Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated withMADVand VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama. / National Institute for Health Research / Revisión por pares

In the Mediterranean basin, human visceral leishmaniasis caused by the protozoan parasite Leishmania infantum is a zoonotic disease that gives rise to 1,200 to 2,000 new cases annually. The domestic dog constitutes its main reservoir, of which some may suffer from a severe chronic disease, canine leishmaniasis (CanL). The sand fly Phlebotomus perniciosus is considered to be the principle vector. Saliva of bloodfeeding vectors of diseases has been used in the past to assess host exposure to vector bites and to evaluate vector control tools. This Ph.D. focused on saliva of P. perniciosus to identify exposure markers that could be used in the preparation of a new vector exposure tool. The first part of this Ph.D. aimed at validating the use of a recombinant salivary protein of P. perniciosus - rSP03B - in endemic settings of CanL. During a cross-sectional study, no significant differences between the antibody (Ab) response against whole saliva or the rSP03B were observed between different regions across the Mediterranean basin. Furthermore, the rSP03B was shown to resemble the native protein. During a subsequent study this protein was used to assess the seasonal dynamics of the canine Ab response to P. perniciosus in an endemic area of L. infantum. This study elucidated that also in a heterogeneous...

Understanding the mechanisms used by HIV-1 to evade antibody neutralisation may contribute to the design of a high-coverage vaccine. This thesis explores the mechanism used by a Tier 3 virus leading to its high antibody neutralisation resistance phenotype. This thesis also describes how the glycans at the base of the V3 loop contribute to (i) breadth and potency in a cohort of unselected HIV-1-infected individuals and (ii) the selective pressures resulting from the V3/glycans shielding the virus from neutralisation and the glycans themselves being targets of broad antibody responses. HIV-1 isolates that are highly resistant to broadly neutralising antibodies could limit the efficacy of an antibody-based vaccine. For this reason, it is important to understand the mechanisms behind high HIV-1 resistance to neutralising antibodies. Chapter 2 and Chapter 3 of this thesis describe virus 253-11, a highly neutralisation resistant virus, which is particularly resistant to commonly-elicited, anti-membrane proximal external region (MPER) antibodies in sera. To further understand its resistance, mutations in the MPER were introduced that are known to delay fusion following CD4-binding and thus increase the time the virus spends in the open conformation. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4-binding by destabilising the closed trimer structure. From these data, we hypothesized that the neutralisation resistance of 253-11 was due to an unusually tight, compact pre-fusion Env trimer that resists transient changes to the open conformation. The open conformation frequently exposes narrowly-neutralising antibody epitopes. Because the unliganded 253-11 Env presumably transitions infrequently into the open conformation, it would be able to evade these responses. 253-11 was sensitive to most but not all of the most potent broadly neutralising antibodies (bnAbs) tested, most likely because those broadly neutralising antibodies can access their epitopes in the pre-fusion Env conformation. To gain further information about the structure of the 253-11 Env, we designed a recombinant 253-11 SOSIP trimer and found it to be stable and predominantly adopt a closed conformation. The crystal structure of the SOSIP trimer revealed structural elements likely responsible for 253-11 Env compactness including the inward disposition of the heptad repeat helices and gp120 protomers towards the trimer axis. Taken together, the data from Chapter 2 and Chapter 3 highlight an underappreciated Env compactness mechanism of HIV-1 resistance to neutralising antibodies and these data may be useful in HIV-1 immunogen design research. Previous candidate HIV vaccines have failed to induce wide-coverage neutralising antibodies capable of substantially protecting vaccinees. A key approach in HIV immunogen development has been to define and model epitopes recognised by anti-HIV bnAbs. Candidate immunogen models identified by bnAbs include the V3/glycans, the V2/apex and the MPER epitopes. Autoreactivity and polyreactivity of anti-V3/glycan and anti-MPER antibodies are thought to pose both direct and indirect barriers to achieving neutralisation breadth. Chapter 4 of this thesis explored which of these bnAb epitopes were associated with breadth and potency in a South African cohort of chronically HIV-infected individuals. The study found that antibodies targeting the V3/glycans were associated with breadth and potency. In contrast, antibodies to the V2/apex were not associated with neutralisation breadth/potency. This suggests that auto/polyreactivity are not critical factors in the development of breadth and potency and that the V3/glycans should remain a high-priority vaccine candidate. Since targeting the V3/glycans was associated with breadth and potency in this cohort, the study continued to look at this epitope to investigate the role of these glycans in neutralisation resistance of Tier 2 viruses. The HIV-1 Env is surrounded by glycans that often prevent antibody neutralisation, leading to the term the "glycan shield", however some bnAbs have evolved to recognise these carbohydrates. Chapter 4 of this thesis describes how the N-linked glycan at position N301 is critical for maintaining neutralisation resistance of one subtype C virus (Du156.12), but not for another subtype-matched virus (CAP45.2.00.G3). Thus, the loss of the N301 glycan may have a substantial antibody-related fitness cost for some viruses but not others. The N301 glycan, as well as glycans at positions 332 and 334, are the primary targets of the anti-V3/glycan class of neutralising antibodies, which may select for loss of the targeted glycan. The evidence presented in Chapter 4 suggests that in some viruses, loss of the N301 glycan may result in evasion of anti-V3/glycan antibody responses while maintaining overall neutralisation resistance. This phenomenon may impair efficacy of passively-infused anti-V3/glycan bnAbs or a therapeutic vaccine.

Indiana University-Purdue University Indianapolis (IUPUI) / Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.

Cancer immunotherapy has revolutionized the field of oncology by giving the possibility to ligands (e.g., antibodies) to selectively target tumor antigens and accumulate at the site of the disease while sparing normal tissues. During the past years, the number of patient eligible for immune-based cancer treatments has seen an exponential increase as these therapies are becoming first line treatment for many cancer indications. A promising anticancer strategy consists in the targeted delivery of bioactive compounds (e.g., cytokines) to the tumor microenvironment with high-affinity ligands specific for tumor-associated antigens. This approach improves the efficacy of the drug by reducing related side effects and increasing the therapeutic index of the payload. Currently, antibodies represent one of the most successful class of ligands used for this purpose as they can be generated against virtually any antigen. Many methodologies have been described for the generation and isolation of antibodies with high antigen-binding specificity. Among these, phage display technology has emerged as a powerful and versatile tool for the in vitro discovery of antibodies and peptides. Since it was invented in mid 1980s, phage-display has paved the way to the generation of more than 70 phage–derived monoclonal antibodies (mAbs) that entered clinical studies, and 14 of which have been approved in the market. Cytokines are proteins capable of modulating the activity of the immune system and some cytokine-products have gained marketing authorization for the treatment of cancer. In order to increase the therapeutic index of cytokine payloads, the generation of fusion proteins with tumor-homing antibodies has been proposed. These so-called “immunocytokine” products constitute a class of “armed” antibody products, in which a tumor-targeting immunoglobulin is fused with a cytokine. In this thesis, we present the generation and characterization of antibodies specific for two tumor microenvironment-associated antigens: Tenascin C and Fibroblast Activation Protein. Both antigens are undetectable in healthy tissues but abundantly expressed in the tumor stroma. In the first part of the thesis, we have isolated antibodies specific for the spliced domain D of Tenascin C from the synthetic phage library “ETH2Gold”. Antibodies were affinity matured randomizing key residues of CDR1 of heavy and light chains. The highest affinity clone, R6N, was characterized in vitro and in vivo showing selective accumulation at the tumor site in mouse models of cancer. An immunocytokine featuring IL12 as payload has been generated and its therapeutic activity evaluated in tumor bearing mice. R6N-IL12 exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. In the second part of this thesis, a monoclonal antibody has been isolated against Fibroblast Activation Protein. After affinity maturation of the CDR2 of heavy and light chains of the parental antibody C5, the selected 7NP2 antibody showed improved affinity and excellent tumor targeting properties in SKRC52-hFAP tumor bearing mice. When fused to IL12, 7NP2 was able to induce tumor growth retardation and tumor remission in mouse models of cancer. Collectively, in this thesis we have isolated and validated two monoclonal antibodies selective for tumor microenvironment-associated antigens. Both antibodies when fused to IL12 induced tumor growth retardation and remission in immunodeficient and immunocompetent mouse models providing a rationale for possible future applications of R6N and 7NP2 antibodies for the treatment of cancer patients.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

Multiple sclerosis is a neurodegenerative disorder affecting the central nervous system (CNS). Currently, the disease is incurable and immunomodulating drugs are the only option to control the disease. CD49d is an adhesion receptor expressed on most immune cells. Antibodies that bind to CD49d and block immune cells from trafficking toward the CNS are being pursued as one class of therapeutics. In this work, by combining recombinant antibody and phage display technologies we isolated 10 anti-CD49d single domain antibodies from a synthetic antibody light chain variable domain (VL) phage display library. Isolated VLs (~ 12 kDa) were expressed in Escherichia coli, purified and analysed for biophysical characteristics. The majority were expressed in good yields and were non-aggregating. All 10 VLs bound recombinant CD49d by ELISA, and 7 bound to CD49d-expressing cells in flow cytometry experiments. To empower the VLs for better therapeutic efficacy (thru increasing avidity and half-life), three of the lead VLs were re-engineered as fusions to fragment crystallisable (Fc) of human immunoglobulin gamma (IgG). The engineered hFc-VL fragments (~ 70 – 90 kDa) retained their specificity for CD49d by flow cytometry. With (i) being less immunogenic due to their human nature, (ii) their efficient access to cryptic epitopes (iii) having half-lives comparable to IgGs’ and (iv) being more cost effective compared to IgGs, these novel antibody fragments (monovalent VLs and bivalent hFc-VLs) provide a promising therapeutic platform against multiple sclerosis.

Poon Kwok Man. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 118-123). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / DECLARATION --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1. --- Antibody structure and diversity --- p.1 / Chapter 1.2. --- Antibody genes --- p.5 / Chapter 1.3. --- The antibody response to phosphorylcholine --- p.10 / Chapter 1.3.1. --- Group I antibodies --- p.11 / Chapter 1.3.2. --- Group II antibodies --- p.14 / Chapter 1.3.3. --- Fine specificity of group I antibodies --- p.14 / Chapter 1.4. --- Anti-phosphorylcholine antibody structure --- p.15 / Chapter 1.5. --- Recombinant antibody --- p.22 / Chapter 1.5.1. --- Phage biology --- p.24 / Chapter 1.5.2. --- Phage-displayed antibodies --- p.29 / Chapter 1.5.3. --- Helper phage --- p.32 / Chapter 1.6. --- Objectives and scope of study --- p.34 / Chapter CHAPTER 2 --- METHODOLGY / Chapter 2.1. --- Antibody --- p.41 / Chapter 2.1.1. --- Hybridoma culture --- p.41 / Chapter 2.1.2. --- Production of antibody by induction of ascitic fluid --- p.41 / Chapter 2.1.3. --- Antibody purification --- p.41 / Chapter 2.1.3.1. --- Ammonium sulfate precipitation --- p.42 / Chapter 2.1.3.2. --- Affinity purification by Protein A-sepharose --- p.42 / Chapter 2.1.4. --- Production of Fab fragment by papain digestion --- p.43 / Chapter 2.2. --- Antigens --- p.43 / Chapter 2.2.1. --- Preparation of TsAg form infected ICR mouse --- p.44 / Chapter 2.2.2. --- Purification of Trichinella spairalis PC antigen --- p.44 / Chapter 2.2.2.1. --- Preparation of Mab2 affinity column --- p.44 / Chapter 2.2.2.2. --- Purification of TsAg --- p.45 / Chapter 2.2.3. --- Preparation of PC-HSA --- p.45 / Chapter 2.2.3.1. --- Preparation of p-diazonium phenylphosphorylcholine (DPPC) --- p.45 / Chapter 2.2.3.2. --- Conjugation of PC to HSA --- p.45 / Chapter 2.2.4. --- Commercial available antigens --- p.46 / Chapter 2.2.4.1. --- Pneumovax® 23 --- p.46 / Chapter 2.2.4.2. --- Lipopolysaccharide --- p.46 / Chapter 2.2.5. --- Standardization of PC-antigens --- p.46 / Chapter 2.3. --- Cloning of Mab2-scFv into phage display form --- p.47 / Chapter 2.3.1. --- Total RNA extraction --- p.50 / Chapter 2.3.2. --- cDNA synthesis --- p.50 / Chapter 2.3.3. --- Heavy chain variable region gene amplification --- p.51 / Chapter 2.3.4. --- Light chain variable region gene amplification --- p.51 / Chapter 2.3.5. --- Joining of heavy and light chain gene with linker --- p.52 / Chapter 2.3.6. --- Ligation of scFv gene with pCANTAB-5E vector --- p.52 / Chapter 2.3.7. --- Transformation --- p.53 / Chapter 2.3.7.1. --- E.coli strains --- p.53 / Chapter 2.3.7.2. --- E.coli cell preparation for electroporation --- p.54 / Chapter 2.3.7.3. --- Electroporation --- p.54 / Chapter 2.3.7.4. --- Competent E.coli preparation by CaCl2 --- p.55 / Chapter 2.3.7.5. --- Heat shock --- p.55 / Chapter 2.4. --- Expression of phage display scFv --- p.55 / Chapter 2.5. --- Enrichment and screening of Mab2-scFv phage --- p.56 / Chapter 2.5.1. --- Biopanning --- p.56 / Chapter 2.5.2. --- Restricition fragment analysis --- p.58 / Chapter 2.5.3. --- PCR screening --- p.58 / Chapter 2.5.4. --- DNA sequencing --- p.58 / Chapter 2.5.4.1. --- Manual sequencing --- p.58 / Chapter 2.5.4.2. --- Auto sequencing --- p.59 / Chapter 2.6. --- Mutagenesis --- p.59 / Chapter 2.6.1. --- Preparation of Uracil containing ssDNA --- p.60 / Chapter 2.6.2. --- Phosphorylation of mutagenic oligonucleotide --- p.60 / Chapter 2.6.3. --- Hybridization and secondary strand synthesis...…… --- p.60 / Chapter 2.6.4. --- Transfection and screening of mutants --- p.61 / Chapter 2.7. --- Expression of soluble scFv-E-tag --- p.61 / Chapter 2.7.1. --- SDS-PAGE analysis --- p.62 / Chapter 2.7.2. --- Anti-E-tag ELISA --- p.62 / Chapter 2.8. --- ELISA binding assay --- p.63 / Chapter 2.8.1. --- Specificity of Mab2 antibody Fab --- p.63 / Chapter 2.8.1.1. --- Carrier specifcity assay --- p.63 / Chapter 2.8.1.2. --- Free hapten inhibition assay --- p.64 / Chapter 2.8.2. --- Specificity of the scFv --- p.64 / Chapter 2.8.2.1. --- Antigen binding assay --- p.65 / Chapter 2.8.2.2. --- Free hapten inhibition assay --- p.65 / Chapter 2.8.2.3. --- Inhibition on Ts2 and Mab2 antibody assay --- p.65 / Chapter 2.9. --- Affinity assay --- p.66 / Chapter 2.10. --- Mutants analysis --- p.66 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1. --- Cloning VH and VL gene of Mab2 into scFv --- p.67 / Chapter 3.1.1. --- Amplification of variable region of H and L chain --- p.67 / Chapter 3.1.2. --- Biopanning --- p.70 / Chapter 3.1.3. --- Genetic composition of isolated clones --- p.70 / Chapter 3.2. --- Mutagenesis --- p.84 / Chapter 3.3. --- Expression and characterisation of wild-type scFv --- p.88 / Chapter 3.3.1. --- ScFv soluble protein --- p.88 / Chapter 3.3.2. --- Phage displayed scFv --- p.91 / Chapter 3.3.3. --- Standardization of PC antigens --- p.91 / Chapter 3.3.4. --- Binding acticity of scFv --- p.94 / Chapter 3.3.4.1. --- Influence of the avidity on carrier specificity binding --- p.96 / Chapter 3.4. --- Antigen specificity --- p.99 / Chapter 3.4.1. --- Free hapten inhibiton --- p.99 / Chapter 3.4.2. --- Inhibition on the binding of Ts2 --- p.102 / Chapter 3.4.3. --- Binding affinity --- p.104 / Chapter 3.5. --- Binding activities of mutants --- p.106 / Chapter CHAPTER 4 --- GENERAL DISCUSSION --- p.109 / REFERENCE --- p.118

In recent years a number of therapeutically useful immunotoxins have been produced using recombinant gene technology. In general, this involves fusion of a toxin gene with sequence encoding a variety of clinically relevant proteins or peptides. Using these techniques a recombinant immunotoxin has been engineered by fusing the genes encoding an antibody fragment with the sequence of a small cytolytic peptide, melittin. The antibody fragment consists of the antigen binding site derived from a murine monoclonal antibody K- 1-21, which binds to human free kappa light chains and recognises a specific epitope (KMA) expressed on the surface of human myeloma and lymphoma cells. The toxic portion of the molecule is melittin, a 26 amino acid, membrane lytic peptide which is a major component of bee venom. Using PCR a single chain Fv (scFv) was constructed by linking VH and VL genes with an oligonucleotide encoding a flexible, hydrophilic peptide. The melittin gene was synthesised as an oligonucleotide and extended by PCR. Nucleotide sequence encoding a linker peptide was added to the 5' end and a primer encoding a FLAG peptide was used to extend the 3' end. This gene construct was then ligated into the recombinant expression vector, pPOW scFv, to create the fusion gene encoding the recombinant immunotoxin. The gene construct was expressed in the periplasm of E.coli (TOPP2) using the secretion signal pelB . Expression of the foreign protein was monitored by western blot using a monoclonal antibody which recognises the FLAG peptide encoded at the carboxy terminal region of the gene construct. Expression of the recombinant immunotoxin was optimised and the resulting protein was purified using anti-FLAG M2 affinity chromatography. Antigen binding activity was assessed by ELISA and flow cytometry using a human myeloma cell line, HMy2, which expresses the KMA antigen.Binding of the immunotoxin to a control human cell line, K562, which does not express KMA on the cell surface was also assessed. The results indicated that the recombinant immunotoxin retained antigen binding specificity and it was cytotoxic towards the target cell line (HMy2).