Nanobody-mediated targeting of Plasmodium falciparum PfPIMMS43 can block malaria transmission in mosquitoes

Abstract

The transition from ookinete to oocyst is a critical step in the Plasmodium falciparum lifecycle and an important target for malaria transmission-blocking strategies. PfPIMMS43, a surface protein of P. falciparum ookinetes and sporozoites, is critical for this transition and aids the parasite in evading mosquito immune responses. Previous studies demonstrated that polyclonal PfPIMMS43 antibodies reduced P. falciparum infection in Anopheles mosquitoes. Here, building on these findings, we have developed high-affinity single-domain VHH antibodies (nanobodies) derived from llama heavy-chain-only antibodies. We have shown that these nanobodies bind both recombinant and endogenous PfPIMMS43 produced by P. falciparum ookinetes in the mosquito midgut. Importantly, they significantly reduce infection intensity and prevalence of laboratory and field strains of P. falciparum in An. coluzzii and An. gambiae, respectively. Epitope mapping has revealed that the nanobodies target conserved regions in the second half of PfPIMMS43, with homology modelling confirming epitope accessibility. These findings establish PfPIMMS43 as a promising transmission-blocking target. To enhance malaria control and elimination efforts, we propose an innovative strategy in which genetically modified mosquitoes express PfPIMMS43-specific nanobodies in their midguts and spread this trait in wild mosquito populations via gene drive technology.

Fig. 1. PfPIMMS43 nanobodies and recognition of recombinant and endogenous protein.

A Multiple sequence alignment of the nine PfPIMMS43-specific nanobodies, highlighting the four framework regions (FR) and three complementarity-determining regions (CDR). Sequence conservation is represented by shades of blue, with complete conservation shown in dark blue and non-conserved regions unshaded. B SDS-PAGE gel analysis of recombinant MYC-6xHis-tagged PfPIMMS43 nanobodies expressed in E. coli and purified by affinity chromatography. C Western blot detection of recombinant thioredoxin-His-tagged PfPIMMS43 using the nine nanobodies. Binding was visualized with an HRP-conjugated anti-VHH antibody. Probing with an anti-His antibody served as a control (HIS). A Coomassie-stained SDS-PAGE gel of recombinant PfPIMMS43 prior to blotting was used as a loading control (COO). D ELISA showing nanobody binding affinities to recombinant PfPIMMS43. E Detection of endogenous PfPIMMS43 in P. falciparum NF54 ookinetes using the four highest affinity nanobodies (G9, E5, C12, and E2). Reduced cell lysates from P. falciparum-infected midguts, collected 18 h post-blood meal, were probed with nanobodies, and binding was detected using an anti-MYC antibody. An anti-Pfs25 antibody served as a positive control. The fluorescence microscopy image shows ookinetes (red, stained with anti-Pfs25) invading midgut epithelial cells (nuclei stained blue with DAPI), corresponding to midgut samples used for western blot analysis. IN, infected midguts; BF, blood-fed midguts. Arrows in the immunofluorescence image indicate Pfs25-stained ookinetes and arrowheads indicate DAPI-stained epithelial cell nuclei. Smaller nuclei correspond to other cells in the midgut epithelium. Scale bar, 5 µM.

Fig. 2. PfPIMMS43 nanobody performance in transmission assays.

A Oocyst counts per midgut at 8–10 days post-blood meal from SMFAs using An. coluzzii N’gousso mosquitoes infected with P. falciparum NF54 gametocytes. Gametocytes were treated with G9, E5, C12, and E2 nanobodies, with PBS-treated gametocytes serving as the control. Data from three biological replicates per nanobody treatment were pooled. Red horizontal lines indicate the median oocyst count. Statistical significance was assessed using the Mann–Whitney test: ns, not significant; *P < 0.05; **P < 0.001; ***P < 0.0001. B Percent transmission-reducing activity (TRA) of the PfPIMMS43 nanobodies G9, E5, C12, and E2 relative to the PBS control, calculated from the results in A. Circles represent individual data. Error bars represent the SEM. Statistical significance was determined using an unpaired t-test with Welch’s correction: ns, not significant; *P < 0.05; **P < 0.001; ***P < 0.0001. C Oocyst counts per midgut at 8–10 days post-blood meal from DMFAs using An. gambiae Ifakara mosquitoes infected with P. falciparum gametocytes collected from children in Tanzania. Gametocytes were treated with G9 and E5 nanobodies, with PBS-treated gametocytes as the control. Data from three biological replicates were pooled. Statistical analyses of oocyst load and TRA were performed as in A, B.

Fig. 3. PfPIMMS43 epitope mapping of VHHs and 3D homology modeling.

A Schematic representation of the full-length (FL) recombinant PfPIMMS43 protein and its subdomains D1-D5, created through progressive C-terminal deletions. Amino acid sequences corresponding to the regions bound by each tested nanobody, as determined by LC-MS/MS following VHH-PfPIMMS43 binding and trypsin digestion, are shown. All constructs were expressed in E. coli as Thioredoxin-His fusion proteins. TRX-Thioredoxin, H-6xHis, T-Thrombin cleavage site, S-S-tag and E- enterokinase cleavage site. B Western blot detection of recombinant Thioredoxin-His-tagged PfPIMMS43 FL and its truncated variants D1-D5 using G9, E5, C12, and E2 nanobodies. Each nanobody was tested individually, and binding was detected using an HRP-conjugated anti-VHH antibody. A Coomassie-stained SDS-PAGE gel of the recombinant PfPIMMS43 proteins, served as a loading control. C Homology model of the G9-PfPIMMS43 complex. PfPIMMS43 is colored based on pLDDT confidence scores: pLDDT > 90 in blue, 90 > pLDDT > 70 in cyan, and pLDDT<70 in fading shades of silver. G9 is represented with framework regions (FR) in black, CDR1 in pale cyan, CDR2 in green, and CDR3 in red. Panels: (i) surface representation of the G9-PfPIMMS43 complex; (ii) cartoon representation of the region indicated in panel (i); (iii) zoom-in of the region indicated in the panel (ii) with side chains of interacting residues from G9 CDR2 (green), CDR3 (red) and PfPIMMS43 (blue) shown; (iv) Back side of the region shown in panel (iii) with side chains of interacting residues shown.