Coronavirus Pandemic Potential Coronavirus Pandemic Potential and Research Response and Research Response Mark R. Denison, Mark R. Denison, M.D. M.D. Departments of Pediatrics, Microbiology & Departments of Pediatrics, Microbiology & Immunology Immunology The Elizabeth B. Lamb Center for Pediatric The Elizabeth B. Lamb Center for Pediatric Research Research Vanderbilt University Medical Center Vanderbilt University Medical Center
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Coronavirus Pandemic Potential and Research Response Mark R. Denison, M.D. Departments of Pediatrics, Microbiology & Immunology The Elizabeth B. Lamb Center.
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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
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
• 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)
• 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.
• 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
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
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:
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
• Structural limitations to reversionStructural limitations to reversion
• Reverse genetics to introduce multiple attenuating and stabilizing Reverse genetics to introduce multiple attenuating and stabilizing mutationsmutations