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Transcript of Subbarao FINAL
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Scientific and technical aspects of
research on Influenza, SARS and MERS
Kanta Subbarao
Laboratory of Infectious Diseases, NIAID, NIHNAS GoF symposium
December 15, 2014
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Topics
The types of Gain of Function (GoF) research
What impact does virological research typically have on
the viruses being studied, in terms of gain or loss of
function?
What do we know or not know about flu, SARS, and
MERS and can GoF research help fill the knowledge
gaps?
Where does virological research cross the line into GoF
research as defined by the U.S. government?
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Questions virologists ask
Why/howdoes the virus infect and kill mammals?
Doantiviral drugs work and howdoes the virus become resistant?
Docurrent or novel vaccines provide protection and canthe virusescape?
Howdoes the virus spread within animals?
Howdoes the virus spread from animals to humans and from
humans to humans?
Couldthe virus cause a pandemic?
Whatis the likelihood of (re)emergence?
Adapted from Paul Duprex; image Russell Kightley Science Photo Library
http://tree.bio.ed.ac.uk/research/influenza/ -
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Reverse Genetics
Generating viruses de novo
cDNA copy of genome
(+)
RNA
recombinant
virus
introduce into cells
Adapted from Paul Duprex
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New Technologies in Virology Research
Reverse genetics: flu, SARS and MERS
Deep sequencing: viral quasispecies at different sites
can be characterized
Virus-host interactions: siRNA screens
Human MAbs generated from phage display libraries or
plasmablasts or by immortalizing B cells
Modifying the host genome: CRISPR/Cas9
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Applications of Gain of Function (GoF) Research
Biology of viruses Identification of novel viral gene products
Identification of receptor(s) used
Virus Ecology Characterizing viruses from animal hosts
Identification of host range determinants
Viral Pathogenesis Identification of virulence determinants
Airborne spread
Virus-host interactions including innate and adaptive immunity
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Applications of Gain of Function (GoF) Research-2
Development of animal models Adaptation to specific animal species to induce clinical signs of
disease e.g. SARS MA15 and MA20
Antiviral drugs
Mechanisms of antiviral drug resistance
Evaluation of the fitness cost of antiviral resistance mutations
Immunoprophylaxis/immunotherapy
Identification of epitope(s) targeted by Abs, especially broadlyneutralizing MAbs
Evaluation of the virulence and fitness of MAb resistance
mutants
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Applications of Gain of Function (GoF) research-3
Vaccine development
Molecular characterization of antigenic variants
Generation of vaccine seed viruses with appropriate properties
including enhanced yield and immunogenicity while preserving
antigenicity; example H1N1pdm vaccine
Elucidating the biological basis for adverse outcomes
associated with vaccine candidates (e.g. SARS vaccines)
Determining the molecular basis for attenuation of vaccine
viruses
Determining the stability of live attenuated vaccine candidates
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How and why do virologists study evolution of
viruses under immune or antiviral pressure?
Sequence their genomes (or target gene segments)
Introduce each substitution individually and in
combinations into a reverse-genetics derived virus
Examine the fitness and resistance phenotypes invitro orin vivo
Why?
To understand the biology of the virus
To map epitopes of antibodies
To develop robust counter measures
(antiviral drugs and vaccines)
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Coronavirus targets for vaccines and
therapeutics
An3
ES M N
nsp12
RdRp
nsp14
Exonuclease
nsp12 nsp14
nsp3
PL-protease
nsp5
proteasensp16
2O MT-ase Spike
Proteases PolymeraseProofreading
ImmuneEvasion
Entry
Host RangeImmunity
5 nsp3 nsp5 nsp16
CoV genome ~30kb +sense RNA
defined by reverse genetics and adaptive experimental evolution
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Topics
The types of Gain of Function (GoF) research
What impact does virological research typically have on
the viruses being studied, in terms of gain or loss of
function?
What do we know or not know about flu, SARS, and
MERS and can GoF research help fill the knowledge
gaps?
Where does virological research cross the line into GoF
research as defined by the U.S. government?
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Gain of Function
The term gain of functionis a vague and unsatisfactory termfor microbiologists
One suggested alternative is aTRIP: an acronym for an
experiment that uses one or more of the DURC agents andproduces, aims to produce, or can be reasonably
anticipated to alter Transmission, Range and resistance,
Infectivity/immunity or Pathogenesis
Another suggestion is Gain of Virulence or Transmissibility
Duprex and Casadevall mBioin press; Mark Denison
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The impact of virological methods on gain of
function in viruses
Research
maneuver
Comment/
Example
GoF? Other
consequences:
LoF?
aTRIP/GO
VT?
Passage in
cellculture/eggs
Yes: usually
desired
Possible for
other cells
no
Serial
passage in
experimental
animals
SARS CoV
MA15
Possible:
often desired
Possible for
other species
RP/V
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The impact of virological methods on gain of
function in viruses
Research
maneuver
Comment/
Example
GoF? Other
consequences:
LoF?
aTRIP/GO
VT?
Passage in
cellculture/eggs
Yes: usually
desired
Possible for other
cells
no
Serial
passage in
experimental
animals
SARS CoV
MA15
Possible:
often desired
Possible for other
species
RP/V
Genetic
reassortment
of influenza
viruses
Influenza
vaccines GoF
yield, LoF
virulence
Yes; often
desired
Possible: often
desired
no
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The impact of virological methods on gain of
function in viruses
Research
maneuver
Comment/
Example
GoF? LoF? aTRIP/GO
VT?
Antiviral or
MAb resistant
mutants
Valuable
information on
suitableantiviral
strategies
Yes: usually
desired
Possible
reduction in
virulence andfitness
no
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The impact of virological methods on gain of
function in viruses
Researchmaneuver Comment/Example GoF? LoF? aTRIP/GOVT?
Antiviral or
MAb resistant
mutants
Valuable
information on
suitable
antiviralstrategies
Yes: usually
desired
Possible
reduction in
virulence and
fitness
no
Site directed
mutagenesis
Used to
conclusively
prove the
molecular
basis of a
phenotype
Possible Possible No or
TRIP/VT
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Topics
The types of Gain of Function (GoF) research
What impact does virological research typically have on
the viruses being studied, in terms of gain or loss of
function?
What do we know or not know about flu, SARS, and
MERS and can GoF research help fill the knowledge
gaps?
Where does virological research cross the line into GoF
research as defined by the U.S. government?
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What do we know about influenza, SARS and
MERS coronaviruses?
Biology of the virus
Ecology; reservoir
Several host range and virulence
determinants
Innate and adaptive immunity
Antiviral drugs and Vaccines for influenza but
not for coronaviruses
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What do we not know about influenza viruses?
Why are some viruses more virulent than others?
Why do some viruses spread efficiently while others dont?
Critical for understanding seasonal influenza and for assessing
pandemic potential of novel viruses
What drives the evolution of antigenic change and antiviral
resistance?
Critical for vaccines and antiviral drugs
Are there viral targets that will not escape under immune pressure?
Critical for the development of universal vaccines
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What do we not know about SARS and MERS-CoV?
Will SARS or a SARS-like CoV re-emerge from bats or other animalhosts?
What are the critical host range and virulence determinants of
MERS-CoV?
Can we develop a candidate vaccine that is safe, immunogenic andefficacious?
Can MAbs be used safely for prevention and treatment?
Can we identify antiviral drugs that are safe and effective?
Critical because of the ongoing outbreak of MERS and in preparing for
possible re-emergence of SARS-CoV
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What safety and oversight measures are in place?
Measures MERS SARSNon-HPAI
influenza
HPAI and
H7N9influenza
BSL3 with
respirators
Medical
surveillance
and support
Vaccines and
antiviralsN/A N/A
Select agent
regulations
DURC
questionnaire
Wh d i l i l h h
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Where does virological research cross the
line into GoF research as defined by the
U.S. government?
The line:
all influenza viruses, SARS-CoV, MERS CoV
Experiments that are reasonably anticipated toincrease
pathogenicity
or
transmissibility
in any mammalian species
Where does virological research cross the line into
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Where does virological research cross the line intoGoF research as defined by the U.S.
government?
Adaptation of MERS CoV to animal models
Elucidating the molecular determinants of transmissibility by the
airborne route (influenza)
Elucidating the biological basis for adverse outcomes associated
with candidate SARS vaccines
Conclusive experiments to demonstrate the biological significance of novel gene products
genetic differences between isolates from animals and/or humans for
newly emerged viruses e.g. H7N9, H5N8, H5N2, H10N7 and H10N8
influenza and MERS CoV
Virulence determinants of newly emerged viruses e.g. H7N9, H5N8,H5N2, H10N7 and H10N8 influenza and MERS CoV
Molecular basis for resistance to antiviral drugs and MAbs
Viral evolution under immune pressure
Viral evolution in the presence of antiviral drugs
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The perspective of many influenza and
coronavirologists
Concerns about aerosol-transmissible highly pathogenicavian influenza viruses have expanded to include
MERS-CoV and SARS-CoV
Research in rodent models
All influenza viruses including laboratory strains that areavirulent for humans
An open scientific discussion is needed about the rationale and
justification for doing so.
Risk assessment should be based on
biosafety measures and PPE that are in use
numbers that represent actual experience and
laboratory exposures and accidents with SARS,
MERS and highly pathogenic avian influenza viruses
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Priorities
Replace GOF with microbiological terms that precisely and
appropriately define the experiments of concern
Lift the restrictions on MERS for 3 critical reasons:
there is an urgent public health need for medical countermeasures with
cases and deaths reported every week
there is no evidence of transmission in any animal model of MERS orSARS
no laboratory acquired infections in the US in over 10 years of work on
SARS and MERS
Lift the restrictions on rodent models because adaptation to rodentsdoes not enhance virulence for humans
Focus only on influenza viruses that are of concern
Lift the restrictions on characterizing escape from antibodies,
antivirals and vaccines
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Approaches to virology research
gain-of-function pandemic threat
universal genetic approach
ferrets are a useful model for human influenza
convergence of results
NAb escape mutants arise in patients
how much selection?
risk mitigation is available engineered strains
vaccination of lab personnel
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Good reasons for it and often no alternatives[e.g. drug resistance mechanisms]
Uses basic methods common to all areas of microbiology
Not a question of whether, but how
- Thorough risk assessment needed
- Emphasis on transmissibility and virulence
Current rule is counterproductive[e.g. MERS-CoV small animal model for vaccine/drug evaluation;
not all flu are alike]
Gain-of-Function Research and Virology
[e.g. passage in different species, reassortment, adaptation, selection]