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Coronavirus Pandemic Potential and Coronavirus Pandemic Potential and Research ResponseResearch Response
Mark R. Denison, M.D.Mark R. Denison, M.D.
Departments of Pediatrics, Microbiology & ImmunologyDepartments of Pediatrics, Microbiology & Immunology
The Elizabeth B. Lamb Center for Pediatric ResearchThe Elizabeth B. Lamb Center for Pediatric Research
Vanderbilt University Medical CenterVanderbilt University Medical Center
• Vanderbilt University Vanderbilt University Department of Pediatrics, Department of Pediatrics, Department of Microbiology & Department of Microbiology & ImmunologyImmunology
• Elizabeth B. Lamb Center for Elizabeth B. Lamb Center for Pediatric Research Pediatric Research
• NIH R01 AI26603 -15 NIH R01 AI26603 -15 R01 AI50083 -R01 AI50083 - 01 (S1)01 (S1)
Disclosures: Disclosures: nonenone
Denison LabDenison Lab
• Xiaotao LuXiaotao Lu• Erik PrenticeErik Prentice• Rachel GrahamRachel Graham
UNC Chapel HillUNC Chapel Hill
• Ralph BaricRalph Baric
• Boyd YountBoyd Yount
• Amy SimsAmy Sims
Course of the PandemicCourse of the Pandemic
• February 2003February 2003
““Dad why don’t you work on an ‘important’ virus”Dad why don’t you work on an ‘important’ virus”
• April 2003April 2003
““Daddy, I think you are getting kind of ‘full of yourself’”Daddy, I think you are getting kind of ‘full of yourself’”
• August 2003August 2003
““Dad, why haven’t you found a cure for SARS yet?”Dad, why haven’t you found a cure for SARS yet?”
ObjectivesObjectives
• Describe the role of basic research in SARS Describe the role of basic research in SARS
• Describe coronavirus life cycle and replicationDescribe coronavirus life cycle and replication
• Discuss genetic systems coronavirus researchDiscuss genetic systems coronavirus research
• Summarize research goals Summarize research goals
SARS, Public Health, and ResearchSARS, Public Health, and Research
SARS - CoV SARS - CoV
Pandemic PotentialPandemic Potential
High mortalityHigh mortality
Worldwide spreadWorldwide spread
Naive populationNaive population
SARS - CoV SARS - CoV
Pandemic potentialPandemic potential
High mortalityHigh mortality
Worldwide spreadWorldwide spread
Immune naive populationImmune naive population
SARS - CoV SARS - CoV
Pandemic PotentialPandemic Potential
High mortalityHigh mortality
Worldwide spreadWorldwide spread
Naive populationNaive population
Public Health ResponsePublic Health Response
SARS, Public Health, and ResearchSARS, Public Health, and Research
SARS - CoV SARS - CoV
Pandemic potentialPandemic potential
High mortalityHigh mortality
Worldwide spreadWorldwide spread
Immune naive populationImmune naive population
No DiseaseNo Disease
New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections
Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention
Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses
SARS, Public Health, and ResearchSARS, Public Health, and Research
• Escape / mutationEscape / mutation
• PersistencePersistence
• Animal reservoirAnimal reservoir
SARS, Public Health, and ResearchSARS, Public Health, and Research
No DiseaseNo Disease
New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections
Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention
Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses
• Escape / mutationEscape / mutation
• PersistencePersistence
• Animal reservoirAnimal reservoir
SARS, Public Health, and ResearchSARS, Public Health, and Research
• Continued human diseaseContinued human disease
• Re-emergence of epizootic, Re-emergence of epizootic, epidemic or pandemic diseaseepidemic or pandemic disease
• New SARS-CoV disease New SARS-CoV disease
No DiseaseNo Disease
New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections
Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention
Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses
• Escape / mutationEscape / mutation
• PersistencePersistence
• Animal reservoirAnimal reservoir
SARS, Public Health, and ResearchSARS, Public Health, and Research
• Continued human diseaseContinued human disease
• Re-emergence of epizootic, Re-emergence of epizootic, epidemic or pandemic diseaseepidemic or pandemic disease
• New SARS-CoV disease New SARS-CoV disease
• Need for research in biology, Need for research in biology, pathogenesis, therapeutics and pathogenesis, therapeutics and vaccinesvaccines
No DiseaseNo Disease
New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections
Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention
Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses
• Escape / mutationEscape / mutation
• PersistencePersistence
• Civet, Raccoon Dog, other?Civet, Raccoon Dog, other?
SARS, Public Health, and ResearchSARS, Public Health, and Research
• Singapore - September 2003Singapore - September 2003
• Re-emergence of epizootic, Re-emergence of epizootic, epidemic or pandemic diseaseepidemic or pandemic disease
• ASx infection animals / humans ASx infection animals / humans
• Need for research in biology, Need for research in biology, pathogenesis, therapeutics and pathogenesis, therapeutics and vaccinesvaccines
No DiseaseNo Disease
New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections
Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention
Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses
Big QuestionsBig Questions• Is SARS-CoV still present in human populations? Is SARS-CoV still present in human populations?
• Is there risk of human animal transmission? Is there risk of human animal transmission?
• Will SARS-CoV adapt to better survive in human Will SARS-CoV adapt to better survive in human populations?populations?
• Will SARS reemerge as a “seasonal disease”Will SARS reemerge as a “seasonal disease”
• Can vaccines and therapeutics for SARS be Can vaccines and therapeutics for SARS be developed, tested and used?developed, tested and used?
ObjectivesObjectives
• Describe the role of basic research in SARS Describe the role of basic research in SARS
• Describe coronavirus life cycle and replicationDescribe coronavirus life cycle and replication
• Discuss genetic systems coronavirus researchDiscuss genetic systems coronavirus research
• Summarize research goals Summarize research goals
CoronaviridaeCoronaviridaeGroup 1Group 1
TGEVTGEV
HCoV229EHCoV229E
Group 2Group 2MHVMHV
BCoVBCoV
HCoV-OC43HCoV-OC43
Group 3Group 3
IBVIBV
Nidovirales OrderNidovirales Order
SARS-CoVSARS-CoV
Snijder et al 2003 J. Mol. Biol. (2003) 331, 991–1004Snijder et al 2003 J. Mol. Biol. (2003) 331, 991–1004
Coronavirus Genome Organization Coronavirus Genome Organization
MHV Genome + RNA 32 kbMHV Genome + RNA 32 kb
genes 2-7genes 2-7Replicase gene 22 kbReplicase gene 22 kbLeaderLeader
AANN
SS EE MM NNORF 1aORF 1aORF 1bORF 1b
SARS Genome + RNA 30 kbSARS Genome + RNA 30 kb
genes 2-9genes 2-9Replicase gene 20 kbReplicase gene 20 kbLeaderLeader
AANN
SS EE MM NNORF 1aORF 1aORF 1bORF 1b
Coronavirus Replicase Coronavirus Replicase
p213p213 polpol helhelp70 p70 3CLpro3CLprop20p20
PLP2PLP2SARSSARS
10001000 20002000 30003000 1400014000 50005000 60006000 70007000aaaa
PLP1PLP1
p210p210 polpol hel hel p65p65 3CLpro3CLprop28p28
PLP-2PLP-2MHVMHV
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
Coronavirus genome and replicationCoronavirus genome and replication
replicase polyproteinreplicase polyprotein
polpol
Genome + RNA 32 kbGenome + RNA 32 kb
SS EE MM NN
genes 2-7genes 2-7gene 1 (replicase)gene 1 (replicase)R CR C
R CR C
LeaderLeader
775'5'GenomeGenomeRNARNA ((++))
3'3'22 33 44 55 66
AAnn
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
775'5'
3'3'
GenomeGenomeRNARNA ((++))
((--))
3'3'22 33 44 55 66
IntermediateIntermediateRNARNA
AAnn
5'5'
UUnn
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
5'5' 3'3'
AAnn
GenomeGenomeRNARNA ((++))
775'5'
3'3'
GenomeGenomeRNARNA ((++))
3'3'22 33 44 55 66
AAnn
AAnn
5'5'
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
775'5'
3'3'
GenomeGenomeRNARNA ((++))
3'3'22 33 44 55 66
AAnn
AAnn
5'5'
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
775'5'
3'3'
GenomeGenomeRNARNA ((++))
3'3'22 33 44 55 66
AAnn
UnUn
5'5'
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
775'5'
3'3'
GenomeGenomeRNARNA ((++))
3'3'22 33 44 55 66
AAnn
UUnn
5'5'
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
Replicase geneReplicase gene genes 2-7genes 2-7
AAnn
((--) strand ) strand subgenomic subgenomic RNARNA
((++) strand ) strand Subgenomic mSubgenomic mRNARNA
Spike (S)Spike (S)
Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication
77RNARNA
5'5'
3'3'
GenomicGenomicRNARNA
LeaderLeaderRNARNA
((++))
((++))
((--))
3'3'
Spike (S)Spike (S)
MM
NN
EE
NSNS
NSNS
Gene 1Gene 1
ReplicaseReplicase
ProteinsProteins
1122 33 44 55 66
Genes 2-7Genes 2-7
RNARNA
33
44
55
66
77
22
5'5' 3'3'
AAnn
AAnn
AAnn
AAnn
AAnn
AAnn
AAnn
AAnn
5'5'
UUnn
SubgenomicSubgenomicRNAsRNAs
(( +/-+/- ))
GenomicGenomicRNARNA ((++)) ReplicaseReplicase
Coronavirus genome and replicationCoronavirus genome and replication
replicase polyproteinreplicase polyprotein
polpol
Genome + RNA 32 kbGenome + RNA 32 kb
SS EE MM NN
genes 2-7genes 2-7gene 1 (replicase)gene 1 (replicase)R CR C
R CR C
LeaderLeader
Coronavirus genome and replicationCoronavirus genome and replication
replicase polyproteinreplicase polyprotein
polpol
Genome + RNA 32 kbGenome + RNA 32 kb
SS EE MM NN
genes 2-7genes 2-7gene 1 (replicase)gene 1 (replicase)R CR C
R CR C
LeaderLeader
Coronavirus genome and replicationCoronavirus genome and replication
replicase polyproteinreplicase polyprotein
polpol
Genome + RNA 32 kbGenome + RNA 32 kb
SS EE MM NN
genes 2-7genes 2-7gene 1 (replicase)gene 1 (replicase)R CR C
R CR C
LeaderLeader
Proteinase inhibitors result in shutoff of viral Proteinase inhibitors result in shutoff of viral RNA synthesis at any time during MHV infectionRNA synthesis at any time during MHV infection
[[33 H] c
pm
H] c
pm
101033
101044
101055
Time (hours p.I)Time (hours p.I)11 22 33 44 55 66 77 88 99 1010
E64-dE64-d
Kim et al 1995Kim et al 1995
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
nucleusmaturationmaturationassemblyassembly
attachmentattachment
entry
releasereleaseReplication ComplexesTranslationProcessingRNA synthesis
Coronavirus Life CycleCoronavirus Life Cycle
Coronavirus Replication and Coronavirus Replication and Targets for InhibitionTargets for Inhibition
• S protein, receptorS protein, receptor
• Fusion, uncoatingFusion, uncoating
• Replicase polyprotein expression and processingReplicase polyprotein expression and processing
• Virus assembly and releaseVirus assembly and release
• Novel functions (polymerase, helicase, methyltransferase, Novel functions (polymerase, helicase, methyltransferase, exonuclease, CoMt)exonuclease, CoMt)
• Cellular functions used by virusCellular functions used by virus
– CholesterolCholesterol
– Membrane TraffickingMembrane Trafficking
– AutophagyAutophagy
ObjectivesObjectives
• Describe the role of basic research in Describe the role of basic research in SARS SARS
• Describe coronavirus life cycle and Describe coronavirus life cycle and replicationreplication
• Discuss genetic systems coronavirus Discuss genetic systems coronavirus researchresearch
• Summarize research goals Summarize research goals
• High theoretical mutation rate: 10High theoretical mutation rate: 1044 per template per template per replication (3 changes per genome)per replication (3 changes per genome)
• RNA-RNA homologous recombination (20%)RNA-RNA homologous recombination (20%)
• Result:Result: rapid adaptation, recovery from rapid adaptation, recovery from deleterious mutations, mechanisms to acquire deleterious mutations, mechanisms to acquire and regain virulence.and regain virulence.
• Genome tolerates deletions, mutations, Genome tolerates deletions, mutations, substitutionssubstitutions
Coronavirus Replication and Molecular Biology: Coronavirus Replication and Molecular Biology:
General ThemesGeneral Themes
Coronavirus Diseases: General ThemesCoronavirus Diseases: General Themes
• Repeat infectionsRepeat infections
• Persistent infectionsPersistent infections
• Disease enhancement in vaccinated animalsDisease enhancement in vaccinated animals
• Trans-species transmissionTrans-species transmission
• Changes in virus transmission, tropism and Changes in virus transmission, tropism and disease - disease - **New Coronavirus Diseases**New Coronavirus Diseases
Coronavirus Vaccines: General Themes Coronavirus Vaccines: General Themes
• Live attenuated, inactivated virus, protein, heterologous Live attenuated, inactivated virus, protein, heterologous virus, DNA have been usedvirus, DNA have been used
• ““Classical” live-attenuated vaccines have been most Classical” live-attenuated vaccines have been most licensed and employedlicensed and employed
• Challenges for vaccines : poor protection, enhanced Challenges for vaccines : poor protection, enhanced disease, altered disease, reversion to virulence, disease, altered disease, reversion to virulence, recombinationrecombination
• Every coronavirus has required different approachesEvery coronavirus has required different approaches
• **Multiple pathways for SARS vaccine development**Multiple pathways for SARS vaccine development
Coronavirus Reverse Genetics Coronavirus Reverse Genetics
• Infectious clones : MHV, IBV, TGEV, HCoV-229EInfectious clones : MHV, IBV, TGEV, HCoV-229E
– In vitro assembly, Vaccinia recombinants, BACIn vitro assembly, Vaccinia recombinants, BAC
• Applicable to entire genomeApplicable to entire genome
• Example of mutations yielding viable virusesExample of mutations yielding viable viruses
– Gene deletions, gene duplicationsGene deletions, gene duplications
– Gene substitution (GFP)Gene substitution (GFP)
– Gene order rearrangement Gene order rearrangement
– Replicase polyprotein cleavage site deletionReplicase polyprotein cleavage site deletion
In vitro ligationIn vitro ligation
RT / PCR RT / PCR cDNA clonescDNA clones
Genome +strand RNA (32 kb)Genome +strand RNA (32 kb)
Transcribe Transcribe + strand RNA+ strand RNA
Transfect cellsTransfect cells
nucleusnucleus
Yount, Denison, Weiss and Baric 2002Yount, Denison, Weiss and Baric 2002
In vitro ligationIn vitro ligation
RT / PCR RT / PCR cDNA clonescDNA clones
Genome +strand RNA (32 kb)Genome +strand RNA (32 kb)
Transcribe Transcribe + strand RNA+ strand RNA
Transfect cellsTransfect cells
nucleusnucleus
Yount, Denison, Weiss and Baric 2002Yount, Denison, Weiss and Baric 2002
In vitro ligationIn vitro ligation
RT / PCR RT / PCR cDNA clonescDNA clones
Genome +strand RNA (32 kb)Genome +strand RNA (32 kb)
Transcribe Transcribe + strand RNA+ strand RNA
Transfect cellsTransfect cells
nucleusnucleus
Yount, Denison, Weiss and Baric 2002Yount, Denison, Weiss and Baric 2002
Mutagenesis of MHV genome Mutagenesis of MHV genome
Replicase gene (22kb)5’
3’
replicase polyprotein
K G Y R G V K P
CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’
K G Y R G V K PK G Y R G V K P
CS1CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’P5 P4 P3 P2 P1 P1’ P2’ P3’
Mut 3 Mut 3 HH NO YESNO YES
Mut 4 Mut 4 AA NO YESNO YES
Mut 5 Mut 5 HH VV NO YESNO YES
Mut 8 Mut 8 AA YES YESYES YES
Mut 9 Mut 9 HH YES YESYES YES
Cleavage VirusCleavage VirusIn VitroIn Vitro
Mutation of CS1Mutation of CS1
MHV replicase cleavage site mutations block MHV replicase cleavage site mutations block cleavage during infection cleavage during infection
wtwt icwticwt
p93p93
p65p65
p28p28
mut8 mut9 mut3 mut4 mut5mut8 mut9 mut3 mut4 mut5
p28p28 p65p65
p93p93
CS1CS1
CleavingCleavingNon-Non-cleavingcleaving
Cleavage site mutants are viable Cleavage site mutants are viable
WildtypeWildtypeVirusVirus
““Assembled”Assembled”WildtypeWildtype
CleavingCleavingMutantMutant
Non-CleavingNon-CleavingMutantMutant
Time (hours post infection)
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
wticwtmut9
mut3mut4mut5
Viru
s tit
er (
Log
PF
U/m
l)Noncleaving mutants grow to lower peak titersNoncleaving mutants grow to lower peak titers
Cleaving
Non-Cleaving
Application of Genetics to Application of Genetics to SARS-CoV Research SARS-CoV Research
• Functions of replicase, structural and “accessory” Functions of replicase, structural and “accessory” genesgenes
• Pathogenic determinantsPathogenic determinants
• Recapitulation of animal virusesRecapitulation of animal viruses
• Deteminants of trans-species adaptationDeteminants of trans-species adaptation
• Capacity for recombination, reversion, and Capacity for recombination, reversion, and resistanceresistance
• Basis for stable live-attenuated virus vaccinesBasis for stable live-attenuated virus vaccines
Genetics Research Needs Genetics Research Needs
• Develop reverse genetic systemsDevelop reverse genetic systems
• Viruses from animals, humans, lab-adapted, and Viruses from animals, humans, lab-adapted, and passagedpassaged
• Rapid sequencing capacity for “natural”, cloned, Rapid sequencing capacity for “natural”, cloned, recovered, and modified viruses.recovered, and modified viruses.
Research NeedsResearch Needs
• GeneticsGenetics
• Animal models Animal models
• Protein structure and functionProtein structure and function
• Emergence / persistenceEmergence / persistence
• Immune response / protectionImmune response / protection
• Vaccines and AntiviralsVaccines and Antivirals
• Animal ModelsAnimal Models
– SARS-CoV animal reservoirSARS-CoV animal reservoir
– SARS-CoV animal modelsSARS-CoV animal models
• Primate and small animalPrimate and small animal
• Replication, pathogenesis, disease, persistenceReplication, pathogenesis, disease, persistence
• Adaptation of SARS-CoV in different speciesAdaptation of SARS-CoV in different species
• Mutant virus studiesMutant virus studies
– Natural and Trans-species Coronavirus ModelsNatural and Trans-species Coronavirus Models
• PRCoV (pigs), BCoV (cattle), MHV (mice)PRCoV (pigs), BCoV (cattle), MHV (mice)
• Pathogenesis, persistence, immune response, protection, reinfectionPathogenesis, persistence, immune response, protection, reinfection
Research Needs: Preparing for SARSResearch Needs: Preparing for SARS
Big QuestionsBig Questions• Is SARS-CoV still present in human populations? Is SARS-CoV still present in human populations?
• Is there risk of human animal transmission? Is there risk of human animal transmission?
• Will SARS-CoV adapt to better survive in human Will SARS-CoV adapt to better survive in human populations?populations?
• Will SARS reemerge as a “seasonal disease”Will SARS reemerge as a “seasonal disease”
• Can vaccines and therapeutics for SARS be Can vaccines and therapeutics for SARS be developed, tested and used?developed, tested and used?
Assumptions, Hypotheses, Myths and RumorsAssumptions, Hypotheses, Myths and Rumors
• SARS is a respiratory diseaseSARS is a respiratory disease
• Global public health response was responsible for control / elimination Global public health response was responsible for control / elimination of SARSof SARS
• SARS is an acute disease SARS is an acute disease
• If SARS-CoV reemerges it will be a “seasonal” diseaseIf SARS-CoV reemerges it will be a “seasonal” disease
• SARS-CoV will adapt and become less virulentSARS-CoV will adapt and become less virulent
• SARS-CoV will adapt and become more virulentSARS-CoV will adapt and become more virulent
• The barriers for trans-species movement of coronaviruses are highThe barriers for trans-species movement of coronaviruses are high
• Immune response equals recovery and protectionImmune response equals recovery and protection
• Response of animals correlates with protection in humansResponse of animals correlates with protection in humans
SARS Clinical DiseaseSARS Clinical Disease
• Prolonged incubation period with late Prolonged incubation period with late development of progressive respiratory diseasedevelopment of progressive respiratory disease
• Lack of upper respiratory prodromeLack of upper respiratory prodrome
• Evidence for systemic infection - multisystem Evidence for systemic infection - multisystem disease, and lab findingsdisease, and lab findings
• Disease in children clinically milderDisease in children clinically milder
• Development of antibody responses 10-20 daysDevelopment of antibody responses 10-20 days
Models for SARS PathogenesisModels for SARS Pathogenesis
SARS as a measles wannabe:SARS as a measles wannabe:
• Respiratory transmission Respiratory transmission
• Systemic infectionSystemic infection
• Severe progressive pulmonary disease in naïve adultsSevere progressive pulmonary disease in naïve adults
• Less severe in childhood primary infectionLess severe in childhood primary infection
• Direct epithelial destruction + immune diseaseDirect epithelial destruction + immune disease
• Multi-system diseaseMulti-system disease
• Concerns about late immune disease, virus mutationsConcerns about late immune disease, virus mutations
• Enhanced disease after killed vaccine?Enhanced disease after killed vaccine?
Targets for Immunity Targets for Immunity
Spike- SSpike- S
– FunctionsFunctions
• Receptor BindingReceptor Binding
• FusionFusion
• Host rangeHost range
• TropismTropism
– ImmunityImmunity
• AntibodiesAntibodies
• Cell mediated immunityCell mediated immunity
• NeutralizationNeutralization
• Fusion inhibitionFusion inhibition
Nucleocapsid- NNucleocapsid- N– FunctionsFunctions
• EncapsidationEncapsidation
• ? Replication? Replication
• ? Nuclear interactions? Nuclear interactions
– ImmunityImmunity
• Cell mediatedCell mediated
• Antibodies- non Antibodies- non neutralizingneutralizing
E
S
M
Targets for Immunity Targets for Immunity
Membrane - MMembrane - M
– FunctionsFunctions
• Virion formationVirion formation
• Incorporation of RNAIncorporation of RNA
– ImmunityImmunity
• CMI - not protectiveCMI - not protective
• Antibodies - non Antibodies - non neutralizingneutralizing
Small Membrane- ESmall Membrane- E
– FunctionsFunctions
• Virion formation / Virion formation / maturationmaturation
– ImmunityImmunity
• ??
E
S
M
Targets for Immunity Targets for Immunity ReplicaseReplicase
SS EE MM NNReplicaseReplicase
• Functions: polymerase, helicase, proteinase, transcription Functions: polymerase, helicase, proteinase, transcription factors, membrane interactions, intracellular movement, ?factors, membrane interactions, intracellular movement, ?exonuclease, exonuclease,
• Immunity: unknownImmunity: unknown
Non-structural proteinsNon-structural proteins
• Functions: ? Pathogenesis, host-specific functions, IFN-Functions: ? Pathogenesis, host-specific functions, IFN-cytokine induction ?cytokine induction ?
• Immunity: unknownImmunity: unknown
Targets for Immunity Targets for Immunity ReplicaseReplicase
SS EE MM NNReplicaseReplicase
• Functions: polymerase, helicase, proteinase, transcription Functions: polymerase, helicase, proteinase, transcription factors, membrane interactions, intracellular movement, ?factors, membrane interactions, intracellular movement, ?exonuclease, exonuclease,
• Immunity: unknownImmunity: unknown
Non-structural proteinsNon-structural proteins
• Functions: ? Pathogenesis, host-specific functions, IFN-Functions: ? Pathogenesis, host-specific functions, IFN-cytokine induction ?cytokine induction ?
• Immunity: unknownImmunity: unknown
Possible Vaccine Strategies for SARS Possible Vaccine Strategies for SARS
• Inactivated whole virusInactivated whole virus
• ProteinProtein
– Spike (S), nucleocapsid (N), membrane (M), others Spike (S), nucleocapsid (N), membrane (M), others
– Protein, DNA, viral (adenovirus, VEE, paramyxovirus)Protein, DNA, viral (adenovirus, VEE, paramyxovirus)
• Live-attenuatedLive-attenuated
– Passage, Passage,
– Chemical,UV, cold adaptationChemical,UV, cold adaptation
– EngineeredEngineered
• Targeted recombinationTargeted recombination
• Infectious cloneInfectious clone
Targeted RecombinationTargeted Recombination
NNMHV - SMHV - S
ReplicaseReplicase FIPV - SFIPV - S NN
ReplicaseReplicase MHV - SMHV - S NN
GrowthGrowth
feline cellsfeline cells
murine cellsmurine cells
recombination vectorrecombination vector
• Takes advantage of high rate of homologous recombination:Takes advantage of high rate of homologous recombination:
• Powerful selection for mutations- growth on specific cellsPowerful selection for mutations- growth on specific cells
• Applicable to 3’ 10 kb of genomeApplicable to 3’ 10 kb of genome
• Includes all stuctural and “non-replicase” nonstructural genesIncludes all stuctural and “non-replicase” nonstructural genes
• Has been well utilized for studies of S, M, N and E genesHas been well utilized for studies of S, M, N and E genes
22 kb22 kb 10 kb10 kb
5’5’ 3’3’MM
MM
MM
EE
EE
EE
Why Should We Pursue SARS Public Why Should We Pursue SARS Public Health Policies and Vaccines Health Policies and Vaccines
ProposedProposed SARS SARS
Non-human reservoirNon-human reservoir Civet, Raccoon Dog, otherCivet, Raccoon Dog, other
Naive populationNaive population 6 billion 6 billion
Variable disease penetrationVariable disease penetration Asymptomatic? - deathAsymptomatic? - death
Nonspecific diseaseNonspecific disease Flu-like, gastrointestinal Flu-like, gastrointestinal
Multiple modes of transmissionMultiple modes of transmission Respiratory, fomites, fecal?Respiratory, fomites, fecal?
Spread by world travelSpread by world travel DocumentedDocumented
Genetic change and adaptationGenetic change and adaptation Mutation, deletion, Mutation, deletion, recombinationrecombination
Vaccines for SARS Vaccines for SARS • The genome organization, proteins, and replication strategy The genome organization, proteins, and replication strategy
likely are similar to other coronaviruses, especially group 2.likely are similar to other coronaviruses, especially group 2.
• Determinants of pathogenesis, immune response and Determinants of pathogenesis, immune response and protection have yet to be determinedprotection have yet to be determined
• Animal models for SARS, both primate and small animal, Animal models for SARS, both primate and small animal, will be critical to development of vaccineswill be critical to development of vaccines
• No single vaccine approach has been highly effective in No single vaccine approach has been highly effective in animal coronavirus diseasesanimal coronavirus diseases
• Inactivated virus, recombinant protein, vectored expression, Inactivated virus, recombinant protein, vectored expression, DNA, heterologous virus, and live-attenuated virus DNA, heterologous virus, and live-attenuated virus approaches all need to be pursued for SARS vaccines approaches all need to be pursued for SARS vaccines
SARS and MHV Replicase Genes: Introduction of SARS and MHV Replicase Genes: Introduction of multiple attenuating and stabilizing mutationsmultiple attenuating and stabilizing mutations
p213p213 polpol helhelp70 p70 3CLpro3CLprop20p20
PLP2PLP2
p210p210 polpol hel hel p65p65 3CLpro3CLprop28p28
PLP-2PLP-2
SARSSARS
MHVMHV
Genes 2-9Genes 2-9S, E,M, NS, E,M, N
Genes 2-7 Genes 2-7
S, E, M, NS, E, M, N
Replicase GeneReplicase Gene
PLP1PLP1
Live-attenuated vaccines for SARS-CoV Live-attenuated vaccines for SARS-CoV
Potential AdvantagesPotential Advantages
• Impaired replication, normal immune determinants and presentationImpaired replication, normal immune determinants and presentation
• Base for live vaccine for emerging variants, serotypesBase for live vaccine for emerging variants, serotypes
• Limited immune selectionLimited immune selection
• Structural limitations to reversionStructural limitations to reversion
• Reverse genetics to introduce multiple attenuating and stabilizing Reverse genetics to introduce multiple attenuating and stabilizing mutationsmutations
Potential DisadvantagesPotential Disadvantages
• Pathogenesis, diseasePathogenesis, disease
• Recombination, reversion Recombination, reversion