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GOF studies in MERS andSARS countermeasures
Mark R. Denison M.D.Vanderbilt University School of Medicine
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SARS-CoV: 32 countries >8000 cases in 2 months in 2003 High mortality in the elderly
High identity SARS-like CoV currently circulating in bats No US Lab associated infections in >10 years of research Already regulated by BSL3, Select Agent and DURC No approved Vaccines or Therapeutics
MERS-CoV: CURRENTLY active epidemic ongoing since 2012 >800 cases > 40% mortality (no reduction in mortality since 2013) Mechanisms for ongoing outbreak unclear No vaccines or therapeutics available No small animal model available
Two natural, zoonotic, potentially pandemic
coronaviruses in 10 years:Coronaviruses do not pause and do not deliberate
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Goals of CoV therapeutics
Broadly active against diverse CoVs: MERS, SARS,zoonotic precursor CoVs
Efficacy in vivo (small animals)
Slow or no resistance during therapeutic use Known mechanism of activity
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Progress at risk in current GOF definition and
pause for MERS-CoV In vitro replication models
Adaptation for improved replication in culture
In vivo (mouse, small animal) models of replication andpathogenesis
Genetic modification of mice
Passage adaptation for replication and pathogenesis
Passage for resistance, sequencing and reversegenetic confirmation
Identify common mechanisms and determinants
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Infidelity is Bad forCoronavirus Relationships:
A Case Study
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Would you take or give a livevaccine with a virus that has
an engineered increasedmutation rate?
Why Not?
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Should we engineermutator strains?
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E An
3S M N
RdRp ExoN
aa1 D E D D aa527motif II motif IIImotif I
3-5 exonuclease (ExoN)
nsp12 nsp14
(Snijder et al. 2003; Minskaia et al.,2006; Chen, et al., 2009)
Coronaviruses proofread using a3-to-5 exonuclease (ExoN)
DE-D-D Exonuclease Superfamily ProofreadingExonucleases in Bacteria and Eukaryotes
Mutations in DE-D-D motifs inactivate ExoN activity(ExoN-)
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aa1 D E D D aa527motif II motif IIImotif I
3-5 exonuclease (ExoN)
What is the effect of ExoN inactivation(ExoN-) on virus replication fidelity?
AAExoN-
ES M N
RdRp ExoN
nsp12 nsp14 An
3
Coronaviruses proofread using a3-to-5 exonuclease (ExoN)
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Eckerle et al., J Virol 2007; PLoSPathogens 2010; Michelle Becker
Substitutionsper genome
SARS-CoV
ExoN +SARS-
ExoN -
20.7x
CoV ExoN- mutants have a mutator
phenotype with a 20-fold increasedmutation rate
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Generate populations with tremendous variation
Increased mutation rate = increased : Adaptation and Fitness Virulence Public Health risk
Assumptions about RNA virus fitness
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SARS-ExoN - mutator strain is less fitthan wildtype SARS-ExoN +
Passage Passage Passage1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Input Ratio1:1
SARS-ExoN-
P e r c e n
t o
f t o t a l
g e n o m e
R N A
Input Ratio 10 :1
Input Ratio 100 :1
0
20
40
60
80
100
SARS-ExoN +
Graham et al Nature Med 2012
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SARS ExoN - mutator virus is attenuatedin mouse model of lethal SARS
SARS-ExoN - 10 2, 10 3, 10 4 PFU
P e r c e n
t s u r v
i v a
l
Time post infection (days)
SARS-ExoN+
10 2 PFU
10 4 PFU
10 3 PFU
0 2 4 6 8 10 12 140
20
40
60
80
100
Graham et al Nature Med 2012
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Wildtype CoVs are resistant to Ribavirin, RNAmutagens, nucleoside analogs
ExoN- mutants are highly susceptible to acuteinhibition and lethal mutagenesis.
Inhibition of ExoN fidelity as combination Rx
with Ribavirin and developed nucleosideanalogs
Targeting nsp14-ExoN proofreading
for therapeutics
Smith et al., PLoS Pathogens 2013
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Increased mutation rate does NOT favor virus fitness:Fidelity is a target for vaccines and therapeutics.
Passage for adaptation and resistance in vitro and animalmodels in vivo are essential components of therapeuticdevelopment.
There are no bioinformatic or predictive safer alternativeapproaches to identify unknown, unpredicted, and
unprecedented targets for countermeasures. Limiting already safe and regulated research does not stop
coronavirus epidemics or adaptation of potential zoonoticviruses to human disease.
Our Assumptions: Are often wrong
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CV-B3 polio EV-71 FMDV ChikV SARS-CoV H5N1-Flu
ExoN-
H5N1influenza
Attenuation of Decreased Fidelity andIncreased Fidelity Mutants of RNA Viruses
Anti-mutator
Mutator
Adapted from Smith Ann Rev Virol 2014
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AcknowledgementsVanderbiltClint SmithNicole SextonMichelle BeckerXiaotao Lu
UNCRalph BaricRachel Graham
Amy SimsVineet Menachery
R01 AI 108197
R01 AI26603U19 AI109680
Elizabeth B. Lamb Center forPediatric ResearchCraig-Weaver Chair in Pediatrics