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Plum pox virus (PPV) es miembro del género Potyvirus, causa la enfermedad de la sharka que tiene un serio impacto agrícola y económico sobre el cultivo de frutales de hueso. Puesto que la utilización de variedades resistentes es la estrategia más duradera y definitiva en la lucha contra virosis de plantas. El objetivo general de esta tesis doctoral sería evaluar la expresión de anticuerpos recombinantes específicos de proteínas de PPV para interferir o inmunomodular la infección viral y servir así como estrategia para obtener resistencia. Para ello se ha utilizado el fragmento de anticuerpo recombinante scFv2A (del inglés "single chain Fv variable fragment) que reconoce específicamente a la proteína NIb replicasa de PPV. Esta proteína se localiza principalmente en el núcleo de células de plantas infectadas, aunque su función de replicación, presumiblemente, tiene lugar en estructuras membranosas derivadas del retículo endoplásmico (RE) en el citoplasma. Se transformaron plantas de Nicotiana benthamiana con tres versiones del fragmento recombinante scFv2A dirigidas a diferentes compartimentos celulares. Una versión citosólica (scFv2A), una versión nuclear (NLS-scFv2A) y otra dirigida a membranas del RE (6K2-scFv2A) por la cara citosólica. Se obtuvieron diversas líneas transgénicas: líneas A, líneas N y líneas K transformadas respectivamente con las construcciones scFv2A, NLS-scFv2A y 6K2-scFv2A. Se realizaron ensayos independientes de desafío, mediante inoculación mecánica con extractos de plantas infectadas con virus o bien inoculando con un clon recombinante de PPV fusionado a GFP (PPV-GFP). Tras los ensayos, se comprobó que las plantas de las diversas líneas transgénicas de N. benthamiana que expresaban los fragmentos scFv2A, 6K2scFv2A o NLSscFv2A presentaban diferentes grados de protección frente a la infección por PPV. Además, se determinó que varias de las líneas transgénicas, tanto líneas A como K y N, presentaban porcentajes de infección significativamente m / Gil Capitán, MT. (2010). Inmunomodulación de la infección de Plum pox virus mediante la expresión estable y transitoria de anticuerpos recombinantes contra la NIb replicasa viral en plantas de Nicotiana benthamiana [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34204

Les anticorps catalytiques sont étudiés pour comprendre leur rôle en conditions physiopathologiques. Ils semblent aussi représenter des outils révolutionnaires pour des études à l'interface entre la chimie, la biochimie, la biologie et immunologie. Par conséquent, la connaissance des relations de structure- fonction représente un grand intérêt. Nous avons exploré deux systèmes d'expression pour la production d'un anticorps catalytique modèle présentant une activité bêta-lactamase. Le fragment scFv recombinant a été produit dans le système d'expression procaryote. Les scFv sont souvent décrits comme des protéines difficiles à produire. Une méthode efficace a été développée pour produire de grandes quantités de scFv solubles et correctement repliés. L'anticorps catalytique entier a aussi été produit en exploitant le système d'expression eucaryote. Des cellules de mammifères ont été utilisées car elles peuvent conserver le repliement original des protéines, leur assemblage et les modifications post-traductionnelles. La structure secondaire du scFv catalytique a été analysée par dichroïsme circulaire pour s’assurer que la renaturation du scFv est en accord avec le repliement des scFv natifs. La fonctionnalité du scFv catalytique et de l'anticorps catalytique entier a été validée par deux approches : (1) le développement d’un test immuno-enzymatique (ELISA) et la résonance plasmonique de surface (RPS) et (2) le développement d'un test catalytique sensible utilisant un substrat fluorogénique. Ce travail amène à considérer de potentielles applications biotechnologiques et thérapeutiques des anticorps catalytiques. / Catalytic antibodies are investigated in order to understand their role under physio-pathological situations. But they also appear to be revolutionary tools to perform studies at the interface between chemistry, biochemistry, biology and immunology. Consequently, the knowledge of structure–function relationships is of great interest. We explored two expression systems for the production of a model catalytic antibody displaying a beta-lactamase activity. The recombinant scFv fragment was produced in the prokaryotic expression system. scFv fragments are often described as proteins being laborious to produce. An efficient method was developed to produce large quantities of refolded soluble catalytic scFv. Whole catalytic antibody was also produced by exploiting eukaryotic expression system. Mammalian cells were used because they are able to retain the original protein folding, assembly and post-translational modifications. The secondary structure of the catalytic scFv has been analyzed by circular dichroism to ensure that the refolded scFv is consistent with a native scFv fold. The functionality of the catalytic scFv and whole catalytic antibody has been validated by two approaches: (1) development of enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (2) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate. This will lead to consider potential biotechnological and therapeutic applications of catalytic antibodies.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Richard 'Dick' Hesse / Carol Wyatt / Over the last two decades molecular laboratory techniques have enabled researchers to investigate the infection, replication and pathogenesis of viral disease. In the early eighties, Dr. George Smith developed a unique system of molecular selection. He showed that the fd bacteriophage genome could be manipulated to carry a sequence of DNA coding for a protein not contained in the phage genome. Infection of the recombinant bacteriophage or phagemid into a specific strain of the bacterium, Escherichia coli, produced progeny phage with the coded protein displayed as a fusion with the phage's coat protein. Antibody phage display utilizes the same technology with the DNA encoding an antibody fragment. The DNA insert can carry the information to produce either a single chain variable fragment (scFv) producing the heavy chain variable and light chain variable (VH-VL) portion or a Fab fragment which also contains the heavy chain constant 1 with the light chain constant (CH and CL) portion of an antibody. Screening an antibody phage display library has the possibility of producing an antibody not produced in the normal course of immune selection. This decade also saw the emergence of a viral disease affecting the porcine population. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has been one of the most costly diseases affecting the pig producer. Molecular investigations found that PRRSV is a single, positive-stranded RNA virus which codes for five structural and 12-13 nonstructural proteins producing an enveloped, icosahedral virus. An interesting characteristic of PRRSV is the ability to produce infective progeny with genomic deletions, insertions and mutations within the nonstructural protein 2 (nsp2). With this knowledge, many researchers have produced marker vaccines containing fluorescent tags with the hope of developing a DIVA (Differentiate Infected from Vaccinated Animals) vaccine. In my Master‟s studies, I studied the techniques of antibody phage display technology and how to apply these methods to producing scFvs which recognize a recombinant PRRSV nsp2 fragment protein and the native protein during infection of MARC-145 cells.

Understanding protein function highly benefits from the knowledge of its three-dimensional structure, especially in the case of protein-ligand complexes. Structural biology methods such as X-ray crystallography, SAXS and NMR are therefore widely used for structural studies of protein-ligand interaction. In this work, these methods were used to understand two biological processes involving protein interactions: X-ray structural analysis was used to study binding of effector molecule to a prokaryotic transcription factor. NMR and SAXS techniques were used to study interaction of a monoclonal antibody with its protein antigen. Transcriptional regulator DeoR negatively regulates the expression of catabolic genes for the utilization of deoxyribonucleosides and deoxyribose in Bacillus subtilis. DeoR comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain (C-DeoR), and its function is regulated by binding of a small-molecular effector deoxyribose-5-phosphate. We determined crystal structures of C-DeoR both in the free form and in complex with deoxyribose-5-phosphate. Structural analysis revealed unique covalent binding of effector molecule through a reversible Schiff-base double bond with an effector-binding-site lysine residue. The physiological nature of this binding mode was...

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.

GPCRs, influencing myriads of cellular functions, are the members of the largest family of the membrane proteins. However, their structures and the signaling mechanisms still remain enigmatic. In case of the Glycoprotein Hormone Receptor (GpHR) family the structure-function relationship is less understood because of a large extra-cellular domain (ECD). This large ECD, consisting of Leucine Rich Repeats (LRRs) and membrane-proximal hinge region, is sufficient for specific binding to the hormone (Ascoli, Fanelli, & Segaloff, 2002), but for receptor activation, hormone binding is translated via a conformation wave starting at hinge region and relayed to the transmembrane domain. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure-function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005; Fox, Dias, & Van Roey, 2001; Wu, Lustbader, Liu, Canfield, & Hendrickson, 1994). However, the complete understanding of the hormone‐receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action or regulating receptor activation in a hormone independent manner. The crystal structure of FSHR ECD (amino acids 17-366) revealed that LRRs form a semicircular palm shaped structure with the C terminus region, designated as the hinge region, protruding out like a thumb. The hinge region, rather than being a separate functional unit, was found to be an integral part of the LRR domain, having two such repeats (LRR11 &12). LRR 11 is connected to LRR12 through a hairpin loop (amino acids 280-344) harboring the invariant sulfated tyrosine residue (sTyr) in YD/EY motif (X. Jiang et al., 2012). The heterodimeric hormones consisting of a common  subunit and a hormone specific  subunit, bind to the primary hormone binding site at LRR 4-6 as reported in the FSHR-FSH co crystal (Fan & Hendrickson, 2005). This primary binding of the hormone at LRR 4-6 creates a pocket (comprising of the residues P16α, L17α, F18α, F74α, L37β, Y39β, and P45β) in the hormone for secondary binding at sTyr residue. This interaction is proposed to initiate conformation change in the hinge region which further leads to FSHR activation (X. Jiang et al., 2012). Thus, the role of hinge region in GpHR activation got evolved from a linker to a switch, which decides the fate of the receptor activity (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). sTyr residue being conserved, presents itself as a potential player in activation mechanism of all the three receptors of the family (Bonomi, Busnelli, Persani, Vassart, & Costagliola, 2006; Kreuchwig, Kleinau, & Krause, 2013). Precise involvement of sTyr in GpHR activation is yet to be explored. The previous studies from the laboratory using the hinge region specific polyclonal and monoclonal antibodies established the unequivocal role of the hinge region in FSHR and TSHR activation (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). However, its function in LHR activation has not been conclusively established. Due to the unavailability of the structural information of LHR ECD/hinge, it is more difficult to study and explain the role of hinge region in LHR activation. The hormone independent signaling by point mutants of LHR also remains poorly understood. In the present study an attempt has been made to understand the role of the hinge region in LHR signaling and modulating role of LRRs in hinge mediated LHR activation. The present study was initiated with an overall objective of understanding the molecular details of LHR activation mechanism keeping hinge at the centre of the picture. To have clarity of this picture with a holistic view of the mechanism, multi-pronged approach was adopted. Initially, ScFvs against LHR hinge region were employed as tools to probe into the hormone‐receptor interactions. Antibodies against glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone‐receptor interactions and signal transduction (Agrawal & Dighe, 2009; Dighe & Moudgal, 1983; Gadkari, Sandhya, Sowdhamini, & Dighe, 2007; Gadkari et al., 2007; Kene, Nalavadi, Dighe, Iyer, & Mahale, 2004; Majumdar, Railkar, & Dighe, 2012a, 2012b). In this study, Single chain Fragment variables (ScFvs) against the hinge region of LH receptor have been employed to understand the mechanism of receptor activation. The effects of LHR ScFvs on hCG-LHR interactions have been investigated and three of the ScFvs, JE10, JE4 and JG1 could bypass the hormone and activate the receptor directly, with JE10 being the most potent one. The effect on the signaling was specific for LHR as no increase in cAMP response was observed for TSHR/FSHR in presence of these ScFvs. JE10 surprisingly was unique and could alter the hCG-LHR interaction by decreasing hormone affinity and simultaneously increasing the Bmax for the hormone. JE10 binding was decreased to the pre-formed hormone receptor complex suggesting that hCG and the stimulatory antibody show stearic hindrance at the binding sites on hinge or hormone binding induces conformational change in the epitope of JE10. The change in affinity and Bmax of the hormone by JE10 could be due to unmasking of new binding sites for hormones or an allosteric effect on the protomer interaction like explained in case of a small TMD specific allosteric modulator of FSHR (Xuliang Jiang et al., 2014). JE10 could also potentiate hCG signaling at sub-saturating concentrations of hCG, the precise mechanism of which is not clear. Through TSHR-LHR chimeric mutants, a stretch from amino acids 313-349, within the hinge region, was identified as the site recognized by JE10. In order to study structural features of the JE10 epitope, LHR ECD was modeled on the basis of FSHRED crystal structure. With most of the motifs being structurally conserved (CF3 and YPSHCCAFF); the major portion of the hinge region was found to be unstructured. This unstructured region harbored the JE10 epitope as well as the functionally important conserved sTyr residue. The CD spectra of LHR hinge in presence of ScFv JE10 suggested a ScFv induced helical conformation and stabilization of the hinge loop region, which was constrained in the homology model into helices. As loop was now constrained in the Mode 2, so was the interaction of sTyr, which was now in contact with positively charged residues, probably stabilizing its charge. The YEY motif mutants further confirmed the indirect essential role of Y331 in activation of LHR by JE10. Another approach followed to study hCG-LHR interactions was use of a series of LHR N-terminal truncation mutants and truncation mutants along with one of the LHR CAM (S277Q/D578Y). The effect of these truncations on hormone binding and receptor activation was investigated. The deletion of Cysteine box (Cb-1) of LHR (present at N-terminus of ECD) leads to abrogation of hCG binding, indicating importance of this region in maintaining ECD conformation required for hormone binding. This is the most unexplored region of the ECD. Though Cb-1 does not bind to the hormone directly (as is evident from the crystal structure) but it is indirectly essential for hormone binding. The basal activity of these truncated mutants was as low as that of the wild type LHR, reconfirming that no region of LHR ECD acts as an inverse agonist for the TMD (Karges, Gidenne, Aumas, Kelly, & Milgrom, 2005). Truncation mutants with CAM (double mutants) also showed low basal activity, suggesting that intact ECD is prerequisite for keeping LHR in a conformation, best suited for hormone binding and binding of G protein for activation. That best conformation still needs to be explored. Truncation mutants did not get stimulated by JE10 also. This observation is opposite to the previous studies in which FSHR/TSHR truncated mutants could be stimulated by hinge specific antibodies (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). This difference points out to the variations in which LHR hinge-TMD interactions prevail and lead to the receptor activation. This variation was also confirmed with a previous report in which the binding of TSHR-ECL specific antisera to wild type LHR and TSHR-LHR 6 chimeric mutant suggested that hinge of LHR does not seem to be constraining the TMD (Majumdar et al., 2012b). Thus the LHR TMD itself possesses all the inhibitory interactions, also indicated by the presence of most of the activating mutations in LHR TMD (Piersma, Verhoef-post, Berns, & Themmen, 2007). Protomer interaction is the newest aspect of GpHR activation mechanism and has not reached any conclusive, physiologically relevant explanations yet. By co-transfection of wild type LHR and ECD truncated mutants, this study suggests the LHR protomer interaction and proposes the involvement of allosteric effect of ECD on LHR protomer interaction. The effect of JE10 on activating and inactivating mutants of LHR were quite interesting. The ScFv could bind to the activating mutant D578Y (associated with precocious puberty). This mutant exhibited higher basal cAMP production, but was activated even further by the ScFv. The inactivating mutant A593P is a completely inactive receptor associated with (associated with pseudo-hermaphroditism. It does not respond to the hormone at all. The ScFv JE10 binds to this receptor and stimulates cAMP production. This observation is rather striking, as it is possible to activate a completely inactive mutant that could not be stimulated by the hormone by a binder specific for the hinge region. It is not clear how the binder that interacts with the hinge region affects the function of the inactive TMD thus providing an interesting tool to investigate the interactions between the hinge region and TMD that are probably key to understand the activation of GpHR. which has been shown to be central to the GpHR activation mechanism, (Agrawal & Dighe, 2009; Majumdar et al., 2012b; Schaarschmidt, Huth, Meier, Paschke, & Jaeschke, 2014). As per the recently suggested model by Deupi et. al., that each mutation and agonist can take a different pathway during activation (Kobilka & Deupi, 2007). The activated state induced by JE10 in D578Y and A593P seems to be different from the wild type LHR, with each activated receptor state having different capacity to bind to the G protein. The difference in G protein capacity in itself reflects the different receptor turnover or different Gs uncouplings or different Gs binding affinities, which needs to be further investigated, opening up another avenue for exploration. There is a lacuna in understanding the signal relay from the hinge to TMD. However, JE10 seems to be activating the wild type LHR and the mutants directly or indirectly by modulating the 6th helix of the TMD, known to be important for hormone independent activation of LHR (Fanelli, 2000; Latronico & Segaloff, 2007; Majumdar et al., 2012b). As evident from the absence of any hinge mediated constrain on LHR TMD and absence of uncharged residues present in LHR LRRD-TMD interface (LHR ECD Model 1), LHR hinge does not seem to be maintaining significant interactions with the TMD in absence of a ligand or in its basal state. Hormone/ agonist binding or activating mutations act as a positive regulator (inducing conformation change in hinge), required to bridge the interactions between LHR hinge and the TMD, which is supported by various studies in the past (Karges et al., 2005; Majumdar et al., 2012b; Nishi, Nakabayashi, Kobilka, & Hsueh, 2002; Osuga et al., 1997; Ryu, Gilchrist, Tung, Ji, & Ji, 1998; Zeng, Phang, Song, Ji, & Ji, 2001). This interaction bridged by the conformational change in the hinge region, seems to isomerize the closed state of LHR into an activated state. The present study supports the conformational induction model for receptor activation in which intramolecular interactions between the two domains (hinge-TMD) lead to the receptor activation. In conclusion, this study presents a possible mechanism of activation of LHR by a partial agonist ScFv, which induces the conformation change in the disordered loop region (a.a.313-349) of the hinge and stabilizes it into helical state. This conformation change is predicted to be important for relaying the activation signal to the TMD. The study also demonstrates the activation of a completely inactive mutant A593P by JE10, suggesting a distinct possibility of its use as a therapeutic tool in treating infertility caused by inactivating mutations in LHR. On a second note, the study extends the role of LRRs, apart from direct hormone binding, to an indirect allosteric role in hormone binding, LHR activation and functional stability. This functional stability does not seem to be restricted to a single LHR but also depends on its interaction with nearby protomers. Though there are evidences for and against each of the above discussed possibilities, as yet there is no accepted model that explains the precise steps of receptor activation, hence, the molecular details of these interactions needs to be investigated in future.

Contraception through a vaccine has been a very attractive proposition and several attempts were made in the past. To achieve contraception through immunological means, several points need to be considered. First, the targeted antigen should be an important component of reproduction and interference in its actions should lead to infertility. Second, the antigen must be highly immunogenic and the antibodies elicited should be able to block the functions of the antigen. Third, the antibody titres should be effective and must sustain for longer periods. Gonadotropins fulfill all the above criteria and therefore, have been attractive targets for developing human contraceptive vaccines. The pituitary gonadotropins- Luteinizing hormone (LH) and the Follicle stimulating hormone (FSH) are the principal regulators of the reproduction process in all the mammalian species (McLachlan et al., 1995c; Moudgal et al., 1992b; Murty et al., 1979a; Selvaraj and Moudgal, 1994a; Weinbauer et al., 1991). In males, LH binds to its specific receptor-LHR, expressed on the Leydig cells and regulates the production of testosterone. This testosterone binds to the androgen receptors expressed in the Sertoli cells and along with FSH, which binds to the specific receptors present on the Sertoli cell membranes, regulate the testicular functions and the spermatogenesis (Simoni et al., 1997; Themmen and Huhtaniemi, 2000; Ulloa-Aguirre and Timossi, 1998). The well documented studies have unequivocally established that the specific immunoneutralization of either hormone by active or passive immunization, leads to disruption of the gonadal functions (Fraser et al., 1986a; Marathe et al., 1995; Moudgal et al., 1992b; Murty et al., 1979b; Shetty et al., 1996; Srinath et al., 1983b) and consequent infertility and this observation formed the basis of the human contraceptive vaccines (Moudgal et al., 1997b; Talwar et al., 2011a; Talwar et al., 2009a). Several studies using testosterone as the main male hormonal contraception method (Matsumoto et al., 1986; Matsumoto et al., 1983a) and anti-hCG vaccine as the female hormonal contraceptive vaccine reached Phase I and II clinical trials (Talwar, 1997; Talwar et al., 1994; Talwar et al., 1997) . However, these human contraceptive vaccines faced several limitations. There was a need to inhibit only particular segments of the entire reproduction process whereas others needed to remain completely unaffected. For example, in males, the FSH regulated functions, the sperm production and spermatogenesis needed to be inhibited whereas the LH/testosterone associated functions should be unaffected. Similarly in females, the functions of hCG alone, elaborated by the conceptus should be blocked without affecting either LH or FSH regulated functions, thus, maintaining the normal reproductive cycle. This however is a difficult task especially when the antigens share a large degree of homology and common subunits (Pierce and Parsons, 1981). Moreover, the issues relating to the development and sustenance of high titres of the bioneutralizing antibodies were major limitations of these human contraceptive vaccines. Therefore, despite reaching Phase I and II clinical trials, these studies did not progress further. However, the same concept of an immunocontraceptive vaccine involving the neutralization of the functions of the gonadotropins is an extremely attractive strategy for controlling the animal populations where the reproduction process could be inhibited in its entirety. The overgrowing populations of the stray animals such as dogs and cats pose problems unlike those experienced with the human overpopulation. Thus, there is an immediate need to develop the methods of controlling the populations of these animals both in the developed and the developing countries. Whereas, in countries like the US, the major emphasis is on the domestic animals, in countries like India, the populations of the stray animals need to be controlled. The current methods employed for reducing the numbers of these animals include either castration or culling of the animals. These methods are however, traumatic, unsafe and not widely accepted by the society. The animal contraceptive vaccines currently available are mostly GnRH vaccines which have high cost of production, are not safe for animal use and elicit unwanted side effects. Apart from these, the animals need multiple administrations of these vaccines to elicit high and effective antibody titres, mostly with the use of conventional but non-approved adjuvants (Boedeker et al., 2009; McCoy, 1994). As mentioned above, the gonadotropins, by virtue of their ability to control the mammalian reproduction process, are attractive targets for achieving contraception. Moreover, the ease of administration of this vaccine to neutralize the functions of the endogenous circulating hormones makes them ideal targets for developing animal immunocontraceptive vaccines. This method of neutralizing the functions of the gonadotropins is also humane and safe for the animals as opposed to the current methods which are employed to reduce their numbers. However, in case of animal contraception, particularly for strays such as dogs, where large numbers of animals need to be treated, the challenge is to develop a method to sustain the high levels of the bioneutralizing antibodies for prolonged periods preferably with a single administration of the immunogen and without the use of conventional adjuvants such as the Freund’s adjuvant. In the present study, an attempt has been made develop a strategy to achieve a sustained immune response to small quantities of the hormonal antigens, preferably with a single administration of the immunogen resulting in complete disruption of the gonadal function for prolonged periods. To achieve this goal, recent developments in the field of immunology and vaccinology have been employed. This involves targeting of the hormonal antigens to the dendritic cells. Targeting the antigens to the dendritic cells for vaccination is becoming an extremely fascinating strategy and is being used extensively to target the antigens involved in several diseases (Escudier et al., 2005; Frankel et al., 1998; Garcia et al., 2005; Nouri-Shirazi et al., 2000a; Nouri-Shirazi et al., 2000b; Steinman and Germain, 1998). Most antigens are targeted to the dendritic cells by coupling them to the antibodies specific for the receptors expressed on the dendritic cell surface. One such receptor is the DEC205, which is expressed on most of the dendritic cells (Jiang et al., 1995) and is being widely used to develop vaccines and vaccination strategies. Targeting the antigens to the dendritic cells provides advantages such as ability to induce hundred fold higher immune response to very low doses of antigen without the use of any conventional adjuvant (Bonifaz et al., 2004a). Therefore, in the present study, these features of the dendritic cells have been harnessed to target the hormonal antigens (hCG and hFSH) to the canine DEC205 receptor to induce a long-term immune response capable of disrupting the gonadal functions. Towards this goal of delivering hormonal antigens to the dendritic cells, a fragment of the canine DEC205 corresponding to the Cysteine Rich Fibronectin II domain (CR/FNII) was expressed and used to isolate several canine DEC205 specific recombinant antibodies in the form of single chain fragment variable (ScFvs) from the Tomlinson’s and the yeast human ScFv display libraries. From a pool of eight unique ScFvs screened from the Tomlinson’s libraries, three ScFvs namely B3, G10 and H4 were characterized. All these ScFvs could bind to the human DEC205 receptor but not to the mouse DEC205. Their inability to recognise the mouse DEC205 suggested that mouse could not be used as the model system for these studies and therefore, a surrogate model system was needed. As the canine CR/FNII shared a high degree of homology with the rabbit counterpart, adult rabbits have been used as the surrogate model for immunization studies after confirming the binding of the ScFvs to the rabbit dendritic cells. Since the goal of the study was to deliver the hormonal antigens to the dendritic cells, each ScFv was translationally fused to a core streptavidin fragment, thus creating bi-functional agents (ScFv-CS) capable of binding to the dendritic cells and also to any biotin-tagged antigen, thus delivering the antigen to the dendritic cells. Of the three ScFvs, the ScFv-CS-H4 which could bind to the canine CR/FNII with the KD of 25nM was used for demonstrating the ability of the ScFv-hormone complex to elicit the bioneutralizing antibody response. The ScFv-CS-H4-biotin-hCG or hFSH or both were administered to adult male rabbits along with poly IC: LC, a Toll-like receptor agonist and the antibody titres were monitored. It was possible to maintain high titres of the bioneutralizing antibodies for more than one year with a single administration of the immunogen. Testicular histology of the immunized animals showed extensive disruption of spermatogenesis with most of the germ cells being TUNEL positive undergoing apoptosis. There was complete absence of elongated spermatids and sperms in the testis indicating infertility caused by immunization with the gonadotropins. These data show that targeting the hormonal antigens to the dendritic cells leads to long-term infertility with minimal immunization. Although the ScFvs from the Tomlinson’s libraries were able to deliver the hormonal antigens to the dendritic cells and produce robust and sustained antibody response capable of disrupting the gonadal functions, the affinities of these ScFvs to DEC205 were moderate. It was felt that increasing the affinities of the ScFvs could enhance the effect with respect to the dose of the antigen that needs to be administered and the duration until which the high antibody titres could be maintained. Therefore, the yeast human ScFv display library offering higher diversity of the human ScFvs displayed, was screened for high affinity DEC205 specific binders. From a pool of several ScFvs, six unique ScFvs were characterized. The amino acid sequences of all ScFvs followed the Kabat's rules for identifying the complimentarity determining regions of the heavy and the light chains of the antibodies. All these ScFvs were unique in their amino acid sequences. The dissociation constants of all these antibodies for the canine CR/ FNII ranged from 10-9 to 10-11 M which was 20-300 fold higher than the ScFvs obtained from the Tomlinson’s libraries. The best ScFv obtained from this library was ScFv-92 with a KD value of 8 x10-11 M. All these ScFvs were able to deliver the payload antigen to both, the mouse DEC205 over-expressing cells and the bone marrow derived dendritic cells. Mice immunized with yeast display ScFvs also yielded antibody response to very small quantities of the immunogen with the highest antibody titres obtained with the ScFv-92. It was further demonstrated that all ScFvs also activated the cell-mediated immunity with significant increase in the antigen stimulated T cell proliferation. These ScFvs could also deliver the antigen to the human dendritic cells differentiated from the human monocytes in vitro, thus emphasising their utility in human vaccine development. An attempt was also made to develop nanoparticle (NP) based strategies of delivering the antigen to the dendritic cells. The PLGA-NPs, encapsulating hCG and coated with the DEC205 ScFv-92 was able to elicit high antibody response to very low doses of the antigen. This response could be sustained for 120 days and was higher than the response obtained with similar doses of hCG encapsulated NPs or hCG complexed to ScFv-92 alone. Targeting of the NPs also elicited antigen specific T cell response thus, potentiating their use in cell mediated immunity along with humoral immune responses. In conclusion, this approach of delivering the gonadotropins to the dendritic cells resulted in the production of bioneutralizing antibodies that could disrupt the gonadal functions for a prolonged period and can be effectively used in the fields for controlling the animal populations. This method fulfils all the criteria for any animal contraception. This strategy also elicits both T cell mediated and humoral immunity and can thus be used for producing vaccine against viral and parasitic infections. It can also be used for cancer immunotherapy. Another exciting feature of the strategy used in this study is the usage of ScFv-CS which allows the delivery of any biotin tagged antigen to the rodent and human dendritic cells. As discussed above, the methods for controlling the animal populations are expected to be effective, humane, safe, simple, non-surgical, single shot with long lasting effects, cheap, applicable in the fields and widely accepted by different societies. The methods presented in this study fulfill all these criteria and should be effective in controlling populations of different animal species.

Amyloid PET imaging with [11C]PIB enabled detection of Aβ for the first time in vivo. However, [11C]PIB is a small molecule that binds only the insoluble Aβ plaque. Rather, the soluble Aβ aggregates are considered the cause of Alzheimer’s disease (AD). As such, a more sensitive and specific PET tracer is needed for tracking longitudinal AD pathology. Soluble Aβ aggregates likely interact with the metabotropic glutamate receptor 5 (mGluR5) to cause neurotoxic effects. However, with [11C]ABP688 PET we were unable to detect aberrant mGluR5 binding in AD mouse models, although we find elevated mGluR5 protein levels with immunoblotting. Antibodies are highly specific large molecules that can bind specifically to soluble Aβ aggregates, thus they can be a good marker for AD pathology. Unfortunately, due to their large size they cannot cross the blood-brain barrier (BBB). However, it is possible to shuttle antibodies into the brain by taking advantage of endogenous transporter systems on the BBB. By creating bispecific antibodies binding both to soluble Aβ aggregates and to the transferrin receptor (BBB target), we successfully transported the antibody into the brain and could visually detect soluble Aβ aggregates with PET. Recombinant expression further improved and optimized antibody design, creating smaller bispecific antibody-based constructs that had better pharmacokinetic properties allowing for earlier PET scanning (1 day instead of 3), and more sensitive signal. Lastly, using TCO-tetrazine click chemistry, we indirectly labeled our antibodies with fluorine-18, and could successfully perform PET already 11 h post-injection with a fluorine-18 labeled antibody.

Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...