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. 2024 Aug 22;15(1):7206.
doi: 10.1038/s41467-024-50770-7.

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites

Dimuthu Angage  1 , Jill Chmielewski  2 , Janesha C Maddumage  1 , Eva Hesping  3   4 , Sabrina Caiazzo  3   4 , Keng Heng Lai  2 , Lee Ming Yeoh  5   6 , Joseph Menassa  1 , D Herbert Opi  5   6   7 , Callum Cairns  1 , Hamsa Puthalakath  1 , James G Beeson  5   7   8 , Marc Kvansakul  1 , Justin A Boddey  3   4 , Danny W Wilson  2   5   9 , Robin F Anders  1 , Michael Foley  10   11
Affiliations

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites

Dimuthu Angage et al. Nat Commun. .

Abstract

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

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Conflict of interest statement

This study was a collaboration between AdAlta Limited and La Trobe University. M.F. is the founding chief scientist and a shareholder in AdAlta Ltd., and R.F.A. is also a shareholder in AdAlta. The other authors have declared no competing interests.

Figures

Fig. 1
Fig. 1. Selection of anti-PfAMA1 i-bodies.
a Outline of the strategy used for haplotype-distance biopanning the phage-displayed i-body library on 3D7 and W2mef recAMA1. The figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b ELISA of phage pools from the biopanning campaign against immobilised 3D7 recAMA1. c ELISA of phage pools from the biopanning campaign against immobilised W2mef recAMA1. In both campaigns, three independent experiments were performed as triplicates. Data are presented as mean values ± SD. d ELISA screening of individual i-bodies for Pf3D7AMA1 binding. Forty-eight clones were randomly selected from each biopanning campaign. i-bodies were expressed in E. coli and assessed for AMA1 binding in an ELISA. The binding of the i-bodies to the different AMA1 isoforms is represented as a heat map. Two independent experiments were performed as triplicates. e Sequence alignment of AMA1-specific i-bodies with the CDR1 and CDR3 sequences highlighted in blue. f Binding profile of i-bodies against Pf3D7, W2mef, 7G8, FVO, HB3, D10 recAMA1 isoforms and reduced and alkylated 3D7 AMA1 represented as a heat map where the darker the shade, the greater the binding. Three independent experiments were performed for each i-body. g Analysis of WD33 and WD34 binding to Pv and PcAMA1. Three independent experiments were performed. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. WD33 and WD34 recognise AMA1 expressed by P. falciparum.
a Immunoblotting of saponin-lysed Pf3D7, W2mef, FVO and 7G8 schizonts. Parasite material was fractionated by SDS-PAGE under reducing or non-reducing conditions. After transfer, membranes were probed with either WD34 or WD33 and then HRP-conjugated anti-myc mAb 9E10. mAb 5G8, which binds a linear epitope near the N-terminus of AMA1, was used as a control (lower panel). At least two independent experiments were performed. Source data are provided as a Source Data file. b Indirect immunofluorescence of fixed 3D7 and W2mef schizonts with pro-domain specific mAb 5G8, and i-bodies WD33 and WD34. At least two independent experiments were performed.
Fig. 3
Fig. 3. Characterisation of the WD33 and WD34 interactions with AMA1.
a ELISA showing the binding of biotinylated RON2 peptide (150 nM) to immobilised AMA1 in the presence of i-body (150 nM). AMA1-bound RON2 peptide was measured using streptavidin HRP. Data are presented as mean values ± SD. b Inhibition of RON2 peptide (150 nM) binding to AMA1 by increasing concentrations of WD34. Data are presented as mean values ± SD. Source data are provided as a Source Data file. c Equilibrium dissociation constants (KD) of WD34 and WD33 interacting with AMA1 determined by surface plasmon resonance. All the generated sensorgrams were analysed by T200 evaluation software. At least three independent experiments were performed to get the final reported values. KD values are reported at the nanomolar level, and the standard deviations are reported in parenthesis. See the Supplementary SPR information. d Dose-dependent inhibition of WD34 binding to AMA1 by mAbs 1F9 and 4G2, which have been previously reported to bind around the hydrophobic cleft (e). Three independent experiments were performed. Data are presented as mean values ± SD.
Fig. 4
Fig. 4. Structural analyses of WD34-AMA1 complexes.
a Crystal structure of the WD34-AMA1Pf complex. Pf AMA1 is in cyan, and WD34 is in purple. b Crystal structure of the WD34-AMA1Pv complex. PvAMA1 is shown in green, and WD34 is shown in purple. c Superimposition of the WD34-AMA1Pf and WD34-AMA1Pv complexes. d WD34 footprint (purple) on PfAMA1 compared to the hydrophobic cleft (green). The shared footprint is shown in grey. e WD34 footprint (purple) on PvAMA1 compared to the hydrophobic cleft (green). f WD34 footprint (purple) compared to the PfRON2 peptide footprint (yellow). The shared region is depicted in orange. g ELISA showing that the WD34CS mutant, in which C22WD34 and C31WD34 were mutated to serine failed to bind to PfAMA1. Three independent experiments were performed, and data are presented as mean values ± SD. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. WD34 inhibits both merozoite and sporozoite life-cycle stages.
a Inhibition of merozoite growth by WD34 was tested using multiple Plasmodium parasites. Assays were performed using two-fold dilutions of WD34 starting with 0.5 mg/ml. Data represents the mean of three independent experiments and error bars represent standard deviations. b Growth inhibition of multiple Plasmodium parasites by WD33. Assays were performed with two-fold dilutions of WD33 starting with 1 mg/ml. Three independent experiments were performed, and data points are represented as mean values ± SD. c IC50 values for each parasite line were calculated by using the non-linear regression model. d, e Effect of i-bodies on sporozoite traversal (d) invasion (e) of HC-04 hepatocytes by P. falciparum sporozoites. WD34 or 21H5 treated FITC-Dextran+HC-04 cells or HC-04 cells were incubated with sporozoites. Cell traversal and invasion were measured at 3 and 24 h, respectively. The mean of n = 3 biological triplicates and standard deviations (a, b) or standard errors of the mean (d, e) are shown. Statistical analysis: 2-way ANOVA with multiple comparisons. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. WD34 suppression of P. berghei parasitaemias in BALB/c mice.
a Experimental design and WD34 treatment schedule (n = 3 for untreated controls, n = 3 for chloroquine-treated controls, and n = 4 for WD34 treatment). Treatments were given once daily (Q.D). One experiment was performed. b Level of suppression of P. berghei infections by WD34. Data are represented as means ± SEM. Statistical analysis: 1-way ANOVA with multiple comparisons. c In vivo efficacy of WD34 against P. berghei infections using Peters' 4-day suppressive test. d Development of parasitaemias in untreated control mice, chloroquine-treated control mice, and WD34-treated mice. Recrudescences were seen with chloroquine due to giving a single dose on day 0. Data are presented as mean values ± SD. Source data are provided as a Source Data file. e Survival of WD34-treated mice was similar to untreated control mice. f Immunoblot with WD34 of uninfected erythrocytes (Ui) and erythrocytes infected with P. berghei expressing mCherry-luciferase (Pb) under reducing and non-reducing conditions. At least two independent experiments were performed.
Fig. 7
Fig. 7. Conservation of residues in Plasmodium AMA1 that interact with WD34.
a Alignment of AMA1 sequences of multiple Plasmodium species showing conservation of WD34 contact residues (<4 Å) identified in the Pf and Pv AMA1-WD34 complexes. Polar interactions are shown in grey boxes and van der Waals bonds are shown in open boxes. b–d Percentage conservation of WD34 contact residues in AMA1 of multiple isolates: b P. falciparum (1054 sequences); c P. vivax (586 sequences); and d Plasmodium species (1745 sequences). Polar interactions are shown in red and van der Waals interactions are shown in blue. A detailed analysis is provided in Supplementary Fig. 12 and Supplementary Data 1.
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