Phage display technology has emerged as the dominant approach in antibody engineering. Here I describe my work in developing a high-throughput method of reliably generating intracellular antibodies. In my first data chapter, I present the first known high-throughput pipeline for antibody-phage display libraries of synthetic diversity and I demonstrate how increasing the scale of both target production and library selection still results in the capture of antibodies to over 50% of targets. In my second data chapter, I present the construction and validation of a novel scFv-phage library that will serve as the first step in my proposed intrabody pipeline. Antibodies obtained from this library will be screened for functionality using a novel yeast-two-hybrid approach and have numerous downstream applications. This high-throughput pipeline is amenable to automation and can be scaled up to thousands of domains, resulting in the potential generation of many novel therapeutic reagents.

Phage display technology has emerged as the dominant approach in antibody engineering. Here I describe my work in developing a high-throughput method of reliably generating intracellular antibodies. In my first data chapter, I present the first known high-throughput pipeline for antibody-phage display libraries of synthetic diversity and I demonstrate how increasing the scale of both target production and library selection still results in the capture of antibodies to over 50% of targets. In my second data chapter, I present the construction and validation of a novel scFv-phage library that will serve as the first step in my proposed intrabody pipeline. Antibodies obtained from this library will be screened for functionality using a novel yeast-two-hybrid approach and have numerous downstream applications. This high-throughput pipeline is amenable to automation and can be scaled up to thousands of domains, resulting in the potential generation of many novel therapeutic reagents.

The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.

Hospital acquired pneumonia is a major problem in the nosocomial environment worldwide. The rise in the number and level of antibiotic resistant strains of bacteria means that conventional therapies are no longer as effective as they once were. Many of the main causative organisms are Gram-negative rods, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae and one that has become a greater problem in the last twenty years Acinetobacter genospecies 13 TU. Lipopolysaccharide (LPS) is a molecule that is found on the cell surface of all Gram-negative organisms. LPS is a vital part of the outer membrane of Gram-negative bacteria and is a major factor in these organisms’ ability to cause serious infection and disease. While many Gram-negative organisms, such as E. coli and Klebsiella pneumoniae, are well characterised, other species that have become potential nosocomial pathogens more recently, such as Acinetobacter genospecies 13 TU, are much less well characterised. It is unknown as to how widespread exposure to Acinetobacter genospecies 13 TU is in a healthy population. Also, little is also about pathogenesis of Acinetobacter genospecies 13 TU such as the capacity for induction of cytokines by the Acinetobacter genospecies 13 TU LPS LPS was extracted with the aqueous phenol method and re-purified by Voegel’s method from eight strains of Acinetobacter genospecies 13 TU, four strains of Haemophilus influenzae, two strains of Pseudomonas aeruginosa, two strains of Klebsiella pneumoniae and two strains of E. coli. These LPSs were used in enzyme linked immunosorbant assays (ELISAs) with serum taken from 475 blood donors from the Southeast Scotland Blood Transfusion Service. The results from the ELISAs were averaged for each individual blood donor across all the species tested. These averaged results were compared across the species. LPS from two strains of each species, ten in all, were used to challenge the THP-1 human monocytic cell line and the mRNA was extracted and used in quantitative polymerase chain reactions to measure cytokine induction. It was seen that exposure to Acinetobacter genospecies 13 TU LPS is about as widespread in a healthy population from Southeast Scotland as exposure to Pseudomonas aeruginosa LPS and somewhat similar to Klebsiella pneumoniae LPS. Antibodies to E. coli LPS and Haemophilus influenzae LPS were similarly widespread in a healthy population from Southeast Scotland. These last two were much more widely spread than the other organisms tested. Some individuals seem to produce antibodies at high levels to all of the LPSs tested. It may be possible to use serum from these individuals to make a hyper-immune immunoglobulin preparation to be used in the immunotherapy of hospital associated pneumonia. The LPS from one of the strains of Acinetobacter genospecies 13 TU was able to induce similar levels of cytokine production as Klebsiella pneumoniae and Pseudomonas aeruginosa. It was able to induce higher levels of cytokine production over a greater number of cytokines than both Haemophilus influenzae and E. coli LPS. LPS from the other strain of Acinetobacter genospecies 13 TU tested induced lower levels of cytokines compared to the other strain. These levels were lower than those developed by Haemophilus influenzae and E. coli LPS as well as those induced by the LPSs from Klebsiella pneumoniae and Pseudomonas aeruginosa. It seems that there is a range of different levels of cytokine production induced by Acinetobacter genospecies 13 TU LPS with some strains inducing high levels and others inducing low levels of cytokines.

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

Rheumatoid arthritis (RA) which affects 0.5-1% of the world population and is characterised by joint erosions and presence of the autoantibodies anti-citrullinated protein antibodies (ACPA) and rheumatoid factor. Collagen II (CII) is a joint-specific antigen and we have shown that antibodies against CII (anti-CII) are present in around 8% of RA patients. RA patients with anti-CII are characterized by acute RA onset with elevated CRP and early joint erosions at the time of RA onset. Polymorphonuclear granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) are abundant in RA synovial fluids, where they can interact with anti-CII, thus forming immune complexes (IC) with CII. In my thesis I have shown that PMN upregulated the cell surface markers CD66b and CD11b and downregulated CD16 and CD32 after stimulation with anti-CII IC. These changes in CD66b and CD16 associated to joint erosions to a larger extent than did PBMC responses to anti-CII IC. PMN cocultured with PBMC and stimulated with anti-CII IC showed augmented chemokine production that was dependent on TLR4 and functionally active PMN enzymes. This mechanism can lead to accumulation of inflammatory cells in joints of RA patients who are anti-CII positive around the time of RA diagnosis, and may thus help explain the acute onset RA phenotype associated with anti-CII. In a large Swedish RA cohort, anti-CII associated with elevations in clinical and laboratory measures of disease activity at diagnosis and until 6 months, whereas ACPA associated with late inflammation. Anti-CII seropositive RA was associated with improvements in clinical measurements and was negatively associated with smoking in contrast to ACPA that was associated with worseneing of clinical symptoms and associated positively with smoking. Anti-CII levels associated to  HLADRB1*03 and  HLADRB1*01 whereas ACPA showed negative association to HLA-DRB1*03. In a Malaysian RA cohort anti-CII also associated to elevated CRP at the time of diagnosis. Anti-CII seropositive RA represents a distinct phenotype, in many respects representing the converse  to the clinical, genetic and smoking associations described for ACPA. Early determinations of anti-CII in parallel to ACPA predict the inflammatory outcome in RA.

El presente trabajo de tesis tuvo como objetivo titular anticuerpos neutralizantes contra una cepa endémica del virus Chikungunya que circula en la costa norte peruana. Para desarrollar esta investigación, se empleó la prueba de neutralización por reducción en placas (PRNT), la cual se realizó en el Instituto Nacional de Salud (INS)– Laboratorio de Aislamiento y Cultivo Celular perteneciente al área de Metaxénicas Virales. La justificación de la investigación se basa en la necesidad de comprobar que los anticuerpos detectados por la prueba de ELISA son neutralizantes y logran inhibir la dispersión del CHIKV en el cuerpo humano. Además, la prueba es necesaria para evaluar drogas antivirales y futuras vacunas que lleguen al Perú. El diseño metodológico utilizado fue analítico y experimental. La cepa y las muestras fueron proporcionadas por el INS y provinieron del departamento de Tumbes. Las muestras positivas fueron previamente diagnosticadas con ELISA utilizando IgM e IgG y las muestras negativas fueron de individuos sanos sin contacto previo con arbovirus. La prueba de PRNT se realizó en 24 horas usando la línea celular VERO CCL-81 en monocapa. La valoración de la prueba se realizó al 50% de neutralización. Se obtuvo como resultado títulos de anticuerpos en el rango de 1/8 y 1/16. Ésta variación responde a una relación inversa con el inicio de los días de síntomas. Por lo tanto, se concluye que la población de la costa norte del Perú sí está desarrollando anticuerpos neutralizantes para contrarrestar el virus Chikungunya endémico en la región.The present thesis aims to titrate neutralizing antibodies against an endemic strain of the Chikungunya virus that circulates in the northern coast of Peru. In order to develop this research, the plaque reduction neutralization test (PRNT) has been used, which has been carried out at the National Institutes of Health (INS) - Isolation and Cell Culture Laboratory belonging to the Viral Metaxenics area. The justification of the investigation is based on the need to verify that the antibodies detected by the ELISA test are neutralizing and manage to inhibit the dispersion of CHIKV in the human body. In addition, the test is necessary to evaluate antiviral drugs and future vaccines that arrive in Peru. The methodological design used was analytical and experimental. The strain and samples were provided by the INS. The strain is endemic to the department of Tumbes. The positive samples were previously diagnosed with ELISA using IgM and IgG and the negative samples were from individuals that had not had contact with any arbovirus. The PRNT test was performed in 24 hours using the VERO CCL-81 cell line in monolayer. The titration of the test was performed at 50% neutralization. Antibody titres were obtained in the range of 1/8 and 1/16. This variation responds to an inverse relationship with the onset of symptom days. Therefore, it is concluded that the population of the northern coast of Peru is developing neutralizing antibodies to counteract the endemic Chikungunya virus in the region.

In North America, Borrelia burgdorferi is the primary causative agent of Lymedisease which is a growing health concern. The ability of B. burgdorferi to maintain chronic infection indicates that they are capable of immune evasion. A distinguishing characteristic of B. burgdorferi is the large number of sequences encoding predicted or known lipoproteins, including outer surface protein F (OspF). This study analyzes the specificity of the humoral immune response to B. burgdorferi B3 IMI OspF proteins during murine and human infection. Immunoblot analyses revealed a temporal expression of OspF proteins during infection and mapped the immunodominant epitopes which lie within the variable domains. To determine if OspF-related proteins are produced by other isolates, immunoblot analyses were performed using sera collected from mice and humans infected with diverse B. burgdorferi strains. Differences in the immunoreactivity profile to OspF proteins were seen among the infection sera tested. To identify the molecular basis of these differences, the ospF gene was isolated from several strains, sequenced and evolutionary analyses were conducted. These analyses revealed that OspF proteins show little diversity despite the separate geographic locations from which isolates originated. The high degree of OspF protein conservation seen in isolates from two distinct regions emphasizes the potential for OpsF proteins as vaccinogens or in serodiagnostic assays. Altogether, this study demonstrates the potential contribution of OspF proteins to immune evasion through its temporal expression during infection which may play specific roles at different stages of infection. Studies are underway to determine if inactivation of ospF genes through allelic exchange mutagenesis impacts on the pathogenicity of the Lyme disease spirochetes.

Currently, an increasing attention is being paid to phytochemicals as one of the most widely used chemopreventive compounds, generally considered as health-promoting and safe. Flavonoids representing a large group of phytochemicals are present in many dietary supplements formulated from natural sources. The consumption of these concentrated phytochemicals has dramatically increased in the recent decade. It appears, however, that the ingestion of flavonoids might be associated with some adverse effects. Some flavonoids are known modulators of enzymes involved in phase I and phase II metabolism of xenobiotics biotransformation, thus their induction may result in an increase of carcinogen activation. In this study, the effects of selected flavonoid compounds -naphthoflavone, - naphthoflavone, myricetin, and dihydromyricetin, and carcinogens (BaP, PhIP) on phase II metabolism enzymes, sulfotransferases (SULT), have been investigated. To determine the induction of SULT, antibodies for their immunodetection have been developed. Peptide antigens derived from sequences of selected rat sulfotransferases rSULT1A1, 1B1, 1C1, 1C2, 1C1/2, 1E1, and 2A1, were used as KLH conjugates for hen immunization to obtain yolk anti-peptide antibody (IgY). Fractions of IgY were isolated from eggs yolks by simple...

<p>The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies. </p><p>After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.</p><p>Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies. </p><p>In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies. </p><p> Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.</p> / Dissertation