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Additional file 1: Figure S1. Genetic diversity of Plasmodium falciparum and Plasmodium malariae length polymorphic genes. Figure S2. False-positive rapid diagnostic tests as a proportion of all positive rapid diagnostic tests. Table S1. Multivariable logistic regression analysis of risk factors associated with false-positive rapid diagnostic tests.

Additional file 1: Figure S1. Trend ofparasite density over time between individuals in selected and unselected groups.Trend of parasite density over time between selected and unselectedparticipants. Parasite density was determined by quantitative polymerase chainreaction assays (qPCR). Figure S2. Overall distribution of parasite densityamong groups of selected and unselected participants. Scatter plots of parasitedensity measured by quantitative polymerase reaction assays (qPCR) betweenselected and unselected data points. Figure S3. Distribution of parasitedensity by individuals participants in selected and unselected groups. Barplotsof parasite density as measured by quantitative polymerase reaction assays(qPCR) between selected vs. unselected individual volunteers.

Additional file 2: Figure S1. Intersection of modular approaches reveals core responsive modules. Proportional venn diagram shows the overlap of significant response modules identified by spline-curve fitting, GSEA, and hypergeometric tests for BTM enrichment in individual response genes identified by mixed modelling.

Introduction Malaria is caused by intracellular organisms that belong to the genus Plasmodium. In 2015, there were an estimated 438,000 deaths and 214 million clinical illnesses due to malaria infection, of which the majority were in sub-Saharan African children below five years of age. Amongst the five species that are known to infect humans, Plasmodium falciparum causes the most severe disease, mostly in children and pregnant women in sub-Saharan Africa. Despite malaria control programs being operational for many years, malaria elimination in most endemic regions is far from being achieved. Vaccination is considered the most cost effective method of preventing infectious diseases. To date, there are no effective vaccines available for parasitic infections, despite the existence of strong evidence of acquired immunity in most parasitic infections studied. It is therefore highly likely that the addition of an effective tool such as a vaccine to the current malaria control strategy would have a strong positive impact on our ability to control this disease. In the first part of this thesis, we aimed to investigate the vaccine efficacy as well as the cellular and humoral immunity of African paediatric volunteers vaccinated with the most clinically advanced malaria vaccine; the RTS, S/AS01. Meanwhile, novel vaccination and testing approaches are being pursued to improve or replace the recombinant subunit malaria vaccine approach to meet the goals formulated in the Malaria Vaccine Roadmap of WHO (http://www.who.int/immunization/topics/malaria/ vaccine_roadmap/en). These goals strategized that by 2030, licensed vaccines targeting Plasmodium falciparum and Plasmodium vivax should encompass the following two objectives, for use by the international public health community: i) First, it should have a protective efficacy of at least 75 percent against clinical malaria and be suitable for administration to appropriate at-risk groups in malaria- endemic areas. ii) Secondly, it should reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria infection; enable elimination in multiple settings and be suitable for administration in mass campaigns. Currently, the most promising candidate seems to be the whole malaria sporozoite approach, which is formed of cryopreserved, purified whole live-attenuated (either by irradiation or genetic attenuation) sporozoites. One of the novel tools used to analyze induced vaccine efficacy in sub-Saharan Africa experimentally vaccinated volunteers is controlled human malaria infection (CHMI). Many CHMIs using infectious mosquito bites or purified sporozoites have been successfully conducted in the USA and Europe over many years, but this approach had not been employed in sub-Saharan Africa until 2012. The aim of the second part of this thesis was to describe the potential of using CHMI as a tool to accelerate malaria vaccine development in sub-Saharan Africa and to dissect malaria- specific immunity induced by CHMI based on our trial conducted in 2012 in Bagamoyo. Methods and findings In the first part of this thesis (Chapter 4), the aim was to investigate safety, efficacy, cellular and humoral immunity in RTS,S/AS01 vaccinated Tanzanian paediatric populations. Adverse events were used to determine the safety of the RTS,S/AS01 vaccine in this age group (paper I), ELISA to measure the vaccine-induced CS-specific antibodies and Luminex to measure vaccine-induced cytokine responses (paper II and III). Furthermore, flow cytometry was used to investigate vaccine-induced cellular immune responses (paper III). We also looked into the implications and practicalities of immunological sampling in the African paediatric population. We did community sensitization and collected blood samples from 400 children for immunological study (paper IV). We showed that in 6-12 week old infants, vaccine efficacy against clinical malaria 14 months after first vaccination was 30.1% (95% CI, 23.6 to 36.1) in the intention-to-treat (ITT) and 31.3% (97.5% CI, 23.6 to 38.3) in the per-protocol (PP) population. Furthermore, the vaccine efficacy against severe malaria was 26.0% (95% CI, −7.4 to 48.6) and 36.6% (95% CI, 4.6 to 57.7) in the ITT and PP populations, respectively. The safety of the vaccine in terms of serious adverse events showed similar trends in both study groups. We identified two main RTS,S/AS01 vaccine induced cellular immune mechanisms:- (i) Th1-related responses such as CS-specific IFN-g, GM-CSF and IL-15 are associated with protection and (ii) Th2-related responses mediated by CS-specific IL5 and RANTES are associated with increased odds of malaria. Moreover, antibody avidity alone did not predict protective efficacy in the current study. The induction of RTS, S/AS01 protective Th1 and pro-inflammatory responses was lower in infants compared to children; a scenario that might explain the lower efficacy observed in the infant cohort. Furthermore, we also showed that immunology studies in the paediatric population can feasibly be conducted in African research institutions. In the second part of this thesis (Chapter 5), we conducted in 2012 the first CHMI using cryo-preserved purified non-attenuated sporozoites in Tanzanian adult volunteers with previous malaria exposure (paper V). In this study, the humoral and cellular immune responses elicited following CHMI were evaluated (paper VI and VII). We used adverse events to determine the safety of the CHMI model in malaria pre-exposed volunteers. We also used blood slide microscopy to define sporozoite infectivity rates, Luminex assays to examine the sporozoite-induced antibodies, B-cell Elispot analysis, single cell RNA sequencing, flow cytometry and cell sorting followed by in vitro stimulation assays to investigate and define the affected innate and adaptive immune responses following CHMI (paper VIII). Our studies showed that: (i) CHMI is safe, tolerable and infective when used in malaria endemic regions, (ii) a single dose of intradermal sporozoite (PfSPZ) challenge elicited long-lived merozoite-opsonizing antibodies and long-lasting innate and innate-like lymphocyte populations, (iii) When we compared Dutch (malaria naïve) and Tanzanian (malaria exposed) subjects undergoing the same challenge study, Dutch subjects responded differently to PfSPZ challenge compared to Tanzanian (malaria pre-exposed) subjects. Conclusion Substantial investment in research and development is needed to develop a highly efficacious malaria vaccine. To date, the recombinant subunit vaccines are yet to give the desired levels of protection for malaria elimination but seem to prevent malaria disease in high transmission settings. Large scale manufacturing, storage and distribution of live whole malaria sporozoite-based vaccines for mass administration need further development. So far, data generated from the PfSPZ vaccine trials conducted in the USA, Europe and in African research institutions imply that malaria naive individuals respond better to malaria vaccines than malaria pre-exposed individuals. The question remains to be, “what exactly constitutes the reason for lack of durable protection against malaria infection in endemic areas?” The most important factor in accelerating future vaccine development is a better understanding of the biology and nature of acquired immunity, which will lead to improved vaccine design. We have established the foundation for using CHMI to assess efficacy of new interventions against malaria and to study the mechanisms of the lack of protection conferred by different malaria vaccines in endemic settings. This study has opened new doors in the field of malaria intervention, whereby malaria vaccine and drug efficacy can be easily tested using CHMI in the target population.

BACKGROUND: The candidate malaria vaccine RTS,S/AS01 reduced episodes of both clinical and severe malaria in children 5 to 17 months of age by approximately 50% in an ongoing phase 3 trial. We studied infants 6 to 12 weeks of age recruited for the same trial. METHODS: We administered RTS,S/AS01 or a comparator vaccine to 6537 infants who were 6 to 12 weeks of age at the time of the first vaccination in conjunction with Expanded Program on Immunization (EPI) vaccines in a three-dose monthly schedule. Vaccine efficacy against the first or only episode of clinical malaria during the 12 months after vaccination, a coprimary end point, was analyzed with the use of Cox regression. Vaccine efficacy against all malaria episodes, vaccine efficacy against severe malaria, safety, and immunogenicity were also assessed. RESULTS: The incidence of the first or only episode of clinical malaria in the intention-to-treat population during the 14 months after the first dose of vaccine was 0.31 per person-year in the RTS,S/AS01 group and 0.40 per person-year in the control group, for a vaccine efficacy of 30.1% (95% confidence interval [CI], 23.6 to 36.1). Vaccine efficacy in the per-protocol population was 31.3% (97.5% CI, 23.6 to 38.3). Vaccine efficacy against severe malaria was 26.0% (95% CI, -7.4 to 48.6) in the intention-to-treat population and 36.6% (95% CI, 4.6 to 57.7) in the per-protocol population. Serious adverse events occurred with a similar frequency in the two study groups. One month after administration of the third dose of RTS,S/AS01, 99.7% of children were positive for anti-circumsporozoite antibodies, with a geometric mean titer of 209 EU per milliliter (95% CI, 197 to 222). CONCLUSIONS: The RTS,S/AS01 vaccine coadministered with EPI vaccines provided modest protection against both clinical and severe malaria in young infants. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619.).