Dr Annwyne HouldsworthBSc, MSc, PhD, PGCME, FHEA, FIBMS, MRSC

Genetic study of innate and immunological mechanisms of protection

against HCV infection in man

CytokineIL-12 p40

Viral receptor

CD81

Study hypothesisGenetic differences may be important in the

disease outcome for patients infected with HCV

AntioxidantSOD-2

PathogenEntry Mechanisms

External defences

Natural immunity (innate)NK cells, IFN, phagocytes, complement

Elimination

Escape

Spread multiplication Adaptive immunity (+memory) Antibody

Cytotoxic T cells, Activation of macrophagesEscape

Persistence

Chronic infection

EliminationRecovery

Disease

Disease

Disease

Overview of Stages and Outcomes of Infection

Transmission

Hepatitis C Virus

• 150-200 million - chronically infected globally

• About 3% of the world population

• 350,000 die each year of HCV-related disease

• 214,000 chronically infected in UK (0.4% population)

Hepatitis C Viral RNA9.4 kb

• Small enveloped single stranded RNA positive sense• Capsid with envelope proteins• Tropism to hepatocytes

Progression and outcome of HCV infection

No infection

AB-veRNA-ve

HCV Exposure

Acute HCV infectionAB+ve

RNA+ve

Chronic HCV infectionAB +veRNA+ve

ResolutionAB+veRNA-ve

25%

weeks

months

75%

Cirrhosis

85%5yr survival

Hepatoma Decompensation2%

Non-progressive

>30% at 30years

4%

Death/transplantation

Polymorphism in the Interleukin-12B Gene and Outcome of HCV

Infection

Interleukin-12• Proinflammatory

cytokine

• Bridges innate and adaptive immune systems

• Immunoregulatory function

• Heterodimeric cytokine (p70)

• Two subunits P35 and p40

T helper Progenitor

T helper-1cytotoxic

IL12

IL4

IL4IL10

Tmemory

T helper 2humoural

INFg

Macrophage

T-helper cell response to HCV

IL12

HCV Infected antibody positive

HCV RNA Positive

HCV RNA Negative

Sustained responders

(SR)Relapsers

(R)

Non-responders

(NR)

Receive antiviral therapy

HCV Exposed

But resistant to HCV

infection(EU)

HCV exposed

Patient Group Categories

Normal controls from cord blood

Controls

IL-12p40 polymorphisms

4kb11kb

3’UTRPromoter Intron 4

3’5’

Polymorphism

• Several polymorphisms have been described for IL-12 p40• We investigated three

Single Nucleotide Polymorphism

http://neuroendoimmune.files.wordpress.com/2014/03/snp.png

C

A

Frequency of IL-12B genotypes in Kings College Hospital patients with HCV EU n=33; RNA+ve n=123; RNA –ve n=72; controls n=105

RNA+/RNA-P*=0.04, c2=4.12 for aaControls vs RNA - p*= 0.03, c2 =4. 83 for aa

Treatment Response in Italian Cohort for IL-12B data

NR/RR/cc p=0.027 x2=4.89

LTR/NR/cc p=0.027 x2=4.85

* *

LTR long term respondersNR non-respondersRR responder/relapsers

*

Expression of IL-12 p40 after SAC stimulation in HCV infected cases

IL-12p40 polymorphisms

4kb11kb

Linkage disequilibriumP = 0.00, c2 = 45.15 for ‘CE’

3’UTRPromoter Intron 4

3’5’

Linkage disequilibriumP =0.05 c2 = 3.70 for ‘1e’ P=0.02, c2 = 5.64 for ‘2e’

P= >0.05

Polymorphism

‘CE’ higher frequencythan expected

‘1E’ higher frequency‘2E’ lower frequencythan expected

Conclusions about IL-12 and HCV

Precise involvement in HCV not fully understood

Th1-cell responses associated with spontaneous viral clearance and treatment response

Different cytokine alleles 3’UTR SNP result in different expression levels

Enhances cytotoxic lymphocyte response Influence immune responses Influence the mechanisms involved in viral clearance Determine chronicity

Publications• Haplotype analysis and linkage study of the IL-12 gene in patients with HCV, Houldsworth A, Metzner M,

Hodgkinson A, Shaw S, Kaminski E, Demaine AG, Cramp ME (In print Journal of Medical Virology 2015)• CD81 sequence and susceptibility to HCV infection, Houldsworth A, Metzner M, Rossol S, Kaminski AG, Demaine

AG, Cramp ME. (Journal of Medical Virology, 86:162-168. Journal of Medical virology, 86:941-947, (2014)• Polymorphisms in the Interleukin-12B Gene and Outcome of HCV Infection, Houldsworth A, Metzner M, Rossol S,

Shaw S, Kaminski E, Demaine AG, Cramp ME, Journal of Interferon and Cytokine Research, Journal of Interferon and Cytokine Research, 25(5) 271-6 (2005)

• CD81 sequence and susceptibility to HCV infection, Houldsworth A, Metzner M, Rossol S, Kaminski E, AG, Demaine AG, Cramp ME., GUT supplement no.11, vol 54, 179, page A47 (2005)

• IL-12B gene polymorphism and the outcome of HCV infection. Houldsworth A, Metzner M, Rossol S, Kaminski AG, Demaine AG, Cramp ME. Hepatology; 38 (supplement 1):314 (2003)

• SOD-2 antioxidant gene and diabetic complications, Houldsworth A, Hodgkinson A, Millward BA, Demaine AG (submitted to Gene 2014)

• Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection Hegazy D, Thurairajah P, Metzner M, Houldsworth A, Shaw S, Kaminski E, Demaine AG, and Cramp ME Clinical and Experimental Immunology Vol 152, Issue 3, Pages 538-541 (2008)

• Interleukin 12B gene polymorphism and apparent resistance to HCV infection. Hegazy DM, Thurairajah PH, Metzner M, Houldsworth A, Kaminski E, Demaine A, Cramp ME. Hepatology 46(4):1456 (2007)

• HCV-specific cellular immune responses in subjects exposed, but uninfected by HCV. Metzner M, Houldsworth A, Demaine AG, Kaminski E, Cramp ME. HCV-specific cellular immune responses in subjects exposed to but uninfected by HCV. GUT. 54: A6-A7. (2005)

• HCV-specific cellular immune responses in subjects exposed, but uninfected by HCV. Metzner MK, Houldsworth A, Demaine A, Kaminski E, Cramp ME. GASTROENTEROLOGY. 128: A712-A712. (2005)

Polymorphism in the SOD-2 Gene and Outcome of HCV

Infection

Targets for ROS damage

What is a free radical?

Free radicals A molecule or ion with an unpaired

electron Very reactive species Undergo dimerisation readily Some are relatively stable Some last for long periods of time

Reactive Oxygen Species

Dioxygen (O2) in its ground state is stable diradical

Oxygen toxicity related to its high affinity for electrons

This produces damaging intermediates such as superoxide and hydroxyl anions

Ionising radiation

HCV and Oxidative Stress Chronically affected HCV cases- higher

oxidative burst and H2O2 production

Oxidative stress can inhibit Th-cell and NK responses

NS5A alters intracellular calcium levels and induces oxidative stress

HCV core protein expression causes oxidative injury- mitochondria

Liver with viral liver cirrhosis, swollen with an uneven whitish surface that is dull and coarsely

nodular showing macronodular cirrhosis.

Superoxide dismutase

SOD converts superoxide radicals to hydrogen peroxide

Cu, Zn, Mn and Fe

Mn in mitochondria

Cu/Zn in extramitochondrial cytosol

Extracellular SOD

SOD-2 In humans localized to chromosome 6 (6q25) Found in the mitochondria in nearly all cells SOD-2 gene are typical of housekeeping genes -9 mitochondrial targeting sequence Affects enzyme transport through mitochondrial

membrane ‘C/T’ substitution Amino acid change alanine/valine Valine considered- less efficiently transported

Key SOD-2 facts

Mice without SOD-2 die shortly after birth In animal cells decreased SOD-2 and catalase

levels were observed in breast cancer, adenomas and leukaemia

Val/Val genotype associated with bladder cancer

Increased SOD-2 in HCV patients

HCV Patient Data for SOD-2

*

** **

% genotype frequency

0

20

40

60

80

100

EU RNA +ve RNA -ve Normalcontrols

Patient groups

% fr

eque

ncy

CC

CT

TT

*

**

*p=0.02

P=0.001**

P=0.005***

***

Hardy Weinberg = 1.00 for controls and all patients

1 RNA positive vs RNA negative were also significantly different for CT (89.3% vs 66.1%) p=0.001, c2=11.87, OR=0.23 (0.09<OR<0.57).

2EU (86.2%) vs RNA negative (66.1%) were significantly different for CT; p=0.02, c2=5.54, OR=3.20 (1.19<OR<8.85), 3 RNA negative (27.4%) vs RNA positive (6.8%) were significantly different for TT; p=0.001, c2=11.64, OR=5.18 (1.85<OR<14.96) No other significant differences were found.

Results

Some conclusions

SOD-2 may determine oxidative stress levels during HCV infection

May determine extent of liver damage post infection

SOD-2 blood levels are significantly reduced in patients with viral hepatitis (regardless of the viral etiology).

Decreased levels may contribute to several diseases

Study Conclusions IL12B 3’UTR Genetic differences may- Influence immune responses

SOD2 genetic differences may- Less efficient form associated with RNA negative cases Less efficient form associated with DN

LEL of CD81- Highly conserved molecule Genetic differences in the coding region do not

determine clinical outcome of HCV

Acknowledgments

This work was supported in part by National Health Service Project Grant

RDW/010/249

Many thanks toHCV team Professor Andy Demaine Prof. Matthew Cramp Dr Andrea Hodgkinson Dr Doha Hegazy

And to Prof. Ed Kaminski as

advisor to the teamDr Magda Metzner as fellow

researcher in the team

Acknowledgments

This work was supported in part by National Health Service Project Grant

RDW/010/249

CD81 Tetraspanin family

Two hydrophilic domains

Large extracellular loop (LEL)

Small extracellular loop (SEL)

Molecular facilitators

Cell surface signalling complexes

Cell to cell adhesion

Why CD81? E2 is widely reported to interact with CD81

E2 Antibodies in chimpanzee sera prevent HCV infection, inhibit the binding of HCV to CD81

Molecular mimicry of E2 to normal molecular functions

CD81 widely thought to play a role in HCV endocytosis

Variable region in AGM alters E2 binding

F

NKD

Q

D

QKV

NNAD

KA

AV

VK

FYDQAL

QQ

AV V D

V

KA

D

I

TFHETL

DC

C

T

GS

STL

A LTTS

VL

KNNL

CP

S G SN I

ISNLF

KED

CHQ

KI

DD

L FS G

KL

Y

Areas of genomic CD 81 sequenced

(long extracellular loop)

Extracellular

Transmembrane

COOH

cDNA Section Sequenced

CD81 findings All four exons were sequenced for 46 cases (15 RNA –ve, 9

RNA +ve and 22 EU)

Entire cDNA CD81 sequence in 23 cases (11 RNA -ve, 5 RNA +ve and 7 EU)

In 7/ 23 cases the nucleotides were confirmed with the genomic sequence, 4 RNA –ve and 3 EU cases

No sequence variation was found in any of the cases studied by either method, including gene sections encoding the residues most important for CD81-HCV E2 binding.