It was proposed that some O-alkyl analogues of the beta-adrenergic agonist clenbuterol would be effective structural and functional congeners of clenbuterol which may then be used for the production of clenbuterol-specific idiotypic antibodies. These antibodies could possibly then be used to generate anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol. Therefore, the aim of this work was to synthesise and characterise these compounds, evaluate their physiological effects, characterise the specificity of antibodies produced in response to protein conjugates of two of the novel compounds, and then use this data to determine the utility of these compounds for the generation of anti-idiotype antibodies which mimic clenbuterol. The target compounds were synthesised in five steps from 3,5-dichloro-4-hydroxyacetophenone in overall yields of 5-28%. A synthetic scheme similar to that which has led to clenbuterol was used to form the phenylethanolamine backbone, with modifications to include the O-alkyl moiety via a modified Williamson ether synthesis, and elimination of a synthetic chlorination step. Overall, 15 new compounds were synthesised, which were characterised and their structure confirmed from proton and carbon-13 NMR, IR and mass spectral data. The two haptenic analogues were then conjugated to carrier proteins using carbodiimide-based chemistries. In conclusion, the results indicated that the O-alkyl analogues, although structurally similar, were ineffective functional mimics of clenbuterol. Therefore, the anti-clenbuterol antobodies produced from the novel O-alkyl analogues would appear to be unsuitable for production of anti-idiotypic antibodies that mimic the energy-repartitioning effects of clenbuterol since the antibodies were unable to distinguish between the compound which demonstrated energy-repartitioning effects (clenbuterol) and those that did not (O-alkyl clenbuterol analogues). / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--University of Western Sydney, Macarthur, Faculty of Business and Technology, 1995. / Includes bibliographical references (p. 151-168).

Seafood including crustaceans, on ingestion, are known to provoke gastrointestinal as well as systemic allergic reactions. Crustaceans are aquatic arthropods with a chitinous exoskeleton and include shrimp, lobster, prawn and crab. Earlier studies in our laboratory have led to the identification and characterization of three allergens from shrimp, designated as Sa-I, Sa-I1 and Sa-III. The former two were shown to be heat stable proteins with a mol. wt. of 8.4 and 34 kDa respectively, while Sa-III was identified as tRNA Arg and TRNATyr ). Sa-II was found to be the major allergen contributing to more than 50% of the allergenic activity. There are several reports on the existence of cross-reactivity among atopic allergens, in particular food allergens. It is well known that individuals with shrimp allergy often complain of adverse reactions following the ingestion of other re1ated crustaceans. Recognition of crustacea as a group causing adverse reactions in sensitive individuals has a basis in the close phylogenetic relationship of shrimp, lobster, crab and prawn. Thus, one could expect appreciable similarity in the IgE binding epitopes of the offending allergens from related crustaceans. The present study was, therefore, aimed towards the identification of the major cross-reacting crustacean allergen and localization of its IgE binding epitopes. Cross-reactivity among a1lergens from shrimp, prawn, crab and lobster was evaluated by immunochemical methods. Antigenic cross-reactivity was established by immunodiffusion using shrimp-specific rabbit IgG. Competitive ELlSA inhibition experiments using sera of shrimp sensitive patients revealed a high degree of allergenic cross-reactivity between different crustaceans. SDSPAGE and immunoblot analysis using the sera of shrimp sensitive patients have identified a 34 kDa protein as the cross-reacting crustacean allergen. Using shrimp as a model system and Sa-II as a representative crustacean allergen, further studies were carried out to get an insight into the structural and molecular basis of allergenic cross-reactivity. The strategies adopted were, (1) to raise allergen specific anti-idiotypic antibodies and explore the possibility of using these anti-idiotypic antibodies as surrogate allergens for diagnosis of crustacea allergy and (2) to identify the IgE binding epitopes on the major shrimp allergen Sa-II, which may be shared by the 34 kDa allergen from the related crustaceans. In order to explore idiotypic, anti-idiotypic and anti-anti-idiotypic responses to Sa-II, Balb/c mice were immunized with affinity purified human idiotypic antibodies directed against the purified allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated using rabbit idiotypic antibodies raised against the same allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp sensitive patients. Immunization of Balb/ c mice with affinity purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. The induction of anti-anti-idiotypic antibodies functionally identical to allergen-specific idiotypic antibodies confirmed that the anti-idiotypic antibodies generated, are indeed a mirror image of the allergen. The present study thus provides evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens. These anti-anti-idiotypic antibodies not only recognized Sa-II, but also the 34 kDa allergen from prawn, lobster and crab. Cross-reactivity studies using polyclonal sera of shrimp sensitive patients and Sa-II anti-anti-idiotypic antibodies have attributed the allergenic cross-reactivity observed among the related crustaceans to the presence of highly conserved IgE binding epitopes on the 34 kDa crossreacting allergen from shrimp, crab, lobster and prawn. In order to identify the igE binding epitopes on Sa-11, it was subjected to limited tryptic digestion and the peptides were separated by reverse phase HPLC. Amino acid sequence analysis of these peptides and several other peptides generated by Asp N and Lys C treatment revealed an 861 homology with the muscle protein tropomyosin from the fruit fly Drosophila melanogaster, suggesting that the major shrimp allergen is tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from shrimp and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8-5.4. In the presence of 6 M urea, the mobility of both Sa-I1 and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp sensitive patients as assessed by competitive ELISA inhibition and immunoblot analysis. Tropomyosin, similar to Sa-I1 was subjected to limited tryptic digestion and the tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were found to be super imposable. Dot blot immunoassay and competitive ELISA inhibition assay using the sera of shrimp sensitive patients identified two peptides, 6 and 9 that exhibited allergenic activity. Both the peptides were purified to homogeneity and sequenced. Peptide 6 is a nonapeptide corresponding to the amino adds 153-161 and peptide 9 has 17 amino acids corresponding to the aminoacid residues 50-66. The peptides individually blocked upto 50% the binding of allergen-specific IgE to hropomyosin. Sa-II specific mouse anti-anti-idiotypic antibodies recognized not only tropomyosin, but also the two allergenic peptides, thus confirming that these peptides represent the major IgE binding epitopes. The IgG binding activity was found to be associated with peptides 6 and 9 as assessed by dot blot immunoassay using the sera of shrimp sensitive patients. Thus, it was found that both IgG and IgE binding epitopes on shrimp tropomyosin are identical. Tropomyosins from both phylogenetically related and unrelated species were assessed for allergenic activity using the sera of shrimp sensitive patients. It was found that allergenic activity was associated with tropomyosins from related crustaceans and from Drosophila melanogaster which shares 86% homology with shrimp tropornyosin. However, tropomyosins from totally unrelated species like yeast, chicken, bovine, rat, rabbit and human did not exhibit allergenic activity. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes with the corresponding regions of bopomyosins from different species confirmed lack of allergenic cross-reactivity. The allergenic peptides 6 and 9 were able to inhibit the binding of tropomyosins from related crustaceans to shrimp tropomyosin-specific IgE antibodies to the same extent, confirming the presence of highly conserved IgE binding epitopes. It has been established for the first time that the major crustacean allergen is the heat stable muscle protein, tropomyosin, and extensive cross-reactivity between different members of crustacea is due to the presence of highly conserved IgE binding epitopes on tropomyosins from these sources. Thus, from the present study, information with respect to the amino acid sequence of tropomyosin and localization of its 1gE binding epitopes, could be used to design synthetic peptides corresponding to the B cell and T cell epitopes which would find application in the diagnosis and desensitization of individuals allergic to crustacea.

The ability to faithfully replicate DNA is dependent upon the maintenance and regulation of its precursors, the deoxyribonucleoside triphosphates. Enzymes encoded by the bacteriophage T4 have been widely used as models of biochemical processes. A body of evidence supports the concept that the bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are associated as a complex within the infected Escherichia coli. This dissertation describes the continued examination of the protein-protein interactions involved in deoxynucleotide biosynthesis of bacteriophage T4. My studies on the protein-protein interactions involved in deoxyribonucleotide biosynthesis focused on two unique phage proteins, the dCMP hydroxymethylase enzyme and the translational regulator RegA. An initial study was undertaken to determine if the generation of anti-idiotypic antibodies would prove useful in the identification of direct interactions between dCMP hydroxymethylase and other proteins of the deoxyribonucleotide synthetase complex. For the initial generation of anti-idiotypic antibodies, polyclonal rabbit antibodies were generated to affinity purified anti-dCMP hydroxymethylase polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the bacteriophage T4 dTMP synthase. A second method to generate anti-idiotypic antibodies was attempted with the translational regulator RegA. The generation of anti-idiotypic antibodies to the RegA protein involved the purification of anti-RegA rabbit Fab fragments and the generation of anti-anti-RegA polyclonal antibodies within mice. This alternative method was determined to be inferior to the initial method for generating anti-idiotypic antibodies. Additional studies involved the examination of RegA protein-protein interactions using affinity chromatography. A number of bacteriophage T4 early proteins were determined to associate with an immobilized RegA column. / Graduation date: 1992

Living amidst a milieu of pathogenic organisms, vertebrates are in constant threat of contracting one or the other disease. As a mechanism of protection against such ‘invasions’, the vertebrate immune system has evolved to serve two main functions. One, to generate a specific immune response against the invading pathogen (in the from of specific antibodies and cell mediated immune responses). And two, to ‘remember’ the pathogen after the first exposure and mount a heightened and quicker immune response upon subsequent encounters. This phenomenon is called immunological memory, or anamnestic response and is achieved by the generation of memory B and T cells. The generation of specific Immunological memory is indeed the most important requirement/purpose of prophylactic vaccination Though different mechanisms are known to operate to maintain memory B and T cells, some aspects are still debatable. The ‘relay hypothesis’ (Nayak etal., Immunology.102(4)(2001); Nayak R etal., Microbes. Infect.(2005)), addresses some of those key issues. It describes that antigen specific memory B cells can be maintained by the interaction of membrane bound idiotypic (Id, Ab1) and anti-idiotypic (α-Id, Ab2) antibodies on B cells. Anti-Ids binding to idiotopes on Abs (Ab1) are known to be potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer as well as viral, bacterial or parasitic infections. The relay hypothesis outlines the mechanism of persistence of memory cells in the absence of persisting antigen. This is achieved through the ‘internal image’ of the antigen on the Ab2 variable region, which serves as surrogate antigen thus helping in maintenance of immunological memory even in the absence of persisting antigen. It also explains that all antigens, protein or nonprotein can be converted to the common “coinage” of internal image peptides, otherwise called peptido-mimics. Peptido-mimics that have similar binding properties to MHC as the antigenic epitope, will ensure that the antigen specific memory T cells are also maintained. Hence T cell activation could also occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. Scope and objectives of this work: To demonstrate the presence of idiotypic and cognate anti-idiotypic antibody for the given antigen  To examine the likelihood of the three dimensional structural similarity between antigen and Ab2 variable region  To demonstrate the presence of peptido-mimics of the antigenic epitope in the Ab2 variable region; and if those peptido-mimics have structural and functional similarity with antigenic peptides when bound to MHC-I To examine the (immunological) memory associated phenotype of thediotypic anti-idiotypic B cells. The antigen of choice for the current study is Heamagglutinin-Neuraminidase (HN) protein of peste des petits ruminants virus (PPRV). Idiotypic (Id, Ab1), antiidiotypic (α-Id, Ab2) hybridoma against a deletion mutant of PPRV HN were generated and characterized. These hybridoma served as surrogate B cells for the study of Id α-Id B cell interactions. Anti-anti-idiotypic (Ab3) lymphocytes were also generated by immunizing syngenic BALB/c mice with Ab2 hybridoma. Results not only indicated the interplay of idiotypic and anti-idiotypic B and T cells in this cascade but also the mimicry of the antigen by Ab2. Ab2 Mab could recognize idiotopes of anti-PPRV HN Ab1 raised in diff species of animals, thus demonstrating that Ab2 was indeed an antigen mimic that interacts with Ab1 paratope irrespective of which species the Ab1 originates from. Ab2 Mab also mimicked the antigen (Hemagglutinin-neuraminidase) in functional assays by bringing about hemagglutination. Similarly, Polyclonal Ab3 which reacts with Ab2 Mab and with antigen, inhibits hemagglutination, just as Ab1 does, albeit to a lesser extent. This suggests Ab3 has functional similarity with Ab1. It is imperative that T cells be involved in this network of B cells for the maintenance of antigen specific immunological memory. This is because B cells require T cell help in the form of cytokines for proliferation and Cytotoxic T Lymphocytes (CTLs) are needed to control the specific population of Id and anti-Id B cells to maintain homeostasis. The Ab2 hybridoma as well as soluble Ab2 stimulated the proliferation of antigen specific T cells. Similarly, Ab3 splenocytes were stimulated to proliferate by the Ab2 as well as the antigen. Peptides generated from monoclonal Ab2 heavy and light chain variable regions (VH and VL) showed structural and functional similarity to the antigenic peptides in terms of p-MHC binding. These peptides stimulated the proliferation of antigen and Ab2 specific T cells, and also triggered 4-5 times higher CTL targeted cell lysis of peptide pulsed RMAS-Kd cells, as compared to a control peptide. VH, VL and antigenic peptides stabilized MHC-I on the cell surface of the TAP deficient, RMAS-Kd cell line for upto six hrs as compared to the ‘empty’ MHC-I, which remained on the surface only for one hr. The presence of peptido-mimics in the Ab2 variable region, which have structural similarity with antigenic peptide (when bound to MHC I), was also established using insilico software tools. Antigenic peptides and VH and VL peptides were modeled onto MHC-I crystal structures using the molecular modeling software InsightII and the minimization program, CNS. Putative MHC-I binding peptides from these sequences were generated using the p-MHC-I binding prediction algorithm, BIMAS. By replacing these peptides in the respective crystal structure of MHC I and superimposing the two structures, we have tried to establish that through structural similarity in binding to MHC-I, peptidomimics have a role in the maintenance of antigen-specific CTL memory. Consequently CTL memory specific to antigenic epitope can be preserved even in the absence of antigen by its peptidomimic. Following long-term immunizations, as expected of a secondary immune response, the serum Ab1 titre was found to be higher than the titer during primary response. It was also noted that though the number of Ab1 and Ab2 cell number was comparable in the total splenocyte population, Ab1 titre in the serum was higher than Ab2, immaterial of Ag/Ab2 booster. The same trend was noticed in prolifertion assay and CTL assays when the splenocytes were stimulated by Ag/Ab2 pulsed bone-marrow derived dendritic cells (BMDCs) as APCs. That is, irrespective of immunization and boost with Ag/Ab2, Ag pulsed BMDCs stimulated the proliferation and CTL lysis of long term immunized splenocytes to a greater extent than Ab2 pulsed BMDCs. Memory markers present on B and T cell surface might help maintain their close interactions in the idiotypic network. CD27/CD70 (CD27L) might play a role in maintaining these cells in a memory state. The Id α-Id B cells in addition to being triggered through the membrane bound Id, α-Id antibodies, can also be activated through CD27/CD70 to differentiate into plasma cells upon activation by antigen. Id and α-Id B cells were demonstrated to possess the CD27 memory marker on their surface in addition to the membrane bound IgM. Antigen specific IgM and CD27 double positive cells were detected in the range of 1-3% in the total splenocyte population. In conclusion: PPRV HN immunization triggered the generation of Ab1, Ab2, Ab3 (Id, α-Id, α-α-Id) cascade through the interaction of membrane bound immunoglobulin of the corresponding B cells. Ab2 was demonstrated to be a significant structural and functional mimic of the antigen. Peptidomimics of the antigenic epitope, present in the Ab2 variable region, can serve the purpose of maintaining antigen specific T cell memory response. These findings re-confirm the importance of anti-id antibodies in the regulation of immune responses. Ever since the concept of antigen mimicry by anti-Id antibody has been confirmed by several laboratories, the utility of anti-Ids as surrogate antigens for the purpose of prophylactic vaccination has received great attention. The results of the current work are especially significant for the purpose of development of vaccines for diseases related to antigens that are very cumbersome to purify (for ex., in case of several cancers) or when it is too dangerous to immunize with the antigen itself (for ex., in case of some pathogenic organisms). The results also signify that immaterial of the nature of the antigen, their respective petidomimics can establish and maintain immunological memory.