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Abstract

HIV-1 affects about 38 million people worldwide, the majority in low income countries. Antiretroviral therapy (ART) is used to control the disease, however life-long ART is expensive, compliance is variable, it can cause severe side effects and resistance to ART is increasing. Broadly neutralising antibodies (bNAbs) offer a new perspective to pre-exposure prophylaxis (PrEP) and post-exposure therapy. However, despite their unique characteristics, several limitations hamper the therapeutic usage of bNAbs, including their cost and half-life.

To address these limitations and improve the efficacy of the bNAb 10-1074, two modifications have been investigated. Firstly, mutations (YTE/LS), that have been shown to increase the affinity to the neonatal receptor (FcRn), were introduced into the Fc-region of the antibody to extend mAb half-life. Secondly, a glycoengineered N. benthamiana line was used as the production host to allow human-like glycosylation, improve binding to FcγRIIIa, and potentially enhance antibody-dependent cell-mediated toxicity (ADCC). A significant negative impact on antibody yield, but no effect on affinity to gp120 or the ability to neutralise HIV-1 was observed upon introduction of the mutations into bNAb 10-1074. Enhanced affinity to FcRn was achieved with the YTE mutation, which translated functionally to increased transcytosis in vitro. In vivo studies further revealed an average 29 % reduction in blood clearance of 10-1074 YTE compared to the non-modified version. Production of a glycoengineered version of bNAb 10-1074 resulted in enhanced affinity to FcγRIIIa and an improvement in ADCC.

Finally, in anticipation of using transgenic tobacco for large scale manufacturing of HIV bNAbs for resource poor regions, inactivation of the α1,3-fucosyltransferase genes of N. tabacum using CRISPR/Cas9, was investigated. Five guide RNAs (gRNA) were included in a polycistronic tRNA-gRNA construct which was transformed into N. tabacum SR1. This resulted in a plant line at T2, with up to 65 percent reduction in fucosylation.

Publication Type:	Thesis (Doctoral)
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology T Technology > TP Chemical technology
Departments:	School of Health & Medical Sciences > Infection and Immunity Research Institute School of Health & Medical Sciences > School of Health & Medical Sciences Doctoral Theses Doctoral Theses

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