WO2004031225A1 - Orthopoxvirus vectors, genes and products thereof - Google Patents
Orthopoxvirus vectors, genes and products thereof Download PDFInfo
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- WO2004031225A1 WO2004031225A1 PCT/IE2003/000131 IE0300131W WO2004031225A1 WO 2004031225 A1 WO2004031225 A1 WO 2004031225A1 IE 0300131 W IE0300131 W IE 0300131W WO 2004031225 A1 WO2004031225 A1 WO 2004031225A1
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
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- G01N2333/065—Poxviridae, e.g. avipoxvirus
Definitions
- the invention relates to a viral protein that is a novel inhibitor of intracellular signalling mediated by the immunologically important interleukin-1 / Toll-like receptor (IL-IR/TLR) superfamily.
- the invention also relates to the mechanism whereby the inhibitor functions, and the use of the inhibitor, or information derived from its mechanism of action, in designing peptides or small molecule inhibitors for use in IL-IR/TLR related diseases and conditions.
- the invention also relates to a recombinant vaccinia virus (NV) as a vaccine candidate for the prevention of smallpox or other infectious diseases, or for the prevention or treatment of cancer.
- NV recombinant vaccinia virus
- TIR Toll/IL-1 receptor
- TLR4 the first TLR to be discovered, is essential for the response to lipopolysaccharide (LPS) (Poltorak, A. et al. Science 282, 2085-2088 (1998); Qureshi, S.T. et al. J. Exp. Med. 189, 615-625 (1999)).
- LPS lipopolysaccharide
- TLR5 recognises and responds to bacterial flagellin (a 55kD monomer of bacterial flagella) (Hayashi, F. et al. Nature 410, 1099-1103 (2001)), while TLR9 is required for the recognition of unmethylated CpG motifs which are present in bacterial DNA (Hemmi, H. et al.
- TLR2 recognises diverse bacteria and their products, including bacterial lipoproteins, peptidoglycan and other Gram-positive molecular patterns, but only when present as a heterodimer in combination with another TLR, such as TLR1 or TLR6 (Brightbill, H.D. et al. Science 285, 732-736 (1999); Aliprantis, A. et al. Science 285, 736-739 (1999);
- TLRs have also been implicated in sensing viral infections.
- TLR4 has been shown to be necessary for the cytokine-stimulating ability of F protein from respiratory syncytial virus (RSN) and also for murine retrovirus activation of B cells (Kurt- Jones, E. A. et al. Nature Immunol. 1, 398-401 (2000); Rassa, J.C. et al. Proc. Natl. Acad. Sci. USA 99, 2281-2286 (2002)).
- TLR3 meanwhile was identified as a receptor activated in response to poly(I:C), a synthetic double-stranded R ⁇ A
- dsR ⁇ A mimic of viral dsR ⁇ A.
- Poly(I:C) activation of cells via TLR3 led to the activation of the transcription factor ⁇ F ⁇ B and the production of type I interferons, which are important in anti-viral innate immunity (Alexopoulou, L. et al. Nature 413, 696-712 (2001)).
- ⁇ F ⁇ B is a homo- or hetero- dimer of members of the Rel family of transcriptional activators that is involved in the inducible expression of a wide variety of important cellular genes.
- the activation of ⁇ F ⁇ B by IL-1, IL-18, TLR2, TLR7 and TLR9 is absolutely dependent on the cytoplasmic TIR domain- containing protein MyD88 (Hemmi, H. et al. Nature Immunol. 3, 196-200 (2002);
- TLR4 activates NF ⁇ B, by a MyD88-dependent pathway, although an alternative MyD88-independent pathway also exists (Kawai, T. et al. Immunity 11, 115-122 (1999)).
- MyD88 is a crucial adaptor molecule for the entire ILIR/TLR superfamily, with the exception of TLR3, where NF ⁇ B activation is MyD88-independent (Alexopoulou, L. et al. Nature 413, 696-712 (2001)).
- the MyD88 dependent pathway is involved in TNF induction by LPS in dendritic cells whereas the MyD88 independent pathway leads to the upregulation of costimulatory molecules required for dendritic cell maturation, and induction of genes dependent on the transcription factor Interferon Regulatory Factor 3 (IRF3)
- TIR Interferon-
- MyD88Adaptor-Like another TIR adapter molecule, MyD88Adaptor-Like (Mai, also known as TIRAP) is involved in the MyD88 dependent pathway (Fitzgerald, K. A. et al. Nature 413, 78-83 (2001); Horng, T., Barton, G. M. & Medzhitov, R. Nature Immunol. 2, 835-841 (2001);
- Activation of NF ⁇ B by the MyD88 dependent pathway can proceed via recruitment by MyD88 of IL-1 receptor-associated kinase (IRAK) and/or IRAK2, while Mai functions via the recruitment of IRAK2 (Fitzgerald, K. A. et al. Nature 413, 78-83 (2001)).
- IRAK or IRAK2 activation in turn leads to recruitment of tumour necrosis factor receptor-associated factor 6 (TRAF6).
- TRAF6 is required for the ubiquitination and activation of the kinase TAK-1, which, in complex with TAB1, phosphorylates I ⁇ B kinase (IKK) leading to NF ⁇ B activation (Wang, C. et al. Nature 412, 346-351 (2001)).
- MAP kinases can be triggered through MyD88 (shown for IL-18, TLR2, TLR9), but also, in the case of LPS and poly (I: C), through MyD88-independent pathways (Adachi, O.
- IRF3 Another important transcription factor activated by some TLR3 and TLR4 is IRF3, which has importance in IFN-dependent anti-viral defense (Servant, M.J.,
- TICAM-1 TIR adapter
- an orthopoxvirus vector such as vaccinia, wherein the A46R protein from vaccinia, or a closely related protein from any orthopoxvirus is not expressed or is expressed but is non-functional.
- part or all of the nucleotide sequence encoding A46R is deleted from the viral genome.
- nucleotide sequence encoding A46R is inactivated by mutation or the insertion of foreign DNA.
- the nucleotide sequence encoding A46R may be changed.
- the A46R gene comprises amino acid SEQ ID NO: 1
- the vector comprises DNA sequences encoding one or more heterologous polypeptides.
- the orthopoxvirus vector of the invention has enhanced immunogenicity and/or safety compared to the wild type orthopoxvirus.
- the invention also provides a medicament comprising an orthopoxvirus vector of the invention.
- the invention provides a vaccine comprising an orthopoxvirus vector of the invention.
- the invention provides a recombinant orthopoxvirus incapable of expressing a native A46R protein.
- a vaccine may comprise such a recombinant virus.
- the invention provides a method of attenuating an orthopoxvirus vector such as vaccinia virus, comprising the steps of:
- the invention provides a method of inhibiting ILIR/TLR superfamily signalling comprising administering an effective amount of vaccinia A46R protein, or a closely related protein from any orthopoxvirus or a functional peptide, peptidometic, fragment or derivative thereof, or a DNA vector capable of expressing such a protein or fragment thereof.
- the invention provides a method of modulating anti-viral immunity in a host comprising administering a vaccinia virus vector of the invention or a functional peptide, peptidometic, fragment or derivative thereof.
- the invention also provides an immunogen comprising a vaccinia virus vector or a recombinant virus vector of the invention.
- the invention provides use of a vaccinia virus A46R protein, or a closely related protein from any orthopoxvirus, or a functional peptide, peptidometic, fragment or derivative thereof, or a DNA vector expressing any of the above in the modulation and/or inhibition of IL-IR/TLR superfamily-signalling.
- the use may be in the modulation and/or inhibition of IL-IR/TLR superfamily- induced NF ⁇ B activation.
- the use may be in the modulation and/or inhibition of IL-IR/TLR superfamily- induced MAP kinase activation.
- the use may be in the modulation and/or inhibition of TLR induced IRF3 activation.
- the vaccinia virus A46R protein or a closely related protein from any orthopoxvirus, inhibits Toll-like receptor proteins.
- the use may be in the modulation and/or inhibition of NF- ⁇ B activity or MAP kinase activation by interaction of A46R with Mai.
- the vaccinia virus A46R protein inhibits MyD88- and/or Mal- dependent signalling.
- the use may be in the modulation and/or inhibition of IRF3 or NF- ⁇ B activity by interaction of A46R with TRIF.
- the vaccinia virus A46R protein inhibits TRIF-dependent signalling.
- the invention also provides a peptide derived from, and/or a small molecule inhibitor designed based on vaccinia virus A46R protein.
- the invention further provides a method of screening compounds that modulate the IL-lR/TLR-induced NF- ⁇ B or MAP kinase pathway comprising measuring the effect of a test compound on the interaction of vaccinia virus A46R protein or a functional peptide, peptidometic, fragment or derivative thereof with MyD88.
- the invention also provides a method of screening compounds that modulate the IL- lR/TLR-induced NF- ⁇ B or MAP kinase pathway comprising measuring the effect of a test compound on the interaction of vaccinia virus A46R protein or a functional peptide, peptidometic, fragment or derivative thereof with Mai.
- the invention further provides a method of screening compounds that modulate the IL-lR/TLR-induced NF- ⁇ B, IRF3 or MAP kinase pathway comprising measuring the effect of a test compound on the interaction of vaccinia virus A46R protein or a functional peptide, peptidometic, fragment or derivative thereof with TRIF.
- the invention also provides a method of identifying signalling pathways that require MyD88 and/or Mai and/or TRIF, comprising measuring their sensitivity to A46R.
- the invention provides use of a functional peptide, peptidometic, or fragment derived from vaccinia virus A46R protein, or a small molecule inhibitor designed based on A46R protein or a DNA vector capable of expressing such a protein or fragment in the treatment and/or prophylaxis of IL-IR/TLR superfamily- induced NF- ⁇ B, IRF3 or MAP kinase related diseases or conditions.
- the NF- ⁇ B related disease or condition is selected from any one or more of a chronic inflammatory disease, allograft rejection, tissue damage during insult and injury, septic shock and cardiac inflammation, autoimmune disease, cystic fibrosis or any disease involving the blocking of Thl responses.
- the chronic inflammatory disease may include any one or more of rheumatoid arthritis, asthma or inflammatory bowel disease.
- the autoimmune disease may include systemic lupus erythematosus. The use may be in the treatment and/or prophylaxis of inflammatory disease, infectious disease or cancer.
- One aspect of the invention also provides use of a viral protein derived from an A46R-like protein or a functional peptide, gene, or peptidometic thereof in the treatment and/or prophylaxis of inflammatory disease.
- the A46R-like protein may be derived from an orthopoxvirus.
- peptide, peptidometic, fragment or derivative as used herein are understood to include any molecule or macromolecule consisting of a portion of the A46R protein, or designed using sequence or structural information from A46R.
- non-functional is understood to mean not functioning in the normal way compared to how the wild-type A52R protein would function.
- the invention is in the field of poxviruses.
- the family name is poxvirus
- the subfamily name is chordopoxvirinae (infect vertebrates)
- the genus is orthopoxvirus which includes species of virus some of which have A46R related proteins.
- the best known species of this genus are vaccinia, variola, camelpox, cowpox, monkeypox and ectromelia (which infects mice).
- the invention relates to any orthopoxvirus vector in which the A46R protein is deleted/modified.
- the invention further relates to the use of a DNA vector expressing A46R protein. Brief description of the drawings
- Fig. 1 is a sequence alignment showing the sequence similarity between A46R and TIR domains from IL-IR/TLR signalling proteins
- Figs. 2a to d are graphs showing the inhibition by A46R of the activation of
- Figs. 3a to d are graphs showing the inhibition by A46R of the activation of NF ⁇ B and MAP kinases by TLR4 in human 293 cells;
- Fig. 4 is a graph showing the inhibition by A46R of the activation of NF ⁇ B by
- TLR4 TLR4
- R-848 TLR7/8
- flagellin TLR5
- Fig. 5 is an immunoblot showing the ectopic expression of A46R in 293T cells
- Figs. 6a and b are immuno-blots showing association of A46R with MyD88, by co-immunoprecipitation and GST pull-down.
- Figs. 7a and b are immuno-blots showing association of A46R with Mai, by co-immunoprecipitation and GST pull-down.
- Figs. 8a to e are graphs showing the inhibition by A46R of the activation of
- Figs. 9a and b are bar graphs showing the inhibition by A46R of TLR3- induced Interferon-Stimulated Response Element (ISRE) and Interferon- ⁇ (IFN ⁇ ) promoter;
- ISRE Interferon-Stimulated Response Element
- IFN ⁇ Interferon- ⁇
- Figs. 10a and b are immuno-blots showing association of A46R with TRIF, by co-immunoprecipitation and GST pull-down.
- Figs. Ila and b are graphs showing the inhibition by A46R of the activation of NF ⁇ B and IFN ⁇ promoter by TRIF;
- Figs. 12 a to c are graphs showing the inhibition by A46R of TLR4-mediated TRIF-dependent pathways.
- Fig 12c also shows inhibition of ISRE by the TLR activators LPS, poly(I:C), R-848 and flagellin;
- Figs. 13a and b are immuno-blots showing the phase during infection at which the A46R protein is expressed, and lack of expression of A46R in cells infected with the VV deletion mutant lacking the A46R gene;
- Figs. 13c and d are graphs showing that deletion of A46R does not affect the growth or replication of VV in cell culture.
- Fig. 14 is a graph showing that deletion of A46R from the vaccinia virus genome attenuates the virus, as measured by weight loss in a murine intranasal model of infection.
- Poxviruses are a family of complex DNA viruses that include variola virus, the causative agent of smallpox, and the antigenically related virus used to eradicate this disease, vaccinia virus (VV).
- Orthopoxvirus such as VV display unique strategies for the evasion of host immune responses such as the ability to produce secreted decoy receptors for cytokines such as IL-1, TNF, and the interferons IFN ⁇ and IFN ⁇ . Often these inhibitors of host immune function display sequence similarity to host proteins.
- the present invention concerns a VV protein A46R, which is known to be an intracellular inhibitor of signalling via IL-1 (Bowie, A. et al. Proc. Natl Acad. Sci. USA 97, 10162-10167 (2000)).
- PROFILESEARCH Genetics Computer Group, Madison, WI
- A46R was based on the standard VV nomenclature of the Copenhagen strain (Goebel, S. J. et al. Virology 179, 247-266 (1990)). A46R was cloned from the laboratory VV strain Western Reserve (WR), where it was previously called SalF9R (Smith, G. L., Chan, Y. S. & Howard, S. T. J. Gen. Virol. 72, 1349-1376 (1991)).
- A46R displays a high degree of sequence conservation between different strains of VV, including WR, Copenhagen, Modified Virus Ankara and Tian Tian, while many other orthopoxviruses also have a closely related version of A46R, namely variola major, variola minor, camelpox, monkeypox and cowpox. This high degree of conservation could reflect an important role for A46R in viral virulence.
- Fig. 1 shows an alignment of A46R with other TIR domains from TLRs.
- Box 1, 2 and 3 regions of important sequence conservation, which have been termed Box 1, 2 and 3 (Bowie, A. & O'Neill, L.A.J. J. Leuk. Biol. 61, 508-514 (2000)).
- Box 1 is particularly strong in A46R, the sequence DTFISY being as closely related to the Box 1 consensus of other proven family members.
- TIR domains for TLR1 and TLR2 have been determined and show that the domain adopts a three-layer ⁇ sandwich conformation, similar to the bacterial protein CheY. Threading the A46R amino acid sequence through secondary structure prediction programmes revealed that A46R could also fold in such a manner. Therefore the A46R protein does appear to contain a bona fide TIR domain.
- A46R inhibits numerous signalling pathways activated by IL-1, including c-Jun N terminal kinase (JNK) and extracellular-regulated kinase (ERK) MAP kinase activation (Fig. 2a to d). Further, A46R is also shown to inhibit NF ⁇ B activation by multiple TLRs including TLR4 (Fig. 3a), TLR7/8 (Fig. 4) and TLR5 (Fig. 4). The fact that A46R could associate with both MyD88 (Fig. 6) and Mai (Fig. 7) provides a rationale for these inhibitory effects.
- A46R was able to block MyD88-independent pathways (Figs 10 and 13) by associating with TRIF (Fig. 11).
- A46R was shown to be expressed early on in cells infected with VV (Fig. 14).
- a deletion mutant VV lacking the A46R gene was shown to be attenuated compared to wild type and revertant controls in vivo (Fig. 15), indicating the importance of A46R in viral virulence.
- IL-IR/TLR IL-IR/TLR
- IL-1 is antagonized by the production of a soluble IL-1 binding protein (B15R) by VV (Alcami, A. & Smith, G.L. Cell 71, 153-167 (1992).
- IL-18 is a potent inducer of IFN- , and administration of IL-18 has been shown to elicit antiviral effects in VV- infected mice (Tanaka-Kataoka, M. et al. Cytokine 11, 593-599 (1999)).
- TLR3, TLR4 and TLR7 are crucial mediators of an innate immune response to viral infection (Kurt- Jones, E. A. et al Nature Immunol. 1, 398- 401 (2000); Rassa, J.C., Meyers, J.L., Zhang, Y., Kudaravalli, R. & Ross, S. Proc. Natl. Acad. Sci. USA 99, 2281-2286 (2002), Alexopoulou, L., Czopik-Holt, A.,
- TLR2 and TLR9 have also been implicated in responding to some viruses (Lund, J. et al J. Exp. Med. 198, 513-520 (2003); Compton, T. et al J. Virol. 11, 4588-4596 (2003). It is possible that other TLRs also have a role in responding to viral infection.
- the TLR family is therefore important in anti-viral host defense. Viral mechanisms to antagonise this family would have valuable therapeutic potential.
- VV protein A46R has been found to be an IL-IR/TLR inhibitor. Deletion of A46R from VV causes the virus to be attenuated in a murine model of infection (Fig. 15). These results further support the emerging role of
- TLRs in the host response to viral infection are TLRs in the host response to viral infection.
- A46R is very similar to the TIR domain and has wide-ranging effects on IL-IR/TLR signalling.
- the inhibitory data suggests that A46R blocks IL-IR/TLR signalling close to the receptors, before the NF ⁇ B and MAP kinase pathways bifurcate, probably by disrupting TIR-dependent interactions necessary for signalling.
- One important target for A46R is likely to be MyD88, since A46R can be co- immunoprecipitated with MyD88 (Fig. 6a), while GST-A46R can pull MyD88 out of a cell lysate (Fig. 6b), probably due to an association between the A46R and MyD88 TIR domains. This is the first demonstration of a viral protein targeting MyD88.
- A46R can also target Mai (as assessed by co-immunoprecipitation and GST pull-down, (Fig. 7) is consistent with the fact that TLR4 inhibition by A46R is particularly potent (compare Fig. 3 to Fig. 2).
- FIG. 9 shows that both Interferon-Stimulated Response Element (ISRE) and Interferon- ⁇ (IFN ⁇ ) promoter induction by TLR3 was potently blocked by A46R. This indicated that A46R might also target the adapter TRIF, since this has been shown to be responsible for most if not all TLR3-dependent.
- Fig. 10 shows that A46R could co-immunoprecipitate TRIF and also that GST-A46R was capable of pulling down TRIF.
- A46R by nature of its similarity to the TIR domain, can target three key adapters involved in signalling by the IL-1 /TLR superfamily, thus allowing A46R to antagonise a huge array of signalling pathways.
- the targeting of TRIF is particularly interesting, since it has recently been shown that TRIF controls the IFN-dependent, and thus anti-viral arm, of TLR3 and TLR4 signalling.
- the present invention relates to a recombinant vaccinia virus in which the gene sequence of A46R is deleted.
- Fig. 13b confirms that the mutant virus does not express the A46R protein, while a revertant virus (in which the A46R gene is inserted into the virus deletion mutant lacking A46R) does. Deletion of A46R did not affect the replication of the virus in cell culture (Fig. 13c and d). However, the absence of A46R led to an attenuation of the virus, in that when mice were infected intranasally, the deletion mutant caused a reduction in the weight loss induced in the animals, compared to wild type and revertant virus (Fig. 14).
- Live vaccinia virus is currently used as the vaccine to immunise against and eradicate smallpox.
- Such recombinant vaccinia viruses can be engineered in which genes derived from other organisms are inserted (Macket, M. & Smith, G.L. J. Gen. Virol. 61,
- the recombinant viruses retain their infectivity and express any inserted genes during the normal replicative cycle of the virus. Immunisation of animals with recombinant viruses containing foreign genes has resulted in specific immune responses against the protein(s) expressed by the vaccinia virus, including those protein (s) expressed by the foreign gene(s) and in several cases has conferred protection against the pathogenic organism from which the foreign gene was derived. Recombinant vaccinia viruses have, therefore, potential application as new live vaccines in human or veterinary medicine.
- the present invention also relates to a vaccinia virus wherein 93.5 % of the nucleotide sequence encoding A46R is deleted. Alteration or deletion of A46R from the vaccinia genome may increase virus safety and immunogenicity. Such a virus or a derivative virus expressing one or more foreign antigens may have application as an improved vaccine against smallpox or other orthopoxvirses, or for the application of recombinant vaccinia viruses as vaccines against other infectious diseases and cancer.
- HEK 293, HEK 293T and RAW 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L- glutamine.
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- TLR4-TLR4 composed of the extracellular domain of CD4 fused to the transmembrane domain and cytosolic tail of TLR4 was a gift from R. Medzhitov, (Yale University, New Haven, CT). AU1-
- MyD88 expression vector was a gift from M. Muzio (Muzio, M., Ni, J., Feng, P. & Dixit, V.M. Science 278, 1612-1615 (1997)).
- the mammalian expression vector pRK5 was kindly provided by Tularik Inc. (San Francisco, CA).
- Flag-TRIF was from S.Akira (Research Institute for Microbial Diseases, Osaka University, Japan).
- A46R, Flag-A46R and HA-Mal expression plasmids have been previously described
- A46R is based on the standard VV nomenclature of the Copenhagen strain (Goebel, S.J et al, Virology 179, 247-266 (1990)).
- A46R was cloned from the laboratory VV strain WR where it was previously called SalF9R (Smith, G.L et al J. Gen. Virol, 72 1349-1376 (1991); Goebel, S.J et al, Virology 179, 247-266 (1990)) into the mammalian expression vector pRK5.
- Any other suitable mammalian expression vector such as pcDNA3.1 (available from Invitrogen) or pEF-BOS (Mizushima et al Nucleic Acids Res. 18, 5322 (1990)) for example may also be used.
- the A46R ORF was cloned by PCR amplification from WR DNA with primers incorporating restriction sites for EcoRI upstream and Hindl ⁇ l downstream of the ORF.
- the primers used were 5' - CGTGAATTCCGAGAA1G.GCGTTTGA (sense) and 5' -CGGAAGCTTTTATACATCCGTTTCCT (antisense).
- the restriction sites and start and stop codons are underlined.
- the resulting Eco Rl-Hindlll fragment was ligated into the multiple cloning site of the mammalian expression vector ⁇ RK5.
- an epitope-tagged A46R expression vector was constructed, employing the same strategy, except that the 8-amino acid Flag coding sequences was inserted into the antisense primer 5' of the stop codon.
- Antibodies Polyclonal antibodies were raised against a purified, bacterially expressed glutathione S-transferase (GST) fusion of A46R, encoded by a plasmid synthesised by inserting full length A46R downstream of GST in the bacterial expression vector GEX4T2.
- GST glutathione S-transferase
- Other antibodies used were anti-AUl monoclonal antibody (BabCO), anti-HA polyclonal antibody (Y-ll, Santa Cruz Biotechnology) and anti-flag M2 monoclonal antibody (Sigma)
- Luciferase reporter gene assays HEK 293 cells (2 x 10 4 cells per well) or RAW 264.7 cells (4 x 10 4 cells per well) were seeded into 96- ell plates and transfected the next day with expression vector, and reporter plasmids. GeneJuiceTM (Novagen) was used for transient transfections, according to the manufacturer's instructions.
- GeneJuiceTM Novagen
- 60 ng of ⁇ B -luciferase reporter gene, ISRE-luciferase reporter gene (Stratagene) or INF ⁇ promoter luciferase reporter (a gift from Prof. Taniguchi, University of Tokyo) respectively were used as previously described (Fitzgerald, K. A. et al. Nature 413, 78-83
- MAP kinase reporter assays the Stratagene Pathdetect SystemTM was used whereby c-jun (2 ng) or Elkl (5 ng) Gal4 fusion vectors were used in combination with 80 ng pFR-luciferase reporter to measure JNK and ERK activation respectively (Fitzgerald, K. A. et al. Nature 413, 78-83 (2001)).
- p65 or IRF3 transactivation assay a p65-Gal4 or IRF3-Gal4 fusion vector was used in combination with pFR-luciferase reporter (Jefferies, C, et al. Mol. Cell. Biol. 21, 4544-4552 (2001)).
- HEK 293T cells were seeded into 100 mm dishes (1.5 x 10 6 ) 24 hrs prior to transfection. Transfections were carried out using GeneJuiceTM (Novagen) according to manufacturers instructions. Four g of each construct was transfected. Where only one construct was expressed the total amount of DNA (8 g) was kept constant by supplementation with vector DNA. Cells were harvested 24 hrs post transfection in 750 1 o f lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 containing
- Lysates prepared as described above were incubated for two hours with GST-A46R, washed three times as above, and subjected to SDS-PAGE and immunoblotting. Plaque Assays. Aliquots from vaccinia virus stocks (WR strain) were frozen and thawed 3 times, sonicated and serial dilutions were made in 2.5% FBS DMEM. Three of these dilutions were inoculated in duplicate onto confluent monolayers of BSC-1 cells in 6-well plates (0.5 ml of each dilution per well).
- CMC carboxymethylcellulose
- Example 1 A46R inhibition of multiple signals induced by the IL-IR TLR superfamily
- A46R inhibits multiple IL-1-dependent signals A46R has been shown to block IL-1 induced NFkB activation, while not affecting
- A46R inhibits multiple TLR4-dependent signals
- TLR4 has a putative TIR domain we determined whether other TIR-dependent signals were sensitive to inhibition by examining the TLR4 pathway.
- Chimeric versions of the TLRs comprising the murine CD4 extracellular domain fused to the cytoplasmic domain of a given human TLR have proved useful in probing TLR signalling pathways (Hayashi, F. et al. Nature 410, 1099-1103 (2001); Ozinsky, A. et al Proc. Natl Acad. Sci. USA 97, 13766-13771 (2000); Medzhitov, R., Preston- Hurlburt, P. & Janeway, C. A. Jr. Nature 388, 394-397 (1997)).
- the extracellular domain of CD4 promotes homodimerisation of the molecules.
- Chimeras composed of the extracellular domain of CD4 fused to the intracellular domain of TLR4 are constitutively active, in that overexpression of CD4-TLR4 induces NF B activation and gene induction (Medzhitov, R., Preston-Hurlburt, P. & Janeway, C. A. Jr. Nature 388, 394-397 (1997)).
- HEK 293 cells (2 x 10 4 cells per well) were transfected with a Renilla-luciferase internal control and either ⁇ B- luciferase construct (Fig. 3a), or plasmids encoding Gal4 fused to p65 (for p65, (b)),
- NF ⁇ B activation was measured as shown in Fig. 3a.
- A46R was found to block other signals induced by CD4-TLR4.
- a single dose of A46R cDNA transfected into cells was capable of blocking the ability of the transactivating subunit of NF ⁇ B, p65, to activate a reporter gene (Fig. 3b), ERK activation (Fig. 3c), and p38 activation (Fig. 3d), respectively.
- Murine macrophage RAW264.7 cells were transfected with an NK ⁇ B luciferase construct and a Renilla-luciferase internal control, together with empty vector (EV) or 100 ng cDNA encoding A46R.
- Cells were stimulated for 6 hours with the TLR agonists 1 M R-848 (TLR7), 1000 ng/ml LPS (TLR4) or 250 ng/ l flagellin (TLR5) before harvesting. 24h after transfection cells were harvested, and the reporter gene activity was measured. Data are expressed as mean fold induction + s.d. relative to control levels, single experiment, performed in triplicate.
- A46R inhibited NK ⁇ B activation induced by R-848, LPS, and flagellin.
- TLR4 (LPS) activation was again particularly sensitive to A46R inhibition.
- R-848 induced NK ⁇ B activation was also very strongly inhibited by A46R.
- A46R is capable of blocking multiple signals emanating from the IL-1R and TLRs.
- the fact that A46R has a putative TIR domain suggests that it might block these TIR-dependent pathways by disrupting the TIR receptor interactions necessary for signalling. Thus A46R probably acts close to the receptors, before signal bifurcation.
- Example 2 - A46R associates with MyD88 and Mai, and can block signals induced by MyD88 and Mai overexpression
- NF ⁇ B The activation of NF ⁇ B by IL-IR/TLR family members is mediated by a common set of signalling molecules.
- MyD88 was a likely target, given its role in these pathways, and the fact that it has a TIR domain. Therefore the ability of A46R to interact with MyD88 was examined.
- HEK 293T cells were seeded at lxlO 5 cells/ml in 6 well plates 24 h prior to transfection. Transfections were carried out using GeneJuiceTM (Novagen) according to manufacturers instructions. Increasing amounts of a plasmid vector encoding A46R was transfected, as indicated in Fig. 5. The total amount of DNA (2 g) w as kept constant by supplementation with vector DNA. Cells were harvested 24 h post transfection and resolved by SDS-PAGE. The blot was probed with an antibody specific for A46R and Fig. 5 shows that a clear dose-dependent pattern of expression was observed, at the correct molecular mass for A46R (near 26kD). .
- HEK 293T cells were transfected with plasmids encoding A46R and AUl-MyD88, and co- immunoprecipitation performed, as described above.
- the results are shown in Fig. 6a, where lanes 1-3 correspond to lysates directly blotted for expression of MyD88, lanes 4-6 correspond to lysates immunoprecipitated with anti-A46R antibody and blotted for the presence of MyD88, while lanes 7-9 correspond to immunoprecipitation using anti-AUl antibody directed towards AUl-MyD88.
- the amounts of cDNA used were 2 ng Mai or 5 ng MyD88 for NF B reporter gene assay (a), 50 ng MyD88 or Mai for p65 transactivation assay (b), 25 ng MyD88 or Mai for JNK assay (d), and 10 ng MyD88 or 25 ng Mai for ERK (c) and p38 (e) assays. These amounts of cDNA were determined as being optimal for activation of the different reporters in previous experiments. Consistent with the targeting of MyD88 and Mai by A46R, both MyD88- and Mal- mediated NF B , p65 and ERK signals are inhibited very potently by A46R (Fig. 8a to c). However, A46R inhibits MyD88 mediated JNK and p38 signals only slightly, when compared to the very strong inhibition of Mai mediated JNK and p38 signals by A46R (Fig. ⁇ d and e).
- A46R can inhibit signals emanating from these adaptors.
- Example 3 - A46R blocks MyD88-independent pathways by associating with TRIF.
- A46R was also capable of antagonising TLR signalling pathways known to be independent of MyD88 and Mai.
- One important example of such a pathway is the activation of ISRE promoter elements and subsequent induction of IFN ⁇ by poly(I:C) via TLR3.
- HEK 293 cells (2 x 10 4 cells per well) were transfected with 0.5 ng TLR3 in the presence or absence of A46R, with an ISRE-luciferase construct (Fig. 9a), or an IFN ⁇ promoter reporter plasmid (b) and Renilla-luciferase internal control.
- Fig. 9 shows that A46R potently blocked both ISRE (a) and IFN ⁇ promoter (b) activation, suggesting that A46R can also target the MyD88-independent, IRF3-dependent pathway. This pathway has recently been shown to be controlled by the TIR adapter TRIF (Hoebe, K. et al Nature doi:10.1038/nature01889 (2003); Yamamoto, M.
- FIG. 10 shows that A46R was indeed able to associate with TRIF in both assays (Fig. 10a, lane 6 and Fig. 10b, lane 3).
- A46R can target TRIF-dependent signalling by association with this TIR adaptor. This was confirmed in other experiments where activation of either NF ⁇ B or induction of the IFN ⁇ promoter by ectopic expression of TRIF was blocked by A46R (Fig. Ila and b respectively). Furthermore, TLR4-induced ISRE or IRF3, which are also dependent on TRIF, were also potently inhibited (Fig. 12a and b). Finally, Fig. 12c shows that TLR ligand induced ISRE induction in murine macrophages by LPS, and other TLR agonists, is also sensitive to A46R inhibition.
- Example 4 Comparison of a W deletion mutant lacking A46R gene with wild type and revertant viruses.
- a mutant virus lacking the A46R gene was constructed in order to determine what contribution A46R might make to VV virulence.
- A46R gene was re-inserted into the mutant, was also constructed.
- BSC-1 cells were mock infected or infected with VV WR at 10 pfu/cell in the absence or presence of 40 mg/ml cytosine arabinoside (an inhibitor of DNA synthesis). At various times p.i., cells were harvested, and extracts were prepared, separated by SDS-PAGE and analysed by immunoblotting. Blots were detected with anti-A46R antibody (diluted 1:1000) and anti-D8L antibody (diluted 1:1000). D8L is known to be expressed late during VV infection. Bound IgG was detected with HRP-conjugates goat anti-rabbit IgG antibody (diluted 1:2500) and ECL reagents (Amersham) and blots were exposed to X-OMAT film.
- Fig 13a shows that in comparison to D8L, which was only detected at 24 h p.i.,
- A46R could be seen after just 6 h. The level of protein continued to accumulate up to 24 h. This profile of expression of A46R in infected cells shows that the protein is expressed quite early in infection, and rapidly accumulates, compared to D8L, a known late expressed VV protein. This is consistent with a role for A46R in suppressing IL-IR/TLR signalling in infected cells, since these receptors are generally involved in the triggering of host immune responses in the early phases of infection.
- A46R The role of A46R in the VV life cycle was investigated by the construction of a deletion mutant lacking the A46R gene and by the comparison with wild type and revertant controls.
- a VV mutant lacking 93.5 % of the A46R gene was constructed by transient dominant selection (Falkner, F.G. & Moss, B. (1991) J. Virol. 64, 3108-3111).
- a plaque purified wild type virus vWT-A46R
- vA46R-RV a revertant virus in which the A46R gene was reinserted at its natural locus were also isolated.
- Fig. 13c is a single step growth analysis of recombinant viruses.
- BSC-1 cells were infected with the indicated viruses at 10 pfu/cell. After 24 h, the virus present in the clarified supematants and cells were determined by plaque assay in duplicate. Results show the mean of duplicate experiments. As can be seen, there is no difference between the viral titre for vWT-A46R, vA4 6 R and vA46R- RV.
- Fig. 13d shows multi-step growth curves for the recombinant viruses. BSC-1 cells were infected with the indicated viruses at 0.01 pfu/cell.
- virus was harvested by scraping cells into the culture supematants. Samples were frozen and thawed 3 times and sonicated. Total virus levels were determined by plaque assay on BSC-1 cells. Results show the mean of duplicate experiments. vWT-A46R, v A 46R and vA46R-RV display the same growth characteristics.
- A46R therefore does not affect the growth or replication of VV in cell culture.
- mice The virulence of the virus was examined in a mouse intranasal model.
- Groups of five female, 6- eek old Balb/c mice were anaesthetized and inoculated with 10 pfu of VV in 20 ⁇ l of phosphate-buffered saline (PBS).
- PBS phosphate-buffered saline
- a control group was mock infected with PBS.
- Each day the weights of the animals was measured as described previously (Alcami, A. & Smith, G.L. (1992) Cell 71, 153-167).
- Data are presented as the mean weight of each group of animals compared to the mean weight of the same group on day 0.
- the deletion mutant caused reduced weight loss in mice, compared to wild-type and revertant viruses.
- the A46R protein contributes to virus virulence and this is likely to be due to the inhibition of IL-IR/TLR signalling.
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AU2003273512A AU2003273512A1 (en) | 2002-10-01 | 2003-09-29 | Orthopoxvirus vectors, genes and products thereof |
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WO2006035066A2 (en) * | 2004-09-29 | 2006-04-06 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Toll-like receptor 3, its signalling associated molecule trif and their use in the prevention and treatment of host inflammation response induced by a rna virus |
EP1755670A2 (en) * | 2004-05-28 | 2007-02-28 | Cedars-Sinai Medical Center | Methods for treating vascular disease |
WO2007107770A1 (en) * | 2006-03-22 | 2007-09-27 | Imperial Innovations Limited | Anti-inflammatory proteins and improved vaccines |
WO2010055500A1 (en) * | 2008-11-17 | 2010-05-20 | The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin | A vaccinia virus protein a46 peptide and use thereof |
US8546324B2 (en) | 2008-09-22 | 2013-10-01 | Cedars-Sinai Medical Center | Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling |
US9512196B2 (en) | 2008-09-22 | 2016-12-06 | Cedars-Sinai Medical Center | Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling |
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Non-Patent Citations (6)
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BOWIE ANDREW ET AL: "A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 97, no. 18, 29 August 2000 (2000-08-29), pages 10162 - 10167, XP002264055, ISSN: 0027-8424 * |
HAGA ISMAR R ET AL: "In vitro and in vivo studies of A46R and A52R, two vaccinia virus proteins involved in inhibition of IL-1 signaling.", JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, vol. 22, no. SUPPLEMENT 1, 2002, & JOINT MEETING OF THE INTERNATIONAL SOCIETY FOR INTERFERON AND CYTOKINE RESEARCH, THE INTERNATIONAL C; TURIN, ITALY; OCTOBER 06-10, 2002, pages S - 112, XP009023785, ISSN: 1079-9907 * |
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YAMAMOTO MASAHIRO ET AL: "Cutting edge: A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling.", JOURNAL OF IMMUNOLOGY, vol. 169, no. 12, 15 December 2002 (2002-12-15), &, pages 6668 - 6672, XP002269637, ISSN: 0022-1767 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1755670A2 (en) * | 2004-05-28 | 2007-02-28 | Cedars-Sinai Medical Center | Methods for treating vascular disease |
EP1755670A4 (en) * | 2004-05-28 | 2008-07-02 | Cedars Sinai Medical Center | Methods for treating vascular disease |
US7959918B2 (en) | 2004-05-28 | 2011-06-14 | Cedars-Sinai Medical Center | Methods for treating vascular disease |
WO2006035066A2 (en) * | 2004-09-29 | 2006-04-06 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Toll-like receptor 3, its signalling associated molecule trif and their use in the prevention and treatment of host inflammation response induced by a rna virus |
WO2006035066A3 (en) * | 2004-09-29 | 2006-09-21 | Inst Nat Sante Rech Med | Toll-like receptor 3, its signalling associated molecule trif and their use in the prevention and treatment of host inflammation response induced by a rna virus |
WO2007107770A1 (en) * | 2006-03-22 | 2007-09-27 | Imperial Innovations Limited | Anti-inflammatory proteins and improved vaccines |
US8546324B2 (en) | 2008-09-22 | 2013-10-01 | Cedars-Sinai Medical Center | Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling |
US9512196B2 (en) | 2008-09-22 | 2016-12-06 | Cedars-Sinai Medical Center | Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling |
WO2010055500A1 (en) * | 2008-11-17 | 2010-05-20 | The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin | A vaccinia virus protein a46 peptide and use thereof |
US8722052B2 (en) | 2008-11-17 | 2014-05-13 | The Provost, Fellows, Foundation Scholars, And The Other Members Of Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin | Vaccinia virus protein A46 peptide and use thereof |
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