2
d 3
d 4
d 6
d 7
d 8
3
3
3
3
3
3
1000 PFU MARV (Ci67)
18 Cynomolgus macaques
i.m. injection
Filovirus Infection of Monocytes/Macrophages and Dendritic Cells
0
2
4
6
8
10
D0 D1 D2 D3 D4 D5 D6
Day after challenge
Ly
mp
ho
cy
te c
ou
nt
X 1
0^
3
0
2
4
6
8
10
0 1 2 3 4 5 6 7 8
Days after challenge
Lym
phocyte
count X 1
0̂3
Ebola
Lymphopenia
Marburg
Lymphocyte Apoptosis - Filoviruses
Control Day 4 Day 4 Day 6
EbolaDay 8
Marburg
Ebola Ebola Marburg
IFN-α
0
1000
2000
3000
4000
5000
0 2 4 6Days pos t infection
MCP-1
0
2000
4000
6000
0 2 4 6Days pos t infection
MIP-1α
0
500
1000
1500
2000
0 2 4 6
Days post infection
IL-6
0
100
200
300
400
500
0 2 4 6
Days pos t infection
TNF-α
0
10
20
30
0 2 4 6Days post infection
Nitrate
0
200
400
600
800
1000
0 2 4 6Days post infection
Cytokine/Chemokine Production
• NO is a vasoactive mediator that causes loss of vascular smooth muscle tone
• High levels of NO are associated with cardiac distress and heart failure
• Hypotension is a prominent feature of Ebola HF and increased levels of NO would contribute to this condition
Disseminated Intravascular Coagulation (DIC)
• Two major mechanisms trigger DIC:– widespread injury to endothelial cells– release of tissue factor or thromboplastic
substances into the circulation
• Consequences of DIC:– Widespread deposition of fibrin within the
microcirculation may lead to ischemia and/or hemolytic anemia resulting from fragmentation of RBCs as they squeeze through narrowed vasculature
– Hemorrhagic diathesis resulting from consumption of platelets and clotting factors and activation of plasminogen
Lack of evidence for role of endothelial cells as a trigger for DIC
Coagulopathy EBOV vs MARV-Infected NHPsD-Dimers
0
100
200
300
400
500
600
700
800
900
-10 0 1 2 3 4 5 6 7 8
Days after challenge
Co
nc
en
tra
tio
n n
g/m
l
D-Dimers
0
10000
20000
30000
40000
50000
0 1 2 3 4 5 6
Days after challenge
Co
nce
ntr
ati
on
ng
/ml
EBOV MARV
Platelets
0
100
200
300
400
500
600
0 1 2 3 4 5 6 7 8
Days after challenge
Pla
tele
t C
ount
x 1
0^3
Platelets
0
100
200
300
400
500
600
0 1 2 3 4 5 6
Days after challenge
Pla
tele
t C
ou
nt
X 1
0^
3
Coagulopathy
Day 3
Day 4
Day 5
Gastroduodenal junction, d3-5 PE.Progressive hemorrhage/congestion of the proximal duodenum beginning at the pyloric sphincter and extending distally through the duodenum.
Duodenal Lesion
Fibrin deposition in EBOV-infected NHPs
Spleen – day 4 Kidney – day 4
Disseminated Intravascular Coagulation (DIC)
• Two major mechanisms trigger DIC:– widespread injury to endothelial cells– release of tissue factor or thromboplastic
substances into the circulation
• Consequences of DIC:– widespread deposition of fibrin within the
microcirculation may lead to ischemia and/or hemolytic anemia resulting from fragmentation of RBCs as they squeeze through narrowed vasculature
– hemorrhagic diathesis resulting from consumption of platelets and clotting factors and activation of plasminogen
Control
The Role of Tissue Factor in Ebola Infections
L32
GAPDH
Tissue Factor
Ebola-infected
1 24 96 48 1 24 48 96
TF+ microparticles (NHP) TF+ PBMC (NHP) Primary human monocytes/macrophages
Primary human monocytes/macrophages
Tissue Factor
VIIaCa2+Ca2+X
Xa
Ca2+
Ca2+
Ca2+
Fibrinogen(I)
Fibrin(Ia)
Cross Linked Fibrin
XII
XIIa
IIa
Thrombin
VaV
II
Prothrombin
Thrombin(IIa)
VIIIa
IXa
XIa
XIIa
XII (Hageman Factor)
HMWK collagenKallikerin
Prekallikerin
XI
VIIIThrombin
(IIa)
IX
Phospolipidsurface
Ca2+Ca2+
Active
Inactive
Intrinsic Pathway Extrinsic Pathway
Common Pathway
Micro-particles
EBOV-infectedmonocyte/macrophage
VII
Tissue Factor
Microparticles
Tissue Factor
VIIaCa2+Ca2+X
Xa
Ca2+
Ca2+
Ca2+
Fibrinogen(I)
Fibrin(Ia)
Cross Linked Fibrin
XII
XIIa
IIa
Thrombin
VaV
II
Prothrombin
Thrombin(IIa)
VIIIa
IXa
XIa
XIIa
XII (Hageman Factor)
HMWK collagenKallikerin
Prekallikerin
XI
VIIIThrombin
(IIa)
IX
Phospolipidsurface
Ca2+Ca2+
Active
Inactive
Intrinsic Pathway Extrinsic Pathway
Common Pathway
Micro-particles
EBOV-infectedmonocyte/macrophage
VII
Tissue Factor
Microparticles
rNAPc2
rNAPc2-treated d10
Placebo control – d9
Summary of Severe Sepsis Pathophysiology
Multiple organ dysfunction
Impaired vascular tone
Infection
Microbial products (endotoxin-LPS/ exotoxin-peptidoglycans)
Inflammatory cellular responses(platelets-neutrophils-monocyte/macrophages)
Nitric oxide free radical formation
PlateletActivation
ComplementTissue factor Release
Cytokines: TNFα, IL-1, -6, -10
Cell adhesion Tissue hypoxiaMicrovascular
thrombusFree radical
damageCapillary
leakApoptosis
Alteredmental status
PaO2/FiO2ratio <300; tachypnea
Urine output< 0.5 ml/kg/hr
Hypotensiontachycardia
Thrombocytopenia↑ D-dimer
Poor capillary
refill
Metabolic acidosis
↑ Lactate
Death
Parallels with Ebola HF
Plasma Levels of Protein C
Activated Protein C
0
20
40
60
80
100
0 1 2 3 4 5 6
Days after challenge
% o
f B
asel
ine
• A rapid decline in plasma protein C levels occurs in EBOV infections• This decline is concomitant with disease progression • Decreases are observed before the development of clinical disease• A drop of 40% of APC in severe sepsis is a significant predictor of poor outcome
Protein C
Bel
ow
Lo
wer
Lim
it o
f N
orm
al (
%)
0
20
40
60
80
100
Gram- Gram+ Mixed Pure Mixed Suspected No Inf Gram Fungal Organisms Inf
EBOV-Infected Macaques Human Severe Sepsis Patients
Recombinant human activated protein C (rhAPC) (Drotrecogin alfa [activated]; Xigris®)
• rhAPC is a complex 60 Kd serine protease with 4 types of post-translational modifications
• The approved Xigris dose for severe sepsis is 24 µg/kg/hr for 96 hrs– Highest NOAEL* from toxicology (in monkeys) and in phase 1 studies is 48
µg/kg/hr• The antithrombotic activity of APC is highly species specific (i.e., rhAPC has much
lower antithrombotic activity in nonhuman primates)– NHP are the most relevant species for testing Xigris besides human
* NOAEL – No Observed Adverse Event Level
Could rhAPC have utility in treating Ebola HF?Activated Protein C has a very short half life ~ 13 min
• This will require the continuous administration of drug to NHP under a BSL-4 setting
Day of surgery Four weeks after surgery
Control
Recombinant Human Activated Protein C Treatment Study
days 0-748 ug/kg/hr
Xigris
Saline
1000 PFU Ebola-Zaire
• 3 separate studies performed• Controls n=3; Treated n=11
0
20
40
60
80
100
0 5 10 15 20 25 30
Days after challenge
Pe
rce
nt
su
rviv
al
Treated Study controls Historical controls
Survival
Mean survival = 12.6 days versus 8.3 days for historical controls (P= 0.049)
d7 d7
d8 d8 d10
d14 d16 d21 d22
Survived
Treated nonresponders
Treated responders
All 3 controls succumbed on day 8
Results similar to rhAPC in humans (relative reduction in risk of death is 19.4% and absolute reduction in risk of death is 6.1%)
Vesicular Stomatitis Virus (VSV) Recombinants
VSV-wt
VSV∆G-EBOVGPN P M EGP L
VSV∆G-MARVGP
VSV∆GN P M L
N P M MGP L
N P M G L
N P M EBOVGP L N P M MARVGP L
Ebola GP Marburg GP
VSV∆G-EBOVGP VSV∆G-MARVGP
A Vaccine as a Postexposure Treatment?
N P M MARGP L
rVSV-Marburg Vaccine as a Postexposure Treatment
Marburg virusControls
N P M EBOVGP L
N P M MARGP L
1000 PFU
rVSV vectors administered i.m. 20-30 min after MARV challenge
rVSV-Marburg Vaccine as a Postexposure Treatment
Marburg virusControls
N P M EBOVGP L
N P M MARGP L
1000 PFU
rVSV vectors administered i.m. 20-30 min after MARV challenge
dy11
Survived
dy11
dy12
rVSV-Ebola Vaccine as a Postexposure Treatment against Ebola-Sudan
1000 PFU Sudan ebolavirus
Control
N P M LassaGP L
rVSV vectors administered i.m. 20-30 min after SEBOV challenge
N P M SEBOVGP L
VSV∆G-SEBOVGP
VSV∆G-LassaGPC
rVSV-Ebola Vaccine as a Postexposure Treatment
1000 PFU Sudan ebolavirus
Control
N P M LassaGP L
rVSV vectors administered i.m. 20-30 min after SEBOV challenge
N P M SEBOVGP L
VSV∆G-SEBOVGP
VSV∆G-LassaGPCdy 17
• Maintenance of D-dimer levels
• Maintenance of protein C activity (> 50%)
• Maintenance of levels of proinflammatory / procoagulant cytokines (e.g., IL-6)
• Low viral load
Common Denominators of Survival in Filovirus-Infected Macaques
Plasma Viremia
0
1
2
3
4
5
6
7
8
9
0 2 3 4 5 6 7 8 9 10
Days after challenge
Lo
g1
0 p
fu/m
l
Importance of Viral Load in Survival - Ebola
Historical controls (x23): BlackrNAPc2 survivors (x3): RedXigris survivors (x2): Blue
Rhesus macaques
rVSV survivors (x4): Green
National Emerging Infectious Diseases LaboratoriesBoston University Medical Center
Acknowledgments
Katie Daddario -DiCaprio
Heinz Feldmann
Ute StroherAllen Grolla
Lisa FernandoFredericke Feldmann
Hideki EbiharaGabi Neumann
Joan Geisbert
Lisa Hensley Liz Fritz
Tom Larsen
Steven Jones
Ed Stevens
Gary Nabel
Nancy Sullivan
Denise Braun
Jeff Brubaker
Yoshi Kawaoka