the cotton rat model of respiratory viral infections pathogenesis

The Cotton Rat Model of The Cotton Rat Model of Respiratory Viral Infections Respiratory Viral Infections Pathogenesis and ImmunityPathogenesis and Immunity

Marina S. Marina S. BoukhvalovaBoukhvalova, Ph.D., Ph.D.VirionVirion Systems, Inc.Systems, Inc.

Rockville, MDRockville, MD

Sigmodon hispidus

• Member of the family cricidae

• In many regions of southern UnitedStates most abundant wild rodent

• Natural host of several viruses (i.e., Hantavirus, arenaviruses)

Advantages as an animal model:

• Much more permissive for most virusesthan mice (for RSV more than 100 fold)

• Inbred

• Reagents are available

Development of Cotton Rat Development of Cotton Rat Commercial Reagents Commercial Reagents

nn cDNAcDNA for >290 cotton rat genesfor >290 cotton rat genesnn Sequences are immediately deposited in Sequences are immediately deposited in

GenBankGenBank, none are being patented, none are being patentednn R&D Systems, Inc. expresses gene product, R&D Systems, Inc. expresses gene product,

produces antibodyproduces antibodynn 71 cotton rat reagents in the current R&D 71 cotton rat reagents in the current R&D

Systems online catalogSystems online catalog

A: recombinant protein; B: Polyclonal antibody; C: Monoclonal antibody; D: ELISA 2/08

Cytokines:IFN-γ. (A, B, C, D)IFN-α. (A,B)IFN-β.IL-1 α. (A, B, C)IL-1 β. (A, B)IL-2. (A, B, C, D)IL-4. (A, B, C, D)IL-5IL-6. (A, B, C)IL-9.IL-10. (A, B, C)IL-12p40. IL-12-p35.IL-13.IL-18.TNFα. (A, B, C, D)TNFβ.TGFβ1.GM-CSF

Chemokines:MCP-5 analogMIP-1α. (A, B, C, D)MIP-1β. (A, B, C)RANTES. (A, B)IP-10. (A, B)GRO/IL-8. (A, C, D)MIP-2/IL-8. (A, C).MCP-1/JE. (A,C)

Cell surface molecules:CCR5 CD3CD4 (C)CD8alpha (C) CD11b CD14CD16 CD18 CD25CD45/B220CD62L (L-selectin)CD74(MHC II)CD83(HB15)CD86(B7-2)Ly-6MHC IMHC II AMHC II ETLR-2β-2 microglobulin

Other genes:IRF-2IRF-8 (ICSBP)Cox-2 Hsp70Mx1 and Mx2

Housekeeping genes:β-actinGAPDH18S rRNA

A: recombinant protein; B: Polyclonal antibody; C: Monoclonal antibody; D: ELISA 2/08

Cotton Rat Genes and Reagents(Selected from a total of 290 cDNAs)

Important Infectious Diseases in the Important Infectious Diseases in the Cotton RatCotton Rat

nn 1937: Endemic typhus1937: Endemic typhusnn 1939: Polio (1, 2 & 3)1939: Polio (1, 2 & 3)nn 1940: 1940: M. bovisM. bovisnn 1940: 1940: C. diphtheriaeC. diphtheriaenn 1942: Epidemic typhus1942: Epidemic typhusnn 1944: Filariasis1944: Filariasisnn 1967: 1967: R. rickettsiiR. rickettsiinn 1970: VEE1970: VEEnn 1971: RSV1971: RSVnn 1981: Parainfluenza (1, 2 & 3)1981: Parainfluenza (1, 2 & 3)

nn 1984: Adenoviruses (2, 4, 5, 7, 8)1984: Adenoviruses (2, 4, 5, 7, 8)nn 1985: HSV1985: HSV--11nn 1987: Lyme disease1987: Lyme diseasenn 1987: Influenza (A & B)1987: Influenza (A & B)nn 1992: Measles1992: Measlesnn 1993: Venezuelan hemorrhagic 1993: Venezuelan hemorrhagic

feverfevernn 1995: Hantavirus1995: Hantavirusnn 2002: Monkeypox2002: Monkeypoxnn 2004: hMPV2004: hMPVnn 2006: HSV2006: HSV--22

Important Infectious Diseases in the Important Infectious Diseases in the Cotton RatCotton Rat

nn 1937: Endemic typhus1937: Endemic typhusnn 1939: Polio (1, 2 & 3)1939: Polio (1, 2 & 3)nn 1940: 1940: M. bovisM. bovisnn 1940: 1940: C. diphtheriaeC. diphtheriaenn 1942: Epidemic typhus1942: Epidemic typhusnn 1944: Filariasis1944: Filariasisnn 1967: 1967: R. rickettsiiR. rickettsiinn 1970: VEE1970: VEEnn 1971: RSV1971: RSVnn 1981: Parainfluenza (1, 2 & 3)1981: Parainfluenza (1, 2 & 3)

nn 1984: Adenoviruses (2, 4, 5, 7, 8)1984: Adenoviruses (2, 4, 5, 7, 8)nn 1985: HSV1985: HSV--11nn 1987: Lyme disease1987: Lyme diseasenn 1987: Influenza (A & B)1987: Influenza (A & B)nn 1992: Measles1992: Measlesnn 1993: Venezuelan hemorrhagic 1993: Venezuelan hemorrhagic

feverfevernn 1995: Hantavirus1995: Hantavirusnn 2002: 2002: MonkeypoxMonkeypoxnn 2004: 2004: hMPVhMPVnn 2006: HSV2006: HSV--22

Cotton Rat Model of Respiratory Viral DiseasesCotton Rat Model of Respiratory Viral Diseases

SemiSemi--permissivepermissive(histopathology and output virus proportional to input virus)(histopathology and output virus proportional to input virus)

Tissue tropismTissue tropism (lungs and nose)(lungs and nose)

No species adaptation is required No species adaptation is required Functional Functional MxMx systemsystem

--antiviralantiviral typetype--II--interferoninterferon--inducible proteinsinducible proteins--important component of important component of innate innate antiviral defense in humansantiviral defense in humans--MxMx genes are genes are defectivedefective in common laboratory strains of in common laboratory strains of micemice

•• lack of appropriate innate responselack of appropriate innate response--MxMx proteins are rapidly induced in infected cotton rats.proteins are rapidly induced in infected cotton rats.

RSV RSV Respiratory Respiratory SyncytialSyncytial VirusVirus

Peak pulmonary replication: day 4, clearance by day 7Peak pulmonary replication: day 4, clearance by day 7

Disease is primarily inflammatoryDisease is primarily inflammatory

Only shortOnly short--term immunityterm immunity

Most important parallels to human disease: Most important parallels to human disease: 1. 1. Antibody efficacy: PAntibody efficacy: Predicted redicted efficacy and doseefficacy and dose of of RespiGamRespiGam® and ® and

SynagisSynagis® in preventing RSV disease® in preventing RSV disease2. 2. VaccineVaccine--induced induced immunopathologyimmunopathology

RSV in cotton rats:RSV in cotton rats:

LOT 100 TRIAL LOT 100 TRIAL (1965)(1965)

nn FormalinFormalin--Inactivated (FIInactivated (FI--RSV)RSV)nn AlumAlum--Precipitated, 100X ConcentratedPrecipitated, 100X Concentrated

Formulation:Formulation:

nn 1616--fold higherfold higher hospitalization rate in RSVhospitalization rate in RSV--infected infected Lot 100 Lot 100 vaccineesvaccinees than in RSVthan in RSV--infected controls infected controls

nn Two RSVTwo RSV--infected Lot 100 infected Lot 100 vaccineesvaccinees dieddiedn Lung Pathology: Characterized by alveolitis, or

cellular infiltrates in alveolar spaces

Results:Results:

Mock FI-RSV

Alveolitis is the Primary Marker ofFI-RSV Vaccine-enhanced Disease in Cotton Rats

Monophosphoryl lipid A (MPL) reverses FI-RSV vaccine-enhanced disease histologic marker

Prince et al., Vaccine 2001 19(15-16):2048-54, Boukhvalova et al., Vaccine 2006 24(23):5027-35

Mock

FI-RSV

FI-RSV+MPL

Monophosphoryl lipid A (MPL) reverses FI-RSV vaccine-enhanced disease histologic marker

Boukhvalova et al., Vaccine 2006 24(23):5027-35

IL-4

0 6h d2 d5 0 6h d2 d5 0 6h d2 d5 0 6h d2 d5 0 6h d2 d505

1015202530

01020304050


IL-2

*

05

1015202530


MIP-1a

mock RSV IN FI FI+MPL IM FI+MPL IN

* *

*

*

mR

NA

leve

ls (r

elat

ive

to b

-act

in)

A

B

C mock RSV IN FI FI+MPL IM FI+MPL IN

mock RSV IN FI FI+MPL IM FI+MPL IN

Boukhvalova et al., J Virol 2007 81(17):9443-9450-35

primsec

5

4

3

2 0 6h 12h d1 d2 d4 d7 d14time post-infection

Vira

l Tite

r (L

og10

pfu/

g)

1

10-1

10-2

10-3

primsec

time post-infection

Gen

ome

Leve

lA

B≤

Real-time RT-PCR analysis of RSV replication in vivo Abortive replication in the re-infection model

0 6h 12h d1 d2 d4 d7 d14

Infectious Virus Release

Viral Genome Replication

Effect of Aging on Cotton Rat Effect of Aging on Cotton Rat Responses to RSVResponses to RSV

day 4 day 62

3

4

5

6

4-6 wks6-7 mnths9-10 mnths12-13 mnths15-16 mnths

Time post-infection

Lung

Vira

l Tite

r (lo

g 10

pfu/

g)

Delayed viral clearance from the lungs of aged cotton rats:

Curtis et al., Exp Biol Med (Maywood) 2002 227(9):799-802

0

5

1 0

1 5

2 0

2 50

2

4

6

8

1 0

1 2

0

5

1 0

1 5

2 0

2 5

3 0

3 5

IFN-γ

0

5

1 0

1 5

2 0

2 5 IL-10

Aged Young

Cyt

okin

e m

RN

A le

vel r

elat

ive

to β-

actin

MCP-1

GRO

d0 d4 d6 d8 d0 d4 d6 d8

*

*

Boukhvalova et al., J Infect Dis 2007 195(4):511-8

Effect of Aging on Cytokine Responses Effect of Aging on Cytokine Responses

hMPVhMPVHuman Human MetapneumovirusMetapneumovirus

Peak pulmonary replication: day 5, clearance by day 12Peak pulmonary replication: day 5, clearance by day 12

Disease is primarily inflammatoryDisease is primarily inflammatory

VaccineVaccine--enhanced diseaseenhanced disease

hMPVhMPV in cotton rats:in cotton rats:

NewlyNewly--discovered discovered paramyxovirusparamyxovirus (2001)(2001)

More severe disease in infants, More severe disease in infants, immunocompromizedimmunocompromized and elderlyand elderly

Only shortOnly short--term immunityterm immunity

hMPVhMPV::

Day 4 Day 7 Day 10

PrimaryInfection

Re-infection

FI-hMPV

mock

VaccineVaccine--enhanced disease in enhanced disease in FIFI--hMPVhMPV--immunized cotton ratsimmunized cotton rats

Yim et al., Vaccine 2007 25(27):5034-40

IL-4

Cd4 d7 d10

mock Re-infection FI-hMPV FI-hMPV FI-hMPVundil 1:10 1:100

d4 d7 d10 d4 d7 d10 d4 d7 d10 d4 d7 d10

10

8

6

4

2

0

IL-4

mR

NA

leve

l (r

elat

ive

to β

-act

in)

*** *

* *

*

****

mock hMPV FI-hMPV FI-hMPV FI-hMPV

IFN-γ

Cd4 d7 d10

undil 1:10 1:100

d4 d7 d10 d4 d7 d10 d4 d7 d10 d4 d7 d10

40

30

20

10

0

IFN

-γm

RN

A le

vel

(rel

ativ

e to

β-a

ctin

)

**

*

** **

FIFI--hMPVhMPV vaccinevaccine--enhancement is enhancement is associated with Th2/Th1 imbalanceassociated with Th2/Th1 imbalance

Yim et al., Vaccine 2007 25(27):5034-40

InfluenzaInfluenza

Infection is possible using Infection is possible using unadaptedunadapted influenza virusesinfluenza viruses

Replication is rapid, inflammatory response lingers after viralReplication is rapid, inflammatory response lingers after viralclearance from the lungsclearance from the lungs

Rapid activation of antiviral Rapid activation of antiviral MxMx response response

Abundant pulmonary cytokine responseAbundant pulmonary cytokine response

HeterosubtypicHeterosubtypic immunityimmunity

Influenza in cotton rats:Influenza in cotton rats:

Type (Subtype) Adaptation Strain Lung Titera Nose Titer

B B/HK/73B/Sichuan/379/99B/HK/330/01

3.65.25.5

6.36.55.2

A (H1N1) MouseTissue culture

A/PR/8/34A/PR/8/34A/Malaya/302/54

N/D6.35.2

4.75.06.3

A (H3N2) X-31A/Wuhan/359/95

4.63.3

6.26.4

a Log10 TCID50/g

Replication of influenza viruses in cotton ratsReplication of influenza viruses in cotton rats

Ottolini et al., J Gen Virol 2005 86 (Pt 10):2823-30.

2

3

4

5

6

7

8

4hr 5hr 7hr 1d 2d 3d 4d 6d 8d

Vira

l Tite

r(L

og10

TCID

50/g

)

LungNose

Time post-infection

60

65

70

75

0 1 2 3 4 5 6 8 10Day post-infection

Wei

ght (

g)

InfectedControl

Viral replication and clinical signs of Viral replication and clinical signs of influenza A infection in cotton ratsinfluenza A infection in cotton rats


Viral Replication

Weight Loss

Induction of Induction of MxMx response during response during influenza A infection in cotton ratsinfluenza A infection in cotton rats

Pletneva et al., J Interferon Cytokine Res 2006 26 (12):914-21.

6h 12h d1 d2 d3 d4 d7 d10 d21 Mx 1Mx 2β-actin

0

1

2

3

4

0

1

2

3

4

Epithelial Damage

Interstitial Pneumonitis

1 2 3 4 6 8 10 14 21 28

Day post-infection

Path

olog

y Sc

ore

Pathology of influenza A infection Pathology of influenza A infection in the cotton rat modelin the cotton rat model


1 2 3 4 6 8 10 14 21 28

3

4

5

6

7

3

4

5

6

7 Wuhan/WuhanNone/WuhanPR8/Wuhan

day 1 day 2 day 4 day 7

day 1 day 2 day 4 day 7

Nose

Lung

Time post-infection

* **

*

Vira

l Tite

r, Lo

g 10T

CID

50/g

HeterosubtypicHeterosubtypic immunity to influenza immunity to influenza in the cotton rat modelin the cotton rat model

Straight et al., Vaccine 2006 24 (37-39):6264-71.

Advantages of the Cotton Rat ModelAdvantages of the Cotton Rat Modelüü Highly permissive to infection with Highly permissive to infection with unadaptedunadapted human human

respiratory viruses, including respiratory viruses, including unadaptedunadapted influenza strainsinfluenza strains

üü Carries functional Carries functional MxMx systemsystem

üü Displays strong Displays strong effect of agingeffect of aging on antiviral responseson antiviral responses

üü Reflects human FIReflects human FI--RSV vaccineRSV vaccine--enhanced disease and enhanced disease and predicts efficacy predicts efficacy andand safetysafety of candidate vaccines of candidate vaccines

üü Allows analysis into the Allows analysis into the mechanismsmechanisms of vaccineof vaccine--enhancement and adjuvant action.enhancement and adjuvant action.

üü PredictsPredicts vaccinevaccine--enhanced disease for FIenhanced disease for FI--hMPVhMPV

üü Displays Displays heterosubtypicheterosubtypic immunity immunity to influenzato influenza

Kevin YimKatherine KuhnPatrick HemmingLayla SoroushDolores HarriganTim StraightMaryna Eichelberger

Gregory PrinceJorge Blanco

David Porter

Virion Systems, Inc. University of Maryland, Baltimore, School of Medicine

Stefanie Vogel

the cotton rat model of respiratory viral infections pathogenesis

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