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

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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

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12000

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16000

18000

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1500

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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.

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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

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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.

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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