Presentation UniBern09 hynaim...
Transcript of Presentation UniBern09 hynaim...
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
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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.
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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
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3
LocalLocallymph nodelymph node
Systemic infection4
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Infection and TransmissionInfection and Transmission
Ductus thoracicus
macrophages
Respiratory tract
Recovery5
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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
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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
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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
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3
4
5
6
Assembly
MM proteinprotein
membrane boundmembrane bound
F and H F and H glycoproteinsglycoproteins
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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
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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
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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
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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
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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
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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
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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
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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.)