A controlled human malaria infection model enabling evaluation of transmission-blocking interventions

Abstract

Background: Drugs and vaccines that can interrupt the transmission of Plasmodium falciparum will be important for malaria control and elimination. However, models for early clinical evaluation of candidate transmission-blocking interventions are currently unavailable. Here, we describe a new model for evaluating malaria transmission from humans to Anopheles mosquitoes using controlled human malaria infection (CHMI).

Methods: Seventeen healthy malaria-naive volunteers underwent CHMI by intravenous inoculation of P. falciparum-infected erythrocytes to initiate blood-stage infection. Seven to eight days after inoculation, participants received piperaquine (480 mg) to attenuate asexual parasite replication while allowing gametocytes to develop and mature. Primary end points were development of gametocytemia, the transmissibility of gametocytes from humans to mosquitoes, and the safety and tolerability of the CHMI transmission model. To investigate in vivo gametocytocidal drug activity in this model, participants were either given an experimental antimalarial, artefenomel (500 mg), or a known gametocytocidal drug, primaquine (15 mg), or remained untreated during the period of gametocyte carriage.

Results: Male and female gametocytes were detected in all participants, and transmission to mosquitoes was achieved from 8 of 11 (73%) participants evaluated. Compared with results in untreated controls (n = 7), primaquine (15 mg, n = 5) significantly reduced gametocyte burden (P = 0.01), while artefenomel (500 mg, n = 4) had no effect. Adverse events (AEs) were mostly mild or moderate. Three AEs were assessed as severe - fatigue, elevated alanine aminotransferase, and elevated aspartate aminotransferase - and were attributed to malaria infection. Transaminase elevations were transient, asymptomatic, and resolved without intervention.

Conclusion: We report the safe and reproducible induction of P. falciparum gametocytes in healthy malaria-naive volunteers at densities infectious to mosquitoes, thereby demonstrating the potential for evaluating transmission-blocking interventions in this model.

Trial registration: ClinicalTrials.gov NCT02431637 and NCT02431650.

Funding: Bill & Melinda Gates Foundation.

Keywords: Clinical Trials; Infectious disease; Malaria.

Figure 1. Study design schematic.

Malaria-naive volunteers (n = 17) were inoculated with pRBCs on day 0 (D0). Blood samples were taken for PCR analysis to measure both asexual parasites and gametocytes from day 4 pi and continued until the end of the study. Piperaquine (PIP 480 mg) treatment was administered on either day 7 or day 8 pi, and any subsequent recrudescent asexual infections were treated with piperaquine (PIP 960 mg). Mosquito-feeding assays were performed between day 17 and day 30 pi by feeding mosquitoes by live bite (direct skin feeding) on the participants or by membrane feeding on venous blood (Aim 1). During the period of gametocyte carriage, participants either received primaquine (15 mg) or artefenomel (500 mg) or did not receive either intervention (Aim 2). All participants received a course of artemether/lumefantrine and, if required, a single dose of primaquine (45 mg) to clear all parasites at the end of the study (Supplemental Table 1).

Figure 2. Trial profile.

All participants received 480 mg piperaquine. Twelve participants also received 960 mg piperaquine to treat recrudescence. ⁱMosquito infectivity not assessed in cohort 1 (n = 6). ⁱⁱAll 6 participants in the EFITA study were not intended to be randomized and 4 participants in the OZGAM study were not randomized due to recruitment and drug availability limitations (as detailed in Methods). ⁱⁱⁱAll 6 participants from EFITA and 2 from OZGAM did not receive any drug during the period of gametocyte carriage (Supplemental Table 1).

Figure 3. Development of parasitemia and gametocytemia.

Participants (n = 17) were experimentally infected with P. falciparum on day 0. (A) Parasite development as measured by 18S rDNA qPCR. Piperaquine (480 mg) was administered on day 7 pi for group 1 (n = 6; vertical red dashed line) or on day 8 pi for groups 2 and 3 (n = 11; vertical gray dashed line). Gametocyte development measured by qRT-PCR for (B) pfs25 (female gametocytes) and (C) pfMGET (male gametocytes). For A–C, the thin lines show individual participant curves and geometric means are in bold. (D) Asexual parasitemia AUC (days 0–10) and (E) gametocytemia AUC (days 10–21), grouped by treatment day (480 mg piperaquine) (compared by Kolmogorov-Smirnov test). Correlation of gametocytemia AUC (from day 10–21) with (F) asexual parasitemia on the day of treatment or (G) asexual parasitemia AUC (from day 0 to day of treatment) assessed using Spearman’s rank correlation.

Figure 4. Male and female gametocyte sex ratios.

Gametocyte development was monitored by qRT-PCR for pfs25 (female gametocytes) and pfMGET (male gametocytes). (A) Comparison of male and female gametocyte densities over time, with box plots indicating the median and whiskers showing the minimum and maximum responses (only includes data at time points where values exist for > 50% of the participants). (B) Proportion of gametocytes that are either male or female over time for 3 participants.

Figure 5. The course of asexual parasitemia and gametocytemia.

Representative graphs from groups 2 and 3 showing all data from qRT-PCR mRNA assays to illustrate the relationship between the parasite populations and the ability of SBP-1 mRNA to identify recrudescent infections. (A) Data from participant 203, (B) participant 306, (C) participant 302, and (D) participant 301 are shown. Male gametocytes (blue squares), female gametocytes (red circles), and ring-stage parasites (black triangles) are shown. Total parasitemia was also quantified by 18S rDNA qPCR (gray lines). Treatment administration is indicated with black arrows as follows: PIP 480, 480 mg piperaquine; PIP 960, 960 mg piperaquine; A/L, artemether/lumefantrine; PQ 45, 45 mg primaquine. Complete set of graphs for all participants is shown in Supplemental Figure 1, A–C.

Figure 6. Infectivity to mosquitoes.

Successful transmission is defined as the presence of oocysts in the mosquito midgut on day 8 or 9 after feeding as determined by 18S rDNA qPCR. Mosquito infection rate is reported as the prevalence of infection (percentage of mosquitoes infected per assay). (A) Prevalence of mosquito infection in all feeding assays performed for each participant in groups 2 and 3. (B) Prevalence of mosquito infection for all assays at each time point (dots represent the median, with error bars indicating the range) (Supplemental Tables 2 and 3).

Figure 7. Gametocyte infectivity and clearance.

(A) The peak gametocytemia (male and female gametocytes) for participants who were noninfectious throughout the study compared with participants who were infectious on at least one occasion. Box plots indicate the median and whiskers show the minimum and maximum responses. Groups compared using Mann-Whitney U test. (B) Percentage reduction in gametocytemia (as measured by 18S rDNA qPCR in samples where ring-stage parasites were not present) between day of drug treatment and 5 days after treatment with 15 mg primaquine (n = 5), 500 mg artefenomel (n = 4), or no drug (negative control, n = 7). One participant from the negative control group is not represented due to the presence of ring-stage parasites precluding analysis of gametocyte clearance. Lines indicate the median response and groups compared by Kruskal-Wallis test with Dunn’s multiple comparison test comparing all groups.

Figure 8. Safety of experimental malaria infection.

(A) Number and severity of AEs per participant with 480 mg piperaquine treatment on either day 7 pi (n = 6) or day 8 pi (n = 11). (B) Comparison of the number and severity of AEs by treatment day. Box plots indicate the median with whiskers showing the minimum and maximum responses. Groups compared using Mann-Whitney U test.