Quasispecies dynamics and antiviral designs

Esteban Domingo

Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Cantoblanco (Madrid)

edomingo@cbm.uam.es

Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz (Madrid)

Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona

CONSENSUS

MUTANT SPECTRUM

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- Viral quasispecies constitute reservoirs of genotypic and phenotypic variants (drug resistance, etc.)

- Intra-mutant spectrum interactions (complementation or interference) can promote or suppress dominance of specific variants

- Replication rate, viral load, genetic heterogeneity and viral fitness are interconnected parameters

- Quasispecies dynamics can affect long-term virus evolution [memory, chance shedding of particular variants, rapid evolution related to pandemic potential (norovirus, enterovirus 71)]

Domingo, E., Sheldon, J., Perales, C., MMBR, 2012

Domingo, E., Sheldon, J., Perales, C., MMBR, 2012

Underlying influences High mutation rates 10-3 to 10-5 subst./nt ~106-fold higher than for cellular DNA

Principles of Darwinian evolution Reproduction with genetic variation Competition Selection

Bottleneck events

Combination therapy (i.e. HAART for AIDS)

Splitting into an induction and maintenance regimen

Targeting of cellular functions

Combined use of immunotherapy and chemotherapy

Lethal mutagenesis

Major strategies developed to overcome the adaptive potential conferred by high mutation rates and quasispecies dynamics

Resistance mutations may jeopardize the efficacy of these strategies

Domingo, E., Sheldon, J., Perales, C., MMBR, 2012

Low viral load and low fitness favor extinction

Transition towards extinction involves a 102- to 103- fold decrease of specific infectivity

No change of the consensus sequence of the population

Rapid movement towards regions of sequence space with biased base composition (A,U- rich genomes upon ribavirin treatment)

Selection of mutagen-resistant mutants can jeopardize extinction

Extinction by lethal mutagenesis

Fitness landscape (Sewall Wright)

Can a virus be trapped in a pit of low fitness?

FMDV genome

L VP4 VP2 VP3 2A 2B 2C VP1 3A 3C 3D

VPg(s)

poly A

A U G A U G

Lab Lb

O

OH OH

HO

N N

N

O

NH2

Ribavirin, mutagenic for FMDV

Guanidine hydrochloride, inhibitor of FMDV replication

H N - C - NH HCl 2 2 . NH

Advantage of sequential inhibitor-mutagen administration

Guanidine prevents interference

Perales C. et al., 2009 PLoS Pathog 5: e1000658

Mutagen removed at different passages

Effect of increasing amount of inhibitor

Different administrations of inhibitor and mutagen

A simple theoretical model (S. Manrubia)

Main features: WT, D, sensitive and resistant to the inhibitor (I) w = mutation rate µ = rate of generation of I-resistant individuals R, r = number of progeny genomes by WT, D Number of infected cells, infectious cycles per cell

Perales C. et al., 2009 PLoS Pathog 5: e1000658

[ I ]

[ I ]

[ I ]

Jc1 GNN

105

104

103

102

10

Vira

l tite

r (TC

ID50

/ml)

106

107

108

… … … p15 p30 p45…

p60 … p80…

p100Jc1

0 10 20 30 40 50 60 70 9080 100

Passage number

p0

105

104

103

102

10

RN

A (m

olec

ules

/ng)

106

107108

0 10 20 30 40 50 60 70 9080 100

C Gluc NS2 NS3 NS4a NS4b NS5a NS5b E1 p7 E2 5’UTR 3’UTR

J6 JFH-1

HCV Jc1, genotype 2a (Charles Rice)

Passage number

RN

A (m

olec

ules

/ng)

Vi

ral t

iter (

TCID

50/

ml)

Combination Sequential

Combination Sequential

Sequential versus combination treatment

Iranzo J. et al., 2011 P.N.A.S. 108(38): 16008-13

I+Mut.

+I +Mut.

+I

+Mut.

I+Mut.

Perales C. et al., 2009 PLoS Pathog 5: e1000658

Therapy involving mutagens and inhibitors

• A mutagen can increase the frequency of inhibitor resistant mutants

• An inhibitor can prevent replication of interfering mutants that contribute to lethal defection

• The mutant spectrum can suppress inhibitor-resistant mutants that affect a trans-complementable protein

• A mutagenized mutant spectrum can suppress high fitness genomes

González-López, et al., 2004 J. Virol. 78: 3319-24 Crowder and Kirkegaard, 2005 Nature Genetics 37: 701-9 Perales et al., 2007 J. Mol. Biol. 369: 985-1000

Thus, a number of interconnected parameters (mutation rate, intensity of inhibition, target of the inhibitor, etc.) must be considered in antiviral designs

Combined

Dose dependant Combined

MUTAGEN M

UTA

GEN

INHIBITOR

INH

IBIT

OR

Mutagen dose In

hibi

tor d

ose

C S

Ext

*

Drug 2 D

rug

1

Perales C. et al., submitted

Case of FMDV with guanidine and ribavirin

Domingo, E., Sheldon, J., Perales, C., MMBR, 2012

Experiments with animal models are needed • Possible tests of inhibitor-mutagen administration in

non-responder patients

Precedents: 5-Fluorouracil prevented the establishment of a persistent LCMV infection in mice (C. Ruiz_Jarabo et al. 2003) The mutagenic pyrimidine analogue KP1461 mutagenized HIV-1 in a clinical assay (J.I. Mullins et al. 2011) In some cases the antiviral action of ribavirin might be exerted in part through lethal mutagenesis (Arenavirus, HCV ?)

Will lethal mutagenesis find a clinical application?

Prospects

Conclusions

Effective antiviral therapies must consider the adaptive potential of viral quasispecies and internal interactions within mutant spectra Lethal mutagenesis aims at extinguishing viruses through an excess of mutations, a process that can be viewed as an irreversible loss of viral fitness When an inhibitor and a mutagenic agent participate in therapy a sequential inhibitor-mutagen administration may have an advantage over the corresponding combination Such an advantage has been documented with several viruses, and it is probably a consequence of the dynamics of selection of inhibitor-escape mutants and of the need of defector genomes to replicate to exert their interfering activity

Julie Sheldon Celia Perales Ana M. Ortega Nathan Beach

Susanna Manrubia Jaime Iranzo

Ana Isabel de Ávila Isabel Gallego María Eugenia Soria

Josep Quer F. Rodríguez-Frías Juan I. Esteban Carlos Briones Jordi Gómez

Charles Rice