This thesis describes studies on the binding of peptides to the murine major histocompatibility complex (MHC) class I molecule H-2D^b (D^b). The expression of the recombinant soluble D^b molecule in Chinese hamster ovary cells and its subsequent purification by nickel affinity chromatography, gel filtration, and preparative native isoelectric focusing are reported. The product is the correct molecule, homogeneous, a dimer of dimers, and free of endogenous peptide. A novel binding assay based on the enhancement of natural tryptophan fluorescence by the binding of peptide is introduced. This assay is used to determine melting curves of the empty and peptide-loaded protein, and to measure association rate constants by stopped-flow fluorescence spectroscopy. Radioligand binding measurements of equilibrium as well as association and dissociation rate constants and their temperature dependence are reported. In agreement with earlier observations, the ratio of association and dissociation rate constants is much larger than the equilibrium association constant. Fluorescence anisotropy decay spectroscopy gives evidence for conformational alterations in the D^b molecule upon peptide binding. The data, possible errors and ways to avoid them, and mathematical models of binding are discussed to obtain an overall picture of the binding process.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

No description available.

No description available.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 134-152) / Microfiche. / xvi, 152 leaves, bound ill. 29 cm

Following the completion of the human genome sequence, post-genomic efforts have shifted the focus towards the analysis of the encoded proteome. Several different systematic proteomics approaches have emerged, for instance, antibody-based proteomics initiatives, where antibodies are used to functionally explore the human proteome. One such effort is HPR (the Swedish Human Proteome Resource), where affinity-purified polyclonal antibodies are generated and subsequently used for protein expression and localization studies in normal and diseased tissues. The antibodies are directed towards protein fragments, PrESTs (Protein Epitope Signature Tags), which are selected based on criteria favourable in subsequent laboratory procedures. This thesis describes the development of novel software (Bishop) to facilitate the selection of proper protein fragments, as well as ensuring a high-throughput processing of selected target proteins. The majority of proteins were successfully processed by this approach, however, the design strategy resulted in a number ofnfall-outs. These proteins comprised alternative splice variants, as well as proteins exhibiting high sequence similarities to other human proteins. Alternative strategies were developed for processing of these proteins. The strategy for handling of alternative splice variants included the development of additional software and was validated by comparing the immunohistochemical staining patterns obtained with antibodies generated towards the same target protein. Processing of high sequence similarity proteins was enabled by assembling human proteins into clusters according to their pairwise sequence identities. Each cluster was represented by a single PrEST located in the region of the highest sequence similarity among all cluster members, thereby representing the entire cluster. This strategy was validated by identification of all proteins within a cluster using antibodies directed to such cluster specific PrESTs using Western blot analysis. In addition, the PrEST design success rates for more than 4,000 genes were evaluated. Several genomes other than human have been finished, currently more than 300 genomes are fully sequenced. Following the release of the tree model organism black cottonwood (Populus trichocarpa), a bioinformatic analysis identified unknown cellulose synthases (CesAs), and revealed a total of 18 CesA family members. These genes are thought to have arisen from several rounds of genome duplication. This number is significantly higher than previous studies performed in other plant genomes, which comprise only ten CesA family members in those genomes. Moreover, identification of corresponding orthologous ESTs belonging to the closely related hybrid aspen (P. tremula x tremuloides) for two pairs of CesAs suggest that they are actively transcribed. This indicates that a number of paralogs have preserved their functionalities following extensive genome duplication events in the tree’s evolutionary history. / QC 20101021

Host antigen presenting cells (APC) are known to be critical for the induction of graft versus host disease (GVHD) after allogeneic bone marrow transplantation (BMT) but the relative contribution of specific APC subsets remains unclear. We have studied the role of host B cells in GVHD by using B cell deficient (ìMT) mice as bone marrow transplant recipients in a model of CD4 T cell-dependent GVHD to major histocompatibility antigens. We demonstrated that acute GVHD is initially augmented in ìMT recipients relative to wild-type (WT) recipients (mortality: 85% v 44%, P<0.01) and that this was the result of an increase in donor T cell proliferation, expansion and inflammatory cytokine production early after BMT. Recipient B cells were depleted 28-fold at the time of BMT by total body irradiation (TBI) administered 24 hours earlier and we demonstrated that TBI rapidly induced sustained IL-10 generation from B cells but not dendritic cells (DC) or other cellular populations within the spleen. Finally, recipient mice in which B cells were unable to produce IL-10 due to homologous gene deletion developed more severe acute GVHD than recipient mice in which B cells are WT. Thus the induction of interlukin-10 (IL-10) in host B cells during TBI attenuates experimental acute GVHD.

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes a life-long presence in the infected host. The adaptive immune response is indispensable in controlling HCMV infection. Consequently, healthy individuals show no or mild symptoms following primary infection. In contrast, immunocompromised individuals who develop primary infection or recrudescence of HCMV can experience severe morbidity, and sometimes mortality. HCMV-specific T cell populations undergo changes in the architecture of their T cell receptor (TCR) repertoire following each episode of viral reactivation. A diverse TCR repertoire is thought to be required to provide the most efficient protection against virus infection. Perturbation to this repertoire, as can occur in immunocompromised individuals following transplantation, can lead to an increase risk of developing virus-associated clinical disease. Therefore, the study of factors influencing TCR selection is critically important in both healthy and immunocompromised individuals. To further understand the factors governing TCR selection in a persistent virus infection, the current thesis examined this process in different settings. CD8+ T cell responses to persistent viral infections are characterised by the accumulation of T cells exhibiting an oligoclonal T cell repertoire, with a parallel reduction in the naïve T cell pool. However, the precise mechanism for this phenomenon remains elusive. Here, we showed that HCMV-specific CD8+ T cells recognising distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T cell repertoire, or a private, diverse T cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public TCR was co-incident with an atypical peptide-MHC (pMHC) structure, whereby the epitope adopted a helical conformation that bulged from the peptide-binding groove, whilst a diverse TCR profile was observed in response to the epitope that formed a flatter, more ‘featureless’ landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared to the T cells expressing diverse TCR. These findings provide new insights into our understanding of how the biology of antigen presentation, in addition to the structural features of the pMHC, might shape the T cell phenotype and its corresponding repertoire architecture. Next, the role of HCMV in shaping the global and antigen-specific TCR repertoire in healthy donors was examined. First, exposure to HCMV led to an inflation of terminally differentiated CD57-expressing T cells. This effect was not seen in HCMV seronegative individuals who showed evidence of exposure to another persistent herpesvirus, Epstein-Barr virus (EBV). More importantly, these terminally differentiated CD8+ T cells in HCMV-exposed individuals displayed a highly skewed architecture of their peripheral blood T cell repertoire, with large monoclonal/oligoclonal expansions. However, ex vivo analyses of HCMV-specific T cells revealed a heterogeneous pattern of CD57 expression that showed no correlation to the antigenic source of its cognate epitope. Based on these observations, we proposed that exposure to HCMV drives the differentiation of not only the global T cell population, but select HCMV-specific T cell populations as well, and that expression of CD57 by these cells was co-incident with an oligoclonal T cell repertoire. Finally, the TCR repertoire was examined in a cohort of solid organ transplant (SOT) recipients, where primary infection or recrudescence of latent virus infection can be manifested either as asymptomatic or symptomatic disease. We examined 18 SOT recipients, and observed that symptomatic HCMV or EBV infection or recrudescence following solid organ transplantation was co-incident with a dramatic skewing of the TCR repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no HCMV/EBV infection or recrudescence showed minimal or no skewing of the TCR repertoire, and maintained TCR repertoire diversity. Interestingly, this disparate repertoire showed no correlation with levels of viral load in the peripheral blood. More importantly, we showed that large monoclonal/oligoclonal repertoire expansions was linked to the loss of antigen-specific T cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, while SOT recipients who maintained peripheral blood TCR repertoire diversity and functional antigen-specific T cell responses could resist clinical symptomatic disease in spite of high levels of viral load. Therefore, the work presented in this thesis provides additional evidence on the factors governing TCR selection in HCMV-exposed healthy individuals, as well as the consequences that perturbation to the TCR repertoire has on the functionality of the T cell compartment in immunocompromised individuals.

The host adaptive immune response to a pathogen infection comprises both cell mediated and antibody dependent components. Antibody mediated neutralization is a key component of protection against viruses and is the primary focus of this thesis. Antibodies recognize structurally defined epitopes within the context of native proteins. These may be represented by a simple linear sequence of amino acids or a discontinuous sequence of residues brought together by the conformational constraints of the protein. Many protein epitopes recognized by antibodies have been shown to be short α-helices of 3-5 turns. However corresponding synthetic peptides of this length have no structure in water because solvent competes strongly for the hydrogen bonding amides otherwise required to hydrogen bond one another to define an α-helix. This thesis is aimed primarily at (1) synthetically constraining short peptide sequences (9-13 residues) into stable α-helices of 3-4 turns; (2) structurally characterizing such constrained α-helical structures by circular dichroism and 1D and 2D NMR spectroscopy; and (3) evaluating these helix mimetics for serum stability, immunogenicity, antigenicity as well as the biological relevance of the antibodies they induce. The overall aim was to demonstrate that constrained short peptides more effectively structurally and functionally mimic known α-helical B cell epitopes from native proteins than unconstrained short peptides of the same lengths. The primary focus of Chapter 2 was to optimize in vitro ELISA conditions and immunization protocols for potentially assessing antibody responses in mice to short peptides corresponding to segments of important dengue virus proteins (NS1 and the envelope fusion protein, E). The NS1 peptide investigated had been suggested to be an α-helical epitope, but my investigations reveal that it is more likely a turn rather than a helix. While the E protein epitope chosen was not a viable epitope for testing a helix-constraining strategy, it was evaluated as a constrained turn mimic of a viral fusion epitope. Although the constrained peptides from both proteins (NS1 and E) elicited stronger antibody responses in mice than their unconstrained analogues, they still induced relatively poor antibody levels. Interestingly, mouse antibodies raised to the constrained peptide (β-turn analogue) from NS1 protein also reacted with the native protein. To evaluate a helix-constraining strategy for short peptides (less than 15 residues) that have no helix structure in water, an epitope of the HPV E7 protein was selected for mimicry. A short peptide sequence corresponding to this B cell epitope had previously been reported to have α-helical propensity but only in trifluoroethanol-water mixtures, and my initial work showed that it had no detectable helical structure at all in water. Chapter 3 presents an example of a short helical peptide as a B cell epitope, constrained into an α-helix by a side chain to side chain lactam bridge. The constraint involved cyclizing the peptide by specifically linking together side chains of lysine and aspartic acid inserted in the sequence three amino acids apart. CD and NMR structural studies highlighted significant α-helicity in the constrained short peptide, whereas the corresponding unconstrained short peptide had no structure in water. Both unconstrained and constrained short peptide epitopes were injected into mice and antibodies raised were quantified ex vivo by peptide ELISA. The helix-constrained epitope elicited higher antibody titres than the unconstrained peptide which was relatively non-immunogenic. Importantly, antibodies raised to the constrained synthetic α-helical peptide also reacted with the native E7 protein, suggesting that the helical constraint conferred on the peptide a structure analogous to that seen in the protein. In Chapter 4 a constrained α-helical peptide corresponding to a crystallographically defined α-helical sequence in the fusion, F protein of respiratory syncitial virus (RSV) was investigated for its potential to induce an antibody response. Again, while the helix-constrained peptide clearly had α-helicity by CD and NMR studies, the unconstrained short peptide had no detectable helical structure in water. To potentially boost antibody responses, relative to those generated against the dengue virus peptides examined in Chapter 3, both unconstrained and constrained peptides were coupled to the carrier protein KLH before immunizing mice. Significant levels of peptide reactive antibody were generated to both the unconstrained and constrained peptides. However, when investigated in a viral neutralization assay, the antibodies raised to the unconstrained peptide showed a higher neutralization potential than those raised to the constrained peptide. We attribute this unexpected difference to the fact that the region of the F protein corresponding to the epitope chosen, undergoes dramatic conformational changes during the viral fusion process and it is only in its post-fusion form that this helix has been observed. It is possible that the inherent flexibility of the linear, unconstrained counterpart of this epitope may more effectively mimic the conformational intermediates of the native structure on presentation to the immune system. Chapter 5 began an examination of the effects of three different adjuvants on antibody induction by short peptides. They were compared using a candidate peptide vaccine for malaria as a model system. As before, a helix-constrained peptide was compared with its unconstrained peptide sequence in immunization experiments. Higher titres of antibodies were raised to the constrained versus unconstrained peptides. In the second part of this chapter, a putative cancer vaccine peptide was similarly constrained via an ester linkage or a helix-inducing lactam bridge but both methods induced only low T-cell responses compared to their corresponding unconstrained sequences, possibly because the incorrect structure had been stabilized. The focus of this thesis was to evaluate a helix stabilization strategy for its possible application to short peptide vaccines. Using extensive circular dichroism and NMR spectroscopy measurements, we have shown in all cases that helix-constrained peptides were much more α-helical in solution than their corresponding unconstrained short peptide sequences that tended to have no or negligible α-helix structure in water. In some examples, we have compared serum stability and found that constrained peptides have higher serum stability than unconstrained peptides, a difference attributed to their greater stability towards proteolytic degradation – proteases being unable to recognize helices. We have also proven that the helix-constrained peptides induced higher mouse antibody titres than unconstrained peptides. Several attempts were made to boost antibody responses to the peptides by varying either immunization protocols, adjuvant or by attaching a carrier molecule. Further work is needed to optimize this promising new approach to short peptide vaccines.