Humanized mice for investigating sustained Plasmodium vivax blood-stage infections and transmission

Plasmodium vivax is the most widespread human malaria parasite. Due to the presence of extravascular reservoirs and relapsing infections from dormant liver stages, P. vivax is particularly difficult to control and eliminate. Experimental research is hampered by the inability to maintain P. vivax cultures in vitro, due to its tropism for immature red blood cells (RBCs). Here, we describe a new humanized mice model that can support efficient human erythropoiesis and maintain long-lasting multiplication of inoculated cryopreserved P. vivax parasites and their sexual differentiation, including in bone marrow. Mature gametocytes were transmitted to Anopheles mosquitoes, which led to the formation of salivary gland sporozoites. Importantly, blood-stage P. vivax parasites were maintained after the secondary transfer of fresh or frozen infected bone marrow cells to naïve chimeras. This model provides a unique tool for investigating, in vivo, the biology of intraerythrocytic P. vivax.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For in vivo infection studies (kinetics, de novo RBC infection), no statistical methods were used to predetermined the experimental sample size but rather it was determined on mice availability. We only kept mice for which the % of human CD45+ cells in the peripheral blood was ! 20 to ensure that mice were correctly engrafted during transplantation procedure. Each experiment was performed on a group of mice agematched reconstituted at 1-4 day-old with 10 000-30 000 CD34+ cells from the same CB donor. Accordingly, sample size was based on the number of mice reconstituted with the same CB cells and thus depended on the number of CD34+ cells. For BM transfer experiments (one donor into one recipient), the aim was to show that parasite could be transferred in variable settings. For mosquito feeding, available infected mice were used. For microscopic images (oocysts, GIEMSA staining), the aim was to qualitatively show parasites both in vertebrate and invertebrate hosts without any quantification purpose. For in vitro qRT-PCR experiments, samples were analyzed as triplicates.
No data has been excluded. For each reconstitution experiment, all pups from the same litters were engrafted with human CD34+ cells from the same CB. Once adults (12-16 week-old), both male and female mice from the same litters were used and when required, distributed randomly in different experimental groups (for ex. different times post-infection), both male and females were used randomly provided they were reconstituted with the same CD34+ cells, provided that they presented a PB % human CD45+ cells ! 20.
In vivo studies were performed in a blinded manner as infected versus non-infected mice were treated (housing, feeding, injection of clodronate liposomes) and analyzed similarly. Data were collected objectively, and analyzed using software with objective standards.

This study did not involve wild animals
This study did not involve samples collected from the field Procedures involving mice were previously approved by local Animal Ethics Committees (CETEA # 089 at Institut Pasteur) and registered with the French authorities # #15341-2018053115015969.
For non commercial CD34+ cells, cord bloods from de-identified from healthy donors (AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, CRB-Banque de Sang de Cordon, Paris, France -authorization number: AC-2016-2759) were used in some cases to process CD34+ cells. Seven clinical P. vivax samples collected in northwestern Brazil in the context of an ongoing cohort study (ClinicalTrials.gov, NCT03689036) were leukocyte-depleted and cryopreserved in liquid nitrogen. All donors were anonymized.
All participants were volunteers with no direct benefits from participation in this study.
For the CD34+ cord blood donors obtained with informed consent, the authorization number was AC-2016-2759. Study protocols have been approved by the Institutional Review Board of the Institute of Biomedical Sciences, University of São Paulo, and by the National Human Research Ethics Committee of the Ministry of Health of Brazil (CAAE: 64767416.6.0000.5467); all patients provided written informed consent.