This thesis is focused on determining the structures of two anti-carbohydrate antibodies to understand how they achieve their specificity toward antigen. First, the structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide (LPS) has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo (3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater avidity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high avidity and specificity independently of CDR H3. Second, the structure of a rabbit, single chain variable fragment against terminal mannose-6-phosphate (Man6P) residues, termed scFv M6P-1, has been determined by x-ray diffraction to 2.7 Å resolution with Man6P in the binding site. The Man6P pathway is the predominant pathway that transports acid hydrolases from the trans-Golgi to endosomes. Newly synthesized hydrolases first require the generation of Man6P markers before they can be transported. Maintaining a full complement of hydrolases within lysosomes is essential as failure to do so results in a number of different lysosomal storage diseases. Due to its specificity, scFv M6P-1 is able to diagnose lysosomal storage diseases mucolipidosis II and mucolipidosis III. scFv M6P-1 is also able to purify Man6P containing proteins which may be useful for enzyme replacement therapies. Additionally, scFv M6P-1 is one of the first structures of an antibody fragment that exhibits high specificity for a single carbohydrate residue and is one of the first structures of a rabbit antibody fragment. The specificity of scFv M6P-1, which gives it these unique attributes, is revealed in the structure where multiple hydrogen bonds are seen between the antibody’s heavy chain and the mannose ring while two salt bridges are observed between the antibody’s light chain and the phosphate moiety. Finally, scFv M6P-1 binds in such a way as to allow binding to proteins possessing terminal Man6P residues. Crystallographic challenges that arose during this research included poor crystal growth as well as twinning and these are explored while the structure of scFv M6P-1 complex with Man6P is analysed. / Graduate / 0487 / 0982 / 0307 / dyl.w.evans@gmail.com

CD20 is a 35kDa surface antigen expressed on B cells from the early pre-B stage through the mature B stage. Moreover, the CD20 antigen is found on a majority of B cell malignancies. Rituximab is a chimeric anti-CD20 monoclonal antibody which has been extensively used alone or in combination in the treatment of CD20+ B cell malignancies including acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL), as well as in the treatment of numerous autoimmune disorders. Despite its emerging use in the clinic, 30% to 50% of patients with low-grade NHL exhibit no clinical response to Rituximab. Previous work to elucidate the mechanisms of Rituximab resistance has established that antibody dependent cellular cytotoxicity (ADCC) is important as a predominant mechanism of lymphoma cell clearance and that Fcγ receptors (FcγRs) are critical for the in vivo actions of Rituximab in NHL. Natural killer group 2D, NKG2D is a major activating receptor on T lymphocytes and natural killer cells. The NKG2D–ligand (NKG2D-L) interaction triggers an activating signal which results in cytotoxic lysis of the cell expressing the ligand. One potential ligand for murine NKG2D is the retinoic acid early 1β (Rae-1β) protein which is expressed during cellular stress and has a high affinity for the NKG2D receptor in mice. We have recently shown that an anti-HER2-IgG3 fused to murine NKG2D Ligand, Rae1β inhibited HER2+ tumor growth significantly more than Herceptin alone. Similarly, our objective is to enhance the performance of anti-CD20 directed therapy through activation of NK cells by an anti-CD20 antibody encoding the same NK activation ligand. Previous results with anti-HER2-IgG3-Raelβ led us to hypothesize that a CD20 specific fusion protein will bind to CD20 expressing tumor cells and deliver an activation signal to local NKG2D receptors on effector cells triggering a non-FcγR dependent anti-tumor response. Here we show that anti-CD20-NKG2D-L can be synthesized and tested for its ability to bind human CD20 and activate NK cells through the NKG2D receptor in vitro.

West Nile virus (WNV) has a worldwide distribution, with this virus having been isolated on all continents except Antarctica. The recent emergence of highly pathogenic strains of WNV associated with increased rates of neurological disease is of great concern given this broad distribution of the virus. Although two vaccines have been licensed for veterinary use, no prophylactic measures have been approved for humans. Similarly, no antivirals are currently available for post-exposure treatment of WNV. Indeed, few therapeutic agents have shown promise when administered after WNV infection in animal models. KUNV is a highly attenuated, Australasian lineage 1 strain of WNV. This attenuation is mediated in part by the limited neuroinvasiveness of this virus. Phylogenetically, KUNV clusters with pathogenic lineage 1 WNV strains, including the isolates which have been associated with 997 deaths in North America since 1999. Recently, it was shown that mice exposed to KUNV were effectively protected from challenge with pathogenic WNV. The KUNV strain used in that study possessed a single amino acid substitution in NS1 protein that affected oligomerization of this protein, resulting in reduced virus replication in vitro and increased attenuation in mice. In the present study, further characterization of this attenuation marker in NS1 protein was undertaken to determine whether it is suitable for inclusion in a live-attenuated KUNV vaccine. Similarly, mapping of the residues that contribute to the dimerization domain surrounding NS1 protein was performed to identify other potential attenuation markers for stabilization of KUNV attenuation. The mutant viruses created in this study also were manipulated to characterize the role of NS1 protein dimerization in flavivirus replication. The results of this work indicate that NS1 protein dimerization is not absolutely required for virus replication or production of secreted oligomers of NS1 protein, which are important for eliciting protective humoral responses. Although replication of KUNV was found to be highly dependent on retention of the conserved amino acid sequence within the dimerization domain, two mutant viruses were generated by introducing substitutions at residue 250 of NS1 protein. The resultant viruses demonstrated reduced replication in vitro and attenuation in mice. Similarly, a non-conservative substitution in NS2A, which was previously shown to reduce the resistance of KUNV to the host interferon response, was able to attenuate KUNV in mice. Inoculation of adult mice with viruses containing mutations at either site afforded complete protection from lethal WNV challenge. However, the substitutions described in the dimerization domain of NS1 protein were unstable, with restoration of virulence being observed in mutant viruses after limited passaging in vitro. Concerns over the stability of attenuating mutations in KUNV and the time taken to characterize new attenuation markers prompted the evaluation of a novel approach to the development of rationally-designed flavivirus vaccines. The introduction of large complements of synonymous codon substitutions reduced KUNV replication in vertebrate cells. Escape mutations were not observed in a KUNV vaccine candidate containing 37 rare codons after repeated passaging in vertebrate cells at a low MOI. Replication of KUNV in C6/36 cells was unaffected by the introduction of large numbers of rare codons, indicating that this cell line exerts limited selective pressure on the codon composition of this virus. This observation indicates that C6/36 cells may be a useful cell line for the propagation of viruses containing this type of mutation. Finally, three monoclonal antibodies (MAbs) which bind to WNV envelope (E) protein were observed to potently neutralize the pathogenic NY99 strain of WNV. Passive administration of one of these antibodies was shown to afford mice protection even when administered seven days after challenge with WNV NY99 strain. Remarkably, this is the same time that mortality is first observed in control groups. These antibodies mapped to the putative receptor binding domain (domain 3) of E protein. However, these antibodies were found to block virus replication at a stage after receptor-binding. Homology modeling was used to propose a mechanism for the blockade of virus infection mediated by MAb binding. This study describes the development and characterization of a promising new vaccine as well as candidate immunotherapeutics for the prophylaxis and post-exposure treatment of WNV disease. This work described herein also has implications for the development of vaccines and antivirals for other flaviviral diseases.

Typescript (photocopy) / viii, 102, xxv leaves, [6] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.) Dept. of Microbiology, University of Adelaide, 1982

Thesis (Ph.D.) Dept. of Microbiology, University of Adelaide, 1982. / Typescript (photocopy).

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1975. / Typescript (photcopy).

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--Northern Michigan University, 2008. / Includes bibliographical references (leaves 75-81).

Thesis (PhD--Cell Biology)--University of Auckland, 1976.

Thesis (Ph. D.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 152-165).