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Studies on Peyer's patch T cell hybridomasPullen, A. M. January 1987 (has links)
The initial objective of this thesis was to generate Peyer's patch T cell hybridomas producing lymphokines that regulate IgA-secreting B lymphocytes. Unprimed Peyer's patch cells were fused with BW5147. Karyotype analysis and fluorescent staining of the thy-1.2 marker confirmed the generation of hybridomas. It was envisaged that these hybridomas would be tested for their effects on IgA production by LPS-stimulated B cells. However, when the panel of hybridomas was available for testing there were technical difficulties with this assay. Sendai virus-primed Peyer's patch T cells were used in a subsequent fusion, which was screened using an antigen-specific <i>in vitro</i> helper assay. A number of hybridomas stimulated the production of anti-Sendai antibodies by primed B cells. The same hybridomas secreted IL-2 on stimulation by syngeneic spleen cells in the absence of added virus. Recombinant IL-2 replaced the hybridomas in stimulating primed B cells in the helper assay. These studies showed that several of the hybridomas had apparent auto-reactivity. This was not due to viral contamination of the animal stocks since spleen cells from isolator-reared syngeneic mice gave similar results. The genes responsible for stimulating the hybridomas were mapped to the I-region of the MHC. It was important to elucidate whether these T cells were truly auto-reactive or whether they were in fact <i>in vitro</i> artefacts. Hybridomas adapted to grow in serum free medium and subsequently tested for their response to syngeneic cells in the abscence of serum, did not produce IL-2. The addition of foetal calf serum restored the response. The component of foetal calf serum which is necessary for the stimulation of the hybridomas has been partially purified. It can be separated from the main serum protein components by HPLC on a DEAE ion exchange column. It is eluted by high salt which suggests that it is highly acidic or is bound strongly by hydrophobic interactions. The material is trypsin sensitive. It is labile at 4<SUP>o</SUP> C and is unstable to freezing and thawing and this has hampered its further purification. The mode of action of the component has been studied using pulsing experiments and it has been shown to act on the stimulator cells and not on the hybridomas. The data suggests that the hybridomas do not recognise a self-antigen alone, but rather that they recognise a component of the xenogeneic serum as antigen with self-MHC restriction. However it has not been formally excluded that the foetal calf serum component stimulates the expression or processing of an auto-antigen.
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Vývoj nových metod pro studium expozice hostitelů vůči flebotomům / Development of a new sand fly exposure test to evaluate vector control toolsWillen, Laura Adrienne André January 2019 (has links)
In the Mediterranean basin, human visceral leishmaniasis caused by the protozoan parasite Leishmania infantum is a zoonotic disease that gives rise to 1,200 to 2,000 new cases annually. The domestic dog constitutes its main reservoir, of which some may suffer from a severe chronic disease, canine leishmaniasis (CanL). The sand fly Phlebotomus perniciosus is considered to be the principle vector. Saliva of bloodfeeding vectors of diseases has been used in the past to assess host exposure to vector bites and to evaluate vector control tools. This Ph.D. focused on saliva of P. perniciosus to identify exposure markers that could be used in the preparation of a new vector exposure tool. The first part of this Ph.D. aimed at validating the use of a recombinant salivary protein of P. perniciosus - rSP03B - in endemic settings of CanL. During a cross-sectional study, no significant differences between the antibody (Ab) response against whole saliva or the rSP03B were observed between different regions across the Mediterranean basin. Furthermore, the rSP03B was shown to resemble the native protein. During a subsequent study this protein was used to assess the seasonal dynamics of the canine Ab response to P. perniciosus in an endemic area of L. infantum. This study elucidated that also in a heterogeneous...
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Immunoassays with defined malarial antigens and their potential for use in sero-epidemiological studies in the SudanOmer, F. M. January 1987 (has links)
No description available.
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Detection of antibody responses to infection with herpes simplex virus and human immunodeficiency virusSimmonds, Peter January 1988 (has links)
No description available.
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The Characterisation of Antibody Responses to Different Herpesvirus Vaccine Vector StrategiesGraham, David 09 1900 (has links)
Herpes simplex virus is man's oldest viral enemy. Infections result in symptoms ranging from mild skin lesions to deadly herpes simplex encephalitis, making HSV one of the most costly of viral diseases to treat. Thus the development of a vaccine is imperative. To this end, several vaccine strategies have been utilized to generate immunity to HSV in rodents. These include the use of recombinant DNA, recombinant adenoviruses, and dendritic cells transduced with either of the former and re-introduced to the host to induce immunity. In this study, different aspects of these vaccine types were examined. Antibody and cytotoxic T-cell (CTL) responses to a DNA vaccine encoding gB of HSV-1 (gB-DNA) were evaluated. This resulted in variable long lived antibody responses to a wide range of dosages and CTL responses which followed dose-response relationships. An adenovirus expressing gB of HSV -1 (AdgB) which is able to generate IgA responses (Gallichan et al., 1993) was utilized to determine the best method of mucosal administration to optimize these responses. It was suggested in a previous report that nasal associated lymphoid tissue (NALT) was the desired target for inducing lgA responses (Heritage et al., 1997). Accordingly, the hypothesis was formulated that NALT can produce IgA responses similar to those produced from a combination of inductive sites. To test this hypothesis, mice were immunized either awake or asleep with AdgB assuming that awake delivery restricts induction to the NALT, whereas asleep administration disseminates AdgB throughout the respiratory system. The results demonstrate participation of lower airways in the induction of immunity is desirable for
generating IgA responses. Lastly, dendritic cells transduced with AdgB were assessed for their ability to generate systemic and mucosal antibody responses, resulting in the inability to generate IgA, but the ability to generate systemic antibody responses. / Thesis / Master of Science (MS)
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Vaccine development strategies applied to the<i> Plasmodium falciparum</i> malaria antigen 332Vasconcelos, Nina-Maria January 2006 (has links)
<p>Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite <i>Plasmodium falciparum</i> is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the <i>P. falciparum</i> asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an <i>in vitro</i> parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to <i>P. falciparum</i>, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude <i>P. falciparum</i> antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.</p>
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Resposta de anticorpos contra proteínas recombinantes baseadas em antígenos de merozoítos de Plasmodium vivax em indivíduos de uma comunidade rural da Amazônia brasileira / Antibody response against recombinant proteins based on Plasmodium vivax merozoite antigens in individuals from a rural community of the Brazilian AmazoniaCumbane, Victória Simão 20 May 2011 (has links)
Nos últimos anos, estudamos vários aspectos da resposta imune naturalmente adquirida em indivíduos de diferentes áreas endêmicas da Região Amazônica expostos à malária. Para isso, utilizamos proteínas recombinantes baseadas em antígenos de formas sanguíneas de P. vivax, os quais têm sido considerados candidatos à vacina contra a malária vivax. No presente trabalho, nossos estudos imunoepidemiológicos concentraram-se em 396 indivíduos de uma comunidade rural da Amazônia ocidental brasileira, localizada no Estado do Acre, com o objetivo de realizar um estudo transversal e longitudinal da resposta de anticorpos contra um painel de proteínas recombinantes derivadas de merozoítos de P. vivax (MSP119, AMA-1, MSP3α e MSP3β). Para isso, os soros desses indivíduos foram testados por ELISA quanto ao reconhecimento das quatro proteínas recombinantes. As proporções de indivíduos na linha de base com anticorpos IgG contra MSP119, AMA- 1, MSP3α e MSP3β foram de 59,8%, 50,0%, 23,5% e 26,5%, respectivamente. Dentre esses indivíduos, apenas 10,9% tinham infecções por P. vivax, P. falciparum ou malária mista. No grupo de infectados por P. vivax, a proporção de respondedores foi maior para as proteínas recombinantes MSP119 e AMA-1, atingindo 78,6%, em ambos os casos. A proporção de indivíduos com anticorpos para cada uma das proteínas associou-se com o maior tempo de exposição à malária, exceto para a MSP3β. Além disso, a positividade foi mais alta nas áreas de maior risco de transmissão. Não observamos associações relevantes entre o genótipo do antígeno Duffy dos indivíduos e presença de anticorpos para as proteínas estudadas. No estudo longitudinal, observamos um aumento da prevalência de respondedores durante a parasitemia patente, sendo de 81,9%, 80,9%, 31,9% e 48,9% para a MSP119, AMA-1, MSP3α e MSP3β, respectivamente. Em conclusão, nossos resultados confirmam a alta antigenicidade dessas proteínas, o que pode ser de grande importância para futuros ensaios clínicos na região. / In recent years we studied various aspects of the naturally acquired immune response in individuals from different endemic areas of the Amazon region exposed to malaria. For this purpose we used recombinant proteins based on P. vivax blood stage antigens considered candidates for a vaccine against vivax malaria. In the present study, we focused on 396 individuals from a rural community in western Brazilian Amazon located at the state of Acre. We conducted a transversal and longitudinal study of the antibody response to a panel of recombinant proteins representing P. vivax merozoites surface antigens (MSP119, AMA-1, MSP3α and MSP3β). The sera of these individuals were tested by ELISA for the recognition of the four antigens mentioned above. The proportions of individuals at the baseline with IgG antibodies to MSP119, AMA-1, MSP3α and MSP3β were 59.8%, 50.0%, 23.5% and 26.5%, respectively. Among these individuals, 10.9% had patent malaria infections with either P. vivax or P. falciparum or both. Among individuals with patent P. vivax infection, the frequency of responders was high for MSP119 and AMA-1, (78.6% in both cases). Except in the case of MSP3β, the proportion of individuals with antibodies to each protein correlated with the time of malaria exposure. Also, the positivity was higher in areas of higher transmission levels. No relevant association was found between the Duffy genotypes and presence of antibodies to the different antigen. In longitudinal study, we observed an increased prevalence of responders during patent parasitemia, 81.9% 80.9% 31.9% and 48.9% to MSP119, AMA-1, MSP3α and MSP3β, respectively. In conclusion, our results confirm the high antigenicity of these proteins, which can be of great importance for future clinical trials in the region.
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Computational Methods to Study Diversification in Pathogens, and Invertebrate and Vertebrate Immune SystemsMunshaw, Supriya Shaunak January 2010 (has links)
<p>Pathogens and host immune systems use strikingly similar methods of diversification. Mechanisms such as point mutations and recombination help pathogens escape the host immune system and similar mechanisms help the host immune system attack rapidly evolving pathogens. Understanding the interplay between pathogen and immune system evolution is crucial to effective drug and vaccine development. In this thesis we employ various computational methods to study diversification in a pathogen, an invertebrate and a vertebrate immune system.</p>
<p>First, we develop a technique for phylogenetic inference in the presence of recombination based on the principle of minimum description length, which assigns a cost-the description length-to each network topology given the observed sequence data. We show that the method performs well on simulated data and demonstrate its application on HIV <italic>env</italic> gene sequence data from 8 human subjects.</p>
<p>Next, we demonstrate via phylogenetic analysis that the evolution of repeats in an immune-related gene family in <italic>Strongylocentrotus purpuratus</italic> is the result of recombination and duplication and/or deletion. These results support the evidence suggesting that invertebrate immune systems are highly complex and may employ similar mechanisms for diversification as higher vertebrates.</p>
<p>Third, we develop a probabilistic model of the immunoglobulin (Ig) rearrangement process and a Bayesian method for estimating posterior probabilities for the comparison of multiple plausible rearrangements. We validate the software using various datasets and in all tests, SoDA2 performed better than other available software.</p>
<p>Finally, we characterize the somatic population genetics of the nucleotide sequences of >1000 recombinant Ig pairs derived from the blood of 5 acute HIV-1 infected (AHI) subjects. We found that the Ig genes from the 20 day AHI PC showed extraordinary clonal relatedness among themselves; a single clone comprised of 52 members, with observed and inferred precursor antibodies specific for HIV-1 Env gp41. Antibodies from AHI patients show a decreased CDR3H length and an increased mutation frequency when compared to influenza vaccinated individuals. The high mutation frequency is coupled with a comparatively low synonymous to non-synonymous mutation ratio in the heavy chain. Our results may suggest presence of positive antigenic selection in previously triggered non-HIV-1 memory B cells in AHI.</p>
<p>Taken together, the studies presented in this thesis provide methods to study diversification in pathogens, and invertebrate and vertebrate immune systems.</p> / Dissertation
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Vaccine development strategies applied to the Plasmodium falciparum malaria antigen 332Vasconcelos, Nina-Maria January 2006 (has links)
Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite Plasmodium falciparum is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the P. falciparum asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an in vitro parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to P. falciparum, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude P. falciparum antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.
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The Role of IgM and Complement in Antibody ResponsesRutemark, Christian January 2011 (has links)
An intact complement system including the complement receptors 1 and 2 (CR1/2) is crucial for the generation of a normal antibody response in animals and humans. Moreover, activation of the classical pathway is thought to be important since deficiency in complement components C1q, C2, C4 or C3 lead to impaired antibody responses. The classical pathway is mainly initiated by antibodies bound to their antigen. It is unclear how classical pathway activation can be crucial for primary antibody responses since the levels of specific antibodies are very low in naïve animals. It has been hypothesized that natural IgM, with high enough affinity, can initiate the classical pathway after immunization. To test this, we generated the knock-in mouse strain Cμ13, producing IgM unable to activate complement. Surprisingly, the antibody response against SRBC and KLH in Cµ13 mice was normal. Thus, the importance of classical pathway activation and natural IgM in antibody responses is not dependent on the ability of IgM to activate complement. SIGN-R1, SAP and CRP are other known activators of the classical pathway, but mice lacking these also had normal antibody responses. Complement activation leads to the generation of C3 split products which are ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC), but it is unclear on which cell-type expression of CR1/2 is needed for the generation of a normal antibody response. Some reports argue that increased antigen retention by CR1/2+ FDC would increase the effective antigen concentration, giving more effective B-cell stimulation. In contrast, several mechanisms involving CR1/2 on B cells are suggested. First, marginal zone B cells could transport complement-coated antigen or IC via CR1/2 into the follicle. Second, different ways of co-crosslinking the B-cell receptor with CR1/2, lowering the threshold for B-cell activation, have been proposed. Finally, CR1/2 on B cells are shown in vitro to facilitate endocytosis and thereby presentation of antigen to T cells. We show that abrogated antibody responses in mice lacking CR1/2 are not due to lack of CR1/2-mediated antigen presentation to T cells. Chimeric mice with CR1/2 expression on both B cells and FDC, on neither B cells nor FDC, or on either B cells or FDC, were generated. The antibody response against SRBC was completely dependent of CR1/2-expression on FDC. However, when this requirement was fulfilled, B cells without expression of CR1/2 were equally efficient antibody producers as wildtype B cells. Antigen-specific IgM together with its antigen can enhance the antibody response to that antigen and CR1/2-expression is crucial for the enhancement. We show that the response to IgM in complex with SRBC is dependent on CR1/2 expression on both B cells and FDC.
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