Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure.Methods: We cleared schoolchildren living in an area  with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1).In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed usingPlasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by18S quantitative PCR were studied.Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition .In vitro hepatocyte invasion was inhibited by naturally acquired antibodies , and there were positive correlations between invasion inhibition and gliding inhibition  and between invasion inhibition and CSP-specific antibodies . Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06).Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed toPlasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responsesin vivo; epidemiological studies powered and designed to address this important question should become a research priority. P. falciparum circumsporozoite protein (CSP), and thereby the pre-erythrocytic stages of malaria, and has been shown to be partially effective in delaying the time to clinical malaria episodes1. Alongside the RTS,S subunit vaccine, several other pre-erythrocytic stage vaccines are under development, based on subunit or whole-parasite vaccination5. Vaccination with the attenuated sporozoite vaccine PfSPZ resulted in a protective efficacy of ~48%, as quantified by differences in time to first positive blood smears in malaria-experienced adults in Mali6. The results of this and other vaccine trials and the efficient immunisation of malaria-naive individuals with multiple infected mosquito bites while receiving chloroquine7 contrast with the limited epidemiological evidence of naturally acquired functional immunity toPlasmodium pre-erythrocytic stages, which could be possibly linked to the lower number of sporozoites in natural parasite inoculations or to the frequency of host-vector contacts. Individuals living in malaria-endemic regions can develop naturally acquired immunity against severe malaria disease and death9, but it is unclear whether immunity that reduces, entirely or partially, the probability of blood-stage infection after parasite inoculation develops following natural exposure9. The high incidence of blood-stage re-infection after effective anti-malarial treatment in adults living in malaria-endemic regions suggests that sterilizing immunity does not develop even after years of repeated infection11. Similarly, cohort studies that have analysed the relationship between age and risk ofP. falciparum infection showed no evidence for complete protection against infection and conflicting evidence on whether naturally acquired immunity can result in a different time to patency13. One of the most detailed studies on this topic reported clear negative associations between age and the risk of clinical malaria or microscopy-detected malaria infection, but similar times to PCR-detected infection for all age groups. The study concluded no or very limited evidence for an age-dependent acquisition of immunity that protects from infection13.The most advanced malaria vaccine, RTS,S , induces immune responses that targetGiven the interest in pre-erythrocytic vaccines, studies are needed to understand natural protective immune responses that target sporozoite and liver-stages of malaria infection. Here, we determine the associations between responses affecting sporozoite gliding motility, hepatocyte invasion and malaria infection risk assessed by weekly quantitative PCR (qPCR) in a cohort of schoolchildren from Burkina Faso exposed to intense malaria transmission.Of the 58 school-aged children who were recruited and received treatment at enrolment, 6 were PCR-positive 3 weeks after dihydroartemisinin-piperaquine (DHA-PQ) administration and were not eligible to continue follow-up. One child who was only followed for one routine visit, when no infection was detected, and who withdrew from the study, did not have immune responses quantified and was not included in this analysis.P. falciparum parasites detected by18S qPCR during follow-up. Malaria infection caused clinical disease in 43/50 children. One child developed symptomatology suggestive of malaria, but no parasites were detected in samples collected before and during the clinical episode; data from this child were censored after the onset of symptoms. The median time from confirmation of the absence of parasites  to infection detection by nested PCR or onset of symptoms was 28 days; one child who did not have parasites detected by nested PCR was not included in this calculation. Similarly, the median time to parasite detection by18S qPCR was 30 days. In18S qPCR positive result and, if applicable, of development of clinical disease are presented for all study participants.Parasitological and immunological data from the remaining 51 children followed intensively were analysed . Every wPlasmodium sporozoites were quantified using fluorescently labelled anti-IgG and anti-IgM antibodies by flow cytometric analysis. Levels of antibodies targeting whole sporozoites were significantly higher in the study participants compared to malaria-naive donors , and between CSP-IgG antibody levels and whole sporozoite IgG antibodies  were also observed.Malaria antigen-specific antibodies to pre-erythrocytic antigens CSP, liver stage antigen (LSA-1) and to asexual lysate were determined in naturally exposed children and malaria-naive European donors by ELISAs. Antibody titres to the CSP pre-erythrocytic antigen were on average low in naturally exposed children and not significantly different from malaria-naive donors , P=0.006in vitro assay. Sporozoites pre-treated with PBS showed an average gliding trail surface of 7,392  pixels. To characterize the effect of human plasma on sporozoite gliding motility that is independent of naturally acquired immunity, sporozoites were incubated in the presence of plasma from malaria-naive individuals (n=5) and showed an average gliding trail surface of 1,427 pixels, significantly greater than the gliding trail surface of sporozoites incubated with 30 μg/ml of a monoclonal anti-CSP antibody, our positive control, 141 (95% CI 71.9-225.4) pixels.The neutralizing activity of naturally acquired antibodies against sporozoite motility was determined in anin vitro sporozoite gliding motility with a median gliding inhibition of 59.6% (IQR 27.8-77.0%); incubation of sporozoites with plasma from three participants resulted in lower gliding trail surfaces than the positive control. We defined two groups: one with poor gliding inhibition  and the other with strong gliding inhibition . The first group had similar gliding trail surface  compared to malaria-naive donors  , but correlated with CSP-specific IgG antibodies , whole sporozoite IgG  and IgM antibodies . While levels of IgG and IgM antibodies targeting whole sporozoite did not significantly differ between poor gliding inhibitors and malaria-naive adults, whole sporozoite antibody levels of strong gliding inhibitors were significantly higher compared to poor gliding inhibitors  and malaria-naive adults . This suggests that quantitative differences in measured immune responses might explain variation in these functional phenotypes.Plasma from the majority of cohort participants reduced 1,652) , whilst  1,652) , P<0.01.in vitro sporozoite invasion of hepatocytes was also assessed in a selected number of samples shown to inhibit gliding motility strongly (n=8) or poorly (n=8).In vitro invasion was inhibited by naturally acquired antibodies , and plasma from children categorized as strong gliding inhibitors (see previous section) also prevented hepatocyte invasion more effectively compared to malaria-naive donors , suggesting thatin vitro gliding inhibition by naturally acquired antibodies might serve as a good surrogate forin vitro hepatocyte invasion inhibition. Sporozoite invasion inhibition correlated with IgG targeting whole sporozoites , but not with whole sporozoite IgM antibody levels . There was a correlation of hepatocyte invasion inhibition with CSP-specific IgG antibodies , but not with LSA-1-specific IgG antibodies .The inhibitory effect of naturally acquired antibodies on donors , P<0.001P. falciparum infection (qPCR-based parasitaemia ≥ 0.1 parasites/μl) later compared to children with lower CSP responses and less efficient gliding inhibitory activity , similar results were obtained . Additional analyses (Extended data14) show that functional responses against pre-erythrocytic stages were also associated with infection risk when different parasite density thresholds are used to define infection.For each immunological assay, ELISA or sporozoite gliding motility assays, children were categorized in two groups: participants with high antibody responses (or high sporozoite gliding inhibition activity) were those with assay values higher than the study population median ; childrectivity , P=0.005in vitro sporozoite gliding motility and time to falciparum infection also did not reach statistical significance after adjustment for blood-stage immunity .Since children with high CSP responses were more likely to have high antibody responses to asexual blood stage parasite lysate compared to low CSP responders , multivariate Cox proportional hazards models were fit to assess the mutually adjusted effects of these immune responses on time to infection detection . In a moin vitro, and (ii) those children with antibodies that more efficiently reducedin vitro sporozoite gliding motility remained uninfected for longer periods of time. Our epidemiological data together with thein vitro data provide evidence that there is partially effective pre-erythrocytic immunity that influences individual level infection incidence, although we cannot exclude the contribution of immunity against asexual blood-stage antigens to this observation.In this longitudinal study in Burkina Faso, we analysed the associations between functional immune responses against pre-erythrocytic stages of malaria and the incidence of qPCR-detected infections in children aged 5–10 years. We observed that (i) schoolchildren develop antibody responses that can interfere with sporozoite motility and infectivity19, but not following natural malaria exposure13. Field studies showed conflicting evidence on whether naturally acquired immunity can result in delays to patency and showed no evidence for sterile immunity completely preventing the appearance of parasites in the blood stream of exposed individuals13. We selected children aged 5–10, who have lower blood stage immune responses  compared to semi-immune adults, and who allow repeated blood sampling compared to toddlers. Since submicroscopic infections are prevalent in the area20, we used a curative dose of antimalarials to clear possible sub-patent infections and, upon confirmation that children were parasite-free at the start of the transmission season, assessed the infection incidence by sensitive molecular assays. Our finding that 98% of the cohort became infected withP. falciparum within 3 months confirms the high force of infection in the area.Sterile protection against malaria can be readily demonstrated in human and animal experimental models24, our results indicate that antibodies toP. falciparum sporozoite and liver-stage antigens are acquired following natural exposure. Antibody titres to the CSP antigen were on average low in our cohort and not significantly different from malaria-naive donors. These low titres could be related to the timing of the study recruitment , which would imply that responses to CSP are short lived. The antibody levels could also have been influenced by repeated blood-stage infections in our cohort, which may have suppressed immune responses against the pre-erythrocytic stages25, although, in theory, responses to both LSA-1 and CSP would be affected. To assess the contribution of pre-erythrocytic humoral immunity to protection, previous studies have related antibodies at baseline with time-to-infection after parasite clearance with antimalarials12. A significant challenge in these studies is that both protective immunity and cumulative exposure increase with age, and so it is often unclear whether measured responses mediate protection or are merely a marker of past exposure27. In an attempt to move beyond indirect epidemiological associations, we explored functional anti-sporozoite immunity by assessing the ability of plasma to inhibit sporozoite gliding motility28 and hepatocyte invasion29 and related thesein vitro phenotypes to field findings. We observed that CSP IgG antibodies showed a strong positive association with sporozoite gliding motility inhibition. It has been demonstrated thatPlasmodium parasites use the system of adhesion-based motility, gliding, to actively penetrate host cells30; and that the invasive ability of sporozoites is associated with their motility31. In agreement with these findings, the data presented here suggest thatin vitro gliding inhibition by naturally acquired antibodies may be a useful surrogate marker forin vitro hepatocyte invasion. In an immuno-epidemiological study undertaken in Indonesia, hepatocyte invasion inhibition was associated with higher anti-CSP antibody titres32. In our cohort, subjects with high CSP responses (i.e. higher than the study population median) and those whose plasma more efficiently inhibited sporozoite gliding movement developedP. falciparum infection later compared to children with lower responses. To our knowledge, this is the first study to show that there are functional antibodies against pre-erythrocytic malaria stages in malaria-exposed children.Consistent with previous sero-epidemiological studies33. Indeed, as pointed out by one of the reviewers of the first version of this manuscript, 17/29 (58.6%) children with high anti-sporozoite responses in either CSP-specific or sporozoite gliding assays had above-median asexual lysate responses versus 8/22 (36.4%) children with below-median anti-sporozoite activities. We believe it is likely that the functionally important pre-erythrocytic antibody responses that we quantified here are acquired alongside anti-blood-stage antibodies. In addition, a recent study in the same geographical area demonstrated that heterogeneity in mosquito exposure contributes considerably to heterogeneity in parasite inoculation risk34. In the current study, we used delayed time to blood-stage infection as a simplistic indicator of partial protection, which fails to take into account variation in exposure. In an ideal approach, we would have been able to quantify malaria exposure at an individual level, which may involve linking of blood meals in household-caught mosquitoes to household occupants and determining sporozoites in the salivary glands of these mosquitoes35. Measuring exposure at individual level in such an approach will help to shed further light on pre-erythrocytic immunity in naturally exposed individuals. With respect to discriminating between pre-erythrocytic and blood-stage immunity, a valuable but laborious approach would be to examine the observed relationships in a larger cohort that allows stratification based on similar blood stage immunity but different levels of pre-erythrocytic immunity at baseline and that provides sufficient power to detect weaker associations. Another limitation of our study is that we were only able to determine humoral responses. Cellular responses to pre-erythrocytic stages have been implicated in malaria protection in multiple studies37. It is conceivable that by quantifying both antibody and cellular responses we would be able to better define natural immunological phenotypes associated with differential malaria risk38. Immunity to mosquito saliva might have also influenced sporozoite invasion although the magnitude of such an effect compared to the established impact of anti-sporozoite responses is currently unknown and it is currently unknown whether this would require responses to antigens that are conserved betweenA. stephensi mosquitoes used for sporozoite production and vectors that study participants were naturally exposed toOur study has several limitations. We observed that high responses in the asexual stage lysate assay with unknown functionality were also associated with longer time to infection. We thus cannot rule out a supportive role for asexual antibody responses in the observed associationsin vitro sporozoite gliding inhibition and hepatocyte invasion inhibition. Children with functional anti-sporozoite antibody responses had a longer time toP. falciparum infection compared to children with lower functional responses, suggesting that thesein vitro assays are relevant to understand natural protection. The partial protection  observed in our study does not prevent individuals from becoming infected during an entire transmission season, but reduces infection incidence and consequently needs to be considered in epidemiological studies aiming to understand malaria risk heterogeneity and in malaria vaccines trials. Identifying host or parasite factors linked to these functional immunological phenotypes and characterizing how these phenotypes change with cumulative exposure to malaria parasites will help the understanding of why natural immunity against pre-erythrocytic stages is incomplete.In summary, in our cohort of children, anti-CSP antibodies were strongly associated withP. falciparum transmission20. Written informed consent was provided by the parent or guardian of each child. The study was approved by the ethics committees of the London School of Hygiene and Tropical Medicine (reference number 9008) and the Ministry of Health in Burkina Faso (reference number 2015-3-033). Children aged 5–10 years with haemoglobin levels above 8 g/dl and noPlasmodium parasites detected by microscopy were eligible. DHA-PQ was used to clear sub-microscopic infections. At 3 weeks (20–22 days) after treatment, finger-prick blood samples were collected to ensure parasite negativity by nested PCR40 prior to formal enrolment into the cohort. Citrated plasma samples were collected before treatment using citrated vacutainer cell preparation tubes , stored at -80°C and used for malaria-antigen-specific IgG ELISAs and sporozoite assays. Peripheral blood mononuclear cells (PBMC) were also collected, but were lost due to the inability to maintain liquid nitrogen supplies during civil unrest in Ouagadougou. Following enrolment, participants were examined during weekly visits, when finger-prick samples were collected forP. falciparum nested PCR that was performed within 48 hours. Following parasite detection, finger-prick blood samples were collected every day for 1 week, and every week afterwards, up to 35 days after parasite detection. Study participants were closely monitored for the development of malaria symptoms. Artemether-lumefantrine was given upon the detection of symptoms or 35 days after initial detection of infection by nested PCR, whichever came first. For the current analyses, only the time to first infection detection was used and related to baseline immunological assays.This study was performed from June to December 2015 in the village of Balonghin in the Saponé health district, Burkina Faso, which is exposed to intense and seasonal18S rRNA41. Genomic DNA from the same extraction was used to test for human haemoglobinopathies haemoglobin S and C42.Nucleic acids from 100 μl whole-blood samples stored in RNAprotect Cell Reagent were extracted using MagNAPure LC automatic extractor  and used for qPCR targetingAnopheles stephensi mosquitoes were infected by standard membrane feeding onP. falciparum NF54 gametocyte cultures43. Salivary glands from infected mosquitoes were dissected, collected in Leibovitz culture medium (Lonza) without serum (supplemented with 1% penicillin-streptomycin and 1% L-glutamine forin vitro gliding motility assays), and homogenized in a homemade glass grinder. The number of sporozoites was counted in a Bürker-Türk counting chamber using phase contrast microscopy18.As source of sporozoites,29 was acquired through MR4 as part of the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). Hepatoma cells (referred to as hepatocytes) were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 nutrient mixture medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum , 1% glutamine and 1% penicillin/streptomycin (GIBCO) at 37°C in an atmosphere of 5% CO2.The HC-04 human hepatoma cell lineP. falciparum NF54 CSP with repeats, produced inE. coli by Gennova Biopharmaceuticals Ltd., Pune, India), LSA-1 (LSA-NRC construct expressed inE. coli) and asexual lysate using previously reported standardized enzyme-linked immunosorbent assays (ELISAs)45 in naturally exposed children (n=51) and malaria-naive European donors (n=9). Antibody levels were calculated in relation to the positive control (hyperimmune plasma pool from Tanzania) that was set at 100 arbitrary units (AU) using Auditable Data Analysis and Management System 19.Levels of antibodies were determined to circumsporozoite protein  and lowest (8) gliding activity were selected for sporozoite opsonization assays and invasion assays, allowing us to investigate potential correlations. Flow cytometric analysis was performed with a LSRII flow cytometer (BD BioSciences); data analysis by FlowJo software .Recognition of whole sporozoites by naturally acquired IgG and IgM antibodies was determined by an47. Written informed consent was obtained from these individuals including for their samples to be stored and used in additional immunological experiments. Pre-immunization samples collected before the CPS-immunization were used for analysis of malaria antigen-specific antibody levels.Malaria-naive donors are healthy malaria-naive European volunteers who participated in CPS-immunization trial  at the Radboud University Medical Center in vitro sporozoite assays, plasma aliquots were heat-inactivated for 30 minutes at 56°C, centrifuged at 13,000 rpm for 5 minutes at room temperature and kept at 4°C. Flat-bottom optical-bottom 96-well plates with cover glass base were incubated overnight at 4°C with an anti-CSP monoclonal antibody  3SP2; 5 μg/ml in PBS). Following incubation, wells were washed twice with 150 µl/well PBS, blocked for 20 minutes at room temperature with 100 μl/well Leibovitz medium (Lonza) supplemented with 1% penicillin-streptomycin (GIBCO), 1% L-glutamine (GIBCO) and 10% heat-inactivated FBS(GIBCO).P. falciparum NF54 sporozoites (100 µl) were pre-incubated with citrated samples  for 30 minutes at room temperature and added to each well in triplicate (50 μl/well) at a concentration of 10,000 sporozoites/well. Sporozoites were allowed to glide for 90 minutes at 37°C, 98% humidity, 93% N2, 4% CO2 and 3% O2. Wells were washed thrice with 100 μl/well PBS and gliding trails were fixed for 15 minutes at room temperature with 4% paraformaldehyde (Affymetrix). Following fixation, wells were washed thrice with 100 μl/well PBS and blocked with 150 μl/well 10% FBS/PBS for 20 minutes at room temperature. Subsequently, gliding trails were stained for 1 hour at room temperature with 50 µl/well 5 µg/ml biotinylated anti-CSP monoclonal antibody , followed by a wash step (thrice with PBS) and a 1 hour incubation at room temperature with 50µl/well 10 µg/ml streptavidin-Alexa Fluor-594 (Life Technologies) diluted in 10% FBS in PBS. Subsequently, wells were washed thrice with 100 μl/well PBS and stored in 150 μl/well PBS at 4°C in the dark until analysis. Gliding trails were imaged automatically with the BioTek Cytation cell imager (25 images per well at 200x magnification) and images were analysed automatically byFIJI software (under ImageJ version 2.0.0-rc-68/1.52h) with Otsu’s thresholding28. Results were plotted in GraphPad Prism version 5.03.The number of pixels present on a stitched image made from 25 individual pictures taken per well is a measure of the amount of shed CSP in that particular well and therefore, differences in the number of pixels can be interpreted as differences in sporozoite gliding trail surface28.Prior toP. falciparum sporozoite hepatocyte invasion by naturally acquired antibodies was assessed in a flow-cytometry-basedin vitro invasion assay, as previously described with small adaptations46. Briefly, freshly dissectedP. falciparum NF54 sporozoites were added to heat-inactivated plasma samples  from malaria-naive or malaria-exposed individuals and pre-incubated for 30 minutes at 4°C. Subsequently, the sporozoite-plasma mixtures (5.104 sporozoites in the presence of 10% plasma) were added to HC-04 hepatocytes in 96-well plates. Following 3 hours of incubation at 37°C in 5% CO2, invaded and intracellular sporozoites were stained with an Alexa Fluor 488-conjugated anti-CSP antibody. Flow cytometric analysis was performed with a Gallios (Beckman Coulter) flow cytometer and data were analysed with FlowJo software . The percentage of CSP-positive hepatocytes was first corrected for background reactivity by subtracting the background (uninfected HC-04 cells in the presence of 3SP2-Alexa Fluor-488 antibody). The percent invasion inhibition was expressed relative to control IgG.Neutralization ofin vitro sporozoite data, comparisons between two (controls versus field samples) or multiple groups were performed using Mann-Whitney U-test and Kruskal-Wallis test followed by Dunn’s test between two groups, respectively. The associations between immune responses and malaria infection risk were assessed using survival analysis methods. Log-rank test was used to compare times to infection incidence for individuals with different values of immune phenotypes. Cox survival models were fit to assess the effect of pre-erythrocytic immunity after adjustment for blood-stage immunity; the proportional hazards assumption was tested using Schoenfeld residuals. In these analyses, study participants were considered to have high or low responses (binary explanatory variables) based on the study population median (see14). The first scheduled weekly visit or intensive follow-up visit when parasitaemia of at least 0.1 parasites per μl was detected by18S qPCR was considered the time of infection incidence. Using this criterion, the median18S qPCR-based parasitaemia at infection detection was 2.2 parasites per μl and the interquartile range was 0.4 – 80.4. This threshold of parasitaemia was chosen to minimise false-positive results. In the supplemental material, sensitivity analyses were included that used different cut-offs of18S qPCR-based density to determine infection positivity. Stata 14  and GraphPad Prism software  were used for statistical analysis. P<0.05 was considered statistically significant.For analysis ofteun.bousema@radboudumc.nl). However, the dataset used in the survival analysis has been de-identified and is available from the Dryad repository, along with raw ELISA results and sporozoite gliding data. DOI:https://dx.doi.org/10.5061/dryad.n1m33qq14.The main dataset relating to the field study contains individual level data and identifying information on participants; as such, this dataset is stored under restricted access and not available through an open-access repository. Requests from researchers to access these data for pooled or meta-analysis should be addressed to the corresponding author (Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of theThe results of sensitivity analyses (see Results section) andFigure S1. Correlation analyses of naturally acquired whole sporozoite IgG and IgM antibodies. (A) Scatter plot of whole sporozoite IgG and IgM antibodies targetingP. falciparum NF54 sporozoites is shown (n=17). (B) Correlation analysis forP. falciparum NF54 sporozoite and CSP-specific IgG antibodies as determined by ELISAs is shown.in vitro gliding inhibition by naturally acquired antibodies.Figure S2. Antibody specificity and (A). Scatter plot ofin vitro gliding inhibition and CSP-specific IgG antibodies as determined by ELISAs is shown (n=51). Samples selected for additional invasion experiments had either poor (orange) or strong (green) gliding inhibitory activity. Correlation analysis for whole sporozoite (B) IgG or (C) IgM antibodies andin vitro gliding inhibition by naturally acquired antibodies was conducted with samples from 16 children. Recognition ofP. falciparum NF54 sporozoites by whole sporozoite(D) IgG and (E) IgM antibodies from naturally exposed children (n= 16) or malaria-naive adults (n=8) was shown as the geometric mean fluorescent intensity (MFI) and divided in subgroups: malaria-naive adults (black), poor (orange)versus strong gliding inhibitors (green). Correlation analyses were conducted with Spearman correlation analysis. Comparisons between multiple groups were tested by Kruskal Wallis test.in vitro sporozoite invasion of hepatocytes by antibodies from children in Burkina FasoFigure S3. Inhibition of. Gliding motility and invasion ofP. falciparum NF54 sporozoites pre-treated with plasma from children from Burkina Faso and malaria-naive adults was determined byin vitro gliding motility and invasion assays in human hepatoma cells. (A) Scatter plot ofin vitro gliding and invasion inhibition by naturally acquired antibodies is shown. Additionally, correlation analyses of the percent invasion inhibition with whole sporozoite (B) IgG, (C) IgM antibodies or (D) CSP-specific IgG antibodies is shown. Children whom had poor or strong neutralizing effect on sporozoite infectivity are shown in orange and green circles, respectively.Figure S4. CSP antibody levels versus immune responses against asexual stage antigens. In this figure, the x-axis shows CSP antibody levels; the y-axis represents antibody responses against asexual stage parasites lysate. Both axes are in log-scale; one child with undetectable CSP response is not included in this graph.Figure S5. Distribution of immune phenotypes. The distributions of log10-transformed antibody responses (x-axes) against CSP, LSA-1 and asexual blood stage lysate (AL) are presented in panels (A), (B) and (C). The y-axes in these panels represent the percentages of study population with various levels of responses. In (A) and (B), one and seven individuals had undetectable responses and, to be included in this figure, were assigned response values equivalent to half of the lower limit of detection. In (D), the results of gliding assays are presented: the left plot presents the distribution of log10-transformed sporozoite gliding surface; the right plot shows gliding inhibition (y-axis) for each study participant . The median gliding inhibition (59.6%) was used to define high and low inhibition in the survival analysis.https://doi.org/10.5061/dryad.n1m33qq.214.DOI:Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the The authors have addressed the points raised in the review.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The manuscript version following the reviewer's suggestions and comments has improved and I have no problems with the current version.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. in vitro in plasma fromP. falciparum exposed children for their capacity to interfere with sporozoite gliding and hepatocyte invasion which are required for progress with their life cycle to liver and blood stages, and these are related to titers. The outcome measured is blood stage parasitemia (delay of appearance of subpatent infections) as no direct measurement of liver parasitemia can be done in humans.The study by Barry and colleagues is important to shed more light into the efficiency of naturally-acquired sporozoite immunity to interfere with the parasite development at the pre-erythrocytic stages. Functional antibodies are assessed Some changes are required to clarify some aspects and make the work more scientifically sound: ABSTRACT In the Abstract background, specify if the statement "it is unclear whether immunity that affects the establishment of infections develops following continuous natural exposure" refers to children or in general, and if it refers to naturally acquired immunity as typically perceived (mostly against blood stages) or here if specific for pre-erythrocytic immunity. This also applies to the main Introduction. Although sterilizing immunity does not develop, parasite densities tend to decrease with age.in vitro. In the Abstract Methods it would be relevant to specify the intensity of malaria transmission and the specific area of study (location) within Burkina Faso, to know how repeatedly are they exposed to Pf. Also clarify if it is qPCR or nested PCR . Liver stage antigen: which one? Specify also that the functional sporozoite assays are In the results, mention if CSP antibody titres correlated with hepatocyte invasion and if the latter correlated to delayed time to qPCR +ve. INTRODUCTION See comments in the Abstract section.Can it be clarified if children did not receive antimalarial treatment, if during the weekly monitoring visits the nested PCR (done 48h after bleeding) was Pf positive but they had no symptoms (at least for 35 days)? If so, this was accepted by ethics committees easily? Were the families communicated of a positive PCR reaction in absence of symptoms?Indicate where the sporozoites were generated.Indicate if and how the patent parasitemias were also monitored and taken into account in the analysis. METHODSWhen presenting data on median invasion inhibition, please comment on whether these % are high, moderate or low, and give some references.in vitro gliding inhibition by naturally acquired antibodies might serve as a good surrogate forin vitro hepatocyte invasion inhibition." The correlation in Fig S3B is clearer.The following statement does not look convincing by looking at the Fig S3A scatter plot despite the P=0.02 and Spearman’s ρ=0.60 that appears driven by exteme values: "There was a positive correlation between gliding and invasion inhibition suggesting thatFig 3A: interesting to see that there is not always overlap of the three metrics, expected to be discussed. Why was hepatocyte invasion inhibition not included?Explain the rationale for categorizing in high and low responders. Was the analysis also done with continuous values but did not yield significant insights?The joint analysis of the antibody levels to CSP and blood stage lysate and the functional pre-erythrocytic responses is helpful, but shouldn't there be a model with the 3-4 responses together? If not, explain why. RESULTSIt is not convincing to say that children 5-10 years in a highly endemic area of Burkina Faso have limited blood stage immunity. They will have quite high antibody responses to blood stage antigens already. Better to reformulate.It is surprising the lack of difference in antibody levels for CSP between these children and naive volunteers. Even if CSP is not as highly immunogenic as many blood stage antigens, the responses are usually significant and detectable, even in children. Could there be a problem with the capture antigen? Seasonality by itself may not totally explain this, or it would imply they are very short lived. The potential inhibition by blood stage has been proposed but it would also affect LSA1.It is not well discussed how the affection of gliding and invasion before liver stage is detected as delay in positive qPCR in the blood, how do authors think this works DISCUSSION As mentioned, the study is valuable due to the lack of data on functional pre-erythrocytic antibody responses in exposed African children.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Comment“The study by Barry and colleagues is important to shed more light into the efficiency of naturally-acquired sporozoite immunity to interfere with the parasite development at the pre-erythrocytic stages. Functional antibodies are assessed in vitro in plasma from P. falciparum exposed children for their capacity to interfere with sporozoite gliding and hepatocyte invasion which are required for progress with their life cycle to liver and blood stages, and these are related to titers. The outcome measured is blood stage parasitemia (delay of appearance of subpatent infections) as no direct measurement of liver parasitemia can be done in humans.Some changes are required to clarify some aspects and make the work more scientifically sound:ABSTRACTIn the Abstract background, specify if the statement "it is unclear whether immunity that affects the establishment of infections develops following continuous natural exposure" refers to children or in general, and if it refers to naturally acquired immunity as typically perceived (mostly against blood stages) or here if specific for pre-erythrocytic immunity. This also applies to the main Introduction. Although sterilizing immunity does not develop, parasite densities tend to decrease with age.”AnswerAnopheles mosquitoes, including in adults. The term “establishment of infections” in that sentence is equivalent to “release of merozoites, from hepatocytes, in the blood”. We now modified two similar sentences in theAbstract andIntroduction section:In the sentence mentioned by the reviewer, we refer to the fact that there is little or no evidence that naturally acquired immunity against liver stage parasites blocks initiation of blood-stage infection after parasite inoculation byimmunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure.” (Abstract)“Individuals living in malaria-endemic regions develop naturally acquired immunity against severe malarial disease, but it is unclear whetherreduces, entirely or partially, the probability of blood-stage infection after parasite inoculation develops following natural exposure.” (Introduction section)“Individuals living in malaria-endemic regions can develop naturally acquired immunity against severe malaria disease and death, but it is unclear whether immunity thatComment“In the Abstract Methods it would be relevant to specify the intensity of malaria transmission and the specific area of study (location) within Burkina Faso, to know how repeatedly are they exposed to Pf. Also clarify if it is qPCR or nested PCR . Liver stage antigen: which one? Specify also that the functional sporozoite assays are in vitro.In the results, mention if CSP antibody titres correlated with hepatocyte invasion and if the latter correlated to delayed time to qPCR +ve.”AnswerAbstract the study area and clarify which liver stage antigen was studied:We now mention in theliving in an area  with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections bynested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-erythrocytic-stage antigens circumsporozoite protein (CSP) andLiver stage antigen1 (LSA-1).In vitro sporozoite gliding inhibition by naturally acquired antibodies was assessed using Plasmodium falciparum NF54 sporozoites; hepatocyte invasion was assessed using the human HC-04 hepatoma cell line and NF54 sporozoites. The associations betweenantibody responses, functional pre-erythrocytichumoral immunity phenotypes and time to infectiondetected by 18S quantitative PCR were studied.”“We cleared schoolchildrenin vitro hepatocyte invasion inhibition and anti-CSP antibody levels:We have also included information on the correlation betweenwere positive correlations between invasion inhibitionand gliding inhibition  and between invasion inhibition and CSP-specific antibodies .In vitro hepatocyte invasion was inhibited by naturally acquired antibodies , and there”“The hepatocyte invasion inhibition phenotype was not included in the survival analysis as only 16 participants had samples tested with this assay.Comment“INTRODUCTIONSee comments in the Abstract section.”AnswerWe addressed these comments in our answer to the first comment of Reviewer 3.Comment “METHODSCan it be clarified if children did not receive antimalarial treatment, if during the weekly monitoring visits the nested PCR (done 48h after bleeding) was Pf positive but they had no symptoms (at least for 35 days)? If so, this was accepted by ethics committees easily? Were the families communicated of a positive PCR reaction in absence of symptoms?”Answerearly, i.e. low density, infections and characterize for the first time the evolution of naturally acquired acute infections in partially immune children. Children with parasites detected by nested PCR were closely monitored for development of symptoms and treated with first line therapy when symptoms developed. Burkina Faso’s national malaria guidelines did not include treatment of asymptomatic parasite carriage, consequently initial detection of parasites, often at low levels, by nested PCR was not sufficient to prompt administration of antimalarials. It should be noted, and we have clarified this in theMethods section, that children received a long-lasting net at the start of the study and were guaranteed access to care 24 hours per day. Moreover, children were clinically examined on a daily basis in the first week of detection of infection. In this manner, the study adhered to the national guidelines but provided improved (access to) care for all study participants.In this study, we monitored children with weekly samples to identify new infections during the transmission season. We used nested PCR to detectUncommon asymptomatic Plasmodium falciparum infections in Gabonese children. Clin Infect Dis 2003). In the study by Missinou and colleagues, children were followed every second week for detection of infection by blood smear, and after malaria infection was diagnosed, children were clinically monitored with daily blood smears. In large cohort studies conducted in the context of the International Centers of Excellence for Malaria Research (ICEMR) program in Uganda, participants of all age groups  are followed every 4 weeks with asymptomatic infections sometimes remaining untreated for more than a year . An important difference between some of these studies and ours is that we used molecular methods to be able to detect infections from the early, sub-patent, phases and we also characterized malaria infectiousness over time, which will be described in a separate manuscript (in preparation). In conclusion, we appreciate the discussion about the necessity to treat asymptomatic infections and believe this is an important discussion to have, but the current study followed national guidelines and is by no means unique in following asymptomatic infections without treatment but with excellent care.Parents and guardians were informed that study participation could involve intensive follow-up if parasites were detected in their child’s blood samples and were oriented to contact the study staff if their child developed symptoms. The study was approved by the ethics committee of the London School of Hygiene and Tropical Medicine (reference number 9008) and the Ministry of Health in Burkina Faso (reference number 2015-3-033). Of note, previous studies with similar design were performed that described the development of malaria symptoms in children after detection of patent infections .The analysis presented in this manuscript focuses on the influence of immune factors on infection establishment, i.e. appearance of falciparum parasites in the blood. Consequently, all different comparisons presented relate to the first visit of each participant when parasites were detected by sensitive molecular methods. Blood smears were also performed for all study visits, before and after infection detection, but results were not immediately available. The relationship between blood smear results and parasite and gametocyte levels quantified by molecular methods after infection detection will be described in detail in a separate manuscript , suggesting that in vitro gliding inhibition by naturally acquired antibodies might serve as a good surrogate for in vitro hepatocyte invasion inhibition.”“Comment“Fig 3A: interesting to see that there is not always overlap of the three metrics, expected to be discussed. Why was hepatocyte invasion inhibition not included?”Answerin vitro hepatocyte invasion assay was only performed in a subset of the study population (16 children) and for this reason was not included in this figure and in the survival analysis. The invasion experiments are highly laborious and resource intensive. We used these as supportive experiments in our study, to confirm the relevance of gliding assays, but invasion assays did not form the core of our analysis. We therefore feel that our approach to perform these experiments on a selective number of samples is justified.TheComment “Explain the rationale for categorizing in high and low responders. Was the analysis also done with continuous values but did not yield significant insights?”AnswerThe survival analysis was only performed with binary variables. The rationale for categorizing the study population in high and low responders, based on the median, was to facilitate interpretation of the results and avoid assumptions about linearity of effect.Comment“The joint analysis of the antibody levels to CSP and blood stage lysate and the functional pre-erythrocytic responses is helpful, but shouldn't there be a model with the 3-4 responses together? If not, explain why.”AnswerResults section), we decided not to include these two variables in the same model. Additionally, the limited sample size also influences the number of predictors that can be reasonably included in our multivariate analyses.Since CSP-specific antibody levels are highly correlated with sporozoite gliding inhibition  compared to semi-immune adults, ….”. We have now modified the sentence to avoid misinterpretation:We agree that children in highly endemic areas will have considerable immunity by the age of 5 years. However, our statement compared immunity in children relative to adults: “lower blood stage immune responses  compared to semi-immune adults, and who allow repeated blood sampling compared to toddlers.”“We selected children aged 5–10, who haveComment“It is surprising the lack of difference in antibody levels for CSP between these children and naive volunteers. Even if CSP is not as highly immunogenic as many blood stage antigens, the responses are usually significant and detectable, even in children. Could there be a problem with the capture antigen? Seasonality by itself may not totally explain this, or it would imply they are very short lived. The potential inhibition by blood stage has been proposed but it would also affect LSA1.”AnswerDiscussion section accordingly:Thank you for these suggestions. The reviewer suggested that suppression of immune responses by blood-stage parasites would, theoretically, affect responses to both antigens, LSA-1 and CSP. We agree and have now modified the, which would imply that responses to CSP are short lived. The antibody levels could also have been influenced by repeated blood-stage infections in our cohort, which may have suppressed immune responses against the pre-erythrocytic stages, although, in theory, responses to both LSA-1 and CSP would be affected.”“These low titres could be related to the timing of the study recruitment The reviewer also suggested that there might have been a problem with the antigen used in the CSP assay. The significant correlations between anti-CSP levels and functional phenotypes (i.e. sporozoite gliding inhibition or hepatocyte invasion inhibition) suggest that this is not the case , and this is supported by the fact that a subset of children had particularly high antibody responses to CSP .Comment “It is not well discussed how the affection of gliding and invasion before liver stage is detected as delay in positive qPCR in the blood, how do authors think this works”AnswerWe observed an association between sporozoite gliding inhibition and time to parasite positivity by qPCR. This could either reflect an ability of these immune responses to completely neutralize some sporozoite inoculums, leading to a delay in patency because this population would have no merozoites being released into the bloodstream from a first sporozoite inoculum. Alternatively, the number of sporozoites that reach hepatocytes can be reduced by immune responses targeting sporozoites and thus, the number of merozoites released upon completion of the hepatic phase of parasite development. This would result in a lower starting point of blood stage infection and plausibly a longer time to reach detectability of infections by PCR. Lastly, and we believe this is well explained in the revised manuscript, it is possible that the number of released merozoites is unaltered in the population with high sporozoite immunity, but the concurrent higher blood stage immunity dampens parasite growth and results in a longer time to reach detectability of infections by PCR. Plasmodium falciparum infections, then attempts to explain delays in time to infection in terms of antibodies against circumsporozoite protein (CSP) and whole parasites.  The authors demonstrate that, in individuals who show greatest delays in time to parasitemia, there were stronger functional antibody responses against sporozoites which reduced gliding motility and liver cell invasionin vitro.  The major question addressed in this ms is whether these anti-sporozoite responses are affecting time to parasitemia in these children.  From the data presented, the answer is “maybe”.  The problem the authors face is that for almost every infection that stimulates anti-sporozoite antibodies, that infection also stimulates blood stage antibodies as the infection progresses.  It is these latter that show the best negative correlation in reduction in time to parasitemia, and while separating out the functional anti-sporozoite effects is informative it is not definitive.  This is illustrated in Figure 3A, where of the 29 participants with anti-sporozoite functional activities in one or both of the assays, 17 also have higher than median anti-blood stage responses.  I do not see a way around this at the participant level and the authors clearly acknowledge this difficulty in their discussion; one is trying to see subtle effects on infection (in this case time to infection no children we uninfected during the study) using markers that autocorrelated and cannot therefore be separated from one another in terms of cause and effect.This ms measures antibody responses in Burkinabe children after drug clearance and subsequentAnopheles stephensi) salivary glands be present in these sera (of children bitten by African anophelines), and if so, could they have affected thein vitro functional antibody assays? One additional complication is not considered as far as I can tell.  In order to have become infected, these children must have been bitten by mosquitoes.  The mosquito bites induce antibody responses.  The sporozoites used in the assays for gliding motility and hepatocyte invasion were from crude homogenates that contain salivary gland material.  It is possible (though I admit not too likely) that there was some functional interaction between these two.  Could antibodies against  cannot be ascribed to them.Other points:Given the comments above and the stronger correlation of blood stage antibodies with delay to parasitemia, the authors should consider changing the abstract (and perhaps the title) to include this information.  It is buried in the paper but should be more explicit.At the start of the introduction, the authors highlight the protective efficacies of RTS,S and PfSPZ Vaccine.  However, they should also note that the outcomes, efficacies are different, RTS,S being time to clinical malaria, PfSPZ Vaccine being time to parasitemia.  This has direct relevance to the ms.Also in the introduction, the authors suggest that there is a discrepancy between protection with sporozoites and naturally induced protection not preventing infection.  However, they do not consider the obvious reason for this – dose.  Mosquitoes deliver a few (median 50 sporozoites per bite) now and again over a transmission season, while the sporozoite vaccines are delivering thousands of sporozoites per dose and defined intervals.The authors say that there are sporozoite-specific IgG and IgM antibodies in the malaria exposed children, but such specificity has not been demonstrated, only reactivity.  The term should be changed.risk-modifying pre-erythrocytic immunity”?What do the authors mean by the title “Evidence of naturalThe authors note an important variable that could not be accounted for in their studies, namely variability in exposure.  One of the authors has worked on antibody responses to mosquito saliva as a marker for exposure risk: would it not be possible to measure antibody responses against mosquito bites here?Minor points:Introduction, paragraph 1. The sentence ending “ but it is unclear whether immunity that prevents or reduces infection incidence develops following natural exposure”. This is a misuse of the term incidence – prevention of infection in one person will reduce incidence in the population.  I think the authors mean “that prevents infection or reduces infection intensity in an individual….”.  In the same paragraph, change the word ‘suggests’ .Results, paragraph 2.  Should read Parasitological and immunological data from the remaining 51 children followed intensively were…”  What does “data from this child were censored after the onset of symptoms” mean?  Could have any one of many interpretations.In the results section “Naturally acquired antibodies in children neutralize in vitro sporozoite gliding motility” the authors should change the term “considerably larger’ for something more scientific (significantly greater”?).Authors should give sample sizes  used in the curves for figure 3B,C.In the discussion, the authors say “To our knowledge, this is one of the first studies to show that there are functional antibodies against pre-erythrocytic malaria stages in malaria-exposed children.” Shouldn’t the other studies be referenced here?The authors should check the axis labeling of figures: for example (% infected) ought to be “Percent children infected with P. falciparum”, in Figure S1 the abbreviations/units are not explained, and so on.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Comment“This ms measures antibody responses in Burkinabe children after drug clearance and subsequent Plasmodium falciparum infections, then attempts to explain delays in time to infection in terms of antibodies against circumsporozoite protein (CSP) and whole parasites.  The authors demonstrate that, in individuals who show greatest delays in time to parasitemia, there were stronger functional antibody responses against sporozoites which reduced gliding motility and liver cell invasion in vitro.  The major question addressed in this ms is whether these anti-sporozoite responses are affecting time to parasitemia in these children.  From the data presented, the answer is “maybe”.  The problem the authors face is that for almost every infection that stimulates anti-sporozoite antibodies, that infection also stimulates blood stage antibodies as the infection progresses.  It is these latter that show the best negative correlation in reduction in time to parasitemia, and while separating out the functional anti-sporozoite effects is informative it is not definitive.  This is illustrated in Figure 3A, where of the 29 participants with anti-sporozoite functional activities in one or both of the assays, 17 also have higher than median anti-blood stage responses.  I do not see a way around this at the participant level and the authors clearly acknowledge this difficulty in their discussion; one is trying to see subtle effects on infection (in this case time to infection no children we uninfected during the study) using markers that autocorrelated and cannot therefore be separated from one another in terms of cause and effect.One additional complication is not considered as far as I can tell.  In order to have become infected, these children must have been bitten by mosquitoes.  The mosquito bites induce antibody responses.  The sporozoites used in the assays for gliding motility and hepatocyte invasion were from crude homogenates that contain salivary gland material.  It is possible (though I admit not too likely) that there was some functional interaction between these two.  Could antibodies against (Anopheles stephensi) salivary glands be present in these sera (of children bitten by African anophelines), and if so, could they have affected the in vitro functional antibody assays?Nevertheless, the ms provides important information concerning anti-sporozoite functional antibodies, even if a clinical outcome (delayed time to parasitemia) cannot be ascribed to them.”AnswerDiscussion section a statement that emphasizes this point:Thank you. We agree with this assessment of the difficulty in separating pre-erythrocytic-stage- and blood-stage-specific immune responses. We have now included in theIndeed, as pointed out by one of the reviewers of the first version of this manuscript, 17/29 (58.6%) children with high anti-sporozoite responses in either CSP-specific or sporozoite gliding assays had above-median asexual lysate responses versus 8/22 (36.4%) children with below-median anti-sporozoite activities.”“We observed that high responses in the asexual stage lysate assay with unknown functionality were also associated with longer time to infection. We thus cannot rule out a supportive role for asexual antibody responses in the observed associations.Immunization with AgTRIO, a Protein in Anopheles Saliva, Contributes to Protection against Plasmodium Infection in Mice. Cell, Host & Microbe 2018) and (not the topic of this paper) the transmission of parasites from man to mosquito . How important such responses are compared to parasite-specific responses is unclear. Our assumption, based on the available literature, is that responses against salivary proteins would have a considerably smaller impact on sporozoite invasion compared to anti-sporozoite responses.The reviewer also suggested that the functional activity of the plasma samples from study participants could be partially related to targeting antigens of salivary gland material rather than targeting sporozoites antigens. This is a very novel idea and an area of research that is both exciting and relatively new. There are indeed some indications that saliva responses may influence both the viability of sporozoites . Whilst many salivary proteins appear to be conserved amongAnophelines, others are distinct betweenStephensi andGambiae s.l. . Responses to conserved salivary proteins would thus have to be functionally important in invasion assays and differentially produced by individuals with high sporozoite responses/low sporozoite responses. Our previous work on the salivary protein gSG6 suggested an early saturation of responses to this protein  and correlations between asexual, sporozoite and salivary gland responses , making it difficult to disentangle the different responses. In conclusion, there is no convincing evidence that salivary gland responses could explain our current findings but it is a theoretical possibility that we cannot rule out completely. We have thus added the following underlined sentence to the paragraph where we describe some of the limitations of this study:Moreover, theImmunity to mosquito saliva might have also influenced sporozoite invasion although the magnitude of such an effect compared to the established impact of anti-sporozoite responses is currently unknown and it is currently unknown whether this would require responses to antigens that are conserved between An. stephensi mosquitoes used for sporozoite production and vectors that study participants were naturally exposed to.”“Another limitation of our study is that we were only able to determine humoral responses. Cellular responses to pre-erythrocytic stages have been implicated in malaria protection in multiple studies. It is conceivable that by quantifying both antibody and cellular responses we would be able to better define natural immunological phenotypes associated with differential malaria risk.Comment“Other points:Given the comments above and the stronger correlation of blood stage antibodies with delay to parasitemia, the authors should consider changing the abstract (and perhaps the title) to include this information.  It is buried in the paper but should be more explicit.”AnswerAbstract:To address this comment and comments from Reviewer 1, we have now modified the, although this association became non-significant after adjustment for blood-stage immunity (P = 0.06).“Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01)Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority.” “In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia.Comment“At the start of the introduction, the authors highlight the protective efficacies of RTS,S and PfSPZ Vaccine.  However, they should also note that the outcomes, efficacies are different, RTS,S being time to clinical malaria, PfSPZ Vaccine being time to parasitemia.  This has direct relevance to the ms.”AnswerIntroduction section. We now included information on the outcome used to assess efficacy of the RTS,S vaccine:The efficacy metric used in the PfSPZ vaccine trial is mentioned in the, and thereby the pre-erythrocytic stages of malaria, and has been shown to be partially effective in delaying the time to clinical malaria episodes.”“The most advanced malaria vaccine, RTS,S , induces immune responses that target P. falciparum circumsporozoite protein (CSP)Comment“Also in the introduction, the authors suggest that there is a discrepancy between protection with sporozoites and naturally induced protection not preventing infection.  However, they do not consider the obvious reason for this – dose.  Mosquitoes deliver a few (median 50 sporozoites per bite) now and again over a transmission season, while the sporozoite vaccines are delivering thousands of sporozoites per dose and defined intervals.”AnswerIntroduction section:We have now modified the following sentence in the, which could be possibly linked to the lower number of sporozoites in natural parasite inoculations or to the frequency of host-vector contacts. Individuals living in malaria-endemic regions can develop naturally acquired immunity against severe malaria disease and death, but it is unclear whether immunity thatreduces, entirely or partially, the probability of blood-stage infection after parasite inoculation develops following natural exposure.”“The results of this and other vaccine trials and the efficient immunisation of malaria-naive individuals with multiple infected mosquito bites while receiving chloroquine contrast with the limited epidemiological evidence of naturally acquired functional immunity to Plasmodium pre-erythrocytic stagesComment“The authors say that there are sporozoite-specific IgG and IgM antibodies in the malaria exposed children, but such specificity has not been demonstrated, only reactivity. T44he term should be changed.”AnswerWe agree with the reviewer that the specificity of these antibodies has not been demonstrated, and have now modified the title of the sub-section in the manuscript:Sporozoite-specific IgG and IgM antibodies in malaria-exposed children”“to:Naturally acquired IgG and IgM antibodies targeting sporozoites”“We also modified the following sentence:Levels of antibodies targeting whole sporozoites were significantly higherin the study participants compared to malaria-naive donors . Strong correlations between IgG and IgM antibodiestargeting whole sporozoites , and between CSP-IgG antibody levels and whole sporozoite IgG antibodies  were also observed.”“In addition to antigen-specific assays, IgG and IgM antibodies recognizing Plasmodium sporozoites were quantified using fluorescently labelled anti-IgG and anti-IgM antibodies by flow cytometric analysis.sporozoite specific” with more accurate expressions in other paragraphs of the manuscript.We also replaced the expression “Comment“What do the authors mean by the title “Evidence of natural risk-modifying pre-erythrocytic immunity”?”AnswerThis sub-section title refers to the observation that functional immune responses against pre-erythrocytic stages were associated with delay in infection appearance. As discussed and addressed in answers to other comments, this evidence is based on the univariate survival analysis. “Risk” here is being used loosely, and not in the epidemiological sense of probability of an event during a specified time period.Comment“The authors note an important variable that could not be accounted for in their studies, namely variability in exposure.  One of the authors has worked on antibody responses to mosquito saliva as a marker for exposure risk: would it not be possible to measure antibody responses against mosquito bites here?”Answer Anopheles mosquitoes generally, as shown in a previous study in the same area , only a small proportion of blood-fed mosquitoes are parasite carriers, and the prevalence of infection in malaria vectors varies from household to household. For these reasons, we believe quantification of immunity against mosquito saliva antigens alone would not adequately adjust for exposure. Moreover, saliva responses are complicated to use in areas of high mosquito exposure. There is considerable evidence for a level of immune tolerance that reduces responses to mosquito saliva proteins in older individuals with high mosquito exposure. This would complicate the interpretation of mosquito salivary gland responses considerably in our cohort experiencing high but heterogeneous mosquito exposure.Although quantification of antibody responses to mosquito saliva antigens would provide some information on exposure toComment “Minor points:Introduction, paragraph 1. The sentence ending “ but it is unclear whether immunity that prevents or reduces infection incidence develops following natural exposure”. This is a misuse of the term incidence – prevention of infection in one person will reduce incidence in the population.  I think the authors mean “that prevents infection or reduces infection intensity in an individual….”.  In the same paragraph, change the word ‘suggests’ .”Answer We modified the sentence below for clarification:reduces, entirely or partially, the probability of blood-stage infection after parasite inoculation develops following natural exposure.”“Individuals living in malaria-endemic regions can develop naturally acquired immunity against severe malaria disease and death, but it is unclear whether immunity thatWe prefer to keep the word ‘suggests’ in that same paragraph as “high incidence of blood-stage re-infection after effective anti-malarial treatment in adults” is a strong but not conclusive evidence that sterilizing immunity does not develop.Comment“Results, paragraph 2.  Should read Parasitological and immunological data from the remaining 51 children followed intensively were…”  What does “data from this child were censored after the onset of symptoms” mean?  Could have any one of many interpretations.”AnswerIn that sentence, we meant that any visits and samples collected after the development of symptoms were disregarded and the child was classified as uninfected during the entire valid follow-up period.We replaced the first sentence of that paragraph with the following sentence:“Parasitological and immunological data from the remaining 51 children followed intensively were analysed.”Comment “In the results section “Naturally acquired antibodies in children neutralize in vitro sporozoite gliding motility” the authors should change the term “considerably larger’ for something more scientific (significantly greater”?).”AnswerThe modified sentence is:significantly greater than the gliding trail surface of sporozoites incubated with 30 μg/ml of a monoclonal anti-CSP antibody, our positive control,141 (95% CI 71.9-225.4) pixels.”“To characterize the effect of human plasma on sporozoite gliding motility that is independent of naturally acquired immunity, sporozoites were incubated in the presence of plasma from malaria-naive individuals (n=5) and showed an average gliding trail surface of 1,427 pixels,Comment “Authors should give sample sizes  used in the curves for figure 3B,C.”AnswerWe have now included this information in the figure legend.Comment “In the discussion, the authors say “To our knowledge, this is one of the first studies to show that there are functional antibodies against pre-erythrocytic malaria stages in malaria-exposed children.” Shouldn’t the other studies be referenced here?”AnswerWe modified the sentence:“To our knowledge, this is the first study to show that there are functional antibodies against pre-erythrocytic malaria stages in malaria-exposed children.”Comment “The authors should check the axis labeling of figures: for example (% infected) ought to be “Percent children infected with P. falciparum”, in Figure S1 the abbreviations/units are not explained, and so on.”AnswerWe have now modified the legends, rather than axes’ labels, of the following figures:Figure 1The black line represents the percentage of the study population that remained uninfected at different time-points (y-axis).”“…Figure 3(N = 51); in (C), curves for participants categorized based on their responses to asexual stage antigens (AL) are shown(N = 51).In both panels (B) and (C), the y-axis corresponds to the percentage of the population uninfected at different time points.”“…. In (B), Kaplan-Meier curves for children with high and low circumsporozoite protein (CSP) responses and gliding inhibition (GI) phenotypes are presented P. falciparum and I make a few comments and suggestions that may improve the current version of the manuscript. In this manuscript Barry, Behet and colleagues address an important question aiming to possibly appoint an effective role in protection from clinical malaria to liver-stage immunity acquired naturally in malaria endemic areas. I believe the work presented is a significant contribution to our understanding of naturally acquired immunity to pre-erythrocytic stages of in vitro suggesting that ABs acquired during natural infections can reduce new liver-stage infections. However, the contribution of a possible similar effect in vivo is, in my view, less clear from the data presented, and more caution may be needed to discuss the results. All the children followed throughout the study became parasite positive by qPCR, indicating that even children with strong inhibiting ABs were unable to block liver-stage infection efficiently. Furthermore, high response to asexual stage lysate might be confounding the analysis. I would add to the manuscript  figures the association of CSP ABs with asexual stage lysate ABs, and also the association of whole SPZ ABs with asexual stage lysate ABs, to give the reader an idea of how close these parameters are. The data shows that ABs developed during past malaria cases can reduce sporozoite motility and hepatocyte invasion Reported bed net use in the multivariate analyses, as this could also be a factor increasing time to PCR positive and clinical malaria.  I also suggest including, if that has not been done already, the 1 where it was shown that, in Mali, time to PCR positive was independent of age, while time to clinical malaria increased with age, and where as stated it was concluded that there no or very limited evidence for an age dependent acquisition of immunity protecting from infection. Similarly, in the present manuscript, in vitro functional data of higher humoral response against pre-erythrocytic stages does not (independently of blood-stage immunity) protect from infection. So, I would rephrase the last sentence in first paragraph of the discussion to add a bit more caution in interpreting what may be causing partial protection.  The authors cite the study by Tran et al.Evidence of natural risk-modifying pre-erythrocytic immunitythe authors should, in my view, clearly state that High gliding inhibition activitydoes not independently associate in a statistically significant way, with protection against falciparum infectionon the multivariate analyses where blood stage immunity was included; the P value is above 0.05 (0.055) and the CI includes 1, making the relative risk not statistically significant. At the end of section 18s qPCR upon first parasite detection and determine if there is a negative association with the inhibitory capacity of the individuals’ ABs. It would also be very interesting to question if time from first PCR positive to time of presentation of symptoms is different between poor and strong in vitro inhibitors. If the in vitro data showing gliding inhibition and reduced hepatocyte invasion are significant in vivo, one would expect a lower inoculum in the liver and thus a lower parasitaemia on the first PCR positive time-point. And then potentially a slower progression to clinical malaria. I believe with the data generated in this manuscript these analyses could be done, and would enrich the story. I believe the manuscript could be improved by presenting the quantitative analysis of the  It is not totally clear to me how individuals were selected for the flow cytometry assays. Survival, gliding inhibition, CSP, LSA1 and asexual lysate ELISAs were performed for the 51 participants, but flow cytometry data presented in fig2 D and E was obtained from 17 Burkinabes only; how were those selected and what is their time to PCR+ in the survival analysis. If they are the 8 poor and 8 strong inhibitors as defined by their gliding inhibition it should be stated in the methods .  I also suggest to pinpoint these 8 poor and 8 strong inhibitors in fig1 so that the reader would be informed of their time to PCR+ and time to malaria symptoms.2 shows that non-motile SPZ are unable to invade, but is not clearly showing a direct association between % of human AB affecting motility and those levels correlating directly with invasion either. I would be more cautious when citing ref 31, I believe the study by Michael Stewart et al. Minor points:Persisting parasites post-treatmentas I do not think that it can be excluded that the children were re-infected after clearance of PQ. In table 1, I would not refer to the 6 children who were PCR positive at the 3 weeks after treatment time-point as In vitro sporozoite infectivity assay of a human hepatoma cell line inmaterial and methods. I recommend adding a brief description of the method in ref 45 in the section field PCR which may be a mistake. On page 3 below table 1 there is mention to in vitro gliding inhibition by LSA IgG seems to be not shown. I think it should be clarified in the text that that is indeed the case. Likewise, if the LSA-1-specific IgG antibodies correlation with sporozoite invasion inhibition is data not shown I would clearly state it in the text. The data from the  In figS2A I would specify that is IgG in the figure x axis and use the label CSP IgG titer instead of CSP antibody titer. Figure S3D is called before Figure S3C, I would call figures in ascending and alphabetical order instead.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We would like to thank the reviewers for their insightful comments and suggestions. The modifications we made to address their comments improved the quality of our manuscript and clarified the interpretation of our results.Reviewer 1Comment“In this manuscript Barry, Behet and colleagues address an important question aiming to possibly appoint an effective role in protection from clinical malaria to liver-stage immunity acquired naturally in malaria endemic areas. I believe the work presented is a significant contribution to our understanding of naturally acquired immunity to pre-erythrocytic stages of P. falciparum and I make a few comments and suggestions that may improve the current version of the manuscript. The data shows that ABs developed during past malaria cases can reduce sporozoite motility and hepatocyte invasion in vitro suggesting that ABs acquired during natural infections can reduce new liver-stage infections. However, the contribution of a possible similar effect in vivo is, in my view, less clear from the data presented, and more caution may be needed to discuss the results. All the children followed throughout the study became parasite positive by qPCR, indicating that even children with strong inhibiting ABs were unable to block liver-stage infection efficiently. Furthermore, high response to asexual stage lysate might be confounding the analysis. I would add to the manuscript  figures the association of CSP ABs with asexual stage lysate ABs, and also the association of whole SPZ ABs with asexual stage lysate ABs, to give the reader an idea of how close these parameters are.”AnswerAbstract to mention that our study did not fully address the correlation between liver-stage- and blood-stage-specific immunities and that additional studies are necessary.In response to this comment and to a comment from Reviewer 2, we modified the, although this association became non-significant after adjustment for blood-stage immunity (P = 0.06).”“Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01) Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority.” “In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia.Discussion section:We have also modified the first paragraph of thepre-erythrocytic immunity that influences individual level infection incidence, although we cannot exclude the contribution of immunity against asexual blood-stage antigens to this observation.”“Our epidemiological data together with the in vitro data provide evidence that there is partially effectiveAdditionally, we agree that the inclusion of a figure showing the correlation between liver-stage immune responses and asexual stage lysate responses would be informative for the readers. We included a scatter plot of CSP-specific immune responses versus asexual stage lysate responses .Figure S4.“ CSP antibody levels versus immune responses against asexual stage antigens. In this figure, the x-axis shows CSP antibody levels; the y-axis represents antibody responses against asexual stage parasites lysate. Both axes are in log-scale; one child with undetectable CSP response is not included in this graph.”We also modified the legend of Figure 3 to refer to this supplementary figure:.To assess the effect of immune responses on infection risk, children were classified based on whether the results of their assays were higher or lower than the study population median (see Results): in ( A), vertically aligned cells represent the same participant, and orange cells indicate that assay results are higher than the median”“Comment“I also suggest including, if that has not been done already, the Reported bed net use in the multivariate analyses, as this could also be a factor increasing time to PCR positive and clinical malaria.”Answerreported bed net use were collected and it is evident that this is an imprecise indicator of personal protection.The analysis in this manuscript concerns time to infection detection, regardless of whether symptoms are present or not. In this cohort, reported bed net use at enrollment had no effect on time to infection . In analyses that included reported bed net use at enrollment and either CSP immune responses or sporozoite gliding inhibition phenotype as binary variables, neither the hazard ratio point estimates nor the confidence intervals changed significantly (data not shown). Of note, only data onComment“The authors cite the study by Tran et al. where it was shown that, in Mali, time to PCR positive was independent of age, while time to clinical malaria increased with age, and where as stated it was concluded that there no or very limited evidence for an age dependent acquisition of immunity protecting from infection. Similarly, in the present manuscript, in vitro functional data of higher humoral response against pre-erythrocytic stages does not (independently of blood-stage immunity) protect from infection. So, I would rephrase the last sentence in first paragraph of the discussion to add a bit more caution in interpreting what may be causing partial protection.”AnswerOur multivariate analyses showed that the effects of functional phenotypes became non-significant after adjustment for blood stage immunity. However, we disagree with the reviewer that we can conclude the opposite, i.e. that “higher humoral response against pre-erythrocytic stages does not (independently of blood-stage immunity) protect from infection”. The study was not designed, and might not have been powered, to assess the separate effects of the correlated pre-erythrocytic-stage and blood-stage immunities on malaria infection establishment. A study with a larger sample size, where a sufficient number of individuals would have relatively high liver-stage immunity and relatively low asexual responses, and vice-versa, would be more suitable to address this question.Discussion section and theAbstract to mention that our approach did not fully separate the effects of these two types of immunity (see response to the first comment).We have modified theComment At the end of section Evidence of natural risk-modifying pre-erythrocytic immunity the authors should, in my view, clearly state that High gliding inhibition activity does not independently associate in a statistically significant way, with protection against falciparum infection on the multivariate analyses where blood stage immunity was included; the P value is above 0.05 (0.055) and the CI includes 1, making the relative risk not statistically significant.Answeret al. Epidemiology 1998; 9:7-8), illustrated also by a recent comment inNature . With our small sample size, we agree that all results should be interpreted with caution . The sentence in theResults section has now been modified to:With the small sample size, we believe a very strong focus on the P-value threshold of 0.05 is potentially problematic. In general, the P-value and strict thresholds for interpreting significance are criticized by many .”“Comment“I believe the manuscript could be improved by presenting the quantitative analysis of the 18s qPCR upon first parasite detection and determine if there is a negative association with the inhibitory capacity of the individuals’ ABs. It would also be very interesting to question if time from first PCR positive to time of presentation of symptoms is different between poor and strong in vitro inhibitors. If the in vitro data showing gliding inhibition and reduced hepatocyte invasion are significant in vivo, one would expect a lower inoculum in the liver and thus a lower parasitaemia on the first PCR positive time-point. And then potentially a slower progression to clinical malaria. I believe with the data generated in this manuscript these analyses could be done, and would enrich the story.”Answer18S qPCR-based parasite levels at the time of infection detection would be informative and have now included the sentence below in theMethods section:Thank you, we agree that presenting data on theUsing this criterion, the median 18S qPCR-based parasitaemia at infection detection was 2.2 parasites per μl and the interquartile range was 0.4 – 80.4. This threshold of parasitaemia was chosen to minimise false-positive results.”“The first scheduled weekly visit or intensive follow-up visit when parasitaemia of at least 0.1 parasites per μl was detected by 18S qPCR was considered the time of infection incidence.18S qPCR parasite density during the first parasite-positive visits of children with higher-than-median sporozoite gliding inhibition activity was 1.0 (interquartile range 0.3 – 28.2) parasites per μl; for those children whose plasma inhibited sporozoite gliding with efficiency below the population median, the parasite density at first detection was 4.7 . The interpretation of associations between these parasite levels and immune phenotypes, however, is not straightforward. Immune responses that reduce liver parasite burden and influence the size of the parasite inoculum in the blood would most likely influence the time required for parasitaemia to reach the detection threshold, but it is not obvious that the parasite levels at which we first detect infection have a direct relation with liver stage immunity. For example, even if after a week (the time between consecutive scheduled visits), parasite levels in blood-stage infections starting on the same day would be higher in children with higher liver-to-blood parasite inocula, if “lower liver-to-blood inoculum” infections remain undetectable, they might be detected at higher levels afterwards.The medianRegarding the evolution of parasite levels during these natural infections, and the eventual development of symptoms, another manuscript, with a more comprehensive analysis of immune responses against blood stage antigens, is being prepared.Comment “It is not totally clear to me how individuals were selected for the flow cytometry assays. Survival, gliding inhibition, CSP, LSA1 and asexual lysate ELISAs were performed for the 51 participants, but flow cytometry data presented in fig2 D and E was obtained from 17 Burkinabes only; how were those selected and what is their time to PCR+ in the survival analysis. If they are the 8 poor and 8 strong inhibitors as defined by their gliding inhibition it should be stated in the methods . I also suggest to pinpoint these 8 poor and 8 strong inhibitors in fig1 so that the reader would be informed of their time to PCR+ and time to malaria symptoms.”AnswerMethods section:We have rectified this. In the original submission 17 individuals were included in the flow assays . For consistency, we now presented data for the 8 poor and 8 strong gliding inhibitors with available invasion data in all figures where a selection of the total study population of 51 individuals was used. These 16 individuals were selected based on highest gliding inhibition (84-99%) and lowest gliding inhibition (-34 – 20%), as illustrated in supplementary figure 2B. To ensure consistency, we updated Figure 2, Figure S2 and Figure S3. This had no impact on any of the effects, significance levels changed marginally and have been updated. We have now included this information in theDue to limited plasma availability and available sporozoite numbers, 16 naturally exposed children with highest (8) and lowest (8) gliding activity were selected for sporozoite opsonization assays and invasion assays, allowing us to investigate potential correlations.”“Recognition of whole sporozoites by naturally acquired IgG and IgM antibodies was determined by an in vitro flow-cytometry-based antibody opsonization assay that was presented in detail elsewhere.The median time to infection detection in the group of children classified as strong inhibitors of sporozoite gliding motility was 41 days ; poor gliding inhibitors (N = 8) had a median time to infection of 34 days (range 13 – 48).Comment “I would be more cautious when citing ref 31, I believe the study by Michael Stewart et al. shows that non-motile SPZ are unable to invade, but is not clearly showing a direct association between % of human AB affecting motility and those levels correlating directly with invasion either.”AnswerWe cite the study by Stewart et al in the following sentence:30; and that the invasive ability of sporozoites is directly associated with their motility31.”“It has been demonstrated that Plasmodium parasites use the system of adhesion-based motility, gliding, to actively penetrate host cellsThis is an accurate statement about that study, where the authors concluded that “sporozoite invasiveness is associated with sporozoite motility”.Comment“Minor points:In table 1, I would not refer to the 6 children who were PCR positive at the 3 weeks after treatment time-point as Persisting parasites post-treatment as I do not think that it can be excluded that the children were re-infected after clearance of PQ.”Answer We modified Table 1 to ‘Presence of parasites post-treatment’.Comment “I recommend adding a brief description of the method in ref 45 in the section In vitro sporozoite infectivity assay of a human hepatoma cell line in material and methods.”Answerwith small adaptations.4 sporozoites in the presence of 10% plasma) were added to HC-04 hepatocytes in 96-well plates. Following 3 hours of incubation at 37 ˚C in 5% CO2, invaded and intracellular sporozoites were stained with an Alexa Fluor 488-conjugated anti-CSP antibody.Briefly, freshly dissected P. falciparum NF54 sporozoites were added to heat-inactivated plasma samples  from malaria-naive or malaria-exposed individuals and pre-incubated for 30 minutes at 4 ˚C. Subsequently, the sporozoite-plasma mixtures  to infection detection by nested PCR or onset of symptoms was 28 days; one child who did not have parasites detected byComment “The data from the in vitro gliding inhibition by LSA IgG seems to be not shown. I think it should be clarified in the text that that is indeed the case. Likewise, if the LSA-1-specific IgG antibodies correlation with sporozoite invasion inhibition is data not shown I would clearly state it in the text.”AnswerResults section:Although no correlation plot is shown for LSA-1 titers and gliding inhibition, the lack of association with gliding inhibition is included in the“In vitro gliding inhibition did not correlate with LSA-1 IgG antibody levels …”Sporozoite invasion inhibition correlated with IgGtargeting whole sporozoites  but not withwhole sporozoite IgM antibody levels . There was a correlation of hepatocyte invasion inhibition with CSP-specific IgG antibodies , but not withLSA-1 specific IgG antibodies .”“In response to the next comment and to comments by another reviewer, we have changed the order of the sentences and removed the word ‘specific’ from the section above and replaced it with “targeting whole sporozoites”.Comment“In figS2A I would specify that is IgG in the figure x axis and use the label CSP IgG titer instead of CSP antibody titer.Figure S3D is called before Figure S3C, I would call figures in ascending and alphabetical order instead.”AnswerWe have now modified Figure S2 and rephrased the text so Figure S3C is mentioned before Figure S3D (see answer to previous comment).