Flu immunology for the non-immunologist

Dr Oanh Nguyen Professor Katherine Kedzierska

Department of Microbiology and Immunology University of Melbourne

At Peter Doherty Institute for Infection and Immunity

Innatecells Bcells Tcells

Day0---3 Day4---10

Days

Num

ber

Immune response to influenza virus infection

Immune protection against influenza virus infection Upper and lower airways

Cytotoxicity

T cells B cells

Secondary lymphoid

organs and blood

Mucosal IgA

Epithelial cells

A/H1N1

A/H3N2

B A/HxNx

Seasonal inactivated

vaccine

Current pandemic vaccines

“UniversalTcell---basedvaccines”

Alveolar ΝΚ cell

Mφ

CD8+ T cells kill virally-infected cells

Cytotoxicity

RANTES

MIP1β IFNγ TNF

CD8+ T cells recognize peptides presented by MHC-I

CD8+ T cell

CD8+ T cells recognize peptides presented by MHC-I

ØØ Differential frequency of HLA alleles across ethnic groups

ØØ Up to 2 different HLA-A and 2 HLA-B alleles per individual HLA-A

HLA-B HLA-C

MHC-I = HLAàà many different alleles

etc

A*01:01 B*07:02 A*02:01 B*08:01 A*03:01 B*18:01 A*24:02 B*27:02

etc

Virus

peptides proteosome

MHC-I replication

IFNγ

ΤΝF Cytotoxicity

pMHC-I=epitope

CD8+ T cells provide broad immunity against influenza viruses

• Influenza in animals H5N1 H7N9

•  Influenza pandemics H1N1-1918

H2N2-1957 H3N2-1968 H1N1-2009

• Seasonal annual epidemics

B cells/ antibodies

Killer CD8+ T cells

Kelso A; Nat Med 2012

In the absence of pre-existing antibodies, memory T cells can provide heterologous immunity and reduce the severity of influenza disease

McMichaelAetal,NEJM1983;EpsteinSL,JID2006;KreijtzJHetal,JVI2008;Leeetal,JCI2008;Wilkinsonetal,NatMed2011;Sridharetal,NatMed2013;WangZetal,NatComm2015;

CD8+ T cells provide broad immunity against IAVs Primary infection with seasonal strain Challenge with a novel strain (pandemic potential)

•  April 1st WHO compiled a report that during Feb-Mar 2013 China’s CDC reported three fatal human infections with a novel avian-origin reassortant A(H7N9)

•  ≈ 1489 cases/588 deaths, mortality rate of 40%

CONCERNS: 1)   Lack of neutralizing Abs

2)   Natural reservoir for the virus, but mild disease in birds

3)   Possibility to acquire human-human transmission

ààneed to understand and control A/H7N9

A novel influenza strain capable of infecting humans: A/H7N9

Shanghai Public Health Clinical Centre -> a first class teaching and research infectious diseases hospital

affiliated to Fudan University

Shanghai Institute for Emerging and Re-emerging Infectious Diseases

Sergio Quinones Parra

Dr Zhongfang Wang

Prof Jianqing Xu

Dr Liyen Loh

Ex-vivo functional assessment of the cellular immunity against H7N9 influenza virus

18 hrs

Peripheral blood mononuclear cells

(frozen) - contain CD8+ T cells

Thaw

IAV Infection

Measure CD8+ T cell response by flow cytometry

TNF

IFNγ

+Virus

R1 Recovered d14 -d18

R3 Recovered

d31 - d35

R2 Recovered

d21 - d27

RD Death

d6-d8 d9-d17

+/-

+/-

d15-d21 d16-d22 d19-d21

+/-

+ +

d22-d30 d20-d26 d22-d35

CD8+ T cells alone

or CD8+ T cells +

MN Abs

CD8+ T cells + MN Abs or

CD8+ + CD4+ T cells or

CD4+ T cells alone

CD8+ T cells + MN Abs and CD4+

+ NK cells or

+ MN Abs alone

Minimal

CD8+ /CD4+ /MN Abs /NK

responses

Num

ber o

f IFNγ+

cel

ls in

1x1

06 P

BM

C

Days after disease onset

Rapid recovery is associated with early CD8+ T cell responses IFNγ+CD8+ IFNγ+CD4+ MN Abs IFNγ+NK cells

Wang et al, Nat Comms 2015

+

Study Aim

• To dissect cross-strain protective CD8+ T cell immunity across distinct influenza strains and different HLAs/ethnicities

• Identification of novel epitopes (IAV & IBV)

ààTo identify novel influenza CD8+ T cell epitopes for universal immunity

T cell responses measured by cytokine production

+ IL-2

9-12 days

Peripheral Blood

Mononuclear Cells

Detecting antigen-specific CD8+ T cell responses in vitro Expansion of antigen-specific memory CD8+ T cells

+ peptide or

+ virus

TNF

IFNγ

No peptide +peptide

Cytokine measurement (ICS) CD107a (degranulation)

IFNγ ΤΝF

MIP1β

CD8+ T cell responses to IAV are well characterized

àà195 potential CD8+ T cell epitopes restricted by 24 different HLA alleles

Immunomic Analysis of the Repertoire of T-Cell Specificities for Influenza A Virus in Humansv†‡

Erika Assarsson,1 Huynh-Hoa Bui,1 John Sidney,1 Qing Zhang,1 Jean Glenn,1 Carla Oseroff,1

Innocent N. Mbawuike,2 Jeff Alexander,3 Mark J. Newman,3 Howard Grey,1 and Alessandro Sette1*

Nucleoprotein of influenza A virus is a major target of immunodominant CD8 þ T-cell responses Emma Grant1,5, Chao Wu2,3,5, Kok-Fei Chan3, Sidonia Eckle1, Mandvi Bharadwaj1, Quan Ming Zou2, Katherine Kedzierska1 and Weisan Chen3,4

Identification of broad binding class I HLA super type epi topes to provide universal coverage of influenza A virus Jeff Alexander a,*, Pamuk Bilsel a, Marie-France del Guercio a, Aleksandra Marinkovic-Petrovic a, Scott Southwood a, Stephani Stewart a, Glenn Ishioka a, Maya F. Kotturi b, Jason Botten c, John Sidney b, Mark Newman a, Alessandro Sette b

with Sneha Sant, Dr. Dhanasekaran Vijaykrishna and Don Teng

Identification of peptides conserved across IAV, IBV and ICV

KoutsakosMetal,inpress

MariosKoutsakos

C

M2 NA

NP

PA

B A

Functional validation of peptides conserved across IAV, IBV and ICV

Conserved epitopes

Conserved epitopes

KoutsakosMetal,inpress

A2/PB1413-421: a promising vaccine target

A/Puerto Rico/8/1934 H1N1

0.4

A/goose/Guandong/01/1996 H5N1 A/Hong Kong/1073/1999 H9N2

Infectious Salmon Anemia

Wellfleet Bay

Thogoto Influenza C Influenza D Influenza B

A/Korea/462/1968 H2N2 A/Shangai/02/2013 H7N9

A/California/07/2009 H1N1pdm09 A/New York/392/2004 H3N2

PB1413

ØØ Universally conserved in all influenza viruses

PB1413 PB1413 PB1413

FluB FluC FluA

PB1413 PB1413

H5N1 H7N9 1918 H1N1 2009 H1N1

PB1413 PB1413

ØØ Presented by HLA-A2, one of the most common HLA alleles

ØØ ~40% worldwide

Calculated by the IEDB population coverage tool

39.8%

CD8+ T cell cross-reactivity across IAV, IBV and ICV in HLA-A*02:01+ humans

KoutsakosMetal,inpress

What IBV-derived CD8+ T cell epitopes?

Lyse cells Y YYY

Affinity purify with Antibody

specific for HLA class I

(w6/32) Elute with Acetic Acid

109 cells: CIR-HLA-A2 cell line – infected or uninfected

Identification of HLA bound ligands •  Search against human

proteome •  Search against human +

IAV proteome

Subject to LC/MS/MS AB SCIEX TripleTOF™ 5600 System

Dr Patricia Illing, Dr Nicole Mifsud, Prof Tony Purcell

Identification of novel HLA-I epitopes for influenza: mass spectrometry

Samples C1R-Parentals – Uninfected C1R-Parentals – IAV/IBV infected C1R-A2– Uninfected C1R-A2 – IAV/IBV Infected

Look for peptides exclusive to the C1R-A2 IAV/IBV

infected cell line

Discovery of new epitopes targeted by CD8+ T cells

KoutsakosMetal,inpress

n=73 peptides

-> T cell-targeted flu vaccine to provide broadly-cross-reactive and protective immunity to unpredicted influenza viruses

Quality CD107a

IFNγ

ΤΝF

MIP1β 0

120

100

80

60

40

20

-6 -7 -8 -9 -10 -11 -12 log[peptide], M

% o

f max

imum

IF

Nγ+

TNF+

CD

8+ T

cel

ls

M 1 H A P B1

FluA FluB FluC

Breadth Protection Universally

cross-reactive A2/PB1413

Broadly cross-reactive B37/NP338

A1/PB1591

Type specific eg: A2/M158, A1/NP44, A3/NP265

Location

Tissue-resident memory

Central/effector memory

What makes a good T cell vaccine?

Designing a flu vaccine that does not require annual reformulation

Current advances and hurdles for T cell-based vaccines •  CD8+ T cell epitopes from this study would cover ~3.5 billion people (HLA-A2+ population)

•  Certain ethnic groups would not be covered

•  Broadly cross-reactive epitopes can be derived from the highly-variable surface glycoproteins •  not only from highly conserved internal proteins

•  Novel vaccine formulations are needed to boost CD8+ T cells against influenza viruses •  Provide longer protection beyond 1 year •  Broad immunity against antigenically diverse strains

•  Immune correlates of protection required for universal protection is still not answered •  Combination of T cells and B cells?

Anti-HA- vaccines

CD8+ T cell- inducing vaccines

Acknowledgements: Kedzierska Lab Katherine

Liyen

Marios

ShanghaiPublicHealthClinicalCentre,FudanUniversityProfJianqingXuandtheTeam

WHOInfluenzaCentreA/ProfAeronHurtProfIanBarr

AlfredHospitalProfAllenChengA/ProfTomKotsimbos

MonashUniversityProfJamieRossjohnDrStephanieGrasProfAnthonyPurcellDrNicoleMifsudDrPatriciaIlling StJudeResearchChildren’sHospital,MemphisA/ProfPaulThomasDrJeremyCrawfordDrEmmaAllen