Measles virus: Measles virus: A pathogen, a vaccine and a vectorA pathogen, a vaccine and a vector

Hussein Y. Naim, Ph.D.

Berna BiotechBern, Switzerland

2

MeaslesMeasles

•• An ancient and highly contagious childhood disease.An ancient and highly contagious childhood disease.

•• First described by the El Rhazi, 840 AD.First described by the El Rhazi, 840 AD.

•• The greatest killer of children in the history.The greatest killer of children in the history.

•• Annually affects ~50 million individuals and causes up to aAnnually affects ~50 million individuals and causes up to amillion deaths primarily in developing world. million deaths primarily in developing world.

•• The virus spreads by aerosol.The virus spreads by aerosol.

•• Complications: immunosuppression, encephalitis, bacterialComplications: immunosuppression, encephalitis, bacterialinfections and the rare but deadly infections and the rare but deadly SSPESSPE (1:10000 cases).(1:10000 cases).

•• In mid 1960's a live attenuated vaccine became available.In mid 1960's a live attenuated vaccine became available.

3

Transmission by aerosol

Basolateral fusion withMacrophages and uptakeof infectious viral RNP

Spread to local lymph node

Transport to the blood streamvia Macrophages and virions

Apical virus infectionand release

1

3

LocalLocallymph nodelymph node

Systemic infection4

2

Infection and TransmissionInfection and Transmission

Ductus thoracicus

macrophages

Respiratory tract

Recovery5

4

Measles VirusMeasles Virus

100nm

PleomorphicPleomorphicDiameter 200Diameter 200--350nm350nm

ENVELOPE GLYCOPROTEINSENVELOPE GLYCOPROTEINS• HH, receptor attachment &

Helper F-function. • FF, fusion at the host cell surface.

• Helical (>1) •• ssRNAssRNA, -ve sense,NN, nucleoproteinPP, phosphoproteinLL, polymerase

MATRIX proteinMATRIX protein ((MM))• Regulates fusion,• Virus release,• Env. protein sorting.

VC

NN HHFF LLMMPP(~16 kb)Non-segmented

ss (-ve) RNA genome

GENOMEGENOME

5

Mononegavirales SuperfamilyMononegavirales Superfamily

Paramyxoviridae Rhabdoviridae

GenusGenus

Sendai virus

Humanparainfluenza

Virus 1, 3

Mumps virus

Human Parainfluenza

Virus 2,4

SimianVirus 5-41 Newcastle

Disease virus

Measles virus

Rinderpest virus

Peste desPetits

Ruminantsvirus

Dolphinmorbillivirus

CanineDistemper

virus

H and BRespiratory

Syncytial virus

VesicularStomatitis

virus

Rabiesvirus

Marburg virus

Ebolavirus

Filoviridae

GenusGenus GenusGenus

Nipha virus

Equinemorbillivirus

Hendra virus

HenipavirusMorbillivirusRubulavirus

Respirovirus Avulavirus

Pneumovirus Vesiculovirus Lyssavirus Filovirus

Paramyxovirinae Pneumovirinae

GenusGenus

Metapneumovirus

Turkeyrhinotracheitis

Lettusnecroticyellow

Potatoyellowdwarf

PlantPlant

Bornaviridae

GenusGenus

Bornavirus

Borna disease virus

6

Protein synthesis

mRNAmRNA

Replication/transcription

HF

CD46 or CD150

AttachmentEntry/Fusion

Release

RNP

Cytosolic:Cytosolic: MM,, NN,, PP,, LL

L, P dependL, P depend

CellCell--cellcellfusionfusion

RNP

interactioninteraction

Attachment and release of measles virusAttachment and release of measles virus

12

3

4

5

6

Assembly

MM proteinprotein

membrane boundmembrane bound

F and H F and H glycoproteinsglycoproteins

7

LLiveive aattenuatedttenuated MMeasleseasles VVaccineaccine (Morbillivirus, Paramyxoviridae)(Morbillivirus, Paramyxoviridae)

- One injection: 104 TCID50 (9-12 months)- High neutralizing antibody titers- Efficient boosting (6-10 years)- Life-long efficacy (NAb + CD8 + CD4)- High genetic stability- Efficacious as an aerosol vaccine (EZ)- Mass vaccination is still needed for long time.

EdmonstonD. Edmonston’s blood to primary human kidney cells (Enders & Peebles, 1954)

HK/24HA/28

Edmonston-Enders

Ed-“wt”

HK/7Vero/6

Schwarz

CEF/8532°CEdmonston A

CEF/1CE/36

Moraten

Rubeovax

CEF/4032°C

CEF/336°C

Edmonston BCEF/3

AIK-C

ZagrebZagreb

HA/12, SK/17, 33°C, CEF/22, 33°C

CE/22, DK/15, WI38/19

HK= human kidneyHA= human amnionCE= chicken embryoCEF= chicken embryo fibroblastsDK= dog kidneyWI-38 = human diploid cellsSK= sheep kidney

VVCC

NN HHFF LLMMPP(16 kb)Non-segmented

ss (-ve) RNA genome

8

LiveLive--attenuated viral vaccines attenuated viral vaccines ..are very efficient at preventing viral diseases..are very efficient at preventing viral diseases

SmallpoxSmallpox(eradicated)(eradicated)

PolioPolio(95(95--100% morbidity reduction)100% morbidity reduction)

MeaslesMeasles(95(95--100% morbidity reduction)100% morbidity reduction)

MumpsMumps(95(95--100% morbidity reduction)100% morbidity reduction)

RubellaRubella(95(95--100% morbidity reduction)100% morbidity reduction)

Yellow fever Yellow fever (95(95--100% efficiency)100% efficiency)

VaricellaVaricella(70(70--80% morbidity reduction)80% morbidity reduction)

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Measles vaccine is safe and induces a broad and longMeasles vaccine is safe and induces a broad and long--lasting immunitylasting immunity

CD8+T-cell

Cytotoxic against tumor & virus

Measles virusvaccine

A P C

MHCII MHCI

CD4+T-CellB-cellY Y

Y

Y

Y

Neutralizingantibody

12MembraneMembrane--boundbound

proteinsproteinsCytosolicCytosolicproteinsproteins

InfectionInfectionProtein Synthesis

10

Break-throughBreakBreak--throughthrough

Rescue of Negative Strand RNA Viruses from cDNARescue of Negative Strand RNA Viruses from cDNA

Measles virus 1993Measles virus 1993--9595Rhabdoviruses 1995Rhabdoviruses 1995--9696RSV 1996RSV 1996--9797PIF 1996PIF 1996--9797

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p(+)MVp(+)MV

293 cell line with293 cell line with•• T7 RNA polT7 RNA pol•• NN andand PP

T7

T7 pEMCLapEMCLa

CotransfectionCotransfection

Syncytia formationSyncytia formationVirus rescueVirus rescue

The technologyThe technology

EMCIRES

L

Rescue of infectious measles virus from cDNARescue of infectious measles virus from cDNA

12

Rescue systemRescue system

MV particle

MV genome as RNP(15894 nt.)

L

6. MV proteins

helper cells(293-3-46)

NPT7

T7

p(+)MVNT7 P M F H L

T

5'3' ( ) _

2. cytoplasmic T7 transcription

5' 3'(+)

A(n)AUG5' 3'ACCA UGGU

T7pEMC-La

LIRES pdA

1. plasmid DNA transfection

3. simultaneous encapsidation

4. MV N, P and L-dependentreplication

5. MV P/L-dependent transcription

N P M F H LA(n)A(n)A(n)A(n)A(n)

A(n)

7. virus assembly

• Transfection of two plasmids p(+)MV and pEMC-La theT7RNA polymerase produces MV antigenomes and the mRNA for the MV L protein.

• The MV antigenome is tightly encapsidated by N and P proteins, stabily expressed in the helper cell line.

• Together with the transiently expressed L, a RNP is formed

template for MV-N,P, L-dependent transcription of all MV mRNAs.

• With increasing amounts of MV mRNAs switch from transcription to replication production of full-length RNPs

virus assembly formation of progeny MV particles.

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Generation of Live rMV and proofGeneration of Live rMV and proof--ofof--principleprinciple

Rec. measles vaccine

Additional

transgene(s)

Measles vaccine

RNARNA

LLPPNN

H

F

M

VVCC

T7

NN HHFF LLMMPP

T7t

332211

eGFP LacZ CAT

Proof-of-principleA- Marker genes

• GFP, LacZ & CAT (Tangy and Naim, Viral immun. 2005; Zuniga, Vaccine, 2007)

B- Heterologous antigens• HBV (Singh, JVI, 1999; Reyes del

Valle, JVI, 2007)

• VSV (Fehr, Nat.Med. 1998; Spielhofer, JVI, 1998)

• Mumps (Wang, Vaccine 2001)

• SARS-CoV (Liniger, Vaccine 2008)

• WNV (Depres, JID, 2005)

• HPV (Cantarella et al, Vaccine2009)

• SIV (Wang, Vaccine 2001; Zuniga, 2007)

• HIV (Lorin, JVI, 2004; Vaccine 2005; Tangy and Naim, Viral immun. 2005; Zuniga et al, Vaccine 2009)

Gene transcription / translation gradient

100

% 1.51525 2034

14

Therapeutic potential of MV-vector

Proof-of-concept

Day 0 Day 6 Day 15

Regression of CTCL tumor growth in Nude mice

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•• Correlates of protection are not definedCorrelates of protection are not defined

•• HIV envelope protein is highly variable, conserved/neutralizing HIV envelope protein is highly variable, conserved/neutralizing epitopes of the surface antigen (env) are almost hidden epitopes of the surface antigen (env) are almost hidden

The development of an HIVThe development of an HIV--vaccine is challenging .....vaccine is challenging .....

Novel approaches are necessaryNovel approaches are necessary......

•• The design of antigensThe design of antigens

•• Elicit broader spectrum of immunity (humoral, cellular and muElicit broader spectrum of immunity (humoral, cellular and mucosal)cosal)

•• The design of delivery vectorThe design of delivery vector

•• Safe, stable and immunogenicSafe, stable and immunogenic

•• Animal modelAnimal model

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Technologies and FormulationsTechnologies and Formulations

•• DNADNA•• Adjuvanted proteinsAdjuvanted proteins

•• LipoproteinsLipoproteins•• Replication deficient recombinant virusesReplication deficient recombinant viruses

•• Bacterial vectorsBacterial vectors•• Live attenuated recombinant virusesLive attenuated recombinant viruses

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Technologies and FormulationsTechnologies and Formulations

•• DNA• Adjuvanted proteins

• Lipoproteins• Replication deficient recombinant viruses

• Bacterial vectors•• Live attenuated recombinant virusesLive attenuated recombinant viruses

•• Live attenuated viruses are particularly suitable for mass immunLive attenuated viruses are particularly suitable for mass immunization.ization.•• Live attenuated viruses are cost effective.Live attenuated viruses are cost effective.•• Induce longInduce long--term memory.term memory.

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GateGate--keeperskeepers•• Clinically approved vector Clinically approved vector •• Stability of antigen expressionStability of antigen expression•• ManufacturingManufacturing•• Immunogenicity (humoral, cellular) in tgImmunogenicity (humoral, cellular) in tg--micemice•• Immunogenicity in NHPImmunogenicity in NHP

Recombinant Measles virus vectorDevelopment of recombinant MVDevelopment of recombinant MV--HIVHIV

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The Retroviral genome and The Retroviral genome and ttarget antigensarget antigens

20

PhC

PhC

Ant

i-gag

Ant

i-env

MV2

b-HI

V-e1

MV2

b-HI

V-e2

MV2

b-HI

V-gp

MV3

b-HI

V-gp

MV2

3b- H

IV-e

1gp

MV2

3b-H

IV-e

2gp

MVb

Recombinant Measles virus vectorExpression of HIV antigens by rMVExpression of HIV antigens by rMV isis efficientefficient

Antigens of the VRC-NIHe1= gp140_dCFI e2= gp140_dV12_dCFI gp= gag-pol fusion

gp140_dCFI

gp140_dV12_dCFI

p(+)MV2b-e1 (17850 nt; 112%)

N P M F H Le1

p(+)MV2b-gp (20292 nt; 128%)

N P M F H Lgp

p(+)MV3b-gp (20292 nt; 128%)gpN P M F H L

p(+)MV23b-e1gp (22248 nt; 140%)

N P Me1 gpF H L

p(+)MVb (15894 nt, 100%)

T7N P M F H L

T7tP2P2 P3P3

e2

e2

MVMV--HIV constructsHIV constructs

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Recombinant Measles virus vectorInduction of sustainedInduction of sustained antianti--HIVHIV--envenv IgG responsesIgG responses

ELISA: Anti-MV ELISA: Anti-HIV-Env

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

5 10 15 20 251.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

5 10 15 20 25

MVb2-HIV-e1 MVb2-HIV-e2

MVb23-HIVe1gp MVb2-HIV-e1 + MVb2-HIV-gp

MVbv

Ant

i-MV

IgG

tite

r

Ant

i-gp1

60 Ig

G ti

ter

Week post immunization Week post immunization

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Recombinant Measles virus vectorInduction of Induction of quantitative cellular immune responses in various formulationsquantitative cellular immune responses in various formulations

0

100

200

300

400

MVb2-gpMVb3-gpMVb23-e1gp

MVb2-e1 + MVb2-gpMVbvMV naive

MVb2-gag

0

100

200

300

400

500

600

MVb2-gag

MVb2-gagpol

MVbv MV naive

IFN

-po

sitiv

e ce

llspe

r 106

sple

enoc

ytes

MediaMVAMVA-gag

Stimulation:

3

Prime

Week 0Splenocytes

N=5

HIVHIV--gaggag--specific IFNspecific IFN--gamma ELISPOT assaysgamma ELISPOT assays

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Vaccinia and rVaccinia HIVVaccinia and rVaccinia HIV--gag induce weight loss in gag induce weight loss in susceptible IFNARsusceptible IFNAR--CD46 tg miceCD46 tg mice

rVaccinia-HIV-gag

Vaccinia

Weight Loss

Weight Loss0-15d

0-15d

IFNARIFNAR--CD46 tg miceCD46 tg miceI.N. application of 5x10I.N. application of 5x1066 pfu of pfu of VacciniaVaccinia oror rVacciniarVaccinia--HIVHIV--gaggag

NaiveNaive

24

rVaccinia-HIV-gag

Vaccinia

Weight loss

NO wt Loss

rMV-HIV-gag

10 days

0-15d

0-15d

IFNARIFNAR--CD46 tg miceCD46 tg miceimmunized with 10immunized with 1044 pfu of rMVpfu of rMV

I.N. pseudoI.N. pseudo--challenge with 5x10challenge with 5x1066 pfupfuofof VacciniaVaccinia oror rVacciniarVaccinia--HIVHIV--gaggag

Vaccinia and rVaccinia HIVVaccinia and rVaccinia HIV--gag induce weight loss in gag induce weight loss in susceptible IFNARsusceptible IFNAR--CD46 tg miceCD46 tg mice

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Recombinant Measles virus vectorrMVrMV--HIVHIV--gag pgag protectrotectss tgtg--micemice from rVV challengefrom rVV challenge

rMV-HIV-gag/Vac-gag

rMV-HIV-gag/Vac

Naive/Vac-gag

Naive/Vac

Line represents the median of wt-loss post rec.vaccinia challenge.Each dot represents 1 mouse.

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GateGate--keeperskeepers• Clinically approved vector • Stability of antigen expression• Manufacturing• Immunogenicity (humoral, cellular) in tg-mice •• Immunogenicity in NHPImmunogenicity in NHP

Recombinant Measles virus vectorDevelopment of recombinant MVDevelopment of recombinant MV--HIVHIV

27

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

0 10 20 30 40 50 60

Recombinant Measles virus vectorInduction of long term antiInduction of long term anti--HIV and antiHIV and anti--MV antibodies in NHP MV antibodies in NHP

HIV

IgG

tite

r (En

dpoi

nt d

ilutio

n)

Time (weeks)

- dotted blue-line: Genscreen HIV kit- solid pink-line: HIVenv assay- dotted pink-line: being verified

Endpoint Titration

0200400600800

10001200140016001800

1 2 3 4 5 6 7 8 9

Animal

Tite

r

Results at Week 49

1.E-01

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

0 10 20 30 40 50 60Time (weeks)

Mea

n M

V-Ig

G T

iter (

Endp

oint

dilu

tion)

MV

IgG

tite

r (En

dpoi

nt d

ilutio

n)

Time (weeks)

Boost (rMVb2-HIV-env)

- standard MV ELISA

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Recombinant Measles virus vectorSummarSummaryy

Experimental Evidence:Experimental Evidence:

•• SStability of genes and protein expression.tability of genes and protein expression.•• MV genome can accomodates large capacity of additional gene(s).MV genome can accomodates large capacity of additional gene(s).

•• ApplicableApplicable for immunization for immunization as a single or a combination of ras a single or a combination of rMVsMVs..

•• rMVs induce sustained IgGs against HIVrMVs induce sustained IgGs against HIV--antigens.antigens.

•• rMVsrMVs iinduce functional cellular immunnduce functional cellular immunee responseresponse..

PotentialPotential

•• Aerosol application.Aerosol application.

•• As a pediatric vaccine.As a pediatric vaccine.

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Thanks....Thanks....

Berna Biotech-CrucellTeldja N. AzzouzMarlyse KnuchelRene Marty Matthias LinigerMarian WiegandArmando ZunigaManuela ZanoniJorge BarcosOrhan IlterJohanna Signer Thomas SteinerKaspar ScherlerSara Weibel

NIAID-NIHPeggy Johnston Jim Bradac Michael PensieroVijay MehraGeetha BansalNancy Miller-- HIVRAD: AIHIVRAD: AI--4600746007-- HVDDT: NOIHVDDT: NOI--AIAI--6001860018

Collaborators/AdvisorsGary Nabel, VRC-NIHSharon Orndorff, ABLSampa Santra, BIDMCNorm Letvin, BIDMCBarny Graham, VRC/NIHPaul Johnson, HarvardDiane Griffin, Johns HopkinsSteve Udem, IAVIPeter Wright, VanderbiltKen Draper, CRLKatarina Radosevic, CrucellJaap Goudsmit, CrucellBehazine Comberdier, CNRSFrederic Tangy, Inst. PasteurMichel Klein, Uni. Montreal

University of ZurichMartin BilleterZili Wang Michael CaballeroLars HangertnerPeter Wild (Vet. Med.)Jovan Pavlovic (Med. Virol.)