A luciferase-based hRBC-NSG mouse model to evaluate anti-CelTOS transmission-blocking antibodies against P. falciparum

BACKGROUND: Reliable experimental platforms are essential for evaluating transmission-blocking interventions against Plasmodium falciparum (P. falciparum). Although the standard membrane feeding assay (SMFA) is widely used to assess transmission-blocking activity, it is performed in vitro and does not fully represent physiological conditions. In this study, we developed and applied a luciferase-based assay using NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Sug/JicTac) mice engrafted with human red blood cells (hRBCs) to establish an hRBC-engrafted NSG (hRBC-NSG) humanized mouse model for evaluating the transmission-blocking activity of anti-CelTOS monoclonal antibodies (mAbs) against P. falciparum. METHODS: A luciferase expression cassette was integrated into the genome of the P. falciparum KF7 parasite line using the piggyBac transposon system. A stable luciferase-expressing parasite clone, PfKF7G4, was selected based on high luciferase activity and efficient gametocyte production. Following serial adaptation in hRBC-NSG mice, PfKF7G4 parasites produced mature gametocytes capable of infecting An. stephensi mosquitoes through a direct feeding assay (DFA). As part of the direct feeding assay, anti-CelTOS mAbs were administered intravenously prior to mosquito feeding. Oocyst development in mosquitoes was quantified using a luciferase-based mosquito assay on day 8 post feeding. Results were compared with previously reported parallel in vitro transmission-blocking data generated using SMFA (Tang et al. in Nat Commun 15, 2024). RESULTS: hRBC-NSG mice infected with PfKF7G4 parasites reached parasitemia levels of up to 10.58% and successfully produced mature gametocytes, with stage V gametocytemia ranged from 0.07% to 0.18%. These gametocytes were successfully transmitted to mosquitoes and developed into luciferase-expressing oocysts. Administration of anti-CelTOS mAbs significantly reduced oocyst development in mosquitoes compared with control groups, as measured by luciferase activity. Similar inhibitory effects were observed in previously reported in vitro SMFA experiments. CONCLUSIONS: This study establishes a luciferase-based in vivo transmission assay using an hRBC-NSG humanized mouse model for quantitative evaluation of P. falciparum transmission to mosquitoes. The platform enables functional assessment of antibody-mediated transmission-blocking activity under physiologically relevant conditions. Using this system, anti-CelTOS mAbs demonstrated inhibitory effects on parasite transmission. This assay provides a useful tool for evaluating transmission-blocking interventions and may support the development of vaccines and therapeutics aimed at reducing malaria transmission.