Session 8: Special Considerations about GOF Research (Baric)
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Transcript of Session 8: Special Considerations about GOF Research (Baric)
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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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SARS Urbani in Ciliated
Airway Epithelial Cells
SZ16 or HC/SZ/61/03
Urbani
D8
D22
Sheahan J.Virol 2008, 2008
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SARS Urbani in Ciliated
Airway Epithelial Cells
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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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
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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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
Treat half animals with Plpro inhibitor
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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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
Treat half animals with Plpro inhibitor
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