1. Dr Annwyne Houldsworth
BSc, MSc, PhD, PGCME, FHEA, FIBMS, MRSC
Genetic study of innate and
immunological mechanisms of protection
against HCV infection in man

2. Cytokine
IL-12 p40
Viral
receptor
CD81
Study hypothesis
Genetic differences may be important in the
disease outcome for patients infected with
HCV
Antioxidant
SOD-2

3. Pathogen
Entry
Mechanisms
External defences
Natural immunity (innate)
NK cells, IFN, phagocytes,
complement
Elimination
Escape
Spread
multiplication
Adaptive immunity (+memory) Antibody
Cytotoxic T cells, Activation of macrophages
Escape
Persistence
Chronic
infection
Elimination
Recovery
Disease
Disease
Disease
Overview of Stages and Outcomes of Infection
Transmission

4. 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)

5. Hepatitis C Viral RNA
9.4 kb
• Small enveloped single stranded RNA positive sense

6. Progression and outcome of HCV infection
No
infection
AB-ve
RNA-ve
HCV
Exposure
Acute HCV
infection
AB+ve
RNA+ve
Chronic HCV
infection
AB +ve
RNA+ve
Resolution
AB+ve
RNA-ve
25%
weeks
months
75%
Cirrhosis
85%
5yr survival
Hepatoma Decompensation2%
Non-
progressive
>30% at 30years
4%
Death/transplantation

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

9. Interleukin-12
• Proinflammatory
cytokine
• Bridges innate and
adaptive immune
systems
• Immunoregulatory
function
• Heterodimeric
cytokine (p70)
• Two subunits P35
and p40

10. T helper
Progenitor
T helper-1
cytotoxic
IL12
IL4
IL4
IL10
T
memory
T helper 2
humoural
INFg
Macrophage
T-helper cell
response to HCV
IL12

11. 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

12. IL-12p40 polymorphisms
4kb11kb
3’UTRPromoter Intron 4
3’5’
Polymorphism
• Several polymorphisms have been described for IL-12 p40
• We investigated three

13. Single Nucleotide Polymorphism
http://neuroendoimmune.files.wordpress.com/2014/03/snp.png
C
A

14. Interleukin-12 B polymorphisms investigated
Region Taq-1 3’UTR Promoter Intron-4
Location (1188A/C )
chromosome 5q 31-33
3kb upstream of the
transcriptional start site
chromosome 5q 31-33
chromosome 5q 31-33
Polymorphism/method single nucleotide polymorphism
(SNP) ‘A’/’C’ substitution
RFLP
4bp insertion
PAGE
4bp deletion
PAGE
Clinical significance ‘C’ allele associated with
increased IL-12 expression
Different IL-12 levels associated
with autoimmune diseases
Heterozygote associated
with IL-12 expression and
more severe form of asthma
In linkage with Taq-1
polymorphism
Reason for
investigation
Increased IL-12B expression may
confer improved resolution of
viraemia
May preferentially drive a
Th2 response thus
associated with RNA positive
patients
Shows similar distribution to
the Taq-1 polymorphism

15. 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
0
10
20
30
40
50
60
70
80
90
EU RNA+ RNA- Control
%Frequency
Patient group
AA
AC
CC
*
*
*
RNA+/RNA-P*=0.04, c2=4.12 for aa
Controls vs RNA - p*= 0.03, c2 =4. 83 for aa

16. Treatment Response in Italian Cohort
for IL-12B data
0
10
20
30
40
50
60
70
80
LTR NR RR
%frequency
Patient Group
aa
ac
cc
NR/RR/cc p=0.027 x2=4.89
LTR/NR/cc p=0.027 x2=4.85
* *
LTR long term responders
NR non-responders
RR responder/relapsers
*

17. Expression of IL-12 p40 after SAC
stimulation in HCV infected cases
Mean IL12 p40 expression
0
1000
2000
3000
4000
5000
6000
RNA- RNA+
Patient Status
MeanlogIL12p40
expression
RNA-
RNA+

18. IL-12p40 polymorphisms
4kb11kb
Linkage disequilibrium
P = 0.00, c2 = 45.15 for ‘CE’
3’UTRPromoter Intron 4
3’5’
Linkage disequilibrium
P =0.05 c2 = 3.70 for ‘1e’
P=0.02, c2 = 5.64 for ‘2e’
P= >0.05
Polymorphism
‘CE’ higher frequency
than expected
‘1E’ higher frequency
‘2E’ lower frequency
than expected

19. Interleukin-12 B polymorphisms investigated
Region Taq-1 3’UTR Promoter Intron-4
Location (1188A/C )
chromosome 5q 31-33
3kb upstream of the
transcriptional start site
chromosome 5q 31-33
chromosome 5q 31-33
Polymorphism/method single nucleotide polymorphism
(SNP) ‘A’/’C’ substitution
RFLP*
4bp insertion
PAGE**
4bp deletion
PAGE**
Clinical significance ‘C’ allele associated with
increased IL-12 expression
Different IL-12 levels associated
with autoimmune diseases
Heterozygote associated
with IL-12 expression and
more severe form of asthma
In linkage with Taq-1
polymorphism
Reason for
investigation
Increased IL-12B expression may
confer improved resolution of
viraemia
May preferentially drive a
Th2 response thus
associated with RNA positive
patients
Shows similar distribution to
the Taq-1 polymorphism
Results
HCV RNA negatives significantly
lower ‘A’ allele than HCV RNA
positives; allele expresses lower
levels of IL-12B
No significant differences
between groups
Linkage disequilibrium seen
with Taq-1 polymorphism

20. 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

21. Publications
• Haplotype analysis and linkage study of the IL-12 gene in patients with HCV, HouldsworthA, 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)

22. Polymorphism in the SOD-2
Gene and Outcome of HCV
Infection

23. Targets for ROS damage

24. What is a free radical?

25. 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

26. 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

28. 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

31. Liver with viral liver cirrhosis, swollen with an
uneven whitish surface that is dull and coarsely
nodular showing macronodular cirrhosis.

32. Superoxide dismutase
 SOD converts superoxide
radicals to hydrogen
peroxide
 Cu, Zn, Mn and Fe
 Mn in mitochondria
 Cu/Zn in
extramitochondrial cytosol
 Extracellular SOD

33. 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

34. 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

39. HCV Patient Data for SOD-2
*
**
**
% genotype frequency
0
20
40
60
80
100
EU RNA +ve RNA -ve Normal
controls
Patient groups
%frequency
CC
CT
TT
*
**
*p=0.02
P=0.001
**
P=0.005
***
***

40. 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

41. Oxidative
stress
Inhibits T-cell
+ NK response
Association between antioxidant function and HCV
resistance
HCV ViraemiaSequestered
phagocytes/macrophages
Release ROS
Imbalance in
redox equilibrium
Antioxidant Defence
Increases in SOD-2 etc
due to HCV
Chronic HCV patients
have
Lower levels of SOD-2
HCV proteins
Stimulates TNFa
NFKB
Ca2+ altered
Produces
ROS
SOD-2 response lower in liver
SOD-2 blunts
NFKB, TNFa
+TGFb activity
Mitochondrial injury
Reduced
antioxidant response on
prolonged
activation
Less efficient immune
response
to HCV infection

42. 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

43. 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

44. Acknowledgments
This work was supported in part by
National Health Service Project Grant
RDW/010/249

45. Many thanks to
HCV team
 Professor Andy Demaine
 Prof. Matthew Cramp
 Dr Andrea Hodgkinson
 Dr Doha Hegazy
And to
Prof. Ed Kaminski as
advisor to the team
Dr Magda Metzner as fellow
researcher in the team

46. Acknowledgments
This work was supported in part by
National Health Service Project Grant
RDW/010/249

47. CD81
 Tetraspanin family
 Two hydrophilic domains
 Large extracellular loop (LEL)
 Small extracellular loop (SEL)
 Molecular facilitators
 Cell surface signalling complexes
 Cell to cell adhesion

48. 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

49. N
K
D
Q
D
Q
K
NN
AD
K
A
A
V
V
K
F
Y
D
Q
A
L
Q
Q
A
V
V
D
V
K
A
D
I
T
F
H
E
T
L
D
C
C
T
G
S
S
T
L
A
L
T
T
S
V
L
K
N
NL
C
P
S G S
N
I
I
S
N
L
F
K
E
D
C
H
Q
K
I
D
D
L
F
S
G
K
L
Y
Areas of genomic CD 81
sequenced
(long extracellular loop)
Extracellular
Transmembrane
COOH

50. cDNA Section Sequenced

52. 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.