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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




function?



gaps?




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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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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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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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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




function?



gaps?




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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]

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