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Adenovirus: an eternal archetype A short history 1953: first adenovirus isolation by Rowe et al., Proc. Soc. Exp.Biol. Med. 1962: oncogenic properties.

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Presentation on theme: "Adenovirus: an eternal archetype A short history 1953: first adenovirus isolation by Rowe et al., Proc. Soc. Exp.Biol. Med. 1962: oncogenic properties."— Presentation transcript:

1 Adenovirus: an eternal archetype A short history 1953: first adenovirus isolation by Rowe et al., Proc. Soc. Exp.Biol. Med. 1962: oncogenic properties of HAdV species A (Ad12) : biochemical characterization : virion assembly : splicing and transcriptional control : viral oncogenes and association with tumor suppresseur N: viral vector in gene therapy

2 Adenovirus  Replication Encapsidation - DNA ds (~36 kb) hexon penton penton base fibre

3 Adenoviruses uptake 1) Membrane attachment RGD hCAR  V integrins... 2) Uptake Modify adenovirus tropism : specific fiber modifications Heparan sulfate

4 Caveolae Non-caveolae non-clathrin endocytosis Clathrin- caoted pit endocytosis Macropinocytosis macropinosome CCV EE LE LY Golgi trans Medial Cis ER Caveosome Lysosomal degradation A549 HEK29 HeLa NLS-machinery hCAR  v  3/5 NPC proteins Central channel Peripheral channel Nuclear basket Outer membrane Inner membrane Nucleus Cytoplasm Viral DNA Transgene expression Endocytosis...

5 Structure of the fiber protein Penton base Shaft  -X-  -X-X-X-X-P-  -X-  -X-X-X  -X-  -X-X-G-X-G-  -X-  -X-X-X Tail Knob

6 IVa2 IIIa pVI pVIII E4 ITR Precursor RNA 1 DAdV-1 Atadenovirus Fiber FAdV-1 Aviadenovirus Fiber 1Fiber 2 HAdV2 Mastadenovirus E3 Fiber E4 E1AE1B Adenoviridae E3E4

7 Species Serotype% GC %homology species Fiberlength (nm) oncogenic properties A 12, 18, high B 3, 7, weak C 1, 2, 5, nul D 8, 9, 10, 13, 15, nul (except Ad 9) E nul F 40, et 23 nul Human adenoviruses receptor hCAR CD46 hCAR + X

8 Tropism modifications of adenovirus Association with antibodies Genetic modifications of the fiber Chimeric fiber transient expression important immun response wide tropism

9 Project and history 1984 cloning of the BAdV4 genome 1997 BAdV-4 based vector ? no permissive cell line bovine cell line obtaining … X vectors with chimeric fibers Project: targeting vectors to B lymphocytes lost of hCAR interaction add new target

10 Project: targeting vectors to B lymphocytes No receptor Lost of hCAR interactionAdd new target

11 FAdV-1-L BAdV10 PAdV-4 HAdV12 HAdV31 HAdV17 HAdV9 HAdV2 HAdV5 HAdV4 SAdV-25 HAdV41-L HAdV40-L HAdV40-S HAdV41- S HAdV7 HAdV3 HAdV16 HAdV35 FAdV-1-S BAdV-3 MAdV-1 FrAdV-1 TAdV-3 BAdV-4 odocoileus OAdV-287 DAdV-1 Siadenovirus Atadenovirus Mastadenovirus non-human Aviadenovirus Mastadenovirus Fiber Knob Phylogenic tree

12 Penton base binding sequence of fiber proteins Mastadenovirus Aviadenovirus Atadenovirus Siadenovirus Conserved the Nter of HAdV fiber protein

13 1 HAdV2(1) ---NDDKLTLWTTPDPSPNCRIHSDN----DCKFTLVLTKC-GSQVLATVAALAVS- HAdV3 (1) NTLWTGPKPEANCIIEYGKQNP-DSKLTLILVKN-GGIVNGYVTLMGASD HAdV4 (1) GNKDYDKLTLWTTPDPSPNCQILAEN----DAKLTLCLTMC-DSQILATVSVLVVRS HAdV5 (1) ---NDDKLTLWTTPDPSPNCRIHSDN----DCKFTLVLTKC-GSQVLATVAALAVS- HAdV7 (1) NTLWTGPKPEANCIIEYGKENP-DSKLTLILVKN-GGIVNGYVTLMGASD HAdV9 (1) ----EDKRTLWTTPDTSPNCKIDQDK----DSKLTLVLTKC-GSQILANVSLIVVDG HAdV12 (1) SNTPYDPLTLWTTPDPPPNCSLIQEL----DAKLTLCLTKN-GSIVNGIVSLVGVKG HAdV15 (1) --KKEDKRTLWTTPDTSPNCKIDQDK----DSKLTLVLTKC-GSQILANVSLIVVDG HAdV17 (1) --PKYDTRTLWTTPDTSPNCRIDKEK----DSKLTLVLTKC-GSQILANVSLIVVSG HAdV31 (1) IATPYDPLTLWTTPDPPPNCTLRQEL----DAKLTLCLTKN-ESIVNGIVSLIGVKG HAdV35 (1) INTLWTGINPPPNCQIVENTNTN-DGKLTLVLVKN-GGLVNGYVSLVGVSD HAdV40-L (1) QTRSVTSLTTIWSISPTPNCSIYETQ----DANLFLCLTKN-GAHVLGTITIKGLKG HAdV40-S (1) SPTTTTPTTLWTTADPSPNATFYESL----DAKVWLVLVKC-NGMVNGTISIKAQKG HAdV41-L (1) SPTTTTPTTLWTTADPSPNATFYESL----DAKVWLVLVKC-NGMVNGTISIKAQKG HAdV41-S (1) RTRSVPSLTTIWSISPTPNCSIYETQ----DANLFLCLTKN-GAHVLGTITIKGLKG BAdV-3 (1) TAPNMVTETLWTGTGSNANVTWRGYTAP--GSKLFLSLTRFSTGLVLGNMTIDSNAS MAdV-1 (1) LQAPQYELTTNGMREITFSFNSTGGLEQPAPVTYWGALDPPPTAKAAEIENQKRVKK FAdV-1-L (1) NTN---AAKVAAYCRLAAAGGVVSGTIQVTSYAG FAdV-1-S (1) TGNFVSSSNNPFNGSYFLQQINTMGMLTTSLYVKVDTTTMGTRPTGAVNE BAdV-4 (1) ----FEVLNDNLALGLNNSLTVESGK---LAVKTAGPIQSTEQGINLSVANPFSISS DAdV-1 (1) PQNK---AAKVAGYVYLTSVGGLVHGTIQIKATAG FrAdV-1 (1) AVSPPFSSTGGLSLLLDPSLRLISGALGLKPSSASCIVSSP-AGVELKTGKGLGVTG TAdV-3 (1) --GIRLAPNSGLQITPNGLAVSVNAVQILSSPLITAASIGPPTTMVTGTVSPGRATN Consensus(1) TLWTT DPSPNC I DAKLTLVLTK GSIVLGTVSLLGV G ABC Sequence homology of fibers knob domain

14 3D structure of the HAdV5 fiber knob

15 BAdV-4 HAdV5 HAdV3 HAdV4 HAdV15 HAdV12 DAdV-1 HAdV40-S HAdV40-L BAdV-3 PAdV-4 HAdV35 ABCDEFGHIJ Putative secondary structure of the fibers knob domain.

16 Construction of modified genomes Homologous recombination in E.coli Swa I (1) HAdV5 WT fiber ITR Swa I (37523) CMV ITR lacZ Chimeric fiber Backbone plasmid Shuttle plasmids transgene ClaIPacI

17 Production of recombinant vectors without hCAR interaction. ITR lacZ ITR modified fiber gene transfection amplification wt fiber HEK-293 wt fiber Virus with modified fiber Modified fiber HEK-293

18 HEK-293 cells expressing HAdV2 fiber - Modification of the sequence around AUG - 5’ UTR: TPL exons 1/2/3 HEK non modified : GCC AUG AAA - consensus : GCC AUG GNG (Kozak) - modified : ACC AUG GCG AAA in vitro transcription/translation G418 selection northern blot western blot HEK E pC3Fb modified ATG TPL exons - 1/2/3 1/2/3 2/3 2/3 1/2/3(wt) 62 kDa

19 6 LSIATKGP ITVSDGK 7 LALQTSAP LSGSDSDT FAdV-1-L GK 21 LEVNTDP SGPLTSGNNG 22 LSLAAVTP LAVSSAG DAdV IVLNLQDP LGVIDGK 5 LGVKLGPG VHINGEGA DAdV LGLTEPAKP LKLADGK 14 LGVNVGLG LAVSNGS FAdV-1-S GK 9 VAAFTSGT IGLSSPTGN 10 FVSSSNNP FNGSYF L HAdV2/BAdV-4 HAdV2 HAdV2/FAdV-1-L HAdV2/FAdV-1-S HAdV2/DAdV-1-1/2 7 LVITSPELP LSTTPEGK 8 LFIQLSNCF ATDNGK Chimeric fiber proteins K L AAR CTN AAG CTT HindIII

20 in vitro trimerization DDNN HAdV2HAdV2/BAdV-4 trimer in vivo trimerization HAdV2 HAdV2/FAdV-1-L HAdV2/FAdV-1-S HAdV2/DAdV-1-1 HAdV2/DAdV-1-2 HAdV2/BAdV-4 trimer Chimeric fibers trimerization MluIHindIIISalI pFIB/HAdV2/XXX PacI NotIXhoI HAdV5 Fiber lITRrITR SwaI BstBI SwaI AscI p5Shut/HAdV2/XXX pRECad KpnI PacI lacZ Viral genome with chimeric fiber gene Homologous recombination in E. coli Top10F’

21 Production of recombinant vectors with chimeric fibers. ITR lacZ ITR Chimeric fiber gene transfection amplification wt fiber HEK E wt fiber HEK-293 Virus with modified fiber Modified fiber HAdV2-ß-Gal HAdV2/DAdV-1-1 HAdV2/DAdV-1-2 HAdV2/BAdV-4 62kDa Patent N° B0151WO HAdV2/XXX : 2x10 12 PP/ml

22 Vectors with knob domain of L- or S- FAdV-1 fiber In vitro trimerization analysis A PP/cell HAdV2 FAdV-1-L % positive cells HAdV2/FAdV-1-S HAdV2/FAdV-1-L No virus HAdV2 FAdV-1-S

23 Tropism of the HAdV2/DAdV-1 and HAdV2/BAdV-4 vectors HAdV2/DAdV-1-2 HAdV2/DAdV-1-1 Shaft repeats HAdV2/BAdV-4 is the best prototype

24 Tropism of the HAdV2/BAdV-4 vector HepG HAdV2 HAdV2/BAdV-4 RFI X 10 3 PP/cell % positive cells HEK 0 40 % positive cells Daudi Jurkat PP/cell Relative Fluorescence Intensity Cell Number HAdV HeLa cells PP (10 3 ) PP (10 3 ) HAdV2/BAdV-4

25 Reduction in cell binding of the HAdV2/BAdV-4 vector %IF Infection (ß-galactosidase) HAdV2 HAdV2/BAdV-4 Uptake HAdV2 HAdV2/BAdV-4 A549 cells

26 hCAR receptor and HAdV2/BAdV-4 infection no competition between HAdV2 fiber and HAdV2/BAdV-4 vector. hCAR independent infection of CHO cells. CHO cells CHO-hCAR cells % positive cells x 10 3 PP/cell Chinese Hamster Ovaries: hamster and/or ovarian cell receptor(s) ? HAdV2 HAdV2/BAdV-4

27 Ovarian cell tropism of HAdV2/BAdV-4 vector % positive cells SKOV3 HAdV2 HAdV2/BAdV-4 A549 cells PP/cell % positive cells 10 3 PP/cell HELACHOOVCAR3IGROV1 % positive cells SKOV3

28 B-CLL specific receptor ? EDPGFFNVE Epstein-Barr virus: Binding to CD21 receptor on B lymphocytes surface using GP 350/220 (Nemerow et al., Cell, 1989 ) (Nemerow et al., Cell, 1989 )

29 RajiDaudiHSB2Jurkat % of positive cells Isotype CD21 Daudi Jurkat Raji HSB2 [K] 7 EDPGFFNVE [K] 7 EDGGFFNVE Daudi Jurkat Targeting of CD21 on B-lymphocytes

30 LB HI loop EDPGFFNVEIPEFPF Penton base Fiber CD21 ? Genetic modifications of adenovirus fiber

31 Endosomolysis CCV 6.5 Clathrin- caoted pit endocytosis Non-modified HAdV EE LE CCV 6.5 Fiber-modified HAdVs ?? Clathrin- caoted pit endocytosis LY

32 HAdVCD21 conserved its membrane lysis ability Early endosomes ,05,56,06,57,07,5 % 3 H release pH HAd5Vfiber wt HAdV3 HAdVCD21

33 1) Membrane attachment X HAdV5-FAM HAdV-CD21HI-FAM A549HSB2RajiDaudi X5.3 X3.7 X0.3 % of positive cells 50 Targeting of CD21 on B-lymphocytes Copies of viral genome per cell A549 HeLa Jurkat HSB2 HAdV5HAdV5 CD21HI ) Infection (  -galactosidase) A549DaudiRaji % of positive cells HAdV5 HAdV-CD21HI X5.5 X10.4 X0.3 MOI : 300

34 4 Enhance positive cells  weak  -gal level Altered intracellular trafficking ? Interaction between HAdVs and hematopoïetic cells: New therapeutic ways ?? % of positive cells HAdV5  gal HAdV5-CD21HI  gal Fluorescence Intensity A549 : MOI 30 FI : 4000 Targeting of CD21 on B-lymphocytes MOI

35 ND: not determined Cell type Binding hCAR CD18 (  2 ) CD11a (  L ) Uptake HEK 293 A549HeLaRehDaudiRajiBC U266 HSB2JurkatK562 ND Epithelial cellsB cell lineages T cells  -gal

36 HEK293 A549 Reh Daudi Raji B C U266 HSB2 K562 %/HEK293 B lineage - trypsin +trypsin Attach.Upt..Upt. Eff. HEK293 K562 HSB2 U266 C B Raji Daudi Reh A Deficiency of HAdV2 uptake into B-lymphocytes

37 Deficiency of Clathrin coated pit endocytosis... HEK HeLa A549 Reh Raji DaudiJurkatK562HSB2U266 CD Clathrin 192 kDa Epithelial cellsHematopoietic cells Total cell extract Clathrin 192 kDa HEK HeLa A549 RehRaji DaudiJurkat K562 HSB2 U266 CD Epithelial cellsHematopoietic cells Membrane Fraction

38 A549 Reh DaudiRajiCDU266HSB2Jurkat Relative Fluorescence Intensity Weak Transferin uptake Control Transferin Clathrin coated pit endocytosis of transferin A549 U266 Raji

39 Is Caveolae endocytosis an alternative to clathrin coated pit endocytosis deficiency ? Associated- membrane receptors Transmembrane receptors Caveolin CAVEOLAE Lipid rafts

40 Lipid raft Caveolin-1 A549 U266 C D Daudi HSB2 Jurkat Raji A549 U266 HSB2 JurkatDaudi D C Raji

41 Top Bottom Dot-Blot anti-Caveolin-1 U266 A549

42 A- B A549HEK293HeLaU266 % positive cells mUnits  -gal/mg of protein (x10 3 )  ethyl  cyclodextrine (mM) A549 HeLa U A549HeLaHEK293 Chlorpromazine (  g/ml) Uptake (% of non treated) Chlorpromazine (  g/ml) U266 mUnits  -gal/mg of protein (x10 3 )  ethyl  cyclodextrin (mM) 20 0

43 U266 (up to 90 %)A549 Colocalization caveolin-1/ HAdV5 Is Caveolae endocytosis an alternative to clathrin coated pit endocytosis deficiency ?

44 Dominant négatif CAV-1-GFP U266 ? A549 U266

45 Non-caveolae non-clathrin endocytosis Clathrin- caoted pit endocytosis Macropinocytosis macropinosome CCV EE LE LY Golgi trans Medial Cis ER Caveosome Lysosomal degradation A549 HEK29 HeLa NLS-machinery hCAR  v  3/5 NPC proteins Central channel Peripheral channel Nuclear basket Outer membrane Inner membrane Nucleus Cytoplasm Viral DNA Transgene expression Endocytosis... ? Plasmocyte ? Caveolae

46 Conclusion: targeting vectors to B lymphocytes and ovarian cells No receptor Lost of hCAR interactionAdd new target X Ovarian cell specific receptor and reduction of hCAR interaction

47 In vitro - CD21+ Lineage CD21 targeting - Interaction hCAR - Primary B-LLC cells Other receptors ? New therapeutic ways Caveolae/phagocytose ? (Primary plasmocytes) In vivo Ex vivo - Primary B-LLC cells ex vivo assays...  doses  cytotoxicity/ immunogenicity Non-specific dissemination Biodistribution transgene HSV-tk, CD154 ? Immune response Cellularantibody Conclusion


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