session 8: special considerations about gof research (baric)

8/10/2019 Session 8: Special Considerations about GOF Research (Baric)

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Ralph S. Baric, PhD

Professor: Epidemiology

University of North Carolina at Chapel Hill

Research: Coronaviruses, Noroviruses and Flaviviruses, host-susceptibility gene mapping, cross species transmission,

pathogenesis, animal model development, vaccine design


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Ralph S. Baric, PhD

Professor: Epidemiology

University of North Carolina at Chapel Hill

Research: Coronaviruses, Noroviruses and Flaviviruses, Host-susceptibility genes, cross species transmission, pathogenesis,

animal model development, vaccine design

Disclosure: Well funded by NIH, Patents CoV

reverse genetics, CoV Vaccine Design,

DENV reverse genetics and vaccine design,

NoV Vaccine Design


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Goals: Coronaviruses and influenza viruses are biologically

distinct organisms

Concepts/Outcomes:Described for influenza virusesmay or may not be relevant to Coronaviruses

GOF:Encompasses a very diverse set of experiments Critical for vaccine and therapeutic design

Pathogenesis and virulence (young/aged models)

Virus-host interaction networks, Susceptibility

Alleles, Host response patterns Host antiviral defense programs

Transmissibility Studies

Regulations Develop: framed to appreciate the

biological complexity of different virus families

Engine for the

Development of

Broad-based

Vaccines andTherapeutics


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Charge

Differences among organisms?

What functionality is being gained or lost?

Are transmissibility, virulence, growth and

functionality all similar in terms of GOF

objectives?

Special considerations about alternative research

methods with less risk?


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CoV Biology

Introduction

CoV Cross Species Transmission Coronavirus Receptor Interactions (SARS/MERS)

Transmission in lethal mouse adapted models (No)

Difficulty in developing transmission models for SARS-CoV/MERS-CoV

Ethical Alternatives to GOF Safe, alternative models for evaluating drug efficacy (Fail)

Challenge: Ease Structure-Guided Predictions (sequence data)

Intangibles/Unintended Consequences

No Approved Vaccines or Therapeutics for SARS-CoV or MERS-CoV

Midst of an ongoing MERS-CoV Outbreak (950+ cases/40% mortality)


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Emerging Coronaviruses

2012 in US

~95% mortality in newborn piglets

>8 million pigs (Dec 2014)


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Emerging Coronaviruses

2012 in US


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Receptor Driven Cross Species Transmission

Paradigm: SARS Evolved from Viruses Circulating in Zoonotic Pools

Palm Civets(Raccoon Dogs)

(critical intermediate host)

Humans

Evolution towards efficient infection of human cells (ACE2)

Rhinolophus pearsoni (28%)Rhinolophus pusillus (33%)

Rhinolophus macrotis (71%)

?

2002-2003 Epidemic: ~8,000 cases/800 deaths Guan et al., 2003; Li et al.,2005, Song et al., 2005


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Gene ORF 1A 1B Spike M X1

Phase STRAIN 549

1021

1121

1136

1663

2116

2222

2269

2746

2971

3047

3072

1389

2532

77

227

239

244

261

344

360

479

487

607

665

701

743

754

778

849

1163

M4

X1-7

X1-8

1

X1-9

3

X1-1

21

Animal SZ16 S A T L I F Y S W A A A E K D K L T K R S K S P S L A A D A E S I S Y GHC/SZ/63 A V T P I L C L C A A A E R D K S T T R S R S S S S T V D T E S I S H G

Early GZ02 A V T P I F Y L W A A A E K D N L T T R F N T S L S T V D T E G F C H CMiddle CUHK-W1 A V I P L L C L C V A A E R D N S T T K F N T S L S T V Y T K G F C H CLate Urbani A V I P L L C L C V V V D R G N S I T K F N T S L S T V Y T K G F C H C

Original residueCivet to HumanEarly to middle

Middle to Late

SARS-CoV Evolution: 2003 Pandemic (16 mutations)

Human ACE2 Receptor

Use

VirusYield/cell

SARS-CoV

S glycoprotein


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Gene ORF 1A 1B Spike M X1

Phase STRAIN 549

1021

1121

1136

1663

2116

2222

2269

2746

2971

3047

3072

1389

2532

77

227

239

244

261

344

360

479

487

607

665

701

743

754

778

849

1163

M4

X1-7

X1-8

1

X1-9

3

X1-1

21

Animal SZ16 S A T L I F Y S W A A A E K D K L T K R S K S P S L A A D A E S I S Y GHC/SZ/63 A V T P I L C L C A A A E R D K S T T R S R S S S S T V D T E S I S H G

Early GZ02 A V T P I F Y L W A A A E K D N L T T R F N T S L S T V D T E G F C H CMiddle CUHK-W1 A V I P L L C L C V A A E R D N S T T K F N T S L S T V Y T K G F C H CLate Urbani A V I P L L C L C V V V D R G N S I T K F N T S L S T V Y T K G F C H C

Original residueCivet to HumanEarly to middle

Middle to Late

SARS-CoV Evolution: 2003 Pandemic (16 mutations)

Human ACE2 Receptor

Use

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0 10 20 30 40 50 60 70

VirusTiter(pfu/ml)

Hours post infection

SARS-CoV infection of HAE

Human

Strains

Civet

StrainsHAE Cultures


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SARS Urbani in Ciliated

Airway Epithelial Cells

SZ16 or HC/SZ/61/03

Urbani

Sheahan et al. J. Virolo 2007Sheahan J.Virol 2008, 2008


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SZ16 or HC/SZ/61/03

Urbani

D8

D22

Sheahan J.Virol 2008, 2008


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SZ16 or HC/SZ/61/03

Urbani

D8

D22

In Vitro Model

Gain of human ACE2 receptor usage

Loss of Civet ACE2 receptor usage

=

Sheahan J.Virol 2008, 2008


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=Ferret Human

IAV Transmission Model

Select for increased 2,6 usage in one, selects for increased 2,6 usage in the other

Debate: selection for increased transmission in one=increased transmission in the other


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=Ferret Human

IAV Transmission Model

Select for increased 2,6 usage in one, selects for increased 2,6 usage in the other

Debate: selection for increased transmission in one=increased transmission in the other

ACE2 Orthologue Receptor Interface is Different/Species


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ACE2 Orthologue Receptor Interface is Different/Species

Possible to select for Generalists that efficiently use multiple ACE2 receptors

Selection by In vitro or in vivo passage in one species, usually selects for specialists

I t f A i l M d l D l t


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Importance of Animal Model Development

to Vaccine Development SARS-CoV replicates poorly in mice (106)

No weight loss or clinical disease Little if any pathology

Every vaccine developed works in the model

Development of Mouse Adapted Strains: 6-10 changes

Poor engagement

of mouse ACE2

receptor

More S Gene MutationsMore in vivo

passages mACE2 usage


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SARS-CoV Mouse Adaptation

MA15 MA20 MA25

Increase SARS-MA interactions with mACE2

Generally decrease interaction fitness with hACE2

RBD

mACE2

Isolate these S gene MA mutations in wildtype SARS-CoV

Does not produce a lethal disease phenotype in mice

You need 2 or more other mutations to produce a lethal phenotype


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SARS-CoV Mouse Adaptation

MA15 MA20 MA25

Increase SARS-MA interactions with mACE2

Generally decrease interaction fitness with hACE2

RBD

mACE2

Does increased replication fitness and virulence

in mice correlate with increased transmissibility

in mice?


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Models of Outbred Human Populations

Collaborative Cross Mouse Resource 8 Founder Lines64 F1 diallel cross combinations

Age: 1 year, vaccinate half with DIV, challenge with virus (~1860 mice)

Mock-vaccinated controls co-housed with challenged animals (n=600+)

No evidence of

transmission In

vulnerable inbred and

outbred populations

N=1820

Increased virulence and replication

efficiency doesnt correlate with

increased transmissibility in mouse

Models (MA15)Most vulnerable aged mouse model

Different genetic backgrounds (model outbred populations)Certain CC Founders LD50=


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Models of Outbred Human Populations

Collaborative Cross Mouse Resource 8 Founder Lines64 F1 diallel cross combinations

Age: 1 year, vaccinate half with DIV, challenge with virus (~1860 mice)

Mock-vaccinated controls co-housed with challenged animals

No evidence of

transmission In

vulnerable inbred and

outbred populations

N=1820

Increased virulence and replication

efficiency doesnt correlate with

increased transmissibility in mouse

Models (MA15)

Handful of these controls

RNAseq

Absence of SARS viral nucleic acid


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SARS-CoV Transmission Models

Have not been established, or attempted by the field

SARS-CoV doesnt replicate in the guinea pig SARS-CoV replicates in ferrets/limited disease

Inefficient usage of the ferret ACE2 receptor

Establish a transmission model? Likely could be established in ferrets

Likely select for receptor mutations that enhance ferret

receptor usage with concomitant reductions in hACE2 usage Select for handful of other mutations (other genes) that

increase virus pathogenesis/transmission in the ferret

Biosafety: Likely safe, especially if conditions are used to

select for specialists; easy to evaluate hACE2 receptor usage


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Value Transmission Studies?

SARS-CoV Model System:

Relevant for Ferret Identify key phenotypes associated with enhanced virus

transmission

Establish biochemical screens for phenotypes associatedwith enhanced transmission (relevant prepandemic strains)

Fundamental insights into mechanisms of

emerging coronavirus transmission

Some argue these data are critical for modeling epidemic

potential (hard metrics/reduce uncertainty)

New strategies for improved safety of live

attenuated vaccines (transmission defective viruses)


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Alternative in vivo Models


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SARS Plpro- Target for protease inhibitors

Mediates removal of ISG15

(deISGylation) from cellular proteins

Simple in vivo screen for activity

Inserted into Sindbis Virus (alphavirus)

Inoculate in IFNR-/- Mice

3e Plpro

Inhibitor

MA15 Mouse Model

IFNR-/- Model

Safer Mouse Models for In Vivo Drug Screening?

Sindbis Virus-alphavirus (nonpathogenic)

Deng et al., JVirol 2014


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Treat half animals with Plpro inhibitor

MA15 Mouse Model

IFNR-/- Model

Safer Mouse Models for In Vivo Drug Screening?

Sindbis Virus-alphavirus


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3e Plpro

Inhibitor

MA15 Mouse Model

IFNR-/- Model

Safer Mouse Models for In Vivo Drug Screening

X

Drug fails

Success!s


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3e Plpro

Inhibitor

MA15 Mouse Model

IFNR-/- Model

Safer Mouse Models for In Vivo Drug Screening

X

Drug fails

Success!sWhy Fail?

Virus Tropism (CNS vs lung)

Bioavailability of Drug (lung?)Virus Titer in Target Organ: Different

In vivo targets: Different

Indirect Models: Misinform


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MERS-CoV

Receptor is human dipeptidyl peptidase 4 (DPP4) High affinity for camel and certain bat DPP4 as well

MERS-CoV cannot use mouse, rat, hamster, ferret

and guinea pig DPP4 receptors for entry

Amino acid variation at receptor interface/species

Glycosylation site at the receptor interface

Lu G, et al., Nature 2013

Figure 2


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g

C

AB

Mouse DPP4

chDPP4 (273-340)

Human DPP4

No DPP4

Numbered relative to moue DPP4

Kayla Peck

Adam Cockrell

mDPP4 Species Specificity Determinants

MERS-CoV RFP

DPP4 Cockrell et al., JVirol 2014


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Modeling Based Predictions

D455

R511

3 Different groups: predicted 10 mutation sets in MERS-CoV increase mDPP4 usage

All failed; while it is possible to use structures to predict pathways/many (all) failed

Figure 3


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A C

D

B

Using these data to develop mouse adapted strains MERS-CoV

MERS-CoV RFP Infection

Figure 3


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A C

D

B

Using these data to develop mouse adapted strains MERS-CoV

MERS-CoV RFP Infection

T330R removes

glycosylation site mDPP4

Transgenic mice hDPP4

CRISPR/CAS to introduce

hT330R and A288L mutations

into mDPP4 receptor

Mouse

models

Potential Glycosylation Sites


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Human

GPFerret

Mouse

Potential Glycosylation Sites


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MERS-CoV Transmission Models

Models have not been developed

Difficult, but not impossible to establish

Biosafety Concerns:

Uncertain Outcomes/SARS-CoV

Possible to evaluate hDPP4 receptor usage/mutant

Its not just about the receptor


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Regulatory Intangibles on CoV, IAV

Research

Students and Postdocs

Grant Review Process

H i th d b t & h ff ti f t l f t i ?


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How is the debate & research pause affecting future plans of trainees?

Survey Monkey poll for students & postdocs (posted 12/13/2014)

Advertised on Twitter, ASV Facebook, emails

Have you heard about

the GOF/PPP debate?

Yes- a lot

Yes

No

Less

Equal

Knowing about the debate,

are you more or less likely

to work on SARS, MERS,

or influenzain the future?

For virologists: Has the

debate changed yourfuture plans?

Yes

No

Respondents (n=126 as of 12/15/2014):79% currently study a virus

52% currently study a respiratory virus; 70% want to in the futuregrowth area

95% know about debate

37% are less likely to work

on SARS/MERS/influenza

53% have changed or

may change their plans


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Subtle: Grant Review Process

GOF Restrictions

Limit animal model development

Limit reverse genetic applications

Limit ability to make causal

associations between genotype and

phenotype

Uncouple: very powerful

experimental continuum Biochemistry and structure-function

studies

Genetic studies (validate biochemistry,

role in virus replication)

Evaluating functionality in viral

th i i h t i t ti

The studies span from

structural biology and cell

culture experiments to

pathogenesis in mice, likelyproviding a comprehensive

understanding.

Structure

Function

Mechanism

P th i

session 8: special considerations about gof research (baric)

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