Virologie

H. STOIBERVIENNA, 19.07.2010

MULTIFACETED INTERACTIONS OF RETROVIRUSES WITH THE COMPLEMENT

SYSTEM

SCHEMATIC OVERVIEW OF IMMUNE RESPONSES UPON VIRAL INFECTIONSVienna, 19.07.2010

Complementactivaton

MACLysis

Infected cellPathogens

Inflammation

Chemotaxis

C3aC5a

IncreasedPermeability

C3aC5a

APCsGranulocytes

BasophilsMast cells

Antigen -Presentation

T cell

DCs

Opsonisation

Immune complexes

CR3

CR1

M

Phagocytosis

YCR2

FDCs

Trapping

Cell activation

BasophilsMast cells

Granulocytes

DCs

YY Y

B cells

C3aC5a

iC3bC3d

CONSEQUENCES OF COMPLEMENT ACTIVATIONVienna, 19.07.2010

(Schacker et al. 2000)

EX VIVO DETECTION OF HIV IN LT OF INFECTED PATIENTS Vienna, 19.07.2010

COMPLEMENT-DEPENDENT TRAPPING OF HIV IN THE LT

Kacani et al. JV

Vienna, 19.07.2010

Species Virus Source Trapping AIDS-like Symptoms

African Green Monkey SIVagm no no

Sooty mangabey SIVsm no no

Rhesus Monkey SIVmac yes yes

Chimpanzees SIVchp/HIV-1 no no

Human HIV-1 yes yes

RELATIONSHIP BETWEEN TRAPPING AND AIDS Vienna, 19.07.2010

Complementactivation

MACLysis

Infected cellsPathogens

Inflammation

Chemotaxis

C3aC5a

C3aC5a

APCsGranulocyte

BasophilsMast cells

Antigen -Presentation

T cell

DCs

Opsonisation

Immune complexes

CR3

CR1

M

Phagocytosis

YCR2

FDCs

Trapping

Cell activation

BasophilsMast cells

Granulozytes

DCs

YY Y

B cells

C3aC5a

iC3bC3d

CONSEQUENCES OF COMPLEMENT ACTIVATIONVienna, 19.07.2010

IncreasedPermeability

IN VITRO PRIME-BOOST EXPERIMENTS: PROTOCOL

Loading of DCs withSEB, HIV, HIV-C autologous, unstimulated CD8+ T cells (Prime)

Boost of primed CD8+ T cells with Ag-loaded DCs (3x)(all cells from the same donor)

Data-Acquisition of Prime-Boost Experiments

Proliferation-assays(CFSE staining)

IFN-g assays (ELISA, Secretions-assay)

Test on Specificity and Functionality ofin vitro generated CTLs

(Tetramer, degranulation, antiviral activity)

Vienna, 19.07.2010

Bánki et a., PLoS Path., 2010 Apr 29;6(4):e1000891.

PROLIFERATION OF HIV-C-DC-PRIMED CD8+ T CELLS (7 DONORS 5 VIRAL STRAINS) Vienna, 19.07.2010

Control (mDCs) HIV-hiC (X4) HIV-C (X4) HIV-C (X4)-P2 HIV-C (R5X4) HIV-C (R5)

0.1%1.5% 7.6% 1.1% 7.2%14.2%

CD8+ T cells

Tetramer-staining

ANTIVIRAL RESPONSE OF HIV-C-DC-PRIMED CD8+ T CELLS(5 DONORS, 4 VIRAL STRAINS)

Vienna, 19.07.2010

Bánki et a., PLoS Path., 2010 Apr 29;6(4):e1000891.

Vienna, 19.07.2010ERYTHROLEUKEMIA INDUCED BY FRIEND VIRUS (FV) IN MICE

1st StageExpansion of erythroblastsin spleen due to gp55

2nd StageMalignant transformation of erythroblasts

LTR gag pol env LTR LTR

env: Recombinationdeletion, insertion

F-SFFV F-MuLV

gp55

LTR gag pol env

(replication competent helper-virus)

1. Helper virus, F-MuLV (murine leukemia virus),  is responsible for growth of the virus complex.

2. F-MuLV can only produce lymphomas if injected into neonatal mice.3. F-SFFV (spleen focus-forming virus) is defective/interfering virus that expresses

gp55 env protein that binds to EpoR to produce erythroblastosis; has no oncogene;

%in

fect

ed c

ells

40

30

20

10

0

0.0405

B6 contro

l

B6C3-/- co

ntrol

B6 7dpi

B6C3-/- 7dpi

10

8

6

4

2

0

CD8+

%FV

gag

tetr

amer

+ 0.0119

B6 contro

l

B6C3-/- co

ntrol

B6 7dpi

B6C3-/- 7dpi

8

6

4

2

0

CD8+

%FV

gag

tetr

amer

+CD

43+

0.0088

B6 contro

l

B6C3-/- co

ntrol

B6 7dpi

B6C3-/- 7dpi

ENHANCED FV-INFECTION OF C3-DEFICIENT MICE CORRELATES WITH LOWER FREQUENCIES OF FV-SPECIFIC CTLS.

Vienna, 19.07.2010

Bánki et a., PLoS Path., 2010 Apr 29;6(4):e1000891.

B6 contro

l

B6C3-/- co

ntrol

B6 4dpi

B6C3-/- 4dpi

5

4

3

2

1

0

CD11

c+ %

infe

cted

cel

ls

0.0025

Complementactivation

MACLyse

Infected cellsPathogens

Inflammation

Chemotaxis

C3aC5a

IncreasedPermeability

C3aC5a

APCsGranulocyte

BasophilsMast cells

Antigen -Presentation

T cell

DCs

Opsonisation

Immune complexes

CR3

CR1

M

Phagocytosis

YCR2

FDCs

Trapping

Cell activation

BasophilsMast cells

Granulocyte

DCs

YY Y

B Zellen

C3aC5a

iC3bC3d

CONSEQUENCES OF COMPLEMENT ACTIVATIONVienna, 19.07.2010

COMPLEMENT ACTIVATION, MAC AND OPSONISATION

Host cells are protected from CML by Regulators of the Complement system

factor I

+ CR1+ factor H+ MCP

P

iC3b

factor I

C3d

P

CD59

P

P

Complement Activation

MAC

C3

P

C3b

LYSE

CD55fH

Why are intact and infectious viruses in the serum?

Vienna, 19.07.2010

EfficientVirolyse

InefficientVirolyse

+ Serum + Serum + Antibody

Cell infected with Retroviruses

“budding”gp120/41 complex

MHC-I

MHC-II

DAF

CD59

fH

Stoiber et al. JEMStoiber et al, Nat. Med.

Vienna, 19.07.2010 BUDDING OF HIV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Factor H

C3b bindingcatalytic region C3b/c binding

heparin binding heparin binding

C3b/d binding

2 7 13 18 19 20Expression

SCR13

Cross-link to mAb

SCR7SCR2 SCR20

SCR18 SCR19

FACTOR H IN DETAIL

Binding site is distinct from the enzymatic active site

Pathogen-specificAntibody

Complement-specificPart

Vienna, 19.07.2010

NMS+Ab

IgepalInput virus

NMS NMS+Ab-SCR

D = 9ct unitseq. 3 logs

IN VITRO LYSIS OF FV BY COMPLEMENT IN THE PRESENCE OF THE CONSTRUCTSVienna, 19.07.2010

Patent: WO 2008/135237 A1

Reduction of the viral titers at about 3logs

105 104 103 102 101

negative control [“no FV“] 1 / / / / /

2 / / / / /

3 / / / / /

positive control [“No Ab, no SCR” ] 4 na na 86 9 5

5 na na 60 11 5

SCR2-Ab#48 8 na na 51 27 6

9 na na 22 5 1

SCR7-Ab#48 13 47 4 / / /

14 na 84 13 2 /

SCR13-Ab#48 15 na 29 / / /

16 73 11 1 / /

SCR18-20-Ab#48 17 61 5 / / /

18 17 1 / / /

INFECTIOUS CENTERS IN THE SPLEENVienna, 19.07.2010

Patent: WO 2008/135237 A1

SUMMARY I Vienna, 19.07.2010

Trapping of HIV in vivo is mainly dependent on Complement-CR interactions;thus, Complement shows responsible for maintaining the largest viral reservoir in HIV-infected individuals

YCR2

Follicular Dendritic Cells

HIV-C

antiviral

Activity high

IFN-g

HIV/FV

HIV-C/FV-C

SUMMARY II

Complement is a natural adjuvant for the induction of retrovirus-specific CTLs

Vienna, 19.07.2010

CD8 TC Infected cells

antiviral

Activity low

retrovirus

C

C‘ CC‘

X

AIM: Reconstitute CML by Pathogen-specific constructsY

constructs

Y

Y Y

Y

YY

Y Y

Putative Targets beside HIV:Other enveloped-Viruses, Bacteria

SUMMARY III Vienna, 19.07.2010

retrovirus

C

C‘ CC‘

Y

Y

YX

X

ACKNOWLEDGEMENTS

INSTITUTE OF APPLIED MICROBIOLOGY, BOKU, VIENNAG. STIEGLERH. KATINGER

BERNHARD-NOCHT-INSTITUTE, HAMBURGK. TENNER-RACZP. RACZ

DEPARTMENT OF MEDICINE, EPPENDORF, HAMBURGJ. VAN LUNZEN

INSTITUTE OF IMMUNOLOGY, HELSINKIS. JOKIRANTA

ROCKEFELLER UNIVERSITY, NEW YORKR. STEINMAN

GERMAN PRIMATE CENTER, GÖTTINGENC. STAHL-HENNING

INSTITUTE OF MED. VIROLOGY, BOCHUMK. ÜBERLA

INSTITUTE OF VIROLOGY, ZÜRICHA. TRKOLA

INSTITUTE OF VIROLOGY, ESSENU. DITTMER

INSTITUTE OF BIOCHEMISTRY, EPALINGENH. ARCHA-ORBEA

SECTION OF IMMUNOLOGY ,ST. GALLENB. LUDEWIG

RETROVIRAL IMMUNOLOGY SECTION, HAMILTONKIM HASENKRUG RON MESSER

Vienna, 19.07.2010

OPSONIZED!THINK

THANK YOU !

SECTION OF VIROLOGY, INNSBRUCKDORIS WILFLINGSEDERWILFRIED POSCH

ZOLTAN BANKIASIM EJAZVERENA OBERHAUSERCHRISTOPH AMMANNGEORG HUBERBRIGITTE MÜLLAUERHERIBERT STOIBER

SECTION OF MICROBIOLOGY, INNSBRUCKCORNELIA LASS-FLÖRL

DEPT. OF HAEMATOLOGY, INNSBRUCK GÜNTER GASTL

EU, , MFI

Vienna, 19.07.2010

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