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XV Congresso Nazionale SIMIT
Baveno, 16-19 ottobre 2016
Immunità antivirale e meccanismi di evasione dei virus
MASSIMO CLEMENTI
Università Vita-Salute San Raffaele
Laboratorio di Microbiologia e Virologia, Ospedale San Raffaele
Hepatitis C Virus [HCV]
A PECULIAR RNA INFECTIOUS
MOLECULAR SYSTEM EVOLVED AND
SPREAD THROUGH THE HUMAN
POPULATION, UNRECOGNIZED FOR
HUNDREDS OF YEARS
Global Burden of Hepatitis C Virus
• Hepatitis C Virus (HCV)
infects persistently ~185 M
individuals worldwide
• HCV causes 3-4 M new
contagions every year
• 35000 patients die annually
for hepatocellular carcinoma
(HCC) and liver cirrhosis
secondary to HCV infection
• HCV-associated cirrhosis is
the leading indication for
liver transplantation
Thomas, 2015, Nature
HCV Phylogeny and Genome Structure
• HCV strains clusterize in the
Hepacivirus genus, part of the
Flaviviridae family
• HCV polymerase does not have
proofreading activity
Adapted from ViralZone
• HCV strains are classified in:
7 genotypes (30-35% variability)
87 subtypes (15% variability)
• Variability is the highest in E1 and E2
Adapted from Kapoor et al., 2011, PNAS
MILESTONES IN HEPATITIS C VIRUS RESEARCH
Description of
non-A, non-B
hepatitis
Description of
HCV genome
organization and
polyprotein
processing
First
infectious
clone of HCV
1975 1989 1993 1996
3-dimensional
structure of
the HCV
protein NS3
Identification
of Hepatitis C
Virus (HCV)
Replicon
system
established
Recombinant
infectious HCV in
tissue culture
Novel
classes of
anti-HCV
compunds
(clinical
trials)
Phase 3
clinical
trials of
NS5A Inh
1997 1998 1999 2003 2005 2009 2011 2012-16
Proof of
concept of
HCV protease
inhibitors
Interferon-a
and ribavirin
therapy
Phase 3
clinical
trials of
NS3 PI
HCV pseudo-particles
(HCVpp)
Novel classes
of anti-HCV
compounds
Anti-HCV\E2
human
monoclonal
antibodies
HCV
neutralization
assays
Preclinic
COMBINATIONS
of ANTIVIRALS
of different
classes
Immune response to HCV (an elusive pathogen) 1
CD4+ and CD8+ T-cells play a central role in the anti-HCV
immune response of infected hosts (particularly in driving HCV
clearance)
Immune response to HCV (an elusive pathogen) 2
The protective role of anti-HCV antibody response is
not completely clarified as yet, and HCV show a
number of potential antibody escape mechanisms,
including
(a) virus variability of antigenically crucial regions
(due to lack of proofreading activity of the NS5B
product; RNA polymerase)
(b) glyscosilation of envelope proteins in order to
hide the important antigenic sites (HCV envelope
is discontinuous and unusually enriched in
phospholipids)
(c) interaction with lipoproteins (LDL, VLDL, and
HDL; HCV hijacks the very-low density lipoprotein
(VLDL) pathway
(d) binding to cell receptors.
(e) cell-to-cell transmission
Immune response to HCV (an elusive pathogen) 3
However, evidence that the induction of neutralizing antibodies
can strongly influence the outcome of HCV infection has been given
HCV Treatment
• First generation therapy:
PEG-interferon α and ribavirin
• Direct-acting antivirals (DAA):
NS3/4A inhibitors
NS5A/5B inhibitors
• To date neither entry inhibitors nor a
anti-HCV vaccine are available
NS3/4A in complex with Telaprevir,
PDB ID: 3SV8
HCV Envelope Proteins
• HCV E1 and E2 are highly
glycosylated and flexible proteins
• Their topology relies on a
complex disulfide connectivity
• E1 and E2 fold and oligomerize
properly only when co-expressed
• They mediate both attachment
and entry
• Their structure remains largely
unknown
Adapted from Krey et al., 2010,
PLoS Pathog.
HCV ligands
HCV Entry
Virus Fusion Proteins
• Virus fusion proteins are
divided in three classes:
• Class I: Trimeric structure,
mainly helical
• Class II: Dimeric structure,
enriched in β-sheets
• Class III: Trimeric structure,
α/β proteins
10
HCV Entry Machinery
• E1 forms a central trimeric structure
covered by E2
• E1 and E2 on viral particles are covalently
bound, forming a heterohexamer
• E2 mediates virus attachment, while E1
mediates fusion
• During fusion, changes in E1E2 disulfide
connectivity drive their conformational
change
• Available E1 and E2 structures are not
conclusive, burdened by artifacts and
cannot be functionally validated
Adapted from Falson et al., 2015, JVI
• Direct structural information on the
oligomerization interfaces is not available
17
E2c Topology
• E2 core (E2c) structures are not homologous with other class II fusion
proteins
40 Å
142 Å
116 Å
PDB IDs: 1SVB, 2YQ2 and 4MWF
HCV E2c
TBEV E
BVDV E2
13
Nature Volume: 509, Pages:381–384 Date published:(15 May 2014) DOI:doi:10.1038/nature13117
HCV Glycoprotein E1
• E1 has 8 cysteines and 5 Nglycosylation sites
• It presents a putative protein
disulfide isomerase (PDI) domain
• E1 is poorly immunogenic
• It is the putative fusion protein, as
demonstrated by:
• Mutagenesis analysis
• In vitro emergence of mutants
resistant to Flunarizine
• It forms trimers on the virus
surface
15
HCV Glycoprotein E2
• E2 has 18 cysteines, 11 N-glycosylation and 6 O-glycosylation
sites
• It has 3 hypervariable regions (HVR1, HVR2 and IgVR)
• E2 is the most immunogenic HCV protein
• It is responsible of the interaction with several host receptors
(mainly CD81 and SRBI)
11
E1 Structure
• E1 N-ter has been recently crystallized
• It highlights two possible homodimerization interfaces
• Similarly to E2c, the construct is not functionally validated
PDB ID: 4UOI
16
E2c Structure
• Both structures present two
structured regions and several
outer loops
• E2c disulfide connectivity is
different in the two structures, as
well as the conformation of the
outer domains
• E2c structures are poorly
validated from both a functional
and an immunological point of
view
Castelli et al., 2014, DDT
14
Anti-E2 Antibodies
E2 elicits both neutralizing and nonneutralizing antibodies with different
breadth of action
• We have previously isolated a panel of
human monoclonal antibodies with
common features:
• Binding to a conformational epitope
• Broad breadth of recognition
• Non-neutralizing mAbs: e8, e10
• Neutralizing mAbs: e20, e137, e301, e509
Model of e137 structure
19
ANTI-HCV E2 ANTIBODIES
•
THE ANTI-HCV-E2 ANTIBODY REPERTOIRE INCLUDES
(i) ANTIBODIES THAT ARE NOT BENEFICIAL TO THE HOST
(NOT INFLUENTIAL)
(ii) NEUTRALIZING ANTIBODIES (VERY LOW
CONCENTRATIONS IN MOST NATURAL INFECTIONS);
•
ANTI- HCV-E2 NEUTRALIZING ANTIBODIES MAY BE ACTIVE
AGAINST DIFFERENT HCV GENOTYPES (CROSS-NEUTRALIZING
ANTIBODIES);
•
ANTI- HCV-E2 NEUTRALIZING ANTIBODIES MAY INTERACT WITH
THE CD81-REACTING SITES OR WITH DIFFERENT REGIONS OF
THE PROTEIN, BEING AN IMPORTANT MODEL SYSTEM TO STUDY
THE COMPLEX PROCESS OF HCV ENTRY;
Generation of E1E2 structural model
• Assessment of indirect structural, immunological and
functional data in literature
• Human monoclonal antibodies as a probe for E1 and E2 native
folding
• In silico analysis
• Secondary structure prediction
• Tertiary and quaternary structure prediction
5
THE E2 TERTIARY FOLDING IS MAINTAINED BY 18 COMPLETELY CONSERVED
CISTEINES INVOLVED IN DISULFIDE BRIDGES
E1E2 Heterohexamer: Epitope Mapping
• The heterohexamer model provides a structure-function correlation of
neutralizing and non-neutralizing epitopes
RESULTS
• The E1E2 heterohexamer undergo drastic conformational changes to
exert its function
• The E1E2 heterohexamer represents the starting point for structurebased drug and vaccine design
• The model provides information for the rational design of constructs for
further structural and functional studies
Is an efficient HCV vaccine possible ?
Despite the advent of increasingly
effective treatments, the need for a
preventive HCV vaccine has not
vaned.
APPROACHES IN THE DEVELOPMENT OF HCV VACCINES
• Recombinant protein-based vaccines
• Peptide-based vaccines
• DNA-based vaccines
• Viral vector-based vaccines
• Dendritic cells-based vaccines
• Others
WHY HAVE VACCINES FOR HCV NOT BEEN DEVELOPED?
a) preclinical
b) Healthy human volunteers
ONLY ONE HCV VACCINE CANDIDATE
AIMED AT ELICITING N-Ab (recombinant
E1 AND E2 glycoproteins) (Chiron Co.)
The E1\E2 vaccine was trialed in healthy
human volunteers and adjuvated with MF59
Preclinical studies in Chimpanzees:high
titers of anti E1 and E2 and no infection
with homologous challenge
21/41 subjects had Abs against the HVR1
region, 23/41 against epitope I, 13/41
against epitope II (Ray, 2005)
Challenge with a closely related
heterologous virus resulted in infection in all
cases
25%-50% of subjetcs with antibodies
neutralized the virus(Ray, 2005)
All but one vaccinee did not progress to
chronic infection, suggesting the vaccine
does induce a degree of protective immunity
(Hougton & Abrignani, 2005)
3 subjects evidenced cross-neutralizing
acitivity (Law, 2013)
Quantification and characterization of humoral response induced by
Ad6 and protein vaccines in guinea pigs
CAR’S STRUCTURE
Copyright © BMJ Publishing Group Ltd & British Society of Gastroenterology. All rights reserved.
e137-CARs redirected T cells are activated by coated HCV/E2 glycoprotein
and not by free HCV/E2 antigen in the medium
Giuseppe A Sautto et al. Gut 2016;65:512-523
Copyright © BMJ Publishing Group Ltd & British Society of Gastroenterology. All rights reserved.
Acknowledgements
Alexander W.Tarr
Jonathan K. Ball
Francesca Cappelletti
Valeria Caputo
Elena Criscuolo
Roberta A. Diotti
Giuseppe Sautto
Nicola Clementi
Nicasio Mancini
Roberto Burioni
Yoshiharu Matsuura
Tatsuo Miyamura
Ania M. Owsianka
Arvind H. Patel
Christophe Bovigny
Vito Genna
Giorgio Tamò
Matteo Dal Peraro
Jennifer Pfaff
Benjamin Doranz
Karin Wisskirchen
Ulrike Protzer
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