Anchoring of a human immunodeficiency virus antigen to the surface of lactobacillus plantarum
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- Master's theses (KBM) 
The aim of this study was to develop the lactic acid bacterium (LAB) Lactobacillus plantarum WCFS1 as delivery vector for a human mucosal vaccine against human immunodeficiency virus (HIV). LAB are known for their wide use in the food industry, especially in food and beverage preservation, and are natural inhabitants of the human gastrointestinal (GI) tract. L. plantarum WCFS1 has probiotic and immunomodulating effects and can persist in the human tract for up to seven days. L. plantarum and other LAB are regarded as safe for oral consumption and are thus subjects of extensive research regarding their potential as in situ delivery vectors for heterologous proteins. In several previous studies on the ability of recombinant bacteria to activate an immune response through the mucosal immune system, specific immune responses have been detected. In the present study, the HIVp24 fusion protein consisting of a HIV antigen and a cytokine, was expressed and anchored to the surface of L. plantarum WCFS1 using different anchors: a lipoanchor, an LPxTG-type anchor and a LysM-type anchor. Expression and surface display of the fusion protein was achieved, but with varying results depending on the anchor type. The variation in results emphasises the need to optimise the combination of signal peptide, anchor type, target protein and host strain for each heterologous protein that one wishes to express and display. A replicon change, presumably leading to higher plasmid copy numbers, was performed to increase production of HIVp24. Production was increased, but surface display of HIVp24 decreased. Surface display was analysed using flow cytometry and confocal microscopy. The surface structure of the various recombinant strains was closer investigated through scanning electron microscopy, which showed that strains producing HIVp24 fused to an anchor sequence, and in particular the strain expressing HIVp24 with the LPxTG-type anchor, had a disrupted, blistery surface. Finally, the recombinant strains were tested in a mouse model. ELISPOT analysis indicated development of tolerance, but final conclusion cannot be made before additional experiments have been done. Our data show that L. plantarum WCFS1 is able to express and anchor the HIVp24 fusion protein to the surface using homologous signal peptides and anchors, which is an important achievement in its own right. So far, immune responses in mice have not been detected for any of the recombinant strains, but further analyses are in progress. There is a great need and potential to optimise the secretion efficiency, delivery dosage and route of immunisation in further development of this promising system for preparing an oral vaccine for HIV.