Session 5: GOF Research and Countermeasures Against SARS/MERS

8/10/2019 Session 5: GOF Research and Countermeasures Against SARS/MERS

1/19

GOF studies in MERS andSARS countermeasures

Mark R. Denison M.D.Vanderbilt University School of Medicine


2/19

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


3/19


4/19

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


5/19

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


6/19

Infidelity is Bad forCoronavirus Relationships:

A Case Study


7/19

Would you take or give a livevaccine with a virus that has

an engineered increasedmutation rate?

Why Not?


8/19

Should we engineermutator strains?


9/19

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-)


10/19

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)


11/19

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


12/19

Generate populations with tremendous variation

Increased mutation rate = increased : Adaptation and Fitness Virulence Public Health risk

Assumptions about RNA virus fitness


13/19

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


14/19

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


15/19


16/19

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


17/19

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


18/19

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


19/19

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

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Session 5: GOF Research and Countermeasures Against SARS/MERS