Browsing by Subject "Phage display"

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    Open Access
    High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria
    (Public Library of Science, 2013) Ngubane, Nqobile A C; Gresh, Lionel; Ioerger, Thomas R; Sacchettini, James C; Zhang, Yanjia J; Rubin, Eric J; Pym, Alexander; Khati, Makobetsa
    Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis ( M. tb ), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections.
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    Open Access
    Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity
    (2021-01-11) Seidel-Greven, Melanie; Addai-Mensah, Otchere; Spiegel, Holger; Chiegoua Dipah, Gwladys N; Schmitz, Stefan; Breuer, Gudrun; Frempong, Margaret; Reimann, Andreas; Klockenbring, Torsten; Fischer, Rainer; Barth, Stefan; Fendel, Rolf
    Abstract Background Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. Material and methods Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1–3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. Results The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. Conclusion We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.