Briefing Document: Nanobody-Mediated Blocking of Malaria Transmission Targeting PfPIMMS43

Source: Excerpts from "s42003-025-08033-8.pdf" (A Nature Portfolio journal; https://doi.org/10.1038/s42003-025-08033-8)
Authors: Chiamaka Valerie Ukegbu, et al.
Date: Received - 04 December 2024 | Accepted - 02 April 2025 | Published - 30 April 2025

Executive Summary:

This study explores a novel strategy to block malaria transmission by targeting the Plasmodium falciparum protein PfPIMMS43 using single-domain VHH antibodies, also known as nanobodies. PfPIMMS43 is a critical surface protein for the parasite's development within the mosquito, specifically during the transition from ookinete to oocyst, and aids in evading the mosquito's immune response. Building on previous research demonstrating the potential of polyclonal antibodies against PfPIMMS43, this study successfully developed and characterized high-affinity nanobodies derived from llamas. These nanobodies were shown to significantly reduce both the intensity and prevalence of P. falciparum infection in Anopheles mosquitoes using both laboratory and field strains of the parasite. The study mapped the binding epitopes of the nanobodies to conserved regions in the second half of PfPIMMS43, confirming epitope accessibility. These findings establish PfPIMMS43 as a promising target for malaria transmission-blocking interventions and propose an innovative strategy utilizing genetically modified mosquitoes expressing these nanobodies in conjunction with gene drive technology for enhanced malaria control and elimination efforts.

Key Themes and Important Ideas:

1. Malaria Transmission as a Target: The study emphasizes the importance of targeting the parasite's development within the mosquito vector to interrupt the human-to-mosquito and mosquito-to-human transmission cycle. This is presented as a crucial approach to complement existing malaria control measures, especially in the face of challenges like insecticide failure, climate change, and funding limitations. The transition from ookinete to oocyst in the mosquito midgut is identified as a "key developmental bottleneck" for the parasite.
2. PfPIMMS43 as a Critical Transmission Target: The research highlights PfPIMMS43 as an "indispensable" surface protein for P. falciparum ookinetes and sporozoites. It is crucial for the ookinete-to-oocyst transition and plays a role in the parasite's ability to "evade the mosquito immune responses," specifically the complement-like system in the hemolymph. Previous studies, including those by the authors, had already indicated the potential of polyclonal antibodies targeting this protein in reducing transmission.
3. Nanobodies as a Promising Intervention Tool: The study focuses on the development and application of VHH domain nanobodies as an alternative and potentially superior approach to conventional antibodies for transmission blocking. Nanobodies, derived from camelids and sharks, are described as "smaller, more easily produced monoclonal, heavy-chain variable (VHH) domain antibodies." Their advantages include:

• "small size (~15 kDa)"
• "structural simplicity"
• "strong binding affinity"
• Easily bioengineered for targeting parasite antigens in mosquito vectors.

1. Development and Characterization of PfPIMMS43 Nanobodies: High-affinity nanobodies targeting PfPIMMS43 were successfully generated by immunizing llamas with recombinant PfPIMMS43. Nine nanobodies were selected based on variations in their antigen-binding regions (CDR1-3). Four nanobodies (G9, E5, C12, and E2) exhibited high nanomolar binding affinities to recombinant PfPIMMS43 (3, 5, 6, and 8 nM, respectively). These four nanobodies were also able to detect endogenous PfPIMMS43 protein expressed by P. falciparum ookinetes in infected mosquito midguts.
2. Significant Transmission Blocking Activity (TRA): The developed nanobodies demonstrated significant transmission-reducing activity in mosquito feeding assays.

• In standard membrane feeding assays (SMFAs) using laboratory P. falciparum NF54 and An. coluzzii mosquitoes, the four high-affinity nanobodies (G9, E5, C12, and E2) significantly reduced oocyst numbers at a concentration of 100 µg/ml, with reductions ranging from 83% to 99%. Oocyst reduction was concentration-dependent.
• In direct membrane feeding assays (DMFAs) using natural P. falciparum isolates from gametocytaemic children in Tanzania and local An. gambiae mosquitoes, G9 and E5 (the two nanobodies with the highest affinities to recombinant PfPIMMS43) also showed significant TRA, with reductions of 99% and 79% at 100 µg/ml, respectively. Both nanobodies significantly reduced mosquito infection prevalence in field conditions.

1. Epitope Mapping and Structural Insights: Epitope mapping revealed that the four nanobodies bind to "conserved regions in the second half of PfPIMMS43," specifically beyond amino acid residue 258. This suggests the C-terminal half of the protein is more immunogenic. G9 and E5 appear to recognize similar conformational epitopes, while C12 and E2 bind to distinct linear epitopes closer to the C-terminus. Homology modeling of the G9-PfPIMMS43 interaction suggests that G9 binds primarily through its CDR2 and CDR3 domains, interacting with residues in a β-sheet and an adjacent α-helix in the PfPIMMS43 structure. The model also supports the hypothesis that PfPIMMS43 is largely intrinsically disordered, potentially facilitating immune evasion.
2. Innovative Future Strategy: The study proposes a "novel approach" for malaria control: "genetically modifying mosquitoes to express nanobodies targeting key post-fertilization proteins such as PfPIMMS43, with the possibility of spreading these traits in wild populations via gene drive technologies." This strategy aims to overcome limitations of vaccine-induced antibodies, such as the challenge of achieving high and sustained titers against mosquito-stage antigens that are not naturally boosted in humans.
3. Conservation and Potential Challenges of PfPIMMS43: While PfPIMMS43 is largely conserved across P. falciparum isolates from Africa, the study notes the presence of single nucleotide polymorphisms (SNPs) in the epitope recognized by G9 and E5. These SNPs (M307I, L311I, L355H, L355P) have "high fixation indices and signatures of positive selection," suggesting potential evolutionary adaptations of the parasite to evade immune detection. Further investigation into the impact of these alleles on nanobody recognition is deemed necessary for field applications.

Supporting Quotes:

• "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."
• "Here, building on these findings, we have developed high-affinity single-domain VHH antibodies (nanobodies) derived from llama heavy-chain-only antibodies."
• "Importantly, they significantly reduce infection intensity and prevalence of laboratory and field strains of P. falciparum in An. coluzzii and An. gambiae, respectively."
• "These findings establish PfPIMMS43 as a promising transmission-blocking target."
• "To enhance mala...