Klapper, PaulValley, PamelaFaqih, LaylaAlatiq, Abdulrahman2024-11-072024-11https://hdl.handle.net/20.500.14154/73512The prevalence of HIV infection remains a serious challenge for public health. HIV infection results in considerable mortality and morbidity, as well as imposing a substantial financial burden on healthcare systems globally. Despite the availability of effective antiretroviral treatments to control infection, an effective protective vaccine remains elusive. This project aimed to develop a practical, affordable, and effective combined vaccine and treatment by combining intracellular antibodies and extracellular immunisation to prevent or reduce HIV viraemia. Specifically, an approach in which DNA-encoding N49P7 broadly neutralising antibodies against the HIV-1 envelope glycoprotein 120 is delivered to cells using recombinant baculovirus or lipid nanoparticle (LNP) transfection. Extracellular antibodies were anticipated to prevent gp120 from attaching to cells, and intracellular antibodies were intended to inhibit the genesis of virions. The expression of N49P7, either as a human IgG antibody or as human Fab, was attempted. The expression of the N49P7 IgG antibody was not achieved in HEK 293 cells despite employing three delivery models for transfections: recombinant baculovirus, MC3-based LNP, or lipofectamine 2000 LNP. While transfection was successfully achieved (monitored via an eGFP gene included within the plasmid design), functional antibody was not achieved. The most likely explanation for the failure was thought to be the use of dual promoters within the expression cassette. Redesign of the plasmid to create a bicistronic vector including the N49P7 Fab region and signal peptide sequences of murine IgG and IL-2 allowed successful expression of N49P7 Fab through recombinant baculovirus or lipofectamine 2000 reagent transfection of HEK 293 cells. However, the expressed N49P7 Fab region was predominantly accumulated intracellularly. A further redesign was implemented to incorporate homogenous signal peptides H7 and L1 into the N49P7 Fab gene, which significantly enhanced the secretion of the antibody fragment. This design maintained functional intracellular and extracellular antibody activity. Lower cellular cytotoxicity was seen with recombinant baculovirus transfection compared to lipofectamine 2000 LNP mediated transfection although both were equally efficient. The selection of an optimum method for transfection will form part of future investigations progressing to animal model testing. This proof of principle study showed that recombinant baculovirus or lipofectamine 2000-mediated transfection systems allowed efficient N49P7 Fab expression both intracellularly and extracellularly in mammalian cells, suggesting that this approach indicates potential for providing a vaccine against HIV infection in addition to a therapeutic intervention for those who already have HIV infection.269enHIVVaccineIntracellular immunisationimmunisationBaculovirusHuman immunodeficiency virusExpressionThesis