Rozprawy doktorskie: „Recombinant vaccine”

1

Hand, Nicholas. "Development of a Recombinant Attenuated Salmonella Cancer Vaccine". Thesis, The George Washington University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10635177.

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New treatments for neuroblastoma are desperately needed; high-risk neuroblastoma patients have a less than 50% five-year survival rate despite intensive treatment. The greatest impact on improving survival rates for cancer patients in recent years is the result of a number of immunotherapeutic approaches. A proportion of high-risk neuroblastoma patients undergo spontaneous regression, possibly mediated by the immune system, indicating the potential of immunotherapies targeting neuroblastoma-associated antigens. Recombinant attenuated Salmonella vaccines (RASV) are cost-effective and have shown efficacy against a number of pathogen-associated antigens and are easily adapted for use as cancer immunotherapies. Here we cloned survivin, a neuroblastoma tumor-associated antigen into RASV expression plasmids to develop 24 RASV candidate vaccines with an array of select phenotypes. While conventional recombinant attenuated Salmonella vaccines are permanently attenuated, the RASV used here are engineered with inducible in vivo attenuation and other delayed phenotypes shown to improve immune responses. Survivin expression did not impact the growth or stability of any of the RASV constructs. Six of the constructs were tested in vivo, the RASV survived in the gut lumen, and all RASV-immunized mice produced anti-Salmonella responses. Protein/adjuvant immunized mice produced humoral and cellular survivin specific immune responses; however two independent in vivo experiments showed that no survivin specific immune responses were induced in survivin-expressing RASV immunized mice. Based on the results, a number of improvements to the future development of the vaccine are suggested.

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2

Cook, Jeremy K. "Recombinant immunotargeting antigen, antibody fusions in vaccine design". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0001/NQ35130.pdf.

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Herr, Roger Alan. "Evaluation of Coccidioides posadasii antigens as recombinantly expressed monovalent, divalent, and chimeric vaccine candidates". Connect to full-text via OhioLINK ETD Center, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1160404292.

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Thesis (Ph.D.)--Medical University of Ohio, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Garry Cole. Includes abstract. Document formatted into pages: ii, 206 p. Title from title page of PDF document. Title at ETD Web site: Evaluation of two homologous Coccidioides posadasii antigens as recombinantly expressed monovalent, divalent, and chimeric vaccine candidates. Bibliography: pages 75-83, 116-120, 165-169, 185-204.

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4

Al-Zarouni, Mansour. "Expression of recombinant antigen in BCG". Thesis, University of Surrey, 2000. http://epubs.surrey.ac.uk/843308/.

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Little is known about the effect of different modes of expression of an antigen in rBCG on immune response. An appropriate wing of the immune system, with different degrees, is activated upon encounter with a foreign antigen. Knowledge of these responses is vital to the development of future recombinant vaccine. Various E. coli-mycobacterial species shuttle vector constructs were made using a combination of mycobacterial promoters and signal sequences. Thus enabling foreign antigens to be expressed cytoplasmically or secreted outside rBCG as native proteins or membrane-associated lipoproteins. A pivotal study using an E. coli beta-lactamase as a reporter gene is described for the evaluation of the strength of promoter and signal sequence constructs both in vitro and most importantly in vivo using the mouse macrophage cell line J-774. Expression of the diphtheria toxin fragment B as a foreign antigen was detected in vitro with all constructed plasmid vectors in rBCG using a western blot as a means of detection. It was observed that all hsp60 promoter-based constructs exhibited a high frequency with variable degree of plasmid DNA deletions Using three different rBCG substrains, the BCG Tokyo was found to be more stable (P < 0.01) and exhibited less degree of deletion (P < 0.001) compared to either BCG Moreau or BCG Pasteur. Sequence analysis of deleted plasmid DNA revealed a specific region common with nearly all plasmid deletions. Such a region of the DNA was found to correspond to the first transcriptional starting site of the hsp60 promoter. Furthermore no differences were observed in the level of expression among the three-rBCG substrains, retaining plasmid DNA, when detected by immunoblotting.

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5

Danet, Nicolas. "Molecular characterisation of the recombinant Vesicular Stomatitis Virus- ZEBOV-GP virus, prototype vaccine against Ebola virus". Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1009/document.

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Ebolavirus (EBOV) est un filovirus responsable de fièvres hémorragiques virales sévères chez l’humain, qui peuvent être létales dans 90% des cas. L’actuelle épidémie en République Démocratique du Congo et l’ampleur démesurée de l'épidémie de 2014-2016 en Afrique de l’Ouest, qui a causé la mort de plus de 11 000 personnes, ont poussé les agences sanitaires internationales à tester plusieurs approches thérapeutiques afin d’essayer d’endiguer rapidement la propagation virale et de limiter la mortalité liée au virus lors de futures épidémies. Parmi toutes les stratégies testées, le virus recombinant réplicatif rVSV-ZEBOV qui exprime la glycoprotéine de surface d’EBOV, semble offrir la meilleur protection, aussi bien en modèle animaliers que sur le terrain. Avant d’être testé chez l’humain, de nombreuses études ont permis de mettre en évidence l’efficacité et l’innocuité de ce vaccin prototype. Pourtant et malgré le fait que de nombreuses études ont démontré l’importance et le rôle de la glycoprotéine GP dans l’efficacité des vaccins contre ce virus, aucune étude n’a encore été réalisé sur la nature des glycoprotéines virales synthétisées par le gène GP d’EBOV inséré dans le génome du virus VSV. Ainsi, les caractérisations moléculaires des protéines virales produites lors de l’infection par le virus rVSV-GP décrites dans ces travaux de thèse offrent de nouvelles perspectives pour comprendre le succès de ce vaccin mais aussi l’origine virales dans les effets secondaires sévères observés lors de la vaccination, et pourront aider à développer un vaccin plus sûr, qui n’est actuellement pas utilisable chez les personnes immunodéprimées
The filovirus Ebolavirus (EBOV) is the causative agent of severe viral hemorrhagic fevers in humans that can be lethal in 90% of cases. The current outbreak in the Democratic Republic of Congo and the extraordinary scale of the 2014-2016 outbreak in West Africa, that caused the death of more than 11 000 disease victims, lead the international public health agencies to test several therapeutic approach to limit viral spreading and mortality. Amongst those, the recombinant replication-competent rVSV-ZEBOV virus, that expressed EBOV GP glycoprotein, appears to offer the best protection in animal models and outbreak settings. While its effectiveness and safety have been widely investigated before human trials and despite numerous studies that showed the importance the nature of the glycoproteins which are produced during the infection from the EBOV GP gene that has been inserted in VSV genome are unknown. In this respect, the molecular characterisations of the viral glycoproteins synthesised during rVSV-GP presented in this thesis, offer new insights with which to understand the success of the rVSV-GP vaccine but also the potential viral origins of the severe adverse side effects observed during vaccination and could help in developing a safer vaccine, which currently cannot be used in an immunocompromised population

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Teixeira, Lais Helena. "Geração e análise da imunogenicidade de proteínas recombinantes baseadas nas diferentes formas do antígeno circumsporozoíta de Plasmodium vivax visando o desenvolvimento de uma vacina universal contra malária". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-11072014-110149/.

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O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes.
The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.

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7

Saubi, Roca Narcís. "Scaling up recombinant BCG based HIV vaccine development. Lessons learned". Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/400667.

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El treball de recerca realitzat en aquesta Tesi Doctoral està dirigit a la millora del desenvolupament de la vacuna contra el VIH basada en BCG recombinant. La Tesi Doctoral inclou tres articles de recerca publicats en revistes “peer-reviewed” i una patent depositada a la Oficina Europea de Patents. Inicialment, hem avaluat la influencia de l’edat i la ruta d’immunització en la inducció de respostes immunes especifiques front a VIH i M. tuberculosis després d’immunitzar ratolins BALB/c recent nascuts i adults amb BCG.HIVA222 i reforçar-los amb Modified Virus Ankara (MVA).HIVA. Les freqüències de cèl·lules CD8+ productores de IFN- γ eren superiors en ratolins adults vacunats per via intradèrmica i menors en ratolins adults i recent nascuts vacunats per via subcutànea. En tots els casos la producció de IFN-γ era significativament superior en ratolins induïts amb BCG.HIVA222 en comparació dels ratolins induïts amb BCGwt. Quan l’activitat Citotòxica dels limfòcits T (CTL) específica pel VIH es va comprovar, les freqüències de toxicitat específica eren superiors en ratolins recent nascuts que en ratolins adults. El programa vacunal d’inducció-reforç heteròleg amb BCG.HIVA222 i MVA.HIVA va ésser segur en ratolins recent nascuts. L’administració de BCG.HIVA222 a ratolins recent nascuts és segura i immunogènica i va augmentar la resposta específica front a VIH induïda per la vacuna MVA.HIVA. Seguidament, vàrem construir el pJH222.HIVACAT, un plasmidi llançadora que expressava l’immunogen de VIH subtipus A HIVA. Aquest vector llançadora utilitza un mecanisme lliure de resistències a antibiòtic basat en un sistema de Titració del Repressor de l’Operador (ORT) per la selecció i manteniment del plasmidi en cèl·lules E. coli i una complementació de lisina en micobactèria. Aquest vector llançadora es va electroporar una soca de BCG auxotròfica per lisina per generar la soca vacunal BCG.HIVACAT. Vàrem demostrar que el plasmidi episomal pJH222.HIVACAT era estable in vivo per un període de 20 setmanes, i vàrem caracteritzar genèticament i fenotípica la soca vacunal BCG.HIVACAT. La vacuna BCG.HIVACAT en combinació amb MVA.HIVA indueix una resposta de cèl·lules T productores de IFN- γ especifiques per VIH i M. tuberculosis. Per altra banda, quan es varen induir ratolins adults amb BCG.HIVACAT i es varen reforçar amb MVA.HIVA.85A, es varen induir cèl·lules T CD8+ especifiques per VIH que produïen IFN-γ, TNF-α, IL-2 i CD107a al ésser estimulades. Per tant, vàrem demostrat la immunogenicitat per cèl·lules T de una nova vacuna, més segura, compatible amb GLP, basada en BCG recombinant que expressa l’immunogen prototipus HIVA. Finalment, hem construït un nou disseny de vacuna basada en micobactèria mitjançant la utilització d’un sistema de selecció de plasmidis lliure de resistència a antibiòtics. Hem construït un nou plasmidi llançadora per E. coli i micobactèria que expressa l’immunogen HIVA. Aquest vector llançadora utilitza un sistema de selecció i manteniment de plasmidis sense resistències a antibiòtics, que es basa en la complementació de glicina en E. coli i la complementació de lisina en micobactèria. Aquest plasmidi primer es va tranformar en una soca d’E. coli auxotròfica per glicina, i llavors es va transformar en una soca de BCG auxotròfica per lisina per generar la soca vacunal BCG. HIVA2auxo. Vàrem demostrar que el plasmidi episomal p2auxo.HIVA era estable in vivo per un període de 7 setmanes, i vàrem fer la caracterizació genètica i fenotípica de la soca vacunal BCG.HIVA2auxo. La vacuna BCG.HIVA2auxo, en combinació amb MVA.HIVA, va ésser segura i va produir cèl·lules T productores de IFN-γ especifiques per VIH i M. tuberculosis en ratolins BALB/c adults. Es varen induir cèl·lules T CD8+ polifuncionals especifiques per VIH que produïen IFN-γ, TNF-α i expressaven el marcador de degranulació CD107a. Així, hem construït una nova vacuna basada en BCG més segura i compatible amb les GLP fent servir l’immunogen HIVA. Aquests sistema de selecció de plasmidis lliure de resistències a antibiòtics basat en la doble complementació d’auxotrofies podria ésser una nova plataforma vacunal micobacteriana per desenvolupar no només BCG recombinant que expressa immunògens de VIH de segona generació, sinó també d’altres patògens d’interès pediàtric per induir resposta protectiva just després de néixer.
The research work of this PhD thesis aims to scale up recombinant BCG based HIV vaccine development. Our main goal is to develop a mycobacterial vaccine design for HIV-TB pediatric vaccine. The PhD thesis includes three research papers published in peer-reviewed journals and one patent filed in the European patent office. Initially, we have evaluated the influence of age and immunization routes for induction of HIV-1- and M. tuberculosis-specific immune responses after neonatal and adult BALB/c mice immunization with BCG.HIVA222 prime and Modified Virus Ankara (MVA).HIVA boost. The frequencies of HIV-specific CD8+ T cells producing IFN-γ were higher in adult mice vaccinated intradermally and lower in adult and newborn mice vaccinated subcutaneously. In all cases the IFN-γ production was significantly higher when mice were primed with BCG.HIVA222 compared with BCGwt. When the HIV-specific Cytotoxic T-lymphocytes (CTL) activity was assessed, the frequencies of specific killing were higher in newborn mice than in adults. The prime-boost vaccination regimen which includes BCG.HIVA222 and MVA.HIVA was safe when inoculated to newborn mice. The administration of BCG.HIVA222 to newborn mice is safe and immunogenic and increased the HIV-specific responses induced by MVA.HIVA vaccine. Secondly, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVACAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph strain of BCG to generate vaccine BCG.HIVACAT. We demonstrated that the episomal plasmid pJH222.HIVACAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVACAT vaccine strain. The BCG.HIVACAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific IFN-γ- producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVACAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8+ Tcells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Finally, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)-mycobacterial shuttle plasmid p2auxo.HIVA expressing the immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with MVA.HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific IFN-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T-cells, which produce IFN-γ and TNF-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

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Ahuja, Sanjay. "Development of a recombinant protein vaccine against Plasmodium falciparum malaria /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-788-X/.

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Okay, Sezer. "Development Of Recombinant Vaccines Composed Of Plpe And Omph From Pasteurella Multocida A:3". Phd thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613980/index.pdf.

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Pasteurella multocida serotype A:3 is a gram-negative bacterial pathogen which is one of the causative agents of shipping fever in cattle. In this study, ompH and two fragments of plpE gene (plpEN and plpEC) were cloned from the genomic DNA of P. multocida P-1062 (ATCC 15743, serotype A:3) and plpEN-ompH and plpEC-ompH fusions were constructed. In vitro expression of the genes was shown in HEK-293 cells. Later, full-length plpE gene was cloned and the recombinant proteins were expressed in E. coli and purified. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and five recombinant protein based vaccines, PlpEN-OmpH, PlpEC-OmpH, OmpH, PlpEC and PlpE were generated. Recombinant proteins were formulated with at least one of the adjuvants: alum, CpG, alum-CpG, oil based and oil based-CpG. BALB/c mice were immunized with these vaccine formulations and their sera were used for the evaluation of antibody and serum IFN-&gamma
titers. Protective capacities of the vaccines were also evaluated via challenge of mice with 10 LD50 of P. multocida A:3. DNA vaccines induced immune responses, but did not provide protection. All protein vaccine formulations increased antibody levels and CpG containing formulations enhanced serum IFN-&gamma
titers. 100 µ
g of PlpEC-OmpH protein adsorbed on alum adjuvant conferred 40% protection while no protection was obtained with PlpEN-OmpH. Next, the effects of CpG, or its alum and oil based combinations as adjuvants were investigated on PlpEC-OmpH mediated protection. The vaccine formulation composed of PlpEC-OmpH and oil based-CpG adjuvant conferred 100% protection. Finally, the mice were vaccinated with recombinant OmpH, PlpEC and PlpE formulated with oil based-CpG adjuvant. OmpH, PlpEC and PlpE formulations provided 50%, 60% and 100% protection, respectively. These findings implicated that recombinant PlpE and PlpEC-OmpH fusion proteins when formulated with oil based-CpG adjuvant are potent acellular vaccine formulation candidates against shipping fever.

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Vaghefi, Negin Gitiban. "The role of innate immunity in protection against respiratory syncytial virus (RSV)". Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1138388518.

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Amaral, Maria Rita Rebocho Lopes do. "Evaluation of a new vaccine based on pDNA and recombinant protein against Helicobacter pylori". Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8520.

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Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
Helicobacter pylori is a bacterium capable of surviving and infecting a healthy human stomach and it is estimated that infect more than a half of world population. Despite of being almost always asymptomatic, in some cases, the infection can evolve to several gastric disease as chronic gastritis, peptic ulcers, gastric cancer and MALT lymphoma. Vaccination against H. pylori is a promising option due to emerging problems of antibiotics treatment. It is thought that oral immunization could be a good approach for a more effective protection against infections by H. pylori, creating a first line of defense in mucosal surfaces. Chitosan nanoparticles are a suitable vehicle for oral vaccines delivery due to its immunogenic and mucoadhesive properties, protecting the DNA and allowing high levels of transfected cells. Thus, this work aims to evaluate a new pDNA- and recombinant protein-based vaccine, with multi epitopes of different H. pylori antigens. Following production and purification of plasmid DNA and recombinant proteins, vaccines were formulated for oral and intramuscular administration with the antigens encapsulated with chitosan nanoparticles. The type of immune response induced and the effectiveness of protective immunity elicited were assessed by ELISA, through analysis of specific IgGs, mucosal SIgA and cytokines levels produced by immunized BALB/C mice. When give by the intramuscular route, the formulated pDNA and recombinant protein-based vaccines efficiently stimulated the production of specific IgG2a and IgG1, which is supported by cytokines levels, revealing a better and balanced systemic immune response than oral immunizations. Nevertheless as expected, oral immunizations with either pDNA vaccines or recombinant protein revealed high levels of SIgA, showing to be effective in gastric mucosal immunization for a more protective immune response, contrasting with intramuscular immunizations which did not induce SIgA. The immunization results showed that both pDNA and recombinant proteins vaccines encapsulated with chitosan nanoparticles are good candidates for the development of a future vaccine to prophylactic and therapeutic use to improve the eradication of H. pylori infections.
Fundação da Ciência e Tecnologia - (PTDC/BIO/69242/2006); FEDER - (PEst-OE/SAU/UI4013/2011)

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Ramamoorthy, Sheela. "Approaches towards vaccine development against Neospora caninum". Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/28054.

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Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-Ã£ and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission.
Ph. D.

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Alharbi, Naif K. "New approaches for improving the immunogenicity of modified vaccinia virus Ankara as a recombinant vaccine vector". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:bbde86fd-ea8f-4e66-b260-f923d7e01e4b.

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Corgozinho, Carolina Nunes Costa. "Desenvolvimento de vacina baseada em sistema de liberação sustentada contendo proteína recombinante". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60137/tde-31072009-083709/.

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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.

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15

Orubu, Toritse. "Generation of multivalent recombinant MVA vaccines for malaria". Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589603.

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Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

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16

Sridhar, Saranya. "Development of recombinant adenoviral vectors as a pre-erythrocytic malaria vaccine". Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497101.

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17

Alkahtani, Saad M. "Recombinant expression and immuno-screening for vaccine candidates in Plasmodium falciparum". Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/12491.

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The main aim of this study was to develop a fast and efficient way of expressing P. falciparum antigenic epitopes as conformationally conserved antigenic structures whose expression can be readily ‘scaled-up’ for clinical testing. The E. coli and P. pastoris systems were used, both singly and as linked production systems using ‘shuttle vectors’ that can be dually expressed in both microorganisms. Initially a proof-of-principle experiment to express the CIDRI domain of PfEMP1 has been conducted. It was expressed and purified in good quantity and used to immunize rabbits. The sequenced 3D7 P. falciparum genome was then exploited to construct degenerate primers for several domains of PfEMPl (namely DBL α, β and γ). These primers were used to amplify targets from FCR3CSA parasites. PCR products were then ligated into the dual expression vector pPICHOLI1 plasmid. Several erythrocyte surface antigen expression libraries were constructed. Development and optimisation of microarray and high-throughput screening assays in malaria vaccine development was carried out to accelerate the process of identifying malaria vaccine candidates. Growth of colonies of P. pastoris on filters on agar plates for high-throughput screening, a novel procedure, was optimized. These libraries were screened using patients’ pooled sera from endemic areas in Sudan, as well as sera from male and pregnant women suffering from the pregnancy malaria syndrome from a holo-endemic malaria transmission zone, Ghana. Different domains from a gene of particular interest, the NF54 var2CSA gene were also amplified for study. The domains of DBL3X and DBL4e have been produced and purified from mid-scale induction experiments.

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18

Taupin, Jean-Luc. "Anticorps monoclonaux dirigés contre la cytokine humaine HILDA/LIF : production au moyen de virus de la vaccine recombinants, caractérisation et mise au point d'un test ELISA pour le dosage du HILDA/LIF". Bordeaux 2, 1992. http://www.theses.fr/1992BOR2P049.

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19

Al, Qublan Hamzeh. "Development and testing of recombinant B. abortus RB51 vaccine strains carrying M. tuberculosis protective antigens". Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73696.

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Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most prevalent infectious diseases inflicting humankind. The World Health Organization estimates that one third of the world's population, approximately 2.2 billion people, is infected with TB with a mortality of 1.7 million people annually. Currently, the WHO estimates that each year more than 9 million people develop TB. Bacille Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only licensed TB vaccine in the world. Clinical studies have shown childhood vaccination with BCG to be protective against disseminating and meningeal forms of TB. However, the efficacy of BCG against pulmonary TB in adults has been variable and inconsistent (0-80%). The objective of this study is to develop and test the efficacy of the B. abortus vaccine strain RB51 as a platform for expression of M. tuberculosis antigens (Ag85B, ESAT6 and Rv2660c) and induction of a protective immune response against M. tuberculosis and B. abortus challenge in mice. Here we report the construction of two recombinant strains of B. abortus vaccine strain RB51 capable of expressing mycobacterial antigens Ag85B, ESAT6 and Rv2660c. Our studies show that expression of mycobacterial antigens in strain RB51 lead to induction of antigen-specific immune responses characterized by secretion of IgG2a antibodies as well as of IFN- and TNF-α. Mice immunized with a combination of two strains of RB51 in equal numbers, one carrying Rv2660c-ESAT6 and another carrying Ag85B, led to a 0.90 log reduction in CFU burden with significance nearly reaching borderline (p = 0.052). However, when mice were primed with the same strains of RB51 and boosted with proteins Ag85B and ESAT6, a significant level of protection (1 log reduction) compared to the PBS vaccinated group was achieved. The protection levels conferred by this vaccination strategy was similar to that conferred by BCG vaccine. In conclusion, we have shown that recombinant RB51 strains expressing mycobacterial protective antigens result in stimulation of antigen specific immune response without altering the vaccine efficacy in protecting against the more virulent strain of B. abortus 2308. These recombinant vaccines could potentially be used to protect against M. tuberculosis infection.
Ph. D.

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20

Bridge, Simon Harwood. "HIV-Neutralising response to recombinant, cross-clade, adjuvanted, VLP-forming vaccine candidates". Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485898.

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The development of a safe, effective and affordable HIV-1 vaccine is urgently needed especially in resource poor settings. It is widely accepted that for a vaccine to confer sterilising immunity to HIV-1 it will have to elicit broadly neutralising antibodies (nAbs) to primary isolates of HIV but this goal has remained elusive. There are two key problems to be addressed in this regard: vaccine candidates have to express the correct epitopes capable of eliciting broadly cross-reactive neutralising antibodies to HIV, and they have to provide potent stimulatory signals to the relevant B cells so that high titres ofneutralising antibodies are obtained. Plasmid DNA and recombinant poxviruses such as modified vaccinia virus Ankara (MYA) and fowlpox virus (FPV) have been safely administered to humans and offer the advantage of accommodating large amounts of additional recombinant DNA. In addition, they have provided promising cytotoxic T-Iymphocyte inducing HIV-l vaccine candidates, especially in heterologous prime-boost combinations. The hypothesis investigated here is that sequentially immunising with envelopes of different clades of HIV might lead to the antibody response being focussed on neutralising epitopes held in common. To overcome the poor immunogenicity of cross-clade neutralising epitopes, and the generally poor antibody responses elicited to priming with DNA, followed by boosting with recombinant poxviruses, B cells were stimulated by the coexpression of cholera toxin B and human complement component C3d. To further stimulate antibody production the candidate vaccines coencoded envelope and capsid components so that there would be in vivo virus-like particle formation. The candidate HIV vaccines proceeded directly to a non-human primate study of immunogenicity because this model most closely resembles the immune response in humans, and the benefits of virus-like particle formation and the adjuvant effect ofhuman C3d were most likely to be demonstrated. The work in this thesis describes the construction and characterisation of complex, adjuvanted poxviral recombinants for use as HIV vaccine candidates in a DNA primeFPV boost-MVA boost in macaques and the subsequent efficacy of macaque sera to neutralise primary isolates ofHIV-l in a validated HIV-l neutralisation assay.

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21

Rajakumar, P. D. "The chloroplast of Chlamydomonas reinhardtii as a platform for recombinant vaccine production". Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1532922/.

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Infectious diseases represent an on-going problem adversely affecting the poultry industries and fish farming. Protein-base vaccines that are currently used for the prevention and spread of diseases have several drawbacks including high cost of recombinant production and delivery; and the needs to eliminate endotoxins when produced in bacterial platforms such E. coli. Therefore, there is an urgent need for effective, cheaper and safer vaccines. The microalga Chlamydomonas reinhardtii could be an ideal candidate for the production of recombinant protein such as vaccines. This is because: i) it has high growth rates; ii) it is generally regarded as safe (GRAS) to eat and therefore does not present a problem of endogenous toxin; iii) it has a genetically tractable chloroplast genome that allows a good expression of recombinant proteins that undergo correct folding to form an active product. Hence, in this research two C. reinhardtii transgenic chloroplast lines expressing the infectious bronchitis virus (IBV) muti-epitope genes (Spike Protein S540-564, Nulceocapsid Protein N67-83 and 120 residues of C terminus of the Nucleocapsid Protein) fused with the adjuvant, CTB and full length nervous necrosis virus (NNV) capsid gene were created. IBV causes respiratory disease in chickens. On the other hand, NNV causes cellular vacoulation and neuronal shape changes in fish. The IBV and NNV antigens were successfully detected anti-HA antibodies by western analysis. The lyophilised C. reinhardtii CTB-IBV that were fed to day 0 chicks, successfully triggered a mucosal and systemic immune response giving rise to antibodies against CTB, IBV and HA epitopes. The NNV fish vaccine trial is currently ongoing in Kasetart University, Thailand. Serum and mucus of NNV vaccinated fish will be analysed in the Purton Lab, UCL. The chloroplast of C. reinhardtii is an attractive organelle for the production of recombinant protein such as vaccines. However, the current level of recombinant protein production in the chloroplast of C. reinhardtii is normally less than 1% of total soluble protein. Therefore, in this study four C. reinhardtii mutant strains (UVM 2, 4, 10 and 11) were created through forward genetics. These mutants exhibit superior expression of recombinant genes in the chloroplast through forward genetics. These strains will be further analysed, so that they can be used to express recombinant genes such as peptide vaccine in the chloroplast of C. reinhardtii in the future.

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22

Labus, Marie B. "The production of a recombinant vaccine against the salmon louse Lepeophtheirus salmonis". Thesis, University of Aberdeen, 1994. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU539163.

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Infestation of commercially reared salmonids with the salmon louse Lepeophtheirus salmonis, is a large problem for the fish farming industries in Scotland, Ireland and Norway. Lice infestations cause high fish mortalities and losses in revenue for the fish farmer. Conventional treatment of lice infestation involves the use of organophosphate insecticides which are limited in their efficacy against lice, as well as being highly toxic to the flora and fauna which surround the sea cages. We have investigated the possibility of producing a recombinant vaccine against L. salmonis based on immunisation of farmed salmonids with 'concealed' antigens from lice. Murine monoclonal antibodies were raised against L. salmonis antigens using standard cell fusion techniques. The antibodies were screened by immunohistochemistry in order to establish the location of the recognised antigen in L. salmonis sections. We used the monoclonal antibodies to screen L. salmonis DNA libraries constructed in gt11 and ZAP in order to isolate the DNA coding for each recognised antigen. A monoclonal antibody was produced which showed strong immunoreactivity with cuticle-associated areas in lice sections. This antibody was subsequently shown to recognise the enzyme chitinase from L. salmonis. We have expressed recombinant proteins from the isolated phage which have been used in immunisation trials on the Atlantic salmon (Salmo salar). Lice feeding on fish immunised with three of the recombinant antigens, displayed abnormal egg development and an overall decrease in egg number. We are currently retesting these antigens in a larger immunisation trial. We have also used polymerase chain reaction technology to look for known genes in L. salmonis and have identified a member of the wnt gene family, wnt-5a.

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23

Chun, Elizabeth M. "Developing a Recombinant Plant Virus Nanoparticle Vaccine for Rift Valley Fever Virus". Scholarship @ Claremont, 2019. https://scholarship.claremont.edu/scripps_theses/1345.

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Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

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24

Nascimento, Ivan Pereira. "Expressão de antígenos de Bordetella pertussis em BCG Recombinante: subunidade 1 da toxina Pertussis e fragmento A da Hemaglutinina Filamentosa (FHA)". Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-15022016-163854/.

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O desenvolvimento de vacinas multivalente constitui urna das prioridades na pesquisa de vacinas modernas. A utilização de vetores vivos para apresentação de antígenos heterólogos mostra-se bastante atraente, e eliminaria a necessidade de várias doses para se alcançar uma proteção máxima. Este tipo de vacina poderia ser administrado em dose única, o que poderia aumentar a cobertura vacinal. Neste trabalho foi explorado o potencial do Mycobacterium bovis BCG recombinante (rBCG) vivo expressando antígenos de Bordetella pertussis, como futuro componente de urna vacina tetravalente rBCG-DTP contra a tuberculose, tétano, difteria e pertussis. Os antígenos de pertussis utilizados foram a subunidade S1 mutada e atóxica da Toxina Pertussis (SI-P1) e o fragmento CRD, um domínio imunogênico da proteína FHA (Adesina Hemaglutinina Filamentosa). PT é o principal fator de virulência de B. pertussis e tanto sozinho como combinado com outros antígenos é o principal componente de todas as vacinas acelulares desenvolvidas até o momento. FHA é um importante fator de aderência da bactéria às células ciliada alvo do hospedeiro, sendo o CRD (domínio de reconhecimento a carboidrato) o responsável pelo reconhecimento de carboidratos em receptores de células do hospedeiro. Os genes detses antígenos foram clonados em vetores de expressão micobacterianos e expressos em BCG sob o controle do promotor mutado da β-lactamase de Mycobacterium fortuitum, pBlaF*, em fusão com o peptídeo sinal da β-lactamase. Estes vetores possuem como marcador de seleção um gene de resistência a kanamicina. Camundongos foram imunizados com rBCG expressando S1-PT e os respectivos esplenócitos mostraram elevada produção de IFN-γ e baixa produção de IL-4, caracterizando uma forte resposta celular Th1 antígeno-específica dominante. rBCG-S1PT induziu uma baixa resposta humoral contra PT. Camundongos imunizados com rBCG-S1PT mostraram elevado nível de proteção contra um desafio intracerebral com uma cepa virulenta de B. pertussis. Animais imunizados com rBCG expressando CRD revelaram a presença de anticorpos anti-FHA no soro. Ensaios com camundongos imunizados com a combinação destas duas vacinas estão sendo realizados. Uma nova abordagem para obtenção de rBCG sem o uso de genes de resistência a antibióticos como marcador de seleção, foi investigada, utilizando a complementação em BCG auxotrófico. Uma cepa de rBCG auxotrófico para lisina foi transformada com vetores de expressão contendo o gene de complementação para lisina e os antígenos de pertussis sob controle do mesmo promotor: a seleção dos recombinantes é realizada em meio sem lisina. Estas construções permitiram a expressão estável dos antígenos e serão avaliadas quanto a indução de uma resposta imunológica efetiva contra pertussis.
The development of combined vaccines constitutes one of the priorities in modem vaccine research. The use of live vectors for heterologous antigen presentation is desirable, as it could eliminate the necessity of several doses to reach a maximum protection and increase vaccine coverage. In this work, the potential of recombinant Mycobacterium bovis BCG (Bacillus Calmette and Guerin) (rBCG), expressing Bordetella pertussis antigens was investigated. The antigens used were the genetically detoxified S1 subunít of pertussis toxin (S1-PT) and the CRD fragment of FHA (Filamentous hemagglutinin. The antigen genes were cloned into mycobacterial expression vectors under control of the upregulated M. fortuitum β-lactamase promoter, pBlaF*, in fusion with the β-lactamase signal sequence. Mice were immunized with rBCG expressing S1-PT and the respective splenocytes induced specific production of INF-γ and low IL-4, characterizing a strong antigen-specific Th1-dominant cellular response. The rSCG-S1 PT induced a low humoral response against PT. Mice immunized with rSCG-S1 PT strains displayed high-level of protection against an intracerebral challenge with live B. pertussis. Animals immunized with rBCG expressing CRD induced anti-FHA antibodies production. Protection induced by the combination of these two strains is being evaluated. A new approach for production of rBCG without the use of antibiotic resistance markers was also investigated, using complementation in auxotrophic BCG. A lysine auxotrophic rSCG strain was transformed with expression vectors containing the complementation gene for lysine and the pertussis antigens: selection of recombinant clones was carried in media without lysine. These constructs allowed steady expression of the antigens and will be evaluated for the induction of an immunological response against pertussis. These strains would be appropriate for clinical evaluation in humans.

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Thomas, Robin. "Recombinant BCG expressing HIV-1 C GAG : selection of the vaccine gene and construction and evaluation as a vaccine candidate". Doctoral thesis, University of Cape Town, 2005. http://hdl.handle.net/11427/2739.

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Branco, Juliana Inês. "Avaliação da imunogenicidade de diferentes formas alélicas da proteína recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/9/9142/tde-12112018-145334/.

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A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax.
Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax.

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Ben, Chehida Regaya Faten. "Etude de la variole ovine en Tunisie et caractérisation des protéines virales impliquées dans la réponse immunitaire anti-capripoxvirus". Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT021.

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Le virus de la variole ovine est omniprésent dans les élevages de petits ruminants dans les pays d’Afrique du Nord et particulièrement en Tunisie malgré les campagnes de vaccination annuelles mises en place par les autorités vétérinaires du pays. L’optimisation de la souche vaccinale utilisée passe par le développement de vaccins dits de nouvelle génération tels que les vaccins sous unitaires utilisant des protéines reconnues pour induire une réponse humorale protectrice chez l’animal immunisé. Ceci pourrait être une alternative aux stratégies de lutte actuelles permettant de limiter la dissémination du virus en Tunisie. Peu de données existent sur les antigènes protecteurs spécifiques des virus du genre Capripoxvirus. Ce travail de thèse a ciblé, par homologie aux protéines du virus de la vaccine, quatre protéines du genre Capripoxvirus appartenant au virus de la dermatose nodulaire contagieuse potentiellement immuno-dominantes nommées LSDV60, LSDV117, LSDV122 etLSDV141 respectivement homologues des protéines L1, A27, A33 et B5. En premier lieu, une analyse structurale in silico a permis d’identifier les domaines essentiels de chaque protéine et de vérifier le taux de conservation de ces protéines parmi différents virus appartenant à la famille des poxvirus. Une analyse structurale approfondie mettant en évidence la structure primaire, secondaire et tertiaire de la protéine A27 a été réalisée. Suite à cette étude structurale, les protéines ont été produites dans deux systèmes d’expression différents ; le système eucaryote et le système baculovirus-cellules d’insectes afin de caractériser leur antigénicité vis-à-vis de sérums provenant d’animaux immunisés ou éprouvés.La reconnaissance des protéines d’intérêt en vecteur d’expression eucaryote n’a pas été concluante. En revanche, le système d’expression BEVS a permis la production de la protéine A27 (L1, A33 et B5 encours) avec succès sous forme soluble qui a été correctement reconnue par des sérums provenant de caprins naïfs challengés. La mise en évidence de formes trimériques et hexamériques confirment sonantigénicité. Une immunodétection des peptides correspondants à la protéine A27 synthétisés surmembranes (PepScan) combinée à une analyse in silico ont permis d'identifier des zones susceptibles de constituer des régions épitopiques reconnues situés majoritairement en partie N terminale de la protéine
The sheep pox virus is omnipresent in small ruminant farms in North African countries andparticularly in Tunisia despite the annual vaccination campaigns set up by the Tunisian veterinaryauthorities. The optimization of the used vaccine strain involves the development of the so-called newgeneration vaccines such as subunit vaccines and this, using proteins recognized to induce a protectivehumoral response in the immunized animal. This could be considered as an alternative to currentcontrol strategies limiting virus spread in Tunisia. Few data exist on protective antigens specific toviruses in the genus Capripoxvirus. By homology to vaccinia virus proteins, this thesis work hastargeted four proteins in the genus Capripoxvirus belonging to the potentially immuno-dominantcontagious nodular dermatosis virus named LSDV60, LSDV117, LSDV122 and LSDV141respectively homologues of proteins L1, A27, A33 and B5. First, an in silico structural analysis hasallowed to identify the essential domains of each protein and to check the conservation rate of theseproteins among different viruses belonging to the poxvirus family. A thorough structural analysisidentifying the primary, secondary and tertiary structure of the A27 protein was conducted. Followingthis structural study, the proteins were produced in two different expression systems, namely theeukaryotic system and the baculovirus-insect cell system, in order to characterize their antigenicity tosera from immunized or proven animals. The recognition of the proteins of interest in the eukaryoticexpression vector has not been conclusive. On the other hand, the BEVS expression systemsuccessfully allowed the production of the A27 protein (L1, A33 and B5 in progress) in a solubleform, which was correctly recognized by sera from challenged naïve goats. Identifying trimeric andhexameric forms confirms its antigenicity. An immunodetection of the peptides corresponding toprotein A27 synthesized on membranes (PepScan) combined with an in silico analysis led to identifyzones capable of constituting recognized epitopic regions located predominantly in part N-terminal ofthe protein

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Piquart, François. "Les vaccins recombinants : données actuelles". Paris 5, 1989. http://www.theses.fr/1989PA05P110.

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Carre, Heather Emily. "Expression and analysis of recombinant mycoplasma hyponeumoniae proteins as potential subunit vaccine candidates". Thesis, Royal Veterinary College (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522182.

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Breau, Cathy. "Development of an oral recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261". Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28079.

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Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of H1V. An effective vaccine against chancroid has yet to be developed. We hypothesize that a Salmonella vector-based vaccine, expressing H. ducreyi antigens, could confer protective immunity in the rabbit model of H. ducreyi infection. The H. ducreyi outer membrane hemoglobin receptor HgbA has been shown to be a suitable vaccine candidate. HgbA was expressed from S. typhimurium SL3261 (pnirBhgbA) but not from the control strain, S. typhimurium SL3261 (pnirB). After a single dose or three doses, at two-week intervals of the vaccine, no antibody response to HgbA was detected in the rabbit model. The vaccine administered was immunogenic and survived in vivo passage. In this small animal trial, we were unable to induce protective immunity against chancroid. We conclude that the vaccine does not confer protective immunity against chancroid.

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Halle, Briana. "Production of a Cost-Effective, TMV-Based Rabies Vaccine through Recombinant DNA Technology". Scholarship @ Claremont, 2018. http://scholarship.claremont.edu/cmc_theses/1816.

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Infectious diseases remain a significant cause of human deaths, as approximately 15 million deaths were attributed to infectious diseases in 2010 (Dye, 2014). One such disease is rabies, which causes around 59,000 human deaths worldwide annually according to some estimates (Kessels et al., 2017). However, 95% of human deaths attributed to rabies occur in Asia and Africa (Singh et al., 2017). Rabies is preventable, yet it is still a major concern in developing, low-income countries that lack access to the medical care necessary to combat it (Hampson et al., 2015). Alternative techniques for low-cost vaccine production have the potential to resolve this issue. This research investigates the use of recombinant DNA techniques and plant biotechnology to produce a more cost-effective vaccine for rabies. Gene sequences from the rabies glycoprotein were inserted at the end of the coat protein portion of the Tobacco Mosaic Virus (TMV) genome. Plants were then infected with this recombinant virus, with hopes that TMV particles would assemble with proteins produced from the inserted glycoprotein sequence fused to the TMV coat protein. Results thus far suggest some of the sequences could be producing recombinant TMV particles, although issues involving successful extraction and reversion to wild type are still a challenge. Additionally, other research suggests that this is an effective method for vaccine development in general and for rabies.

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32

Judd, Fairbanks Lyndon. "Construction of a recombinant fowlpox virus as a vaccine against avian lymphoid leukosis". Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315077.

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Dennehy, Maureen. "Recombinant BCG expressing rotavirus VP6 : construction and evaluation as an anti-rotavirus vaccine". Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/2725.

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Tabar, Gholam Reza Hashemi. "Selection of peptides from random peptide libraries for a recombinant vaccine against dermatophilosis". Thesis, Tabar, Gholam Reza Hashemi (1998) Selection of peptides from random peptide libraries for a recombinant vaccine against dermatophilosis. PhD thesis, Murdoch University, 1998. https://researchrepository.murdoch.edu.au/id/eprint/53219/.

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Dermatophilosis commonly known as "lumpy wool" in sheep in Australia, is a skin disease of animals and man caused by Dermatophilus congolensis. In sheep dermatophilosis causes significant wool production losses both directly and indirectly due to down grading of wool and hides, low meat production and high mortality. particularly in young sheep. Control of the disease by vaccination is desirable but has only been partially successful and antigens are difficult to produce in sufficient quantity from D. congolensis. Dr T.M. Ellis and colleagues (Agriculture WA) have developed a vaccine that can produce a significant reduction in ovine dermatophilosis against several strains of the bacterium. In pen and field studies, the haemolysin based vaccine did not prevent initial infection but approximately 70% were protected against the development of lumpy wool. Their previous research showed that a vaccine prepared from a serine protease produced by D. congolensis also gave some protection against the disease. This antigen was expensive to prepare by conventional culture. An alternative approach is to use recombinant protein as an antigen. However, this is not a simple task if the protective antigens have not been identified and even if they have been, it can be difficult to prepare a recombinant protein in high yield. Random peptide libraries provide an alternative approach to the identification of peptides which might be useful in a vaccine. While these libraries have been used to identify epitopes and prepare diagnostic tests, their potential to produce antigens which could generate a protective immune response has still to be explored. The main aim of this thesis was to use random peptide libraries to identify new antigens which could be used in the future to immunise sheep and other animals against dermatophilosis. Because of the commercial availability of large random peptide libraries displayed on phage and flagellin there is an opportunity to produce low cost and immunologically potent peptide vaccines. To explore the effectiveness and reliability of random peptide libraries (Ph.D.™ and FliTrxTM libraries) for the selection of peptides, polyclonal antibodies against a recombinant serine protease from D. congolensis were used to pan the libraries. This recombinant serine protease was produced using a pQE expression vector and purified by immobilised metal affinity chromatography. Clones selected from the libraries were sequenced and the peptides aligned with the original amino sequence of serine protease. Many of the peptides aligned with varying homology to the serine protease sequence which demonstrated that the antibodies were selecting specific sequences from the libraries rather than just random peptides. To obtain peptides, which might be associated with a protective immune response to D. congolensis sheep which had been immunised with a crude enzyme preparation form D. congolensis by Dr T.M. Ellis were used as a source of antibodies. Four peptides from Ph.D.TM and three peptides from FliTrx™ libraries were chosen for vaccination of sheep. Sheep were given two doses of vaccine one month apart. Twentyone days after the second vaccination each sheep was challenged with a zoospore suspension of the MB and W14 strains of D. congolensis and the presence of lesions and their severity were measured at 7, 14 and 21 days after challenge. The immune response to D. congolensis antigens was also studied. There was a striking production of antibodies to antigens from D. congolensis induced by the peptides selected from the random peptide libraries. Vaccination with recombinant serine protease and with the peptides selected from the Ph.D.™ library increased the rate of resolution of the lesions caused by one strain of D. congolensis. The results in this thesis provide the first demonstration in large animals that phage displayed peptides can induce a specific immune response against an infectious agent.

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O’Meara, Kellie Marcella. "Evaluation of Recombinant Salmonella Expressing CD154 for Enhanced Immune Responses in Commercial Turkeys". The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1246567532.

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Teixeira, Aline Rodrigues Florencio. "Avaliação e caracterização de candidatos vacinais voltados para o controle da leptospirose". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-25082016-100745/.

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A leptospirose é uma doença sistêmica, causada por bactérias patogênicas do gênero Leptospira. O desenvolvimento de novas estratégias para prevenir a doença é necessário. Vacinas surgem como fortes candidatas para contornar o problema. As pesquisas atuais têm interesse em identificar antígenos conservados que estão envolvidos nas interações patógeno-hospedeiro.O presente projeto selecionou três proteínas hipotéticas de L. interrogans para serem caracterizadas quanto ao seu papel na patogênese e avaliadas quanto ao seu potencial protetor. Os genes foram amplificados por PCR e clonados no vetor de expressão PAE. As proteínas recombinantes foram purificadas por cromatografia de afinidade e foram reconhecidas por soro de indivíduos infectados. As proteínas LIC13479 e LIC10050 foram capazes de se ligar a laminina, plasminogênio e fibronectina plasmática. Em relação à LIC10537, dois fragmentos recombinantes foram gerados. Apenas o fragmento 2 foi capaz de interagir com PLG. As proteínas que interagiram com o PLG foram capazes de gerar plasmina As proteínas foram capazes de estimular uma resposta imune e LIC13479 e LIC10050 exerceram proteção parcial no modelo de leptospirose em hamsters.
Leptospirosis is a systemic disease caused by pathogenic bacteria of genus Leptospira. The development of new strategies to prevent the disease is needed. Vaccines emerge as strong candidates to fight the problem.Currently research has focused to identify conserved antigens This project selected three hypothetical proteins of L. interrogans. Thesecoding sequences were characterized for their possible role in pathogenesis and their potential to protect animals against challenge with virulent leptospires. Genes were amplified by PCR and cloned into the expression vector pAE. The recombinant proteins were purified by metal affinity chromatography and were recognized by confirmed human leptospirosis serum samples.LIC13479 and LIC10050 proteins were able to bind with laminin, plasminogen and plasma fibronectin. The coding sequence LIC10537 was cloned in two fragments. Fragment 2was able to interact with plasminogen. All proteins were able to generate active plasmin. The recombinant proteins were able of inducing an immune response. Evaluation of immunoprotection in leptospirosis hamster model followed by challenge with virulent bacteria showed that the recombinant proteins conferred partial protection.

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Mendes, Jéssica Mariane Ferreira. "Produção e Avaliação de antígenos recombinantes candidatos a componente de uma vacina contra leishmaniose visceral canina". reponame:Repositório Institucional da FIOCRUZ, 2016. https://www.arca.fiocruz.br/handle/icict/14161.

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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
Uma vacina efetiva contra a leishmaniose visceral (LV) canina pode contribuir para o controle da doença no homem. Visando o desenvolvimento de uma vacina contra LV canina, antígenos recombinantes de L. infantum foram selecionados em nosso laboratório pelo uso de uma mistura de soros de seres humanos ou cães naturalmente infectados pela L. infantum. Alguns destes antígenos foram testados em diversos protocolos de imunização, incluindo uso de diferentes adjuvantes, em camundongos ou cães. A imunização de camundongos ou cães com um dos antígenos recombinantes (rLci2B) usado isoladamente ou em associação com saponina induziu resposta imune Th2 ou Th1/Th2, respectivamente, não protetoras contra a infecção experimental. Com a determinação da sequência deduzida de aminoácidos notou-se que a maioria dos antígenos selecionados apresenta um segmento com sequência de aminoácidos única (domínio não repetitivo) e segmentos com sequência de aminoácidos com motivos repetitivos (domínios repetitivos). Possivelmente a incapacidade dos antígenos recombinantes de induzir uma resposta imune predominantemente Th1, protetora contra a LV, seria por conta da presença de domínios repetitivos, que favorecem a apresentação antigênica por linfócitos B e, consequentemente, estimulam uma resposta imune Th2. Para avaliar o direcionamento da resposta imune pelos dois tipos de domínio, novas construções de DNA foram concebidas de modo a codificar apenas domínio(s) não repetitivo(s) ou domínio(s) não repetitivo(s) e domínios repetitivos. OBJETIVOS: Produzir quatro proteínas recombinantes com domínios não repetitivos (rLci2-NT-CT, rLci3-NT-CT, rLci10-NT e rLci12-NT-CT) e avaliar a capacidade desses polipeptídios de induzir resposta imune celular in vitro em cães assintomáticos inoculados por via dérmica com L. infantum. MATERIAIS E MÉTODOS: Foram realizadas: a) a subclonagem de construções de DNA (Lci3-NT-CT, Lci10-NT e Lci12- NT-CT) em um plasmídeo apropriado para expressão em Escherichia coli, b) a determinação de condições apropriadas para produção das proteínas recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R e rLci10- NT) c) a purificação das proteínas recombinantes por cromatografia de afinidade e d) avaliação da capacidade dos polipeptídios de induzir estimulação de células mononucleares sangue periférico (PBMC) de cães assintomáticos inoculados por via dérmica com L. infantum. RESULTADOS: Três (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3- NT-CT, rLci3-NT-2R-CT, rLci10-NT e rLci10-NT-2R) dos quatro pares de polipeptídios recombinantes foram expressos, produzidos e purificados. Três antígenos recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT e rLci3-NT-2R-CT) promoveram a linfoproliferação in vitro utilizando PBMC de cães assintomáticos inoculados por via dérmica com L. infantum CONCLUSÕES: Três das seis proteínas produzidas induziram a linfoproliferação, sendo a maior linfoproliferação encontrada para PBMC estimulado com a proteína sem domínios repetitivos (rLci2-NT-CT). Avaliações adicionais são necessárias para comprovar a utilidade destas moléculas em formulação de vacina contra leishmaniose visceral canina.
An effective vaccine against visceral leishmaniasis (VL) dog can help to control the disease in man. Aiming at development of a vaccine against canine VL, recombinant antigens of L. infantum were selected in our laboratory by using a mixture of sera from humans or dogs naturally infected with L. infantum. Some of these antigens were tested in different immunization protocols, including use of different adjuvants in mice or dogs. The immunization of mice or dogs with a recombinant antigens (rLci2B) used alone or in combination with saponin induced Th2 response or Th1 / Th2, respectively, did not protective against experimental infection. With the determination of the deduced amino acid sequence it was noted that most of the antigens selected segment has a unique amino acid sequence (non-repetitive domain) and segments of amino acid sequence with repetitive motifs (repetitive domains). Possibly the inability of recombinant antigens to induce an immune response predominantly Th1 protective against LV would be due to the presence of repetitive domains that promote antigen presentation by B cells and thus stimulate an immune response Th2. To assess the direction of the immune response by two types of domain, new DNA constructs were designed to encode only the domain (s) not repetitive (s) or domain (s) not repetitive (s) and repetitive fields. MATERIALS AND METHODS: Were performed: a) subcloning DNA constructs (rLci3-NT-CT, rLci10-NT and rLci12-NT-CT) into a suitable plasmid for expression in Escherichia coli, b) determining the appropriate conditions for the production of proteins recombinant (rLci2- NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R and rLci10-NT) c) purification of recombinant proteins by chromatography affinity d) evaluating the ability of polypeptides rLci2-NT-CT, rLci3-NT-CT and rLci10-NT to induce stimulation of peripheral blood mononuclear cells (PBMC) from healthy dogs inoculated dermal with L. infantum. RESULTS: Three (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3-NT-CT, rLci3-NT-2R-CT, rLci10- NT and rLci10-NT-2R) of the four pairs of recombinant polypeptides are expressed, produced and purified. Three recombinant antigens (rLci2-NT-5R-CT, rLci2-NT-CT and rLci3-NT-2R-CT) promoted lymphocyte proliferation in vitro using asymptomatic dogs PBMC inoculated dermal L. infantum CONCLUSIONS: Three of the six proteins produced induced lymphoproliferation, most lymphoproliferation was found to PBMC stimulated with the protein without repetitive domains (rLci2-NT-CT). Additional evaluations are necessary to confirm the utility of these molecules against canine visceral leishmaniasis vaccine formulation

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38

Poobalane, Saravanane. "Aeromonas hydrophila vaccine development using immunoproteomics". Thesis, University of Stirling, 2007. http://hdl.handle.net/1893/195.

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Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

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Kalicharran, Kishna Kumar. "Studies on the stability, production and microencapsulation of a recombinant human adenovirus-rabies vaccine". Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/7579.

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The recombinant human adenovirus type 5 (rHAd5-RG1) tested in this study contains the rabies virus glycoprotein gene. This virus is being considered as a possible substitute for the attenuated rabies virus in the oral immunization of wildlife in Ontario. This study examined the stability of the virus indoors and outdoors, and tested technically simple ways of concentrating the virus and its microencapsulation. The findings of this study show that the recombinant virus has the potential for release into the environment and there is no evidence that the virus will rapidly decay under the outdoor conditions experienced during the fall season. The virus can probably be microencapsulated and packaged for oral delivery. However, more studies are needed to assess the actual immunizing potential of these microencapsules. The use of detergents for increasing the virus yield has potential. (Abstract shortened by UMI.)

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Kuzyk, Michael Allan. "Development of an efficacious recombinant vaccine for the obligate intracellular salmonid pathogen Piscirickettsia salmonis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0010/NQ52764.pdf.

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Lu, Dongmei Hickey Anthony J. "Aerosol delivery of recombinant antigen 85B in microparticle vaccine systems for protection against tuberculosis". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1388.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmaceutical Sciences." Discipline: Pharmaceutical Sciences; Department/School: Pharmacy.

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42

Silva, Maria Elizabeth. "Development of a Recombinant Attenuated Salmonella Vaccine System for Taenia Solium Cysticercosis in Pigs". Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/biology_diss/81.

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Taenia solium is a cestode that has a two-hosts life cycle. The adult tapeworm causes an asymptomatic disease known as taeniasis whereas the larval stage causes a disease called cysticercosis. In humans, the most common localization for the larvae is the central nervous system where it produces the neurological disorder neurocysticerco-sis. Previous works by several research groups around the world have shown that T. so-lium is a potentially eradicable parasite. Control programs have included treatment of human and pig populations with antihelmintics in conjunction with health education and are now considering vaccination of naïve piglets. The potential of a live vector vaccine system to deliver Taenia solium Tsol18, a proven protective antigen, to prevent transmission of cysticercosis was investigated. An attenuated strain of Salmonella enterica serovar Typhimurium χ9402 was used to develop an oral delivery system. Tsol18 gene was cloned downstream from the β-lactamase signal sequence in a multicopy asd + plasmid vector pYA3620 to yield plasmid pYA3620/Tsol18 and then transformed into the vaccine strain. The recombinant atte-nuated salmonella vaccine construct was stable for 50 generations and expressed rTsol18. Immunization of mice either with one or two doses of 109 CFU of the recombi-nant vaccine strain carrying plasmid pYA3620/Tsol18 elicited specific antibody response to Salmonella self antigens and to rTsol18. Moreover, oral immunization of piglets with 1012 CFU of the vaccine construction significantly reduced the numbers of viable cysts after challenged. The development of a quantitative assay to detect specific antibodies against Tsol18 is also presented here. The Falcon assay screening test –enzyme linked immu-noabsorbant assay (FAST-ELISA) format was used to develop a quantitative antibody detection assay. We have cloned, expressed and purified rTsol18. With purified porcine IgGs we constructed a standard curve that can be used to quantify the immune re-sponse. Our Fast-ELISA was able to follow the kinetics of the immune response in vac-cinated pigs from an experimental trial. The data we present here provides the basis for a safe, affordable and easy vaccine delivery system that can be used as an adjunct in control programs.

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Dar, Nosheen. "Development of replication defective recombinant adenoviruses for the purpose of HIV-1 vaccine delivery". Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429507.

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Sheih, Tianna. "Development of a Dengue Fever Vaccine from Recombinant DENV2 Protein and Tobacco Mosaic Virus". Scholarship @ Claremont, 2016. http://scholarship.claremont.edu/scripps_theses/810.

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Dengue fever is a rapidly growing concern to human health and is currently the most prevalent mosquito-borne viral disease worldwide. Although there are several vaccine candidates being tested in clinical trials, there are no vaccines publicly available to prevent this disease. Plant-based vaccines are rapidly becoming viable alternatives to traditional animal-based vaccines because they are safe, easy to manufacture, and more cost-efficient. The purpose of this project is to develop a vaccine against the dengue virus by producing a recombinant DENV2 protein, engineered by Dr. David Lo and his lab at University of California Riverside, in Nicotiana benthamiana plants through Tobacco Mosaic Virus (TMV) infection. Initial attempts to ligate the complete DENV2 epitope, a combination of hybrid flagellin sequences and the envelope protein from dengue viral serotype 2, into the pJL TRBO vector were incompatible with established protocols. However, a proof of concept test that replaced the DENV2 envelope protein with Green Fluorescent Protein (GFP) successfully inserted the new sequence into pJL TRBO. In the future, the DENV2 envelope protein sequence will be re-inserted into the construct and updated protocols will be repeated for DENV2 protein expression. The recombinant DENV2 proteins will be extracted from the plants after signs of infection become apparent and tested for their ability to induce an immunogenic response that produces pathogen-specific antibodies.

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Ralikhwatha, Tovhowani Daphney. "Development and evaluation of a recombinant vaccine against Clostridium perfringens type D epsilon toxin". Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/79304.

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Enterotoxemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens Type D. To combat the disease, a vaccine consisting of formalin-inactivated Type D toxin is available, but several concerns regarding its production have been raised. The development of efficacious recombinant subunit vaccines can provide a means whereby many of the production problems may be eliminated or minimized. However, the production of purified antigens is often associated with weakened antigen immunogenicity. New vaccine strategies therefore rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. An increasing number of studies have demonstrated that flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. Consequently, the aim of this investigation was to produce different recombinant vaccine candidates, inclusive of a flagellin-epsilon toxin fusion protein, for preventing Type D enterotoxemia. Attempts at expressing the native gene sequence for the Type D epsilon toxin in Escherichia coli have been characterized by low levels of expression. This may be due to differences in the codon bias between the heterologous gene and E. coli. Therefore, in this study, a completely synthetic codon-optimized gene encoding the epsilon toxin was obtained. The full-length etx gene (etx0), an etx gene lacking the first nine amino acids of the signal peptide sequence (etx1), as well as a flagellin-toxin fusion gene composed of the etx1 gene and a truncated hag gene of Bacillus halodurans Alk36 (Δhag::etx1) were expressed in E. coli BL21-Gold(DE3) cells. Expression of the respective recombinant proteins, designated ETX0, ETX1 and NC-ETX1, respectively, were confirmed by Western blot analysis using anti-epsilon toxin antibodies. The ETX0 protein was expressed in a soluble form, whereas the ETX1 and NC-ETX1 proteins were insoluble and accumulated in inclusion bodies. In an attempt to optimize expression of the recombinant proteins, different cultivation media and concentrations of the promoter inducer were evaluated. Under optimized expression conditions, the yields of ETX0, ETX1 and NC-ETX1 were determined to be 828 mg/l, 395 mg/l and 525 mg/l, respectively. Despite having expressed the recombinant proteins with an N-terminal hexahistidine tag, the recombinant ETX0 protein could not be purified with nickel (Ni2+) chelate affinity chromatography. This was due to removal of the affinity tag as a consequence of intracellular processing of the N-terminal signal peptide sequence. The recombinant ETX0 protein was consequently purified to near homogeneity by ion exchange chromatography. The recombinant ETX1 and NC-ETX1 proteins were purified from inclusion bodies by affinity chromatography under denaturing conditions and then refolded in TBS buffer. The yield of purified ETX0, ETX1 and NC-ETX1 were determined to be 430 mg/l, 400 mg/l and 340 mg/l, respectively. The cytotoxicity of these purified recombinant proteins was assayed in vitro against Madin-Darby canine kidney (MDCK) cells, the results of which indicated that NC-ETX1 was not toxic towards the cells and both ETX0 and ETX1 displayed moderate toxicity towards the cells. Cumulatively, the results indicated that the respective proteins were expressed to high levels in E. coli and large amounts of purified proteins could be obtained. These recombinant proteins can in future be evaluated as vaccine candidates against C. perfringens Type D enterotoxemia.
Dissertation (MSc)--University of Pretoria, 2013.
Microbilogy and Plant pathology
MSc
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46

au, T. La@murdoch edu, i Tom La. "Characterisation, Recombinant Expression and Immunogenicity of BHLP29.7, An Outer Membrane Lipoprotein of Brachyspira Hyodysenteriae". Murdoch University, 2006. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070830.141953.

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Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative 10 and 35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7. The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system. An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis. Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.

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com, tickle_me_patty@hotmail, i Patrick Leslie Shearer. "Development of Novel Diagnostic and Vaccine Options for Beak and Feather Disease Virus". Murdoch University, 2009. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20090720.142800.

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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.

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48

Kremer, Courtney J. "Evaluation of Recombinant Salmonella Expressing the Flagellar Protein FliC for Enhanced Immune Responses in Commercial Turkeys". The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1246568225.

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Eldaghayes, Ibrahim Mohamed. "Use of chicken interleukin-18 as a vaccine adjuvant with a recombinant fowlpox virus fpIBD1, a subunit vaccine giving partial protection against IBDV". Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419211.

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Cavalcanti, Marcos Antônio Rocha. "Efeito de vacinas alopática e homeopática frente a Mycobacterium spp em diferentes modelos animais". Universidade Federal de Goiás, 2013. http://repositorio.bc.ufg.br/tede/handle/tede/3148.

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The development of new vaccines in the control of various diseases in cattle has been increasing the marketing of animals and animal products. Thus we tested two vaccines, a biotherapy homeopathicvaccine and other recombinantallopathic vaccine,using mycobacteria in their formulations that were subsequently tested in mice and cattle. In order to study the prophylactic effect of homeopathic vaccine and the potency to be used as a vaccine, we used a model of immunizations and infections. To this end, we used mice female of BALB / c lineage which were distributed in 15 groups of three animals each. To assess the possible immune mechanisms involved in homeopathic biotherapy vaccinations we used Mycobacterium massiliense. The biotherapics were prepared from mycobacterial M. massiliense. After infections and immunizations, the animals were euthanized and their livers and spleens were harvested, macerated, homogenized, plated and incubated at 37 ° C for five days. Then we did the counting of colony forming units (CFU) of bacteria recovered from organs and according to the results obtained were selected the potencies11cH and 19cH to be tested as vaccine, because they have shown more homogeneous results. In the animals that were immunized with 19 cHthere were induction of IgG2a class antibodies specific to M. massiliense similar to (0.18 ± 0.07) infections alone (0.19 ± .02). To assess allopathic vaccine protection was used mycobacterium (Mycobacterium smegmatis mc2155 with PLA71/Fusion) in cattle. After allopathic vaccinations, blood was collected and serum was removed for ELISA test. Animals that received the recombinant live vaccine expressing protein of fusion showed greater levels of specific antibodies (p <0.01). This study evaluated the effect ofhomeopathic biotherapy vaccine composed of M. massiliense and allopathicvaccine formulated with M.smegmatis recombinant in different animal models, thus concluding that both vaccines and vaccines homeopathic and allopathic using different kinds of mycobacteria can induce humoral immune response in an animal model.
O desenvolvimento de novas vacinas no controle de várias doenças na bovinocultura vem incrementando a comercialização de animais e produtos de origem animal. Com isso testaram-se duas vacinas, uma vacina bioterápica homeopática e outra vacina alopática recombinante, utilizando micobacterias em suas formulações que posteriormente foram testadas em camundongos e bovinos. Com o objetivo de estudar o efeito profilático da vacina homeopática e a potência a ser utilizada como vacina, foi empregado um modelo de imunizações e infecções. Para tal, utilizou-se camundongos fêmeas da linhagem BALB/c as quais foram distribuídas em 15 grupos com três animais cada. Para avaliar os possíveis mecanismos imunológicos envolvidos nas vacinações bioterápicas homeopáticas utilizou-se Mycobacterium massiliense. Os bioterápicos foram preparados a partir de micobactérias M. massiliense. Após as imunizações e infecções, os animais foram eutanaziados e deles colheram-se os fígados e baços, os quais foram macerados, homogeneizados, plaqueados e incubados a 37ºC durante 5 dias. Em seguida, fez-se a contagem de unidades formadoras de colônias (UFC) das bactérias recuperadas dos órgãos e de acordo com os resultados obtidos foram selecionadas as potências 11cH e 19cH para serem testadas como vacina, por apresentarem resultados mais homogêneos. Nos animais imunizados com 19 cH houve indução da produção de anticorpos da classe IgG2a específicos para M. massiliense semelhantes à (0,18 ± 0,07) infecção sozinha (0,19 ± 0,02). Para avaliar a proteção da vacina alopática, foi utilizada a micobactéria (Mycobacterium smegmatis mc2155 com PLA71/Fusão), em bovinos. Após as vacinações alopáticas foi coletado sangue e retirou-se o soro para o teste de ELISA. Animais que receberam a vacina viva recombinante expressando a proteína de fusão apresentaram níveis maiores de anticorpos específicos (p< 0,01). Com este estudo avaliou-se os efeitos da vacina bioterápica homeopática composta de M. massiliense e de vacina alopática formulada com M. smegmatis recombinante em diferentes modelos animais, concluindo assim que tanto as vacinas homeopáticas e vacinas alopáticas usando diferentes tipos de micobactérias podem induzir resposta imune humoral em modelo animal.

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