-Centro de Biología Molecular

“Severo Ochoa”(CSIC-UAM),

Cantoblanco, Madrid

-Centro de Astrobiología

(CSIC-INTA), Torrejón de

Ardoz, Madrid

-Centro de Investigación Biomédica

en Red de Enfermedades Hepáticas

y Digestivas (CIBERehd), Barcelona

Esteban Domingo

Respuesta del virus de la hepatitis C a inhibidores.

Influencia de los espectros de mutantes y fitness

replicativo

edomingo@cbm.csic.es

Quasispecies dynamics and viral fitness

Mechanisms of antiviral drug resistance

Antiviral strategies and lethal mutagenesis

New antiviral options for hepatitis C virus

infections

Problems, prospects and conclusions

CONSENSUS

MUTANT

SPECTRUM

**

* *

* **

* **

*

*

* **

* *

**

*

*

*

*

Intra-host heterogeneity

Experimental evolution

High mutation rates10-3 to 10-5 subst./nt

~106-fold higher than for cellular DNA

Rapid genome replication and high virus turnoverin infected hosts

Principles of Darwinian evolutionReproduction with genetic variationCompetitionSelection

Intra-population interactions: complementation

and interference

Bottleneck events

Basis of quasispecies dynamics of RNA viruses

Extensions:

Cellular communities (tumor cells,

eukaryotic parasites, bacteria)

and prionsDomingo, Sheldon, and Perales, 2012 MMBR 76:159-216

Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

Transmission and passage conditions can affect viral fitness

11 to 1010-1012

1

Presence of inhibitor-escape mutants (generally

in the range of 10-3 to 10-5, depending on the

genetic and phenotypic barriers to resistance)

Genetic barrier: number and types of

mutations needed to acquire resistance

Phenotypic barrier: fitness cost imposed by the

resistance mutations

Fitness can be a multidrug resistance

determinant in HCV, independent of the presence

of specific resistance mutations

Mechanisms of antiviral drug resistance

C GlucNS2 NS3 4A 4B 5A 5B

HCVcc (Genotype 2a)E1 p7E2

5’UTR 3’UTR

GDDJ6 JFH-1

GNN (Control -) GNN

From Charles Rice

p0 p1 p2…

1 passage ~ 3-4 days

Perales et al., 2013 J. Virol. 87:7593-7607

Cell culture system for HCV

Human hepatoma

Huh-7.5 cells

Sheldon et al., 2014 J.Virol. 88:12098-12111

Serial passage of HCV in

Huh-7.5 cells resulted in

increased resistance to

inhibitors that target viral

or cellular proteins,

despite no prior exposure

of the virus to the drugs

Sheldon et al., 2014 J.Virol. 88:12098-12111

The MOI-independent kinetics of HCV production excludes that resistance is due to the

presence of inhibitor resistance mutations in the HCV populations, in agreement with

mutant spectrum analyses

Sheldon et al., 2014 J.Virol. 88:12098-12111

HCV biological

clones maintain the

resistance phenotype

of the parental

population

Resistance is dependent on population complexity,

size, and history (molecular memory in viral

quasispecies)

High mutant spectrum complexity can favor the

presence of inhibitor resistance mutations or can be a

marker of high replicative fitness

In HCV, high fitness is a determinant of resistance,

independent of the presence of resistance mutations.

Whether this mechanism applies to other viruses is an

open question

Drug resistance is a major factor of treatment failure

Multiple facets of resistance to antiviral inhibitors

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 quasispecies dynamics

Resistance mutations may jeopardize the efficacy of

these strategies

Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

Lethal defection: interference by RNA replication-competent defective

genomes is involved in virus extinction

Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

Low viral load and low fitness favor extinction

Transition towards extinction involves decreases of

specific infectivity, and no change of the consensus

sequence of the population

Mutagen-resistant mutants appear to be less frequent

than inhibitor-resistant mutants. However, selection of

mutagen-resistant mutants can jeopardize extinction

Mutagens and inhibitors used together can be more

effective than either of them alone. However, experiments

and a model system developed with replicative parameters

of FMDV indicated that a combination therapy may not

be the best option

Extinction by lethal mutagenesis

Sequential versus combination treatment

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

I+Mut.

+I +Mut.

+I

+Mut.

I+Mut.

Perales et al., 2009 PLoS Pathog 5: e1000658

A

Vir

al ti

ter

(TC

ID50/m

l)

105

104

103

102

10

Vir

al ti

ter

(TC

ID50/m

l)

106

Control

(no virus)

No drugs

IFN-a 1 IU/ml, Rib 50 mM

IFN-a 2 IU/ml, Rib 50 mM

IFN-a 4 IU/ml, Rib 50 mM

[IFN-a 1 IU/ml+Rib 50 mM]

[IFN-a 2 IU/ml+Rib 50 mM]

[IFN-a 4 IU/ml+Rib 50 mM]

105

104

103

102

10

106

B

Vir

al

tite

r (T

CID

50

/ml)

105

104

103

102

10

Vir

al ti

ter

(TC

ID50/m

l)

106

107

Control

(no virus)

No drugs

TPV 400 nM, Rib 50 mM

TPV 600 nM, Rib 50 mM

TPV 800 nM, Rib 50 mM

[TPV 400 nM+Rib 50 mM]

[TPV 600 nM+Rib 50 mM]

[TPV 800 nM+Rib 50 mM]

105

104

103

102

10

106

107

Combination

Combination

Sequential

Sequential

C

Vir

al ti

ter

(TC

ID5

0/m

l)

105

104

103

102

10

Vir

al

tite

r (T

CID

50

/ml)

106

107

Control

(no virus)

No drugs

DCV 100 pM, Rib 50 mM

DCV 200 pM, Rib 50 mM

DCV 500 pM, Rib 50 mM

[DCV 100 pM+Rib 50 mM]

[DCV 200 pM+Rib 50 mM]

[DCV 500 pM+Rib 50 mM]

105

104

103

102

10

106

107

D

Vir

al

tite

r (T

CID

50

/ml)

105

104

103

102

10

Vir

al

tite

r (T

CID

50

/ml)

106

Control

(no virus)

No drugs

CsA 400 nM, Rib 50 mM

CsA 600 nM, Rib 50 mM

CsA 800 nM, Rib 50 mM

[CsA 400 nM+Rib 50 mM]

[CsA 600 nM+Rib 50 mM]

[CsA 800 nM+Rib 50 mM]

105

104

103

102

10

106

Combination

Combination

Sequential

Sequential

Increased mutagenesis is a natural defense mechanism (APOBEC,

ADAR, RIP)

5-Fluorouracil prevented the establishment of a persistent LCMV

infection in mice (Ruiz-Jarabo et al. 2003)

Some anti-cancer drugs (or derivatives) are under investigation as

virus-specific mutagens. The mutagenic pyrimidine analogue KP1461

mutagenized HIV-1 in a clinical assay (Mullins et al. 2011)

In some cases the antiviral action of ribavirin might be exerted in

part through lethal mutagenesis [Arenavirus, HCV (Dietz et al. 2013)]

The antiviral T-705 (favipiravir) induces lethal mutagenesis of

influenza virus (T. Baranovich et al. 2013) and norovirus in vivo (A.

Arias et al. 2014)

Experiments with animal models and clinical trials with non-

responder patients are needed

Prospects: Will lethal mutagenesis find a clinical application?

….It is essential that CDC, NIH and WHO be supported at the

levels needed to provide the public health response to this

international health crisis...

….Ebola will not be the last virus to evolve into a major health

and security threat. The only way to stay ahead of the rapid

evolution of microorganisms and the inevitable emergence of new

diseases is to support infectious disease research and public

health agencies that can respond rapidly when outbreaks occur.

This requires adequate and predictable funding, training of

personnel, and safe and secure facilities.

Timothy J. Donohue, Ph.D. Ronald M. Atlas, Ph.D.

President, ASM Chair, Public and Scientific Affairs

American Society for Microbiology Statement on Ebola Response

S.K. Gire et al., 2014 Science 345: 1369-1372

The Spanish Research Council loses 15% of its personnel in 2.5 years.

What wonderful news! Thank you! (has stated Ebola virus thankful for

this contribution to the austerity virus)

Viral fitness is a multidrug resistance determinant in

HCV. New antiviral protocols based on lethal

mutagenesis are under investigation. They take into

consideration the interplay between inhibitors and

mutagenic agents that may favor or impede virus

extinction

Model studies with HCV replicating in human

hepatoma cells in culture suggest that an inhibitor-

mutagen sequential administration can have an

advantage over the corresponding combination in

reducing the viral load

Conclusions

Celia PeralesJulie SheldonVerónica MartínNathan Beach

Héctor MorenoIgnacio de la HigueraAna M. OrtegaElena Moreno

Susanna ManrubiaJaime Iranzo

Ana Isabel de ÁvilaIsabel Gallego

Josep QuerJosep GregoriF. Rodríguez-FríasJuan I. EstebanCarlos BrionesJordi Gómez

Charles Rice

Pablo GastaminzaAntonio Mas

Jean-Pierre VartanianSimon Wain-Hobson Juan Carlos de la Torre