In silico design of an H7N7 avian
influenza vaccine with potential
long-term applicability through
the alteration of immunodominant
epitopes
Carlo Lapid and Eduardo Padlan
Asia-Pacific Advanced Network
Jan 24, 2007
Background photo courtesy of Yoshihiro Kawaoka, University of Wisconsin-Madison
Outline
1.
2.
3.
4.
5.
Introduction
H7N7 avian influenza
Prediction of antigenic sites
Validation of results
Design of vaccine
What is influenza?
• Influenza, or flu, is a prevalent contagious
disease of the upper airways and lungs
• Caused by members of the
Orthomyxoviridae family of RNA viruses
– Influenza A: affects birds, mammals, humans
– Influenza B: affects only humans, uncommon
– Influenza C: causes only mild disease
• Influenza affects 5-15% of the world
population, and 250,000 to 500,000
deaths worldwide
The influenza virus
The three-dimensional structure of
influenza virus from electron
tomography. Harris A, et al. PNAS
2006;103(50):19123-19127.
Whittaker GR. Expert Rev Mol Med. 2001 Feb
8;2001:1-13.
The influenza virus
http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html
Hemagglutinin and Neuraminidase
HA
NA
• The two large glycoproteins
found on the virion surface
• Responsible for infectivity and
virulence
• Hemagglutinin = HA
(16 subtypes)
• Neuraminidase = NA
(9 subtypes)
• They determine the
subtype of the virus
http://en.wikipedia.org/wiki/Influenza
Possible pandemic?
• Most humans have no
immunity against
influenza subtypes that
circulate among birds
• If an avian influenza subtype:
– Transferred from birds to humans,
– Gained the ability to spread easily among
humans,
– And was highly pathogenic…
Outbreak!
Hemagglutinin and Neuraminidase
• Nearly all human infections are by H1, H2,
H3, N1, and N2
– H1N1 – 1918 “Spanish flu” (40M dead)
– H2N2 – 1957 “Asian flu” (1-1.5M dead)
– H3N2 – 1968 “Hong Kong flu” (0.75-1M dead)
• All highly pathogenic avian flu outbreaks
are by H5 and H7 subtypes
– H5N1 – currently in Asia, Africa, and Europe
– H7N7 – Netherlands outbreak in 2003
H7N7 influenza
• In late February 2003, poultry in a large
number of farms in the Netherlands were
stricken with an outbreak of highly
pathogenic avian flu.
• Sequencing of the HA and NA genes
identified the cause to be H7N7 influenza.
• Soon after, symptoms were reported in
453 people; 89 were confirmed to have the
H7N7 virus.
H7N7 influenza
• People with no direct contact with poultry
were infected.
• There was one fatal case of a 57-year-old
veterinarian, who died from acute
respiratory distress syndrome. Tests
showed H7N7 to be the cause.
• In a follow-up study, an improved assay
showed that about 1000-2000 people were
infected, though many lacked symptoms.
• The virus later spread to Belgium and
Germany.
H7N7 influenza
• Recap:
– This strain of H7N7 is highly pathogenic
among birds.
– It transferred from birds to humans, and
circulated efficiently among humans.
– It produced one fatal case.
– It is (or at least was) spreading.
• The World Health Organization has
recommended enhanced surveillance.
How to protect ourselves
• Influenza is a constantly mutating
pathogen.
– As populations develop immunity against it,
the virus mutates and evolves in order to
evade immune response.
– This necessitates continuous vaccine design.
How to protect ourselves
Treanor J. 2004. Influenza Vaccine – Outmaneuvering Antigenic Shift
and Drift. N Engl J Med. 350:218-220.
How to protect ourselves
Maybe we can design a vaccine that would
be effective even if the virus is constantly
mutating.
Vaccine design strategy
• Antibodies can be generated against any
accessible part of a macromolecule.
• But some parts are more antigenic (bind to
antibodies more readily) than others. These
are called immunodominant epitopes.
• If we can predict and alter immunodominant
epitopes, antibodies will be generated
against many other regions, producing a
broader, more effective defense.
Vaccine design strategy
1. Specify a target protein as a basis for the
vaccine.
2. Locate the immunodominant epitopes.
3. Identify the residues which are
responsible for the high antigenicity of
those epitopes.
4. Replace those residues with amino acids
that would contribute less to the
antigenicity, while (hopefully) preserving
the structure.
1. Specify a target protein
• Hemagglutinin is…
– Expressed five times more abundantly than
neuraminidase
– The main determinant for host range
restriction
– Plays a greater role in determining infectivity
– More dominant than neuraminidase in
inducing an immune response
– An ideal target for vaccines
Hemagglutinin function
http://www.reactome.org/figures/influenza_life_cycle_overview.jpg
• Recognition of and binding to vertebrate red-blood cells
• Fusion of viral and endosomal membranes, allowing
entry of viral genome into target cell
Hemagglutinin structure
View from side
View from top
http://www.rcsb.org/pdb/explore/images.do?structureId=1HGE
• Rod-shaped trimer, with stem and globular regions
• Membrane-proximal region (bottom) and receptorbinding site (top)
Hemagglutinin structure
• Monomer structure
• Composed of two
subunits: HA1 and
HA2
• HA1 = membranedistal globular region
• HA = membraneproximal stem region
HA1
HA2
http://www.rcsb.org/pdb/explore/images.do?structureId=1TI8
2. Locate immunodominant
epitopes
• 3D analysis using known values of physicochemical properties of amino acids
– Size, charge, polarity, hydrophilicity, etc.
• Padlan EA (1985) Quantitation of the
immunogenic potential of protein antigens. Mol
Immunol 22, 1243-1254.
– “… The method can be used to locate the
immunodominant regions of a molecule …”
(Patent pending)
Sequence and structure
• Sequence: A/Netherlands/219/03 (H7N7)
– Virus responsible for Netherlands fatality
– Accession code ABG57092
• Structure: H7 hemagglutinin
– RCSB PDB code 1TI8
Antigenicity plot
Antigenicity plot
1.5
Antigenicity
1
0.5
0
0
100
200
300
400
-0.5
-1
Location in sequence
Fig. 1: Antigenicity plot for a single H7 hemagglutinin monomer
Identified epitopes
2
A
B
4
1
1
2
4
3
1.
2.
3.
4.
Fig. 2: Identified epitopes on H7 structure. A side view; B – top view. Blue – HA1; red – HA2;
green – identified core epitope residues.
Loop: S130, G131
Tip: G187
Hinge: N265, C266
Interface: N159, T160, R161,
K162, S196, N197, L226,
N227, P228
Number of antigenic sites
“Monoclonal antibodies to the haemagglutinin (HA)
molecule of A/Seal/Mass/1/80 (H7N7) have been
prepared and used to establish an operational
antigenic map. Four nonoverlapping antigenic
areas on the HA of seal influenza viruses were
defined.”
Kida H, et al. 1982. Biological activity of monoclonal
antibodies to operationally defined antigenic regions on
the hemagglutinin molecule of A/Seal/Massachusetts/1/80
(H7N7) influenza virus. Virology 122:38-47.
Comparison with Discotope
Physico-chem.
DiscoTope
ICLGHHAVSN GTKVNTLTER GVEVVNATET VERTNVPRIC SKGKRTVDLG
ICLGHHAVSN GTKVNTLTER GVEVVNATET VERTNVPRIC SKGKRTVDLG
QCGLLGTITG PPQCDQFLEF SADLIIERRE GSDVCYPGKF VNEEALRQIL
QCGLLGTITG PPQCDQFLEF SADLIIERRE GSDVCYPGKF VNEEALRQIL
RESGGIDKET MGFTYSGIRT NGTTSACRRS GSSFYAEMKW LLSNTDNAAF
RESGGIDKET MGFTYSGIRT NGTTSACRRS GSSFYAEMKW LLSNTDNAAF
PQMTKSYKNT RKDPALIIWG IHHSGSTTEQ TKLYGSGNKL ITVGSSNYQQ
PQMTKSYKNT RKDPALIIWG IHHSGSTTEQ TKLYGSGNKL ITVGSSNYQQ
SFVPSPGARP QVNGQSGRID FHWLILNPND TVTFSFNGAF IAPDRASFLR
SFVPSPGARP QVNGQSGRID FHWLILNPND TVTFSFNGAF IAPDRASFLR
GKSMGIQSEV QVDANCEGDC YHSGGTIISN LPFQNINSRA VGKCPRYVKQ
GKSMGIQSEV QVDANCEGDC YHSGGTIISN LPFQNINSRA VGKCPRYVKQ
ESLLLATGMK NVPEGLFGAI AGFIENGWEG LIDGWYGFRH QNAQGEGTAA
ESLLLATGMK NVPEGLFGAI AGFIENGWEG LIDGWYGFRH QNAQGEGTAA
HA1
HA2
DYKSTQSAID QITGKLNRLI EKTNQQFELI DNEFTEVERQ IGNVINWTRD
DYKSTQSAID QITGKLNRLI EKTNQQFELI DNEFTEVERQ IGNVINWTRD
SMTEVWSYNA ELLVAMENQH TIDLADSEMN KLYERVKRQL RENAEEDGTG
SMTEVWSYNA ELLVAMENQH TIDLADSEMN KLYERVKRQL RENAEEDGTG
CFEIFHKCDD DCMASIRNNT YDHSKYREEA
CFEIFHKCDD DCMASIRNNT YDHSKYREEA
Fig. 3: Comparison
of predicted core
epitope residues
obtained using
physico-chemical
properties (top) and
Discotope (bottom).
Predicted antigenic
regions are
highlighted. Those
predicted by both
methods are boxed.
One antigenic region
was predicted
through physicochemical properties,
but not by Discotope
(encircled).
Comparison with H3 epitopes
• Among the well-studied hemagglutinin
subtypes, H3 is structurally the most
similar to H7.
Fig. 4: Phylogenetic tree of 15
hemagglutinin subtypes according
to structure. Figure is taken from
Russell RJ, et al. H1 and H7
influenza haemagglutinin
structures extend a structural
classification of haemagglutinin
subtypes. Virology. 325:287-296.
Comparison with H3 epitopes
H7
predicted
antigenic
regions
2
A
B
1
1
2
4
H3
actual
antigenic
regions
4
3
3
Fig. 5: Comparison of A) predicted antigenic sites in A/Netherlands/219/03 H7N7 hemagglutinin
and B) actual antigenic sites in A/Hong Kong/1/1968 (H3N2) hemagglutinin (PDB code 1HGE).
Highlighted residues in H3 according to Wilson IA, Skehel JJ, Wiley DC. 1981. Structure of the
haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289:366-373.
“Fixed” substitutions
• An alignment of 29 H7 HA1 protein
sequences isolated in Europe from 20012004:
– 4 from Italy, 2001-2002
– 14 from Sweden, 2002
– 4 from Netherlands, 2003 (actual outbreak)
– 1 from Germany 2003,
– 6 from Italy, 2004
• “Fixed” substitutions appearing in multiple
sequences were identified.
“Fixed” substitutions
• Twelve positions underwent repeated
substitutions:
T61
R65
K69
S152
K189
I195
T208
I252
P255
E286
V289
L331
• Eleven of these twelve matched with or
were in close proximity to predicted
antigenic sites.
3-4. Identify and replace antigenic
residues
Antigenicity plot
Vaccine antigenicity plot
1.5
1.5
1
0.5
0
0
100
200
300
-0.5
400
Antigenicity
Antigenicity
1
0.5
0
-0.5 0
100
200
300
-1
-1.5
-2
-1
Location in sequence
Location in sequence
Fig. 6: Antigenicity plot for H7 before and after vaccine design.
400
Next step
• H7 protein with the designed vaccine
sequence can be synthesized and tested.
• Possible Applications:
– Vaccination of poultry located in countries with
H7N7 outbreaks
– Vaccination of humans in the (unlikely) event
of a human epidemic or pandemic
The End