Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies. / QC 20100907

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.   In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.   The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors. / QC 20101102

Mycoplasma hyopneumoniae is the etiologic agent causing chronic pneumonia of swine. The lung lesions of swine produce the slower growth rate and lower feed conversion ratio and finally cause economic loss. Although four genome projects of mycoplasma species had been completed, the genome-sequencing project of M. hyopneumoniae also closed to the finished stage. However, only a few genes and proteins of M. hyopneumoniae have been studied, the molecular pathogenic mechanism remains elusive. The research of molecular vaccine is still preliminary. In order to obtain more information about epitope structures as the basis to develop molecular vaccine against this pathogen, two phage-displayed random heptapeptides libraries were used to identify epitopes recognized by purified IgG of rabbit anti-M. hyopneumoniae hyperimmune serum in this study. Individual phage clones were isolated and verified the binding specificity to the purified IgG by Western blot analysis and competitive ELISA after three rounds of biopanning. The selected clones were further characterized by DNA sequencing analysis and deduced to amino acid sequences. There are six consensus sequences contained tri- to hepta-peptide existing among the selected phage clones by aligning the sequences of foreign amino acids displaying on pIII protein. The consensus sequences may be serving as crucial epitopes of M. hyopneumoniae. By searching the protein database of M. hyopneumoniae deposited in NCBI, some surface proteins were matched by the selected mimotopes. Like P97, the essential protein for attaching to cilia of swine, the deduced epitopes mainly located at a.a. from 365 to 382, 395 to 403 and 436 to 452, the R1 and R2 repeated sequences also matched by the mimotopes. To evaluate the potential of these mimotopes as effective vaccine, several phage clones were chosen to immunize mice by intraperitoneal and intranasal administration. There are specific antibody responses to these mimotopes existing in serum IgG, fecal extracts and bronchoalveolar lavage fluids IgA. The serum IgG subclass profiles analysis reveals that these are mainly existed in IgG1 subclass. Base on the results of IgG subclass profiles analysis in sera, the results suggest that the phage-derived vaccines mainly activate Th2 cellular immunity pathway with the strategy used in this study. The similar results were found in the inactivated vaccine. The Th2 activation will promote the elimination of extracellular microorganism. Western blotting analysis showed that each serum raised by the phage clones could recognize 2 to 5 mycoplasma proteins. With the results of growth inhibition assay, we found that the selected phage clones CS4 and 58 are better vaccine candidates and some proteins (97 kDa¡B56 kDa and 30 kDa) may play crucial roles in block the bacteria growth. The advantage was taken of the natural property of M13 phage to infect E. coli, which is initiated by the N terminal of pIII coat protein binding with the F pili of E. coli. Plaque reduction tests were proposed to demonstrate the humoral immunity responses induced by phage-derived vaccine containing the antibodies specifically against the foreign peptide displayed on pIII coat protein. The present results provide more epitope information of M. hyopneumoniae. The mice immunization results reveal that the phage-displayed mimotopes can be used as potential vaccine candidates. The strategy presented in this study can shorten the time course for vaccine development and provide an alternative pathway for searching vaccine candidates against M. hyopneumoniae.

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy. This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry. Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments. / <p>QC 20141111</p>

Specific antibodies are invaluable tools for proteomics research. The availability of thoroughly validated antibodies will help to improve our understanding of protein expression, localization and function; fundamental processes and features of all living organisms. The objectives of the studies in this thesis were to develop high-throughput methods to facilitate the generation and purification of monospecific antibodies, and to address problems associated with antigen selection for difficult target proteins and subsequent validation issues. In the first of the studies, it was demonstrated that antibodies specific to human proteins could be generated in a high-throughput manner using protein epitope signature tags (PrESTs) as both antigens and affinity ligands. A previously developed purification process was adapted to a high-throughput format and this, in combination with the development of a protein microarray assay, resulted in monospecific antibodies that were used for profiling protein expression in 48 human tissues. Data obtained in these analyses suggest that a complete Human Protein Atlas should be attainable within the next ten years. In order to reduce the number of animals needed for such a massive project, and improve the cost-efficiency of antibody generation, a multiplex immunization strategy was developed in a further study. Antisera from rabbits immunized with mixtures of two, three, five and up to ten different PrESTs were successfully purified and analyzed for specificity using protein arrays. Almost 80% of the animals immunized with up to three PrESTs yielded antibodies towards all the PrESTs administered, and they yielded comparable immunohistochemical staining patterns (of consecutive human tissue sections) to those of antibodies obtained from traditional single PrEST immunizations. Proteins with highly similar sequences to other proteins present a major challenge for the proteome-wide generation of antibodies. In another study, Cytokeratin-17 which displays high sequence similarity to closely related members of the intermediate filament family, was used as a model and the specificity and cross-reactivity of antibodies generated against this target were investigated using epitope mapping in combination with comparative IHC analyses. Antibodies identified by epitope mapping as binding to the most unique parts of the Cytokeratin-17 PrESTs also showed the most Cytokeratin-17-like staining pattern, thus further supporting the strategy of using sequence identity scores as the main criteria for PrEST design. An alternative antigen design strategy was investigated for use in raising antibodies towards G-proteincoupled receptors (GPCRs). The extracellular loops and N-terminus of each of three selected GPCRs were assembled to form single antigens and the resulting antibodies were analyzed by flow cytometric and confocal microscopic analyses of cell lines over-expressing the respective receptors. The results from both flow cytometric and immunofluorescence analyses showed that the antibodies were able to bind to their targets. In addition, the antibodies were used successfully for the in situ analysis of human brain and pancreatic islet cells. / QC 20100727

This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins.  In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes. In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis. Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry. Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes. / QC 20120111

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Pestiviruses such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) are some of the most economically important livestock diseases in the world. The antigenic similarities between members of the pestivirus genus allow for both BVDV and CSFV to infect swine. Infections with heterologous pestiviruses in swine can interfere with diagnostic tests for CSFV. The identification of cross-reactive and cross-neutralizing epitopes between CSFV and BVDV for the development of improved diagnostics and vaccines that allow for the differentiation of infected animals from vaccinated animals (DIVAs) are necessary to accurately detect and control CSFV. The overall goal of this research was to identify epitope regions recognized by antibodies that can differentiate between CSFV and BVDV. The approach was to use serum neutralization assays to confirm the presence of neutralizing antibodies to BVDV in swine serum collected from animals immunized with one of three separate Alphavirus vaccine constructs: BVDV-1b, CSFV E2, and CSFV E[superscript]rns. Results showed that animals immunized with the Alphavirus BVDV-1b construct had high neutralizing titers against BVDV-1a and animals immunized with Alphavirus CSFV E2 and E[superscript]rns constructs had low, but detectable, neutralizing activity. Polypeptide fragments of CSFV and BVDV E2 were then expressed in E. coli and purified using affinity chromatography. Serum from a pig immunized with the CSFV E2 Alphavirus construct was tested against two fragments of CSFV E2, 2/4 and 4/4, and four fragments BVDV E2, 1/4, 2/4, 3/4, and 4/4, using western blot analysis. Reactivity to fragments CSFV E2 2/4 and 4/4 and BVDV E2 1/4 and 4/4 was observed. The results of this study identified CSFV amino acid positions 774 through 857 and BVDV amino acid positions 783 through 872 as the regions that contain the epitopes recognized by cross-reactive antibodies between BVDV and CSFV E2. These results provide more specific sequence regions to improve CSFV diagnostic assays and DIVA vaccines.

abstract: The healthcare system in this country is currently unacceptable. New technologies may contribute to reducing cost and improving outcomes. Early diagnosis and treatment represents the least risky option for addressing this issue. Such a technology needs to be inexpensive, highly sensitive, highly specific, and amenable to adoption in a clinic. This thesis explores an immunodiagnostic technology based on highly scalable, non-natural sequence peptide microarrays designed to profile the humoral immune response and address the healthcare problem. The primary aim of this thesis is to explore the ability of these arrays to map continuous (linear) epitopes. I discovered that using a technique termed subsequence analysis where epitopes could be decisively mapped to an eliciting protein with high success rate. This led to the discovery of novel linear epitopes from Plasmodium falciparum (Malaria) and Treponema palladium (Syphilis), as well as validation of previously discovered epitopes in Dengue and monoclonal antibodies. Next, I developed and tested a classification scheme based on Support Vector Machines for development of a Dengue Fever diagnostic, achieving higher sensitivity and specificity than current FDA approved techniques. The software underlying this method is available for download under the BSD license. Following this, I developed a kinetic model for immunosignatures and tested it against existing data driven by previously unexplained phenomena. This model provides a framework and informs ways to optimize the platform for maximum stability and efficiency. I also explored the role of sequence composition in explaining an immunosignature binding profile, determining a strong role for charged residues that seems to have some predictive ability for disease. Finally, I developed a database, software and indexing strategy based on Apache Lucene for searching motif patterns (regular expressions) in large biological databases. These projects as a whole have advanced knowledge of how to approach high throughput immunodiagnostics and provide an example of how technology can be fused with biology in order to affect scientific and health outcomes. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2014

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups. / <p>QC 20170828</p>

Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.   The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.   In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.   In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value. / <p>QC 20170905</p>