Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)

Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage. Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low. Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. / Canada Research Chair Program, OMAFRA,

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo. / The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors \"enzootic hematuria\" and upper digestive tract tumors \"caraguatá\" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed.

No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86. / In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.

The remarkable metabolic capacity of the soil-dwelling bacterium Sinorhizobium meliloti is encoded on its three circular replicons: the chromosome and two large megaplasmids, pSymA and pSymB. Despite making up 45% of the genome, the pSymA and pSymB megaplasmids can be cured from S. meliloti. This unique attribute provides an opportunity to study the essentiality of chromosomal genes in the presence or absence of nearly half the genome. By interrogating chromosomal genes via massively parallel transposon insertion sequencing (Tn-seq) in the presence and absence of pSymA and pSymB, we identified 307 genes as being essential for viability regardless of the genomic context and 104 genes as being essential specifically when the megaplasmids are absent. We also found that ten percent of genes encoded on the chromosome genetically interact with genes on pSymA and pSymB. In addition, Tn-seq data were utilized to significantly refine a metabolic model of S. meliloti, facilitating more accurate fitness predictions in user-defined nutrient and genetic contexts. Furthermore, the development of a library of barcoded transposon insertion (BarSeq) mutants has enabled us to identify genes that are essential for robust growth in hundreds of nutrient environments simultaneously. This will greatly assist efforts to assign more specific functions to the ~30% of S. meliloti genes that have remained uncharacterized over the years. S. meliloti has been studied for decades as a model organism for symbiotic communication. Its legume host, Medicago truncatula, provides fixed carbon for the bacteria in order to receive fixed nitrogen in return. The molecular dialogue between S. meliloti and M. truncatula, initiates and controls each stage of symbiotic development. When inside host cells, intracellular bacteria are subjected to an arsenal of plant-derived Nodule-specific Cysteine-Rich (NCR) peptides that induce significant morphological changes prior to nitrogen fixation. It was previously shown that a bacterial peptidase, HrrP, present in about 10% of S. meliloti isolates, could degrade host-derived peptides and give the bacterial symbionts greater fitness at the expense of the host. In a screen through peptidases conserved throughout the core S. meliloti genome, we identified one peptidase (sapA) that, when overexpressed, significantly modulates symbiotic outcome. In a manner similar to HrrP, SapA degrades NCR peptides in vitro. Additionally, expression of sapA seems to occur specifically inside the plant host providing compelling evidence that some rhizobial peptidases may have evolved away from housekeeping and toward symbiotic functions.

Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method. / Screening av nya rekombinanta proteiner som ska användas till läkemedel sker oftast i batch-odling. Men själva odlingen av proteinläkemedlet sker sedan under fed-batch förhållanden. Dessa två typer av odling är olika och då cellerna växer olika kan detta leda till att fel, eller den inte mest lämpade kandidaten väljs. Därför vore det önskvärt att ta fram en fed-batch liknande metod i skakkolvar. Biosilta har tagit fram en metod där ett enzym bryter ned stärkelse till glukos som påminner om fed-batch. Denna metod har testats och undersökts i detta examensarbete. Det har visat sig att cellerna som växer under dessa förhållanden är begränsade på glukos men producerar stora mängder ättiksyra under den senare fasen av odlingen och att pH varierar mycket. Systemet bygger mycket på att en booster-tablett tillsätts, vad denna tablett innehåller är okänt. Men om tabletten inte tillsätts slutar cellerna att växa, detta tyder på att det finns någon mer begränsning än glukos. Det visade sig även att protein produktionen blev mycket lägre än vid odling i batch-fas. Det skulle av anledning av ovanstående inte vara bra att använda sig av EnBase som en screening metod.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20450号 / 農博第2235号 / 新制||農||1050(附属図書館) / 学位論文||H29||N5071(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM