La disponibilidad de un sistema de multiplicación del virus de la hepatitis C (VHC) infeccioso en cultivos celulares está permitiendo investigar nuevos factores de respuesta a tratamientos antivíricos en condiciones controladas. Se presentará evidencia de que el fitness vírico puede ser un factor de multiresistencia a inhibidores y quese pueden obtener eficientes reducciones de carga viral empleando diseños secuenciales de administración de inhibidores que incluyan ribavirina. Se discutirán posibilidades de aplicación clínica.

1. Respuesta del virus de la hepatitis C a inhibidores.
Influencia de los espectros de mutantes y fitness
replicativo
Esteban Domingo
-Centro de Biología Molecular
“Severo Ochoa”(CSIC-UAM),
Cantoblanco, Madrid
edomingo@cbm.csic.es
-Centro de Astrobiología
(CSIC-INTA), Torrejón de
Ardoz, Madrid
-Centro de Investigación Biomédica
en Red de Enfermedades Hepáticas
y Digestivas (CIBERehd), Barcelona

2. 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

3. MUTANT
SPECTRUM
CONSENSUS
*
*
* *
* *
*
* *
*
*
*
* *
*
* *
*
*
*
*
*
*

4. Intra-host heterogeneity
Experimental evolution

5. Basis of quasispecies dynamics of RNA viruses
High mutation rates
10-3 to 10-5 subst./nt
~106-fold higher than for cellular DNA
Rapid genome replication and high virus turnover
in infected hosts
Principles of Darwinian evolution
Reproduction with genetic variation
Competition
Selection
Intra-population interactions: complementation
and interference
Bottleneck events
Extensions:
Cellular communities (tumor cells,
eukaryotic parasites, bacteria)
and prions
Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

6. Transmission and passage conditions can affect viral fitness
Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

7. 1 to 10110-1012
1

8. Mechanisms of antiviral drug resistance
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

9. Cell culture system for HCV
HCVcc (Genotype 2a)
5’UTR 3’UTR
C E1 E2 p7
NS2 Gluc NS3 4A 4B 5A 5B
GDD
J6 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
Human hepatoma
Huh-7.5 cells

10. 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

11. 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

12. HCV biological
clones maintain the
resistance phenotype
of the parental
population
Sheldon et al., 2014 J.Virol. 88:12098-12111

14. Multiple facets of resistance to antiviral inhibitors
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

15. Major strategies developed to overcome the adaptive
potential conferred by quasispecies dynamics
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
Resistance mutations may jeopardize the efficacy of
these strategies
Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

16. Lethal defection: interference by RNA replication-competent defective
genomes is involved in virus extinction
Domingo, Sheldon, and Perales, 2012 MMBR 76:159-216

17. Extinction by lethal mutagenesis
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

18. Sequential versus combination treatment
I+Mut.
+I +Mut.
Iranzo et al., 2011 P.N.A.S. 108(38): 16008-13
+I
+Mut.
I+Mut.
Perales et al., 2009 PLoS Pathog 5: e1000658

19. A
Viral titer (TCID50/ml)
6
5
10
4
10
3
10
2
10
10
Viral titer (TCID50/ml)
10
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]
6
10
5
10
4
10
3
10
2
10
10
B
Viral titer (TCID50/ml)
7
10
6
5
10
4
10
3
10
2
10
10
Viral titer (TCID50/ml)
10
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]
7
10
6
10
5
10
4
10
3
10
2
10
10
Combination
Combination
Sequential
Sequential

20. C
Viral titer (TCID50/ml)
7
10
6
5
10
4
10
3
10
2
10
10
Viral titer (TCID50/ml)
10
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]
7
10
6
10
5
10
4
10
3
10
2
10
10
D
Viral titer (TCID50/ml)
6
5
10
4
10
3
10
2
10
10
Viral titer (TCID50/ml)
10
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]
6
10
5
10
4
10
3
10
2
10
10
Combination
Combination
Sequential
Sequential

22. Prospects: Will lethal mutagenesis find a clinical application?
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

23. American Society for Microbiology Statement on Ebola Response
….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
S.K. Gire et al., 2014 Science 345: 1369-1372

24. 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)

25. Conclusions
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

26. Celia Perales
Julie Sheldon
Verónica Martín
Nathan Beach
Héctor Moreno
Ignacio de la Higuera
Ana M. Ortega
Elena Moreno
Ana Isabel de Ávila
Isabel Gallego
Susanna Manrubia
Jaime Iranzo
Josep Quer
Josep Gregori
F. Rodríguez-Frías
Juan I. Esteban
Carlos Briones
Jordi Gómez
Pablo Gastaminza
Antonio Mas
Charles Rice
Jean-Pierre Vartanian
Simon Wain-Hobson Juan Carlos de la Torre