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  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications

Definitions

• the invention relates to the field of immunology, in particular it relates to infection of cells with HPV and to methods to eliminate infected cells from an immune-competent host.
• HPV infections are now recognized as the major cause of cervical cancer. In 2007, it was estimated that 11,000 women in the United States would be diagnosed with this type of cancer and nearly 4,000 would die from it. Cervical cancer strikes nearly half a million women each year worldwide, claiming a quarter of a million lives. Moreover several studies suggest that HPVs may also play a role in some cancers of the anus, vulva, vagina, and penile cancer (cancer of the penis).
• oral HPV infection is a strong risk factor for oropharyngeal cancer (cancer that forms in tissues of the oropharynx, which is the middle part of the throat and includes the soft palate, the base of the tongue, and the tonsils).
• An oral HPV infection and past HPV exposure increase the risk of oropharyngeal squamous cell cancer, independent of the two other important risk factors for this disease, tobacco and alcohol use.
• HPV types that are more likely to lead to the development of cancer are referred to as “high-risk” (hrHPV).
• high-risk HPVs include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73. These high-risk types of HPV cause uncontrolled growth of the epithelia that are usually flat and nearly invisible, as compared to the external lesions caused by low-risk types HPV- 6 and HPV-Il.
• HPV types 16 and 18 together cause about 70 percent of all cervical cancers. It is important to note, however, that the great majority of high-risk HPV infections are cleared by the hosts immune system and do not cause cancer. To date it is still unknown how these viruses evade immunity in order to persist in an immune-competent host.
• HPV high risk HPV
• hrHPV high risk HPV
• 1-3 current preventive vaccines
• HPV is the most common sexually transmitted disease
• a vast number of individuals are already infected and as such can not profit from this preventive vaccination.
• Alternative strategies to combat HPV infection are, therefore, most welcome.
• the present invention provides for such an alternative combat strategy since it shows a new mechanism through which viruses, in general, can evade immunity and provides methods and means for counteracting the evasion.
• CD8+ T-cells fail to migrate into the lesions of many patients during the later stages of the HPV-specific disease (15, 16), whereas, in other cases HPVl 6- specific regulatory T-cells are induced which co-migrate into the lesions and suppress immune effector cells (5, 16-18).
• protected individuals mount an effective HPV- specific immune response (9, 19, 20) which can be reproduced in some of the patients diagnosed with progressive HPV-infections by the means of vaccination and is associated with full regression of lesions and loss of HPV (21). This indicates that even in patients that previously failed to properly deal with an HPV infection the immune system is in principle capable of successfully combating an HPV infection.
• the immune system is deregulated during an HPV infection and progression of the HPV infection into low grade lesions.
• a locally altered cytokine environment with an increase in IL-10 and a decrease in pro-inflammatory cytokines (22-24) is related to the failure of the immune system to control HPV infection.
• pro-inflammatory cytokines (22-24)
• infection of epithelial cells such as keratinocytes by viruses results in the production of antiviral cytokines via the activation of the type I interferon (IFN) response genes and the attraction of immune cells via the production of pro-inflammatory cytokines through activation of the canonical NFkB pathway.
• IFN interferon
• Both pathways can be activated when the intruding virus is recognized by intracellular pathogen recognition receptors (PRR; e.g. Toll like receptors (TLR), DAI- RIG-I, and MDA-5.
• PRR pathogen recognition receptors
• TLR Toll like receptors
• DAI- RIG-I DAI- RIG-I
• MDA-5 MDA-5
• HPV is a DNA virus, therefore, our first studies concentrated on the interference of HPV with TLR9.
• the HPV16 E6 DNA sequence contains a CpG motif which can activate TLR9, indicating that this virus is able to initiate innate responses (34).
• TLR9 is not expressed in undifferentiated keratinocytes of the basal cell layer of epithelia. However, in the layers comprising differentiated keratinocytes in which TLR9 is expressed, HPV is able to suppress TLR9 signalling (35).
• undifferentiated and differentiated keratinocytes do not express TLR7 or 8 but do express TLR3, RIG-I and MDA-5 ( Figure 2ab) all which can sense viral infections through the recognition of viral RNA (36-39) and DNA (40, 41).
• TLR3, RIG-I and MDA-5 Figure 2ab all which can sense viral infections through the recognition of viral RNA (36-39) and DNA (40, 41).
• HPV can not persist by simply interfering with TLR9 alone because TLR3, RIG-I and MDA-5 also can sense an HPV infection, even in the basal layer of epithelia.
• WO2007/111998 describes double stranded RNA molecules against the expression of the HPV target genes El, E6 and the human E6AP gene. These genes are different from the human UCH-Ll gene targeted in the present invention, moreover, the E6AP gene is a ubiquitin ligase whereas UCH-Ll has a dual activity, i.e. a ligase and a hydrolase activity.
• hrHPV infection of cells such as primary keratinocytes significantly reduced the capacity of cells to produce anti-viral cytokines, and/or to produce pro-inflammatory cytokines to attract adaptive immunity upon stimulation of their intracellular pathogen recognition receptors (PRR).
• PRR pathogen recognition receptors
• TNF3 tumor necrosis factor receptor-associated factor 3
• hrHPV- infected cells such as keratinocytes
• UCH-Ll also termed MSYl, neuron cytoplastic protein 9.5, ubiquitin thiolesterase; UBLl; pgp9.5; parkinson disease 5; park ⁇
• the invention therefore provides a method for enhancing and/or stimulating the immunogenicity of a cell, comprising
• an epithelial cell comprising an infectious human papilloma virus (HPV) with an inhibitor of UCH-Ll thereby - increasing expression of an anti-viral cytokine; and/or,
• epithelial cell comprising an infectious human papillomavirus refers to an epithelial cell that is infected with HPV.
• the infection is preferably latent, meaning that no infectious virions are produced.
• limited copies of the HPV genome remain in the cell nucleus and replication of the viral DNA is coupled to replication of the cellular genome.
• Latent HPV infections tend to be transient and of relatively short duration. Only a small proportion of women exposed to HPV become persistently infected and continue to have detectable levels of HPV DNA in the genital epithelium. These women have an increased risk of developing invasive cervical carcinoma, due to transformation of the HPV-infected cell.
• epithelial cell comprising an infectious human papillomavirus preferably refers to a cell that is persistently infected with HPV.
• epithelial cell comprising an infectious human papillomavirus does not refer to a transformed, tumorigenic cell.
• said epithelial cell is a human epithelial cell.
• Enhancing and/or stimulating immunogenicity of a cell preferably comprises enhancing and/or restoring the capacity of infected epithelial cells to produce a anti-viral cytokine, and/or to produce a pro-inflammatory cytokine to attract adaptive immunity that was previously suppressed by HPV, preferably upon stimulation of their intracellular pathogen recognition receptors, in kind not necessarily in amount.
• the findings in the present invention connect UCH-Ll to the regulation of PRR-mediated pathways of immune signalling in epithelial cells that are infected by HPV.
• said epithelial cell is a basal epithelial cell.
• said epithelial cell is a primary keratinocyte.
• said primary keratinocyte is a keratinocyte from the basal cell layer epithelium.
• said keratinocyte is a human keratinocyte.
• hrHPV-induced UCH-Ll interferes with TRAF3 and/or TRAF6 and/or TRAF3 and TRAF6 mediated activation of the type I interferon and canonical NFkB signalling either by tagging these proteins for degradation or by prevention of activating-ubiquitination.
• UCH-Ll not only the innate immune response but also the adaptive immune response is affected by UCH-Ll as both TRAF3 and 6 are also essential for CD40-mediated NFkB signalling and CD40 is expressed on the surface of keratinocytes where it can interact with CD40 ligand expressing T cells.
• the invention is particularly suited to enhance and/or stimulate the immunogenicity of epithelial cells, such as keratinocytes, that are infected with HPV.
• epithelial cells such as keratinocytes
• HPVs human papilloma viruses
• Each type of HPV has the potential to cause an abnormal growth, such as warts and condylomas.
• the HPV is a low-risk HPV such as, for example, HPV6 and HPVIl.
• HPV infections of types 6 and 11 are the main causes of genital warts. Whereas these "low grade" abnormalities do not progress to cancer they can persist for long periods and cause great discomfort.
• high-grade abnormalities are less likely to resolve, and some will advance to cancer of the cervix, vagina or vulva.
• high-grade abnormalities are infections of high risk-type HPV (hrHPV), especially of types 16 and 18.
• hrHPV high risk type HPV
• hrHPV high- risk type HPV
• hrHPV high- risk type HPV
• said hrHPV is selected from HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, and HPV73.
• epithelial cells such as keratinocytes
• viral infection of epithelial cells can be recognized by the PPR mediated signalling pathway and results in the activation of anti-viral cytokines and the activation of pro-inflammatory cytokines/chemokines.
• HPV-infected epithelial cells such as primary keratinocytes evade the immune system, not by altered expression of PPRs but rather by altering the capacity of the PPR pathway to transduce the danger signal to the nucleus.
• this capacity is enhanced by contacting the epithelial cell with an UCH-Ll inhibitor.
• said inhibitor of UCH-Ll enhances an intracellular pathogen recognition receptor (PRR) pathway mediated signalling in said infected epithelial cell.
• PRR pathogen recognition receptor
• said inhibitor of UCH-Ll enhances PPR mediated signalling through the PRR Toll like receptor 1, 2, 3, 5, 6, MDA-5 and/or RIG-I ( Figure 2a). These receptors are found to be expressed in epithelial cells such as undifferentiated keratinocytes, mainly in undifferentiated keratinocytes of the basal layer.
• said inhibitor of UCH-Ll enhances and/or restores PPR mediated signalling through Toll like receptor 1,2, 3, 5, 6, 9, MDA-5 and/or RIG-I ( Figure 2a), which are found to be expressed in epithelial cells such as differentiated keratinocytes.
• said inhibitor of UCH-Ll enhances PPR mediated signalling through the PRR Toll like receptor 3 (TLR3), MDA-5, and/or RIG-I.
• An enhanced capacity of the PPR pathway results in activation of anti-viral cytokines and the activation of pro-inflammatory cytokines/chemokines.
• said enhancement results in a restoration of the capacity of the PPR pathway in the infected cell to a level that is similar to the capacity of the PPR pathway in a cell that is not infected with a virus such as HPV.
• said inhibitor of UCH-Ll enhances Tumor Necrosis Factor Receptor pathway- mediated signalling in an infected epithelial cell. In another preferred embodiment said inhibitor of UCH-Ll enhances and/or restores PPR mediated signalling and/or Tumor Necrosis Factor Receptor pathway-mediated signalling in an infected epithelial cell.
• said HPV is a low-risk HPV, preferably selected from HPV types 6 and 11.
• said HPV is a high-risk HPV, preferably selected from HPV types HPVl 6, HPVl 8, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, or HPV73.
• the capacity of the PPR pathway is enhanced by contacting the epithelial cells, for example keratinocytes, with an UCH-Ll inhibitor and an additional stimulus.
• Cells can be contacted with said additional stimulus together, before, or after contacting the epithelial cells with an UCH-Ll inhibitor.
• Said additional stimulus is preferably selected from inflammation inducing agents that are able to activate innate immune cells such as dendritic cells and Natural Killer cells.
• the additional stimulus is selected from known MDA- 5 ligands, TNF- receptor ligands, and TLR-ligands, such as, but not limited to, CpG (TLR9 ligand; Imgenex); Flagellin (TLR5 ligand; Imgenex); MALP-2 (TLR6/TLR2 ligand; Imgenex); polyinosinic:polycytidylic acid (poly I:C; TLR3 ligand ); and Pam3CSK4 (TLRl and TLR2 ligand; Imgenex).
• CpG TLR9 ligand; Imgenex
• Flagellin TLR5 ligand; Imgenex
• MALP-2 TLR6/TLR2 ligand; Imgenex
• polyinosinic:polycytidylic acid poly I:C; TLR3 ligand
• Pam3CSK4 TLRl and TLR2 ligand; Imgenex
• a preferred additional stimulus is selected from interferon gamma, TNF-alpha, poly(LC)], LPS, inactivated influenza virus, activating anti-CD40 antibodies, and hyperoxic conditions, and any combination thereof.
• said additional stimulus is poly(LC), including variants of poly(LC), such as, for example, poly (Lpoly C12U) (Ampligen; HEMISPHERx Biopharma of Philadelphia) and Hiltonol (Oncovir, Washington, USA).
• Interferons are natural proteins produced by the cells of the immune system of most vertebrates in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferons are produced by a wide variety of cells in response to the presence of double- stranded RNA, a key indicator of viral infection. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing major histocompatibility complex glycoprotein classes I and II, and thus presentation of peptides to T cells, and inducing the resistance of host cells to viral infection. There are various interferons that can be expressed by cells.
• said anti-viral cytokine comprises a type I interferon (IFN-I).
• Type I interferons present in humans are IFN- l ⁇ , IFN- l ⁇ and IFN- l ⁇ .
• said type I interferon is IFN- l ⁇ .
• said interferon is expressed by said cells upon inhibition of UCH-Ll.
• said interferon is IFN- l ⁇ , which is enhanced in an HPV-infected cell that is contacted with an inhibitor of UCH-Ll, as compared to a cell that is infected with HPV but not contacted with an inhibitor of UCH-Ll.
• pro-inflammatory cytokine/chemokine comprises a pro-inflammatory cytokine/chemokine of the canonical NFKB pathway.
• said proinflammatory cytokine/chemokine of the canonical NFKB pathway comprises MIP- l ⁇ , MIP-3 ⁇ , IL- IB, RANTES and/or IL-8. Enhancing the expression of this pro-inflammatory cytokine/chemokine of the canonical NFKB pathway reduces the immune evasive effect mediated by infection by HPV in an epithelial cell of the invention.
• pro-inflammatory cytokines/chemokines of the canonical NFKB pathway is enhanced upon UCH-Ll inhibition. It is preferred that the expression of at least two pro-inflammatory cytokines/chemokines of the canonical NFKB pathway is enhanced. Preferably the expression of at least three is enhanced. Preferably the expression of at least four pro-inflammatory cytokines/chemokines is enhanced. Preferably these four pro-inflammatory cytokines/chemokines are selected from MIP-Ia, MIP-3 ⁇ , IL-IB, RANTES and IL-8.
• an anti- viral cytokine of the invention is enhanced together with at least one proinflammatory cytokine/chemokine of the canonical NFKB pathway.
• the expression of one or more type I interferons with at least one pro-inflammatory cytokine/chemokine of the canonical NFKB pathway is enhanced, as compared to a cell that is infected with HPV but not contacted with an inhibitor of UCH-Ll.
• the non-canonical NFKB pathway is responsible for the activation of plOO/RelB complexes ( Figure 1).
• ligand induced activation results in the activation of NFkB-inducing kinase (NIK), which phosphorylates and activates the IKKa complex, which in turn phosphorylates plOO leading to the processing and liberation of the p52/RelB active heterodimer.
• NIK NFkB-inducing kinase
• UCH-Ll Various methods exist in the art to inhibit the action of UCH-Ll. Many different methods for reducing the expression and/or activity of a protein are known to the person skilled in the art. In principle all such methods are suitable for reducing expression of UCH-Ll in a cell of the invention. Either the activity of the UCH-Ll protein can be inhibited or the level of expression of UCH-Ll can be inhibited, or both.
• the activity of UCH-Ll can, for instance, be inhibited by interfering with its binding partner(s), for instance, but not limited to an intracellular antibody specific for UCH-Ll.
• the intracellular antibody can inhibit binding to a binding partner of UCH- Ll in the cell or it can deregulate correct intracellular localization of the protein.
• said UCH-Ll inhibitor comprises a (small) molecule UCH-Ll inhibitor.
• small molecules are presently known that interfere with the activity of UCH-Ll.
• Small molecules are typically organic substances comprising between 5 to 100 carbon atoms, preferably between 10-50 carbons atoms.
• said small molecule UCH-Ll inhibitor is selected f the UCH- Ll inhibitors 1-7, depicted in figure 4a and 4b, or any combination thereof.
• said small molecule is compound 1-3, or 7 of figure 4.
• an isatin-O-acyl oximes based UCH-Ll small molecule inhibitor as described in reference (64) is preferred.
• UCH-Ll inhibitors are also known from WO2005089518. Such UCH-Ll inhibitors are also preferred.
• said UCH-Ll inhibitor comprises an anti-sense oligonucleotide UCH-Ll inhibitor.
• An anti-sense oligonucleotide UCH-Ll inhibitor of the invention preferably comprises an RNAi, a shRNA (short hairpin), a siRNA and/or a miRNA.
• RNAi UCH-Ll inhibitor this RNAi is therefore a preferred UCH-Ll inhibitor of the invention.
• a preferred RNAi UCH-Ll inhibitor comprises a sequence selected of ⁇ '-GCCAAUGUCGGGUAGAUGA; ⁇ '-CCGAGAUGCUGAACAAAGU, 5'-GCUGAAGGGACAAGAAGUU, and ⁇ '-CAAGGUGAAUUUCCAUUUU.
• RNAi UCH-Ll inhibitors are combined, selected from 5'-GCCAAUGUCGGGUAGAUGA and ⁇ '- CCGAGAUGCUGAACAAAGU, ⁇ '-GCCAAUGUCGGGUAGAUGA and ⁇ '- GCUGAAGGGACAAGAAGUU, ⁇ '-GCCAAUGUCGGGUAGAUGA and ⁇ '- CAAGGUGAAUUUCCAUUUU, CCGAGAUGCUGAACAAAGU and l ⁇ ⁇ '-GCUGAAGGGACAAGAAGUU, CCGAGAUGCUGAACAAAGU and ⁇ '-CAAGGUGAAUUUCCAUUUU, and ⁇ '- GCUGAAGGGACAAGAAGUU, and ⁇ '-CAAGGUGAAUUUCCAUUUU.
• at least three RNAi UCH-Ll inhibitors are combined, selected from ⁇ '-GCCAAUGUCGGGUAGAUGA; ⁇ '-
• RNAi UCH-Ll inhibitors are all four RNAi UCH-Ll inhibitors.
• said UCH-Ll inhibitor is an shRNA specific for RNA encoding UCH-Ll.
• said shRNA is expressed in a cell by means of an expression cassette comprising a coding 30 region for said shRNA.
• An expression cassette for expressing an shRNA preferably comprises a nucleic acid encoding said shRNA; at least one promoter in operable linkage therewith.
• Such expression cassettes are also provided by the invention.
• two or more expression cassettes encoding two or more different shRNA specific for RNA encoding UCH-Ll are used to inhibit UCH-Ll expression in said cell.
• said shRNA UCH-Ll inhibitor is encoded by a virus or viral vector comprising one or more of said expression cassettes.
• said viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a herpes virus vector.
• said shRNA is encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 85% sequence identity with a nucleic acid sequence
• TRCN0000007273 CCGGGTGTGAGCTTCAGATGGTGAACTCGAGTTCACCATCTGAAGCTCAC ACTTTTT.
• TRCN0000007276
• Such viral vectors and/or virus are also provide by the invention.
• said sequence identity is at least 90%, more preferred at least 95%, more preferred at least 98%, most preferred 100%.
• said shRNA is encoded by a nucleic acid molecule having at least 85% sequence identity, preferably at least 90%, more preferably at least 95%, more preferably 98%, most preferably 100% with the nucleic acid sequence
• TRCN0000011079 (LV079) CCGGCAGTTCTGAAACAGTTTCTTTCTCGAGAAAGAAACTGTTTCAGAA
• % sequence identity is defined herein as the percentage of nucleotides in a nucleic acid sequence that is identical with the nucleotides in a nucleic aid sequence of interest, after aligning the sequences and optionally introducing gaps of at most 3 nucleotides, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art. Because of the hairpin-loop character of shRNA it is preferred that the sequences complementary to each other have a high degree of sequence identity, preferably at least 90%, whereas this is less important for the sequence forming the loop.
• the invention further provides a method of silencing a gene encoding UCH- Ll in an epithelial cell for enhancing and/or restoring immunogenicity of said cell, comprising the steps of: a) introducing into said epithelial cell one or more expression cassettes for expressing one or more UCH-Ll RNA specific shRNAs, wherein each of said one or more expression cassettes comprises one or more coding regions for UCH-Ll RNA specific shRNAs; and b) allowing for said shRNAs to be expressed from said one or more expression cassettes, thereby silencing said gene encoding UCH-Ll in said epithelial cell.
• said coding region for UCH-Ll RNA specific shRNA comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85% sequence identity with a nucleic acid sequence
• GTTTTT (TRCN0000011079), or any combination thereof.
• said sequence identity is at least 90%, more preferred at least 95%, more preferred at least 98%, most preferred 100%.
• said coding region for UCH-Ll RNA specific shRNA comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 95%, more preferably 98%, most preferably 100% with the nucleic acid sequence
• said antisense oligonucleotide is designed to interfere with the splicing of the UCH-Ll pre-mRNA such that the normal exon structure is not present in the mature mRNA.
• the structure is typically disrupted to create a coding region that does not encode a functional UCH-Ll protein, typically though not necessarily through the introduction of a premature translation stop.
• Exon-skip inducing antisense oligonucleotides is typically directed toward a splice signal such as a splice acceptor site, splice donor site or splice enhancer and/or splice silencer site.
• the antisense oligonucleotide is typically directed toward a sequence in the mRNA of UCH-Ll.
• intronic sequences can also be the target for exon-skip oligonucleotides.
• An anti-sense oligonucleotide typically contains between 10-50 nucleotides.
• the sequence of the antisense oligonucleotide is capable of hybridizing to the target RNA under stringent conditions.
• the target cDNA sequence is depicted in Figure 5. This sequence corresponds to the target RNA sequence, except that RNA nucleotides contain ribose while DNA contains deoxyribose, and RNA has the base uracil replacing thymine.
• the antisense oligonucleotide comprises a sequence that is the reverse complement of the sequence of the target RNA over a stretch of at least 20 consecutive nucleotides.
• the antisense oligonucleotide may contain one, two, or more nucleotide analogues that have the same base pairing characteristics in kind not necessarily in amount as the nucleotide that they replace.
• the antisense oligonucleotide may also contain other nucleotides such an inosine, at one or two positions.
• the antisense oligonucleotide can be RNA, DNA, a locked nucleic acid (LNA), a morpholino, a peptide nucleic acid or another form as long as it can hybridize under stringent conditions with the target RNA. The latter forms are sometimes also referred to as nucleic acid analogues.
• the antisense oligonucleotide may be modified to provide an additional property to the oligonucleotide, for instance it may be chemically modified for increased or decreased resistance of the oligonucleotide/target hybrid to RNAseH, depending on the type of inhibition. For exon-skipping strategies increased resistance is desired, whereas for RNAi applications decreased resistance or enhanced sensitivity is desired.
• the invention discloses that infection of epithelial cells such as keratinocytes with HPV results in overexpression of UCH-Ll and indicates that this protein exerts a suppressive action on the intracellular signalling pathways stimulated by receptors of both the innate and adaptive immune system as inhibition of UCH-Ll expression reliefs the block on gene activation of proinflammatory cytokines. Reversal of this suppressive action by inhibiting the action of UCH-Ll in these cells stimulates and/or increases the capability of both the innate and the adaptive immune system to recognize and/or clear the infected cells.
• the invention therefore further provides a UCH-Ll inhibitor or a composition comprising said inhibitor, for use in the treatment of an individual suffering from an HPV infection, or of an individual at risk of suffering there from.
• a method for the treatment of an individual suffering from an HPV infection or an individual at risk of suffering there from comprising administering an effective amount of a UCH-Ll inhibitor to the individual.
• an UCH- Ll inhibitor for stimulating the immunogenicity of an epithelial cell such as a primary keratinocyte infected with HPV.
• a pharmaceutical composition comprising a UCH-Ll inhibitor for use in the treatment of an individual suffering from an HPV infection, or an individual at risk of suffering therefrom.
• a UCH-Ll inhibitor for use in the treatment of an HPV infection. Further provided is a UCH-Ll inhibitor for use in the treatment of an individual suffering from an HPV infection by enhancing and/or stimulating the immunogenicity of an epithelial cell such as a primary keratinocyte infected with said HPV in said individual. Further provided is a UCH-Ll inhibitor for use in the treatment of an individual suffering from an HPV infection, wherein the immunogenicity is stimulated by activating the canonical NFkB signalling pathway in an HPV infected cell of said individual.
• the invention further comprises a UCH-Ll inhibitor for use in a treatment of an individual suffering from an HPV-infection, wherein said individual is also provided with a compound for stimulating PRR-mediated signalling in a cell.
• the invention further comprises a UCH-Ll inhibitor for use in the treatment of an HPV-infection, wherein said HPV infection is further treated with a compound for stimulating PRR-mediated signalling in a cell.
• the invention provides a UCH-Ll inhibitor for use in the treatment of an HPV infection, wherein said inhibitor is selected from the UCH-Ll inhibitors 1-7, depicted in figure 4a and 4b, or any combination thereof.
• the invention provides a UCH-Ll inhibitor for use in the treatment of an HPV infection, wherein said inhibitor comprises an anti- sense oligonucleotides, preferably an RNAi, a shRNA, an siRNA and/or a miRNA.
• said antisense oligonucleotide is encoded by a nucleic acid sequence molecule comprising a nucleic acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 95%, more preferably 98%, most preferably 100% with a nucleic acid sequence CCGGGTGTGAGCTTCAGATGGTGAACTCGAGTTCACCATCTGAAGCTCAC ACTTTTT (TRCN0000007273),
• said sequence identity is at least 90%, more preferred at least 95%, more preferred at least 98%, most preferred 100%.
• said antisense olignucleotide is encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 95%, more preferably 98%, most preferably 100% with the nucleic acid sequence CCGGCAGTTCTGAAACAGTTTCTTTCTCGAGAAAGAAACTGTTTCAGAACT GTTTTTTT (TRCN0000011079).
• said HPV infection is caused by a low-risk HPV, preferably selected from HPV types 6 and 11.
• said HPV infection is caused by a high-risk HPV, preferably selected from HPV types HPVl 6, HPVl 8, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, or HPV73.
• the present invention further provides a method for enhancing intracellular pathogen recognition receptor (PRR) pathway mediated signalling in an epithelial cell comprising, providing a UCH-Ll expressing cell wherein said PRR mediated signalling is impaired and providing to said cell an inhibitor of UCH-Ll activity.
• PRR pathogen recognition receptor
• a method for enhancing the expression of IFN-B, MIP-Ia, MIP-3 ⁇ , IL-l ⁇ , RANTES and/or IL-8 in an epithelial cell that is infected with an HPV comprising providing said epithelial cell with a UCH-Ll inhibitor.
• the present invention further provides a method for further enhancing intracellular pathogen recognition receptor (PRR) pathway mediated signalling in a cell comprising, providing a cell wherein said PRR mediated signalling is impaired with an inhibitor of UCH-Ll activity combined with an additional stimulus to further enhance intracellular PRR pathway- mediated signalling in said cell.
• Said additional stimulus is preferably selected from inflammation inducing agents that are able to activate innate immune cells such as Dendritic Cells and Natural Killer cells.
• the additional stimulus is selected from known MDA-5 ligands, TNF-receptor ligands, and TLR-ligands, such as, but not limited to, CpG (TLR9 ligand; Imgenex); Flagellin (TLR5 ligand; Imgenex); MALP-2 (TLR6/TLR2 ligand; Imgenex); polyinosinicipolycytidylic acid (poly I:C; TLR3 ligand ); and Pam3CSK4 (TLRl and TLR2 ligand; Imgenex).
• CpG TLR9 ligand; Imgenex
• Flagellin TLR5 ligand; Imgenex
• MALP-2 TLR6/TLR2 ligand; Imgenex
• polyinosinicipolycytidylic acid poly I:C
• TLR3 ligand TLR3 ligand
• Pam3CSK4 TLRl and TLR2 ligand; Imgenex
• said additional stimulus is poly(LC), including variants of poly(LC), such as, for example, poly (Lpoly C12U) (Ampligen; HEMISPHERx Biopharma of Philadelphia) and Hiltonol (Oncovir, Washington, USA).
• a method according to the invention is provided, wherein said UCH-Ll inhibition is combined with an additional stimulus to further enhance the pathogen recognition receptor (PRR) and/or pathway mediated signalling
• a method for increasing the expression of IFN- ⁇ , MIP- l ⁇ , MIP-3 ⁇ , IL- l ⁇ , RANTES and/or IL-8 in an epithelial cell such as a primary keratinocyte that is infected with an HPV comprising providing said epithelial cell with a UCH-Ll inhibitor and said additional stimulus.
• the invention further provides a method for increasing the ratio of depolyubiquitinated/total TRAF3 in a TRAF3 expressing cell, said method comprising increasing the level of active UCH-Ll in said cell.
• the level of active UCH-Ll can be increased in a cell by for instance introducing an expression cassette comprising a coding region for UCH-Ll into said cell.
• the expression cassette of course comprises all necessary sequences for allowing transcription and translation of the UCH-Ll coding region in said cell.
• the level of active UCH-Ll can also be increased by decreasing the level of UCH-Ll inhibitor in a UCH-Ll expressing cell.
• UCH-Ll for increasing the ratio of depolyubiquitinated/total TRAF3 in a TRAF3 expressing cell.
• a proinflammatory cytokine producing and/or type I interferon, such as IFN ⁇ producing cell decreases the production of IFN ⁇ by that cell.
• said cell is a proinflammatory cytokine producing and/or interferon type I, such as IFN ⁇ , producing cell.
• a compound capable of increasing the ratio of depolyubiquitinated/total TRAF3 in a TRAF3 expressing cell for use in the treatment of an undesired immune reaction, such as for instance GVHD and autoimmune disease.
• said compound is depolyubiquitinated TRAF3.
• said compound is capable of increasing the level of active UCH-Ll in said cell.
• said compound is UCH-Ll.
• the invention further provides a method for decreasing the ratio of deubiquitinated/total TRAF3 in a TRAF3 and UCH-Ll expressing cell, said method comprising decreasing the level of active UCH-Ll in said cell.
• the level of active UCH-Ll is preferably reduced by providing said cell with an inhibitor of UCH-Ll.
• an inhibitor of UCH-Ll for decreasing the ratio of deubiquitinated/total TRAF3 in a TRAF3 and UCH- Ll expressing cell.
• Decreasing the ratio deubiquitinated/total TRAF3 in a TRAF3 and UCH-Ll expressing cell induces and/or increases the production of proinflammatory cytokines and/or type I interferon such as IFN ⁇ by that cell, particularly when that cell is a virus infected cell.
• This embodiment is particularly useful for enhancing and/or stimulating the immunogenicity of a virus infected cell.
• said virus is a human papilloma virus as described herein.
• said cell is an epithelial cell. More preferably said cell is a keratinocyte.
• a compound capable of decreasing the ratio of deubiquitinated/total TRAF3 in a TRAF3 and UCH-Ll expressing cell for use in the treatment of an HPV infection.
• said compound is polyubiquitinated TRAF3.
• said compound is capable of decreasing the level of active UCH-Ll in said cell.
• said compound is an UCH-Ll inhibitor.
• the invention further provides a method for increasing the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 and UCH-Ll expressing cell, said method comprising increasing the level of active UCH-Ll in said cell. Also provided is the use of UCH-Ll for increasing the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 expressing cell. Increasing the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 expressing cell decreases the activity of the canonical NFkB pathway in a cell wherein said pathway is activated.
• This embodiment is useful when the activity of the immune system should be less active against said cell, for instance in a transplantation setting where a host versus graft reaction is expected.
• said compound is non-K63 polyubiquitinated TRAF6.
• said compound is capable of increasing the level of active UCH-Ll in said cell.
• said compound is UCH-Ll.
• a compound according to the invention for use in the treatment of an HPV infection, wherein said HPV infection is further treated with a compound for stimulating PRR-mediated signalling in a cell.
• the invention further provides a method for decreasing the ratio of non- K63 polyubiquinated/total TRAF6 in a TRAF6 and UCH-Ll expressing cell, said method comprising decreasing the level of active UCH-Ll in said cell. Also provided is the use of an inhibitor of UCH-Ll for decreasing the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 and UCH-Ll expressing cell. Decreasing the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 and UCH-Ll expressing cell induces and/or increases the activity of the canonical NFkB pathway in that cell, particularly when that cell is a virus infected cell.
• This embodiment is particularly useful for enhancing and/or stimulating the immunogenicity of a virus infected cell.
• said virus is a human papilloma virus as described herein.
• said cell is an epithelial cell. More preferably said cell is a keratinocyte.
• said compound is non-K63 depolyubiquitinated TRAF6.
• said compound is capable of decreasing the level of active UCH-Ll in said cell.
• said compound is an UCH-Ll inhibitor.
• the invention further provides a method for determining the activity of UCH-Ll in a cell comprising determining the ratio of depolyubiquitinated/total TRAF3 in a TRAF3 expressing cell and/or the ratio of non-K63 polyubiquinated/total TRAF6 in a TRAF6 expressing cell, wherein an increase in at least one of said ratios is indicative for UCH-Ll activity in said cell.
• UCH-Ll binds TRAF3 and TRAF6 in a cell. It is therefore one aspect of the invention to interfere with this binding and thereby interfere with the activity of UCH-Ll on these two TRAFs.
• a method according to the invention is provided, wherein said inhibitor of UCH-Ll interferes with the binding of UCH-Ll to TRAF3 and/or with the binding of UCH-Ll to TRAF6.
• said interfering with the binding of UCH-Ll to TRAF3 and/or to TRAF6 results in increased ubiquitination of TRAF3 and/or decreased non- K63 ubiquitination of TRAF6.
• Intracellular antibodies or intrabodies are typically antibody fragments that are used inside cells for interaction with target antigens and either for interference with function or in some cases to mediate cell killing following antigen binding.
• Intracellular antibodies are typically formulated as single chain Fv (scFv) fragments which comprise immunoglobulin variable (V) domains of heavy (H) and light (L) chains held together by a short linker.
• single chain antibodies consisting only of the heavy chain or heavy chain variable regions are used.
• Such single heavy chain antibodies are typically though not necessarily derived from the family Camelidae and particularly the Camelids of that family.
• antigen-specific hybridomas have been used as a source of antibody genes from which scFv have been made for in-cell expression as intrabodies, and successes have been reported in which cellular phenotypes have been obtained due to scFv-antigen binding.
• Several different methods have been used to directly develop intrabodies without the need of hybridomas. These include genetic screening for intrabody— antigen interaction based on two- hybrid screening and use of fixed scFv frameworks for intrabodies.
• the intracellular antibody capture (IAC) technology facilitated the identification of consensus frameworks comprising residues from VH and VL which are most commonly found in selected intracellular antibodies.
• IAC consensus frameworks can also be used to convert poor intracellular antibodies into efficient ones by mutating framework residues to the IAC consensus whilst leaving the complementarity determining regions (CDRs) intact, which is the part most important for antigen binding.
• Another way of interfering with the binding of UCH-Ll to TRAF3 and/or TRAF6 is to provide the cell expressing the proteins with UCH-Ll peptides that mimic the binding site of UCH-Ll on TRAF3 or on TRAF6.
• a molecule that interferes with the binding of UCH- Ll to TRAF3 or to TRAF6, for instance an intracellular antibody or a peptide as described herein above is a preferred inhibitor of UCH-Ll activity in a cell.
• a UCH-Ll inhibitor of the invention is a UCH-Ll specific intracellular antibody as described herein above.
• said intracellular antibody is specific for human UCH-Ll.
• a UCH-Ll inhibitor of the invention is a peptide that mimics the binding site of UCH- Ll on TRAF3.
• a UCH-Ll inhibitor of the invention is a peptide that mimics the binding site of UCH-Ll on TRAF6.
• said peptide comprises a peptide of between 6- 12 consecutive amino acids of UCH-Ll.
• said UCH-Ll is human UCH-Ll.
• a composition comprising an inhibitor of UCH-Ll as an active ingredient may be prepared by using pharmaceutically and physiologically acceptable additives besides the active ingredient.
• additives may be excipient, disintegrating agent, sweetener, binder, coating agent, blowing agent, lubricant, glidant, flavoring agent, solubilizer, etc.
• the UCH-Ll inhibitor of the present invention is preferably administered to said individual in the form of a pharmaceutical composition.
• the pharmaceutical composition of the present invention comprising an inhibitor of UCH-Ll as an active ingredient may further comprise one or more pharmaceutically acceptable carriers besides the active ingredient, to be formulated to pharmaceutical composition appropriately.
• the composition comprise the pharmaceutically acceptable carriers which have biocompatibility and are sterilized, for example, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, malto dextrin solution, glycerol, ethanol and a mixture thereof. If necessary, other common additives such as antioxidant, buffer, bacteriostatic agent, etc. may be added in the composition.
• the composition may be formulated to injectable forms such as solution, suspension, emulsion, etc., or into a capsule, granule or tablet.
• the present composition may be formulated in any desirable forms according to disease or ingredient, by using conventional methods or the written text of Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).
• the pharmaceutical composition of the present invention may be prepared in any form such as granule, powder, coated tablet, tablet, capsule, suppository, syrup, juice, suspension, emulsion, drop or injectable liquid formulation, and sustained release formulation of the active ingredient(s), etc.
• composition of the present invention comprising an inhibitor of UCH- Ll as an active ingredient may be administered via various routes including intravenous, intra- arterial, intraperitoneal, intramuscular, intrathoracic, transdermal, intranasal, inhalation, topical, rectal, oral, ocular and intradermal introduction, according to conventional method of administration.
• the dosage of the present composition comprising an inhibitor of UCH-Ll as an active ingredient represents an amount of active ingredient or pharmaceutical composition that is required to increase expression of an anti-viral cytokine, increase expression of a pro-inflammatory cytokine/chemokine of the canonical NFKB pathway; and/or decrease expression of a protein of the non-canonical NFKB pathway in an infected epithelial cell such as an infected primary keratinocyte in the individual.
• the dosage can be determined by various factors such as the severity of patient's symptom, the content of active ingredient, the nature and content of other ingredients, the type of formulation, patient's age, body weight, health status, gender and food, dosing time, administration route, the secretion ratio of composition, time of treatment, and other co- administrated drug.
• the dosage is preferably 0.01 ng/kg - 10 mg/kg in case of RNAi, 0.1 ng/kg - 10 mg/kg in case of compounds, 0.1 ng/kg - 10 mg/kg in case of a binding body that binds UCH- Ll, such as a monoclonal antibody, a single chain antibody, or a small antibody mimetic such as an avimer or an anticalin.
• the UCH-Ll inhibitor is administered topically, preferably in the form of a gel or cream that is applied to the site of the infected cells, for example the HPV infected keratinocytes.
• said pharmaceutical composition is a composition for vaginal administration.
• said gel or cream preferably further comprises liposomes.
• iontophoresis, electroporation or low-frequency ultrasound are used to deliver the active molecules at clinically useful rates.
• Fig. 1 Signal transduction routes leading to stimulation of IFN ⁇ genes or NFkB signaling and the impairment of innate immunity by infection by hrHPV.
• PRR refers to different cytoplasmic sensors for viral RNA and DNA (e.g. TLR3, DAI, RIG-I, MDA-5). Activation via these PRR result in the ubiquitination of TRAF3 and TRAF6 and subsequent downstream signaling via the indicated molecules towards the production of IFN ⁇ and the proinflammatory cyto/chemokines IL- l ⁇ , IL-8, RANTES, MIPIa and MIP3 ⁇ .
• DUBA and CYLD are known deubiquitinating enzymes interacting with TRAF3 and TRAF6, respectively, blocking the function of these proteins.
• TRAF6 canonical pathway of NFkB
• TRAF2/5 non- canonical pathway of NFkB
• TRAF3 controls the non-canonical pathway of CD40 signaling by degrading unbound NIK. Accumulation of NIK leads to non-canonical NFkB signaling and the production of for instance IL-IO and IDO.
• HPV infected keratinocytes are impaired in their capacity to respond to PRR signals with the production of IFN ⁇ , ILl ⁇ , IL-8, RANTES, MIPIa and MIP3 ⁇ , suggesting that both the IFN ⁇ and canonical pathway of NFkB signaling is affected. Furthermore, we showed that TRAF3 was degraded and the non-canonical NFkB was activated upon PRR activation in HPV- infected cells.
• UCHLl is a deubiquitinating enzyme of which the expression was found to be highly upregulated in HPV-infected keratinocytes.
• RNAi- mediated knock-down of UCHLl restores type I IFN and canonical NFkB signaling implying that its expression affects TRAF3 and TRAF6 signaling.
• the potential effect of overexpressed UCHLl on TRAF3 and TRAF6 is indicated.
• HPV has also been shown to interact with IRF-3 and CPB/p300. The putative sites were HPV interferes with are indicated in red.
• Fig. 2 High-risk HPV infected keratinocytes are impaired in type I and canonical NFkB signaling by UCH-Ll.
• RNA of the indicated cells was subjected to RT-PCR (35 cycles) with specific primers for human TLRl-IO or GAPDH (top panel) or for RIG-I and MDA-5 (bottom panel). Data show the absence of TLR7, 8 and 9 in undifferentiated (basal) cells. TLR3, RIG-I and MDA-5 are present in undifferentiated and differentiated keratinocytes whereas TLR9 is only present in differentiated keratinocytes.
• TLR3 is expressed at similar levels in HPV-negative (ESG2, HVK) as in HPV16+ cells.
• ESG2, HVK HPV-negative
• Total RNA was isolated and TLR3 mRNA expression was analyzed by quantitative TaqMan RT-PCR. TLR3 expression was normalized against GAPDH mRNA levels.
• Uninfected and HPVl 6 infected keratinocytes were stimulated with poly LC for 24 hours.
• Total RNA was isolated and IL-8 and MIP-3 ⁇ mRNA expression were analyzed by quantitative TaqMan RT-PCR. Gene expression was normalized against GAPDH mRNA levels.
• the secretion of IL-8 and MIP-3 ⁇ was analyzed in the supernatants of poly I:C stimulated keratinocytes by ELISA.
• cytosolic TRAF3 is degraded in high risk HPV+ KC (top panel).
• Cells were either unstimulated or stimulated with PoIy(LC) for 3 hours.
• Cytoplasmic proteins (40 ⁇ g) were subjected to Western blot for TRAF3 expression.
• the use of the proteasome inhibitor MG132 partially prevents TRAF3 degradation in HPV+KC following poly LC stimulation.
• Control or MGl32-treated (overnight) cells were subjected to either no stimulation or stimulated with PoIy(LC) for 3 hours.
• Cytoplasmic proteins (40 ⁇ g) were subjected to Western blot for TRAF3 expression.
• RNAi of UCH-Ll restores the poly LC mediated gene activation of proinflammatory cytokines in hrHPV-infected KC.
• Control RNAi or UCH- Ll RNAi transfected HPV+KCs were either unstimulated or stimulated with PoIy(LC) for 24 hours.
• Total RNA was isolated and IL-8, MIP-3a and MIP-Ia mRNA expression were analyzed by quantitative TaqMan RT-PCR. Gene expression was normalized against GAPDH mRNA levels.
• Fig. 3 Expression of CD40 and HLA class II by keratinocytes and CD40L by T-cells.
• HPV-specific CD4+ tumor-infiltrating T cells express CD40 ligand (CD 154) at their surface when stimulated with cognate antigen.
• HPV-specific CD8+ tumor-infiltrating T cells express CD40L as well as 4- IBB at their surface after stimulation with cognate antigen.
• Fig. 4. Improvement on isatins identified in an initial screen of 42,000 compounds led to the development of 1-3. These three inhibitors show a strong preference for UCH-Ll (IC50 values in black) over UCH-L3 (IC50 values in red). Three compounds (4-6) from the initial screen were selective for UCH-L3 (IC50 values in red) over UCH-Ll (IC50 values in black) by as much as 100-fold in the case of 6. (b) UCH-Ll inhibitor LDN-91946 (7). See reference 72 for further details.
• Fig. 5 Nucleotide sequence of UCH-Ll cDNA.
• Fig. 6 Expression of UCH-Ll in HPV16-infected keratinocytes suprainfected with recombinant Antiviruses expressing a short- hairpin RNA against UCHL-I.
• RNA isolated from either LV079 (anti-UCHLl) or SHC004 (control) lentivirus infected HPVl 6 cells was tested for the expression of UCH-Ll by Taqman PCR.
• the relative expression of UCH-Ll was calibrated using GAPDH as calibrator gene.
• the infection with LV079 resulted in strong downregulation of UCH-Ll indicating that LV079 works independently of poly I:C stimulation.
• Fig. 7 Expression of type I interferon, cytokines and cheniokines following the lentiviral-niediated shRNA blockade of UCH-Ll expression in HPV16-infected keratinocytes.
• Fig. 8 Overexpression of UCH-Ll reduces polyubiquitination of TRAF3.
• 293T cells were transfected with a) 22 ⁇ g empty vector (pCDNA3)+6 ⁇ g HA-Ubiquitin (Ha-Ub), b) 10 ⁇ g Flag-TRAF3, 6 ⁇ g HA-Ub and 12 ⁇ g empty vector, or c) 10 ⁇ g Flag-TRAF3, 6 ⁇ g HA-Ub and 12 ⁇ g UCH-Ll vector by calcium-phosphate method. After 48 hours of transfection, an immunoprecipitation with anti-Flag antibodies followed by a Western blot with anti-HA antibodies was performed.
• TOP: TRAF3, with an original size of 67Kb displays strong poly ubiquitination as reflected by a smear.
• 293T cells were transfected with a) 10 ⁇ g Flag-TRAF6, 6 ⁇ g HA-Ub and 12 ⁇ g empty vector (pcDNA3), or b) 10 ⁇ g Flag-TRAF6, 6 ⁇ g HA-Ub and 12 ⁇ g UCH-Ll vector by calcium-phosphate method.
• TOP After 48 hours of transfection, an immunoprecipitation with anti-Flag antibodies followed by a Western blot with anti-HA antibodies (left) or anti-K63 ubiquitine (right) was performed.
• TRAF6 displays only low ubiquitination while the addition of UCH-Ll results in a larger smear indicating polyubiquitination of TRAF6.
• UCH-Ll binds directly to TRAF3 and TRAF6 293T cells were transfected with a) 12 ⁇ g empty vector (pcDNA3), b) 12 ⁇ g UCHL-I, c) 10 ⁇ g Flag-TRAF3 and 12 ⁇ g UCH-Ll vector or d) 10 ⁇ g Flag- TRAF6 and 12 ⁇ g UCH-Ll vector by calcium-phosphate method.
• Cells were stimulated for 1 hr with 25 ⁇ g poly I:C and then an immunoprecipitation with anti-Flag antibodies followed by a Western blot with anti-UCHLl antibodies was performed.
• As a positive control a whole cell extract of UCH- Ll transfected 293 cells was taken along.
• TOP Only the lanes with TRAF 3 and TRAF6 are positive for UCHLl.
• BOTTOM Ponceau-S staining of blot indicating that similar amount of product were loaded onto the gel, except for TRAF3 where a much lower amount was loaded. However, UCHLl can still be detected in this lane. Examples
• infection of keratinocytes by viruses results in the production of antiviral cytokines via the activation of the type I interferon (IFN) response genes and the attraction of immune cells via the production of proinflammatory cytokines through activation of the canonical NFkB pathway.
• IFN interferon
• Both pathways are activated when the intruding virus is recognized by intracellular pathogen recognition receptors (PRR; e.g. Toll like receptors (TLR), DAI- RIG-I, and MDA-5.
• PRR pathogen recognition receptors
• TLR Toll like receptors
• DAI- RIG-I DAI- RIG-I
• MDA-5 MDA-5
• MDA-5 primers used were CTG CTG C AG AAAAC AATGG A and TGCCCATGTTGCTGTTATGT. PCR products were visualized on 1.8% agarose gels.
• TLR9 primers and probe were used as described previously (76).
• Pre-designed, pre-optimized primers and probe mix for TLR3, IL-8, MIP-3 ⁇ , MIP- l ⁇ , UCH-Ll and GAPDH were from Applied Biosystems (Foster City, USA).
• TaqMan PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems). Threshold cycle numbers (Ct) were determined with 7900HT Fast Real-Time PCR System (Applied Biosystems) and the relative quantities of mRNA per sample were calculated using the ⁇ Ct method as described by the manufacturer using GAPDH as the calibrator gene.
• 5xlO 5 cells were plated in 1 ml in each well of 24-well flat bottom plate.
• Cells were left unstimulated or stimulated with Pam3CSK4 (5 ⁇ g/ml), PoIyLC (25 ⁇ g/ml), LPS (3.33 ⁇ g/ml ), flagellin (150ng/ml), R848 (l ⁇ g/ml), CpG (l ⁇ M) or TNF ⁇ (50 ng/ml) for 24 hours.
• flagellin was a kind gift from Jean-Claude Sirard, Institut Pasteur de Lille, France
• human recombinant TNFD was from R&D Systems (Minneapolis, USA) and other TLR ligands were purchased from Invivogen (San Diego, USA).
• the supernatants were harvested and IL- 8, MIP- 3 ⁇ , IL- 16 and RANTES concentration was determined using the IL8, MIP-3 ⁇ , IL- 16 and RANTES Quantikine ELISA kits (R&D
• cytoplasmic or nuclear extracts were either left unsimulated or stimulated with polyLC (25 ⁇ g/ml) for 3 hours or 24 hours. Where indicated, cells were treated with MG132 (25 ⁇ M or 50 ⁇ M ) for overnight and afterwards were stimulated with poly I:C (25 ⁇ g/ml) for 3 hours. Cytoplasmic or nuclear extracts were prepared as described previously (77) and the amounts of protein were determined (BCA protein assay kit; Pierce, Rockford, IL). 40 ⁇ g of protein was mixed with 4X SDS-
• PAGE sample loading buffer 20% glycerol, 4% SDS, 12OmM Tris-HCl pH 6.8, 0.1% bromophenol blue, 10% beta-mercaptoethanol
• Protein extracts were separated on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose transfer membrane (Optitran BA-S-85, Whatman, Piscataway, NJ).
• the proteins were blotted with anti-TRAF3 antibody (C-20, Santa Cruz, 1:200), anti-RelB antibody (C- 19, Santa Cruz, 1:200) (78) followed by horseradish peroxidase-conjugated anti-IgG antibodies.
• the blots were developed using an enhanced chemoluminescence Western blotting detection system (SuperSignal West Pico, Pierce) according to the manufacturer's recommendations.
• Non-targeting RNAi oligos ON-TARGETp/ws Non-targeting Pool, catalogue D- 001810-10-20
• oligos targeting UCHLl ON-TARGETplus SMARTpool, catalogue L-004309-00
• Dhramacon Cholicgo, IL
• Cells were transfected with RNAi using N-TER Nanoparticle siRNA Transfection System (Sigma-Aldrich, St. Louis, MO) according to manufacturer's instructions. 24 hours after transfection, cells were stimulated with PoIyLC (25 ⁇ g/ml) for another 24 hours and experiments were performed.
• TLR, RIG-I and MDA-5 are not impaired in HPV infected keratinocytes.
• Primary human keratinocytes were cultured in defined serum-free medium, and partial differentiation was induced by the addition of Ca 2+ , while terminal differentiation was induced by the addition of Ca 2+ and methylcellulose.
• Expression of the small proline-rich protein 2A (SPRR2A), a molecular marker of KC differentiation and of TLRs 1-10, RIG-I and MDA-5 were analyzed at the mRNA level by RT-PCR.
• TLR9 showed striking changes upon KC differentiation, in that it was undetectable in undifferentiated and partially differentiated KCs, while being readily detectable in fully differentiated KCs (Figure 2a) (35).
• TLR3 is expressed at all stages of the keratinocyte, we determined the mRNA expression level of TLR 3 in non- infected primary keratinocytes as well as in HPV-infected keratinocytes and observed no difference (Figure 2b).
• TLR9 is not expressed in undifferentiated keratinocytes of the basal cell layer of epithelia.
• undifferentiated and differentiated keratinocytes do not express TLR7 or 8 but do express TLR3, RIG-I and MDA- 5 ( Figure 2ab) all which can sense viral infections through the recognition of viral RNA (36- 39) and DNA (40, 41).
• TLR3, RIG-I and MDA-5 also can sense an HPV infection, even in the basal layer of epithelia.
• PRRs pathogen recognition receptors
• Activation of keratinocytes through stimulation with CpG, poly I:C, flagellin or TNFoc reveals a block in the production of proinflammatory cyto/chemokines when HPV is present.
• TLR9 as expressed in differentiated keratinocytes, by the capacity of CpG oligodeoxynucleotides (CpG ODN) to trigger by the expression of pro-inflammatory cytokines and chemokines.
• IFN- ⁇ and TNF- ⁇ expression levels in mRNA samples from control and CpG ODN-stimulated KC cultures were examined by semi-quantitative TaqMan real-time PCR. Uninfected and infected keratinocytes were either left undifferentiated or differentiated with methylcellulose containing Ca 2+ . After 24 hours, differentiated cells were harvested, methylcellulose was washed out, and cells were then stimulated with CpG (10 ⁇ M) for 7 hours.
• IFN- ⁇ and TNF- ⁇ mRNA expression were normalized using GAPDH mRNA as an internal control.
• differentiated HPV-positive KCs failed to induce IFN ⁇ and TNF ⁇ gene expression upon incubation with TLR9-ligand CpG (35), pointing at a defect in the TLR9 pathway downstream of the receptor.
• TLR2 PAM3CK4
• TLR3 poly LC
• TLR4 poly LC
• TLR4 LPS
• TLR5 Flagellin
• R848 TLR7,8
• CpG TLR9
• TNF ⁇ TNF ⁇
• IFNb type I IFN
• pro-inflammatory cytokines and chemokines such as MIPIa, MIP-3a, ILlB, IL8, and RANTES
• TRAF3-dependent signaling is impaired in HPV-infected keratinocytes.
• HPV E6 protein could bind to interferon regulatory factor 3 (IRF3), a transcription factor downstream of PRRs, and thereby inhibit its transactivation function to stimulate the type I interferon pathway (42).
• IRF3 interferon regulatory factor 3
• HPV16E7 and p300/CBP thereby abolishing the p300/CBP-mediated transactivation, was observed (43).
• HPVl 6 E6 and E7 repressed the promoter activity of the pro-inflammatory cytokine IL-8 via binding to CBP (44).
• TLR3, 4, 7 and 9 mediated induction of type I interferon is abrogated in TRAF3 knock out cells (reviewed in (28)).
• the non canonical immunosuppressive NFkB pathway was activated, as this is also under tight control of TRAF3 (25, 26) ( Figure 1). Therefore we isolated cytoplasmic proteins from normal and HPV-infected keratinocytes either nonstimulated or stimulated with PoIy(LC) for 3 hours and analyzed TRAF3 expression by Western blot.
• This experiment revealed that TRAF3 was degraded in hrHPV-infected keratinocytes after poly I: C stimulation ( Figure 2f) whereas it was upregulated in non-infected keratinocytes ( Figure 2f). Degradation is most likely proteasome-dependent as the use of the proteasome inhibitor MG132 partially prevents TRAF3 degradation in HPV+KC following poly I:C stimulation ( Figure 2f).
• Ubiquitin carboxyl-terminal esterase Ll (UCH-Ll) is overexpressed in HPV-infected keratinocytes.
• DUBA Deubiquitinating enzyme A
• the second enzyme is the tumor suppressor CYLD which can deubiquitinate TRAF2 and TRAF6, thereby preventing the activation of IKK (Figure 1) (46) .
• Figure 1 the tumor suppressor CYLD which can deubiquitinate TRAF2 and TRAF6, thereby preventing the activation of IKK ( Figure 1) (46) .
• the hrHPV-infected keratinocytes showed strong and constitutive increase of a transcript encoding a similar enzyme which is designated ubiquitin carboxyl-terminal esterase Ll (UCH-Ll; not shown).
• UCH-Ll has an important function in cytoplasmic protein degradation. It has two opposing enzymatic activities of which one is a hydrolase activity which prevents lysine-63-linked ubiquitination (needed for the activation of TRAF 3 and 6) and the second is a dimerization- dependent ubiquityl ligase that plays a role in proteasomal protein degradation (48). Therefore, it is highly likely that the HPV-induced overexpression of UCH-Ll affects TRAF-mediated activation of the type 1 IFN and the canonical NFkB signalling pathways.
• RNAi of UCH-Ll restores the poly I:C mediated gene activation of proinflammatory cytokines in hrHPV-infected KC.
• HPV-infected keratincoytes were transfected with control RNAi or with UCH-Ll RNAi and then stimulated with PoIy(LC) or as a control not stimulated. After 24 hours of culture total RNA was isolated and the mRNA expression of IL- 8, MIP-3a and MIP-Ia was analyzed by quantitative TaqMan RT-PCR.
• Non- stimulated HPV-infected keratinocytes did not show activation of the genes encoding IL-8, MIP3 ⁇ or MIPIa, neither did the HPV-infected keratinocytes which were transfected with control RNAi.
• the use of UCH-Ll RNAi restored the activation of genes encoding for the pro- inflammatory cytokines IL-8, MIP3 ⁇ or MIPIa ( Figure 2h).
• UCH-Ll has been described to have similar function as DUBA - which selectively cleaves the lysine- 63-linked polyubiquitin chains on TRAF3 - and CYLD, which can deubiquitinate TRAF2 and TRAF6, UCH-Ll most likely interferes with the TRAF proteins.
• UCH-Ll has also been connected to TRAF6 in a large-scale mapping of protein-protein interactions by co-immunoprecipitation (49).
• TRAF3 and TRAF6 which are directly downstream of PRRs, can block the signal transduction from PRR to the type 1 IFN and canonical NFkB signalling pathways and, therefore, impair immunity against HPV. This could explain how HPV may deregulate the induction of a strong adaptive immune response.
• TRAF3 may not only influence the PRR-mediated activation of the innate immune response, but it may also affect the function of the adaptive immune response. TRAF3 acts as a molecular switch between two NFkB- signaling pathways originating from CD40.
• CD40 is a member of the TNF receptor family and is - besides its well known expression on professional antigen presenting cells of the immune system — also widely expressed on normal epithelial cells and HPV transformed cervical cells (51, 52).
• TRAF3 through the degradation of NIK (25, 26, 32) suppresses TRAF2/5- mediated NF-kB, while TRAF3 also enhances the transcriptional activity of TRAF6-mediated NF-kB (33).
• CD40 is expressed on the basal cells (the target cell for HPV) of epithelia ( Figure 3b, adapted from ref. 52) and ligation of CD40 by CD40 ligand (CD40L or CD 154) inhibits proliferation and increases the secretion of cytokines including interferons of normal skin keratinocytes in vitro (51, 53), suggesting that in normal keratinocytes CD40 ligation may amplify proinflammatory responses as well as limit cell growth.
• EBV-transformed B-cells (B-LCL) - serving as antigen presenting cells - were loaded overnight with 5ug/ml HPV- 16 peptides.
• HPV16-specific CD4+ T cells or CD8+ T cells isolated from tissue-infiltrating lymphocytes of patients with HPV16-induced lesions (15) were seeded into a 96-wells plate at 200.000 cells per well and 40,000 B-LCL were added to each well. After one hour Brefeldin A lOug/ml was added to the culture and left overnight.
• HLA class II may allow CD4+ T-cells to recognize their cognate HLA class II presented epitope (e.g. HPV antigens) on these keratinocytes and upregulate cell surface CD40L. Indeed when HPV16- specific CD4 T cells were stimulated with their cognate antigen they upregulate CD40L (CD 154; Figure 3c). This allows the interaction between T-cell expressed CD40L and CD40 on keratinocytes and subsequent activation of the CD40 signaling pathway in keratinocytes.
• HLA class II presented epitope e.g. HPV antigens
• HPV16-specific CD4 T cells were stimulated with their cognate antigen they upregulate CD40L (CD 154; Figure 3c). This allows the interaction between T-cell expressed CD40L and CD40 on keratinocytes and subsequent activation of the CD40 signaling pathway in keratinocytes.
• HPV-specific CD4+ T cells as well as CD8+ T cells are readily isolated from patients with past infection of HPV (14) or progressive HPV infections (5) as well as patients with cancer (15, 16).
• these T cell Upon recognition of their cognate antigen in either HLA class II or HLA class I, respectively at the cell surface of an HPV-infected keratinocyte or HPV-induced tumor cell, these T cell will express CD40L allowing the interaction with CD40 on the keratinocytes. While under normal situations the interaction between
• CD40L (T cells) and CD40 (keratinocytes) is expected to boost immunity, the interaction with hrHPV-infected keratinocytes — through deregulation of the canonical NF-kB pathway and activation of the immunosuppressive noncanonical NFkB pathway - may result in suppressive immune responses similar to what was observed for the interaction between melanoma- specific CD4+ T-cells following cognate interaction with HLA class II expressing melanoma cells (57). This would also explain why HPV- specific CD4+ T-cells isolated from patients have a low production of IFN ⁇ and often are of a regulatory T-cell type (5, 15, 17).
• HPV16 infected keratinocytes were cultured for two passages on Mitomycin C treated 3T3J2 mouse fibroblast cells in E-medium and afterwards adapted to serum-free Keratinocyte Serum Free growth Medium (K-SFM) for one passage before they were used in experiments.
• HPV16 cells were seeded 7.5x104 cells/well to a 12-wells plate in K-SFM and were allowed to attach over night. Medium was replaced by infection medium (K-SFM + 30% virus supernatant), containing either the lentivirus expressing shRNA against UCHLl (LV079 virus in IMDM 5% FCS) or as a control a lentivirus expressing shRNA against TurboGFP (SHC004).
• HPVl 6 cells were infected over night after which infection medium was replaced by K-SFM containing 1000 ng/ml puromycin to select for HPV16 cells that were successfully infected with lentivirus. After 48 hours the medium was replaced by K-SFM without puromycin and cells were allowed to grow for another 24 hours.
• lentivirus-infected HPVl 6 cells were given K-SFM containing either no PoIy(LC) (two wells) or 25 ug/ml PoIy(LC) and were cultured for 21 hours. Then one of the two non- stimulated wells received 25 ug/ml PoIy(LC) and all cells were cultured for another 3 hours. Cells were harvested and total RNA was isolated. RNA expression analyses
• TaqMan PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems). Threshold cycle numbers (Ct) were determined with 7900HT Fast Real-Time PCR System (Applied Biosystems) and the relative quantities of mRNA per sample were calculated using the ⁇ Ct method as described by the manufacturer using GAPDH as the calibrator gene.
• Ct Threshold cycle numbers
• HPV infected cells display upregulated expression of UCH-Ll and a downregulated response of the poly I:C-activated PRR-pathways as reflected by a decreased response of the type I IFN (e.g. IFN ⁇ ) and the NFkB-pathways (e.g. IL-8, MIP3 ⁇ , RANTES, IL-l ⁇ ).
• the signals of the PRR are mediated via TRAF3 and TRAF6 and the downstream signaling pathways.
• TRAF3 is thought to primarily trigger the IFN ⁇ response genes, whereas TRAF6 triggers the NFkB-pathway ( Figure 1).
• the shRNA's used were obtained from the MISSION TRC-library of SIGMA-ALDRICH.
• the base vector pLKO.l-puro was developed at the Broad Institute as part of TRC.
• the MISSION shRNA clones are sequence-verified shRNA lentiviral plasmids (pLKO.l-puro) provided as frozen bacterial glycerol stocks (Luria Broth, carbenicillin at 100 ⁇ g/ml and 10% glycerol) in Escherichia coli for propagation and downstream purification of the shRNA clones.
• pLKO.l contains the puromycin selection marker for transient or stable transfection.
• the plasmids may be used to generate lentiviral particles in packaging cell lines.
• TRCN0000007273 The following validated constructs against UCH-Ll (NM_004181) were used: TRCN0000007273:
• TRCN0000007274 CCGGCGGGTAGATGACAAGGTGAATCTCGAGATTCACCTTGTCATCTACC CGTTTTT,
• CTGTTTTT As a control we made use of:
• SHC004 (MISSION TRC2-pLKO puro TurboGFP shRNA Control vector) CCGGCGTGATCTTCACCGACAAGATCTCGAGATCTTGTCGGTGAAGAT CACGTTTTT
• the DNA plasmids are mixed, CaCk (100 ⁇ l) is added, the sample is adjusted with MQ to 1000 ⁇ l and then slowly added to 1000 ⁇ l HEBS (2x, filtered pH 7.04) and gently mixed. After 10 minutes this was added to the mediumand the cells are incubated overnight in 37°C / 5% CO2 incubator.
• the virus-containing supernatant was harvested by collecting medium to a sterile 50 ml tube.
• the medium with the lentivirus is filtered through a sterile 0.45 ⁇ m filter.
• the viruses are, aliquotted in tubes of ⁇ 10 ml each and stored at -80 0 C. Additionally a small aliquot (volume 10 ⁇ l), is used for determination of the virus stock by p24 Elisa assay. When needed virus stocks are concentrated (10 - 200 fold) by ultracentrifugation.
• P24 ELISA assay is performed by using the RETRO-TEK HIV-I p24 Antigen ELISA from ZeptoMetrix (ZMC Catalog *: 0801200; ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacture's protocol;
• HPV16-infected keratinocytes HPV16-infected keratinocytes (HPVl 6 cells).
• HPV16-infected keratinocytes HPV16-infected keratinocytes (HPVl 6 cells).
• UCH-Ll was effectively downregulated by lentivirus expressing shRNA against UCHLl (LV079) and not by the control lentivirus SCH004 ( Figure 6).
• LV079 resulted in strong downregulation of >80% of UCHL-I. With respect to this targeted downregulation it did not matter if the HPV16 cells were stimulated with poly I:C or not.
• LV079-infected HPVl 6 cells were able to respond again towards poly I:C stimulation by producing both the type I interferon ⁇ as well as several products of the NFkB pathway known to be important for direct antiviral responses (e.g. RANTES) or to support the attraction and activation of adaptive immunity (IL-8, MIP3 ⁇ , IL-l ⁇ ).
• LV079-infected HPV16 cells were treated for 0, 3 or 24 hours with poly I:C before RNA was isolated and tested.
• SHC004-infected HPVl 6 cells were used.
• suppression of UCH-Ll results in the strong reexpression of IFN ⁇ quickly after poly I:C stimulation.
• UCH-Ll may act as a deubiquitinating enzyme as well as a ligase.
• Deubquitation of K63-ubiquitines can result in loss of function whereas ligase of K48-ubiquitines may target a protein for proteasomal degradation.
• UCH-Ll binds directly to TRAF3 and TRAF6.

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