1 Scientific RepoRts | 6:30357 | DOI: 10.1038/srep30357 www.nature.com/scientificreports production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system Kathryn A. Hjerrild 1 , Jing Jin 1 , Katherine e. Wright 2 , Rebecca e. Brown 1 , Jennifer M. Marshall 1 , Geneviève M. Labbé 1 , sarah e. silk 1 , Catherine J. Cherry 1 , stine B. Clemmensen 3 , thomas Jørgensen 3 , Joseph J. Illingworth 1 , Daniel G. W. Alanine 1 , Kathryn H. Milne 1 , Rebecca Ashfeld 1 , Willem A. de Jongh 3 , Alexander D. Douglas 1 , Matthew K. Higgins 2 & simon J. Draper 1 the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS 2 , based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that difered in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identifed quantitative and qualitative diferences in terms of the functional IgG antibody response against the P. falciparum parasite. the antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifes Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difcult-to-make’ proteins from Plasmodium parasites, and identifes a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. Plasmodium falciparum parasites are the causative agent of the most severe form of human malaria, and the development of an efective vaccine remains a key strategic goal to aid the control, local elimination and eventual eradication of this disease. Next-generation vaccine strategies are now seeking to improve on the moderate levels of efcacy reported for the RTS,S/AS01 malaria vaccine which targets the pre-erythrocytic stages of the para- site’s lifecycle 1 . One leading strategy is to move towards a multi-antigen, multi-stage vaccine formulation, which will necessitate the development of efective vaccine components against the pathogenic asexual blood-stage of infection 2 and the subsequent sexual-/mosquito-stages 3 . Blood-stage vaccines seek to induce antibodies against the merozoite form of the parasite that invades erythrocytes 2 , and could complement pre-erythrocytic immunity aforded by RTS,S/AS01, protect against disease severity and/or reduce transmission by accelerating the control and clearance of blood-stage parasitemia. 1 The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK. 2 Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. 3 expreS 2 ion Biotechnologies, SCION-DTU Science Park, Agern Allé 1, Hørsholm DK-2970, Denmark. Correspondence and requests for materials should be addressed to M.K.H. (email: matthew.higgins@bioch.ox.ac.uk) or S.J.D. (email: simon.draper@ndm. ox.ac.uk) received: 18 March 2016 Accepted: 04 July 2016 Published: 26 July 2016 OPEN