. 18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription–polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2–3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1–8.1 days) than blood smears (11.0 days; 95% CI: 10.3–11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6–13.3 days). Discrepant analysis showed that the risk of a blood smear–positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.

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.

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.