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<p>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.</p><p>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.</p><p>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 (<i>Populus trichocarpa</i>), 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 (<i>P</i>. <i>tremula x tremuloides</i>) 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.</p>

Thesis (MTech (Biomedical Technology))--Peninsula Technikon, 2003. / Word processed copy. Summary in English. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 178-187).

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Delivery of protein therapeutics is restricted to intravenous infusions due to protein-dependent problems including low solubilities, high viscosities, and physical instabilities. The ability to inject high concentrations of proteins via subcutaneous injections would increase accessibility and compliance. Large particles of a protein in a non-aqueous solvent can decrease the viscosity over a solution of equally concentrated individual protein molecules. The lower viscosity of a particle suspension is due to decreased surface area resulting in reduced electroviscous effects, solvation and deviations of the particle shape from a spherical geometry. Additional studies show that aqueous-based dispersions of antibody nanoclusters can be formed by increasing the attractive interactions between protein molecules using the excluded volume effects of extrinsic crowding agents. These novel, equilibrium, nanoclusters are maintained by a balance of highly attractive interactions and weak electrostatic repulsive interactions near the protein’s pI. These protein nanoclusters are ideal for subcutaneous delivery as they have low interactions between the colloids, are reversible in nature, and dissolve rapidly upon dilution in a buffer media. Through in vivo mouse studies, the bioavailability of a monoclonal antibody in the dispersion is prolonged and higher doses can be administered versus a solution. Overall, these studies with high concentration, low viscosity subcutaneous injections of protein therapeutics open new opportunities in biotechnology. For oral delivery of itraconzole, controlled flocculation of individual polymerically-stabilized nanoparticles is used to increase supersaturation. Flocculation of these nanoparticles is achieved by desolvating the polymer by changing the pH. The flocculated dispersions can then be easily filtered. The final amorphous powder maintains high supersaturation with simulated stomach and small intestine conditions and improves bioavailability of itraconazole, over the commercial product, Sporanox®. / text

Specific and sensitive recognition of foreign agents is a critical attribute of the overall effective immune system required for maintaining host protection against challenge from pathogenic cells. In the humoral arm of the immune system, this recognition attribute is carried out by the cell surface bound immunoglobulin-like receptors (BCR) and its soluble forms i.e. antibodies. Over several million years of evolution, the immune system has adopted several strategies for diversifying the antibody sequence and thus its ability to recognize an astronomical variety of molecules through the combinatorial assembly of a small number of DNA segments or genes. Among these immunoglobulin gene diversification strategies, antibody somatic VDJ recombination and junctional diversity are the fundamental mechanisms in generating a broad range of antibody specificities. Understanding how the genetic diversity of antibodies is affected in health and disease is critical for a wide range of medical applications, from vaccine evaluation to diagnostics and therapeutics discovery. Because of the very large number of distinct antibodies encoded by the more than 100 billion B cells in humans, it is essential to use high throughput next generation sequencing technologies in order to obtain an adequate sampling of the sequences and relative abundance of different antibodies expressed by B cells in clinical samples. The process requires rigorous methods for first, experimentally determining the sequences of antibodies in a sample and for second, informatics tools designed for distilling this information for practical purposes. This dissertation describes a variety of experimental approaches and informatics tools developed for the determination and mining of the antibody repertoire. The information from this work has led to major conclusions regarding the nature of the antibody repertoire in healthy individuals, in volunteers following vaccination, and in HIV-1 patients. / text

The properties of human immunodeficiency virus type 1 (HIV-1) and its simian counterpart SIV that enable persistent replication in the face of robust cellular, antibody, and innate immune responses have complicated efforts to develop a safe and effective vaccine. Vaccine protection against HIV-1 infection may require a combination of immune mechanisms. However, the types of immune responses that can be induced by vaccination to prevent HIV-1 infection remain unclear. The features of the viral envelope glycoprotein (Env) that confer inherent resistance to neutralization by antibodies also interfere with the development of antibody responses. We therefore vaccinated rhesus macaques with single-cycle SIV (scSIV) strains expressing Env proteins mutated to remove features that interfere with the induction of antibody responses. Antibodies capable of neutralizing Env-modified but not wild-type SIV were selectively enhanced. Identifying the immune responses underlying complete protection by live-attenuated SIV against pathogenic SIV challenge may provide guidance for HIV-1 vaccine design. To test the hypothesis that antibodies not measurable by assays for virus neutralization correlate with protection by live-attenuated SIV, we developed a novel assay for antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity increased progressively over time after inoculation, and was measurable against viruses expressing heterologous Env proteins from independent SIV isolates when neutralization was undetectable. Two separate pathogenic \(SIV_{mac}251\) challenge experiments took advantage of either the strain specificity or the time-dependent development of immunity to overcome complete protection by live-attenuated SIV. In both experiments, macaques inoculated with live-attenuated SIV that remained uninfected by \(SIV_{mac}251\) had significantly higher ADCC activity than those that became infected. We also measured ADCC for the primary immune correlates analysis of a recent HIV-1 vaccine clinical trial in Thailand (RV144) that reported modest vaccine protection (31%). There was a nonsignificant trend towards lower risk of infection among vaccinees with high versus low relative ADCC activity. However, Env-specific IgA correlated with risk, prompting an analysis stratified by IgA levels. Among vaccinees with low Env-specific IgA, there was lower risk of infection among those with higher ADCC activity. These observations suggest that antibodies that direct ADCC may contribute to vaccine protection against SIV and HIV-1 infection.

The cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins. This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins. Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale. / text

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