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
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.