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No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) has been used as a vaccine, yet safety concerns remain due to its viral immunoevasive properties. Among these, VV infection of antigen presentation cells (APC) perturbs MHC class II-mediated antigen (Ag) presentation. The goals of this project include: 1) to define mechanisms by which VV disrupts class II presentation; and 2) to examine whether disruption of the class II pathway by VV alters T cell responses in vitro and in vivo. A significant reduction in the expression of the class II chaperone, invariant chain (Ii), was observed during the late stage of VV infection. Yet surface expression of MHC class II molecules was maintained along with cell viability. To examine whether VV acts solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation-cycloheximide (CHX). Like VV, CHX negatively regulated Ii protein expression and class II presentation. Ii proteolysis also contributed in part to reduce Ii expression in VV infected and CHX treated APC. Yet only VV infection altered lysosomal protease expression, potentially influencing Ii degradation. Over-expression or ectopic-expression of Ii partially protected cells from VV-induced class II dysfunction. These studies suggest VV destabilizes class II molecules by disrupting Ii expression. To examine the presentation of viral Ags by class II, CD4 T cells from VV-primed mice were used. Viral proteins were presented by class II shortly after APC exposure to low concentrations of VV. The presentation of VV Ags correlated temporally with reductions in exogenous peptide presentation. At higher MOI (≥ 1), class II presentation of VV Ags was reduced. To examine the in vivo effects of VV on Ag presentation, a mouse model of ovalbumin-induced airway hypersensitivity was used. Th2 cytokine production was reduced, while a novel inflammatory cytokine Interleukin-17 (IL-17) production was enhanced in asthmatic VV-infected mice. In health mice, repeated VV infections lead to enhanced CD8 T cell production of Interferon-γ (IFN-γ) and IL-17. Finally, antibodies to a viral protein H3 were generated and shown to preserve class II presentation. Together these studies suggest VV disruption of the class II pathway may blunt T cell responses to VV.

No description available.

Cells process a wide range of signals by means of multi-component receptors that span the plasma membrane. Our knowledge about the individual proteins involved in these signaling cascades has grown considerably over recent years. However, critical information about the detailed mechanisms of receptor activation, and the quantitative relationships between stimulus and biological response, is still missing. Here, I used the RET receptor tyrosine kinase (RTK), together with its glycosylphosphatidylinositol-coupled co-receptor GFRα3 and their activating growth factor artemin (ART), as a model system to investigate the quantitative and mechanistic features of receptor activation and signaling. I used a set of anti-RET agonist antibodies to induce different extents of receptor clustering on the cell surface, and studied how this factor affects the amplitude and kinetics of membrane-proximal and downstream signaling events, as well as the biological response of neurite outgrowth. Using simulations of the RET-GFRα3-ART system, I studied the effect of co-receptor involvement in the activation mechanism, as well as the importance of the specific activation pathway for the RET system’s response to variations in the expression levels of different components. The principal findings of my work include the following: 1) Higher order receptor clustering is required for full RET activation, as well as for the biological response of neurite outgrowth. 2) The activated forms of the receptor brought about by the agonist antibodies and by ART plus GFRα3 are identical with respect to the ability to activate the transient extracellular signal-regulated kinase (ERK) and Akt responses, but the antibodies show a reduced ability to induce sustained activation of ERK, Akt or c-Jun N-terminal kinase (JNK). 3) The involvement of GFRα3 co-receptor in the activation mechanism of RET provides cells with the ability to regulate their sensitivity to ligand without affecting the maximum amplitude of the pRET response. 4) This effect is limited if the co-receptor GFRα3 is pre-dimerized. Overall, my work aims to elucidate broad principles that underlie the quantitative relationships between RET activation, signaling, and the resulting cellular functional response, that can be applied to other receptor systems.

Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the `clearing-up¿ process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies. / Covance Laboratories Ltd.

The development of safer and more accessible contraceptive options is necessary to reduce the high number of unintended pregnancies worldwide. As monoclonal antibody engineering continues to revolutionize drug development, a variety of strategies are being harnessed to establish antibody-based contraceptives. Human Contraception Antibody (HCA), an immunoglobulin G1 (IgG1) monoclonal antibody that potently agglutinates human sperm, is a promising candidate for nonhormonal immunocontraception in women. Our group recently established the safety and efficacy of a topical IgG1 HCA-formulated dissolvable vaginal film. Though successful, we are currently working to further optimize and improve the HCA product. In this study, we characterized engineered variants of HCA. Bioactivities, specifically agglutination and effector functions, of multimeric and fragment crystallizable (Fc)-mutated variants were compared and inform further engineering of an optimal clinical profile. We then established an atomized mRNA mechanism for delivery of HCA to the female reproductive tract (FRT). The use of mRNA could provide several advantages including: efficiency, reversibility, safety, durability, and cost-effectiveness. mRNA-encoded HCAs were expressed in several models of the FRT and were functional, sperm-specific, and safe. We also analyzed Fc N-glycans at the conserved glycosylation site on IgGs that regulate effector functions and compared the site-specific glycosylation on antibodies generated by two HCA expression platforms of interest, namely Nicotiana benthamiana and mRNA-transfected vaginal cells. Disparities in glycan site occupancy and glycoform populations between the two platforms were observed. Platform-specific HCA glycans resulted in differing levels of sperm phagocytosis, an Fc function. In summary, these studies provide a clearer understanding of engineered variants and delivery platforms to further advance the development of HCA as a novel, antibody-based female contraceptive.

Influenza is one of the most common diseases worldwide, yet the vaccines against influenza are only 35% effective at protecting against infection. Creating a more effective vaccine requires an understanding of the foundation and the factors that contribute to a strong and protective adaptive immune response. T-bet [TBX21] is a transcription factor that plays an instrumental role in the orchestration of the type 1 immune response, which is the specialized response used by the immune system for a cell-mediated response against intracellular pathogens, such as influenza. It has yet to be explored in an influenza setting on the role T-bet in the production of antibodies. The aim of this study is to understand T-bet's role in production of antibody isotypes and identify whether expression of T-bet is more important for antibody production in T cells or B cells. We expected T-bet knockout (KO) mice to have IgG2a and that T-bet expression would be more important in T cells for antibody production. An enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of virus-specific antibody in T-bet KO versus wild type (WT) mice infected with influenza. The results show that the T-bet KO and WT mice have relatively the same amount of IgG and IgG1, but the T-bet KO have a significantly lower level of IgG2a, confirming T-bet's importance for its production. To distinguish the importance of T-bet expression while T-bet expression in T cells was constant, a model was developed to allow us to control expression of T-bet in B cells. The results however were inconclusive, and the experiment will have to be repeated to make a firm conclusion on the roles of lymphocytes in the control of IgG isotypes. Overall, these results indicate that the manipulation of T-bet expression can be used as a vector to control IgG antibody levels, which holds potential for the improvement of vaccines.

The autoimmune disease known as autoimmune thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. Anti-platelet antibodies bind to glycoprotein molecules on the membranes of platelets and result in their dysfunction and destruction. Despite a growing body of information about ITP, it is difficult to isolate and characterise anti-platelet antibodies, because only limited monoclonal antibodies are available from ITP patients. This study used a phage display system to recognise Fab anti-platelet antibodies. Anti-platelet Fab-expressing phage was isolated by sequential panning of an ITP Fab library against normal non-ITP platelets. After isolation, the anti-platelet Fab-expressing phage was characterised by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using Ml3 phage as a negative control. 12 colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Colony S7 showed highest the greatest degree of binding to both the lysate and the whole platelet preparation. The specificity of the four colonies (S2, S7, S8 and S9) that had strong positive reactions against platelet antigens was determined for the glycoprotein component GP Ilb/IIIa. Further characterisation of the proteins in the lysate preparation was carried out using blotting techniques. The protein content of the four Fab-bearing phage colonies was quantified under the non-reducing conditions of Western blotting to evaluate their ability to recognise platelet antigens. Three of the four colonies showed three bands representing proteins with different molecular weights. Each of these three colonies had one band that corresponded to a protein of molecular weight 92 kD. The fourth colony showed only a single band, but this band also corresponded to a 92-kD protein.