Combining immunotherapy and vaccination to treat ch ronic infectionsLes Pensières - Fondation Merieux Conference Centre
Veyrier-du-Lac - June 15-17, 2009
Human CMV and atherosclerosis
Claudio LunardiClinical Immunology
Department of Clinical and Experimental MedicineUniversity of Verona - I
Atherosclerosis
Atherosclerosis is the leading cause of morbility and mortality in Western countries
Both genetic and enviromental factors play a role in the pathogenesis of the disease
Classical risk factors include hypercholesterolemia, arterial hypertension, smoking, obesity, diabetes mellitus
Several data (C reactive protein, fibrinogen, soluble adhesion molecules) suggest that atherosclerosis is a chronic inflammatorydisease reflecting a condition of endothelial cell damage and dysfunction.
Atherosclerosis∗ Inflammation
∗ - endothelial cell activation∗ - lymphomonocyte chemoattraction∗ - high C-reactive protein levels
∗ Infections∗ - Chlamydia pneumoniae
- Helicobacter pylorii∗ - Mycobacteriae∗ - Cytomegalovirus
∗ Autoimmunity
∗ Infectious agents seem to be involved in endothelium damage by inducing an autoimmune response to Heat Shock Proteins (HSP).
∗ -antibodies against HSP60 are present in most patients with CAD and their titre correlates with disease severity
ENDOTHELIAL CELL AND ATHEROSCLEROSIS
• Endothelial cell (EC) injury is considered to be an initial event in the development of atherosclerosis (endothelial-leukocyte adhesion molecules)
• Endothelial injury may lead to EC apoptosis
• EC apoptosis is detected in atherosclerotic lesions
INFECTIONS and ATHEROSCLEROSIS
• Seroepidemiological data: several candidate pathogens may causally relate to atherosclerosis (hCMV, hepatitis A virus, HSV1, HSV2, Chlamidia pneumoniae)
• Identification of viruses and bacteria in atherosclerotic plaques
• Strong association between specific infections such as hCMV withtransplant atherosclerosis
• Experimental models
• Ability of infectious organisms to induce responses in cells relevant to atherosclerosis: endothelial cells, T cells , monocyte-macrophages, smooth muscle cells
Human Cytomegalovirus
hCMV has been implicated in vascular disorders characterized by endothelial cell apoptosis, inflammatory cells infiltration and smooth muscle cells proliferation in the intima of vascular walls, defined as neointima formation
HSP60, hCMV e ATHEROSCLEROSIS
• We have shown that autoantibodies against HSP60 are present in most atherosclerotic patients and that these autoantibodies are directed to the sequence at position 153-163 of HSP60 (AELKKQSKPVT)
• The aminoacid sequence 153-163 of HSP60 shows homology with two hCMV-derived proteins, UL122 and US28:
-UL122 is early expressed during infection-US28, is a C-C chemokines receptors-like, involved in the reactivation from
latency
• Antibodies anti-HSP60153-163 cross-react with the viral proteins
HSP60 153-163 A E L K K Q S K P V T: : * : : *
UL122 152-162 G P R K K K S K R I S
HSP60 153-163 A E L K K Q S K P V T* * : : : : :
US28 26-36 T D V L N Q S K P V T
Bason C et al. Lancet 2003
HSP60, hCMV e Atherosclerosis
• Antibodies purified against the HSP60153-163 peptide and against the two hCMV-derived peptides bound non-stressed endothelial cells upon interaction with cell surface molecules through a mechanism of molecular mimicry. Such antibodies induced apoptosis of endothelial cells.
• Indeed both viral peptides, UL122 and US28, show sequence homology with molecules normally expressed on endothelial cell surface: – US28 shows homology with integrin alpha 6 (CD49f)– UL122 show homology with CD151 and with connexin 45
Connexin 45 G P R E K K A K V G S: : * : : * : :
UL122 G P R K K K S K R I S: : : : : * *
CD151 Q L R K K A S G R V A
US28 T D V L N Q S K P V T: * : : : * :
CD49f F R V I N L G K P L T
CONCLUSION
We showed that hCMV infection can be involved in the pathogenesis of atherosclerosis: the virus can induce an autoimmune response that leads to endothelium damage and apoptosis through a mechanism of molecular mimicry involving the engagement of molecules normally expressed on the cell surface. The same (auto)antibodies cross-react with hHSP60, thus amplifying endothelial cell damage.
Lunardi et al. Trends Immunol 2005; 26: 19-24
� To dissect the molecular basis of the interaction between anti-Cytomegalovirus antibodies and endothelial cells.
GENE ARRAY
We analyzed the gene expression profiles in endothelial cells using the Human Genome U133A GeneChip®
(Affymetrix). The GeneChip® Human Genome U133A is a single array representing 14,500 well-characterized human genes.
– We stimulated endothelial cells with antibodies purified against either the UL122 or the US28 peptide (test samples) or with antibodies purified against an irrelevant peptide (control samples) for 6 and 12 hours.
– The different gene expression patterns were analyzed using the Array Assist software version 2.0 (Stratagene, La Jolla, California, United States)
MODULATED GENES IN HUVECs
Down-regulated
Up-regulated Up-regulated
Down-regulated
907 genes 186 genes
anti-US28 anti-UL122
1389 genes 140 genes
0
1
2
3
4
5
6
7
SELE VCAM1 ITGA2 ITGB3 ICAM1
Fol
d c
han
ge
0
1
2
3
4
5
CCL2 CCL20 CXCL1 CXCL2 CXCL3 CCR3 CXCR4
Fo
ld c
han
ge
HUVEC stimulated with anti-US28 antibodies
Adhesion moleculesendothelial adhesion molecule 1 SELE 5,95 1,79vascular cell adhesion molecule 1VCAM1 3,76 2,06integrin, alpha 2 ITGA2 2,61 2,69integrin, beta 3 ITGB3 1,72 1,72intercellular adhesion molecule 1 (CD54) ICAM1 1,60 1,33
Inflammationchemokine (C-C motif) ligand 2CCL2 2,46 1,98chemokine (C-C motif) ligand 20CCL20 1,63 1,42chemokine (C-X-C motif) ligand 1 CXCL1 3,32 1,63chemokine (C-X-C motif) ligand 2CXCL2 4,21 2,72chemokine (C-X-C motif) ligand 3CXCL3 1,62 1,40chemokine (C-C motif) receptor 3CCR3 1,49 1,77chemokine (C-X-C motif) receptor 4 CXCR4 2,24 1,99chemokine-like factor super family 6CKLFSF6 2,07 1,69tumor necrosis factor (ligand), member 4 TNFSF4 1,96 1,36interleukin 6 signal transducer IL6ST 3,61 4,36interleukin 7 receptor IL7R 2,04 2,01
Adhesion molecules
Chemokines
0
0.5
1
1.5
2
2.5
3
EIF5A
CCND1TNFSF10
CASP6CASP7
GAS2GAS7DFFA
NFKBIA
Fo
ld c
han
ge
0
1
2
COL4A3BP HPSE TGIF2 TGFBR3
Fol
d ch
ange
HUVEC stimulated with anti-US28 antibodies
Growth inhibition and apoptosis
eukaryotic translation initiation factor 5A EIF5A 22,93 17,78
tumor necrosis factor (ligand) member 10 TNFSF10 2,03 2,53
caspase 6, apoptosis-related cysteine protease CASP6 2,65 2,35
caspase 7, apoptosis-related cysteine protease CASP7 1,61 1,31
growth arrest-specific 2 GAS2 1,84 1,19
growth arrest-specific 7 GAS7 1,78 1,95
DNA fragmentation factor, 45kDa, DFFA 1,23 1,66
nuclear factor of kappa light polypeptide gene NFKBIA 2,40 1,75
transducer of ERBB2, 2 TOB2 1,92 1,99
Collagenes and extra-cellular matrix
collagen, type IV, alpha 3 binding protein COL4A3BP 1,76 1,87
heparanase HPSE 1,74 1,33
TGFB-induced factor 2 (TALE family homeobox) TGIF2 1,69 1,13
transforming growth factor, receptor III (betaglycan) TGFBR3 1,55 1,61
Apoptosis Extra-cellular matrix
0
5
10
15
20
25
30
35
SELE VCAM1 ICAM-1
fold
cha
nge
02468
101214161820
CCL2
CXCL2
CXCL3
IL8
CX3CL1
LTB
TRAF1 CSF2R
BCXCR4
Fol
d ch
ange
HUVEC stimulated with anti-UL122 antibodies
Adhesion molecules
selectin E (endothelial adhesion molecule 1)SELE 31,15 15
vascular cell adhesion molecule 1 VCAM1 18,8 6,1
intercellular adhesion molecule 1 (CD54)ICAM-1 12,7 7,2
integrin, alpha V ITGAV 1,54 1,32
Inflammation
chemokine (C-C motif) ligand 2 CCL2 19 16
chemokine (C-C motif) ligand 20 CCL20 7,6 4,7
chemokine (C-X-C motif) ligand 1 CXCL1 6,2 4,5
chemokine (C-X-C motif) ligand 2 CXCL2 9,5 6,8
chemokine (C-X-C motif) ligand 3 CXCL3 10 6
interleukin 8 IL8 2,8 2,3
chemokine (C-X-C motif) ligand 11 CXCL11 5,5 6
chemokine (C-X3-C) ligand 1 (fractalkine)CX3CL1 3,11 2,9
TNF receptor-associated factor 1 (TRAF1)TRAF1 1,6 1,37
colony stimulating factor 2 receptor, betaCSF2RB 1,52 1,2
chemokine (C-X-C motif) receptor 4 CXCR4 1,83 2,17
Adhesion molecules
Chemokines and inflammation
0
2
4
6
8
10
12
PLA2 G4C PTGS2 F2RL1 SOD 2 ACE2
Fol
d c
han
ge
HUVEC stimulated with anti-UL122 antibodies
0
0.5
1
1.5
2
2.5
RELB
TCF4
TCF
8 NFK
BIE KIT
TANK
ETS2
Fo
ld c
han
ge
Transcription
v-rel reticuloendotheliosis viral oncogene homolog BRELB 2 2
transcription factor 4 TCF4 1.6 1.5
transcription factor 8 TCF 8 1.4 1.8
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilonNFKBIE 2 1.8
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogeneKIT 2.3 2.2
TRAF family member associated NFKB activator TANK 1.6 1.4
v-ets erythroblastosis virus E26 oncogene homolog 2 ETS2 1.26 1.53
Miscellaneous
CD69 antigen CD 69 6 3.7
ephrin-A1 EFNA1 2.1 1.8
prostaglandin-endoperoxide synthase 2 (COX-2) PTGS2 1.9 1.3
Thrombin receptor like 1 F2RL1 1.72 1.63
Claudin 5 CLDN5 1.17 1.7
superoxide dismutase 2, mitochondrial SOD 2 5.5 4.6
angiotensin I converting enzyme (peptidyl-dipeptidase A) 2ACE2 1.2 1.5
ubiquitin D UBD 2.7 3.8
guanylate binding protein 1, interferon-inducible, 67kDaGBP1 1.8 1.7
Transcription
Miscellaneous
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0
500
1000
1500
2000
2500
3000
3500
1
0
100
200
300
400
500
600
700
800
0
100
200
300
400
500
600
1
A
C D
B
1 2 3 4 1 2 3 4
1 2 3 4 1 2 3 4
MCP-1 (A-B) and E-selectin (C-D) secretion in the medium after 6 and 12 hrs incubation with anti-UL122 (A-C) and anti-US28 (B-D) antibodies
Summary:
• Gene expression analysis demonstrated that both subsets of anti-hCMV antibodies modulated genes encoding for molecules involved in the atherosclerotic process
• Anti-US28 antibodies modulated a higher number of genes (907 up- and 1389 down-regulated) than anti-UL122 antibodies (186 up- and 140 down-modulated genes)
• This observation suggested that a different mechanism could be involved in the endothelial damage induced by the the two subsets of anti-hCMV antibodies.
0
2
4
6
8
10
1
0
2
4
6
8
10
1
3 6 9 12 15 18 21 3 6 9 12 15 18 21
Apo
ptot
ic
Inde
x
A B
Time (h) Time (h)
Apoptosis induced by anti-UL122 Abs occurs at an earlier stage than that induced by anti-US28 Abs
(a) control (b) anti-UL122 (c) anti-US28
ANTI-US28 ANTIBODIES TRASLOCATED HSP60 ON THE CELL SURFACE
…and released the soluble molecule (sHSP60) in the medium
0
4
8
12
16
20
1 2 3 4
Soluble HSP60 in the supernatant of treated HUVEC cells:
-Red line: anti-UL122 treated
cells;
-Blu line: anti-US28 treated cells.
0
10
20
30
40
50
1
Line 1: stimulation with supernatant of untreated cells;Line 2 e 3: stimulation with supernatant of anti-US28 treated cells for 6 and 12 hours;Line 4: stimulation with supernatant of anti-US28 treated cells and after sHSP60 removal.
ACTIVATION OF TLR4 BY SUPERNATANT OF ANTI-US28 TREATED CELL CONTAINING sHSP60
1 2 3 4
Anti-US28 antibodies can induce endothelial cell damage and traslocation of HSP60 on cell surface. Moreover HSP60 is released in the medium.
HSP60 is an endogenous ligand of TLR4, a receptor expressed on endothelial cells, macrophages and dendritic cells. The binding of TLR4 activates innate immunity and promotes the inflammatory response.
ANTIBODIES ANTI-US28: A STRESS FACTOR
Conclusion
• Anti-US28 and anti-UL122 affinity purified antibodies induce apoptosis of non stressed endothelial cells; anti-UL122 antibodies induce apoptosis earlier than anti-US28 antibodies do
• Both anti-hCMV antibodies modulated genes encoding molecules known to be involved in the inflammatory process and in the pathogenesis of atherosclerosis:
• Adhesion molecules (ICAM-1, VCAM-1, Selectina E)
• Chemokines (MCP-1, MIP2α)• Melecules involved in cell activation (MAPK, NF-kB)
• Growth factors (PGF, EGFR)
• Extra-cellular matrix deposition
Conclusion
• Anti-US28 antibodies induce traslocation on the membrane and release in the supernatant of sHSP60
• The sHSP60 released from endothelial cells is able to trigger signalling through TLR4
• TLR4 activation may be considered a bridge between innate and acquired immunity in the pathogenesis of atherosclerosis.
APOPTOSIS
hCMV
Integrins
Legend:
HSP60US28 UL122
Y
Y
Y
YY Y
Y AbsHSP60 TLR4
ACQUIRED IMMUNITY:Anti-US28 e anti-UL122 Ab.
HSP60 EXPRESSION AND SECRETION INNATE IMMUNITY:TLR4 activation
CONCLUSION
Association of anti-UL122 and anti-US28 antibody levels and CAD
-100 CAD free pts -100 pts with CAD and not MI-100 pts with CAD and MI
-The three groups were age and sex matched
- No differences between CAD pts with and without MI, therefore the statistical data were evaluated comparing the group of 200 pts with CAD and the 100 sex and age matched CAD free subjects
A B
C D
Cheng et al. PLoS Pathogens, 2009
Dieudè M et al. J Throm Haemost. 2009
Blasi C, Atherosclerosis, 2008
Potential role of HSP60 in atherogenesis
Genetic and Epigeneric Factors, Risk Factors, oxLDL, infections (hCMV)
Toxinse.g.LPS
HSPs molecular mimicry
AUTOIMMUNITY
INNATE IMMUNITY
INFLAMMATION
ATHEROSCLEROSIS
ADAPTIVE IMMUNITY
Thrombus formation
InvestigatorsInvestigators
• University and Institute G. Gaslini, Genova
• Marzia Dolcino• Riccardo Navone• Antonio Puccetti
• University of Verona• Caterina Bason• Ruggero Beri• Oliviero Olivieri• Nicola Martinelli• Roberto Corrocher
THANK YOU FOR YOUR ATTENTION