Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies. Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the specialty of Virology, June 2018 / The discovery of broad and potent neutralizing HIV antibodies (bNAbs) has opened up new opportunities of passive immunization for HIV-1 prevention. In this study, we have engineered CAP256-VRC26.25, a V1V2 bNAb that neutralizes 70% of clade C viruses, as a single domain antibody (sdAb). These small antigen binding entities are derived from naturally occurring heavy chain only antibodies present in members of the dromedary families, and are characterized by the absence of a light chain, long complementarity-determining regions (CDR) heavy (H) chain 3 and high stability. Since CAP256.25 contains a highly charged and protruding CDR-H3 that binds mainly through its heavy chain, we hypothesized that it may function well as an sdAb. Multiple camelization approaches to engineer CAP256.25 as a sdAb were tested in silico utilising structural modelling software. Parameters such as germline sequence homology, hydrophobicity and solubility, folding energy, torsion angles and native conformation of CAP256.25 in complex with its binding epitope were major factors considered during the modelling process. Four CAP256.25 sdAb derivatives were generated from parental antibody, the mut_0 or a wild type (WT), which was used as a base line for downstream optimization. CAP256.25 mut_4 in which residues involved in LC interactions were replaced with residues strongly conserved in camel sdAbs, which minimize hydrophobic interface of the sdAb. Mut_8 variant, which included four additional substitutions to increase solubility and mut_9 contained a single additional mutation at the base of CDR-H3 to improve the energetic landscape of sdAb. All genes were synthesized and sub-cloned into a mammalian expression vector and recombinant proteins expressed in HEK293T cell line, and purified by Immobilized Metal Ion Affinity Chromatography (IMAC) and Fast Protein Liquid Chromatography (FPLC). CAP256.25_mut0 expression was below the detectable level and whilst mut_4 expressed at low levels, it showed no neutralization activity. CAP256.25 sdAb mut_8 and mut_9 expressed at significantly lower levels compared to m36, a previously described sdAb used a positive control. Nevertheless CAP256.25mut_8 sdAb showed neutralization capability although it lost significant potency in comparison to the parental antibody, yet still within the therapeutic window of the VRC01 bNAb. Importantly, CAP256.25 sdAb was unable to neutralize the K169E mutant confirming that it retained specificity for the V2 epitope. These data suggest that camelization of human antibodies is possible although further engineering is required to increase expression and improve stability. As such, sdAb engineering could be an encouraging step for the generation of small antigen binding fragments for future therapeutic purposes including topical delivery at mucosal surfaces, to interrupt or block sexual transmission of HIV. / XL2018

A panel of monoclonal antibodies were raised against the 1,4-dihydropyridine sensitive Ca2+ channel of rabbit skeletal muscle. When tested on immunoblot assay of denatured and reduced transverse tubule membranes, four of the antibodies specifically recognized a polypeptide of Mr 140,000. This component co-migrated with the large glycoprotein ?2 subunit of purified Ca2+ channel preparations. On immunoblots of nonreducing gels the antibodies detected a component that migrated more slowly in the gel, with a Mr of 170,000, consistent with the disulphide-linkage of the ?2 subunit to a small component of Mr 30,000. Additionally, three of the antibodies also recognized high molecular weight components of Mr 310,000-330,000 under these conditions. Crossreactive polypeptides of similar apparent molecular weight were detected in immunoblot assays of rabbit heart and brain membranes and of skeletal muscle membranes from different species. Further similarities between the ?2 components of Ca2+ channels from different species were investigated by immunoblot assay, following the limited tryptic digestion of the skeletal muscle membranes. A similar pattern of immunoreactive peptides were detected in each case, suggesting that the ?2 subunits of Ca2+ channels from different species are similar, not only in terms of antibody binding sites but also with respect to similarly positioned trypsin cleavage sites. The extent of glycosylation of the ?2 component was investigated using enzymatic and chemical deglycosylation techniques. Chemical deglycosylation resulted in a core polypeptide of Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments, although insufficient to completely deglycosylate the ?2 component, reduced the maximal 1,4-dihydropyridine binding capacity of transverse tubule membranes by 73-77%. The co-development of the ?2 subunit with 1,4- dihydropyridine binding activity was shown in rat skeletal muscle. These results indicate that the '2 subunit is an integral structural component of the 1,4-dihydropyridine sensitive Ca2+ channel.

Having passed all pre-clinical safety testing, the superagonistic anti -CD28 therapeutic monoclonal antibody (mAb ) TGN 1412, intended for the treatment of rheumatoid arthritis and B-cell chronic lymphocytic leukaemia, was approved by German and UK regulatory authorities for first-in-man Phase One clinical trial. Shortly after infusion, all six healthy trial volunteers suffered unexpected and profound systemic pro-inflammatory cytokine release, later termed a 'cytokine storm,' causing multi-organ failure. This unexpected and near fatal cytokine release syndrome (CRS) publically highlighted the failure of current pre-clinical safety testing procedures, emphasising an urgent need for novel cytokine release assays (CRAs) capable of predicting adverse properties of therapeutic mAbs. A wet coat mAb immobilisation approach, developed here, has proven predictive of clinical outcome and would have anticipated TGN1412 immunotoxicity in man. This approach IS now being widely applied by the pharmaceutical industry and contract research organisations (CROs). Comparative studies, testing TGN 1412 against a panel , of therapeutic mAbs, identified a unique mechanism of TGNl412-driven cytokine release. Substantial concentrations of the cytokine lL-2 were subsequently found to be a hallmark for the cytokine storm observed in-vivo, signifYing IL-2 as a TGN1412-like response biomarker. Multiple pro-inflammatory cytokine release was also shown to be principally effector memory T cell (T EM) derived in man. Human and macaque comparative immunophenotyping crucially identified macaque T EM cells as lacking CD28 expression, explaining pre-clinical animal model testing failures. A more physiologically relevant aqueous phase co-culture assay using monocyte-derived dendritic cells is also shown capable of eliciting a TGN1412-like cytokine release profile equivalent to that detected in-vivo and in-vitro using wet coat immobilisation; implying presentation in-vivo likely involved dendritic cells. This thesis describes the most likely mechanisms of action responsible for TGN1412 immunotoxicology in man and provides a plausible explanation for the pre-clinical safety testing failures, findings vital to the fields of immunomodulatory therapeutic mAb development and immunotoxicology.

No description available.

No description available.

No description available.