Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance.Placental malaria (PM) is associated with a higher susceptibility of infants to Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age.Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM , and such infants displayed a lower frequency of non-classical monocytes  that overexpressed LILRB2 . Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection.Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants’ monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages. Plasmodium falciparum (Pf) is estimated to cause up to 100,000 infant deaths every year 1 and LILRB2 are inhibitory receptors that play an important role in the regulation of immune responses that modulate progression or control of infectious diseases , 13. LIL infants , 17.Pf infection with potential implications for the design of new therapeutic strategies against malaria.In the present study, we aimed to define the role of PM on immune profiles as well as on the level of anti-malarial antibodies in infant with a particular focus on LILRB1, LILRB2, IL-10 and HLA-G expression. These data bring a better understanding of the dynamics of monocyte subsets and LILRB1/LILRB2 inhibitory receptor expression during Malaria in Pregnancy Preventive Alternative Drugs, a randomized trial of intermittent preventive treatment (IPTp) in Benin  standard operating procedures developed by the African Malaria Network Trust was used to assess antibody concentrations to MSP1, MSP2, MSP3, AMA1 and GLURP antigens. All details about the recombinant proteins and the procedure are described by Dechavanne et al. .Concentrations of IL-4, IL-10, TNF-α and IFN-γ were determined using BD OptEIA ELISA Set  following the manufacturer’s protocol and as previously described by our team . The lev+CD4+), T cytotoxic cells (CD3+CD8+), T regulatory cells (CD3+CD4+CD25highCD127-), T effector cells (CD3+CD4+CD25+CD127+), γδ T cells (CD3+Vd2+) and B cells (CD19+). NK cell subsets were characterized by expression of CD56 and CD16 among the mononuclear lymphoid cells. Blood monocyte subpopulations were characterized by expression of CD14 and CD16 markers in the population of mononuclear cells. Neutrophils and eosinophils are respectively CD16+ and CD16- in the polymorphonuclear cell population. A classical gating strategy was used to investigate monocyte dysregulation during Plasmodium falciparum infection in children. A second analysis by contour plot was also applied to validate the gates of our flow cytometry analysis for all samples. Due to the inter-individual variability in the proportions of monocyte populations or the variations induced by parasite infection, the selection of monocyte subsets was adapted for each sample. To define positive and negative events, isotype-matched control antibodies were used. Flow cytometer data were analyzed using FlowJo software version 10. The gating strategy is presented in Phenotype analysis was performed on infant peripheral blood (1 mL) the day of collection. The expression of LILRB1 and/or LILRB2 inhibitory receptors on the surface of the following cell populations was analyzed: T helper cells  and TOLIMMUNPAL programs. Both projects were approved by the Ethics Committee of the Faculté des Sciences de la Santé de Cotonou. The TOLIMMUNPAL study protocol and informed consent were also approved by the Comité Consultatif de Déontologie et d’Éthique (CCDE) of the Institut de Recherche pour le Développement .The study was conducted in the context of the MiPPAD “Malaria in Pregnancy Preventive Alternative Drugs,”  the effect of PM on the frequency of immune cell populations and on LILRB1 and LILRB2 expression and (ii) the effect of active malaria infection on LILRB1 and LILRB2 expression, on cytokines and HLA-G levels.® Software, Version 13  and the graphs were done using Graph Pad Prism (Version 8.1.2).For multivariate analysis, a mixed linear model was used, taking into account the measurement at 18 and 24 months of age together in one analysis. The effect of PM or of an active malaria infection was assessed on the frequency of monocytes, LILRB1 and LILRB2 expression, cytokines, HLA-G and anti-malarial antibody concentrations. All models were adjusted on potential confounders and on the frequency of each subset of immune cell in the LILRB1 and LILRB2 expression analyses. For antibody and cytokines levels analysis, an interaction term was included to determine if the effect of an active malaria infection at the moment of blood draw depends on whether the infant is born to mother with or without PM. Data were analysed with StataFunders had no role in study design; collection, analysis and interpretation of data; writing of manuscript; and in the decision to submit the paper for publication.Raw experimental data associated with the figures presented in the manuscript are available from the corresponding author upon reasonable request.Pf-infected or an uninfected placenta (The objective of this study was to investigate the immune response at 18 and 24 months of age in infants born to mothers with or without PM. None of the main characteristics of the studied population were found to differ between infants born to mothers with a placenta Table 1.++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++)  Figure 1Pf infection . A decrease of LILRB1 expression was also observed with age on γδ T cell surface in the mixed model .LILRB1 and LILRB2 receptors were expressed at higher levels on intermediate and non-classical monocytes in infants with active nfection 4. In a Pf infection except for IL-10 levels. IL-10 levels were higher in those with active malaria infections [febrile and/or asymptomatic  and soluble HLA-G concentrations were quantified. No differences were observed as a function of PM or of active ptomatic Figure 6Pf infection, we introduced an interaction term  cell functions. Taken together, those data suggest impaired opsonizing phagocytic capacity in infants born from women with PM. Further studies are needed to confirm this hypothesis of an effect of PM on monocyte functions in infants.Monocytes play a crucial role in controlling parasite loads and protecting the host against malaria. Their protective roles include phagocytosis, production of cytokines and antigen presentation. Monocyte-antibody cooperation also generates a major defence mechanism against malaria, known as ADCI (Antibody Dependent Cellular Inhibition) , in whicin utero  were affected in 2 years old-infants by maternal PM. Our observations raise questions about the reprogramming of neonatal monocytes/macrophages in PM. Recent studies demonstrate that innate immune cells can build a “memory”. Depending on the nature of the components responsible for the first challenge, monocytes can be trained inducing non-specific attenuated or enhanced innate cell responses and functions after a second challenge , 32. Thiin utero . Based oin utero , we coulin utero . Therefoin utero , 38. FurPf infection at time of blood collection. However, we found a higher level of IL-10 during active malaria at both visits and for symptomatic and asymptomatic infections. The concomitant presence of IL-10 and a higher expression of LILRB1 and LILRB2 on non-classical monocytes suggests a down regulation of the cells involved in inflammatory mechanisms and therefore a potentially decreased ability to protect the infants from malaria. Moreover, in vitro studies indicate that IL-10 enhances the expression of LILRB2 on monocytes . Two PhD scholarships were awarded by IRD and the French Embassy, Cotonou to IS and RA. The clinical trial in which the study was nested was funded by the European Developing Countries Clinical Trials Partnership . This work was also supported by the Brazil-France research cooperation program USP/COFECUB (grant no. Uc Me 169-17).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malaria remains common among native Amazonians, challenging Brazil′s elimination efforts.Plasmodium vivax accounts for > 80% of cases. We examined the epidemiology of malaria in riverine populations of the country′s main hotspot - the upper Juruá Valley in Acre state, close to the Brazil-Peru border, where cytochrome b (cytb) gene-based polymerase chain reaction. Positive samples were further tested with quantitative TaqMan assays targeting P. vivax- and P. falciparum-specific cytb domains. We used multiple logistic regression analysis to identify independent correlates of P. vivax infection. Participants (n = 262) from 10 villages along the Azul River were screened for malaria parasites by microscopy and genus-specific, P. vivax and two P. falciparum infections. TaqMan assays detected 33 P. vivax infections , 78.1% of which asymptomatic, with a median parasitaemia of 34/mL. Increasing age, male sex and use of insecticide-treated bed nets were significant predictors of elevated P. vivax malaria risk. Children and adults were similarly likely to remain asymptomatic once infected. Microscopy detected only one  Our findings are at odds with the hypothesis of age-related clinical immunity in native Amazonians. The low virulence of local parasites is suggested as an alternative explanation for subclinical infections in isolated populations. Plasmodium vivax.Malaria transmission has decreased substantially over the past 2 decades in Latin America and the Caribbean,-,P. vivax carriers often harbour mature gametocytes,-,Since the early 2000s, traditional populations scattered along the margins of the Amazonian rivers are known to harbour low-density malarial infections, often missed by microscopy and rapid diagnostic tests  but detected by sensitive molecular techniques such as the polymerase chain reaction (PCR).P. vivax infections remain frequent in isolated riverine villages of the upper Juruá Valley region, a transmission hotspot in northwestern Brazil that contributes nearly 18% of the country′s malaria cases. Adults are not more likely than young children to remain asymptomatic once infected, contrary to the expectations from the age-related clinical immunity hypothesis. We suggest an alternative explanation for the high frequency of subclinical malarial infections in these and similar isolated Amazonian populations.Here we show that low-density and asymptomatic Study area and population - This study took place in the municipality of Mâncio Lima, in the upper Juruá Valley region of Acre state, westernmost Brazil, close to the border with Peru (Anopheles (Nyssorhynchus) darlingi is the primary vector and P. vivax accounts for > 80% of local malaria cases.ith Peru . With a We carried out cross-sectional malaria prevalence surveys in 10 riverine villages or “communities” on the banks of the Azul River, a tributary of the Moa River, all of them originated from former rubber-tapper settlements: Três Unidos, Valparaíso, Barro Vermelho, Belo Horizonte, Buritis, Nova Lição, Bom Sossego, Bela Vista, Bom Jesus and Queimadas . These vDuring the first survey, carried out between 9 and 12 July, 2018 (hereafter “2018 survey”), our field team carried out house-to-house visits in seven villages. We used a structured questionnaire to obtain sociodemographic and morbidity information from 246 individuals aged between 1 and 72 years, who were invited to provide a finger-prick blood sample for malaria diagnosis and haemoglobin measurement using a HemoCue Hb 301 haemoglobinometer . Anaemia was defined using the age- and sex-specific haemoglobin cut-off levels recommended by the World Health Organization (WHO).During the second survey, in 9-12 July, 2019 (hereafter “2019 survey”), residents in the area were invited to attend medical and dental care, routine vaccination and cervical cancer screening in health posts or schools situated in one of three largest villages . A total of 244 attendants aged < 1-75 years, coming from nine villages, were interviewed using the same structured questionnaire applied in 2018; 150 (61.5%) participants provided finger-prick capillary blood for malaria diagnosis by microscopy and the same number by molecular methods . Overall) According to the individuals’ perception, each clinical manifestation was classified as absent, mild, moderate or severe.At enrolment, participants were asked whether they had any clinical sign or symptom that might have been caused by malaria. Participants reporting recent signs or symptoms at or up to 7 days prior to the interview were classified as “symptomatic”. We next applied a semiquantitative questionnaire addressing 13 common symptoms  to all symptomatic individuals.Laboratory diagnosis of malaria - A total of 298 thick blood smears were prepared, stained with Giemsa and had at least 100 fields examined for malaria parasites, under 1,000x magnification, by an experienced microscopist.P. vivax infections diagnosed by onsite microscopy were treated with chloroquine (25 mg/kg of body weight over 3 days) and primaquine (3.5 mg/kg over 7 days); Plasmodium falciparum infections were treated with a 3-day course of artemether (2 to 4 mg/kg/day) plus lumefantrine (12 to 24 mg/kg/day) and a single dose of 0.25 mg/kg primaquine for gametocyte clearance.cytochrome b (cytb) gene of human-infecting malaria parasites,Fifty-millilitre capillary blood samples (n = 295) kept in liquid nitrogen in the field and later stored at -20°C were used for parasite DNA extraction, with QIAsymphony DNA Investigator kits , on an automated QIASymphony platform (Qiagen). The final DNA elution volume was 100 µL. Molecular screening for malaria was carried out with a genus-specific, SYBR Green-based quantitative PCR. The primer pairs  target a conserved sequence of the cytb gene fragments. The P. vivax protocol used the primers 5’-TTT GGT GGT ACT ACA GGA GTA ATA TTA GGT-3’ and 5’-GAA ATG AGC GAT TAC ATA GTA AGT ATC ATG-3’ and the probe 5’-VIC-TGC AGC TAT TGA TAT TGC AT-MGB-NFQ-3’ ; the P. falciparum protocol used the primers 5’-CAT TAT GAT TAC AGC TCC CAA GCA-3’ and 5’-GGT CTG ATT TGT TCC GCT CAA TA-3’ and the probe 5’ FAM -TAC AAG ATT GTG ATA AGA TGA C-NFQ-MGB-3’ . Each 20-µL reaction for both assays, carried out in in 96-well microplates, contained 5 µL of DNA solution and 10 µL of 2 TaqMan Universal Mater Mix II, no-UNG (Thermo Fisher Scientific). In the P. vivax protocol, we used 0.2 µM of each primer and 0.1 µM of the probe per reaction; in the P. falciparum protocol, we used 0.1 µM of each primer and 0.08 µM of the probe. The amplification cycles were identical for both species and comprised a 2-min step at 50°C followed by a 10-min denaturation at 95°C and 45 cycles at 95°C for 15 s and at 60°C for 1 min, carried out on a QuantStudio 6 real-time PCR system (Thermo Fisher Scientific). Standard curves were prepared with nine serial 10-fold dilutions of the respective target sequences and tested in each microplate to allow for species-specific quantitation of parasite loads (number of copies/µL of blood). The detection threshold was estimated at one amplicon copy per µL (approximately 20 parasites per mL) for P. vivax and 10-3 amplicon copies per µL (approximately 0.2 parasites per mL) for P. falciparum.Positive samples were further tested with newly designed TaqMan assays that target species-specific Data analysis - Data were analysed with STATA version 15.1 software . Statistical significance was defined at the 5% level (two-tailed tests). Proportions were compared by applying standard Fisher or chi-square tests to contingency tables. Multiple logistic regression models were run to identify independent correlates of TaqMan-confirmed P. vivax infection in both surveys combined, while adjusting for potential confounders. Individual-level variables included in the model were age , sex, bed net use past night , overnight stays in the forest in the last month for hunting or fishing (yes/no) and presence of any symptom within the past 7 days (yes/no) and presence of specific symptoms . The household-level variables were indoor residual spraying with insecticides within the past 6 months (yes/no) and wealth index (stratified into quartiles). Due to the nested structure of the data, we used the “melogit” STATA command to build mixed-effects logistic regression models that included the grouping variables as random factors. We had repeated observations for some participants , who were clustered within households . Covariates were introduced in the models following a stepwise forward approach, and only those that were associated with the outcome at a significance level of at least 20% were retained in the final model. Participants with missing information were excluded from adjusted analysis, except for those with missing symptom information, for which a missing-information category was created. We next used a similar multiple logistic modelling strategy to test whether anaemia was significantly associated with TaqMan-diagnosed P. vivax infection, while adjusting for age  and sex and excluding participants with P. falciparum infection diagnosed by microscopy or TaqMan assays. Ethics - The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Study protocols have been approved by the Institutional Review Board of the University Hospital of Universidade de São Paulo and by the National Committee of Ethics in Research, Brazilian Ministry of Health (CAAE number 64767416.6.0000.5467). Written informed consent was obtained from all study participants or their parents/guardians. Prevalence of malarial infection and disease - Overall, 31.8 and 24% of the participants tested for malaria parasites in the 2018 and 2019 surveys, respectively, reported recent clinical signs or symptoms that might be associated with malaria. This information was missing for six participants in 2018 and eight participants in 2019. cytb-based TaqMan assays during the surveys. Only three infections were diagnosed by microscopy . However, TaqMan assays detected 33 P. vivax infections , 78.1% of which were asymptomatic. Interestingly, no additional P. falciparum infections missed by microscopy was diagnosed by molecular methods. Parasitaemia was typically very low, with a median of 1.7 amplicon copy per µL . The highest parasitaemia was 12,143 copies per µL (corresponding to 243 parasites/µL), found in the only study participant with microscopy-positive P. vivax infection - an 8 years-old male child who lived in Barro Vermelho and reported eight past malaria episodes and no recent travel. This was also the only participant with a P. vivax density above the detection threshold of field microscopy, estimated at 100 parasites per µL,cytb gene copies per µL. P. vivax infection diagnosed by TaqMan, with Yates corrected chi-square = 0.576, 2 degrees of freedom (d.f.), p = 0.448 (missing information for 14 participants). Only three of the 32 P. vivax-infected participants with complete information reported recent fever , none reported chills and five reported headache  (missing information for one participant). Other symptoms were rarely reported. A single participant, the one with P. vivax infection diagnosed by both microscopy and TaqMan during the 2019 survey, had a full-blown malaria paroxysm at the time of enrolment. Similar proportions of participants who reported  or did not report recent clinical symptoms  had a P. vivax infections was similar .The proportions of infections that were symptomatic vs. asymptomatic did not vary significantly with age: these were 0 vs. 2 (0%) in the 0-5 years age group, 2 vs.7 (22.2%) in 6-15 years age group, 4 vs. 13 (23.5%) in the 16-49 years age group and 1 vs. 3 (25%) in the ≥ 50 years age group . Moreover, the mean age of participants with symptomatic (26.0 years) and asymptomatic (27.4 years) P. vivax did not vary significantly across villages: 7 vs. 62 in Três Unidos, 1 vs. 1 in Valparaíso, 3 vs. 29 in Buritis, 0 vs. 7 in Belo Horizonte, 4 vs. 36 in Nova Lição, 5 vs. 14 in Barro Vermelho, 10 vs. 84 in Bom Sossego, 0 vs. 7 in Bela Vista, 3 vs. 21 in Bom Jesus and 0 vs. 1 in Queimadas .The relative proportions of positive vs. negative TaqMan assay results for P. vivax positivity by TaqMan assays, after adjusting for age and sex, with an adjusted odds ratio (OR) of 0.59 and a 95% confidence interval (95%CI) of 0.27-1.29, p = 0.136 . Haemoglobin measurements were available for 262 participants who were screened for malaria. We found no significant association between anaemia and Correlates of P. vivax infection - We next sought to identify independent correlates of TaqMan-diagnosed P. vivax infection by using multivariable analysis (P. vivax infection was positively associated with age (p = 0.019). Other significant predictors of being infected were male sex  and use of an insecticide-treated bed net past night , after adjusting for age, sex and bed net use  To test this hypothesis, we are currently analysing the genetic connectivity between malaria parasites that circulate in rural and urban areas of the Juruá Valley.This study provides further evidence that clinically silent P. vivax infections persisted while P. falciparum infections are now infrequent in riverine villages? P. vivax may be less susceptible than P. falciparum to the malaria elimination strategies implemented in Brazil over the past 2 decades, which are focused on early microscopic diagnosis and prompt treatment of clinically apparent infections.P. vivax infections are often overlooked by routine microscopy and antigen-based rapid diagnostic tests because of their low parasitaemia,P. vivax infections, often before the laboratory diagnosis can be confirmed and antimalarial therapy is administered.P. vivax-infected individuals are likely to infect mosquitoes even when prompt diagnosis and treatment are widely available. Third, P. vivax sporozoites originate hypnozoites, dormant liver stages that can cause one or more relapses several weeks or months after the primary infection.P. vivax infections are estimated to relapse within 12 months, despite routine prescription of 3.5 mg/kg of primaquine over 7 days.) Therefore, high-dose primaquine regimens  may be required to eradicate hypnozoites in this region.,Why have ,,) First, we found a trend towards “increased” (instead of “decreased”) prevalence of P. vivax infection with increasing age (Table III). Such a trend is typically seen in non-immune migrants who have recently arrived in frontier farming settlements, where malaria affects mostly adult males and under-five children are usually spared.,) However, this is “not” the typical age-related prevalence gradient found among native Amazonians living in traditional villages, who are exposed to malaria since birth and become gradually less susceptible to infection.,,,P. vivax, but as likely as children to have symptoms once infected.Some of our current findings are at odds with previous observations suggestive of strong antimalarial immunity among rural Amazonians.P. vivax infections found in children and adults from isolated villages along Azul River. Virulent parasites are more likely to survive the within-host competition between co-infecting clones in high-transmission settings, where super- or co-infection events are common. In contrast, nonvirulent lineages may have a selective advantage in remote low-transmission settings, where parasite diversity is low due to the limited gene flow between villages and hosts are more likely to harbour single-clone asymptomatic infections.,36 are expected to remain undiagnosed and untreated and thus contribute to transmission over several weeks.-P. falciparum-dominated areas,P. vivax strains in co-infections with two or more lineages.) Larger studies with molecular analysis of the multiplicity of P. vivax infection are clearly needed to test this hypothesis in the Amazon and other settings.We hypothesise that low parasite virulence, in addition to acquired immunity, might also contribute to the high proportion of asymptomatic P. vivax infections and carriers with very low parasitaemia are expected to remain asymptomatic.P. vivax resistance factors, such as the Duffy-negative (Fy) phenotype.P. vivax infection may also be conferred by the Fya polymorphism,,Alternatively, human genetics, in addition to restricted reticulocyte tropism, may also contribute to limit parasite growth across all age groups. The risk of presenting symptoms is directly (but not linearly) proportional to blood-stage parasite density in P. falciparum infections precluded between-species comparisons of malaria risk factors in the study population.The present study has some limitations. First, we screened a relatively small population sample for malaria parasites. Participants may not be representative of the entire population of Azul River villages. Logistic limitations prevented house-to-house visits to be carried out in the smallest and most remote villages in the region. Second, individuals with current illnesses, either infectious or not, were overrepresented in our population sample, as they were more likely to seek health care during the field surveys. Third, we had no detailed information on key individual and contextual predictors of malaria risk, such as occupation and behaviour, cumulative exposure to infection and housing quality . Fourth, as both cross-sectional surveys were carried out at the end of the rainy season, we could not estimate the seasonal variation in malaria prevalence. Finally, the infrequency of P. vivax infections remain common, but are substantially underdiagnosed by microscopy in riverine villages in the main malaria transmission hotspot of Brazil. The vast majority of infections are asymptomatic, due to either clinical immunity or low parasite virulence. Periodic active case detection is required to identify parasite carriers who remain asymptomatic and are likely to fuel residual malaria transmission in this and other similar hard-to-reach communities across the Amazon.We conclude that


Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens , and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions . In addition, Principal Components Analysis (PCA) indicated that the first three components  accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication. Plasmodium falciparum is acquired among individuals living in malaria-endemic regions  were used to probe individual serum samples obtained from residents of a malaria endemic region in Uganda. Protein immunoreactivity was observed at 54% with 128 antigens inducing antibody responses that significantly associated with reduced risk to clinical malaria episodes  during a 12-months follow-up period. Of these antigens, 53 were down-selected as the most viable vaccine candidates by virtue of having a signal peptide (SP) and/or transmembrane domain (TM)  coupled with a homogeneous high-throughput AlphaScreen platform for antibody profiling and mechanistic characterization of proteins that have a role in merozoites invasion of erythrocytes, or induction of protective immunity in malaria naturally exposed individuals –11. Leveain (TM) . These sain (TM) .Previous studies with our follow-up cohort in Uganda  and simiSerum samples used in this study were obtained from residents of Lira Municipality, Northern Uganda, that were taking part in a prospective study of 66 non-vaccinated participants aged 6–20 years. The region was characterized by high malaria transmission , 20–23. P. falciparum  (CIDR: n = 108), Duffy binding–like domains of PfEMP1 (DBL: n = 163)  , repetitn = 163) . All pron = 163) .P. falciparum 3D7 reference strain. Briefly, the DNA sequences representing the ectodomains while excluding the SP and/or TM domains were amplified by using high fidelity PrimeSTAR Max DNA polymerase  and cloned into the WGCFS dedicated pEU plasmid vector  using the In-Fusion HD Cloning Kit (Takara Bio). A semi-automated GenDecoder1000 robotic protein synthesizer (CellFree Sciences) was used for in vitro transcription and mono-biotinylated recombinant protein synthesis with WGCFS.All the proteins were expressed from sequences derived from the P. falciparum protein. Specifically, the proteins were dispensed into a 384-well OptiPlate using a JANUS Automated Workstation dispenser  and mixed with 10 μl of 4000-fold diluted sera in reaction buffer . After 30 min incubation at 26°C, 10 μl of detection mixture containing streptavidin-coated donor beads (PerkinElmer) and protein G  conjugated acceptor beads (PerkinElmer) were added, to make a final concentration of 12 μg/ml for both beads. The plate was then incubated in the dark for 1 hr at 26°C to allow optimal binding of the donor and acceptor beads to the biotinylated protein and human antibody, respectively. Luminescence emitted by acceptor beads upon excitation of the donor beads was detected using an EnVision plate reader (PerkinElmer) and captured as AlphaScreen Counts (ASC). To account for day-to-day and plate-to-plate assay variability, serially diluted biotinylated rabbit IgG (PerkinElmer) was included in each plate, and subsequently used to generate a 5-parameter logistic standardization curve. The assays were randomized to minimize experimental bias.We used the AlphaScreen platform to assess serum antibodies in malaria exposed individuals as described , 24. TheAll data analyses were performed using R software . As previously described, protein seropositivity cut off point to human sera was set at half the lowest non-negative ASC value from that of the assayed samples , and a pFor survival analysis, the time-to-first clinical malaria episode from baseline  was used as the endpoint . For detTo simultaneously evaluate antibodies developed in response to different proteins among patients with and without clinical malaria, we used Principal Component Analysis (PCA). PCA essentially reduces the dimensionality of antibody responses by generating fewer composite variables to capture as much variance in that dataset. The model then assesses and compares the relationship between the scores obtained for each of these components per subject. To generate the global antigen derived principal components, all immunoreactive antigens were used without prespecified groupings  to determine group-based variance. To identify PCA derived clusters of antibody responses that were involved in protection against clinical malaria, we selected principal components where at least three variables were loaded, and the eigenvalue was greater than 2 or the proportion of variance explained was >5% . Individp = 0.001) with STEVORs having significantly lower median levels, and SURFINs the highest compared to other groups. Although this may represent cross-reactivity within the assessed families  suggests that antibodies to different antigens are co-acquired in this population.We assessed the human antibody reactivities to the protein library Table S1families , a stronTo gain further insight into the relationship between individual protein reactivity and clinical malaria outcomes, we determined the hazard ratios based on time-to-event using three parasite thresholds for clinical malaria in univariate analysis. Based on the lower threshold of 1,000 parasites/µl blood, antigen-specific antibodies to 43 proteins associated with protection. This changed to 26 and 32 proteins when assessed at 2,500 parasites/µl blood and a higher threshold of 5,000 parasites/µl blood, respectively Table 1 Since the antibody responses and the corresponding semi-protective immunity involve numerous antibodies acting simultaneously but targeting different proteins , 29, we When assessing the representation of antibody responses to different protein groups based on Dim1 and Dim2, we observed that most clinical malaria cases clustered together irrespective of the protein group or family  but not with Dim2  or Dim3   Figure 4Here, using antibody response data from our previous publications on multi-gene families , 11, 13 We observed strong immune responses with high seroprevalence in the different protein families. This could be due to high level of exposure to these abundantly expressed proteins or cross-reactivity among the conserved regions of the domains . AlthougP. falciparum isolates  family member consisting of a combination of short tandems that is associated with severe malaria –34. Antiisolates . IdentifP. falciparum glutamate-rich protein (GLURP)  –44. PfMA (GLURP) . RecentlThe need for continued development of robust malaria intervention tools including vaccines across different malaria endemicities  calls foWe investigated whether a specific combination of antibody responses to the recombinant proteins evaluated could be associated with key malaria severity factors such as age. PCA is useful for exploring multi-dimensional data, mixed infections and complex host-parasite interactions. To this end, and to capture the joint effects of all antibodies in a single analysis in biological conditions, we investigated the relationship between the overall antibody responses by PCA, a factor analysis approach accommodating the fact that protective antibody responses are likely to involve a number of antibodies working together. With the current dataset, the first three principal components accounted for the majority of the outcomes observed Table 2.P. falciparum protein microarrays have been designed over the past decade to identify malaria vaccine candidate antigens. One of the most important characteristic in this study is the use of WGCFS for recombinant protein production. In contrast to E. coli- , and Uganda National Council for Science and Technology . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ET and BK conceived and designed experiments. HN, MM, and IH conducted experiments. ET, BK, and TT analyzed the data. ET, BK, NP, TE, TH, JG, and TT wrote the manuscript. All authors discussed and edited the manuscript.This work was funded in part by JSPS KAKENHI  and in part by Strategic Promotion of International Cooperation to Accelerate Innovation in Africa by MEXT, Japan. BK is an EDCTP Fellow under EDCTP2 programme supported by the European Union grant number TMA2020CDF-3203. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.