Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits

Abstract

Summary One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood‐stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 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 PfAMA1 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 PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 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 × 106 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 IC50 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.

Characterization of a novel inhibitory human monoclonal antibody directed against Plasmodium falciparum Apical Membrane Antigen 1

“…Only few human antibodies have been disclosed which possess K D values of as low as ≤10 −10  M7172. Moreover, as compared to 1F9 and 4G2 exhibiting K D values of 1.3 × 10 −9  M and 3.3 × 10 −9  M, respectively73, the affinity of humAbAMA1 to AMA1 (3D7) is 7- to 18-times stronger.…”

“…Very efficient murine mAbs directed at other plasmodial targets such as reticulocyte-binding protein homologue 5 (Rh5) show IC 50 values of 50–100 μg/ml44, and AMA1-specific polyclonal rabbit IgG generated by hyperimmunization protocols could achieve IC 50 values in a similar range of 34–180 μg/ml7375. A direct comparison between polyclonal and monoclonal antibodies is difficult as in vivo, an entire spectrum of antibodies contributes to the defense against plasmodial infections and a successful control is likely due to synergistic effects of these antibodies.…”

“…All measurements were performed by using a Biacore T200 instrument (Biacore, GE Healthcare) and CM5-S-Series sensor chips at 25 °C with HBS-EP (10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid, 150 mM NaCl, 3 mM EDTA, 0.005% (w/v) polysorbat-20) as running buffer. (A) The kinetic analysis of the humAbAMA1 interaction was performed as previously described73. (B) To roughly map the epitope of humAbAMA1 in relation to the binding sites of previously described AMA1 specific monoclonal antibodies 1D7, 1F9 and 4G2, AMA1 (3D7) was captured (1600 RU) using a CM5 surface functionalized by EDC-NHS chemistry with 7000 RU of the rat monoclonal 1D7.…”

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies

“…Crosnier et al recently evaluated the relative contributions of codon-optimization, an exogenous signal peptide, and mutation of N-glycosylation sites on expression of functional asexual blood-stage protein PfRH5 in the HEK293E cell system, reporting the relative increases in protein expression to be equivocal, ~3.5-fold, and ~5.5-fold, respectively, compared with the native sequence [22]. By contrast, several other reports exist, albeit in other platforms, wherein retaining native glycans has not made a difference to yield or function [27,[45][46][47][48]. A recent study from our group using a viral-vectored heterologous prime-boost vaccination regime to deliver Pfs48/45, a protein known to have a complex 6-cysteine domain fold, reports a disruption of transmission-blocking epitopes following mutation of N-glycan sequons relative to the native construct [13].…”

Immunization with the Malaria Diversity-Covering Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 DiCo in Complex with Its Natural Ligand PfRon2 Does Not Improve the In Vitro Efficacy

“…The three DiCo variants and six Pf AMA1 alleles ( Pf AMA1-3D7, Pf AMA1-FCR3, Pf AMA1-HB3, Pf AMA1-Dd2, Pf AMA1-7G8, and Pf AMA1-RO33) were purified by immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) on a Superdex 75 column (GE Healthcare Life Sciences, Little Chalfont, UK) as previously described (39). …”