Ralph S.

Baric, PhD
Professor: Epidemiology
University of North Carolina at Chapel Hill

Research: Coronaviruses, Noroviruses and Flaviviruses, hostsusceptibility gene mapping, cross species transmission,
pathogenesis, animal model development, vaccine design

Ralph S. Baric, PhD

Professor: Epidemiology
University of North Carolina at Chapel Hill

Research: Coronaviruses, Noroviruses and Flaviviruses, Hostsusceptibility 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

Goals:
Coronaviruses and influenza viruses are biologically
distinct organisms
Concepts/Outcomes: Described for influenza viruses
may 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 and
Therapeutics

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?

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 SARSCoV/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)

Emerging Coronaviruses

2012 in US

~95% mortality in newborn piglets

>8 million pigs (Dec 2014)

Emerging Coronaviruses

2012 in US

Receptor Driven Cross Species Transmission

Paradigm: SARS Evolved from Viruses Circulating in Zoonotic Pools

?
Rhinolophus pearsoni (28%)
Rhinolophus pusillus (33%)
Rhinolophus macrotis (71%)

Palm Civets

Humans

(Raccoon Dogs)
(critical intermediate host)

Evolution towards efficient infection of human cells (ACE2)

2002-2003 Epidemic: ~8,000 cases/800 deaths

Guan et al., 2003; Li et al.,

2005, Song et al., 2005

SARS-CoV Evolution: 2003 Pandemic (16 mutations)

Gene

Early
Middle
Late
Original residue
Civet to Human
Early to middle

Middle to Late

STRAIN
SZ16
HC/SZ/63
GZ02
CUHK-W1
Urbani

1B

Spike

X1

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
X1-81
X1-93
X1-121

Phase
Animal

ORF 1A

S
A
A
A
A

A
V
V
V
V

T
T
T
I
I

L
P
P
P
P

I
I
I
L
L

F
L
F
L
L

Y
C
Y
C
C

S
L
L
L
L

W
C
W
C
C

A
A
A
V
V

A
A
A
A
V

A
A
A
A
V

E
E
E
E
D

K
R
K
R
R

D
D
D
D
G

K
K
N
N
N

L
S
L
S
S

T
T
T
T
I

K
T
T
T
T

R
R
R
K
K

S
S
F
F
F

K
R
N
N
N

S
S
T
T
T

P
S
S
S
S

S
S
L
L
L

L
S
S
S
S

A
T
T
T
T

Human ACE2 Receptor

Use

A
V
V
V
V

D
D
D
Y
Y

A
T
T
T
T

E
E
E
K
K

S
S
G
G
G

I
I
F
F
F

S
S
C
C
C

Y
H
H
H
H

Virus
Yield/cell

SARS-CoV
S glycoprotein

G
G
C
C
C

SARS-CoV Evolution: 2003 Pandemic (16 mutations)

Gene

Early
Middle
Late

STRAIN
SZ16
HC/SZ/63
GZ02
CUHK-W1
Urbani

1B

S
A
A
A
A

A
V
V
V
V

Original residue
Civet to Human
Early to middle

T
T
T
I
I

L
P
P
P
P

I
I
I
L
L

F
L
F
L
L

Y
C
Y
C
C

S
L
L
L
L

W
C
W
C
C

A
A
A
V
V

A
A
A
A
V

A
A
A
A
V

E
E
E
E
D

K
R
K
R
R

D
D
D
D
G

K
K
N
N
N

L
S
L
S
S

T
T
T
T
I

K
T
T
T
T

R
R
R
K
K

S
S
F
F
F

K
R
N
N
N

S
S
T
T
T

P
S
S
S
S

S
S
L
L
L

L
S
S
S
S

A
T
T
T
T

SARS-CoV infection of HAE

Middle to Late

Human ACE2 Receptor

Use

1.E+06

Human
Strains

Virus Titer (pfu/ml)

1.E+05

1.E+04

1.E+03

1.E+02

HAE Cultures

Spike

X1

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
X1-81
X1-93
X1-121

Phase
Animal

ORF 1A

10

20

30

40

50

Hours post infection

60

70

Civet
Strains

A
V
V
V
V

D
D
D
Y
Y

A
T
T
T
T

E
E
E
K
K

S
S
G
G
G

I
I
F
F
F

S
S
C
C
C

Y
H
H
H
H

G
G
C
C
C

SARS Urbani in Ciliated

Airway Epithelial Cells
Urbani

SZ16 or HC/SZ/61/03

Sheahan et al., J. Virology 2007,

Sheahan J.Virol 2008, 2008

SARS Urbani in Ciliated

Airway Epithelial Cells
Urbani
D22
D8

SZ16 or HC/SZ/61/03

Sheahan J.Virol 2008, 2008

SARS Urbani in Ciliated

Airway Epithelial Cells
Urbani
D22
D8

In Vitro Model
Gain of human ACE2 receptor usage
SZ16 or HC/SZ/61/03

Loss of Civet ACE2 receptor usage

Sheahan J.Virol 2008, 2008

IAV Transmission Model

Ferret

Human

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

IAV Transmission Model

Ferret

Human

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

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

ACE2 Orthologue Receptor Interface is Different/Species

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

Poor engagement
of mouse ACE2
receptor

Development of Mouse Adapted Strains: 6-10 changes

More in vivo
passages

More S Gene Mutations

mACE2 usage

SARS-CoV Mouse Adaptation

MA15

MA20

MA25

RBD

mACE2

Increase SARS-MA interactions with mACE2

Generally decrease interaction fitness with hACE2
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

SARS-CoV Mouse Adaptation

MA15

MA20

MA25

RBD

mACE2

Increase SARS-MA interactions with mACE2

Generally decrease interaction fitness with hACE2

Does increased replication fitness and virulence

in mice correlate with increased transmissibility
in mice?

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+)

Increased virulence and replication

efficiency doesnt correlate with
increased transmissibility in mouse
Most vulnerable
Models (MA15)

3 infected
1 uninfected
5 days

No evidence of
In
aged mouse model transmission
vulnerable inbred and
outbred populations
backgrounds (model outbred populations)

Different genetic
Certain CC Founders LD50=<102 (3 days)

N=1820

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

Increased virulence and replication

efficiency doesnt correlate with
increased transmissibility in mouse
Models (MA15)

No evidence of
transmission In
vulnerable inbred and
outbred populations

Handful of these controls

RNAseq
Absence of SARS viral nucleic acid
N=1820

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

Value Transmission Studies?

SARS-CoV Model System:

Relevant for Ferret
Identify key phenotypes associated with enhanced virus
transmission
Establish biochemical screens for phenotypes associated
with 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)

Alternative in vivo Models

Safer Mouse Models for In Vivo Drug Screening?

Sindbis Virus-alphavirus (nonpathogenic)

IFNR-/- Model

3e Plpro
Inhibitor

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

Deng et al., JVirol 2014

MA15 Mouse Model

Safer Mouse Models for In Vivo Drug Screening?

Sindbis Virus-alphavirus

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

IFNR-/- Model

MA15 Mouse Model

Safer Mouse Models for In Vivo Drug Screening

IFNR-/- Model

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

Success!s

3e Plpro
Inhibitor

MA15 Mouse Model

Drug fails

Safer Mouse Models for In Vivo Drug Screening

IFNR-/- Model

Why Fail?
Virus Tropism (CNS vs lung)
Bioavailability of Drug (lung?)
Virus Titer in Target Organ: Different
SARS PlproIn vivo targets: Different
Target for protease inhibitors
Indirect Models: Misinform

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

Success!s

3e Plpro
Inhibitor

MA15 Mouse Model

Drug fails

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
A

mDPP4 Species Specificity Determinants

B

MERS-CoV RFP

Human DPP4

Mouse DPP4

No DPP4

chDPP4 (273-340)

Kayla Peck

DPP4
Adam Cockrell

Numbered relative to moue DPP4

Cockrell et al., JVirol 2014

Modeling Based Predictions

R511

D455
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

MERS-CoV RFP Infection

Using these data to develop mouse adapted strains MERS-CoV

Figure 3

MERS-CoV RFP Infection

T330R removes
glycosylation site mDPP4
Mouse
models
Transgenic mice hDPP4

Using these data to develop mouse adapted strains MERS-CoV

CRISPR/CAS to introduce
hT330R and A288L mutations
into mDPP4 receptor

Potential Glycosylation Sites

Ferret

GP
Human

Mouse

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

Regulatory Intangibles on CoV, IAV

Research
Students and Postdocs
Grant Review Process

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
Respondents (n=126 as of 12/15/2014):
79% currently study a virus
52% currently study a respiratory virus; 70% want to in the future growth area

Have you heard about

the GOF/PPP debate?

Knowing about the debate,

are you more or less likely
to work on SARS, MERS,
or influenza in the future?

No

For virologists: Has the

debate changed your
future plans?
Yes

Less

Yes
Yes- a lot

95% know about debate

Equal

37% are less likely to work

on SARS/MERS/influenza

No

53% have changed or

may change their plans

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
pathogenesis, virus-host interactions

The studies span from

structural biology and cell
culture experiments to
pathogenesis in mice, likely
providing a comprehensive
understanding.
Structure
Function
Mechanism
Pathogenesis