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. Introduction

2. H7N7 avian influenza

3. Prediction of antigenic sites

4. Validation of results

5. 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• The two large glycoproteins

found on the virion surface• Responsible for infectivity and

virulence

HA

NA

• 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.

Treanor J. 2004. Influenza Vaccine – Outmaneuvering Antigenic Shift and Drift. N Engl J Med. 350:218-220.

How to protect ourselves

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

• Recognition of and binding to vertebrate red-blood cells• Fusion of viral and endosomal membranes, allowing

entry of viral genome into target cell

http://www.reactome.org/figures/influenza_life_cycle_overview.jpg

Hemagglutinin structureView 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 receptor-

binding site (top)

Hemagglutinin structure

• Monomer structure• Composed of two

subunits: HA1 and HA2

• HA1 = membrane-distal globular region

• HA = membrane-proximal stem region

HA1

HA2

http://www.rcsb.org/pdb/explore/images.do?structureId=1TI8

2. Locate immunodominant epitopes• 3D analysis using known values of physico-

chemical 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

Fig. 1: Antigenicity plot for a single H7 hemagglutinin monomer

Antigenicity plot

-1

-0.5

0

0.5

1

1.5

0 100 200 300 400

Location in sequence

An

tig

enic

ity

Identified epitopes

1. Loop: S130, G131

2. Tip: G187

3. Hinge: N265, C266

4. Interface: N159, T160, R161, K162, S196, N197, L226, N227, P228

Fig. 2: Identified epitopes on H7 structure. A - side view; B – top view. Blue – HA1; red – HA2; green – identified core epitope residues.

A 2

1

4

3

B

12

4

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 DiscotopePhysico-chem. ICLGHHAVSN GTKVNTLTER GVEVVNATET VERTNVPRIC SKGKRTVDLG DiscoTope 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

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

HA1 HA2

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 physico-chemical 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

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.

BH7 predicted antigenic regions

A 2

1

4

3

1

2

3

4 H3 actual

antigenic regions

“Fixed” substitutions

• An alignment of 29 H7 HA1 protein sequences isolated in Europe from 2001-2004:– 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 K189 P255

R65 I195 E286

K69 T208 V289

S152 I252 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

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0 100 200 300 400

Location in sequence

Antig

enic

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Vaccine antigenicity plot

-2

-1.5

-1

-0.5

0

0.5

1

1.5

0 100 200 300 400

Location in sequenceAn

tigen

icity

Fig. 6: Antigenicity plot for H7 before and after vaccine design.

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