Final Report Summary - ORALANTINOROVIRUS (Development of an oral anti-norovirus therapy)
Project context and objectives
The main aim of the present project was to establish new antiviral approaches to control human norovirus (HuNoV) infections. With this aim I planned to develop an invasive bacteria methodology to deliver therapeutic molecules in vivo against norovirus. Since HuNoV infections are not cultivable, murine norovirus (MNV) is typically employed as a surrogate. The recent discovery of MNV-3 which establishes persistent infections in wild type mice and symptoms resembling gastroenteritis in STAT -/-mice encouraged us to make use of this new viral strain for our experiments.
Project work and results
To this aim, we firstly had to establish a combined reverse genetics approach and animal model system unavailable at the moment.
We have been able to recover infectious MNV-3 by transfecting a plasmid containing the full MNV-3 cDNA preceded by a T7 promoter sequence into BHK-21 derived cells (BSR-T7 cells). The cells were previously infected with a helper fowl-pox virus expressing recombinant T7 RNA polymerase to allow viral transcription. BHK-21 cells support viral transcripts replication but not successive rounds of infection so this approach has generated a low-titre MNV-3 stock resulting from a single cycle of replication. The viral sample recovered has been amplified through serial passages in RAW264.7 cells which are susceptible to infection. MNV-3 recovered by reverse genetics has been used to orally infect four to five-week-old C57BL/6 male mice (Arias et al 2012, JGV doi:10.1099/vir.0.042176-0). We have determined by different approaches (titrations, RT-PCR) that MNV-3 establishes persistent infections in mice for at least 56 days (although preliminary results suggest that can persist in vivo for at least nine months). The minimum infectious dose has been shown to be less than 10 TCID50, which highlights the high infectivity in vivo of the sample recovered by reverse genetics. Interestingly, we have identified that MNV-3 persists mainly in the colon and the caecum, in contrast with previous data for acute MNV-1 infection (mesenteric lymph node, liver, small intestine and spleen). This is a relevant result in the light of recent studies which have related ulcerative colitis, which affects the colon, and HuNoV persistent infections in patients suffering from exacerbation of inflammatory bowel disease (Khan et al 2009, J Pediatr Gastroenterol Nutr, 48:328). The analysis of full genome sequences recovered from persistently infected mice has identified that the molecular determinants for MNV persistence in vivo lie mainly in the major and minor capsid proteins as well as in the polymerase gene (Arias et al. 2012).
Development of invasive bacteria methodology to inhibit MNV-3 in vivo: we have prepared inhouse a TRIP plasmid (from trans-kingdom RNAi plasmid) required for conferring invasive properties to non-invasive E. coli strains and facilitate the delivery of therapeutic RNAs in target cells. With this aim, we have introduced listeriolysin O (Hly) and invasin (Inv) genes from a low-copy number plasmid (pGB2#hly-inv) provided by Prof. Patrice Courvalain into a high-copy number pET26 plasmid. The invasive plasmid prepared has conferred E. coli BL21 (DE3) strain with 10-100 larger invasiveness than bacteria containing pET26 without Hly and Inv. This has been demonstrated by colony-counting assays and DAPI staining of cells infected with bacteria. We have also observed an increase of ~100-fold in bacterial reporter RNA in cells treated with invasive bacteria with respect to cells treated with non-invasive bacteria.
Hly and Inv genes contained in TRIP plasmid allow the invasion and delivery of bacterial RNA into the cytoplasm of invaded cells. Inv allows the invasion of mammalian cells while Hly is contained in the lumen of invasive bacteria and form pores in the endosome engulfing the bacteria when this is lysed. The pores allow bacterial nucleic acids (i.e. shRNAs) being released into the cytoplasm. This approach has been successfully used to down-regulate beta-catenin in vivo, overexpressed in Familial Adenomatous Polyposis (colon cancer). The potential application against colon cancer is now in a clinical phase of study (Marina Biotech).
We are now aiming to use therapeutic RNAs encoded by TRIP to inhibit MNV-3 replication. In the course of this project we have identified three out of six siRNA sequences tested that efficiently inhibit MNV-3. We are now designing different TRIP encoding shRNAs based on these siRNA sequences identified. We are also designing shRNAs against various host factors that have been identified to be required for virus replication (e.g. PTB). In addition, in collaboration with Dr Nicola Stonehouse we have identified various RNA aptamers selected against MNV VPg that can be cloned as well in TRIP and tested for their inhibitory capacity upon MNV-3 replication. All these different molecules are going to be tested in cell culture to make a shortlist of the best candidates to be further investigated in vivo.
Expected results and impact
The establishment of combined reverse genetics and a mouse model system presented here constitute an important step forward in the study of norovirus molecular biology in vivo. This new animal model system also provides a more adequate animal model to be used as surrogate of HuNoV in vivo than previous models based on MNV-1 lethal infection. Moreover, this system will permit investigations making use of a genetically defined MNV. Since the phenotype of a given RNA virus in vivo is largely dependent on its genome stability it is important to make use of genetically defined MNV genomes that are provided by the reverse genetics system established here. Hence, the relevance of this new combined reverse genetics/in vivo model system that we have set up in Imperial College London.
in addition, we are working on the development of an invasive bacteria approach to control murine norovirus infections in vivo which might later be applied to different translational research projects such as:
-Control of human norovirus infections in vivo. Given the large magnitude of norovirus outbreaks in hospitals, we envisage that the administration of therapeutic invasive bacteria to affected patients can control the dissemination and calibre of the outbreak. Moreover, we foresee that the prophylactic administration of invasive bacteria on persons in contact with affected patients (e.g. non-infected patients, nurses, hospital maintenance workers, etc.) can limit the impact of a potential outbreak in a hospital.
-Therapeutics for patients suffering from chronic exacerbation of inflammatory bowel disease (EIBD). Since a direct correlation between EIBD and persistent shedding of HuNoV has been recently established (Khan et al 2009), we hypothesise that the administration of therapeutic invasive bacteria can potentially result in clearing the infection and/or alleviate the symptoms.
-Expression of human norovirus capsid in vivo as a possible vaccine candidate. Therapeutic bacteria expressing RNA coding for HuNoV capsid under an IRES sequence could be delivered into intestine cells in humans to elicit an immune response against HuNoV. No vaccines are currently available to control HuNoV infections.
-Treatment of other enteric pathogens. Possible applications of therapeutic invasive bacteria commented above could be transferred for the treatment of other important enteric pathogens such as rotavirus, enterovirus 71, and Coxsackie virus.
The main aim of the present project was to establish new antiviral approaches to control human norovirus (HuNoV) infections. With this aim I planned to develop an invasive bacteria methodology to deliver therapeutic molecules in vivo against norovirus. Since HuNoV infections are not cultivable, murine norovirus (MNV) is typically employed as a surrogate. The recent discovery of MNV-3 which establishes persistent infections in wild type mice and symptoms resembling gastroenteritis in STAT -/-mice encouraged us to make use of this new viral strain for our experiments.
Project work and results
To this aim, we firstly had to establish a combined reverse genetics approach and animal model system unavailable at the moment.
We have been able to recover infectious MNV-3 by transfecting a plasmid containing the full MNV-3 cDNA preceded by a T7 promoter sequence into BHK-21 derived cells (BSR-T7 cells). The cells were previously infected with a helper fowl-pox virus expressing recombinant T7 RNA polymerase to allow viral transcription. BHK-21 cells support viral transcripts replication but not successive rounds of infection so this approach has generated a low-titre MNV-3 stock resulting from a single cycle of replication. The viral sample recovered has been amplified through serial passages in RAW264.7 cells which are susceptible to infection. MNV-3 recovered by reverse genetics has been used to orally infect four to five-week-old C57BL/6 male mice (Arias et al 2012, JGV doi:10.1099/vir.0.042176-0). We have determined by different approaches (titrations, RT-PCR) that MNV-3 establishes persistent infections in mice for at least 56 days (although preliminary results suggest that can persist in vivo for at least nine months). The minimum infectious dose has been shown to be less than 10 TCID50, which highlights the high infectivity in vivo of the sample recovered by reverse genetics. Interestingly, we have identified that MNV-3 persists mainly in the colon and the caecum, in contrast with previous data for acute MNV-1 infection (mesenteric lymph node, liver, small intestine and spleen). This is a relevant result in the light of recent studies which have related ulcerative colitis, which affects the colon, and HuNoV persistent infections in patients suffering from exacerbation of inflammatory bowel disease (Khan et al 2009, J Pediatr Gastroenterol Nutr, 48:328). The analysis of full genome sequences recovered from persistently infected mice has identified that the molecular determinants for MNV persistence in vivo lie mainly in the major and minor capsid proteins as well as in the polymerase gene (Arias et al. 2012).
Development of invasive bacteria methodology to inhibit MNV-3 in vivo: we have prepared inhouse a TRIP plasmid (from trans-kingdom RNAi plasmid) required for conferring invasive properties to non-invasive E. coli strains and facilitate the delivery of therapeutic RNAs in target cells. With this aim, we have introduced listeriolysin O (Hly) and invasin (Inv) genes from a low-copy number plasmid (pGB2#hly-inv) provided by Prof. Patrice Courvalain into a high-copy number pET26 plasmid. The invasive plasmid prepared has conferred E. coli BL21 (DE3) strain with 10-100 larger invasiveness than bacteria containing pET26 without Hly and Inv. This has been demonstrated by colony-counting assays and DAPI staining of cells infected with bacteria. We have also observed an increase of ~100-fold in bacterial reporter RNA in cells treated with invasive bacteria with respect to cells treated with non-invasive bacteria.
Hly and Inv genes contained in TRIP plasmid allow the invasion and delivery of bacterial RNA into the cytoplasm of invaded cells. Inv allows the invasion of mammalian cells while Hly is contained in the lumen of invasive bacteria and form pores in the endosome engulfing the bacteria when this is lysed. The pores allow bacterial nucleic acids (i.e. shRNAs) being released into the cytoplasm. This approach has been successfully used to down-regulate beta-catenin in vivo, overexpressed in Familial Adenomatous Polyposis (colon cancer). The potential application against colon cancer is now in a clinical phase of study (Marina Biotech).
We are now aiming to use therapeutic RNAs encoded by TRIP to inhibit MNV-3 replication. In the course of this project we have identified three out of six siRNA sequences tested that efficiently inhibit MNV-3. We are now designing different TRIP encoding shRNAs based on these siRNA sequences identified. We are also designing shRNAs against various host factors that have been identified to be required for virus replication (e.g. PTB). In addition, in collaboration with Dr Nicola Stonehouse we have identified various RNA aptamers selected against MNV VPg that can be cloned as well in TRIP and tested for their inhibitory capacity upon MNV-3 replication. All these different molecules are going to be tested in cell culture to make a shortlist of the best candidates to be further investigated in vivo.
Expected results and impact
The establishment of combined reverse genetics and a mouse model system presented here constitute an important step forward in the study of norovirus molecular biology in vivo. This new animal model system also provides a more adequate animal model to be used as surrogate of HuNoV in vivo than previous models based on MNV-1 lethal infection. Moreover, this system will permit investigations making use of a genetically defined MNV. Since the phenotype of a given RNA virus in vivo is largely dependent on its genome stability it is important to make use of genetically defined MNV genomes that are provided by the reverse genetics system established here. Hence, the relevance of this new combined reverse genetics/in vivo model system that we have set up in Imperial College London.
in addition, we are working on the development of an invasive bacteria approach to control murine norovirus infections in vivo which might later be applied to different translational research projects such as:
-Control of human norovirus infections in vivo. Given the large magnitude of norovirus outbreaks in hospitals, we envisage that the administration of therapeutic invasive bacteria to affected patients can control the dissemination and calibre of the outbreak. Moreover, we foresee that the prophylactic administration of invasive bacteria on persons in contact with affected patients (e.g. non-infected patients, nurses, hospital maintenance workers, etc.) can limit the impact of a potential outbreak in a hospital.
-Therapeutics for patients suffering from chronic exacerbation of inflammatory bowel disease (EIBD). Since a direct correlation between EIBD and persistent shedding of HuNoV has been recently established (Khan et al 2009), we hypothesise that the administration of therapeutic invasive bacteria can potentially result in clearing the infection and/or alleviate the symptoms.
-Expression of human norovirus capsid in vivo as a possible vaccine candidate. Therapeutic bacteria expressing RNA coding for HuNoV capsid under an IRES sequence could be delivered into intestine cells in humans to elicit an immune response against HuNoV. No vaccines are currently available to control HuNoV infections.
-Treatment of other enteric pathogens. Possible applications of therapeutic invasive bacteria commented above could be transferred for the treatment of other important enteric pathogens such as rotavirus, enterovirus 71, and Coxsackie virus.