No description available.

No description available.

The rational design of an HIV-1 vaccine immunogen able to induce potent, cross-reactive, neutralising antibodies remains one of the single greatest challenges in the field of vaccine research today. Roughly a dozen broadly neutralising monoclonal antibodies have been isolated to date, and their epitopes represent important vaccination targets. Interestingly, apart from three that identify over-lapping epitopes in gp41, all of the broadly neutralising monoclonal antibodies target epitopes apparent on different conformations of gp120 (including the epitopes of PG9/PG16). Thus the gp120 monomer remains the most ideal template for immunogen design. Recently, epitopes in the C3-V4 region of gp120 have been shown to be major targets for early strain-specific neutralising antibodies in subtype C infected individuals. Autologous neutralising antibodies identify vulnerable sites on the envelope, and understanding the nature of antigenic “hotspots” on gp120 will help to guide rational vaccine design. This study sought to confirm in four individuals that the C3-V4 epitope was in fact apparent on monomeric gp120, and thereafter to better characterise the nature of viral escape from these antibodies. Using magnetic beads coated with one of 16 different recombinant gp120 proteins it was confirmed that the C3-V4 response was aimed at a monomer-specific epitope in all four cases. In two instances these antibodies were shown to contribute to autologous neutralisation, while in a third the existence of quaternary structure specific antibodies that could not be adsorbed with monomeric gp120 made this link impossible. In the forth instance transfer of the C3-V4 region was shown to expose a normally occluded epitope in the CD4 binding site. This research also provided evidence for other epitopes for autologous neutralising antibodies in C3, overlapping with the CD4 binding site and V5. Lastly, by introducing relevant escape mutations into the parental recombinant gp120s and then comparing the ability of these proteins to adsorb out anti-C3 antibodies, it was shown that while these mutations conferred complete resistance to neutralisation they did not prevent the antibodies from binding to their respective epitopes. The extensive characterisation of C3-related epitopes such as those described in this research should no doubt contribute to the rational design of a gp120 based vaccine immunogen aimed at eliciting broad and potent neutralising antibody responses.

abstract: Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation. / Dissertation/Thesis / M.S. Biological Design 2011

No description available.

No description available.

Devido a existência de 2 alphavírus artritogênicos no Brasil, os vírus Mayaro (MAYV) e Chikungunya (CHIKV) tornou-se importante desenvolver testes diagnósticos eficazes para discriminar suas infecções. No presente trabalho, desenvolvemos ELISAs indiretos para diagnóstico de CHIKV e MAYV utilizando proteínas de envelope viral E2 recombinantes, produzidas em Escherichia coli, as rE2-CHIKV e rE2-MAYV ELISAs. As proteínas E2 recombinantes tiveram suas antigenicidades verificadas nos ensaios utilizando anticorpos policlonais oriundos de camundongos hiperimunizados com CHIKV, MAYV e outros alphavírus. O rE2-CHIKV ELISA detectou anticorpos murinos de forma homotípica e não produziu reações cruzadas evidenciáveis utilizando anticorpos murinos específicos contra outros Alphavírus. O rE2-MAYV ELISA detectou anticorpos murinos homotípicos e também, reagiu cruzadamente com anticorpos murinos anti-CHIKV, mas não para outros Alphavírus. Esses ELISAs, também, foram usados na detecção de anticorpos em soros de pacientes com suspeita de infecção arboviral. Pelo o rE2-CHIKV ELISA, testaram-se 59 soros, resultando em 26 amostras IgG positivas. Resultados desse ELISA, quando comparados aos obtidos por teste de neutralização, demonstraram sensibilidade de 89,66% e especificidade de 100%. Soros humanos IgG positivos foram detectados em altas diluições pelo rE2-CHIKV ELISA. Quanto a detecção de IgM, o rE2- CHIKV ELISA apresentou moderada concordância com outros ensaios sorológicos. Com rE2- MAYV ELISA, testaram-se 68 soros resultando em 23 amostras IgG positivas, das quais 11 também mostraram-se positivas em teste de neutralização, demonstrando sensibilidade de 100% e especificidade de 78,95%. Portanto, os rE2-CHIKV e rE2 MAYV ELISAs, particularmente para detecção de IgG, mostraram-se adequadamente sensíveis e específicos para serem validados em estudos com maiores números de amostras e serem aplicados ao diagnóstico de pacientes infectados com CHIKV e MAYV. / Due the existence of 2 arthritogenic alphaviruses in Brasil, the viruses Mayaro (MAYV) and Chikungunya (CHIKV), it became important the development of efficient diagnose tests to discriminate their infections. In the present work, we developed indirect ELISAs for CHIKV and MAYV diagnosis using viral recombinant envelope proteins E2, produced in Escherichia coli, the rE2-CHIKV and rE2-MAYV. The recombinant E2 proteins had their antigenicity confirmed in the assay by using polyclonal antibodies produced in hyperimmunized mice with CHIKV, MAYV and other alphaviruses. The rE2-CHIKV ELISA detected homotypic murine antibodies and did not produced detectable cross-reactivity signal when using murine antibodies from other alphaviruses. The rE2-MAYV ELISA detected homotypic antibodies and also cross-reacted with murine anti-CHIKV antibodies, but not to other alphaviruses. These ELISAs were also tested for the detection of human antibodies, using patient sera suspected of arboviral infection. For rE2- CHIKV ELISA, it were tested 59 sera, resulting in 26 positive IgG samples. These ELISA results, when compared to those of a neutralizing assay, demonstrated a sensibility of 89.66% and specificity of 100%. The IgG positive human sera were detected in high dilutions by rE2-CHIKV ELISA. Regarding the detection of IgM, the rE2-CHIKV ELISA showed a moderate samples detection agreement when compared to other serologic assays. For rE2-MAYV ELISA, it were tested 68 sera, resulting in 23 positive IgG samples, of which 11 demonstrated to be positive by the neutralization assay, demonstrating a sensibility of 100% and specificity of 78.95%. Therefore, the rE2-CHIKV and rE2-MAYV ELISAs, especially for IgG detection, demonstrated to be properly sensitive and specific to be validated in studies using a greater number of samples, and also to be applied in the diagnosis of infected CHIKV and MAYV patients.

A dengue é uma doença causada por um dos quatro sorotipos de vírus da dengue (DENV 1-4), e representa a principal arbovirose que atualmente atinge seres humanos. Dentre as maiores dificuldades para o desenvolvimento de uma vacina eficaz contra o DENV, correlaciona-se a falta de conhecimento mais preciso sobre os epítopos presentes na superfície do vírus, responsáveis para indução de anticorpos neutralizantes sem promoverem a exacerbação da infecção viral. Desta forma, o objetivo do presente trabalho foi identificar epítopos presentes na superfície do DENV2 capazes de gerar anticorpos neutralizantes sem promover a amplificação viral frente a células que expressem receptores FcR. Avaliamos também a influência dos adjuvantes (LT, LT-K63 e LTB), na modulação da resposta de anticorpos para epítopos presentes nos domínios I, II e III da proteína de envelope do DENV2. Observamos que a administração das LTs como adjuvantes proporcionaram a potencialização da resposta de anticorpos IgG EDI/II ou EDIII-específicos nos animais imunizados, em relação aos outros grupos vacinais. Em relação à qualidade da resposta humoral proporcionada pelas imunizações, os anticorpos antígeno-específicos gerados com LT, LT-K63 ou LTB apresentaram maior capacidade de neutralização viral em comparação com aqueles obtidos dos demais grupos vacinais. Demonstramos de forma inédita através da análise de imunoassinatura dos anticorpos IgG EDIII-específicos, que a administração de LT e LTB como adjuvante vacinal, permitiu a identificação de um epítopo localizado na alça EF e FG da proteína EDIII de DENV2. Além disso, por meio da utilização de um peptídeo 47 contendo a sequência correspondente ao epítopo identificado, foi capaz de inibir a infecção do DENV2, tão bem quanto, a proteína EDIII em ensaios in vitro. Podemos perceber, que a utilização do adjuvante LT e seu derivado atóxico LTB em formulações vacinais, possibilitaram a modulação de anticorpos capazes de reconhecer epítopos presentes no EDIII, importantes para a neutralização viral. Esses resultados, permitem o desenvolvimento de novos antígenos alvos para estratégias vacinas voltadas para o controle dos DENV. / Dengue is a disease caused by one of four dengue virus serotypes (DENV 1-4), represents the main arbovirose that currently affects humans. Among the greatest difficulties for the development of effective vaccine against DENV is the lack of more precise knowledge about the epitopes present at the surface of the virus, responsible for the induction of neutralizing antibodies without promoting the exacerbation of viral infection. Thus, the objective of the present work was to identify epitopes present on the surface of DENV2 capable of generating neutralizing antibodies without promoting viral amplification against cells expressing the FcR receptors. We also evaluated the influence of adjuvants (LT, LT-K63 and LTB) on the modulation of the antibody response to epitopes present in domains I, II and III in envelope protein of DENV2. We have observed that administration of LTs as adjuvants provided potentialization of the antibodies response IgG EDI/II or EDIII-specific in the immunized animals, relative to the other vaccine groups. Regarding the quality of the humoral response provided by the immunizations, the antigen-specific antibodies generated with LT, LT-K63 or LTB presented higher viral neutralization capacity compared to those obtained from the other vaccine groups. We demonstrated through the immunoassay analysis of EDIII-specific IgG antibodies that the administration of LT and LTB as a vaccine adjuvant allowed the identification of an epitope located in the EF and FG loop of the EDIII protein of DENV2. Furthermore, by using a peptide 47 containing the sequence corresponding to the identified epitope, was able to inhibit DENV2 infection as well as the EDIII protein in vitro assays. It can be seen that the use of the LT adjuvant and its non-toxic derivative LTB in vaccine formulations enabled the modulation of antibodies capable of recognizing epitopes present in EDIII, which are important for viral neutralization. These results allow the development of new target antigens for vaccine strategies aimed at the control of DENV.

As células dendríticas (DCs) são consideradas como apresentadoras de antígenos profissionais, iniciando e modulando as respostas imunológicas. O direcionamento de diferentes antígenos para essas células têm sido uma estratégia promissora para a avaliação das respostas imunológicas humoral e celular. Nesse trabalho, o alvo de direcionamento é o subtipo de DCs CD8α+ DEC205+, por apresentarem características importante, como por exemplo, a apresentação cruzada para linfócitos T CD8+ via molécula de MHC II. Os antígenos de escolha para serem direcionados foram diferentes domínios da proteína do envelope (E) do vírus da dengue tipo 2, assim como a proteína na sua forma inteira. Os resultados mostraram que o direcionamento dos diferentes domínios da proteína do envelope viral foi capaz de induzir uma celular robusta nos animais quando comparada com o não direcionamento das mesmas proteínas. Além disso, com o direcionamento para DCs CD8α+ DEC205+ foi possível mapear regiões importantes da proteína E durante a indução da resposta imune. Portanto, os dados obtidos nesse trabalho, sugerem que o direcionamento da proteína do E e dos seus domínios ( EDI/II e EDIII) foi eficiente em induzir resposta imune celular contra o vírus da dengue tipo 2. / Dendritic cells (DCs) are considered to present professional antigens, initiating and modulating immune responses. The targeting of different antigens to these cells has been a promising strategy for the evaluation of humoral and cellular immune responses. Thus, the antigen of interest is directed to the population of DCs desired. In this work, the targeting target is the subtype CD8α + DEC205 + DCs, because they present important characteristics, such as cross-presentation to CD8 + T lymphocytes via the MHC II molecule. The antigens of choice for targeting the CD8α + DEC205 + DCs subtype were different domains of the envelope protein (E) of dengue virus type 2 as well as the protein in its entire form. The results showed that the targeting of the different domains of the viral envelope protein was able to induce a robust cell in the animals when compared to the non-targeting of the same proteins. Furthermore, with the targeting for CD8α + DEC205 + DCs it was possible to map important regions of the E protein during the induction of the immune response. Therefore, the data obtained in this work suggest that the targeting of E protein and its domains (EDI / II and EDIII) was efficient in inducing cellular immune response against dengue virus type 2.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.