A Plasmodium vivax experimental human infection model for evaluating efficacy of interventions

Abstract

BACKGROUNDInterventions that interrupt Plasmodium vivax transmission or eliminate dormant P. vivax liver-stage parasites will be essential for malaria elimination. Development of these interventions has been hindered by the lack of P. vivax in vitro culture and could be accelerated by a safe and reproducible clinical model in malaria-naive individuals.METHODSHealthy, malaria-naive adults were enrolled in 2 studies to assess the safety, infectivity, and transmissibility of a new P. vivax isolate. Participants (Study 1, n = 2; Study 2, n = 24) were inoculated with P. vivax-infected red blood cells to initiate infection, and were treated with artemether-lumefantrine (Study 1) or chloroquine (Study 2). Primary endpoints were safety and infectivity of the new isolate. In Study 2, transmission to mosquitoes was also evaluated using mosquito feeding assays, and sporozoite viability was assessed using in vitro cultured hepatocytes.RESULTSParasitemia and gametocytemia developed in all participants and was cleared by antimalarial treatment. Adverse events were mostly mild or moderate and none were serious. Sixty-nine percent of participants (11/16) were infectious to Anopheles mosquitoes at peak gametocytemia. Mosquito infection rates reached 97% following membrane feeding with gametocyte-enriched blood, and sporozoites developed into liver-stage schizonts in culture.CONCLUSIONWe have demonstrated the safe, reproducible, and efficient transmission of P. vivax gametocytes from humans to mosquitoes, and have established an experimental model that will accelerate the development of interventions targeting multiple stages of the P. vivax life cycle.TRIAL REGISTRATIONACTRN12614000930684 and ACTRN12616000174482.FUNDING(Australian) National Health and Medical Research Council Program Grant 1132975 (Study 1). Bill and Melinda Gates Foundation (OPP1111147) (Study 2).

Keywords: Infectious disease; Malaria; Vaccines.

Conflict of interest statement

Conflict of interest: SC and JJM are employed by Medicines for Malaria Venture.

Figures

Figure 1. Study design schematic.

Malaria-naive volunteers were inoculated with P. vivax–infected RBCs (pRBCs) on day 0 (D0). Asexual parasitemia and gametocytemia were evaluated from day 4 and continued until the end of study. Participants in Study 1 started artemether-lumefantrine treatment on day 8 (n = 2). Participants in Study 2 started chloroquine treatment on day 8 (n = 8), day 9 (n = 1), or 10 (n = 15). For Study 2, mosquito feeding assays were performed between day 6 and day 10 by direct feeding (allowing mosquitoes to feed on participants by live bite), or by membrane feeding on venous blood. D, day relative to inoculation (day 0); pRBC: P. vivax parasite–infected RBCs.

Figure 2. Study profile.

All participants were inoculated with P. vivax on day 0. D, day relative to inoculation; pi, postinoculation.

Figure 3. Parasitemia and gametocytemia.

Participants (n = 26) were experimentally infected with P. vivax on day 0. Parasitemia was measured by 18S qPCR and gametocytemia measured by pvs25 qRT-PCR for Study 1 (n = 2) (A), and Study 2 (n = 24) (D–F). Grey lines, parasitemia; red lines, gametocytemia. Thin lines show individual participant data and thick lines show the geometric mean. Initiation of treatment is indicated by the vertical lines. Treatment was initiated on day 8 for Study 1 (n = 2) and Study 2 cohort 1 (n = 8), or day 10 for Study 2 cohorts 2 and 3 (n = 15). Participant 205 (cohort 2; black lines) was treated on day 9 (vertical solid line). (B) Gametocytemia at time of treatment for Study 2 (n = 23) (compared by Mann-Whitney test). (C) Spearman’s correlation of peak asexual parasitemia and peak gametocytemia (n = 24). Participant 205 represented in gray.

Figure 4. Infectivity to mosquitoes.

Successful transmission was defined as at least 1 oocyst-positive mosquito as determined by 18S qPCR. Mosquito infection rate is reported as prevalence of infection (percentage of mosquitoes infected per feeding assay). (A) Prevalence of mosquito infection in all feeding assays in Study 2 at each time point (n = 113). (B) Prevalence of mosquito infection in successful feeding assays, by feeding assay type (n = 37). Groups compared by Kruskal-Wallis test with Dunn’s multiple comparison test. (C) The gametocytemia for participant samples that were infectious compared with samples that were noninfectious (n = 54). Groups compared by Mann-Whitney test. Box plots indicate the median and whiskers show the minimum and maximum. (D) Representative image from of a P. vivax liver-stage schizont stained with UIS4 and Hoechst33342 following incubation of sporozoites with HC-04 culture for 7 days (left, white channel, Hoechst33342; middle, red channel, Alexa Fluor 488–conjugated UIS4 antibody; right, merge). Image taken at ×40 magnification. Scale bar: 20 μm. Sporozoites were obtained by feeding mosquitoes on enriched gametocytes collected on day 10 from participants in cohort 3 (Supplemental Material).