Dissertations / Theses on the topic 'Vector viruses'

In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels.
Ph. D.

7 p.
There are a number of disease-causing viruses transmitted to people primarily through the bite of infected mosquitoes. Female mosquitoes take blood meals to produce eggs. A mosquito that bites an infected animal may pick up a virus within the blood meal. If the mosquito is the appropriate species, and conditions inside the insect and the surrounding environment are supportive, the virus reproduces within the mosquito. Later, the mosquito may pass the virus on to other animals (including humans) as they feed again.

Potato is one of the most important food crops in the world, and viruses are largely responsible for the degeneration of this vegetatively propagated crop. At least 35 viruses have been reported to infect potato naturally. The majority and the most economically important ones are vectored by aphids. The objective of this study was to conduct molecular and biological investigations into virus transmission mechanisms, including developing diagnostic methods to help to control the spread of aphid-borne potato viruses, and disrupting the vectoring ability of their aphid vectors by insecticide spray. One way to control the spread of aphid-borne viruses is to control their aphid vector, but this is not always feasible as the majority of aphid-borne potato viruses, including the most important ones, are transmitted non-persistently, being acquired within a very short time before agrochemicals can act. Thus an alternative approach to controlling this class of viruses is through a full understanding of the interaction between the virus, the host plant and the aphid vector, which was the first objective of this project. In this respect, some aphid cuticle proteins were identified to interact with potato virus Y helper component (HC-Pro) through screening of an aphid cDNA expression library, and their potential role in virus transmission was discussed. Moreover, the concept of short retention of non-persistent viruses inside their aphid vectors was challenged; the results show that PVY can be retained inside its aphid vector for a long time but it is not transmissible. This novel finding together with binding to aphid cuticle proteins, generated some new ideas about transmission mechanisms that were proposed and discussed. In addition, the effect on aphid vectoring ability of the plants used to rear aphid colonies, as a virus source, and as a virus recipient was investigated. From laboratory studies of aphid transmission, it was concluded that the transmission efficiency of PVY was significantly affected by the host plant species used to rear M. persicae, or that used as a virus recipient plant. The availability of sensitive and cheap virus detection methods is critical for early detection and control of potato viruses. In this project a sensitive fully recombinant ELISA was developed and validated for routine testing of potato leafroll virus. This technology can be applied to detect other potato viruses and has the potential to replace the commonly used immune reagent antisera.

Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied. A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV. To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP). Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants. Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones. To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea. Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows: * pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene * pFLP1gfp, pFL with gj27 gene fused to the 3' end of the ORF 1, * pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable and gene cloned in tandem between the CP gene and the 3' NCR of SCMoV, * pFLCPVsgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and * pFLREPsgprogfp, pFL with the ORF2b, a putative sgPro sequence and a translatable gfP gene cloned in tandem between a truncated CP gene and the 3' NCR These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.

Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied. A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV. To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP). Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants. Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones. To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea. Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows: pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene, pFLP 1 gfp, pFL with gj27 gene fused to the 3 ' end of the ORF 1, pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable & gene cloned in tandem between the CP gene and the 3' NCR of SCMoV, pFLCPVsgprogf$, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and pFLREPsgprog@, pFL with the ORF2b, a putative sgPro sequence and a translatable &fP gene cloned in tandem between a truncated CP gene and the 3' NCR These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.

Ribonuclease T1 oligonucleotide fingerprint (ONF) analysis was used to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following oral ingestion and replication) from the natural mosquito host, Aedes triseriatus. Genomic changes were not detected during transovarial passage of virus through two generations of mosquitoes or in virus isolated from suckling mice infected by transovarially infected mosquitoes. These results demonstrate that the La Crosse virus genome can remain relatively stable during transovarial transmission (TOT) in the insect host and during transfer between insect and vertebrate hosts. ONF analysis was used to demonstrate TOT of reassortant California serogroup bunyaviruses in Aedes treiseriatus. Mosquitoes were simultaneously inoculated with temperature sensitive mutants of La Crosse and Snowshoe hare viruses able to replicate at 33°C but not at 40°C. Putative reassortants, selected by replication at 40°C, were isolated from progeny mosquitoes and mice infected by these mosquitoes. ONF analysis confirmed that they were reassortants. Approximately 75% of the M segment and 25% of the L segment nucleotide sequences of Inkoo virus (Bunyaviridae) were determined by Sanger dideoxynucleotide sequencing of cDNA clones. Comparison of the M segment nucleotide and deduced amino acid sequences with those of four other bunyaviruses, representing two serogroups, revealed greater conservation of nucleotide than of amino acid sequence between serogroups. Areas of the sequences representing nonstructural protein(s) were less conserved than glycoprotein regions. The L segment nucleotide sequence begins with the known 3' end of the viral L segment and contains an open reading frame encoding the amino terminal 505 amino acids of the viral L protein. The amino acid sequence contains the glycine-aspartate-aspartate motif which is conserved in many RNA-dependent RNA polymerases. Comparison of the L segment sequences with those in the GEN Bank Data Base revealed no significant similarities with any other sequence.

Gene therapy is the introduction or alteration of genetic material with the intention to treat disease. To support this aim, viruses have been modified, with elements linked to viral pathogenicity removed from their genome and replaced by the genetic material to be delivered. Gene therapy vectors based on lentiviruses have many advantages, such as the ability to transduce non-dividing cells and to target specific cell types via pseudotyping. They have been successfully used in ex vivo clinical trials for several haematopoietic stem cell disorders. Lentiviral vectors, however, suffer from substantially lower titres than the more popular adeno-associated virus (AAV)-based vectors and therefore have limited applicability for in vivo gene therapy which requires much greater quantities of virus. The main aim of this thesis was to investigate strategies to improve lentiviral vector productivity during manufacture, in order to increase the likelihood of lentiviruses being adopted for disease treatment. Initial experiments were based on the lentiviral vector manufacturing process currently being developed by the United Kingdom Cystic Fibrosis Gene Therapy Consortium for the generation of highly concentrated, purified lentivirus for clinical use. Supplementation of FreeStyle 293 Expression Medium used during upstream processing was attempted, but none of the assessed supplements led to significant increases in lentiviral vector production. Investigation into intrinsic immunity to viral infection indicated that over-expression of the protein kinase RNA-activated (PKR) led to lower production titres, but over-expression of its inhibitors was not successful at increasing titres. The focus then shifted to reducing, or 'knocking-down', inhibitory factors present in the host cells, which could adversely affect viral titres. Investigation of the published HIV-1 literature revealed a possible 152 candidate inhibitory factors described as having a negative impact on HIV-1 replication in the late stages of the life cycle of the virus. A novel siRNA screen was developed to assess the effect of ‘knock-down' of inhibitory factors on lentiviral vector titre. Application of the screen to 89 candidate inhibitory factors identified nine genes which, when knocked-down, resulted in increased lentiviral vector production by more than 40%. Further work will be necessary to understand the role of the inhibitory factors in lentiviral vector production, but novel cell lines in which genes encoding these factors have been permanently deleted from producer cells could lead to higher titres, reducing costs in the manufacture of lentiviral vectors and making in vivo gene therapy more feasible from a health economics perspective.

L’atàxia de Friedreich (FRDA) és una malaltia neurodegenerativa, hereditària autosòmica recessiva, causada per la deficiència de la proteïna frataxina (FXN) deguda a l’expansió patològica del triplet GAA intrònic en el gen FXN. Es caracteritza principalment per una atàxia progressiva, pèrdua sensorial i cardiomiopatia hipertròfica. La neurodegeneració s’identifica en les neurones dels DRG, els nervis sensorials perifèrics, les columnes posteriors i el nucli dentat cerebel·lós. En l’actualitat no existeix tractament, però diverses proves de concepte han evidenciat el potencial terapèutic de la teràpia gènica amb AAV per a la FRDA. No obstant, aquests estudis han utilitzat vies d’administració dels AAV ineficients per tractar la neurodegeneració i han utilitzat promotors sintètics que proporcionen nivells molt elevats de FXN. Dades recents assenyalen la importància de mantenir els nivells de frataxina dins d’un rang fisiològic a causa de la toxicitat cel·lular que ha demostrat la seva sobreexpressió. En aquest estudi s’ha desenvolupat un nou vector de teràpia gènica AAV9 (rAAV9-FXN) amb una seqüència d’ADN que codifica per a la frataxina, regulada pel promotor hPGK1 i l’element WPRE. Una única administració intratecal de rAAV9-FXN en ratolins amb FRDA de 10 setmanes d’edat ha demostrat una àmplia biodistribució i transducció en els teixits neurològics afectats en la FRDA com els DRG, els nervis perifèrics, la medul·la espinal i també en els teixits sistèmics com el cor i el fetge. Aquesta estratègia de teràpia gènica proporciona una expressió de frataxina recombinant sostinguda i de llarga durada, amb nivells segurs, no tòxics, similars als nivells endògens de frataxina en ratolins WT durant almenys 22 mesos després de l’administració. Els ratolins amb FRDA tractats amb rAAV9-FXN han demostrat la prevenció significativa de les característiques patològiques de la malaltia com la coordinació motora, els potencials d’acció sensitius dels nervis perifèrics, el reflex d’agafada, la neurodegeneració en els DRG, la medul·la espinal i el nucli dentat cerebel·lós, juntament amb la correcció de la cardiomiopatia, la reducció de l’acumulació de dipòsits de ferro i la preservació de la funció mitocondrial mantenint l’activitat de la cadena de transport d’electrons. En conclusió, aquest estudi demostra l’eficàcia terapèutica de l’administració intratecal del vector rAAV9-FXN per al tractament a nivell preclínic d’un model de ratolí amb la malaltia i proporciona evidencia del potencial terapèutic per al tractament de l’atàxia de Friedreich.
La ataxia de Friedreich (FRDA) es una enfermedad neurodegenerativa, hereditaria autosómica recesiva, causada por la deficiencia de la proteína frataxina (FXN) debida a la expansión patológica del triplete GAA intrónico en el gen FXN. Se caracteriza principalmente por una ataxia progresiva, pérdida sensorial y cardiomiopatía hipertrófica. La neurodegeneración se identifica en las neuronas de los DRG, los nervios sensoriales periféricos, las columnas posteriores y el núcleo dentado cerebeloso. En la actualidad no existe tratamiento, pero varias pruebas de concepto han evidenciado el potencial terapéutico de la terapia génica con AAV para la FRDA. Sin embargo, estos estudios han utilizado vías de administración de los AAV ineficientes para tratar la neurodegeneración y han utilizado promotores sintéticos que proporcionan niveles muy elevados de FXN. Datos recientes señalan la importancia de mantener los niveles de frataxina dentro de un rango fisiológico debido a la toxicidad celular que ha demostrado su sobreexpresión. En este estudio se ha desarrollado un nuevo vector de terapia génica AAV9 (rAAV9-FXN) con una secuencia de ADN que codifica para la frataxina, regulada por el promotor hPGK1 y el elemento WPRE. Una única administración intratecal de rAAV9-FXN en ratones con FRDA de 10 semanas de edad ha demostrado una amplia biodistribución y transducción en los tejidos neurológicos afectados en la FRDA como los DRG, los nervios periféricos, la médula espinal y también en los tejidos sistémicos como el corazón y el hígado. Esta estrategia de terapia génica proporciona una expresión de frataxina recombinante sostenida y de larga duración, con niveles seguros, no tóxicos, similares a los niveles endógenos de frataxina en ratones WT durante al menos 22 meses después de la administración. Los ratones con FRDA tratados con rAAV9-FXN han demostrado la prevención significativa de las características patológicas de la enfermedad como la coordinación motora, los potenciales de acción sensitivos de los nervios periféricos, el reflejo de agarre, la neurodegeneración en los DRG, la médula espinal y el núcleo dentado cerebeloso, junto con la corrección de la cardiomiopatía, la reducción de la acumulación de depósitos de hierro y la preservación de la función mitocondrial manteniendo la actividad de la cadena de transporte de electrones. En conclusión, este estudio demuestra la eficacia terapéutica de la administración intratecal del vector rAAV9-FXN para el tratamiento a nivel preclínico de un modelo de ratón con la enfermedad y proporciona evidencia del potencial terapéutico para el tratamiento de la ataxia de Friedreich.
Friedreich's ataxia (FRDA) is a recessive inherited neurodegenerative disease caused by frataxin (FXN) deficiency due to the pathological intronic GAA-repeat expansion within the FXN gene. FRDA is characterised by progressive ataxia, sensory loss and hypertrophic cardiomyopathy. Neurodegeneration is identified in DRG neurons, peripheral sensory nerves, the posterior column, and cerebellar dentate nucleus. To date, there is no treatment available, but proof-of-concept approaches have highlighted the therapeutic potential of AAV-gene therapy in FRDA. However, these studies have used inefficient AAV delivery routes to treat the neurodegeneration, and used synthetic promoters providing high-yields of FXN levels. Recent data point to the importance of maintaining frataxin levels within a physiological range due to cellular toxicity that cause its overexpression. In this study, a new AAV9-gene therapy vector (rAAV9-FXN) has developed including a DNA sequence that codify for frataxin regulated by the hPGK1 promoter and the WPRE element. A single intrathecal administration of rAAV9-FXN into ten-week-old FRDA mice has demonstrated a wide biodistribution and transduction into affected neurological tissues in FRDA like DRG, peripheral nerves, spinal cord, and also systemic tissues as liver and heart. This gene therapy strategy provides sustained and long-term frataxin expression, with safe non-toxic levels, similar to endogenous frataxin levels in the WT mice at least 22 months after administration. rAAV9-FXN treated FRDA mice has demonstrated significant prevention of pathological characteristics of the disease as the motor coordination, the sensory action potentials of peripheral nerves, the hindlimb clasping reflex, prevents the neurodegeneration of the DRG, spinal cord and cerebellar dentate nucleus, along with cardiomyopathy correction, reduction of iron deposits accumulation and preservation of mitochondrial function by maintaining the electron transport chain activity. In conclusion, this study demonstrates the therapeutic efficacy of intrathecal administration of the rAAV9-FXN vector for the preclinical treatment of a mouse model with the disease and provides evidence for the clinical therapeutic potential for the treatment of Friedreich ataxia.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

There is an urgent need for improvements to existing vaccine delivery technologies to run parallel with the development of new-generation vaccines. The burdens of needle-based immunisation strategies are exacerbated by poor resource provision in such areas as sub-Saharan Africa, where annual malaria mortality stands at 860,000. Needle-free delivery of vaccine to the skin holds promise for improved immunogenicity with lower doses of vaccine, in addition to significant logistical advantages. Various methods have been described for the transcutaneous delivery of vaccines, including the use of microneedles to overcome the outer stratum corneum of the skin for efficient delivery of liquid or solid, microneedle-coated vaccines into underlying strata rich in antigen-presenting cells. This thesis aims to evaluate two transcutaneous silicon microneedle and microprojection patch technologies for the delivery of live recombinant Adenovirus and Modified Vaccinia Ankara-vectored vaccines encoding pre-erythrocytic malaria antigens in mice. Cellular immunogenicity directed against a well-documented epitope of the Plasmodium berghei circumsporozoite protein is evaluated, as is protection against lethal P. berghei sporozoite challenge. Immunological and logistical benefits of each technology are assessed, as well as mechanisms underlying differences in the generation of a patch-induced immune response to vaccination. These data inform the future development of transcutaneous microneedle patches for the delivery of live vaccine.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 230 p.; also includes graphics. Includes bibliographical references (p. 200-230). Available online via OhioLINK's ETD Center

Age stratified seroepidemiological studies play a crucial role in the design and assessment of vaccination strategies. An existing multiplex bead immunoassay for measles, mumps, rubella and varicella zoster virus antibodies together with a newly developed multiplex bead immunoassay for herpes simplex virus type 1 and type 2 antibodies were used to investigate the age-related seroepidemiology of these viruses in England during 2012.To develop the HSV-1 and HSV-2 antibody assay, attempts were made to produce full length of HSV-1 and HSV-2 glycoprotein G using a baculovirus vector expression system. While HSV-1 gG protein was produced, the proteins were extensively aggregated. Native glycoprotein G molecules undergo partial removal of HSV-1 signal sequence and HSV-1 short membrane anchor sequence during post translational modification. It is possible that such post translational modification is not performed when protein is processed in insect cell culture. Attempts to produce an HSV-2 glycoprotein G were not successful. It is possible that the high GC-content of HSV-2 glycoprotein G led to poor fidelity of copying the PCR amplification sequence. Commercially available truncated HSV-1 gG and HSV-2 gG were therefore used to develop a duplex microbead immunoassay for the simultaneous detection of specific HSV antibodies in human sera. The resultant assays performed with low sensitivity and specificity (HSV-1 of 89% and 66%, respectively and for HSV-2 of 79% and 85%, respectively) compared to the reference HerpeSelect ELISA.The MMRV multiplex bead immunoassay proved rapid, and required minimal sample volume to semi-quantify MMRV specific antibodies. The seroepidemiology of MMR results was compared with previous seroepidemiological studies performed in 1996 in England. The comparison showed an increase in the proportion of individuals who were positive for mumps and measles antibodies in the 2012 survey. The proportion of individuals positive for rubella was essentially unchanged. The increase in the proportion of individuals positive for mumps and measles antibodies in 2012 show the effectiveness of the change in MMR vaccination policy for England from 1996 onward. For VZV, the proportion of individuals who were positive for varicella antibodies between the 1996 and 2012 serological surveys were essentially unchanged. The comparison showed that most young children are susceptible to VZV. At this level of immunity, it can be expected that varicella will continue to produce epidemics of infection in the population, unless varicella vaccination is implemented as a part of routine childhood vaccination.

Les polérovirus infectent une large gamme de plantes d’intérêt économique. Ils sont transmis par un insecte vecteur, le puceron, selon le mode circulant non-multipliant. Le virus, acquis par le puceron lors de l’ingestion de sève sur une plante infectée, traverse l’épithélium des cellules intestinales puis celui des glandes salivaires par un mécanisme de transcytose impliquant des récepteurs encore inconnus. Le récepteur de l’éphrine (Eph) est une protéine membranaire dont un domaine est capable de se lier dans la levure aux protéines structurales des polérovirus. En développant des techniques basées sur l’ARN interférence, nous avons montré que l’acquisition orale d’ARN double brin ciblant Eph chez le puceron Myzus persicae permet de réduire de manière reproductible l’internalisation des polérovirus dans le corps du puceron. Les pucerons ainsi traités transmettent le virus avec une efficacité réduite. Eph pourrait donc assurer la fonction de récepteur des polérovirus chez M. persicae
Poleroviruses infect a wide range of economically important plants. They are transmitted in a circulative and non-propagative mode by an insect vector, the aphid. The virus particles are acquired by aphids when ingesting the sap from an infected plant and cross successively the epithelia of the midgut and the salivary gland cells by a transcytosis mechanism that relies on the presence of unknown receptors.The ephrin receptor (Eph) is a membrane protein which contains a domain able to bind in yeast to the structural proteins of poleroviruses. By developing methods based on RNA interference, we have shown that oral acquisition of double-stranded RNA targeting Eph in the aphid Myzus persicae can reproducibly reduce polerovirus internalization into the aphid's body. Such treated aphids transmit the virus to plants with a lower efficiency. Eph could therefore function as a receptor for poleroviruses in M. persicae

Barley yellow dwarf (BYD), caused by Barley yellow dwarf virus and Cereal yellow dwarf virus, and is a yield limiting disease of small grains. A research study was initiated in 2015 to identify the implications of BYD on small grain crops of North Dakota. A survey of 187 small grain fields was conducted in 2015 and 2016 to assess cereal aphid diversity; cereal aphids identified included, Rhopalosiphum padi, Schizaphis graminum, and Sitobion avenae. A second survey observed and documented field absence or occurrence of cereal aphids and their incidence. Results indicated prevalence and incidence differed among respective growth stages and a higher presence of cereal aphids throughout the Northwest part of North Dakota than previously thought. Field and greenhouse screenings were conducted to identify hard red spring wheat and durum responses to BYD. Infested treatments in the greenhouse had significantly lower number of spikes, dry shoot mass and yield.

El MNGIE és una malaltia mitocondrial d’herència autosòmica recessiva causada per mutacions en el gen TYMP, que codifica l’enzim timidina fosforilasa (TP). La TP catalitza la degradació de timidina (dThd) i desoxiuridina (dUrd), i la seva absència en pacients causa l’acumulació sistèmica d’aquests metabòlits, tòxica per la funció mitocondrial. A dia d’avui les úniques teràpies efectives són el transplantament de cèl·lules mare hematopoètiques i el transplantament de fetge. No obstant, els transplantaments estan limitats per la necessitat d’un donant compatible i tenen una taxa de mortalitat i morbiditat associada especialment elevada en pacients de MNGIE. Per a superar aquests inconvenients, fa temps vam proposar la teràpia gènica mitjançant vectors adenoassociats (rAAV) dirigits al fetge com a alternativa terapèutica, i vam demostrar la seva viabilitat en el model animal de MNGIE, el ratolí doble knockout (dKO) Tymp-/-Upp1-/-. Però aquest model només presenta fenotip bioquímic, de manera que només vam poder demostrar la correcció d’aquest fenotip. Al 2014 es va descriure que augmentant el desequilibri metabòlic mitjançant l’administració oral de dThd i dUrd al model dKO durant tota la seva vida provocava l’aparició d’alguns trets que reproduïen la simptomatologia clínica dels pacients. En aquesta tesi hem estudiat l’ús de la teràpia gènica en aquest model millorat, mitjançant el tractament amb tres rAAVs que expressaven la seqüència codificant de TYMP sota el control de 3 promotors hepàtics a diferents dosis. El tractament amb rAAV va incrementar l’activitat TP en fetge i va disminuir la concentració sistèmica de nucleòsids dels ratolins dKO, que sense tractament eren 30 vegades superiors als valors normals. A nivell fenotípic, en la majoria dels ratolins, el tractament també va prevenir l’increment del volum dels ventricles cerebrals i el deteriorament motor observats en els ratolins no tractats. Quan vam comparar els tres vectors utilitzats, el rAAV amb el promotor de l’alfa-1-antitripsina (AAT) va ser el més eficaç. Aquests resultats confirmen que la teràpia gènica mediada per rAAV dirigida al fetge restaura l’homeòstasi bioquímica i demostren la prevenció de l’aparició del fenotip clínic del model animal millorat de MNGIE. Un altre aspecte important per a la translació de la teràpia a la pràctica clínica és optimitzar el vector per tal de reduir-ne la dosi efectiva. En aquest sentit, hem testat dues aproximacions: la modificació de la seqüència codificant (ADNc) del gen segons la freqüència d’ús de codó per a incrementar la seva expressió, i l’eliminació dels dinucleòtids CpG de l’ADNc del gen per a reduir la immunogenicitat del vector. Vam dissenyar quatre seqüències optimitzades de l’ADNc de TYMP amb 4 algoritmes diferents. Vam generar vectors lentivirals per transduir 4 línies cel·lulars humanes i determinar l’eficiència d’expressió de cada seqüència comparada amb la seqüència salvatge, tenint en compte el grau d’activitat TP, el número de còpies del vector i els nivells relatius d’ARNm. De tots els experiments, només una seqüència optimitzada va millorar el grau d’expressió de TYMP respecte el de la seqüència salvatge en la línia cel·lular hepàtica Huh7. Pel que fa a la reducció de la immunogenicitat del vector, vam eliminar els dinucleòtids CpG de les seqüències dissenyades i vam analitzar el grau d’expressió de TYMP. Només la seqüència salvatge sense dinucleòtids CpG es va acostar a l’expressió de la seqüència natural. Tot i que s’hi observa una reducció d’expressió aproximada del 20%, es compensa per l’avantatge que aporta en termes de reducció de la resposta immunològica de cara a l’ús clínic del vector. En conclusió recomanem aquesta versió sota la regulació del promotor AAT per a ús eventual en la pràctica clínica.
El MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) es una enfermedad mitocondrial de herencia autosómica recesiva causada por mutaciones en el gen TYMP, que codifica la enzima timidina fosforilasa (TP). La TP cataliza la degradación de timidina (dThd) y desoxiuridina (dUrd), y su ausencia en pacientes causa la acumulación sistémica de estos metabolitos, tóxica para la función mitocondrial. Hoy en día, las únicas terapias efectives son el trasplante de células madre hematopoyéticas y el trasplante de hígado. No obstante, los trasplantes están limitados por la necesidad de un donante compatible y tienen una tasa de mortalidad y morbididad asociada especialmente elevada en pacientes de MNGIE. Para superar estos inconvenientes, hace tiempo propusimos la terapia génica mediante vectores adenoasociados (rAAV) dirigidos al hígado como alternativa terapéutica, y demostramos su viabilidad en el modelo animal de MNGIE, el ratón doble knockout (dKO) Tymp-/-Upp1-/-. Pero este modelo solo presenta fenotipo bioquímico, de forma que solo pudimos demostrar la corrección de este fenotipo. En 2014 se describió que aumentando el desequilibrio metabólico mediante la administración oral de dThd y dUrd al modelo dKO durante toda su vida provocaba la aparición de algunos rasgos que reproducían la sintomatología clínica de los pacientes. En esta tesis hemos estudiado el uso de la terapia génica en este modelo mejorado, mediante el tratamiento con tres rAAVs que expresan la secuencia codificante de TYMP bajo el control de tres promotores hepáticos a distintas dosis. El tratamiento con rAAV incrementó la actividad TP hepática y disminuyo la concentración sistémica de nucleósidos de los ratones dKO, (sin tratamiento eren 30 veces superiores a los valores normales). A nivel fenotípico, en la mayoría de los ratones, el tratamiento también previno el aumento del volumen de los ventrículos cerebrales y el deterioro motor observados en los ratones no tratados. Cuando comparamos los tres vectores utilitzados, el rAAV con el promotor de la alfa-1-antitripsina (AAT) fue el más eficaz. Estos resultados confirman que la terapia génica por rAAV dirigida al hígado restaura la homeóstasis bioquímica y demuestran la prevención de la aparición del fenotipo clínico del modelo animal mejorado de MNGIE. Otro aspecto importante para la translación de la terapia a la práctica clínica es optimizar el vector para reducir la dosis efectiva. En este sentido, hemos testado dos aproximaciones: la modificación de la secuencia codificante (ADNc) del gen según la frecuencia de uso de codón para aumentar su expresión, y la eliminación de los dinucleótidos CpG del ADNc del gen para reducir la immunogenicidad del vector. Diseñamos cuatro secuencies optimizadas del ADNc de TYMP utilitzando 4 algoritmos diferentes. Generamos vectores lentivirales para transducir 4 líneas celulares humanas y determinar la eficiencia de expresión de cada secuencia comparada con la secuencia natural, teniendo en cuenta el grado de actividad TP, el número de copias del vector y los niveles relativos de ARNm. De todos los experimentos, solo una secuencia optimizada mejoró el grado de expresión de TYMP comparado con el de la secuencia natural, en la línea celular hepática Huh7. En cuanto a la reducción de la immunogenicidad del vector, eliminamos los dinucleótidos CpG de las secuencias diseñadas y analizamos el nivel de expresión de TYMP. Solo la secuencia natural sin dinucleótidos CpG se acercó a la expresión de la secuencia natural. Aunque se observa una reducción de expresión aproximada del 20%, se compensa con la ventaja que aporta en términos de reducción de la respuesta inmunológica de cara al uso clínico del vector. En conclusión, entre las opciones testadas, recomendamos el rAAV que contiene el ADNc natural de TYMP sin dinucleótidos CpG bajo el control del promotor AAT para un uso eventual en la práctica clínica.
MNGIE (Mitochondrial NeuroGastroIntestinal Encephalomyopathy) is an autosomal recessive disease caused by mutations in TYMP, which encodes for the enzyme thymidine phosphorylase (TP). TP catalyses the first step of the catabolism of the nucleosides thymidine (dThd) and deoxyuridine (dUrd). The lack of TP activity causes systemic nucleoside accumulation which is toxic for mitochondrial function. Nowadays, the only available therapies for MNGIE are allogeneic hematopoetic stem cell transplantation or liver transplantation. However, these treatments are limited by the need of a compatible donor and are associated to high mortality and morbidity rates in MNGIE patients. To overcome these limitations, our laboratory proposed adenoassociated virus (rAAV) mediated gene therapy targeted to the liver for MNGIE and demonstrated its feasibility in the Tymp-/- Upp1-/- (dKO) mouse model of the disease. However, dKO mice show biochemical phenotype only, therefore we could only demonstrate the efficacy of our approach at the biochemical level. In 2014 it was reported that increasing the biochemical imbalances of the dKO model by chronic oral administration of dThd and dUrd to dKO mice over their entire life induced some features that recapitulate part of the clinical manifestations observed in patients. In this thesis we have studied the effect of gene therapy in this enhanced model, by treating the animals with three rAAVs expressing the human TYMP coding sequence under the control of three different liver-specific promoters. rAAV treatment increased liver TP activity and limited the systemic nucleoside concentration present in the dKO enhanced model, which was 30-fold higher as compared with non-exposed wild-type mice. AAV-treatment also prevented, in most animals, the enlarged brain ventricles and the motor impairment observed in the exposed dKO mice. When we compared the three promoters tested, the rAAV containing the AAT promoter showed the best efficacy. These results confirm that AAV-mediated gene therapy restores the biochemical homeostasis and demonstrate that this treatment prevents the clinical phenotype developed by the enhanced murine model of MNGIE. Another key point for the clinical translation of gene therapy is the optimization of the therapeutic gene expression to reduce the vector dose. For this reason, we tested two approaches: modification of the coding sequence of TYMP based on the codon use frequency to increase the expression of the gene, and CpG dinucleotide removal from the coding sequence to reduce immunogenicity of the vector. We designed four different codon-optimized sequences of the TYMP coding sequence, using four different algorithms. We cloned each optimized sequence in a lentiviral vector and transduced 4 different human cell lines. We analysed the degree of expression achieved with each sequence, as compared with the non-optimized wild-type TYMP sequence through evaluation of TP activity, vector copy number, and mRNA levels in the cell lines. Among all the sequences studied, only one optimized sequence resulted in higher TP activity when expressed per vector copy number in the hepatic cell line Huh7. To reduce the immunogenicity of the vector we removed the CpG dinucleotides from the wild-type coding TYMP sequence and two codon-optimized TYMP sequences. The wild-type sequence without CpG was the only one showing expression levels similar to those of the wild-type sequence. Even though the CpG free sequence shows a reduced expression of about 20% of that observed in the wild-type sequence, it is compensated by the advantages associated to the absence of CpG sites in terms of reduction of the immunological response when considering the vector for clinical use. In conclusion, among the different options tested, we recommend the rAAV vector containing the wild-type coding TYMP CpG-free sequence under the control of the AAT promoter for its use in the clinical practice.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Viruses are powerful vectors for the delivery of nucleic acids, with applications in gene therapy and vaccination. However, major challenges for this technology include mis-targeting of the vector and neutralization by host antibody responses. Here we show that chemical addition of synthetic glycans to adenovirus (Ad) increased resistance to neutralizing antibodies and other critical host clearance mechanisms. Carbohydrate ligands were synthesised and functionalised with a cyanol methyl thiol group, which was subsequently activated to the amine reactive 2-imino- 2-methoxyethyl (IME). Ads were coated with ligand whilst maintaining virus cogency, with up to 58% of total Ad lysine resides glycosylated. The viral tropism of glyco-Ads was switched to target macrophages and dendritic cells (DCs), with high selectivity for the complementary sugar receptors. In vaccination studies, DC targeted glyco-virus enhanced antigen-specific T cell responses. Thus, chemical glycosylation of the Ad capsid allows modulation of tropism and shielding from sequestration and neutralizing antibodies. Since manipulation of this process is facile, it provides a flexible and potentially universal solution to key obstacles facing the utilization of viral vectors in therapeutic and vaccination contexts.

Doctor of Philosophy
Division of Biology
Kristin Michel
The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

Replication defective adenoviruses have emerged as promising vectors for delivery of vaccine antigens. The development of new vaccine vectors has recently focused on serotypes t, which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This thesis describes the construction and optimisation of ChAdOX1, a new vector based on chimpanzee adenovirus Y2S, which has recently been manufactured to clinical grade for a Phase 1 human trial. Comparative immunogenicity studies between vectors of different serotype were performed in mice, with careful consideration of the infectious titer of vector preparations, since this parameter can confound studies based solely on viral particle estimation. Aft intramuscular administration, HAdV-S (Human adenovirus C) based vectors elicited superior transgene product specific T cell and antibody responses compared to a selection of chimpanzee adenovirus vectors (from Human adenovirus EJ including ChAdOX1. The construction of ChAdOXl in a bacterial artificial chromosome (BA C), enabled precise, and flexible modification of the genome by recombmation mediated genetic engineering. (recombmeering). Reverse genetics was performed to identify vector determinants of immunogenicity. Chimeric ChAdOXl vectors were created by replacement of native virus associated RNA (VA-RNA) and fiber sequences with the corresponding sequences from HAdV-5 Using these chimeric vectors, the importance of innate immunity and vector transduction in determining vector immunogenicity was investigated. Though the mechanisms responsible ultimately remain unclear, superior transgene product specific immune responses with HAdV-5 correlated with higher levels of transgene expression in vivo after vector administration. The current study has conclusively demonstrated that neither VA-RNA sequences, nor the fiber protein, are responsible for differences in immunogenicity between vectors, contrary to hypotheses based on previous studies.

Human norovirus (NoV) is the leading cause of acute non-bacterial gastroenteritis worldwide. Despite the significant health, emotional, and economic burden caused by human NoV, there are no vaccines or therapeutic interventions for this virus. This is due in major part to the lack of a cell culture system and an animal model for human NoV infection.

Thus, a vector-based vaccine may be ideal for controlling this disease. The major capsid gene (VP1) of a human NoV was inserted into the VSV genome at the glycoprotein (G) and large (L) polymerase gene junction. Recombinant VSV expressing VP1 protein (rVSV-VP1) was recovered from an infectious cDNA clone of VSV. Expression of the capsid protein by VSV resulted in the formation of human NoV virus-like particles (VLPs) that are morphologically and antigenically identical to the native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 stimulated a significantly stronger humoral and cellular immune response compared to baculovirus-expressed VLP vaccination. These results demonstrated that that the VSV-based human NoV vaccine induced strong humoral, cellular, and mucosal immunity in a mouse model.

To further improve the safety and efficacy of the VSV-based human NoV vaccine, the gene for the 72kDa heat shock protein (HSP70) was inserted into rVSV and rVSV-VP1 vectors as an adjuvant, which resulted in construction of recombinant VSV expressing HSP70 (rVSV-HSP70) and VSV co-expressing human NoV VP1 protein and HSP70 (rVSV-HPS70-VP1), respectively. At the same inoculation dose, both rVSV-HSP70-VP1 and rVSV-VP1 triggered similar levels of specific immunity, even though VP1 expression by rVSV-HSP70-VP1 was approximately five-fold less than that of rVSV-VP1. To compensate for the reduced VP1 expression levels, the inoculation dose of rVSV-HSP70-VP1 was increased five-fold or same dosage of rVSV-VP1 and rVSV-HSP70 was combined vaccinated. Mice immunized with five does of rVSV-HSP70-VP1 or those receiving combined vaccination generated significantly higher mucosal and/or T cell immunity than those immunized with rVSV-VP1 alone (P<0.05). Therefore, this data indicates that insertion of HSP70 into the VSV vector further attenuates the VSV-based vaccine and HSP70 enhances the human NoV-specific immunities.

To determine whether the VSV-based human NoV vaccine confers protection from human NoV challenge, a gnotobiotic pig model was developed. Newborn gnotobiotic piglets vaccinated intranasally with rVSV-based vaccine (rVSV-VP1) produced high levels of specific serum IgG and fecal and vaginal IgA antibody. Three weeks after vaccination, piglets were orally challenged with human NoV. All three piglets in the unvaccinated challenged group developed histopathologic lesions typical of human NoV infection in the duodenum and proximal jejunum on day 5 post-challenge. However, only one of five vaccinated piglets exhibited focal epithelium loss and villous atrophy, and mild edema in the small intestines. Immunofluorescent assay showed that a large amount of human NoV antigens were detected in duodenum, jejunum, and ileum of the challenge control group but not vaccinated group. These results demonstrate that the rVSV-based human NoV vaccine triggered partially protective immunity in swine and protected gnotobiotic pigs from challenge by human NoV.

Les moustiques sont colonisés par un virome très peu étudié. Comme les bactéries, le virome influence probablement la biologie et l'immunité des populations de moustiques vecteurs, mais les modèles expérimentaux sont rares. Nous avons récemment découvert deux nouveaux virus chez le virome des vecteurs sauvages du paludisme, anophèles et des colonies d’Anopheles coluzzii : Anopheles C virus (AnCV) et Anopheles cypovirus (AnCPV). L’un ou les deux virus sont présents dans toutes les colonies de laboratoire d’An. gambiae et An. coluzzii. La prévalence des virus varie en fonction des stades du moustique. L'abondance des deux virus est négativement corrélée chez les moustiques individuels. L'analyse fonctionnelle révèle l'implication des voies de signalisation immunitaire des moustiques sur la réplication du virus, avec une influence différentielle sur les deux virus. Un modèle expérimental a été développé pour l'infection d’AnCPV chez les anophèles non porteurs de ces virus, en utilisant du sang infecté afin d'étudier les réponses antivirales chez ces moustiques. Les séquences de l'AnCPV sont hautement polymorphiques chez les moustiques individuels, alors que l'AnCV est pratiquement dépourvue de mutations. AnCPV entraine une plus grande mortalité chez An. stephensi, mais certaines mutations semblent impliquées dans son adaptation à cette espèce. AnCPV peut être potentiellement transmis comme un arbovirus à travers un hôte mammifère à des moustiques non infectés, ce qui suggère une voie évolutive relativement simple. Le virome d’An. stephensi contient un chaq-like virus et un partiti-like virus. Ce dernier appartenant à la famille des Partitiviridae a des formes d’ADN
Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. One or both viruses are present in all tested laboratory colonies of An. coluzzii and An. gambiae. Viral abundance varies reproducibly during mosquito development. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. Functional genomic analysis reveals the implication of mosquito immune signaling pathways on virus replication, with differential influence on the two viruses. An experimental model was developed for AnCPV infection of Anopheles by bloodmeal, in order to study mosquito antiviral responses. Sequences of AnCPV are highly polymorphic in individual mosquitoes, while AnCV is virtually devoid of variation. AnCPV is pathogenic to An. stephensi but some viral mutations seem to be involved in its adaption to this species. AnCPV can be transmitted like an arbovirus through a vertebrate host to uninfected mosquitoes, suggesting that the evolutionary pathway from vertical “insect specific” to infective blood transmission may be remarkably simple. The Anopheles stephensi virome harbors a chaq-like virus and partiti-like virus. This latter belonging to Partitiviridae is present in An. stephensi as DNA forms of the virus genome

L'objectif de mon sujet de thèse était de développer un nouveau type de vecteur viral pour le transfert de gènes, et de faire la preuve de concept de l'efficacité de ce vecteur en culture de cellules et chez la souris. L'idée centrale est de se servir de la très grande capacité transgénique du virus de l'herpès simplex de type 1 (HSV-1) pour transformer le génome de ce virus en une sorte de plateforme, ou rampe de lancement, qui permettra la production in vivo d'un vecteur herpétique secondaire de type amplicon, qui portera le transgène thérapeutique à d'autres cellules, augmentant ainsi significativement le nombre de cellules qui recevront le transgène. Cette plateforme pourra également être utilisée en culture de cellules, pour améliorer la production de vecteurs amplicons non contaminés par du virus auxiliaire. De plus, les vecteurs amplicons ainsi produits, in vivo comme in vitro, ne porteront pas de séquences d'ADN bactériennes. L'élimination de l'ADN bactérien est très importante, tant pour diminuer la réponse cellulaire innée induite par la pénétration du vecteur, que pour éviter la répression de l'expression transgénique. Dans le système de vecteurs que nous nous proposons de développer, le génome d'un vecteur herpétique de type amplicon, entouré par deux sites loxP en orientation parallèle, se trouvera intégré dans le génome d'un virus herpétique recombinant. Dans les cellules infectées par le vecteur herpétique recombinant, et sous l'action de la recombinase Cre, le génome de l'amplicon sera dissocié du génome herpétique le contenant, générant ainsi deux génomes séparés dans le noyau de chaque cellule. Après dissociation, le génome du virus recombinant deviendra défectif à cause de la perte des signaux d'encapsidation (qui seront excisés avec le génome amplicon) et servira en tant que génome auxiliaire, permettant l'amplification et l'encapsidation du génome amplicon, portant le transgène, dans des particules herpétiques. Dans la mesure où chaque cellule infectée par le vecteur recombinant deviendra une usine à produire des amplicons, le nombre de cellules recevant le transgène thérapeutique porté par les amplicons devrait être considérablement élargi. Puisque le génome amplicon sera dissocié du génome HSV-1 recombinant suite à un événement de recombinaison spécifique de site, nous avons donné à ce système le nom de "Site-specific Excision-Dependent" (SED) vectors. Ce nouveau système pourra être utilisé de deux manières différentes en fonction de la localisation du gène codant pour la recombinase Cre. D'une part, il sera utilisé pour produire des stocks de vecteurs amplicons in vitro (virus SEDVITRO), auquel cas la recombinase Cre sera exprimée par les cellules dans lesquelles se fera la production d'amplicons. D'autre part, il sera utilisé pour générer des vecteurs amplicons in vivo (virus SEDVIVO), au sein de l'organisme inoculé, auquel cas la recombinase Cre sera exprimée à partir du génome du virus recombinant
A major obstacle to gene therapy is the inability to transduce sufficient numbers of cells in a target organ or tissue to achieve improvement from the disease state. This applies particularly to the neuromuscular system, considering both the disseminated nature of muscles and the challenges involved in scaling up results from small animal models to the clinical treatment of human disorders. The aim of this project was to develop and validate (in vitro and in vivo) a new concept on HSV-1-based viral vectors, conceived to significantly expand the number of target cells that will receive the vectors expressing the therapeutic transgene. The novel vectors should retain the high safety standards that are common to replication incompetent vectors, and avoid the incorporation of bacterial DNA sequences, which had been identified as responsible for gene silencing and activation of immune responses. This new vector system is based on a replication-incompetent HSV-1 recombinant vector carrying a secondary HSV-1 amplicon vector genome integrated in its own genome. Upon infection of target cells, the secondary vector genome will be dissociated from the carrier vector genome as a subgenomic fragment through Cre/loxP-based site specific recombination, therefore generating two progeny genomes, a defective amplicon vector genome and the remaining of the replication-incompetent HSV-1 genome. This latter genome will act as a defective helper, expressing the proteins required to allow amplification and packaging of the amplicon vector genome into virus particles. In this way, the incoming recombinant HSV-1 vector can be described as a vector platform that will launch a secondary vector, the amplicon vector carrying the therapeutic transgene, to interconnected neurons or muscle cells. Since the secondary vector will be excised from the carrier genome through a site-specific recombination event, we will refer to this system as Site-specific Excision-Dependent (SED) vectors. Two major improvements over currently used HSV-1-based vectors will be introduced in SED vectors. First, they will allow to produce high amounts of high-titre helper-free amplicon vector stocks in vitro without using bacterial plasmids, therefore highly improving the methods to prepare vectors stocks both in quantitative, qualitative and biosafety terms (SEDVITRO). Second, they will allow generating and launching amplicon vectors in vivo, in the SED-vector infected cells, therefore significantly expanding the number of transduced target cells and increasing the penetration of the therapeutic transgene, while retaining the safety level of replication-incompetent vectors (SEDVIVO)

Foamy viruses (FVs) are unique ancient retroviruses that infect all non-human primates, but do not cause disease. We aimed to understand the FV pre-integration complex by isolating it from infected cells and characterising its protein constituents. Using a PCR to quantify integration in infected cells, we determined that integration occurs from 10 hours post-transduction. In synchronised cells, the peak of integration correlated well with cells passing through mitosis. However, we were unable to detect in vitro strand-transfer activity to indicate that active pre-integration complexes had been isolated. We conclude that FV pre-integration complexes are likely to be inactive in the conditions tested. A further aim was to optimise FV vectors for use in mesenchymal stem cells and test this vector in mouse models of sphingolipidoses, namely metachromatic leukodystrophy and Krabbe disease. We permitted transduction of cells at a high multiplicity of infection by exchanging the envelope from the prototype FV to that of the macaque. We tested various FV vectors in mesenchymal stem cells and determined that the non-toxic macaque envelope increased transduction efficiency from under 65% to over 95% in a single round of transduction. We achieved high and sustained transgene expression using the phosphoglycerate kinase promoter. Transduced MSCs delivered to the brains of the mouse model for metachromatic leukodystrophy caused only a modest improvement in sulphatide storage, the primary biochemical output for efficacy, although results are inconclusive. In the mouse model for Krabbe disease, transduced MSCs delivered to the brain or the peritoneum had no effect on disease progression. In conclusion, FV vectors are suited to gene therapy of MSCs since they offer the highest transduction efficiency from a single round of transduction, while MSC based gene therapy strategies for Krabbe disease or metachromatic leukodystrophy are unlikely to offer clinical benefit.

L'empaquetage des huit segments du génome des virus influenza de type A est une des étapes clef du cycle viral. Il intervient également dans l'apparition de virus réassortants, les virus pandémiques par exemple, ce qui en fait un enjeu fondamental de la recherche actuelle.Nous avons étudié ce mécanisme au cours de deux études, la première portant sur les vaccins antigrippaux (réassortiment), la seconde visant à construire un virus vecteur (incorporation d'un segment hétérologue). Les semences vaccinales sont obtenues par co-infection d'oeufs de poule embryonnés avec deux souches virales une donneuse (souche circulante de référence) et une accepteuse (A/Puerto Rico/8/34 (H1N1) (PR8)). L'analyse de la composition génétique de treize semences vaccinales H3N2 montre que le segment PB1 de la souche donneuse est présent dans plus de 50 % des semences analysées et qu'une grande variété de réassortants,allant de 6:2 à 2:6 (PR8:H3N2), peut résulter de ces coinfections. Des expériences de compétition d'encapsidation de segments à l'aide de la génétique inverse révèlent que l'encapsidation sélective du segment PB1 dépend de son environnement génétique notamment l'origine virale des segments HA et NA. La seconde partie de mon travail de thèse a été consacrée à la construction d'un vecteur réplicatif sur la base d'un virus influenza H3 naturel sans segment NA. Aucune des constructions contenant le transgène gfp n'a été incorporée dans les particules virales, contrairement à ce qui a été décrit dans la littérature. Bien que les mécanismes moléculaires régissant l'incorporation des segments des virus influenza A demeurent très complexes, le fond génétique semble être déterminant pour ce processus.

[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species.
[ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas.
[CAT] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora
Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477
TESIS

Includes bibliographical references (leaves 131-137).
Of the many vaccine trials which have taken place, the most promising results have been obtained from the recent phase 3 clinical trial which tested the ability of a dual protein and Canarypox virus recombinant to protect humans against HIV-1 infections. Because poxviruses are being developed as vaccine vectors against a number of diseases, it is important to continue the search for novel poxvirus vectors, in particular, those that do not cross-neutralize one another. This thesis describes the preliminary work performed on the development of Penguinpox virus (PEPV) as a vaccine vector.