Clinical trial

This single-centre, single-blinded randomised controlled trial was conducted at the Radboud university medical centre (Nijmegen, The Netherlands) from August 2016 until February 2017. Prior to inclusion, study volunteers were medically screened as described previously37 and provided written informed consent. The trial was approved by the Central Committee on Research Involving Human Subjects (CCMO NL56222.091.15) of the Netherlands, performed according to the Declaration of Helsinki and Good Clinical Practice and prospectively registered at ClinicalTrials.gov (NCT02692963).

Twenty healthy, BCG-naive volunteers (age 18–35 years) without a history of malaria or residence in a malaria-endemic area in the 6 months before study entry were included and randomly assigned to two groups. Male and female volunteers were allocated separately, to ensure an equal distribution between groups. Volunteers had not received any other vaccinations within 3 months of enrolment. Ten subjects received standard dose (0.1 mL of the reconstituted vaccine) of intradermal BCG vaccination (BCG Bulgaria, Intervax) 5 weeks prior to challenge infection. Ten controls (group 2) received no vaccination.

Five weeks after BCG vaccination, both groups (BCG vaccinated, n = 9; 1 excluded after BCG vaccination and controls, n = 10) were exposed to bites of five Plasmodium falciparum NF54 strain infected Anopheles stephensi mosquitoes (sporozoite challenge). Details on the challenge infection are provided in Supplementary Table 3. Subjects and investigators were not blinded, whereas those performing the qPCR analysis were blinded until after the last qPCR data had been collected. qPCR was performed prospectively, once daily from day 6 after CHMI until day 3 after antimalarial treatment, according to previously published protocols22,38,39. All volunteers were treated with a curative regimen of antimalarial drugs (atovaquone/proguanil) once the treatment threshold of 100 parasites/mL blood was exceeded detected by qPCR or presumptively on day 21 after challenge if qPCR remained below treatment threshold.

Recording of adverse events

Subjects recorded clinical symptoms in a diary, from the time of BCG vaccination until 37 days after the CHMI. Both solicited and unsolicited adverse events were recorded after questioning by the investigators at set time points: prior to BCG vaccination, prior to the CHMI, daily from day 6 after infection until 3 days after antimalarial treatment, and on day 37 post CHMI21,40. Adverse events were graded according to criteria defined in the Clinical Trial Protocol: mild (grade 1): awareness of symptoms that are easily tolerated and do not interfere with usual daily activity; moderate (grade 2): discomfort that interferes with or limits usual daily activity; severe (grade 3): disabling, with subsequent inability to perform usual daily activity, resulting in absence or required bed rest. Relatedness was assessed by the investigator, also on the bases of pre-defined criteria: probable: an adverse event that follows a reasonable temporal sequence from the challenge procedure and cannot be reasonably explained by the known characteristics of the subject’s clinical state; possible: an adverse event for which insufficient information exists to exclude that the event is related to the study procedure; not related: an event for which sufficient information exists to indicate that the aetiology is unrelated either because of the temporal sequence of events or because of the subject’s clinical state or other therapies.

Oral temperature was measured by volunteers and recorded in the symptom diary every morning and more frequently during symptoms. Tympanic temperature was measured by the study physician at every follow-up visit. Fever was scored as follows: mild (grade 1): 37.6–38.0 °C; moderate (grade 2): 38.1–39.0 °C; severe (grade 3): ≥39.1 °C.

Whole blood flow cytometry

One-hundred microlitres(lymphocytes) or 50 µL (monocytes and neutrophils) of fresh EDTA blood was stained directly with antibodies. For lymphocyte analysis, samples were stained with CD3-AlexaFluor700 (Biolegend; clone OKT3; catalogue number 317340; final dilution 1:640), pan-γδTCR−PE (Beckman Coulter; clone IMMU510; catalogue number COIM1349; final dilution 1:160), CD56-Brilliant Violet(BV)421 (Biolegend; clone HCD56; catalogue number 318328; final dilution 1:320), CD16-APC-eFluor780 (eBiosciences; clone CB16; catalogue number 47–0168–42; final dilution 1:640), CD69-PerCP-Cy5.5 (Biolegend; clone FN50; catalogue number 310926; final dilution 1:640). For monocyte analysis, samples were stained with a lineage mix containing CD3-PerCP-Cy5.5 (Biolegend; clone HIT3a; catalogue number 300328; final dilution 1:400), CD19-PerCP-Cy5.5 (Biolegend; clone HIB19; catalogue number 302230; final dilution 1:200) and CD56-PerCP-Cy5.5 (Biolegend; clone HCD56; catalogue number 318322; final dilution 1:100), CD14-FITC (Biolegend; clone HCD14; catalogue number 325604; final dilution 1:80), CD16-PE-Cy7 (Biolegend; clone 3G8; catalogue number 302016; final dilution 1:1280), HLA-DR−APC-Cy7 (Biolegend; clone L243; catalogue number 307618; final dilution 1:160) and CD86−Pacific Blue (Biolegend; clone IT2.2; catalogue number 305423; final dilution 1:100). For neutrophil analysis samples were stained with CD14-PerCP (Biolegend; clone HCD14; catalogue number 325632; final dilution 1:30), HLA-DR-APC (Biolegend; clone L243; catalogue number 307610; final dilution 1:80), CD16−APC-eFluor780 (eBiosciences; clone CB16; catalogue number 47–0168–42; final dilution 1:1280), CD62L-PE-Cy7 (eBioscience; clone DREG-56; catalogue number 25–0629–42; final dilution 1:1280) and CD11b-BV510 (Biolegend; clone ICRF44; catalogue number 301334; final dilution 1:180). Samples were stained for 30 min at 4° C (C) in the dark. After staining, erythrocytes were lysed for 5 min at 4 °C with 1 mL BD FACS Lysis buffer, followed by centrifugation. Cell pellets were washed once with 1 mL FACS buffer (0.5% bovine serum albumin (BSA) in PBS) and resuspended in PBS with 1% paraformaldehyde (PFA) and analysed on a Gallios flow cytometer (Beckman Coulter) the same day. Flow cytometry data was analysed using Flow Jo software (version 10.0.8 for Apple OS). The gating strategy and representative plots are shown in Supplementary Fig. 10.

PBMC isolation, cryopreservation and thawing

Blood samples for peripheral blood mononuclear cell (PBMC) isolation were collected at inclusion (incl), prior to challenge (C−1) and 37 and 121 days after challenge infection (C + 37, C + 121). PBMC were isolated by density gradient centrifugation from citrate anti-coagulated blood using vacutainer cell preparation tubes (CPT; BD Diagnostics). Following four washes in ice-cold phosphate buffered saline (PBS), cells were counted and cryopreserved at a concentration of 10 × 106 cells/mL in ice-cold foetal calf serum (Gibco)/10% DMSO (Merck) using Mr. Frosty freezing containers (Nalgene). Samples were stored in vapour-phase nitrogen. Immediately prior to use, cells were thawed, washed twice in Dutch-modified RPMI 1640 (Gibco/Invitrogen) and counted in 0·1% Trypan blue with 5% Zap-o-Globin II Lytic Reagent (Beckman Coulter) to assess cell viability.

PBMC restimulation

For lymphocyte responses, PBMCs taken at inclusion, C-1, C + 37, and C + 121 were stimulated with purified NF54 strain schizonts or uninfected erythrocytes. Cells were cultured in RPMI 1640 (Dutch Modification; Gibco) with 5 mg/mL gentamycin (Centraform), 100 mM pyruvate (Gibco), 200 mM glutamax (Gibco), supplemented with 10% heat-inactivated pooled human A + serum (obtained from Sanquin Bloodbank, Nijmegen, The Netherlands). Anti-CD107a-Pacific Blue antibody (Biolegend; clone H4A3; catalogue number 328624; final dilution 1:400) was added throughout co-culture. Brefeldin A (10 µg/mL; Sigma-Aldrich) and monansin (2 µM; eBioscience) were added after 20 h. After an additional 4 h of stimulation, cells were washed and stained with a fixable viability dye labelled with eFlour780 (eBioscience) for 30 min at 4° C. After washing cells were stained with antibodies against surface markers: CD3-ECD (Beckman Coulter; clone UCHT1; catalogue number A07748; final dilution 1:100), CD4-FITC (BD Biosciences; clone SK3; catalogue number 340133; final dilution 1:20), CD8-AlexaFluor700 (Biolegend; clone HIT8A; catalogue number 300920; final dilution 1:2000), pan-γδTCR-PE (Beckman Coulter; clone IMMU510; catalogue number COIM1349; final dilution 1:160), and CD56-PerCP-Cy5.5 (Biolegend; clone HCD56; catalogue number 318322; final dilution 1:100), for 30 min at 4 degrees. Cells were washed and fixed with Foxp3 fixation/permeabilization buffer (eBioscience) for 30 min at 4 degrees. After washing with permeabilization buffer (eBioscience) cells were stained for intracellular cytokines with IFN-γ-PE-Cy7 (Biolegend; clone 4 S.B3; catalogue number 502528; final dilution 1:200) and granzyme B-AlexaFluor647 (Biolegend; clone GB11; catalogue number 515406; final dilution 1:200) for 30 min at 4 degrees. After washing with permeabilization buffer, cells were taken up in PBS with 1% PFA. Cells stimulated PMA (10 ng/mL; Sigma) and ionomycin (1 µg/mL; Sigma) for 4 h were used as a positive control.

Samples were analysed on a Gallios flow cytometer (Beckman Coulter) the same day. Flow cytometry data was analysed using Flow Jo software (version 10.0.8 for Apple OS). CD107a and cytokine responses to PfRBC were corrected for uRBC at every time point (thus, defined as percent increase over background), and then corrected for baseline (pre-vaccination) responses. Gating strategy and representative plots are shown in Supplementary Fig. 11.

Circulating cytokines and granzyme B

Plasma concentrations of TNF-α, IL-1β, (detection range 0.98–4000 pg/mL) IL-6 (0.36−1500 pg/mL), IL-8 (0.62–2500 pg/mL), IL-10, (2.92–12,000 pg/mL) IFN-γ (1.22–5000 pg/mL) and granzyme B (2–10.000 pg/mL) were measured in citrate plasma using a Luminex assay according to the manufacturer’s instructions (Milliplex, Merck Millipore, Billerica, MA, USA).

High sensitivity C-reactive protein

Automated hsCRP measurements were performed on citrated plasma samples with immunonephelometry with a Behring Nephelometer Analyser following the manufacturers’ instructions, using reagents and calibrators specifically designed for high sensitivity measurements. The detection limit was 0.16 mg/L.

Malaria-specific antibody ELISA

Malaria-specific antibody levels were determined by standardised ELISA as described previously41. In short, plates were coated with circumsporozoite protein (CSP), liver stage antigen-1 (LSA1) protein or lysed ring stage parasites. Citrated plasma from volunteers was diluted 50x and 150x and analysed in duplicate. A standard curve was generated by serial twofold dilutions of serum from a pool of 100 Tanzanian adults living in an endemic area (HIT serum). ELISA data analysis was performed with Auditable Data Analysis and Management System for ELISA (ADAMSEL, version 1.1). Post-challenge plasma samples were corrected for pre-challenge responses.

Sporozoite invasion assay

HC-04 human hepatoma cells (obtained from MR4) were seeded in collagen coated 96-well plates (coated with 0.056 mg/mL for 1 h; Collagen from Rat Tail, Sigma-Aldrich) at 50,000 cells per well. Sixteen hours after seeding, NF54 P. falciparum sporozoites were pre-incubated on ice for 30 min with 10% heat-inactivated pre- or post-challenge citrate plasma from volunteers and 10% heat-inactivated serum from non-immune adult. Sporozoites incubated with 10% heat-inactivated serum from highly immune Tanzanian adults and 10% non-immune serum, or 20% non-immune serum served as positive and negative control, respectively. Following pre-incubation, 50,000 sporozoites were added per well in triplicate. Plates were centrifuged at 3000 rpm for 10 min (Eppendorf Centrifuge 5810R) and incubated for 3 h on 37 °C, 5% CO 2 .

After three hours, wells were washed three times with PBS to remove medium, antibodies and non-invaded sporozoites. Subsequently, cells and any extracellular adherent sporozoites were dissociated by incubating with 0.05% trypsin with EDTA (ThermoFisher) for 5 min at 37 °C, followed by neutralisation with an equal volume 10% heat-inactivated human serum in PBS. Cells were transferred into 96-well V-bottom plates, spun down at 1700 rpm for 4 min at 4 °C.

Cells were washed with PBS and fixed with Foxp3 fixation/permeabilization buffer (eBioscience). After washing with permeabilization buffer (eBioscience), intracellular sporozoites were stained with FITC-labelled 3SP2 antibody (monoclonal antibody against CSP, published previously42) for 30 min at 4 °C. After washing in permeabilization buffer cells were taken up in 1% paraformaldehyde and analysed on a Gallios flow cytometer (Beckman Coulter) the same day.

Flow cytometry data was analysed using Flow Jo software (version 10.0.8 for Apple OS). Live cells were gated based on forward scatter/sideways scatter characteristics and percent invasion was defined as percentage of live cells positive for FITC. Post-challenge samples were compared to pre-challenge samples.