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