Ethics statement: study approval and ethics

All non-human primate infections were carried out in accordance with European and Dutch law after positive advice from the ethical committee (DEC). The Council of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) has awarded BPRC full accreditation.

Non-human primates were used because no other models (in vitro or in vivo) were suitable for the aims of this project. The local independent ethical committee constituted conform Dutch law (BPRC Dier Experimenten Commissie, DEC) approved the research protocol (agreement number DEC# 751B) prior to the start, and the experiments were all performed according to Dutch and European laws. Thus, BPRC is fully compliant with the international demands on animal studies and welfare as set forth by the European Council Directive 2010/63/EU, and Convention ETS 123, including the revised Appendix A as well as the ‘Standard for humane care and use of Laboratory Animals by Foreign institutions’ identification number A5539-01, provided by the Department of Health and Human Services of the United States of America’s National Institutes of Health (NIH) and Dutch implementing legislation. The rhesus monkeys (male Indian Macaca mulatta, age 6–15 years) used in this study were captive-bred and socially housed. Animal housing was according to international guidelines for non-human primate care and use. Besides their standard feeding regimen and drinking water ad libitum via an automatic watering system, the animals followed an environmental enrichment programme in which, next to permanent and rotating non-food enrichment, an item of food enrichment was offered to the macaques daily. All animals were monitored daily for health and discomfort. All intravenous injections and large blood collections were performed under ketamine sedation, and all efforts were made to minimize suffering.

Study designResearch objective

Our first objective in this proof-of-concept vaccination experiment is to assess the protective effect of the vaccination protocol, and we expect all animals to be protected by the treatment, while we expect all control animals to become infected. With four animals per group and based on the expected outcome, the following 2 × 2 contingency table (Table 1) can be constructed.

Table 1 Statistical power calculation and sample size determination using 2 × 2 contingency table (right) and Fisher’s exact test.Sample size

The results were evaluated using Fisher’s exact test, with the data in the contingency table (Table 1) above the Exact P-value (Fisher’s exact test) would be 0.014, with a power of 81% (1-β) to detect a statistically significant difference in treatment effect (development of parasitemia in control versus total protection in the treatment group) and a type I error of 0.05 (α). As relapses cannot be detected under continuous chloroquine coverage and we want to additionally assess the role of blood stages in this model, a further four animals were included for this purpose. This allows making the best use of the additional animals as we both monitor the relapses, which is needed to assess timelines of the vaccination protocol and the role of blood stages through this single group.

Animal selection and randomization

A total of twelve healthy male Indian rhesus macaque (Macaca mulatta) animals (N = 12) were selected, and ten of them were randomized (matching criteria: age and weight) over three groups (N = 4 per group; N = 2 for the control group): the CPS (hypnoboost-CPS), blood-stage exposed hypnoboost (hypnoboost-BS) and control groups. Two of the twelve animals (R07095, R08100) had previously seen a P. cynomolgi sporozoite infection three years before this study and were, therefore, specifically assigned to the control group. All animals were humanely sacrificed at the end of the study.

Obtaining sporozoites for infection

In order to obtain P. cynomolgi M strain sporozoites for the infection of the hypnoboost-BS groups, An. stephensi mosquitoes were fed using membrane feeding on infected blood derived from a P. cynomolgi-infected rhesus macaque (donor monkey)28. Briefly, rhesus macaques were infected with 1 × 106 P. cynomolgi M strain blood-stage parasites and bled at peak parasitemia. Approximately 300 female Anopheles stephensi mosquitoes strain Sind-Kasur Nijmegen were fed with this blood. An. stephensi adult mosquitoes were obtained from the Radboud University in Nijmegen. Sporozoites were isolated from the salivary glands of An. stephensi mosquitoes and used as detailed below.

Experimental design of CPS in the P. cynomolgi-Macaca mulatta model

After a baseline bleeding (t = 0) was taken to collect plasma and PBMC, the hypnoboost-CPS and hypnoboost-BS groups were infected i.v. with 106 freshly isolated P. cynomolgi M strain sporozoites. From day seven post-infection onwards, the hypnoboost-CPS and hypnoboost-BS groups were monitored for parasitemia using thigh prick (thin film smears). Additionally, on day seven post-infection, the hypnoboost and control groups were treated with 7,5 mg/kg i.m. chloroquine (CQ), while, in the blood-stage exposed hypnoboost group, animals were treated with CQ after primary parasitemia was detected starting on day 13 to eliminate the blood stages. At the time of treatment, parasitemia in this group ranged from 0.13 to 2.2% (Supplementary data: table 1). Following the CPS chloroquine administration schedule3, hypnoboost and control groups were treated with i.m. chloroquine (CQ) to provide continuous coverage, while treatment of the blood-stage exposed hypnoboost group with i.m. CQ was stopped after primary parasitemia was cured to allow for the monitoring of relapses and exposure to blood stages. To allow for more detailed monitoring of the immune response, on day 14 post-infection (t = 14), another blood sample was taken from all animals (plasma, PBMC).

The blood-stage exposed hypnoboost group was monitored for a total of three relapses before all animals (including the control group) were radically cured by administering 1.8 mg/kg primaquine (PQ) per os in combination with chloroquine (CQ) i.m. for seven days. The animals were trained to drink syrup containing primaquine, which masked the bitter taste of the drug. Compliance was high in all animals; in rare cases in which the animal refused to drink the medication, it was given through gavage. After the radical cure, all animals rested for a period of at least 15 days to allow for the complete washout of residual drug. Thereafter, following a large bleed to collect plasma and PBMC, all animals were challenged with 200 freshly isolated P. cynomolgi sporozoites. Starting on day eight after the challenge, the animals were monitored daily for parasitemia through a thigh prick (thin film smears). In terms of relapse, animals were called positive when at least one parasite was seen in slides on two subsequent days.

Thin smears and blinding

Thin smears were obtained using a drop of blood, fixed with methanol and stained with Giemsa (Sigma–Aldrich) according to the manufacturer’s specifications. At each time point, at least 20,000 uninfected RBCs were counted to ensure the accuracy of the count. Blood smears to determine parasitemia development after the challenge were read by multiple staff (blinded to the treatment group), to determine the day of first parasitemia in every monkey.

Data inclusion and exclusion criteria

No inclusion or exclusion criteria were applied; possible outliers are also shown.

Statistical method applied to the evaluation of challenge results

The log-rank test (R survival package) was used to compare the time to parasitemia pair-wise between the three groups, revealing that there is a statistically significant difference between the control and hypnoboost-BS group (p = 0.006). Details in Supplementary Fig. 1.

Harvesting of plasma

The whole blood of adult rhesus macaques was collected in heparin by saphenous vein puncture under ketamine anaesthesia. Plasma was collected by centrifugation (10 min at 524 × g, room temperature (RT)) of the heparin blood using an Allegra XR-15 Centrifuge (Beckman Coulter) and stored at −80 °C.

Isolation and concentration of plasma-derived IgGs used in immunofluorescence assay

IgGs were isolated from 1 mL blood plasma collected at four different time points from each of the 12 animals. Twelve Bio-Rad’s Econo-Column Chromatography columns fitted with Bio-Rad’s Econo-Column Flow adaptors were run simultaneously, 1 column per animal. For each animal, the four different time points were run over one column in chronological order. Each column was rinsed with PBS and 0.02% NaN3 in between the runs29.

Antibody determination

The antibody determination was carried out by three independent IgG ELISAs for each antigen (PcyAMA1, PcyMSP1, PcyRON4, PcyRON5, PcyHSP70 or PcyEXP1) in half area microlon 600 microplates (Greiner), each coated overnight at +4 °C with the specific antigen at concentrations of 1 µg/mL (0.05 µg/well). Briefly, after blocking with 100 µl/well 3% BSA/PBST for 2 h at 37 °C, the plasma samples in 0.05% Tween-20/1% BSA/PBS were added in a two-fold dilution series (starting at a dilution of 1:50 for t = 0, at 1:125 for t = challenge and at 1:250 for the plasma harvested at the end of the study) for the IgG ELISAs plasma dilution started at 1:50 for t = 0, at 1:125 for t = 14, t = challenge and at 1:250 for the plasma harvested at the end of the study. The ELISAs were incubated 2 h at 37 °C. As positive control a pool of end sera was used and was used in a two-fold dilution series starting at 1:2000. Antibodies in the plasma samples were detected using the conjugates Rabbit anti-Rat-HRPO (DAKO cytomation cat#P0450) 1:8000 and Goat anti-Hu-IgM-HRP (Southern Biotech #2020-05) 1:2000, 1 h at 37 °C. The ELISA was developed using 50 µl TMB (DIAsource ImmunoAssays S.A. cat# SB04/B), stopped with 50 µl 0.2 M H2SO4 (DIAsource ImmunoAssays S.A. cat# SS02-01) and measured on a plate reader (BioRad iMark) at 450 nm. Antibody titres were calculated with Excell and Adamsel, graphs were made in Prism30. The different P. cynomolgi protein antigens were produced as reported below.

Antigen production and purification

Synthetic genes optimized for the indicated microorganisms were purchased from Genscript (Piscataway,NJ).

The gene coding for the ectodomain of PcyAMA1 M-strain (PlasmoDB accession nr. PcyM_PlasmoDB PcyM_0938200, aa 43–487) was expressed in Pichia pastoris using the pPicZ alpha A vector and purified using the vector’s encoded hexa-histidine tag31.

A gene encoding the C-terminal of PcyMsp1 (PcyMSP119) [PlasmoDB PcyM_0731200, aa1686–1778] was expressed in Pichia pastoris using the pPicZ alpha A vector and purified using the vector’s encoded hexa-histidine tag32.

The C-terminal of PcyHSP70 [Genbank AAA29625, aa 350–686] was expressed in E.coli33. An N-terminal hexa-histidine tag was used to purify the protein in a single step using a nickel-activated IMAC (HiTRAP IMAC FF, VWR, Amsterdam, The Netherlands).

The leader sequence-less PcyEXP1 protein, equipped with an N-terminal hexa-histidine tag, [Genbank accession nr XP_004222438, aa 19–133], the N-terminal end of PcyRON4 (PlasmoDB accession nr. PcyM_0918500, aa 1 to 219) and the gene coding for a part of the N-term of the PcyRON5 gene (PcyM_0518100, aa 95–199, Mw 11 kDa) both in frame with the C-terminal vector-encoded hexa-histidine tag were all cloned into the PjExpress412 vector (DNA20/ATUM, Newark, CA), transformed to and expressed in electrocompetent E. coli DE3 cells (Thermofisher, Landsmeer, The Netherlands34).

Immunofluorescence assay (IFA)

IFA were carried out on slides (10-wells slides) coated with P. cynomolgi M strain blood stage or sporozoites. Briefly, IFA slides were fixed in cold MeOH for 20 s and air-dried. Of the protG purified monkey IgGs a five-fold dilution series was made, starting at 200 µg/mL in PBS/1%FCS to 1,6 µg/mL. Wells on the IFA slides were incubated with the primary Abs for 1 hr @RT in a moist box. Slides were washed with PBS and incubated with DAPI (D3571; Life Technologies) and Goat-a-Monkey IgG-FITC (ab112766, Abcam; @ 1/1000). Slides were washed, and surface was gently dried. A few drops of Citifluor AF1 Mountant Solution (#17970-25; Electron Microscopy Sciences) were applied before the coverslip was added. IFAs were viewed using the Leica Fluorescence Microscope35.

Flow cytometric analysis of peripheral blood mononuclear immune cell (PBMC) subsets

PBMCs were profiled by flow cytometry to monitor the frequencies of major subsets (see Supplementary Table 3 for the antibody-conjugate panels used).

Cryopreserved PBMCs were thawed quickly and washed twice in a culture medium with 10% FCS. Cells were counted and brought to a concentration of 0.5 × 106 cells/ml in 0.5% FCS/PBS. Aliquots of 150 µL (1.5 × 106 cells) were seeded in a 96-wells plate, washed with PBS and stained for 20 min at RT with 50 µL Live/Dead™ Fixable Blue dead cell stain (Supplementary Table 3). Thereafter, the cells were washed again with PBS and stained for 30 min at 4 °C with the antibody mix in brilliant-stain buffer (Supplementary Table 3). After staining, all samples were washed twice with 0.5% FCS/PBS and fixed with 2% paraformaldehyde for 1 h before the flow cytometric measurement was performed using an Aurora spectral analyzer (Cytek Biosciences). All analyses were performed using the FlowJo 10.8 software (BD Biosciences). The cytometry gating strategy is specified in Supplementary Fig. 2.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.