Session 2: What is virology and what do we know about SARS, MERS, and flu?

Scientific and technical aspects of research on Influenza, SARS and MERS

Kanta SubbaraoLaboratory of Infectious Diseases, NIAID, NIH

NAS GoF symposiumDecember 15, 2014

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 GoFresearch as defined by the U.S. government?

Questions virologists ask

• Why/how does the virus infect and kill mammals?

• Do antiviral drugs work and how does the virus become resistant?

• Do current or novel vaccines provide protection and can the virus escape?

• How does the virus spread within animals?

• How does the virus spread from animals to humans and from humans to humans?

• Could the virus cause a pandemic?

• What is the likelihood of (re)emergence?

Adapted from Paul Duprex; image Russell Kightley Science Photo Library

Reverse Genetics

Generating viruses de novo

cDNA copy of genome

(+)

RNA

recombinantvirus

introduce into cells

Adapted from Paul Duprex

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

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

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 broadly

neutralizing MAbs

• Evaluation of the virulence and fitness of MAb resistance mutants

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

Virus ALethal for mice

Virus BNot lethal for mice

How do influenza virologists identify viral determinants of virulence?

Generate single gene reassortants to decide which gene(s) to compare

Compare viral gene sequences

Test virulence of mutant viruses in mice

Determine role of specific amino acids by introducing single or combinations of mutations by site directed mutagenesis

HANA

PB2PB1PA

MNP

NS

x

HANA

PB2PB1PA

MNP

NS

x

HANA

PB2PB1PA

MNP

NS

x

Transmissibility

Indonesia/2005 H5N1 virus: Ron Fouchier (2012)

Is the virus transmissible? Yes Is it more virulent than the original virus? No

infection in the respiratory tract

mutations in 7 segments, 2 new changes (red) in HA

Q222LG224SQ226LN224K

T318I

T156AN158D

H103Y

10 times

Adapted from Paul Duprex

How and why do virologists study evolution of viruses under immune or antiviral pressure?

How? • Generate a panel of ‘escape’ mutants that replicate in the

presence of MAbs or antiviral drugs

Virus

+ Antibody/Antiviral

Cell monolayer X 2 h

+ Antibody/Antiviral4 days 37°C

30 min 37°C

Escape Mutants

Virus

+ Antibody/Antiviral

Recover virus

Sequence to identify Escape Mutants

Experimental animal model

OR

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 in vitro or in vivo

Why?

• To understand the biology of the virus

• To map epitopes of antibodies

• To develop robust counter measures (antiviral drugs and vaccines)

Coronavirus targets for vaccines and therapeutics

An3’

EESS MM NN

nsp12RdRpnsp12RdRp

nsp14Exonuclease

nsp14Exonuclease

nsp12 nsp14

nsp3PL-protease

nsp3PL-protease

nsp5protease

nsp5protease

nsp16 2’O MT-ase

nsp16 2’O MT-ase SpikeSpike

ProteasesProteases PolymeraseProofreadingPolymeraseProofreading

ImmuneEvasionImmuneEvasion

EntryHost Range

Immunity

EntryHost Range

Immunity

5’5’ nsp3nsp3 nsp5nsp5 nsp16

CoV genome ~30kb +sense RNACoV genome ~30kb +sense RNA

defined by reverse genetics and adaptive experimental evolution

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 GoFresearch as defined by the U.S. government?

‘Gain of Function’

• The term gain of function is a vague and unsatisfactory term for microbiologists

• One suggested alternative is aTRIP: an acronym for an experiment that uses one or more of the DURC agents and produces, 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 mBio in press; Mark Denison

The impact of virological methods on ‘gain of function’ in virology

Research maneuver

Comment/ Example

GoF? Other consequences: LoF?

aTRIP/GOVT?

Passage in cell culture/eggs

Yes: usually desired

Possible for other cells

no

The impact of virological methods on gain of function in viruses

Research maneuver

Comment/ Example

GoF? Other consequences: LoF?

aTRIP/GOVT?

Passage in cell culture/eggs

Yes: usually desired

Possible forother cells

no

Serialpassage in experimental animals

SARS CoV MA15

Possible: often desired

Possible for other species

RP/V

The impact of virological methods on gain of function in viruses

Research maneuver

Comment/ Example

GoF? Other consequences: LoF?

aTRIP/GOVT?

Passage in cell culture/eggs

Yes: usually desired

Possible for other cells

no

Serialpassage in experimental animals

SARS CoV MA15

Possible: often desired

Possible for other species

RP/V

Genetic reassortmentof influenza viruses

Influenzavaccines GoFyield, LoFvirulence

Yes; often desired

Possible: often desired

no

The impact of virological methods on gain of function in viruses

Research maneuver

Comment/ Example

GoF? LoF? aTRIP/GOVT?

Antiviral or MAb resistantmutants

Valuable information on suitableantiviral strategies

Yes: usually desired

Possible reduction in virulence and fitness

no

The impact of virological methods on gain of function in viruses

Research maneuver

Comment/ Example

GoF? LoF? aTRIP/GOVT?

Antiviral or MAb resistantmutants

Valuable information on suitableantiviral strategies

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

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 GoFresearch as defined by the U.S. government?

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

What do we not know about influenza viruses?

• Why are some viruses more virulent than others?

• Why do some viruses spread efficiently while others don’t?

• 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

What do we not know about SARS and MERS-CoV?

• Will SARS or a SARS-like CoV re-emerge from bats or other animal hosts?

• What are the critical host range and virulence determinants of MERS-CoV?

• Can we develop a candidate vaccine that is safe, immunogenic and efficacious?

• 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

What safety and oversight measures are in place?

Measures MERS SARS Non-HPAI influenza

HPAI and H7N9

influenza

BSL3 with respirators

Medical surveillance and support

Vaccines andantivirals N/A N/A

Select agent regulations

DURC questionnaire

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 CoVExperiments that are reasonably anticipated to increase • pathogenicity or • transmissibility • in any mammalian species

Where does virological research cross the line into GoF 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

The perspective of many influenza and coronavirologists

• Concerns about aerosol-transmissible highly pathogenic avian influenza viruses have expanded to include • MERS-CoV and SARS-CoV• Research in rodent models• All influenza viruses including laboratory strains that are

avirulent for humansAn 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

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 or SARS

• 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 rodents does 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

Relevant outbreaks in 2014

H5N2

H7N9H10N8

MERS CoVH10N7H5N8

Approaches to virology research

• gain‐of‐func on ≠ 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

• 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]