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.