Plasmodium falciparum Liver Stage Infection and Transition to Stable Blood Stage Infection in Liver-Humanized and Blood-Humanized FRGN KO Mice Enables Testing of Blood Stage Inhibitory Antibodies (Reticulocyte-Binding Protein Homolog 5) In Vivo

Abstract

The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages of Plasmodium falciparum. In contrast, the current models to directly study blood stage infection in vivo are extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection of P. falciparum-infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressing P. falciparum parasite, resulting in noninvasively measurable liver stage burden by in vivo bioluminescent imaging (IVIS) at days 5–7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activity in vitro) was capable of blocking blood stage infection in vivo when parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressing P. falciparum will be a useful tool to study P. falciparum preerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection.

A Humanized Mouse Model for Plasmodium vivax to Test Interventions that Block Liver Stage to Blood Stage Transition and Blood Stage Infection

“…Here, we developed this model further in order to allow exo-erythrocytic merozoite infection of reticulocytes and asexual blood stage development. We modified our protocol previously developed for P. falciparum ( Foquet et al., 2018 ) using immunomodulation with Clodronate liposomes and Cyclophosphamide to deplete mouse macrophages and mouse neutrophils, respectively, which prevents clearance of infected human red blood cells. We combined this immunomodulation protocol in FRGN KO huHep mice with intravenous (i.v.)…”

Malaria Vaccines: Recent Advances and New Horizons

“…In this case, anti-PfRH5 serum IgG antibody concentration and functional activity, measured using purified IgG in the standardized cell-independent in vitro assay of growth inhibition activity (GIA), were both associated with protective outcome ( Douglas et al., 2015 ). Two further NHP studies have identified the assay of GIA as an in vitro correlate of vaccine-induced in vivo protection ( Mahdi Abdel Hamid et al., 2011 , Singh et al., 2006 ), with passive transfer of a GIA-positive anti-PfRH5 human mAb also showing in vivo protection in a humanized mouse model ( Foquet et al., 2018 ). These data suggest a threshold level of GIA is required to protect and provide a benchmark to aim for in future human clinical studies.…”

A replication-competent late liver stage–attenuated human malaria parasite

“…Transition to blood stage infection is also the most sensitive measure for detecting possible parasite breakthrough because very few infectious exoerythrocytic merozoites are required to successfully initiate the blood stage infection of the life cycle. As previously reported, in FRG huHep mice, P. falciparum NF54 liver stages can transition to a blood stage infection when human RBCs are injected toward the completion of liver stage development (47)(48)(49). These infected RBCs can be transferred to in vitro culture, where parasites undergo asexual schizogony and can be detected by Giemsa-stained thin blood smears within 1 to 5 days of culture (48).…”

“…To enable liver stage-to-blood stage transition, we intravenously (i.v.) injected human RBCs on days 6 and 7 as previously described (47). Blood samples (50 μL) were then removed for 18S qRT-PCR every day from days 7 through 10.…”