Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), one of the most important bacterial diseases of farmed channel catfish in the USA. Use of live attenuated vaccines (LAVs) is an effective strategy for combating mortalities in catfish farms. Our research group has developed three live attenuated E. ictaluri strains [EiΔevpB, EiΔgcvPΔsdhCΔfrdA (ESC-NDKL1), and EiΔhemRΔfrdAΔsdhC (triple-hemR)] that provide various levels of protection against ESC. However, the protective mechanisms of these vaccine candidates are mostly unknown. The overall objective of my study was to investigate protective mechanisms of these LAVs. To accomplish this, catfish fry were immersion challenged with EiΔevpB, ESC-NDKL1, and triple-hemR. Additional catfish fry were immersion challenged with Aquavac-ESC and E. ictaluri wild-type (EiWT) as controls. The internalization of antigens through the mucosal surfaces as well as the pathology and molecular immune responses were studied. The investigations showed that EiΔevpB and ESC-NDKL1 were highly safe and efficacious compared to Aquavac-ESC and EiWT. Pathologically, vaccination with EiΔevpB and ESC-NDKL1 decreased the pathological lesions, EiWT replication in catfish, and increased the ability of the immune system to resist and kill EiWT. On the other hand, triple-hemR was not safe causing severe tissue damage similar to EiWT. The gene expression data showed significantly high expression of innate and adaptive immune genes following vaccination and challenge with EiWT. Additionally, EiΔevpB and ESC-NDKL1 induced immune proliferation in pronephros after exposure to EiWT. Taken together, EiΔevpB and ESC-NDKL1 vaccine strains performed better to induce immune responses and reduce damage in the host tissues during EiWT challenge compared to Aquavac-ESC.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T14:46:42Z (GMT). No. of bitstreams: 1 Milanez_GuilhermePaier_M.pdf: 1651986 bytes, checksum: 4681c88f937b4fe6f4d915fce0eb3f20 (MD5) Previous issue date: 2012 / Resumo: A Salmonelose e a malária são duas doenças infecciosas negligenciadas de grande prevalência no mundo, mas acometendo particularmente populações humanas que vivem em regiões e países subdesenvolvidos ou em desenvolvimento. Apesar dos esforços ainda não existem formulações vacinais totalmente efetivas no controle destas enfermidades. Desta forma, nosso grupo tem buscado aprimorar a expressão do Domínio M2 do antígeno MAEBL de Plasmodium yoelii, que apresenta similaridade com a proteína de Plasmodium falciparum, em linhagens de Salmonella enterica Typhimurium, atenuadas para virulência. Em estudos prévios constatou-se que a expressão em níveis baixos não seria suficiente para a indução de uma resposta imune protetora. Com isso, ficou clara a necessidade de se aprimorar os níveis de expressão deste antígeno nas linhagens a serem testadas. Para tanto, o vetor de expressão anteriormente utilizado foi modificado, os codons da seqüência a ser expressa foram otimizados para expressão em salmonela e um sinal de secreção periplasmática foi adicionado no início da seqüência a ser expressa. Adicionalmente, modificamos novas linhagens atenuadas de S. enterica desenvolvidas por nosso grupo de tal forma a utiliza-las na expressão de antígenos heterólogos, empregando um sistema letal balanceado (Asd) de estabilização da expressão gênica. Apesar do sucesso na construção dos novos vetores, e da otimização de codons para a expressão, constatamos um possível efeito deletério do domínio M2 quando expresso em grande quantidade, o que impossibilitou a obtenção de algumas construções planejadas. Entretanto, os resultados obtidos na estabilização de plasmídios em novas linhagens vacinais significam um avanço no teste e uso de tais linhagens no desenvolvimento de vacinas multifatoriais / Abstract: Salmonellosis and malaria are two infectious diseases of high prevalence in the world, particularly affecting human populations living in underdeveloped regions and countries or developing countries. Despite several efforts, there is not a vaccinal formulation totally effective to control these diseases. This way, our group has sought to enhance the expression of M2 MAEBL's antigen Domain of Plasmodium yoelii, which is quite similar to same protein of P. falciparum, in attenuated strains of Salmonella enterica Typhimurium. In previous studies we found that the expression at low levels was not sufficient to induce a protective immune response. Thus, there was a clear need to improve M2 expression levels in the new strains to be tested. The expression vector previously used was modified, the m2 sequence was codon-optimized for expression in Salmonella, and a periplasmic secretion signal was added at the beginning of the sequence to be expressed. Additionally, new attenuated S. enterica strains developed by our group were modified in a way to use them in the expression of heterologous antigens, employing a balanced lethal system (ASD) for expression plasmid stabilization. Despite of the new vectors successfully built as well as codon optimization for m2 expression, we found a possible deleterious effect of the M2 domain when expressed in large quantities, making it impossible to obtain some planned constructs. However, the results obtained on stabilization of expression vectors in the new vaccine strains are an important advance for the use of such strains as multifactorial vaccines / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Vaccination against intracellular pathogens requires generation of pool of memory T cells, which can respond upon infection and mediate immune responses by either killing of infected host cells or induce killing mechanisms in infected cells. T cell-inducing vaccines aim to deliver the antigen to antigen presenting cells (APCs) by presenting on MHC molecules thus bridging innate and adaptive immunity. The intracellular pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is a devastating disease in catfish industry. E. ictaluri can survive in professional phagocytes and use them as an infection source. Two new live attenuated vaccine (LAV) strains, EiDELTAevpB and ESC-NDKL, were developed by our group. However, the role of LAVs in phagocytosis, bacterial killing, and antigen presentation is unexplored. Therefore, further research is necessary to determine immune responses in channel catfish against LAVs. The long-term goal of this project is to identify immunological APC-dependent mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for ESC. The overall goal of this project is to assess the role of three professional APCs, dendritic cells (DCs), macrophages and B cells in the LAV-induced innate and adaptive immune responses in catfish. The central hypothesis is that efficacious LAV strains will enhance phagocytosis and microbial killing, and promote the generation of T cells that regulate and control protective B cell-mediated immunity. The rationale for this research is that more detailed knowledge about phenotype and function of catfish APCs will not only help gain insight into the evolution of vertebrate adaptive immune system but will provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use. In this study, we first identified DC-like cells in immune-related organs of catfish and assessed their expression patterns in lymphoid organs of catfish in E. ictaluri infection. Although WT strain induces the functional inability of DC-like cells in migration and maturation, LAVs strains promote the migration and maturation of DC-like cells for antigen presentation. Two LAVs enhanced the phagocytosis and killing activity in catfish macrophages and B cells. Also, LAVs induce high expression of T cell-related genes without causing inflammation.

Uma das estratégias mais promissoras para a biotecnologia de vacinas é o desenvolvimento de linhagens precisamente atenuadas, e que possam ser usadas como carregadoras de antígenos heterólogos. Mutantes de <i}>Salmonella Typhimurium têm sido extensivamente utilizados com essa fmalidade. A flagelina, monômero constituinte do filamento flagelar, vem sendo empregada como carregadora de antígenos heterólogos, inseridos na região central, hipervariável (região IV). Inserções nessa região são freqüentemente funcionais, e levam à exposição do epitopo na superfície do filamento. O presente trabalho explora o potencial de outras regiões da molécula para a inserção de epitopos. Nós inserimos a mesma seqüência usada anteriomente (epitopo da proteína M de S. pyogenes, Tipo 5) em regiões com diferentes níveis de homologia (III e VI), e em região totalmente conservada (VIII). Também foram feitas inserções duplas em regiões que se mostraram toleráveis (III e IV; IV e VI). Todas as proteínas híbridas foram sintetizadas pela Salmonella, como demonstrado em imunoblots, usando anticorpo contra a flagelina e contra o peptídeo. Todas as regiões, exceto a VIII, aceitaram a inserção sem perda de motilidade, apesar de, em alguns casos, ela ter sido extremamente reduzida. A imunogenicidade foi avaliada pela imunização de camundongos com bactérias vivas, inativadas ou, quando possível, flagelina purificada. Os resultados foram similares aos descritos na literatura para inserções envolvendo a região IV, obtendo-se um elevado título de anticorpos contra flagelina. Um baixo nível de anticorpo contra o peptídeo também foi detectado para todas as novas linhagens testadas. Nossos resultados com imunização de bactérias vivas sugerem uma resposta levemente melhor ao peptídeo quando duas cópias estão presentes, mas os dados não são conclusivos. / One of the most promising strategies for the biotechnology of vaccines is the development of precisely attenuated strains, which could be used as carriers of heterologous antigens. Mutants of Salmonella Typhimurium have been extensively explored to this effect, since the infection ofmice by S. Typhimurium mimics the infection of humans by S. Typhi, and the genetics of the species is extremely well known, making it easy the obtention of defined mutants with reduced pathogenicity. Mutants with auxotrofy in genes of the aromatic pathway are particularly attractive, since they need PABA and DHB to grow, and these compounds are unavailable in mammalian tissues. Flagellin, the monomer which constitutes the flagellar filament, has been used as a carrier for heterologous epitopes, inserted in a central, hypervariable region (region IV). Insertions in this region are often functional, and lead to exposition of the epitope at the filament\' s surface. The present work explored the potential of the other regions ofthe molecule for the insertion of epitopes. We inserted the same reporter sequence (MS epitope from S. pyogenes M protein) in regions with different levels of homology (III and VI), and totally conserved (VIII). We also made double insertions in regions shown to be permissive (III and IV; IV and VI). All hybrid proteins were synthesized by Salmonella, as demonstrated by immunoblots using antibody against flagellin and against the synthetic peptide. All regions, except the highly conserved region VIII, accepted the insertions without loss of motility, albeit, in some cases, motility was seriously reduced. Immunogenicity of the hydrids was evaluated by immunization with live bacteria, killed bacteria, and purified flagellin (when possible). Results obtained with the new constructs were similar to the ones published for insertions involving region IV, in the sense that antibody titers to the carrier protein were very high. A low level of antibody to the inserted peptide was also detected in all groups of animals. Our results with live immunization suggest a slightly better response to the peptide when two copies are present, but the data are not conclusive.

Uma das estratégias mais promissoras para a biotecnologia de vacinas é o desenvolvimento de linhagens precisamente atenuadas, e que possam ser usadas como carregadoras de antígenos heterólogos. Mutantes de <i}>Salmonella Typhimurium têm sido extensivamente utilizados com essa fmalidade. A flagelina, monômero constituinte do filamento flagelar, vem sendo empregada como carregadora de antígenos heterólogos, inseridos na região central, hipervariável (região IV). Inserções nessa região são freqüentemente funcionais, e levam à exposição do epitopo na superfície do filamento. O presente trabalho explora o potencial de outras regiões da molécula para a inserção de epitopos. Nós inserimos a mesma seqüência usada anteriomente (epitopo da proteína M de S. pyogenes, Tipo 5) em regiões com diferentes níveis de homologia (III e VI), e em região totalmente conservada (VIII). Também foram feitas inserções duplas em regiões que se mostraram toleráveis (III e IV; IV e VI). Todas as proteínas híbridas foram sintetizadas pela Salmonella, como demonstrado em imunoblots, usando anticorpo contra a flagelina e contra o peptídeo. Todas as regiões, exceto a VIII, aceitaram a inserção sem perda de motilidade, apesar de, em alguns casos, ela ter sido extremamente reduzida. A imunogenicidade foi avaliada pela imunização de camundongos com bactérias vivas, inativadas ou, quando possível, flagelina purificada. Os resultados foram similares aos descritos na literatura para inserções envolvendo a região IV, obtendo-se um elevado título de anticorpos contra flagelina. Um baixo nível de anticorpo contra o peptídeo também foi detectado para todas as novas linhagens testadas. Nossos resultados com imunização de bactérias vivas sugerem uma resposta levemente melhor ao peptídeo quando duas cópias estão presentes, mas os dados não são conclusivos. / One of the most promising strategies for the biotechnology of vaccines is the development of precisely attenuated strains, which could be used as carriers of heterologous antigens. Mutants of Salmonella Typhimurium have been extensively explored to this effect, since the infection ofmice by S. Typhimurium mimics the infection of humans by S. Typhi, and the genetics of the species is extremely well known, making it easy the obtention of defined mutants with reduced pathogenicity. Mutants with auxotrofy in genes of the aromatic pathway are particularly attractive, since they need PABA and DHB to grow, and these compounds are unavailable in mammalian tissues. Flagellin, the monomer which constitutes the flagellar filament, has been used as a carrier for heterologous epitopes, inserted in a central, hypervariable region (region IV). Insertions in this region are often functional, and lead to exposition of the epitope at the filament\' s surface. The present work explored the potential of the other regions ofthe molecule for the insertion of epitopes. We inserted the same reporter sequence (MS epitope from S. pyogenes M protein) in regions with different levels of homology (III and VI), and totally conserved (VIII). We also made double insertions in regions shown to be permissive (III and IV; IV and VI). All hybrid proteins were synthesized by Salmonella, as demonstrated by immunoblots using antibody against flagellin and against the synthetic peptide. All regions, except the highly conserved region VIII, accepted the insertions without loss of motility, albeit, in some cases, motility was seriously reduced. Immunogenicity of the hydrids was evaluated by immunization with live bacteria, killed bacteria, and purified flagellin (when possible). Results obtained with the new constructs were similar to the ones published for insertions involving region IV, in the sense that antibody titers to the carrier protein were very high. A low level of antibody to the inserted peptide was also detected in all groups of animals. Our results with live immunization suggest a slightly better response to the peptide when two copies are present, but the data are not conclusive.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a devastating disease in the channel catfish industry. Our research group has developed several E. ictaluri live attenuated vaccine (LAV) candidates (EiΔevpB, EiΔevpBΔfur, EiΔevpBΔhfq, EiΔevpBΔfurΔhfq), which were able to stimulate an immune response in vaccinated channel catfish and reduce ESC. However, innate, and adaptive immune responses in the lymphoid tissues of channel catfish to these LAVs are not known well. The overall goal of the project is to determine the role of adaptive and innate immune responses in catfish after vaccination with LAVs. Analysis of innate and adaptive immune-related gene expressions showed that the LAVs induced expression of adaptive immune-related genes in lymphoid tissues with less inflammation compared to wild type control. Also, the LAVs induced the expression of IgM in the sera of catfish.

<p>Инфективна болест бурзе (Гамборо болест) је контагиозна вирусна болест младих пилића. Узрочник је РНК вирус који припада фамилији Birnaviridae. Испољава тропизам према Фабрицијусовој бурзи доводећи до оштећења лимфоцита и атрофије. Болест се контролише вакцинацијом родитељских јединки и пријемчивих пилића. Употреба атенуираних вакцина може довести до смањења неких технолошких параметара. Циљ овог рада је био да се докаже које од примењених атенуираних вакцина, интермедијарних Гумбокал, Гумбокал D78 и интермедијарне-плус Гумбокал 228Е, индукује најбољи развитак имнунолошког одговора и доводе до најмањих оштећења у ткиву Фабрицијусове бурзе и слезене. Пратили смо да ли делују имуносупресивно на вакцину против Њукастл болести и да ли утичу на смањење телесне масе бројлерских пилића. Основне огледне групе су вакцинисане четрнаестог дана старости против Гамборо болести, а затим су од основних огледних група оформљене подгрупе које су вакцинисане против Њукастл болести у различитим временским периодима: О1-1, О2-2, О3-1 после седам дана, О1-2, О2-2, О3-2 после четрнаест дана, О1-3, О2-3, О3-3 после двадесетједан дан од вакцинације против Гамборo болести .Од првог до четрдесетдругог дана експеримента сваких седам дана је вађена крв пилићима, мерена њихова телесна маса и узорковане бурзе и слезене. Крвни серуми су испитивани на висину титра антитела ЕЛИСА тестом за Гамборо болест, а методом инхибиције хемагутинације за Њукастл болест. Највишу висину титра на Гамборо болест је показала огледна група О1, затим група О2 и најнижи О3. У огледним подгрупама О3-1, О3-2 и О3-3 се испољио имуносупресивни ефекат на имунолошки одговор према Њукастл болести. Фабрицијусовим бурзама је одређивана релативна маса и бурзални индекс да би се установило да ли је дошло до атрофије након примене вакцина. Најнижи бурзални индекс је установљен у огледној групи О3, затим у О2 и у О1. Таквим редоследом су устновљене и патохистолошке промене у бурзи. Добијени резултати указују да употреба интермедијане-плус вакцине индукује најбољи имунолошки одговор, али и најнижу релативну масу и вредност бурзалног индекса, као и нижу телесну масу у односу на инермедијарне вакцине. Примењене вакцине нису утицале на релативну масу слезене и нису довеле до појаве патохистолошких промена у њој.</p> / <p>Infektivna bolest burze (Gamboro bolest) je kontagiozna virusna bolest mladih pilića. Uzročnik je RNK virus koji pripada familiji Birnaviridae. Ispoljava tropizam prema Fabricijusovoj burzi dovodeći do oštećenja limfocita i atrofije. Bolest se kontroliše vakcinacijom roditeljskih jedinki i prijemčivih pilića. Upotreba atenuiranih vakcina može dovesti do smanjenja nekih tehnoloških parametara. Cilj ovog rada je bio da se dokaže koje od primenjenih atenuiranih vakcina, intermedijarnih Gumbokal, Gumbokal D78 i intermedijarne-plus Gumbokal 228E, indukuje najbolji razvitak imnunološkog odgovora i dovode do najmanjih oštećenja u tkivu Fabricijusove burze i slezene. Pratili smo da li deluju imunosupresivno na vakcinu protiv NJukastl bolesti i da li utiču na smanjenje telesne mase brojlerskih pilića. Osnovne ogledne grupe su vakcinisane četrnaestog dana starosti protiv Gamboro bolesti, a zatim su od osnovnih oglednih grupa oformljene podgrupe koje su vakcinisane protiv NJukastl bolesti u različitim vremenskim periodima: O1-1, O2-2, O3-1 posle sedam dana, O1-2, O2-2, O3-2 posle četrnaest dana, O1-3, O2-3, O3-3 posle dvadesetjedan dan od vakcinacije protiv Gamboro bolesti .Od prvog do četrdesetdrugog dana eksperimenta svakih sedam dana je vađena krv pilićima, merena njihova telesna masa i uzorkovane burze i slezene. Krvni serumi su ispitivani na visinu titra antitela ELISA testom za Gamboro bolest, a metodom inhibicije hemagutinacije za NJukastl bolest. Najvišu visinu titra na Gamboro bolest je pokazala ogledna grupa O1, zatim grupa O2 i najniži O3. U oglednim podgrupama O3-1, O3-2 i O3-3 se ispoljio imunosupresivni efekat na imunološki odgovor prema NJukastl bolesti. Fabricijusovim burzama je određivana relativna masa i burzalni indeks da bi se ustanovilo da li je došlo do atrofije nakon primene vakcina. Najniži burzalni indeks je ustanovljen u oglednoj grupi O3, zatim u O2 i u O1. Takvim redosledom su ustnovljene i patohistološke promene u burzi. Dobijeni rezultati ukazuju da upotreba intermedijane-plus vakcine indukuje najbolji imunološki odgovor, ali i najnižu relativnu masu i vrednost burzalnog indeksa, kao i nižu telesnu masu u odnosu na inermedijarne vakcine. Primenjene vakcine nisu uticale na relativnu masu slezene i nisu dovele do pojave patohistoloških promena u njoj.</p> / <p>Infectious bursal disease (Gumboro disease) is a contagious viral disease of young chickens. The causative agent is an RNA virus that belongs to the family Birnaviridae. It exhibits tropism toward bursa of Fabricius causing damage to cells and atrophy. The disease is controlled by vaccination of susceptible broiler breeders and chickens. The use of attenuated vaccines may lead to the reduction of certain production parameters. The aim of this study was to prove which of the applied attenuated vaccines, intermediate Gumbokal, Gumbokal D78 and intermediate-plus Gumbokal 228E, induces the best immunological response and leads to fewest damages to burza of Fabricijus tissue and spleen. We tracked whether it influences on immunosuppressive vaccine against Newcastle disease and if it leads to reduce of body mass of broiler chickens. Basic experimental groups were vaccinated on the fourteenth day of age against Gamboro diseases, and sub-groups were formed of the basic experimental groups which have been vaccinated against Newcastle disease in different time periods: O1-1, O2-2, O3-1 after seven days, O1-2 , O2-2, O3-2 after fourteen days, O1-3, O2-3, O3-3 after twenty-one days of vaccination against Gumboro disease. From the first to the fourty second day of the experiment, blood was extracted form the chicks every seven days, their body weight was measured and burza and spleen were sampled. Blood serum were assayed for the amount of the antibody titer by enzyme-linked immunosorbent assay test (ELISA) for Gumboro disease, and with Hemagglutination-inhibition test (HI) for Newcastle disease. The biggest titer for Gamboro disease were showed by experimental group O1, then O2 and group O3 the lowest. In the experimental subgroups O3-1, O3-2 and O3-3 immunosuppressive effect was exhibited on the immune response to Newcastle disease. Relative weight and bursal index were determined for burza of Fabricius to determine whether there was atrophy after administration of the vaccine. The lowest bursal index was established in the experimental group O3, followed by O2 and O1. Such sequence was establilshed also for histopathological changes in the burza. The obtained results indicate that the use of the intermediate-plus vaccine induces the best immune response, but also the lowest relative value of the mass and burzal index, as well as a lower body weight compared to the inermediate vaccine. Applied vaccines did not affect the relative weight of the spleen and have not led to the appearance of histopathological changes in it.</p>