Plasmodium spp. impacts human DC function in vivo and in vitro. We have highlighted the heterogeneity of the data observed in these studies, compared and critiqued the models used to generate our current understanding of DC function in malaria, and examined the mechanisms by which Plasmodium spp. mediate these effects. This review highlights potential research directions which could lead to improved efficacy of existing vaccines, and outlines novel targets for next-generation vaccine strategies to target malaria.Malaria remains a serious threat to global health. Sustained malaria control and, eventually, eradication will only be achieved with a broadly effective malaria vaccine. Yet a fundamental lack of knowledge about how antimalarial immunity is acquired has hindered vaccine development efforts to date. Understanding how malaria-causing parasites modulate the host immune system, specifically dendritic cells (DCs), key initiators of adaptive and vaccine antigen-based immune responses, is vital for effective vaccine design. This review comprehensively summarizes how exposure to  Plasmodium species of Apicomplexans, which have a complex life cycle spanning multiple organ sites   . At 34 hPf EMP1) , where tP. falciparum trophozoites will commit to sexual replication, where the process of schizogony is replaced by the formation of sexual stages known as gametocytes  of 0 months . The sub0 months –23. To ePlasmodium life stages, namely the skin, blood, bone marrow, spleen, and liver  –26, yet nd liver .−HLA-DR+CD11c−CD123+) and conventional DCs  populations. The pDCs are the body's major producers of the anti-viral interferon (IFN)-α, though they constitute only 0.35% of PBMCs (+/CD141+) and cDC2 (BDCA-1+/CD1c+) subsets, while pDCs express BDCA-2 (CD303) and BDCA-4 (CD304)  –30.4 –30.−HLGiven the central role of DCs in sensing infection and orchestrating immune responses, it is not surprising that many pathogens have evolved immune evasion strategies which specifically target DCs in order to interfere with innate and adaptive immune responses –34. ThusPlasmodium parasites occur at every stage of the parasite life cycle within the human host: skin  studying peripheral blood DCs from currently or previously infected individuals, or (2) measuring DC responses to parasite stimuli in vitro. In the first method, DCs were isolated from the blood of individuals who were naturally or experimentally infected with malaria. The surface phenotype and function of these DCs was compared to uninfected controls, either the same individuals prior to or post-infection, or a matched control group  and were defective at antigen uptake and induction of naïve T cell proliferation in allogeneic T cell activation assays  anchors for merozoite surface proteins  or when parasitaemia reached 1,000 parasites per mL (low-dose group). Despite the low parasite biomass of the inoculum in the low-dose group, an estimated 20 times lower than the number of merozoites released from an infected hepatocyte after sporozoite replication  has produced valuable insights into antimalarial immunity. In one CHMI model which has been used to study DC in malaria, healthy volunteers who are typically malaria-naïve were inoculated with an ultra-low (<180) or low  dose of lication , DC numblication . Intriguost-cure . HLA-DR ost-cure . This st+ cDC2 activation  and ultra-low (150 pRBCs) dose to more closely study BDCA-1tivation  and pDC tivation  after coreatment . Howeverreatment , 92. A preatment .+ cDC2 from individuals in the high-dose group were impaired in their capacity to upregulate HLA-DR and CD86 in response to stimulation with TLR1/2, TLR4, and TLR7 ligands or whole pRBCs. This impairment was DC-specific, as monocytes' capacity for activation marker expression was unaltered by malaria exposure  upregulated caspase-3 during acute infection and after treatment, suggesting that the reductions in DC numbers in the periphery could also be due to increased apoptosis. Overall, DC impairment by P. vivax CHMI was largely similar to what was observed with P. falciparum  clarifying the phenotype of DC suppression, (2) determining precisely how malaria modulates DC function, and (3) identifying whether this is through direct interaction with DCs or indirectly through soluble mediators, including cytokines such as TNF-α.In light of this, Plasmodium spp. and human DCs in vitro. The majority of these studies have examined the responses of human monocyte-derived DCs (moDCs), since they can be easily generated in large numbers from CD14+ PBMCs or BM monocytes by co-culture with GM-CSF ± IL-4 , and cDC1 (BDCA-3+) cDC subsets and therefore do not accurately represent the diversity of DC populations or their functions in vivo , both of which secreted high levels of IFN-γ. TREG induced in this fashion also secreted high levels of IL-10 and TGFβ. Pre-incubating moDCs with parasite lysate did not affect their ability to undergo LPS-driven maturation ..H1 and rOne explanation proposed by Elliott et al.  for the bona fide DCs in future studies. A more rigorous definition of moDCs and how “activation” and “inhibition” are defined in these cells, particularly given how different groups have used different activatory cytokine stimulation methods to drive DC-like cells to begin with, will be imperative to resolve existing conflicts.In summary, while a dose-dependent relationship between pRBC dose and moDC inhibition is suggested, this relationship must be substantiated by further studies examining the individual roles that different parasite stimuli, strains, and methodological factors have on the final DC phenotype, preferably focusing on Plasmodium life stages have been poorly investigated. Only one study has investigated moDC responses to P. falciparum merozoites  did not . Howeverhemozoin . These chemozoin . The althemozoin  and DNAshemozoin  indicatedium DNA .P. falciparum DNA secreted high levels of RANTES, IL-12, and TNF-α. In this system, hemozoin assisted in trafficking parasite DNA to intracellular endosomes, and activated DCs via a TLR9- and MyD88-dependent signaling pathway  molecules, membrane anchors for Plasmodium surface proteins, are known ligands for TLR2 (Plasmodium infection (Plasmodium spp., it remains interesting that the DC response to malaria is not always activatory. While further studies should continue to identify Plasmodium ligands that drive DC activation, this must be studied in combination with the factors that underlie DC suppression in malaria.Murine studies have identified a number of other immunostimulatory for TLR2 , expressfor TLR2 . Parasitfor TLR2 , and TLRfor TLR2 , the majnfection , 147. Thnfection  and humanfection  macrophaPlasmodium is complex, and many aspects of cellular immunity remain poorly understood, particularly the impact of malaria on DC function. A better understanding of DC responses to Plasmodium will provide insight into the low efficacy and relative short duration of protection of the current malaria vaccine RTS,S, as well as the slow acquisition of natural immunity in malaria-exposed individuals. One of the most important unresolved questions is one of DC “suppression”: namely, whether exposure to Plasmodium spp., particularly P. falciparum, inhibits the ability of DCs to initiate and orchestrate effective immune responses. Determining precisely how DCs are modulated by P. falciparum is crucial for understanding the development of immunity to malaria.Immunity to Some of the most interesting insights into the effects of malaria-induced DC impairment come from field studies that stratified patients by severity of infection. Studies that analyzed mild vs. severe malaria cases separately did not observe significant differences in DC phenotype between the groups , 102, suSome valuable insights could be obtained by investigating differences in DC impairment between asymptomatic and symptomatic malaria cases. The systemic inflammation characteristic of symptomatic malaria undoubtedly contributes to DC dysfunction. While asymptomatic cases seem to experience similar loss of DC function short-term , 88, 99,Plasmodium parasites have on DCs in vitro. Particularly, care must be taken when stating that P. falciparum “suppresses” DC function in vitro: levels of activation lower than that seen in response to positive controls is not necessarily indicative of suppression. True suppression should be defined by an inability of DCs to become activated by known activatory stimuli, particularly pattern recognition receptor ligands. Analyses should also always account for increased cell death, which may result in false reports of suppression. Overall, the data indicates that DC function is not universally suppressed (Plasmodium appears to target specific pathways, among them the ones crucial for inducing naïve T cell proliferation (H1-like phenotype (H1 polarization will be important to understand the uniquely Plasmodium factors that delay the development of antimalarial immunity.Variations in methodology have made it difficult to ascertain the effects ppressed . Rather,feration , 104, whhenotype , 106. RePlasmodium spp. without grouping them into a monolithic conglomeration of HLA-DRhiLin− cells. In particular, elevation of the circulating BDCA-3+ cDC1 subset is a commonly reported phenomenon in field studies but, due to the low frequencies of this rare population, its function in malaria has not been extensively investigated. More rigorous strategies, including cell sorting, should be employed to study how purified subsets respond to malaria. Secondly, pDC cross-talk is essential for production of cytokines such as CXCL10 and IFN-α. CXCL10, also known as IP-10, is majorly implicated in malaria pathogenesis (There also appears to be an important role for DC cross-talk between pDC and cDC subsets , 107. Thogenesis –153, andogenesis , 155. Asogenesis , 155, stogenesis , and sinogenesis , detectiex vivo and in vitro studies depict a complex effect of malaria on the pDC subset. Numbers of circulating pDCs were reduced during natural infection (In vitro studies have observed IFN-α production when pDCs were directly stimulated with parasite products (Moreover, nfection , 97, 99 nfection , 95, thonfection . Whethernfection , suggestproducts , 85, 107Plasmodium life cycle, understanding the type of responses induced by each life stage is essential for designing new therapeutic interventions to protect the host against damaging immunopathology. A two-pronged “big data” approach could be particularly informative, with the use of RNAseq or proteomics on the DC side to examine immune pathways induced by each parasite life stage, and conversely a proteomics or other—omics-based approach examining the potential immunogens expressed by each parasite life stage. In particular, understanding the pattern recognition receptor signaling pathways which are inhibited or activated by pRBCs would enable more targeted therapies to reverse their suppressive effects. This would inform functional studies, and therefore form a roadmap for vaccine strategies or other therapeutic approaches that could induce potent, long-lasting antimalarial immunity.When interpreting existing literature, it must be kept in mind that the majority of studies have focused on pRBCs and only a minority have examined responses to extracellular forms of the parasite such as merozoites. Considering that DC responses change depending on the parasite life stage , 107, evPlasmodium life cycle is still out of reach, recent technological improvements have enabled development of models that allow study of immunity to specific life stages . For exCryptosporidium (Organoids, miniature models of organ function, have proven useful in studying tissues such as the liver  and inteporidium . Developporidium . Liver ain vitro studies using moDCs as a model may not be representative of the interactions between Plasmodium and steady-state DCs. Both moDCs and bona fide DCs show pro- and non-inflammatory responses to Plasmodium stimulation, but phenotypic and transcriptomic differences between them highlight that the moDC phenotype is pronouncedly different and may not necessarily be generalizable (+ DCs. However, recent findings outline that moDCs and CD16+ DCs exist as separate populations in the steady-state (Plasmodium infection, moDCs may not accurately reflect the behavior of steady-state DC populations.Finally, data from alizable . It was dy-state , 125, anex vivo and in vitro translate into effectiveness, duration, and quality of antimalarial responses. Including DC studies in vaccine trials would help to address which elements best describe a beneficial DC response. This should be supplemented by more studies of DCs in natural infection against comparable non-exposed donors. Thirdly, there is a need for better models to examine DC function in malaria. DC studies of the future should focus as much as possible upon bona fide DCs, and seek to develop new models that will permit a more in-depth study of how DC function is altered by the malaria parasite.This review has outlined many facets of DC function in malaria that are not well understood . Firstlyex vivo and in vitro Plasmodium-exposed DC phenotypes correlate with effective immunity, and the mechanisms that regulate DC interactions with Plasmodium will grant valuable insight into the acquisition of immunity, and form a basis for the development of better vaccines.To conclude, the complexities of DCs make them a relatively understudied cell type in the context of malaria, where they have a potentially pivotal role in the regulation of antimalarial immunity. Many gaps in knowledge remain to be addressed, and there is a prominent need for novel technologies to bridge the gap. A deeper, more rigorous understanding of how XZY wrote the first draft of the manuscript, which was reviewed and edited by RJL, JGB, and MOK. XZY and RJL prepared tables and figures. All authors have read and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present data from 3 cohorts in 1 geographical area with varying levels of malaria transmission. The results show that asymptomatic parasitemia is modified by transmission level and age, which alter the risk of developing febrile malaria. Plasmodium falciparum infections lead to febrile illness unless the host has sufficient immunity, in which case infection may cause no immediate symptoms . Previous studies are conflicting on the role of asymptomatic parasitemia in determining the risk of developing febrile malaria.We monitored 2513 children  by blood smears in 17 cross-sectional surveys to identify asymptomatic parasitemia and used active surveillance over 11325 child-years of follow-up to detect febrile malaria. We evaluated the interaction between transmission intensity, age, and asymptomatic parasitemia in determining the risk of developing febrile malaria.In the moderate and high transmission intensity settings, asymptomatic parasitemia was associated with a reduced risk of febrile malaria in older children (> 3 years), while in the lower transmission setting, asymptomatic parasitemia was associated with an increased risk of febrile malaria in children of all ages. Additionally, the risk associated with asymptomatic parasitemia was limited to the first 90 days of follow-up.Asymptomatic parasitemia is modified by transmission intensity and age, altering the risk of developing febrile episodes and suggesting that host immunity plays a prominent role in mediating this process. Plasmodium falciparum infections are characterized by disease manifestations that range from asymptomatic infection, mild febrile episodes, through to severe malaria. Asymptomatic parasitemia is widespread, affecting 24% of the population in sub-Saharan Africa [In 2016, there were an estimated 216 million new malaria cases and approximately 445000 deaths globally, making malaria a significant health problem [n Africa , 4, lowen Africa , low birn Africa , and anen Africa . These dn Africa .There are reports that asymptomatic infections are associated with reduced febrile malaria in infected individuals. Premunition is one mechanism by which individuals with asymptomatic parasitemia have been proposed to develop partial immunity to superinfection and resulting febrile malaria by long-term asymptomatic carriage of parasites , 12. In Individuals living in high malaria transmission settings have been shown to have higher levels of immunity , 22 compSome previous studies have found that asymptomatic parasitemia is associated with increased risk but other studies found a reduced risk of subsequent febrile malaria. In a study of 316 children aged 6 months to 5 years in an area of moderate malaria transmission in Uganda, children with asymptomatic parasitemia were at a higher risk of febrile malaria compared to the uninfected children within the first 30 days of detection . LikewisOverall, in the high transmission settings there was a reduced risk of developing febrile malaria and this could be stratified by age, while the moderate transmission settings showed an increased risk of developing febrile malaria. However, the limitations of these studies are that they were conducted using differing methodologies and therefore conclusions based on the assumption that differences in transmission intensity and age explain the differing results require confirmation.We therefore sought to evaluate the role of malaria transmission intensity and age as potential effect modifiers on the risk of children with asymptomatic parasitemia developing febrile malaria, using data from 3 cohorts of children in different transmission intensities in Kilifi, Kenya.The studies presented here were covered under the Kenya Medical Research Institute Scientific Ethics Research Unit protocol number 3149. The data used for this analysis were based on 3 cohorts in Kilifi of varying malaria transmission intensities, comprising Ngerenya (low transmission), Junju (moderate to high transmission), and Chonyi (high transmission). The data were prospectively collected between 1998 and 2014 for Ngerenya, 2005 and 2010 for Junju, and 1999 and 2001 for Chonyi. In these cohorts children were recruited at birth for weekly clinical malaria monitoring until the age of 15 years .Although the transmission intensity was on a general decline during the study period , 29, it P. falciparum parasites by microscopy (thick and thin smears) and having an axillary temperature less than 37.5°C and no history of fever during the cross-sectional survey. Additionally, the children should not have had a recent febrile malaria episode within the month prior to the survey or had a fever within the subsequent 7 days from the date of the survey. Uninfected children were defined as those without P. falciparum parasitemia and no fever during the cross-sectional survey.We defined asymptomatic parasitemia as having any detectable A febrile malaria episode was defined as having ≥ 2500 parasites/µL and an axillary temperature greater than 37.5°C. This definition is based on parasite cutoffs previously defined for these cohorts ; howeverSurvival analysis was used to compare the risk of developing febrile malaria between the asymptomatic and uninfected children and survival times were estimated as follows: the baseline (time 0) was defined as the date of cross-sectional survey while the end of follow-up was set at the last visit or at 365 days if follow-up continued to the subsequent annual cross-sectional survey; the time to first febrile malaria episode was defined as the interval between the baseline date and the first febrile malaria episode. Children who did not complete follow-up or did not get a febrile episode by the end of follow-up were censored.We used Kaplan-Meier curves and log-rank test to compare time to first febrile malaria episode and the Cox proportional hazards model using transmission intensity and asymptomatic parasitemia as categorical variables, and age as a continuous linear and nonlinear variable . We adjusted for calendar year as a continuous variable to account for falling transmission intensity and the nonindependence of repeated observations using the robust cluster Huber-White method. We tested the proportional hazards assumption using Schoenfeld residuals and included interactions with time for the covariates that showed nonproportional hazards.Data cleaning, plotting of Kaplan-Meier curves, and log-rank tests were carried out in R v3.5.1  using thA total of 2644 children were recruited into the study over 17 cross-sectional surveys. Twenty children were excluded due to missing data on age, bringing the person-years of follow-up to 12543. A total of 1482 person-years of follow-up were excluded from the cross-sectional surveys for the following reasons: 249 lacked data on parasitemia or temperature, 220 had a febrile nonmalaria episode, 99 had febrile malaria, 139 asymptomatics who did not meet our criteria for defining an asymptomatic infection (see Methods), and 511 lacked weekly surveillance data. Consequently, we analyzed data on 2513 children, representing 11325 person-years of follow-up . There wP value <.0001).We compared the risk of developing febrile malaria between children with asymptomatic parasitemia and uninfected children at baseline. Uninfected children were at a higher risk compared to children with asymptomatic infections from the start of follow-up. This difference in rates of developing episodes was more distinct after 90 days, when the 2 survival functions diverged to the end of follow-up  in the moderate to high and in the high transmission settings. However, among the older age groups (> 3 years) in the moderate-high and high transmission settings, asymptomatic parasitemia was associated with a reduced risk of developing febrile malaria .There were some variations in effect over time. The risk among children with asymptomatic infection decreased over time relative to the risk among uninfected children. In settings where asymptomatic infection was associated with increased risk  the survival functions of asymptomatic children compared to uninfected children diverged before 90 days, and then became parallel after 90 days. This contrasts with settings where asymptomatic parasitemia was associated with reduced risk , in which the survival functions of asymptomatic and uninfected individuals were similar before 90 days, and then showed an increased risk among children with asymptomatic parasitemia.P < .0001; P < .0001), between transformed age and transmission intensity (P < .0001), between asymptomatic parasitemia and transformed age (P < .0001), as well as between asymptomatic parasitemia and year of survey (P < .0001). The variations in the effects of infection status and year of survey over time were statistically significant , transformed age, sex , year of survey and time cutoffs , and their interactions. Age as a nonlinear transformation, using the multiple fractional polynomial approach, was found to be a better fit than a linear variable  to evaluate risk . The effAsymptomatic parasitemia before the malaria transmission season predicted the risk of developing febrile malaria. This effect was significantly modified by transmission intensity and age. In the moderate to high and high transmission settings, asymptomatic parasitemia was associated with a reduced risk of febrile malaria in older children (>3–15 years). There was, however, no association between asymptomatic parasitemia and risk in younger children in the moderate to high and high transmission settings. In contrast, in children of all ages in the lower transmission setting, asymptomatic parasitemia was associated with an increased risk of febrile malaria. The effect of asymptomatic parasitemia was modified by time since ascertainment. At low transmission, asymptomatics were at a higher risk than uninfected children until 90 days after ascertainment, following which asymptomatics were at similar risk to uninfected children. On the other hand, at higher transmission and older age, asymptomatics were at a similar risk as uninfected children until 90 days after ascertainment, following which asymptomatics were at a lower risk than uninfected children.This study is in agreement with studies conducted in high transmission areas where asymptomatic parasitemia has previously been associated with a reduced risk of febrile episodes , 25, 26,Children living in malaria endemic regions with high transmission acquire immunity at a faster rate than those living in low endemic regions, as they are repeatedly exposed to infections . While iThe 90-day period of increased risk following the ascertainment of asymptomatic parasitemia suggests there is a period during which parasitemia may lead to febrile malaria, but thereafter the likelihood of the parasite escaping immune control is reduced, hence also reducing the risk of febrile infections. Alternatively, the increased risk of febrile episodes may reflect increased exposure associated with the asymptomatic parasitemia  or exposWe found in our cohorts that every 10-fold increase in parasite density was associated with an increased risk of developing a febrile episode. We speculate that higher-density asymptomatic parasitemia indicates less host immunity and therefore a greater risk that parasites will evade immune control.Limitations of this study include the fact that it was conducted in a single geographical area . However, we included 3 different sites at different transmission settings and our findings are in agreement with those from other studies , 23–26. Our study cannot be used to infer the effects of treating asymptomatic parasitemia. Randomized trials of intermittent presumptive treatment show benefits across a range of transmission intensities and ages , despiteAdditional studies are required to examine whether episodes of febrile malaria are associated with parasites of a similar genotype to those involved in the prior asymptomatic infection, or whether the increased risk applies to all parasites. The former would imply a specific risk of asymptomatic parasitemia limited to a specific infection episode, and the latter would imply a general increase in susceptibility not limited to any specific episode.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiy591_suppl_Supplementary_Table_01Click here for additional data file.jiy591_suppl_Supplementary_Table_02Click here for additional data file.jiy591_suppl_Supplementary_Table_03Click here for additional data file.jiy591_suppl_Supplementary_Table_04Click here for additional data file.jiy591_suppl_Supplementary_Table_05Click here for additional data file.jiy591_suppl_Supplementary_Table_06Click here for additional data file.jiy591_suppl_Supplementary_Table_07Click here for additional data file.jiy591_suppl_Supplementary_Table_08Click here for additional data file.jiy591_suppl_Supplementary_Table_09Click here for additional data file.jiy591_suppl_Supplementary_Table_10Click here for additional data file.jiy591_suppl_Supplementary_Table_11Click here for additional data file.jiy591_suppl_Supplementary_MaterialClick here for additional data file.jiy591_suppl_Supplementary_FigureClick here for additional data file.


The MORDOR study, a cluster randomized clinical trial, showed that single-dose azithromycin (20 mg/kg) administered biannually for 2 years to preschool children reduced mortality; a study was conducted to determine its effect on clinical symptomatic episodes of malaria as a potential mechanism for mortality benefit.A randomized control trial (RCT) was conducted, whereby 30 randomly selected communities in Kilosa District, Tanzania were randomized to receive 6-monthly treatment of children ages 1–59 months with single-dose azithromycin (20 mg/kg) vs. placebo. A prospective cohort study was nested within the RCT: children, aged 1 to 35 months at baseline, were randomly selected in each community and evaluated at 6-monthly intervals for 2 years. At each visit, the children were assessed for recent or ongoing fever and anti-malarial treatment; a rapid diagnostic test (RDT) for malaria was performed. The two major outcomes of interest were prevalence of RDT positivity and clinical malaria. The latter was defined as RDT-positivity with fever at time of evaluation and/or reported fever in the 3 days prior to evaluation. Methods that account for correlations at community level and within individuals over time were used to evaluate associations.At baseline, the prevalence rates in the children in the azithromycin and placebo arms were 17.6% vs. 15.5% for RDT positivity (p = 0.76) and 6.1% vs. 4.3% (p = 0.56) for clinical malaria. There was a decline in both RDT-positivity and clinical malaria over time in both arms. The difference by treatment assignment was not significant for clinical malaria; it was significant for RDT-positivity with greater odds of decline in the placebo arm (p = 0.01).Lack of evidence for a significant difference in the prevalence of clinical malaria in children at any visit following treatment suggests that the effect of single-dose azithromycin on malaria is at best transient and limited in scope. Chance overrepresentation of non-seasonal transmission in the communities in the azithromycin arm may account for higher rates of RDT-positivity and less decline over time.Trial registration Clinicaltrials.gov NCT02047981 The MORDOR study, a multinational cluster randomized clinical trial found that biannual mass treatment of preschool children with azithromycin, was associated with a reduction in all-cause mortality . The mecIn the morbidity component of the MORDOR study, a cohort of children, residing in 30 randomly selected communities in Kilosa, Tanzania, were recruited to determine the effect of azithromycin on morbidity indices that included malaria. Pre-school age children that were living in the selected communities, were randomized to undergo biannual treatment with azithromycin or placebo, were evaluated longitudinally every 6 months for 2 years. The purpose of this study was to determine if there was a preferential decline in malaria in the children residing in the communities in the azithromycin arm.The MORDOR trial was a cluster-randomized, placebo-controlled, double-masked clinical trial to evaluate the effect of biannual, single dose azithromycin (20/mg/kg single dose) on mortality in children aged 1–59 months  for Change in the prevalence of RDT positivity and clinical malaria in the cohort over time were the outcomes of interest. Recent exposure to malaria was defined as positivity on the RDT. Clinically symptomatic malaria was defined as being RDT positive with either the presence of fever OR a report of fever in the 3 days preceding evaluation. Those children who were taking an anti-malarial drug at the time of the survey, were included in a subgroup of clinical malaria.Bivariate analyses were performed to evaluate cross-sectional differences in malaria indices by treatment assignment, and the Generalized Estimating Equation approach was used to test for differences while accounting for clustering at the community level. The presence of separate outcomes of RDT positivity, and clinically symptomatic malaria were examined for each of the biannual visits. Mixed effect models that include age, time, treatment arm, and the interaction of treatment arm and time as independent predictors were used to evaluate differences between treatment arms in the cohort of children over time. The models account for clustering at community level and repeated measures in the same children over time. At baseline we show the distribution of RDT positivity by community (not at child level) just to show the heterogeneity of the prevalence. This was calculated using the proportion of RDT positivity in the cohort for each community; the results were displayed graphically by treatment arm Analyses were conducted in SAS (Carey NC) using the GEE and GLIMMIX procedures.Ethical approval was obtained from the Tanzanian National Institute for Medical Research and the Institutional Review Boards of the Johns Hopkins School of Medicine and University of California San Francisco. Children were included in the study on the basis of documented written informed consent from guardians. The study is registered at clinical trials.gov (NCT02048007). A data and safety monitoring committee provided trial oversight.At baseline, there was no significant difference by age, gender, or any of the malaria indices in the children residing in communities randomized to azithromycin or placebo Table . The folAt baseline, there was significant variation in the proportion of RDT positive children by community. There were some communities in both arms that had zero prevalence, while two communities in the placebo- and four communities in the azithromycin arm had > 30% prevalence of RDT positivity Fig. .Fig. 2NuRDT positivity in the cohort of children showed a seasonal decline at 6- and 18-months for those in the placebo arm; a comparable decline was not shown in the same periods in the children in the azithromycin arm Table . Of all Overall, accounting for repeated measures over time, there were higher rates of RDT positivity over time in children residing in the communities randomized to azithromycin compared to children residing in the placebo-treated communities Table . HoweverAdjusting for age as well as the interaction of treatment arm and time, the children residing in the placebo-treated communities showed a modest but significant decline in both RDT positivity and clinical malaria over time Table . The chiThere was no difference in the months or seasons that the surveys were conducted between azithromycin vs. placebo communities at each 6-month interval. A map of the location suggested clustering of intervention villages in north-east Kilosa where malaria was uncommon, and to be in the hills where non-seasonal (i.e. year-round) malaria transmission is more common Fig. .Fig. 3MaGlobally, malaria remains the foremost parasitic infection in humans. In 2016 alone, there were an estimated 216 million clinical cases of malaria and 445,000 deaths. The overwhelming majority of clinical cases (85%) and malaria deaths (90%) occur in sub-Saharan Africa, of which children under five are disproportionately affected . DespiteIn a cohort of preschool children in Tanzania, who had been randomized to biannual treatment with azithromycin or placebo, a difference in clinically symptomatic malaria was not shown by treatment arm, 6 months following treatment over a period of 2 years. There was wide variability in the prevalence of RDT positivity across the communities, suggesting absent transmission in some communities yet high rates of transmission in others. Variations in malaria prevalence in Kilosa district has been reported, previously , 22. ThiP. falciparum histidine-rich protein may persist for weeks following treatment [Only a small proportion of those who were RDT positive had symptoms (i.e. fever). While RDTs are specific, they fail to discriminate recent infection with absent parasitaemia, from active parasitaemia. This can be challenging in areas of high endemicity, particularly given that reatment . PertineA decline in clinical malaria was demonstrated in the children in both arms of the study, which was notable at the last visit. While this may reflect aging of the cohort, it could also reflect the previous year’s active district programme in distribution of insecticide-treated bed nets to pregnant women and children through antenatal clinics . Data were not collected on bed net coverage or use, but the decline in RDT positivity and clinical malaria suggest this may be a factor. A Kilosa-based study in 2011 of malaria preventive measures in pregnancy showed that almost all (98%) women of reproductive age had an insecticide-treated bed net in their households , suggestUnlike RDT positivity, a significant difference was not found in the rates of clinical malaria in children by community treatment assignment at each 6-month survey after treatment. As described above, despite the higher rates of RDT positivity in the children in the azithromycin arm, it may be that the azithromycin resulted in a lower rate of symptomatic malaria. A previous study found a 66% reduction in the odds of PCR determined infection following mass drug administration for trachom . NonetheP. falciparum, Plasmodium malariae, febrile parasitaemia and rates of palpable splenomegaly in those who received the three doses of azithromycin. Similarly, a cluster randomized trial in Niger showed significantly lower rates of parasitaemia in those communities that underwent two vs. single mass distribution of azithromycin 4–5 months post-treatment [Mass drug administration with azithromycin for trachoma control has shown benefit elsewhere. One study in Gambia, evaluated malaria indices in children aged 5–14 years following MDA of azithromycin for trachoma . Four vireatment .The study has limitations. Foremost, the parent study was conceived primarily to test effect of mass treatment with azithromycin on all-cause mortality. In the event that a benefit was found, the morbidity arm was intended to offer insight into the mechanism for such an effect, specifically on bacterial infections. As such, while the morbidity arm allowed for ancillary study of malaria, rigorous microscopic review was not performed. Microscopy is ideally performed by two skilled microscopists, with a third microscopist used to adjudicate discordant results. Instead, microscopy for this study was undertaken by a clinical technologist at the local hospital in Kilosa. Given resource constraints coupled with the primary scope of the morbidity study, review was restricted to RDT positive cases alone. As such, the findings of a relatively modest rate of smear positivity were not surprising and highlight variable sensitivity of microscopy, which is an observation that has been well documented by others –29.In this regard, RDTs have been pivotal to advancing control strategies for malaria . Their lSecond, even with randomization of communities into the placebo and azithromycin arm, chance selection of more villages with year around transmission in the azithromycin arm complicated interpretation of the RDT/clinical symptom results. Even though the district is endemic for malaria, there is clearly significant heterogeneity across and even within communities. This was reported previously, where significant variation in the prevalence of malaria was found at sub village level in Kilosa .Other limitations include some attrition, which resulted in incomplete data on the cohort. However, those with clinical malaria were noted to be more likely to attend the next study visit; this could be attributed to provision of treatment to clinical cases at the time of the survey. Another is the follow-up interval, 6 months after treatment, which is acknowledged to be long in terms of observing an immediate benefit. However, it is uncertain as to whether there might be a longer-term benefit of treating twice a year for 2 years, such that there would be cumulative differences apparent at 2 years. This did not seem to be the case, as seen over time in the cohort analysed by treatment arm at each time point. Finally, there are challenges around the definitions of clinical malaria that were used for this study: for one, fever is non-specific, and it is possible that children were RDT positive and had fever due to another cause, but which was ascribed to malaria. However, if this were the case, it would affect both arms of the study, and should not bias the findings; it would only increase the number of clinical cases. In addition, RDT testing can be negative in those who are already on anti-malarials at time of evaluation. In such cases, the level of antigenaemia may have fallen below the RDT detection limit leading to a false negative despite having recent exposure to malaria. For this reason, this group was included under an alternative definition of clinical malaria and still found no differences.While previous research suggests that azithromycin has modest anti-malarial activity, a significant effect on clinical malaria or prevalence of RDT positivity was not demonstrated in these cohorts. Further, no evidence was found to support a reduction in risk of RDT positivity or clinical symptoms over time following multiple 6-monthly azithromycin dosing.


Baseline CSP IgG levels are elevated in malaria cases than controls (p < 0.001). Both, IgG magnitude to NANP  and avidity to C-terminus (0.07 [0.05–0.90]) post-vaccination are significantly associated with vaccine efficacy. IgG avidity to the C-terminus emerges as a significant contributor to RTS,S/AS01E-mediated protection.RTS,S/AS01E has been tested in a phase 3 malaria vaccine study with partial efficacy in African children and infants. In a cohort of 1028 subjects from one low (Bagomoyo) and two high  malaria transmission sites, we analysed IgG plasma/serum concentration and avidity to CSP  after a 3-dose vaccination against time to clinical malaria events during 12-months. Here we report that RTS,S/AS01E induces substantial increases in IgG levels from pre- to post-vaccination ( RTS,S/AS01E has been tested in a phase 3 malaria vaccine trial and has shown partial efficacy in children and infants. Here, the authors analyze IgG concentration and avidity to CSP in ~1000 participants and show that IgG avidity to the C-terminus of CSP is significantly associated with vaccine-mediated protection. Vaccine efficacy against clinical malaria over a 12-month follow-up period estimated in the African phase 3 randomised controlled trial was 55.8% in children (5–17 months old at study start)1 and 31.3% in infants (6–12 weeks old at study start)12. The vaccine is composed of virus-like particles consisting of the hepatitis B virus surface antigen (HBsAg) and the malarial target antigen, a fragment of the Plasmodium falciparum circumsporozoite protein (CSP) spanning the central region containing blocks of four amino acid repeats (NANP) and the C-terminal (C-term) part and formulated with AS01E adjuvant13. Previous paediatric phase 1/215 and the phase 316 clinical trials in Africa have shown that RTS,S in its various adjuvant formulations induces high IgG antibody titres to CSP NANP repeat that remain above naturally-acquired levels for years17. Presently, it is unclear how the vaccine protects against clinical malaria, and why and how the immune mechanism of protection might be affected by age at first immunisation and malaria transmission intensity (MTI)18.The RTS,S/AS01E is the most advanced malaria vaccine, consistently providing partial protection against clinical malaria in African children16. Humoral responses to the subdominant C-term domain have been evaluated in healthy malaria-naïve adults but not in malaria-pre-exposed populations19. The C-term domain is structurally conserved20 and B cell epitopes in this region have not been characterized. Upon immunisation with P. falciparum irradiated sporozoites, monoclonal antibodies against the C-term are rarely induced in malaria naïve volunteers and thus far have not demonstrated to mediate protection21. The C-term domain contains a well-known promiscuous CD4+ T-cell epitope that is conserved among all parasite isolates22.Thus far, vaccine immunogenicity measurements have focused mainly on the NANP repeat as the immunodominant B cell epitope16, others have shown that this response is not associated with protection against clinical malaria24. The biological function of IgG against the dominant and subdominant CSP epitopes differs, which subsequently could have an impact on protection. Analyses of RTS,S/AS01-induced antibodies in phase 1/2a trials suggested that protected individuals had higher anti-NANP-specific IgG titres but of low opsonisation activity. In contrast, antibodies targeting the C-term in humans were associated with phagocytic activity19. Loss of C-term specificity in the overall antibody response to CSP greatly impaired protective efficacy to P. berghei in mice25. The C-term has been shown to take part in the initial entry of sporozoites into hepatocytes26 and therefore, antibodies to this CSP fragment may play a role in protection. There is recent evidence from ongoing studies in our group of an association between post-vaccination HBsAg IgG levels and malaria protection27 unreported in previous RTS,S field trials. This might be explained partly by correlation with CSP IgG levels16 thus further investigation is required.Although the magnitude of anti-NANP repeat antibodies elicited by RTS,S has been associated with vaccine efficacy in some studies, including the phase 3 trial29. Changes in NANP IgG avidity and concentration following second and third doses, however, were associated with reduction in clinical malaria risk. A recent phase 2a trial in malaria naïve adults showed that a delayed fractional dose boosting regimen increased antibody avidity and sustained higher protection upon rechallenge30. Therefore, these outcomes merit further evaluation as surrogate markers for RTS,S efficacy. Prior phase 2b trials suggested that IgG avidity was higher in children with high malaria exposure compared to those with low malaria exposure one-month post third vaccine dose28. The phase 3 trial conducted under a wide range of MTI provides a unique opportunity to address knowledge gaps of RTS,S immunogenicity, immune mechanisms mediating protection, and their determinants.In addition to understand epitope fine-specificity of RTS,S/AS01E-induced IgG, it is essential to evaluate quality and quantity of these IgG as a possible indicator of their biological function. Studies conducted in prior phase 2 trials evaluated the avidity of anti-NANP IgG responses in RTS,S vaccinees and found no correlation between avidity post-third dose and vaccine efficacyThis study aims to search for antibody correlates of RTS,S/AS01E vaccine-induced protection against malaria by analysing IgG concentrations and avidity to two CSP epitopes (NANP and C-term), and IgG concentrations to HBsAg while considering the effect of age at first vaccination, MTI, and malaria IgG concentrations at baseline. We hypothesise that vaccine efficacy also relies on the contribution of anti-C-term domain binding IgG. Here we show that the magnitude of IgG responses to CSP NANP and C-term and HBsAg, and CSP antibody avidity, are significantly associated with RTS,S/AS01E vaccine efficacy. Our data show that IgG avidity to CSP C-term is important for RTS,S-induced protection against clinical malaria.p-Schonfeld test = 0.04) and, as in the phase 3 RTS,S/AS01E trial, varied across age groups .RTS,S/AS01E and comparator vaccinees  binding to CSP NANP and C-term at M3 compared to M0  . The concentration of CSP IgG in RTS,S compared to comparators at post-vaccination (M3) was on average 6000 times higher for NANP and 1600 times higher for C-term (P < 0.001 for both ratios). IgG concentration (geometric mean [GM]) and avidity index (AI) at M3 were 1652 and 0.39 for CSP NANP, respectively, and 1241 and 0.10 for C-term. In spite of these differences, the concentrations of IgGs to both CSP antigens were highly correlated (Pearson-r = 0.93)  and post-vaccination in RTS,S/AS01E vaccinees (Pearson-r = 0.66), possibly due to the narrower range of concentrations  between NANP and C-term AI  Fig. . Percent93) Fig. . Correlaons Fig. . There w AI Fig. .Fig. 1EfAge at first vaccination and study site significantly affected RTS,S/AS01E immunogenicity Fig. . The magp for all pairwise comparison of trajectories between sites < 0.001). Among all other candidate predictors, only weight-for-age Z-score (WAZ) had a significant association with immunogenicity (Supplementary Table Volunteers from Nanoro (highest MTI) had higher concentrations of anti-NANP and anti-C-term IgG than those from Kintampo at baseline, and at both sites antibody levels were higher than in Bagamoyo (lowest MTI) . In models including both antigens, NANP IgG concentrations continued to be independently associated with first clinical malaria but not C-term , among other factors17.We demonstrate here that anti-CSP IgG concentrations and avidity are correlates of RTS,S induced-protection and that these are affected by age, site and baseline. Our findings confirm data from previous phase 3 analyses showing that RTS,S/AS01E vaccination elicits a potent IgG response to the immunodominant NANP and at higher concentrations in children than infantsWe expanded the analysis of CSP epitope targets by including the C-term domain. We demonstrated that RTS,S/AS01E vaccination induces IgG to this subdominant region and that despite their lower concentrations compared to anti-NANP, these antibodies were also associated with vaccine efficacy.29. A challenge study in naïve adults showed an association between higher avidity and protection30. Here, we showed that malaria-exposed children generated vaccine-induced antibodies with higher AI than infants. This suggests that, in addition to high concentrations, affinity matured antibodies that probably exert better biological functions confer protection against malaria. When multivariable Cox models were adjusted for the other CSP construct and/or the other metric (concentration or AI), the relationship between IgG recognising NANP and malaria protection was different from that of IgG to C-term. Notably, the association between C-term AI and malaria protection remained significant even after adjusting for NANP IgG concentration. Taken together, data indicated that for the NANP region, antibody concentration might be more important in protection than avidity, whereas for the C-term region, the opposite was observed. Indeed, exploratory data analyses pointed to different response patterns in concentrations vs avidities for C-term and NANP. Therefore, the association of one metric with malaria risk was not necessarily confounded by the other metric/antigen.In addition, we assessed not only the magnitude but also the quality of vaccine-induced antibodies and their relevance to protection. Two field studies of RTS,S evaluated the avidity of anti-NANP CSP IgG in African children and found no correlation between M3 avidity and vaccine efficacy27, or might also reflect an impact of the HBsAg antigen on the affinity maturation of CSP-specific antibodies resembling the carbohydrate-based conjugate vaccines. As the biological relevance of such association remains unclear, mechanisms and implications for potential interactions between HBsAg and CSP specific responses will be the focus of future studies.The magnitude of the HBsAg IgG response was also correlated with time to malaria event, in the first six months after vaccination. This finding could either reflect vaccine take as HBsAg is a component of RTS,S/AS0E, thus related to the correlation of anti-CSP and anti-HBsAg IgG titers (rho ≅ 0.7 in this and other studies)3. Here, in the analysis of factors affecting the association between antibody concentrations and malaria events, infants had an increased risk compared to children to acquire malaria. The higher antibody concentrations and AI in children compared to infants could partially explain why RTS,S is more efficacious in older ages3. The nature and kinetics of IgG affinity maturation to dominant (NANP) vs subdominant (C-term) epitopes could be different, and this may also impact the age-related pattern of association between antibodies and malaria protection.We confirmed that RTS,S/AS01E immunogenicity was higher in the children than in infants. Previous correlates analyses within the phase 3 trial found an association between high tertile M3 NANP IgG titres and vaccine efficacy over the long term follow-up (M30) in infants but not in childrenP. falciparum antigens—therefore representing markers of malaria exposure27. In children older than 10 months, baseline CSP IgGs would likely be acquired upon infection and increase with age and MTI. A prior analysis of the effect of anti-NANP CSP IgG within the phase 3 trial reported that pre-vaccination titres were associated with lower RTS,S immunogenicity in infants and higher immunogenicity in children16. We confirm a significant correlation between higher M0 and lower M3 CSP NANP IgG concentrations and expand this for C-term IgG. These associations were significant when infants and children were analysed together, but they were more prominent for NANP IgGs and for infants. Subjects who developed malaria during the 12-month follow-up period had higher concentrations of IgG CSP at baseline and induced lower concentrations of IgG at M3, thus the increments from M0 to M3 in the non-protected subjects were lower than in protected volunteers. Thus, naturally-acquired CSP antibodies  were markers of exposure, while RTS,S/AS01E-induced CSP (M3) are markers of protection. Malaria episodes during the vaccination period were also associated with increased malaria risk post-vaccination. As malaria exposure may affect the phenotype and function of immune cells needed for antibody production, less exposed children at M0 appear able to generate higher responses at M3, and may benefit most from RTS,S vaccination. This may be important for the deployment of vaccines in general as prior parasite clearance may increase vaccine take.Furthermore, we assessed if the concentration of baseline antibodies to RTS,S antigens affected the outcome of vaccination at the individual level. Presence of CSP antibodies at time of first vaccination due to maternally-transferred antibodies during the first months of life and/or to malaria exposure could impact RTS,S immunogenicity, and this may affect infants and children differentially. Indeed, baseline concentrations of anti-CSP IgG were higher in subjects from Nanoro than those in Kintampo or Bagamoyo. We have shown previously that baseline levels of CSP IgG in infants aged less than 10 months (thus presumably transferred in utero from the mother) correlate positively and significantly with baseline levels of IgG to Finally, we explored the effect on vaccination and protection of other anthropometric variables not analysed thus far. We found some associations for sex and WAZ, but patterns were not consistent to draw conclusions. The impact of nutrition on vaccine take merits further investigation in larger studies in case an intervention could improve effectiveness.P. falciparum antigens at baseline in relation to M3 peak and long-term maintenance of responses.One limitation of our study design was that some baseline sera were missing, particularly for children from Nanoro, and thus we could not evaluate the baseline effects at the individual level in the whole study population. In addition, only 5–17-months-old children were enrolled at Bagamoyo for the immunology study as per local protocol. Thus, the findings related to site could be confounded by the effect of age. Therefore, further studies are warranted to assess in more depth the effect of maternally-transferred antibodies and exposure to other In conclusion, we generated data on RTS,S/AS01E immunogenicity including the C-term of CSP and evaluated antibody avidity as a quality index essential for immune effector mechanisms. While our current efforts are devoted to better define the type and subclasses of antibodies elicited by RTS,S vaccination, next steps include the assessment of the functionality of CSP-specific IgGs in regards to protection, and the evaluation of the response kinetics with and without a fourth booster dose at month 20. The elucidation of the mechanisms underlying RTS,S/AS01E-induced partial protection is essential for guiding rational development and deployment of next generation vaccines.16. These three sites were prioritized because they collectively fulfilled the above criteria due to availability of sufficient numbers and volumes of samples to address the study objectives. The follow-up by passive case detection (PCD) for clinical malaria events (fever ≥ 37.5 °C and parasitaemia of any density) was defined for the 12 months starting 14 days after the third vaccine dose. The authors have complied with all relevant ethical regulations for work with human participants. Written informed consent was obtained from parents or guardians before the start of the work. The study protocol was approved by the following relevant institutional or national Ethics Committees: National Institute for Medical Research, Ifakara Health Institute (IHI IRB), Tanzania; Ethics Committee of the University and State of Basel (EKBB), Switzerland; Kintampo Health Research Centre (KHRC) Institutional Ethics Committee (IEC), and Noguchi Memorial Institute for Medical Research IRB, Ghana; Institutional EC IRSS and Comite d’Ethique pour la Recherche en Sante, Burkina Faso; Comitè Ètic d’Investigació Clínica , Barcelona, Spain; Research Ethics Committee (REC), PATH, USA.This ancillary immunology study (MAL067) was nested in the phase 3 RTS,S/AS01E randomised controlled trial  conducted between 2009 and 2014 in sub-Saharan African countries with differing MTIs, including two age cohorts: infants aged 6–12 weeks and children aged 5–17 months at first vaccine dose. Volunteers were vaccinated with either a comparator vaccine or a course of three doses of RTS,S/AS01E given at study months 0, 1 and 2 . Serum or plasma was collected at baseline (M0), and approximately 30 days after the third vaccine dose (M3). Samples selected were from a subset of 1028 subjects enrolled in 3 out of 7 trial sites participating in the MAL067 study  and had (i) one-year cumulative incidence high enough to yield 80% power to detect moderate HRs, (ii) available M3 samples from infants and children , and (iii) varying MTI 2NNANPNANPNANPNANPNANPNANP-C-COOH). The C-term peptide covered 66 amino acids (Ahx-EPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCS-NH2). Both antigens were sourced by IAVI-HIL from JPT peptide Technologies . Antigen coating concentration was at 1 μg mL−1.The NANP synthetic peptide used for this study was based on 6 copies of the CSP central repeat sequence (H−1) (provided by PATH-MVI) specific to either C-term or NANP. Curves were assayed in duplicate and 9-step 3-fold serial dilutions starting at 1:200. Positive, negative and blank controls were assayed in each plate in duplicate. Negative control sera were obtained from healthy adults living in non-endemic malaria areas (seronegative for CSP). The positivity threshold was defined as the mean + 2 standard deviations (SD) of the negative controls . Positive controls were a pool of serum samples from RTS,S/AS01-vaccinated individuals (provided by GSK). Acceptance limits were established prior to processing samples based on Westgard rules for this positive control.IgG titration and avidity assays were performed on the same plate at IAVI-HIL. Each plate included 6 samples run in two sets of duplicates and 2-step 12-fold serial dilutions . Antigen-specific standard curves were implemented using monoclonal antibodies of known concentration  provided by IAVI-HIL (for NANP > 1.43 EU mL−1 and for C-term > 2.79 EU mL−1).The indirect ELISA was performed using 1:6000 rabbit anti-human IgG HRP conjugate , Sureblue TMB Peroxidase substrate  and TMB Stop Solution . For the avidity assay, 1 set of test samples and positive control duplicate wells were incubated for 30 min with a chaotropic agent , whilst the 2nd set of the same samples in duplicate wells on the plate were incubated with buffer alone. IgG concentration was reported in EU mLt-tests or Wilcoxon rank-sum tests, as appropriate. Graphical and basic exploratory comparisons between vaccination groups were done through boxplots and t-tests (Welch corrected). Associations between antibodies to different antigens were sought through linear regression and correlation coefficients.For the descriptive analysis of demographic, clinical, and other characteristics of subjects between RTS,S/AS01E and comparator vaccinees, categorical variables were compared through summary statistics, Pearson Chi-square or Fisher tests, and continuous variables through 10-transformed IgG concentrations for each antigen at M3 and change of concentrations from M0 to M3 through tests of the corresponding fixed effects (via combinations of the estimated coefficients), including random intercept and interaction terms. Additionally, in those models, we considered adjustments for age cohorts and sites and compared the effect of vaccination on changes of IgG concentrations from M0 to M3 and on concentrations at M3 across age cohorts and sites through interaction tests.Mixed linear models investigated the effect of the vaccine on the logarithmt-tests of log10 AI.The impact of age cohort and site on AI focused on antibodies at M3, only in RTS,S vaccinees with anti-CSP IgG concentrations greater than the LLOQ. Analysis was based on correlation, linear regression, and 31. Given prior publications suggesting that vaccine efficacy started declining 3–6 months after vaccination32, we were concerned that the protection conferred by IgGs could wane and be time-varying, assuming that antibodies mediated the effect of RTS,S/AS01E vaccine efficacy (or were surrogates). Therefore, the assumption of proportional hazards or constant hazard ratio over follow-up time was assessed through tests of Schoenfeld residuals. When the proportional hazards assumption was rejected, the functional representation of the hazard over time that resulted in the best model fitness was chosen. Optimality of model fitness was based on Akaike Information Criterion (AIC). In analysis of time to first event, only IgG C-term concentration would require use of time-varying hazards and, thus, for simplicity of reporting, a constant hazard was forced in. In analyses of recurrent events, an exponential hazard over time attained the lowest AIC and, thus, yielded optimal model fitness. Moreover, the HR between age cohort and recurrent clinical malaria as a function of time categorized at 167–186 days, attained an optimal fit. We chose to adjust models for age categorized at 180 days or 6 months in all models, for simplicity. We reported graphically the time-varying hazard ratio for recurrent events of increased antibody responses.Assessment of the impact of IgG concentration and AI on protection was primarily based on time to first clinical malaria event following the parent trial main endpoint but, as a secondary endpoint, recurrent malaria events were analysed. First, Kaplan–Meier time-to-event estimates were compared across tertile-based categories of antibody responses within vaccination groups through log-rank tests. In Kaplan–Meier analysis of AI, only samples with IgG concentration above the LLOQ were included. Second, the effect of each M3 continuous IgG response  on the prevention of clinical malaria was assessed by proportional hazard models that used Anderson and Gill approach when including recurrent events as the outcomeConcentration of IgG to HBsAg from participants with M3 samples available (162 [52%] from Kintampo and 277 [62%] from Nanoro) was obtained from the MAL055 trial database and used to analyse the association between HBsAg antibody titres and malaria vaccine efficacy in this study. Analyses of the hazard of recurrent clinical malaria episodes attained optimal fitness per AIC with HBsAg as a piecewise exponential function split at around 180 follow-up days, depending on the model. Again, for simplicity and given reports of waning vaccine efficacy at about 180 days, we chose to standardize models in a split at 180 follow-up days.Z-score (HAZ), reported clinical malaria prior to vaccination, and malaria transmission season, defined according to the rainy months at the time of M3 sample collection: high for Bagamoyo March–May and November–December, for Nanoro July–December, and for Kintampo April–October. Linearity of associations with continuous covariates was evaluated through penalized splines in generalized additive models (GAM) and no variable was deemed non-linearly associated. Covariates statistically significant to a 0.05 level or that impacted the coefficient of IgG concentration or AI were retained in final adjusted models. As a result, no covariate other than age and site, were kept in proportional hazards models. All analyses were stratified by vaccination group and, in regression models we considered stratification by age cohorts and site when interaction terms were significant to a 0.05 alpha-level. When analysing immunogenicity and comparing sites, p-values were corrected for multiple testing through Benjamini-Hochberg and considered significant if <0.05. Analyses were conducted using R v3.4.0 using the functions detailed under code availability below. Further statistical methodological details are provided in the Supplementary Methods.In all regression models we considered adjustments for candidate confounders: sex, WAZ, height-for-age Further information on research design is available in the Supplementary InformationReporting Summary


Antibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure.Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria.Six-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins , erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by At 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months.Antibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination.Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org Plasmodium falciparum infection is responsible for nearly all malaria deaths. The World Health Organization estimated that in 2017 there were 435,000 deaths due to malaria of which around 61% were in children below 5 years of age [Malaria is one of the leading causes of childhood morbidity and mortality, and Antibodies play a crucial role in mediating acquired immunity to malaria. Blood stage merozoite antigens and variant surface antigens (VSA) expressed on infected erythrocytes (IE) are important targets of this protective immunity. Antibodies to merozoite antigens inhibit invasion of red blood cells (RBCs), prevent intra-erythrocytic growth , and proAntibodies to VSA diminish malaria risk by obstructing cytoadhesion to different host receptors , 21 and This study examines the dynamics of antibody acquisition to multiple merozoite antigens, schizont extract and VSA in young children in response to ongoing exposure to malaria. The study was part of the International Lipid-based Nutrient Supplement (iLiNS) Project DYAD-Malawi randomized controlled trial . The original study reported that nutrient supplementation did not have any significant impact on anthropometric indices in 18 months old children . AnotherThe study participants were 601 infants of 18 months of age from rural Malawi, participants in the iLiNS Project DYAD-Malawi trial. Detailed description of the trial design and supplements has been published elsewhere . Briefly® Malaria Combo  which detects the proteins P. falciparum lactate dehydrogenase and histidine-rich protein 2.Children were followed up weekly at home and febrile episodes were investigated for malaria. Clinical malaria was defined as fever with axillary temperature above 37.5 °C and parasitaemia was confirmed by microscopy or rapid diagnostic test (RDT). For microscopy, slides were examined under 100× magnification and parasites were counted against 200 leucocytes. The RDT was the ClearviewBlood from participants was collected at the 18-month study visit. Plasma was separated by centrifugation and stored at − 80 °C prior to shipping on dry ice to Australia. Samples were thawed, heat inactivated at 57 °C for 45 min, and were stored at − 80 °C until assayed.P. falciparum lines were cultured as previously described [var genes [var genes from group A [var gene as its dominant transcript, but its binding phenotype was not defined [Three escribed . The E8Bescribed , and expar genes . Rosetti group A . A 3D7-d defined . Culture defined . To seleP. falciparum reticulocyte binding protein homologue 2 (Rh2A9) were expressed and purified as previously described [Merozoite antigens MSP119kD, MSP2 (FC27 clone), EBA 175, and escribed , 39, 40,escribed . ELISAs escribed .IgG antibody levels against VSA were measured by flow cytometry as previously described , and floData were analysed using Stata version 13.0  and graphed using GraphPad Prism version 5 . Antibody levels were measured as optical density (OD) for schizont and merozoite antigens, or as geometric mean fluorescence intensity (MFI) for VSA. They were expressed relative to the positive control which was a pooled plasma sample from malaria exposed individuals from Africa.Seroprevalence was specified as the percentage of children whose relative antibody level was greater than the mean plus three standard deviations of the negative controls’ antibody levels. Negative controls were a panel of malaria-naïve Melbourne blood donors. Socio-economic status (SES) was derived from an inventory of key household assets (HHA) adapted from . Chi squThe association between malaria episodes and antibody seroprevalence in 18 months old children was investigated using logistic regression, and linear regression was performed to study the association of malaria episodes with antibody levels. Linear regression was done by transforming antibody levels to their natural logarithm; these were back transformed for reporting descriptive results. Multivariate regression adjusted for the covariates duration of gestation, HIV infection, gender of the child and maternal anaemia.P. falciparum parasitaemia at the 18-month visit was 6.5% by microscopy and 9.9% by RDT . The nutrient supplements received by their mothers were IFA in 33.6%, MMN in 33.4%, and 32.9% received LNS. The mean haemoglobin level was 10.8 ± 1.5 g/dl with a prevalence of anaemia (haemoglobin level ≤ 10.9 g/dl) of 46.3%. The percentage of children having Antibody seroprevalence against merozoite antigens, schizont extract and VSA was measured for all the tested children Table . The serChildren who were parasitaemic (n = 60) by RDT (9.9%) at the time of sample collection at 18 months had higher antibody levels than aparasitaemic children, and differences were statistically significant for all the tested antigens except Rh2A9. The seroprevalence data were also in accordance with the antibody level data and parasitaemic children were more frequently seropositive for all the antigens than the aparasitaemic children (Table One-hundred and forty-four children 23.9%) had one or more malaria episodes before 18 months of age. Antibody seroprevalence was higher in the children with previous clinical malaria episodes than those without malaria for all the tested antigens, and differences were statistically significant for MSP1, MSP2 and schizont extract and IgG against E8B VSA . One-hundred and nineteen children had single episodes of malaria whereas 25 children had multiple episodes recorded. Antibody seroprevalence was higher in children having a single episode or multiple episodes than those without any episode detected. For a single episode, differences in prevalence were statistically significant for MSP1, schizont extract (p ≤ 0.051) and IgG against VSA expressed by the E8B parasite line (p = 0.001). In the smaller number of children having multiple episodes, prevalence of antibody to MSP1 (0.003), EBA175 (0.047) and schizont extract (0.023) was significantly higher than in children without malaria. There were no significant differences in antibody seroprevalence between those with single or multiple episodes.Logistic regression revealed that children with any episode of malaria from birth to 18 months of age had significantly higher odds of being seropositive for MSP1, MSP2, schizont extract and VSA against E8B parasite line in both unadjusted (p ≤ 0.034) and adjusted analysis (p ≤ 0.049) than children without any episode.The levels of antibody to malaria antigens were compared between infants who did and did not have previous malaria episodes Figs. , 2. As oLevels of IgG against VSA were significantly higher for all the tested parasite lines in children having single malaria episode than those who had none .Linear regression Table  indicateTo investigate whether age at the time of clinical malaria affected antibody levels or seroprevalence at 18 months, antibody levels and seroprevalence were compared between children who had malarial episodes prior to 6 months and those who had them between 6 and 18 months of age Table . Both chNaturally acquired immunity to malaria is achieved with ongoing exposure to infections and subsequent acquisition of anti-malarial antibodies. Antibodies against merozoite antigens and VSA are thought to play key roles in conferring immunity against malaria , 44. ThePrevious studies have shown that newborn babies and young infants are relatively protected from symptomatic malaria , 46, andThe present study examined the effect of clinical malaria episodes and parasitaemia on acquisition of antibodies by 18 months of age. At this time, little or no placentally transferred antibody remains, so antibodies elicited are likely to reflect naturally acquired immunity . AntibodChildren who were parasitaemic at the time of sample collection at 18 months had significantly higher levels of antibodies and seroprevalence to all the tested VSA and merozoite antigens (except Rh2A9) than the aparasitaemic children. This finding is consistent with studies from Kenya in which antibodies to VSA  and to mThe relationship between antibody levels or seroprevalence and history of clinical malaria infections was also investigated. Both measures were higher in children who had experienced clinical malaria episodes before 18 months of age than children who did not, and the differences were significant for MSP1 19 kd, MSP2, schizont extract, and  for IgG against all tested parasite lines. This is in accordance with other studies that report antibody boosting following exposure to clinical malaria , 53–55, When the cohort was divided into children with single or multiple malaria episodes, those with single malaria episodes had significantly higher levels of antibody against MSP1, MSP2, schizont extract (≤ 0.0015) and IgG against VSA for all three tested parasite lines (≤ 0.028) compared to those without episodes. Fewer children had multiple episodes, limiting statistical power, but these children also had significantly higher antibody levels against MSP1, MSP2 and schizont extract (≤ 0.0418) than those who did not have malaria. There was no relationship between the number of clinical episodes and either antibody levels or prevalence. These findings suggested that antibodies to MSP1, MSP2 or schizont extract were useful markers of history of clinical malaria, but antibodies to EBA175 and Rh2A9 were not. In Kenyan children 1–8 years old, MSP1, MSP2, Rh2A9 and schizont extract also showed moderate induction following malaria episodes in the previous year, while EBA175 did not . In sum,Infants’ immune systems are rapidly developing, and responses to infection may differ between younger and older children in magnitude or longevity. To investigate this, antibody levels and prevalence at 18 months were compared between children having malaria before 6 months of age, or from 6 to 18 months. Symptomatic malaria is uncommon in very young infants , and onlThe strengths of this study include the longitudinal design, with home-based monitoring of children with weekly home visits to 18 months of age and prompt detection and diagnosis of clinical malaria, and the number of antibody assays performed in a large group of over 600 well-characterized infants. The study provides unique insight into the development of antibody responses to VSA in relation to clinical malaria in infancy. Possible study weaknesses include the infrequent blood sampling of asymptomatic children, which gave only limited insights into the relationship between asymptomatic infection and antibody acquisition.The study provides a considerable insight on the acquisition of antibodies in early infancy in response to concurrent parasitaemia and clinical malaria. This study found that antibodies to tested merozoite and IE surface antigens increased following infection in early childhood and in response to concurrent parasitaemia at 18 months of age, but neither age at first infection nor number of malaria episodes substantially affected the antibody acquisition. The result provides strong evidence that antibodies to blood stage malaria antigens may be biomarkers of infection in early life and this knowledge is relevant to informing vaccine development and strategies for sero-surveillance of malaria.


This opinion article deals with the diagnostic clinical challenges faced by clinicians or health care workers in malaria-endemic areas when a severely sick child presents to the clinic with fever, coma or respiratory distress. Indeed, the coexistence of malaria with other severe infections like meningitis, invasive bacterial infection or pneumonia makes appropriate treatment allocation a matter of life and death. The use of biomarkers has been proposed as a potential solution to this problem. The arrival of high-throughput technologies allowed thousands of molecules  to be been screened in clinical samples from large cohorts of well/characterised patients. The major aim of these studies was to identify biomarkers that inform important decisions: should this child be referred to hospital? Should antibiotics, anti-malarials, or both, be administered? There is a large discrepancy between the number of biomarker discovery studies published and the number of biomarkers that have been clinically validated, let alone implemented. This article reflects on the many opportunities and obstacles encountered in biomarker research in malaria-endemic areas. Plasmodium falciparum parasitaemia with other severe diseases like pneumonia, bacteraemia and meningitis complicates the definitive diagnosis of severe malaria [P. falciparum on the blood smear, severe malaria might not be the primary diagnosis—especially in regions where asymptomatic parasitaemia is common. To further complicate the picture, malaria infection predisposes to bacterial infection and may co-occur with more than half of all cases of bacteraemia in malaria-endemic areas [In malaria-endemic areas, the coexistence of  malaria –4. Notabic areas . The preic areas . This diThe identification and validation of novel biomarkers with high sensitivity and specificity for severe malaria (SM) may contribute to the development and implementation of new diagnostic tools, for example, in a rapid diagnostic test (RDT) format that can optimize treatment allocation and reduce mortality from severe childhood illness.P. falciparum infection. Rapid identification of danger signs by specifically trained non-medical personnel helps to identify those children with malaria at the highest risk of dying. However, despite improved access to treatment and declining trends of malaria observed worldwide [The current case definition of severe malaria proposed by the World Health Organization (WHO) is based on the observation of parasites on a blood smear accompanied by one or more severe clinical manifestations, namely prostration, impaired consciousness, respiratory distress, multiple convulsions, severe anaemia, jaundice or shock . This deorldwide , 9, the orldwide ), mostlyP. falciparum on a Giemsa-stained thick smear (gold standard) or more frequently, through indirect methods, such as the detection of parasite proteins using a RDT. In practice, these guidelines are only partially followed, even when RDTs are available [For any clinical condition, the major consequence of a poorly specific case definition is the administration of unnecessary treatment and, in the context of malaria, the potential for accelerated development of parasite drug resistance. According to the WHO recommendation, the administration of anti-malarials in malaria endemic areas should only be used after the presence of parasites has been confirmed through visualization of the parasite vailable .The diagnostic complexity of severe malaria is a reflection of its pathophysiological complexity, illustrated by three potentially overlapping clinical syndromes of SM: cerebral malaria, respiratory distress and severe malarial anaemia , 12. TheP. falciparum or non-falciparum malaria), administration of antibiotics and anti-malarials, to indicate urgent referral to hospital [The use of biomarkers has been proposed to facilitate a syndrome-based management approach. In a hypothetical scenario, in a malaria-endemic area a drop of blood could be obtained from a child presenting with fever and respiratory distress and analysed with a panel of biomarkers that have the ability to inform a clinical decision see Box : to admihospital –15, or sWhich biomarkers should be used? And do these biomarkers exist? The quest to identify the perfect panel of biomarkers to inform clinical decisions was galvanized by the advent of high-throughput “omics” technologies see Box . When thversus healthy controls) may not yield relevant information and the candidate biomarker(s) might just be expensive alternatives to measuring axillary temperature. Instead, a more useful approach might be to compare severe malaria versus mild malaria, or severe malaria versus pneumonia, to identify biomarkers that inform clinical decision-making.Candidate selection is problematic. The identification of a minimal set of informative biomarkers relies heavily on machine-learning algorithms. Briefly, an algorithm is given two phenotypes, i.e. mild malaria versus severe malaria, and it identifies discriminating features from a broad dataset . Some of these features are then selected and tested in silico on a different set of patients not used in the training phase. In a successful test, the algorithm will correctly assign the unknown patients to the correct clinical phenotype based on a minimal set of informative features. The list of features can vary in length depending on the type of biomarker and the study design. In proteomic and transcriptomic studies, it is not uncommon to identify hundreds of discriminant features. The more distinct the clinical phenotypes analysed, the longer the list of discriminating biomarkers. It is important that the study design mirror the diagnostic challenge to be resolved. A study design that includes only extreme phenotypes . Next, independent, adequately powered, clinical studies will be required, followed by at least one larger, ambitious prospective study where clinical decisions are based on the biomarker concentration and patient outcomes are compared to those achieved with standard practice. This phase also depends on the existence of suitable intellectual property and the availability of a diagnostic product or a prototype to measure the biomarker. In reality, few examples of successful biomarkers have successfully emerged from this process. Even when efficacy is demonstrated, cost-efficacy may prevent incorporation of a biomarker into routine practice. Procalcitonin (PCT), a biomarker that detects bacterial infection, can be used to illustrate this point (reviewed in ). A largNot all good biomarkers are molecules. A working definition of “biomarker”  refers to objective indications of a medical state observed from outside the patient . AlthougCerebral malaria (CM) provides a good example. CM is classically defined in children as a deep level of unconsciousness  in the presence of asexual parasitaemia, after the correction of hypoglycaemia and exclusion of other encephalopathies, especially bacterial meningitis and locally prevalent viral encephalitis . NotablyMolecular biomarkers that discriminate SM syndromes from other infections are urgently needed. Although some of the biomarker research show promise (reviewed in ), a majoRespiratory distress is a common presentation of SM, but practically impossible to distinguish from severe pneumonia , 26–28. haptoglobin and lipocalin-2) to discriminate acute respiratory infection from severe malaria with respiratory distress [haptoglobin, tumour necrosis factor receptor 2 or IL-10, and tissue inhibitor of metalloproteinases-1) discriminated bacterial diagnosis from malaria with high sensitivity and specificity .In 2014, Huang and colleagues conducted a proteomic discovery study in 390 Gambian children and identified a panel of two biomarkers . In this context, the combination of (1) depletion of the most abundant proteins, (2) pre-analytical sample fractionation and (3) protein digestion and MS/MS-based peptide sequencing (shotgun proteomics) can identify peptides at concentrations of ng/mL, which falls three orders of magnitude short of a complete proteome analysis (pg/mL) . However