Melanie Seidel-Greven1, Otchere Addai-Mensah1,2, Holger Spiegel1, Gwladys Nina Chiegoua Dipah1, Stefan Schmitz1, Gudrun Breuer1, Margaret Frempong3, Andreas Reimann1, Torsten Klockenbring1, Rainer Fischer1,4,5, Stefan Barth1,6,7, Rolf Fendel8,9. 1. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr.6, 52074, Aachen, Germany. 2. Department of Medical Diagnostics, Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. 3. Department of Molecular Medicine, School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. 4. Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. 5. Purdue University, West Lafayette, IN, 47907, USA. 6. Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Pauwelsstraße 20, 52074, Aachen, Germany. 7. South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, and Medical Biotechnology & Immunotherapy Research Unit, Institute of Infectious Disease & Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. 8. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr.6, 52074, Aachen, Germany. rolf.fendel@uni-tuebingen.de. 9. Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany. rolf.fendel@uni-tuebingen.de.
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.
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.
Authors: A Sabchareon; T Burnouf; D Ouattara; P Attanath; H Bouharoun-Tayoun; P Chantavanich; C Foucault; T Chongsuphajaisiddhi; P Druilhe Journal: Am J Trop Med Hyg Date: 1991-09 Impact factor: 2.345
Authors: Amed Ouattara; Shannon Takala-Harrison; Mahamadou A Thera; Drissa Coulibaly; Amadou Niangaly; Renion Saye; Youssouf Tolo; Sheetij Dutta; D Gray Heppner; Lorraine Soisson; Carter L Diggs; Johan Vekemans; Joe Cohen; William C Blackwelder; Tina Dube; Matthew B Laurens; Ogobara K Doumbo; Christopher V Plowe Journal: J Infect Dis Date: 2012-11-29 Impact factor: 5.226
Authors: A Trkola; M Purtscher; T Muster; C Ballaun; A Buchacher; N Sullivan; K Srinivasan; J Sodroski; J P Moore; H Katinger Journal: J Virol Date: 1996-02 Impact factor: 5.103
Authors: Clemens H M Kocken; Chrislaine Withers-Martinez; Martin A Dubbeld; Annemarie van der Wel; Fiona Hackett; Augusto Valderrama; Michael J Blackman; Alan W Thomas Journal: Infect Immun Date: 2002-08 Impact factor: 3.441
Authors: Prakash Srinivasan; Adam Yasgar; Diane K Luci; Wandy L Beatty; Xin Hu; John Andersen; David L Narum; J Kathleen Moch; Hongmao Sun; J David Haynes; David J Maloney; Ajit Jadhav; Anton Simeonov; Louis H Miller Journal: Nat Commun Date: 2013 Impact factor: 14.919
Authors: Xiangguo Qiu; Gary Wong; Jonathan Audet; Alexander Bello; Lisa Fernando; Judie B Alimonti; Hugues Fausther-Bovendo; Haiyan Wei; Jenna Aviles; Ernie Hiatt; Ashley Johnson; Josh Morton; Kelsi Swope; Ognian Bohorov; Natasha Bohorova; Charles Goodman; Do Kim; Michael H Pauly; Jesus Velasco; James Pettitt; Gene G Olinger; Kevin Whaley; Bianli Xu; James E Strong; Larry Zeitlin; Gary P Kobinger Journal: Nature Date: 2014-08-29 Impact factor: 49.962
Authors: Kristian Daniel Ralph Roth; Esther Veronika Wenzel; Maximilian Ruschig; Stephan Steinke; Nora Langreder; Philip Alexander Heine; Kai-Thomas Schneider; Rico Ballmann; Viola Fühner; Philipp Kuhn; Thomas Schirrmann; André Frenzel; Stefan Dübel; Maren Schubert; Gustavo Marçal Schmidt Garcia Moreira; Federico Bertoglio; Giulio Russo; Michael Hust Journal: Front Cell Infect Microbiol Date: 2021-07-07 Impact factor: 5.293