Sporozoite immunization of human volunteers under mefloquine prophylaxis is safe, immunogenic and protective: a double-blind randomized controlled clinical trial

Abstract

Immunization of healthy volunteers with chloroquine ChemoProphylaxis and Sporozoites (CPS-CQ) efficiently and reproducibly induces dose-dependent and long-lasting protection against homologous Plasmodium falciparum challenge. Here, we studied whether chloroquine can be replaced by mefloquine, which is the only other licensed anti-malarial chemoprophylactic drug that does not affect pre-erythrocytic stages, exposure to which is considered essential for induction of protection by CPS immunization. In a double blind randomized controlled clinical trial, volunteers under either chloroquine prophylaxis (CPS-CQ, n = 5) or mefloquine prophylaxis (CPS-MQ, n = 10) received three sub-optimal CPS immunizations by bites from eight P. falciparum infected mosquitoes each, at monthly intervals. Four control volunteers received mefloquine prophylaxis and bites from uninfected mosquitoes. CPS-MQ immunization is safe and equally potent compared to CPS-CQ inducing protection in 7/10 (70%) versus 3/5 (60%) volunteers, respectively. Furthermore, specific antibody levels and cellular immune memory responses were comparable between both groups. We therefore conclude that mefloquine and chloroquine are equally effective in CPS-induced immune responses and protection. Trial registration: ClinicalTrials.gov NCT01422954.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Study flow diagram.

Thirty-six subjects were screened for eligibility, of whom twenty were included in the trial and randomized over three groups. One control subject was excluded after initiation of chemoprophylaxis but before the first immunization because of an unexpected visit to a malaria-endemic area during the study period. In a double-blind fashion, fifteen subjects received either CPS-CQ or CPS-MQ immunization and four control subjects received bites from uninfected mosquitoes and mefloquine prophylaxis. Subjects received a challenge infection by bites of five infected mosquitoes sixteen weeks after discontinuation of prophylaxis.

Figure 2. Parasitemia during CPS immunization.

Parasitemia was determined retrospectively, once daily from day 6 until day 10 after each immunization, by real-time quantitative PCR (qPCR). Each line represents an individual subject from the CPS-MQ (dashed blue lines) or CPS-CQ group (red lines). The number of subjects with a positive qPCR/total number of volunteers in the CPS-MQ (blue) and CPS-CQ (red) groups after each immunization are shown above the graph. Values shown as 17.5 on the log-scale were negative (i.e. half the detection limit of the qPCR: 35 parasites/ml).

Figure 3. Adverse events during CPS immunization.

Percentage of volunteers in each group experiencing possibly or probably related AE after the first (I), second (II) and third (III) immunization. AEs were evaluated at each visit and graded for severity as described in the methods paragraph: mild (light grey), moderate (dark grey) and severe (black). Only the highest intensity per subject is listed. No Serious Adverse Events occurred.

Figure 4. Parasitemia after challenge infection.

Parasitemia was assessed retrospectively by real-time quantitative PCR (qPCR) twice daily from day 5 until day 15 and once daily up until day 21 after challenge. Each line represents an individual subject. Red lines represent CPS-CQ immunized volunteers (n = 5), dashed blue lines CPS-MQ immunized subjects (n = 10) and dotted grey lines malaria-naive control subjects (n = 4). Values shown as 17.5 on the log-scale were negative (i.e. half the detection limit of the qPCR: 35 parasites/ml).

Figure 5. Antibody responses induced by CPS-CQ and CPS-MQ immunization.

Antibodies against CSP (A and B; in AU), LSA-1 (C and D), and MSP-1 (E and F) were analyzed at baseline (B), 28 days after the first (I1) and second (I2) immunization and one day before challenge (C-1; 20 weeks after the last immunization) for all CPS-CQ (A, C and E, n = 5) and CPS-MQ (B, D and F, n = 10) immunized volunteers. Data are shown as individual titers with medians. Open squares indicate protected subjects, filled circles indicate unprotected subjects. Differences between the time points were analyzed by Friedman test with Dunn’s multiple comparison post-hoc test. Significant differences are indicated by asterices with * (p

Figure 6. Cellular immune responses: CD107a expression by CD4 T cells and granzyme B production by CD8 T cells.

CD107a expression by CD4 T cells after PfRBC re-stimulation, corrected for uRBC background in CPS-CQ (A) and CPS-MQ (B) groups; granzyme B production by CD8 T cells after PfRBC re-stimulation, corrected for uRBC background in CPS-CQ (C) and CPS-MQ (D) groups. Symbols and lines represent individual subjects before immunization (B) and one day before challenge (C-1). Open squares indicate protected subjects, filled circles indicate unprotected subjects. Differences between B and C-1 for all subjects were tested by Wilcoxon matched-pairs signed rank test.