Open AccessDetailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate Pf AMA 1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits
Author(s) -
Boes Alexander,
Spiegel Holger,
Edgue Gueven,
Kapelski Stephanie,
Scheuermayer Matthias,
Fendel Rolf,
Remarque Edmond,
Altmann Friedrich,
Maresch Daniel,
Reimann Andreas,
Pradel Gabriele,
Schillberg Stefan,
Fischer Rainer
Publication year - 2015
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12255
Subject(s) - nicotiana benthamiana , biology , glycosylation , epitope , plasmodium falciparum , glycan , malaria vaccine , immunogenicity , antigen , immune system , virology , microbiology and biotechnology , biochemistry , immunology , glycoprotein , gene , malaria
Summary One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 ( Pf AMA1). Several studies have shown that this blood‐stage antigen can induce strong parasite growth inhibitory antibody responses. Pf AMA1 contains up to six recognition sites for N‐linked glycosylation, a post‐translational modification that is absent in P. falciparum . To prevent any potential negative impact of N‐glycosylation, the recognition sites have been knocked out in most Pf AMA1 variants expressed in eukaryotic hosts. However, N‐linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated Pf AMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both Pf AMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high‐mannose‐type N‐glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation‐sensitive ligand‐binding studies. Specific titres of >2 × 10 6 were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC 50 values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N‐glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.