**5.1 Small molecules and natural products with anti measles activity**

Ribavirin. This pro-drug analogue of ribonucleosides with a broad antiviral spectrum has been used alone or combined with Interferon-alfa by intra-ventricular administration for SSPE patients with variable results, transient benefit at best, and undesired effects. Experimental results in MV intra-cranial infected hamsters and mice have shown that complexation of ribavirin with cyclodextrin-alfa reduced five-fold the 50% inhibitory dose and improved crossing of the brain-blood-barrier (Jeulin et al., 2009), and could improve the treatment with ribavirin in MV encephalitis.

Vitamin A. Supplements of Vitamin A significantly reduce measles mortality and morbidity, especially in children younger than 2 years of age, and it is the treatment recommended by WHO for children suffering from acute measles (Joint WHO-UNICEF statement-1987- Vitamin A for measles. Wkly Epidemiol Rec 19,133-134). There are indications of Vitamin A playing a role in the innate immune response, particularly in Interferon I signalling pathway, and it has been reported that retinoids directly inhibit MV replication in cultured cells (Trottier et al, 2009). On the other hand, it has been reported about one third of a SSPE (6 of 21) and (0 of 20 matched controls) showed low levels (<20micrograms per dL) of vitamin A (Gugor et al, 2007). It remains an open question whether Vitamin A supplements might implement the Intereferon treatment in some SSPE patients.

Inhibitors of MV entry or MV RNA-polymerase. During the last decade a number of small molecules strong inhibitors of MV entry or viral RNA-dependent RNA polymerase as AS-136A have being designed by R. Compans, R. Plemper & collaborators. Targets in MV RNApolymerase L protein catalytic subunit were identified studying in cell cultures MV escape mutants to the antiviral. The emergence of MV escape mutants could be a draw-back in

Subacute Sclerosing Panencephalitis and Other Lethal Encephalitis

Caused by Measles Virus Infection: Pathogenesis and New Approaches to Treatment 175

Fig. 8. Inhibition by small interfering RNAs (siRNAs) of MV gene expression and viral replication during acute and persistent infections. Based on conserved sequences among MV primary isolates from patients with acute measles or SSPE we have designed siRNAs

Hemagglutinin (siRNA H86 and H11). Human epithelial 293 cells lytically infected with

chemically synthesized siRNAs using Lipofectamin 2000. Upper panel. Quantitative assay to measure hemagglutinin gene silencing by siRNA. MV-H mRNA was assayed by quantitative reverse-transcriptase-polymerase-chain-reaction using SYBR Green core reagents from Applied Biosystems after primer optimization and Actine or GAPDH as endogenous controls in a ABI Prism7000 Sequence Detection System. Each column shows the relative quantification for Hemagglutinin mRNA following transfection of 293-FV-P persistently infected infected cells with the indicated siRNA.Lower panel. Effects of siRNA-P12 and siRNA H86 on production of infective extracellular MV during acute and persistent infections. Cell supernatants were titrated by plaque assay on B95 cells adapted

ds-oligonucleotides complementary to MV Phosphoprotein (siRNA P12) and

in our laboratory to grow in monolayers.

Edmonston virus or persistently infected with MV isolates were transfected with

potential long treatments as for SSPE. It remains to test these drugs in MV persistent infections in animal models for SSPE (for a review, Plemper & Snyder, 2009).

#### **5.2 Interferon-α**

In SSPE patients treatments with intraventricular Interferon-α alone or in combination with ribavirine or isoprinosine produce at best transient effects, and in some cases severe toxic effects (for review, Garg, 2008; Nacagawa et al 2009). Possibly, the low antiviral effect of Interferon-α treatment could be explained by the MV inhibition of antiviral response to exogenous Interferon I in the infected cell (Ortego, 1994 Fernandez-Muñoz et al, 2000). We found that MV inhibited the antiviral response to IFN I by blocking the signal transduction from the IFN I-receptor (Liton, 2001), and that MV non-structural protein V is associated with this inhibition (Celma and collaborators, unpublished results; Palosaari et al. 2003)). Silencing the V protein expression by anti-sense RNA oligonucleotides or RNA interference may be a way to increase IFN I antiviral effect in SSPE treatment. With this aim we have designed siRNAs that block MV P gene expression that could render MV persistently infected cells sensitive to IFN-α (see below). Another possible factor for the low antiviral response to Interferon I in SSPE patients could be the functional MxA promoter polymorphisms associated with SSPE (Torisu et al, 2004).

#### **5.3 RNA interference to control progression of SSPE and MIBE**

The gene suppression effects mediated transiently by short interfering RNA molecules (siRNA) or stably by intracellular expression of short hairpin RNAs (shRNAs) wich are processed by the cellular RNAi machinery (for a review Dykxhoorn et al. 2008) into effective siRNAs, are currently being tested as therapy for acute virus infections such as RSV end for chronic infections as HIV, Hepatitis B virus and Hepatitis C virus. To determine whether exogenous siRNA could inhibit the expression of MV genes and suppress viral replication during acute and persistent infections, we have designed siRNA molecules targeting conserved sequences in the genome of MV in brain of SSPE patients which inhibit the expression of MV Phosphoprotein gene, involved in viral RNA transcription, replication, and IFN response, and Hemagglutinin gene(H), playing a critical role in adsorption, cell fusion, assembly and budding of viral particles (Martín-Cortes et al, 2004 and Celma and coll. unpublished results). As shown in Figure 8 these siRNAs efficiently inhibit the production of MV infective particles in acute and persistently infected cells and indicates could be an useful tool for antiviral therapies by themselves or in combination with others MV specific siRNAs (Reuter et al. 2006; Otaki et al. 2007; Keita et al. 2008). For an efficient siRNA therapy besides a high gene target specificity it will be necsesary to solve problems of siRNA delivery and undesirable toxic site effects, as discussed by Rossi et al 2009.

#### **5.4 Inducers of apoptosis in MV infected cells as a potential early treatment of SSPE and MIBE**

Formation of syncitia by inducing cell fusion is the prominent cytopathic effect of MV in cultured cells (Enders, 1954) and in patients with measles giant cell pneumonia. In our laboratory by infecting a series of human lymphoblastoid cell lines with a MV strain showing low cell fusion activity, we observed that infected MOLT3 cell line underwent an atypical rapid cytopathic effect without a significant formation of syncytia that we described at the 1988 Negative Strand Viruses Conference (Fernandez-Muñoz et al. 1988).

174 Non-Flavivirus Encephalitis

potential long treatments as for SSPE. It remains to test these drugs in MV persistent

In SSPE patients treatments with intraventricular Interferon-α alone or in combination with ribavirine or isoprinosine produce at best transient effects, and in some cases severe toxic effects (for review, Garg, 2008; Nacagawa et al 2009). Possibly, the low antiviral effect of Interferon-α treatment could be explained by the MV inhibition of antiviral response to exogenous Interferon I in the infected cell (Ortego, 1994 Fernandez-Muñoz et al, 2000). We found that MV inhibited the antiviral response to IFN I by blocking the signal transduction from the IFN I-receptor (Liton, 2001), and that MV non-structural protein V is associated with this inhibition (Celma and collaborators, unpublished results; Palosaari et al. 2003)). Silencing the V protein expression by anti-sense RNA oligonucleotides or RNA interference may be a way to increase IFN I antiviral effect in SSPE treatment. With this aim we have designed siRNAs that block MV P gene expression that could render MV persistently infected cells sensitive to IFN-α (see below). Another possible factor for the low antiviral response to Interferon I in SSPE patients could be the functional MxA promoter

The gene suppression effects mediated transiently by short interfering RNA molecules (siRNA) or stably by intracellular expression of short hairpin RNAs (shRNAs) wich are processed by the cellular RNAi machinery (for a review Dykxhoorn et al. 2008) into effective siRNAs, are currently being tested as therapy for acute virus infections such as RSV end for chronic infections as HIV, Hepatitis B virus and Hepatitis C virus. To determine whether exogenous siRNA could inhibit the expression of MV genes and suppress viral replication during acute and persistent infections, we have designed siRNA molecules targeting conserved sequences in the genome of MV in brain of SSPE patients which inhibit the expression of MV Phosphoprotein gene, involved in viral RNA transcription, replication, and IFN response, and Hemagglutinin gene(H), playing a critical role in adsorption, cell fusion, assembly and budding of viral particles (Martín-Cortes et al, 2004 and Celma and coll. unpublished results). As shown in Figure 8 these siRNAs efficiently inhibit the production of MV infective particles in acute and persistently infected cells and indicates could be an useful tool for antiviral therapies by themselves or in combination with others MV specific siRNAs (Reuter et al. 2006; Otaki et al. 2007; Keita et al. 2008). For an efficient siRNA therapy besides a high gene target specificity it will be necsesary to solve problems

of siRNA delivery and undesirable toxic site effects, as discussed by Rossi et al 2009.

**5.4 Inducers of apoptosis in MV infected cells as a potential early treatment of SSPE** 

Formation of syncitia by inducing cell fusion is the prominent cytopathic effect of MV in cultured cells (Enders, 1954) and in patients with measles giant cell pneumonia. In our laboratory by infecting a series of human lymphoblastoid cell lines with a MV strain showing low cell fusion activity, we observed that infected MOLT3 cell line underwent an atypical rapid cytopathic effect without a significant formation of syncytia that we described at the 1988 Negative Strand Viruses Conference (Fernandez-Muñoz et al. 1988).

infections in animal models for SSPE (for a review, Plemper & Snyder, 2009).

polymorphisms associated with SSPE (Torisu et al, 2004).

**5.3 RNA interference to control progression of SSPE and MIBE** 

**5.2 Interferon-α** 

**and MIBE** 


Fig. 8. Inhibition by small interfering RNAs (siRNAs) of MV gene expression and viral replication during acute and persistent infections. Based on conserved sequences among MV primary isolates from patients with acute measles or SSPE we have designed siRNAs ds-oligonucleotides complementary to MV Phosphoprotein (siRNA P12) and Hemagglutinin (siRNA H86 and H11). Human epithelial 293 cells lytically infected with Edmonston virus or persistently infected with MV isolates were transfected with chemically synthesized siRNAs using Lipofectamin 2000. Upper panel. Quantitative assay to measure hemagglutinin gene silencing by siRNA. MV-H mRNA was assayed by quantitative reverse-transcriptase-polymerase-chain-reaction using SYBR Green core reagents from Applied Biosystems after primer optimization and Actine or GAPDH as endogenous controls in a ABI Prism7000 Sequence Detection System. Each column shows the relative quantification for Hemagglutinin mRNA following transfection of 293-FV-P persistently infected infected cells with the indicated siRNA.Lower panel. Effects of siRNA-P12 and siRNA H86 on production of infective extracellular MV during acute and persistent infections. Cell supernatants were titrated by plaque assay on B95 cells adapted in our laboratory to grow in monolayers.

Subacute Sclerosing Panencephalitis and Other Lethal Encephalitis

Caused by Measles Virus Infection: Pathogenesis and New Approaches to Treatment 177

Fig. 9. Apoptosis in MV persistently infected cells treated with recombinant TRAIL.

cell caveolin was used as control of protein loading.

DNA fragmentation detection techniques.

cells.

Left panel. To measure the sensitivity of cells to recombinant TRAIL (tumour-necrosisfactor-related apoptosis inducing ligand) the expression of the uncleaved poly (ADP-ribose) polymerase (PARP)(113KD) substrate for apoptosis specific ICE-family proteases, and its cleaved product 89KD fragment were used as specific markers for apoptosis. Cells were untreated or treated with soluble human recombinant TRAIL with cross-linking enhancer or the killer TRAIL (His-tag) from Alexis, at concentrations and time indicated. Western blot analysis were performed in RIPA-cell extracts, normalized for protein concentration, run in SDS-PAGE , transferred to membranes end developed with specific antibodies. Staining of

293 cells were less sensitive to PARP cleavage induced by TRAIL than their persistent infected cell lines established with a vaccine strain or with a primary isolate. These finding in epithelial cells were further extended to human lymphoid B (Dakiki) and T (MOLT3)

Right panel. To study the sensitivity to exogenous TRAIL of 293 and 293 cells persistently infected with primary (FV) and attenuated (Ed) MV strains, cell apoptosis was stimated as chromatine condensation in more than 200 nuclei after acridine orange staining. The specificity of acridine orange assay has been previously established in MV infected cells by

Attempting to characterize this previously un-recognized MV cytopathic effect we found in MOLT3 infected cells chromatin condensation and DNA inter-nucleosoma fragmentation, hallmarks of the cell death mechanism described and named apoptosis by J.Kerr and collaborators in 1972. Measles virus can induce apoptotic cell death in cultured human cells and this process is mediated by over-expression of Fas membrane protein in MV infected lymphoid cells (Fernandez-Muñoz et al., Ninth International Conference on Negative Strand Viruses, Estoril, 1994). At this conference apoptosis induced by MV was also present by Dr D. Griffin in Vero cultured cells (Esolen et al, 1995; Caballero et al, 1996). Based in our previous observation of Fas (CD95) involvement in apoptosis caused by MV, we studied the effect of Fas ligand (FasL) and other analogs as TRAIL (TNF-related apoptosis-inducing ligand) on acute and persistently infected human cells. We observed that MV persistently infected cells were more sensitive to apoptosis induced by exogenous TRAIL than uninfected cells (Figure 9). This sensitization could be explained by the up-regulation of functional TRAIL receptors TRAIL-R1 and TRAIL-R2, and down-regulation of antiapoptotic factor bcl-2 and activation of protein-kinase Akt and NFkB (Duque et al, 2007, and unpublished results by Celma and collaborators). Since has been generally observed that cancer cells are more sensitive than normal cells to apoptosis induced by recombinant TRAIL this molecule has been object of numerous clinical trials. Although phase I trials have shown low TRAIL toxicity, the efficiency tests got mixed results, largely due to the development of tumours resistence to the action of TRAIL (Yagita et al 2004 for a review, Kim et al. 2008; Eaton et al, 2011). Given the lack of efficient therapies for the encephalitis caused by persistent infection for MV, MIBE and SSPE, we have proposed the potential use of TRAIL as an early treatment of these diseases with the object to kill selectively the cells where MV resides before the virus disseminates across the brain.

TRAIL and its receptors have been shown to play important roles in the immune response to viral infections and in immune surveillance of tumours and metastasis (Falschlehener et al, 2009). During the last decade several studies have shown that different viral infections sensitize cells to apoptosis induced by TRAIL. Thus, TRAIL-resistant fibroblasts could be sensitized to TRAIL-induced apoptosis by infection with human cytomegalovirus (Sedger et al. 1999). On the other hand, it was observed and strong up-regulation of TRAIL, TRAIL-R1, and TRAIL-R2 in response to respiratory syncytial virus in primary tracheal-bronchial cells, A549 and HEP-2 cells and, RSV-infected cells could be eliminated by TRAIL-expressing immune cells in vivo (Kotelkin et al 2003). Furthermore, TRAIL has been implicated in chronic HCV infection and HCV has been shown to sensitize human hepatocytes to TRAIL induced apoptosis (Lan et al, 2008). Thus, the approach of an early treatment with TRAIL could help to control persistent infections by different viruses. However, there are some motives for concern after recent results showing that TRAIL, in addition to anti-tumour activity, has immunomodulatory functions and it has been demonstrated that TRAIL can eliminate plasma cells in vitro and suppress antibody production in vivo. Therefore, it should be noted that a strategy to over-express endogenous TRAIL, as well as administration of rTRAIL may impair host defense against infection (Faschlehener et al, 2009). For treatments of SNC diseases, some findings in cultured brain slices raise concern about neuro-toxicity and argue against the use of TRAIL for therapy of human brain tumours (Nitsch et al, 2000). A recent study obtained successful results combining antipapillomavirus E6/E7 siRNA and TRAIL induction of apoptosis in cancer cells being refractory to TRAIL treatment (Eaton et al 2011).

Attempting to characterize this previously un-recognized MV cytopathic effect we found in MOLT3 infected cells chromatin condensation and DNA inter-nucleosoma fragmentation, hallmarks of the cell death mechanism described and named apoptosis by J.Kerr and collaborators in 1972. Measles virus can induce apoptotic cell death in cultured human cells and this process is mediated by over-expression of Fas membrane protein in MV infected lymphoid cells (Fernandez-Muñoz et al., Ninth International Conference on Negative Strand Viruses, Estoril, 1994). At this conference apoptosis induced by MV was also present by Dr D. Griffin in Vero cultured cells (Esolen et al, 1995; Caballero et al, 1996). Based in our previous observation of Fas (CD95) involvement in apoptosis caused by MV, we studied the effect of Fas ligand (FasL) and other analogs as TRAIL (TNF-related apoptosis-inducing ligand) on acute and persistently infected human cells. We observed that MV persistently infected cells were more sensitive to apoptosis induced by exogenous TRAIL than uninfected cells (Figure 9). This sensitization could be explained by the up-regulation of functional TRAIL receptors TRAIL-R1 and TRAIL-R2, and down-regulation of antiapoptotic factor bcl-2 and activation of protein-kinase Akt and NFkB (Duque et al, 2007, and unpublished results by Celma and collaborators). Since has been generally observed that cancer cells are more sensitive than normal cells to apoptosis induced by recombinant TRAIL this molecule has been object of numerous clinical trials. Although phase I trials have shown low TRAIL toxicity, the efficiency tests got mixed results, largely due to the development of tumours resistence to the action of TRAIL (Yagita et al 2004 for a review, Kim et al. 2008; Eaton et al, 2011). Given the lack of efficient therapies for the encephalitis caused by persistent infection for MV, MIBE and SSPE, we have proposed the potential use of TRAIL as an early treatment of these diseases with the object to kill selectively the cells

TRAIL and its receptors have been shown to play important roles in the immune response to viral infections and in immune surveillance of tumours and metastasis (Falschlehener et al, 2009). During the last decade several studies have shown that different viral infections sensitize cells to apoptosis induced by TRAIL. Thus, TRAIL-resistant fibroblasts could be sensitized to TRAIL-induced apoptosis by infection with human cytomegalovirus (Sedger et al. 1999). On the other hand, it was observed and strong up-regulation of TRAIL, TRAIL-R1, and TRAIL-R2 in response to respiratory syncytial virus in primary tracheal-bronchial cells, A549 and HEP-2 cells and, RSV-infected cells could be eliminated by TRAIL-expressing immune cells in vivo (Kotelkin et al 2003). Furthermore, TRAIL has been implicated in chronic HCV infection and HCV has been shown to sensitize human hepatocytes to TRAIL induced apoptosis (Lan et al, 2008). Thus, the approach of an early treatment with TRAIL could help to control persistent infections by different viruses. However, there are some motives for concern after recent results showing that TRAIL, in addition to anti-tumour activity, has immunomodulatory functions and it has been demonstrated that TRAIL can eliminate plasma cells in vitro and suppress antibody production in vivo. Therefore, it should be noted that a strategy to over-express endogenous TRAIL, as well as administration of rTRAIL may impair host defense against infection (Faschlehener et al, 2009). For treatments of SNC diseases, some findings in cultured brain slices raise concern about neuro-toxicity and argue against the use of TRAIL for therapy of human brain tumours (Nitsch et al, 2000). A recent study obtained successful results combining antipapillomavirus E6/E7 siRNA and TRAIL induction of apoptosis in cancer cells being

where MV resides before the virus disseminates across the brain.

refractory to TRAIL treatment (Eaton et al 2011).

Fig. 9. Apoptosis in MV persistently infected cells treated with recombinant TRAIL.

Left panel. To measure the sensitivity of cells to recombinant TRAIL (tumour-necrosisfactor-related apoptosis inducing ligand) the expression of the uncleaved poly (ADP-ribose) polymerase (PARP)(113KD) substrate for apoptosis specific ICE-family proteases, and its cleaved product 89KD fragment were used as specific markers for apoptosis. Cells were untreated or treated with soluble human recombinant TRAIL with cross-linking enhancer or the killer TRAIL (His-tag) from Alexis, at concentrations and time indicated. Western blot analysis were performed in RIPA-cell extracts, normalized for protein concentration, run in SDS-PAGE , transferred to membranes end developed with specific antibodies. Staining of cell caveolin was used as control of protein loading.

293 cells were less sensitive to PARP cleavage induced by TRAIL than their persistent infected cell lines established with a vaccine strain or with a primary isolate. These finding in epithelial cells were further extended to human lymphoid B (Dakiki) and T (MOLT3) cells.

Right panel. To study the sensitivity to exogenous TRAIL of 293 and 293 cells persistently infected with primary (FV) and attenuated (Ed) MV strains, cell apoptosis was stimated as chromatine condensation in more than 200 nuclei after acridine orange staining. The specificity of acridine orange assay has been previously established in MV infected cells by DNA fragmentation detection techniques.

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