Utilization of DNA vaccines in aquaculture has been gaining interest and recent efforts have been focused on methods of delivering DNA vaccines to fish. In the present study, a methodology was sought that could protect DNA vaccines such that they could be orally administered. The main objective of the study was to determine if a DNA vaccine could be effectively compounded into an orally administrable formulation with chitosan or polylactide-co-glycolide (PLG). The immune response of hybrid striped bass (Morone saxatilis x Morone chrysops) following oral delivery of a DNA vaccine containing Mycobacterium marinum Ag85A plasmid in either chitosan or PLG nanoparticle encapsulation was evaluated. Hybrid striped bass were divided into four experimental groups: IM immunization of the DNA vaccine as a positive control, oral delivery of uncomplexed DNA vaccine, oral delivery of chitosan or PLG alone as a negative control, and oral delivery of complexed chitosan or complexed PLG DNA vaccine. Fish were bled at regular intervals and an ELISA was used to evaluate antibody levels in individual fish. While the chitosan /plasmid DNA complex containing the Mycobacterium marinum Ag85A gene failed to produce a significant antibody response, the PLG/plasmid DNA matrix stimulated humoral immune response in the fish. / Master of Science

Japanese encephalitis virus (JEV), a member of the family flaviviridae, is one of the most important pathogens of the developing countries, causing high mortality and morbidity amongst children. The present study is aimed at the development of a DNA vaccine for Japanese Encephalitis (JE). As a first step towards developing a DNA vaccine for JE, an eukaryotic expression plasmid encoding the envelope (E) glycoprotein of Japanese Encephalitis Virus (pCMXENV) was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. Several independent immunization and intracerebral (i.e.) JEV challenge experiments were carried out and the results indicate that 51% and 59% of the mice are protected from lethal i.e. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV, which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-y (EFN-y) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Thl sub type. These results demonstrated that immunization with a plasmid DNA expressing intracellular form of JEV E protein confers significant protection against i.e. JEV challenge even in the absence of detectable antiviral antibodies. We then examined the potency of JEV DNA vaccines as well as that of the inactivated mouse brain derived BIKEN vaccine in the i.e. challenge model. The results indicate that all the mice immunized with BIKEN JE vaccine were protected against i.e. JEV challenge while 50% protection was observed in case of mice immunized with pJME or pJNSl and 38% protection was observed in pCMXENV-immunized mice. Immunization with both pJME and pJNSl resulted in 66% protection. These results indicate that the BIKEN JE vaccine confers better protection against i.e. JEV challenge than DNA vaccines. The fact that the BIKEN vaccine conferred better protection against i.e. JEV challenge than DNA vaccines indicated that the i.e. JEV challenge model can be exploited further to examine the potency of different DNA vaccine constructs. Towards this goal, we constructed plasmids that encode secretory or nonsecretory forms of JEV E protein and examined their potency in the i.e. JEV challenge model. Our results indicate that i.m. immunization of mice with plasmid encoding secretory form of JEV E protein confers higher level (75%-80%) protection than those encoding nonsecretory forms. Cytokine analysis of splenocytes isolated from DNA immunized mice after stimulation in vitro with JEV revealed that immunization with plasmid encoding secretory form of JEV E protein induces both Thl and Th2 responses while those encoding nonsecretory forms induce only Thl type of response. Thus, synthesis of secretory form of JEV E protein results in an altered immune response leading better protection against i.e. JEV challenge. Based on our studies, we propose that both cellular and humoral immune responses play a key role in protective immunity against i.e. JEV challenge and DNA vaccines that can induce higher levels of neutralizing antibodies will be as efficient as the BIKEN vaccine in conferring protection against i.e. JEV challenge.

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.

A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.

Orientador: Alexandrina Sartori / Banca: Ângela Maria Victoriano de Campos Soares / Banca: Virmondes Rodrigues Júnior / Resumo: A única vacina disponível contra a tuberculose (TB) é o bacilo de Calmette- Guérin (BCG), que é constituída por uma cepa de Mycobacterium bovis vivo atenuado, mas que possui variabilidade de proteção de 0 a 80%. Portanto é necessário o desenvolvimento de novas vacinas para o controle dessa infecção. Dentre as novas formulações profiláticas em teste para TB destacam-se as vacinas gênicas, sendo a DNAhsp65 a vacina investigada por nosso grupo. A DNAhsp65 é uma construção que contém o gene que codifica a proteína de choque térmico de 65KDa do M. leprae. Em virtude da elevada homologia entre a hsp65 micobacteriana e a hsp60 dos mamíferos, pode ocorrer mimetismo antigênico, ou seja, reatividade cruzada entre anticorpos e células T específicas para hsp65 micobacteriana e hsp60 humana. Nesse contexto, nosso grupo tem estudado o possível efeito colateral da DNAhsp65 no desenvolvimento de doenças autoimunes. Neste projeto foi investigado o efeito (proteção, exacerbação ou inocuidade) de diferentes formulações vacinais para TB na encefalite autoimune experimental (EAE). Os objetivos foram caracterizar a resposta imune específica induzida pela DNAhsp65 em ratos Lewis e avaliar o efeito imunomodulador tanto da estratégia clássica de imunização (3 doses de DNAhsp65 por via im) quanto da estratégia prime-boost BCG / DNAhsp65, nas características clínicas, imunológicas e histológicas da EAE. Quanto à imunogenicidade da DNAhsp65, foram testadas duas doses da vacina administradas via intramuscular (100 e 300 μg), sendo que somente a maior dose induziu IgG2b específica para hsp65 e produção de IFN- em cultura de células esplênicas estimuladas in vitro com rhsp65. Para avaliar o efeito da imunização com DNAhsp65 ou do prime-boost BCG /DNAhsp65 no desenvolvimento da EAE, os animais foram previamente imunizados com 3 doses de 300 μg de DNAhsp65 ou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: BCG is the only accepted vaccine against tuberculosis (TB) but its protective ability is very limited. Many new vaccines are therefore being evaluated. Our group has been working with DNAhsp65 that is a genetic construction containing the gene of hsp65 from Mycobacterium leprae. In previous experimental work we demonstrated that both, DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with 3 doses of DNAhsp65 or primed with BCG followed by 2 DNAhsp65 boosters. The animals were then immunized with myelin associated with Complete Freund Adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, inflammation at the central nervous system (CNS) and immune response against myelin. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and inflammation at the CNS did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN- and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate MS, being therefore... (Complete abstract click electronic access below) / Mestre

Das West-Nil-Virus (WNV) ist eine Zoonose mit weltweit zunehmender Verbreitung. Natürliches Reservoir dieses Flavivirus sind Vögel, aber auch Säugetiere wie z.B. Menschen können infiziert werden. In einigen Fällen führt eine WNV-Infektion zu schweren neurologischen Erkrankungen. Infolgedessen werden effektive und biologisch sichere Impfstrategien gegen dieses Virus benötigt. Eine Alternative zu herkömmlichen Impfmethoden beschreibt die DNA-Immunisierung. In dieser Arbeit wurde ein potentieller DNA-Impfstoff gegen das WNV hergestellt. Die Immunisierung des DNA-Vektors induzierte starke zelluläre und humorale Immunantworten in Mäusen. Zudem waren die Tiere gegen eine WNV-Infektion geschützt. Zusätzliche Impfungen mit rekombinantem WNV-Protein führten zu einer weiteren Steigerung der Immunogenität des DNA-Impfstoffkandidaten. Des Weiteren sollte der nicht-virale Gentransfer im Allgemeinen optimiert werden. Ein neu entwickeltes Transportsystem für Plasmid-DNA, bestehend aus natürlichen Histonextrakten und Polyethylenimin, resultierte in einer verbesserten Proteinexpression in in vitro transfizierten Zellen und wurde von diesen sehr gut toleriert. Daher wäre diese Strategie auch für zukünftige DNA-Impftechniken denkbar. Der Einfluss von WNV auf die Expression zellulärer miRNAs in Wirtszellen wurde bisher noch nicht untersucht. Dennoch könnten auf diese Weise potentielle molekulare Biomarker für eine frühe WNV-Diagnose identifiziert werden. Mittels Microarray-Technik wurde die Expression zellulärer miRNAs analysiert. Verschiedene miRNA-Spezies waren infolge einer WNV-Infektion leicht herunter- bzw. hochreguliert und stellen mögliche diagnostische Biomarker für das Virus dar.

A tuberculose (TB) é uma doença infecciosa causada pelo Mycobacterium tuberculosis, que necessita de uma vacina mais efetiva, pois a única vacina licenciada apresenta eficácia variando entre 0 a 80%. Entre as estratégias em desenvolvimento destaca-se a vacina DNAhsp65, que consiste de um plasmídeo carregando o gene hsp65 de Mycobacterium leprae, que demonstra eficácia na profilaxia da TB. Como as HSPs são proteínas altamente conservadas e podem desencadear respostas auto-imunes, seria interessante o desenvolvimento de uma vacina baseada na utilização apenas dos epítopos da proteína Hsp65 reconhecidos por células T. Estudos com vacinas de DNA baseadas na fusão de peptídeos à glicoproteína D (gD) do Herpes Vírus Tipo-1 têm mostrado maior ativação de linfócitos T e B peptídeos-específicos. Dessa forma, o presente trabalho teve como objetivo a construção e avaliação da imunogenicidade de vacinas de DNA constituídas pelo gene da proteína gD e a seqüência gênica que codifica os cinco epítopos da Hsp65. Para a obtenção da seqüência codificadora dos epitopos, denominada Vac1, foi realizada uma síntese gênica e em seguida, essa seqüência foi fusionada ao gene que codifica a gD em dois sítios presentes em seu interior, no sítio da enzima ApaI e entre os sítios das enzimas PvuII e ApaI, com a retirada de uma porção central da gD. Além dessas construções, também foi realizada a construção da Vac2 pela ligação de fragmentos Vac1 que em seguida foi fusionada ao gene da gD no sítio de ApaI. Essas construções, gDVac1AA, gDVac1PA e gDVac2 foram clonadas no vetor pVAX1 e avaliadas quanto a expressão das proteínas. Após a caracterização, camundongos foram imunizados com quatro doses das vacinas e a imunogenicidade avaliada após trinta dias da última dose. Os ensaios ex vivo foram realizados com o soro para dosagem de anticorpos e com as células do baço, que foram estimuladas com as proteínas Hsp65, Vac1 e Vac2. Como resultado, obtivemos duas construções vacinais, pVAXgDVac1PA e pVAXgDVac2, eficientes em induzir anticorpos do subtipo IgG2a específicos a proteína e aos epitopos da Hsp65 e as três vacinas, pVAXgDVac1AA, pVAXgDVac1PA e pVAXgDVac2, foram capazes de induzir proliferação de linfócitos T e produção de IFN- após estímulo ex vivo. As vacinas foram, portanto, eficazes em desencadear um padrão de resposta Th1 importante no combate ao bacilo M. tuberculosis. / Tuberculosis (TB) is an infectious disease, caused by the infection with Mycobacterium tuberculosis and needs a vaccine more effective, for the only current permitted vaccine shows its effectiveness varying of 0-80%. DNAhsp65 vaccine is among the strategy in development, it consists of a plasmid loading the Mycobacterium leprae hsp65 gene and has been efficient in the prophylaxy of TB. As the HSPs are conserved and they can induce autoimmune disease, a vaccine based only in the epitopes of the Hsp65 protein recognized for T cells could be more interesting. Studies with DNA vaccines based on the fusion of peptides to Herpes Type Virus-1 D glycoprotein (gD) have improved the activation of peptide-specific T and B cells. In this context, the aim of this study was the construction of DNA vaccines encoding gD protein plus Mycobacterium leprae Hsp65 protein epitopes and the evaluation of its immunogenicity. The gene sequence encoding the five Hsp65 epitopes, called Vac1, was obtained by synthetic gene and, after that, this sequence was fusioned in two sites inside gene that enconding the gD, in the ApaI enzyme site and between the PvuII and ApaI enzyme sites with the withdrawal of a gD central portion. In addition, Vac2 was contructed through the linking of Vac1 fragments followed by its insertion in the ApaI site inside gD gene. These constructions, gDVac1AA, gDVac1PA and gDVac2 were cloned in pVAX1 vector and they were evaluated to protein expression. After the characterization, mice were immunized with four doses of vaccine and the immunogenicity was evaluated after thirty days from the last immunization. The ex vivo assays were carried by quantification of antibodies in the serum and the splenocytes were stimulated with the Hsp65, Vac1 and Vac2 proteins. As result, two vaccine constructions, pVAXgDVac1PA and pVAXgDVac2 were efficient in the induction of IgG2a subtype antibodies specific to Hsp65 protein and its respective epitopes. All the three vaccines pVAXgDVac1AA, pVAXgDVac1PA and pVAXgDVac2 were capable to induce T cell proliferation and IFN- production after stimulation. Therefore, the vaccines were efficient to induce a Th1 profile which is important in the combat to Mycobacterium tuberculosis bacillus.

Die Entwicklung eines effektiven HIV-Impfstoffes ist bis heute nicht gelungen.Konventionelle Immunisierungsstrategien mit rekombinant hergestellten Hüllproteinen des Virus in verschiedensten Formen induzierten keine subtypenübergreifende, protektive Immunantwort gegen HIV. Die Gewinnung und Charakterisierung der gp41-spezifischen breit neutralisierenden monoklonalen Antikörper 2F5 und 4E10 bildete die Grundlage einer Reihe neuer epitopgerichteter Ansätze für die HIV-Impfstoffentwicklung. Bisherige Immunisierungsstudien basierten auf der Verwendung des linearen Hauptepitopes (E2) der beiden Antikörper aus dem C-terminalen Teil der Ektodomäne von gp41. Nach neueren Erkenntnissen, reicht für eine effektive Neutralisation durch 2F5 oder 4E10 die Bindung dieser Antikörper an ihr lineares Epitop in der membran proximalen externen Region (MPER) von gp41 allein nicht aus. Vielmehr wurde die Beteiligung einer N-terminalen Domäne (E1) von gp41 an der neutralisationsaktiven Bindung von 2F5 bzw. 4E10 postuliert. In dieser Arbeit wurden die beiden 2F5 und 4E10 spezifischen Epitopbereiche E1 und E2 des gp41 erstmals in das strukturell verwandte transmembrane Hüllprotein des Koala Retrovirus (KoRV) eingebracht. Die Applikation der hergestellten Antigene erfolgte sowohl in Form der codierenden DNA mittels ballistischer Immunisierung (GeneGun®) als auch durch bakteriell exprimierte Proteine. Mit beiden Strategien konnten für drei Hybridproteine in den ersten Studien eine HIV-1 gp41 spezifische, breit neutralisierende humorale Immunantwort induziert werden. Diese Ergebnisse konnten jedoch in späteren Studien nicht reproduziert werden. Die Analyse der induzierten Immunantworten zeigte eine Verlagerung der Hauptimmunantwort als deren Ursache eine bakterielle Fremdinfektion der Versuchtiere diskutiert wurde. Zur Evaluierung der Immunisierungsstudien wurde ein neuartiger real time PCR basierter in vitro Neutralisationstest um Kontrollen zur Virusspezifität und Cytotoxizität erweitert. / The development of an effective HIV vaccine is considered the to play a key role in controlling the HIV pandemic. Conventional immunisation strategies using recombinant envelope proteins of the virus did not lead to the induction of a broad range protective immunity. A new target sequence for the induction of a broadly neutralising humoral immune response has been discovered through the characterization of the gp41 specific broadly neutralising monoclonal antibodies 2F5 and 4E10. Until now all attempts to induce 2F5/4E10 like neutralising antibodies failed. So far only the linear main epitope (E2) of 2F5 and 4E10, located in the C-terminal part of the gp41 ectodomain was used as the target sequence. However, it was recently shown that an N-terminal domain (E1) of gp41 increases the avidity of 2F5 to its epitope. The E1 domain may therefore be involved in the mediation of a neutralisation active binding. For the first time immunisation strategies have been developed that target both previously identified domains (E1 and E2) of gp41. The sequences corresponding to E1 and E2 have been introduced at homologous positions in the structurally related transmembrane envelope protein p15E of the Koala Retrovirus (KoRV). These generated hybrid antigens have been used for immunisation of wistar rats. They were applied as recombinant proteins expressed in E.coli and as DNA using a ballistic immunisation (GeneGun®) approach. Although in first trials neutralising antibodies specific for gp41 of HIV-1 were induced, these results could not be reproduced. Analysis of the induced antibodies showed a shift of their binding specifity. A bacterial infection of the used animals was identified as the cause of the unexpected shift in the antigen specific humoral immune response. For evaluation of the immunisation studies a new neutralisation assay based on the measurement of provirus integration by duplex real time PCR has been extended for controls of virus specifity and cytotoxicity.

Puumala viruses, a member of the Hantavirus genus in the Bunyaviridae family, are enveloped by a lipid bilayer and possesses a tripartite single stranded RNA genome with negative polarity. The hantaviruses encode four proteins: a nucleocapsid protein (N), two membrane spanning glycoproteins (GN and GC) and a RNA dependent RNA polymerase (RdRp). Hantaviruses cause two forms of diseases, hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus pulmonary syndrome (HPS) in the Americas. The hantaviruses are mainly rodent borne, and humans are mostly infected by inhalation of aerosolized rodent secrete. Human Puumala virus infection results in nephropathia epidemica (NE), a mild haemorrhagic disease. It is of importance to have a good understanding of the epidemiology and genetics of these viruses for the development of new diagnostic methods and for future vaccine development. In this thesis we determined the complete viral genome sequence and characterized the structural proteins based on studies of expression and glycosylation patterns, for a unique human virus isolate; performed a genomic analysis of local Puumala viruses and their individual rodent host, Clethrionomys glareolus, from six different locations was performed. It was seen that the virus genetic variation between different locations could be stable over relatively large distances while there could be large variation over a short distance. For the bank voles no such variation could be seen; developed and evaluated Genetic vaccines, based on PCR-generated linear DNA. We showed that it was important to protect these fragments against nuclease degradation at that attachment of a nuclear localization signal peptide further improved the immune response. We also designed, fabricated and evaluated a 2000 probe cDNA-microarray for identification and differentiation of hantaviruses. The chips was based on 12 different strains of six hantaviruses and could differentiate between both different hantaviruses and strains within one hantavirus serotype.

O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control.